EPA-600/8-78-017

December 1978

MICROBIOLOGICAL METHODS FOR MONITORING

THE ENVIRONMENT

Water and Wastes

Edited by
Robert Bordner and John Winter
Environmental Monitoring and Support Laboratory-Cincinnati
Cincinnati, Ohio 45268
and Pasquale Scarpino, University of Cincinnati
Cincinnati, Ohio 45219

Prepared in part under EPA Contract No. 68-03-0431

Project Officer

John Winter
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268

ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268

@ Printed on Recycled Paper

 DISCLAIMER

This report has been reviewed by the Environmental Monitoring and Support Laboratory-
Clnclnnati, U.S. Environmental Pr:otection Agency and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.

Ii &EPA MICROBIOLOGICAL MANUAL 1978

 .FOREWORD

Environmental measurements are required to determine the quality of ambientwaters and the
character of waste effluents. The Environmentar Monitoring and Support Laboratory (EMSL}-:-
Cincinnati conducts research to: ' ·

• Develop and evaluate techniques to measure the presence and concentration of

physical, chemical, and radiological pollutants in water, wastewater, bottom
sediments, and solid waste.

• Investigate methods for the concentration, recovery, and identification of viruses,

bacteria and other microorganisms in water.

• Conduct studies to determine the responses of aquatic organisms to water quality.

• Conduct an Agency-wide quality assurance program to assure standardization and

quality control of systems for monitoring water and wastewater.

This publication of EMSL-Cincinnati, contains the methods selected by consensus of EPA

senior microbiologists for parameters of interest to the Agency. Federal agencies, states,
municipalities, universities, private laboratories, and industry should find this manual of assistanpe
in monitoring and controlling microbiological pollution in the environment.

Dwight G. Ballinger
Director, EMSL-Cincinnati

''..
iii
 PREFACE

The Federal Water Pollution Control Act Amendments of 1972, the Marine Protection,
Research, and Sanctuaries Act of 1972, and the Safe Drinking Water Act of 1974, require that EPA
develop and select methods for environmental monitoring and research on public and private
water supplies, rivers, lakes, ground waters, wastewaters and the marine environment for the
purposes of setting and enforcing environmental standards and ultimately enhancing the quality of
the environment. This manual of methodology supports these needs.

Under the direction of a Steering Committee formed for the development of an Agency
microbiology manual, a seminar was held among representative Agency microbiologists in San
Francisco, January, 1973. Assignments were made to committee members for the preparation of
first draft material. The basic design, format and content of the manual, were established and the
first drafts presented and reviewed at the sP.cond meeting of the Committee in January, 197 4 at
Cincinnati.

The drafts submitted by the Steering Committee members were formatted and developed into
the initial version under EPA Contract No. 68-03-0431 by Dr. Pasquale Scarpino, Professor of
Environmental Engineering, Department of Civil and Environmental Engineering, University of
Cincinnati, working with the two EPA editors: Robert Bordner, Chief Microbiology Section,
Biological Methods Branch and John Winter, Chief, Quality Assurance Branch, both of EMSL-
Cincinnati. Subsquently, these editors added technical detail and the necessary information
reflecting Agency policies. Valuable source documents for This Manual were Current Practices in
Water Microbiology, National Training and Operational Technology Center and Handbook for
Evaii:iatlng Water Bacteriological laboratories, Municipal Environmental Research Center, both of
U.S. EPA, Cincinnati, Ohio. The refined product is presented here.

Comments or questions concerning the manual should be directed to:

Robert Bordner or John Winter

U.S. Environmental Protection Agency
EMSt.:-Cincinnati
Cincinnati, OH 45268

iv &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE OF CONTENTS

Page

Foreword iii
Preface iv
Figures vii
Tables x
Acknowledgements xv

PART INTRODUCTION 1

PART II GENERAL OPERATIONS

A. Sample Collection, Preservation and Storage............................................... 5

B. Laboratory Equipment, Techniques and Media............................................. 32
C. Isolation and Enumeration of Bacteria ........................... ,................................ 59
D. Selection of Analytical Methods......................................................................... 91

· PART Ill ANALYTICAL METHODOLOGY

A. Standard Plate Count ......... ,.................................................................................... 101

B. Total Coliforms ............................................................................·............................... 108
C. Fecal Coliforms .......................................................................................................... 1 24
D. Fecal Streptococci ................................................................................ ,................... 135
E. Salmonella.................................................................................................................... 15 4
F. Actinomycetes............................................................................................................. 186

PART IV QUALITY CONTROL

A. Laboratory Operations............................................................................................. 194

B. Statistics for Microbiology.................................................................................... 225
C. Analytical Quality Control Procedures.............................................................. 231

PART V LABORATORY MANAGEMENT

A. Development of a Quality Control Program.................................................. 244

B. Manpower and Analytical Costs......................................................................... 246
C. Safety............................................................................................................................ 259
D. Legal Considerations................................................................................................ 277

v
 APPENDICES

A. Microbiological Activities under the Water Laws........................................ 289

B. Certification of Water Supply Laboratories.................................................... 297
C. Bibliography................................................................................................................. 324

INDEX 325

vi &EPA MICROBIOLOGICAL MANUAL 1978

 •I •,•
:·,·

FIGURES
.~ .

Number Page

11-A-1 Suggested Sample Containers ........................................................................ 7

11-A-2 Demonstration of Technique Used in Grab Sampling of Surface

Waters ...................................................................................................................... 9

11-A-3 Weighted Bottle Frame and Sample Bottle for Grab Sampling ..... . 10

11-A-4 Zobell J-Z Sampler .............................................................................................. 11

11-A-5 Niskin Depth Sampler ........................................................................................ 12

11-A-6 New York State Dept. of Health Depth Sampler ................................. . 13

11-A-7 ·Kemmerer Depth Sampler ................................................................................ 15

11-A-8 Van Donsel-Geldreich Sediment Sampler ................................................... 16

11-A-9 Example of a Sample Label ........................................................................... 18

11-A-10 Field Data Record ................................................................................................ 18

ll-A-11 Sample Log Sheet ............................................................................................... 20

ll-A-12 Chain of Custody Record ................................................................................. 21

ll-A-13 Sampling a Water Supply Reservoir ........................................................... 23

ll-A-14 Sampling a Lake or lmpoundment............................................................... 26

. ll-A-15 Sampling a Large Stream ................................................................................ 27

11-B-1 Finger-mounted Pipetting Device .................................................................... 35

11-B-2 Enlargement of Pipetting Device Tip ........................................................... 35

11-C-1 Microbiological Bench Cards for MF Analyses ....................................... . 60

11-C-2 Bench Cards for MPN Analyses .................................................................... 60

11-C-3 Combined Microbiological Bench Card 61

vii
 11-C-4 Preparation of Decimal Dilution ..................................................................... 63
11-C-5 Suggested Pattern' for Preparing a Streak Plate ............................ ,•...... 67
11-C-6 Membrane Filtration Units Made by Various Manufacturers for
Detection of Bacteria in Aqueous Suspensions ...................................... 72

11-C-7 Exploded View of a Stainless Steel Membrane Filtration Unit 73

11-C-8 Colony Counting Pathway ..................................................................,......... .. 76

11-C-9 Enlarged Portion of Grid-Marked Square of Filter ........................ T ....... . 76

11-C-10 Packaging and Labelling of Microbiological Cultures for

Shipment ........................................................................................................,........ . 89

111-A-1 Typical Dilution Series for Standard Plate Count .......................... ,.,... .. 103

111-B-1 Verification of Total Coliform Colonies on the Membrane Fiiter 115

111-B-2 Flow Chart for the Total Coliform MPN Test ........................,.............. .. 116

111-C-1 Verification of Fecal Coliform Colonies on the Membrane Fllt~r .... 131

111-C-2 Flow Chart for the Fecal Coliform MPN Test ...................,........... ,...... . 133

111-D-1 Verification Procedure for Fecal Streptococci ...............:........,....... ,... 1... ,. 140

111-D-2 Isolation and Identification of Fecal Streptococcci, Genera!

Scheme .....................................................................................................••....,•.. ,..... 141

111-D-3 Identification of Fecal Streptococci, Separation of Enterococcµs

Group by Species and by Original Source of Culture ........ :·•'"'!'":'" 148

111-D-4 Identification of Fecal Streptococci, Separation of Enterococ:ci

from Vegetation, Insect and Animal Sources .......................................... 151

111-E-1 Scheme for the Concentration, Isolation and Identification of

Salmonella ............................................................................................,.. ,, .. ,.... ,...... 15 6

111-E-2 Simplified Scheme for Concentration, Isolation and Identification

of Salmonella ......................................................................................................... 167

111-E-3 Dimensions of the Gauze Swabs .................................................................. 158

111-E-4 The Gauze Swab in Position ......................................................,......... ,.. ,. .... . 159

111-F-1 A Plate Containing Bacterial and Actinomycete Colonies ............... ,.. . 189

viii SEPA MICROBIOLOGICAL MANUAL 1978

111-F-2 An Actinomycete Colony Showing the Branching Filaments that
Cause the Fuzzy Appearance of its Border ......................................... . 190

111-F-3 A Bacterial Colony with its Relatively-Distinct, Smooth Border ...... ~. 190

IV-.A-1 Equipment Operation Temperature Record ................................................ 217

IV-B-1 Normal Distribution Curve ................................................................................ 228

IV-B-2 Positively-Skewed Distribution Curve ............................................................ 228

V-C-1 Laminar Flow Cabinet ...................................................................................,... . 270

V-C-2 Example of Biohazard Sign .:......... :..... :.................................. :........................ 271

ix
 'j

'I

TABLES.

Number Page

11-A-1 Sampling Frequency Based on Pop~lation ................................................ 25

11-B-1 Relationship of Steam Pressure to T~mp~rature in the Autoclave. 37

11-C-1 Recommended Filtration Volumes of Samples in MF Ana.lyses ...... .

11-C-2 Number of Significant Figures (S.F.) Reported ..............................:........ .

11-C-3 Acceptable Limits ...... :.......................:..................................................................

11-C-4 Most Probable Number Index and 95% ·Confidence Limits for
Five Tube, Three Dilution Series ..:................... :............................................ 82

11-C-5 Most Probable Number Index and 95% Co~fidence Limits for
Testing Potable Waters ........ ,.............................................................................. 83

11-C-6 Selection of Code. Results, ,Five Tube Series ........................................... . 85

11-D-1 Approved Test Proce~ures for the Analyses of Pollutants (40

CFR 136) ..................................................,.............................................................. 93
.. . ., \. - . .. ,, .
11-D-2 Water Quality Standards ................................................................................... 94
11-D-3 Water Quality Criteria ........................................................................................ 95

11-D-4 Selection of Methods for P'roblem Samples ..................... :................ :.... . 98

111-8-1 Differentiation of the Cqliform and Related Organisms Based on

Biochemical Reactions.'............................. :., ............... .'....... :...................... :....... . 120

111-C-1 Suggested Range of Sample Volumes for Fecal Coliform Tests

Using the Memb.rane .Filter Methods.~ ................... :.. ,................................. . 127

111-E-1 Colonial Appearance of Salmonella and Other Enterics .on

Isolation Media ..................
. ~ ..'.. :.... .'................................. :......... :............................
. . 166 .

111-E-2 Production Rate and Time Requirements of Multitest Systems ...... . 174

x &EPA MICROBIOLOGICAL MANUAL 1978

111-E-3 Reported Shelf-Life of Multitest Systems With or Without .............. .
Refrigeration ............................................................................................................ 174

111-E-4 Cost and Source of Multitest Systems ...................................................... 175

111-E-5 Biochemical Characteristics of the Enterobacteriaceae ....................... .. 176

IV-A-1 Monitoring Laboratory Equipment.: ...... !........................................................ . 212

IV-A-2 Glassware Maintenance ...................................................................................... 218

IV-A-3 Laboratory Pure Water for Bacteriological Testing .............................. .. 219
IV-A-4 Quality Control of Media .................................................................................. 220
Quality Control of Biochemical
: .·
Tests .........................................................
... . 222

IV-B-1 Microbiological Results, count/ 100 ml ......................................................

. . { .. . . 225

IV-B-2 Coliform Counts and Their. Logarithms ....................................................... 229

IV-B-3 Comparison of Frequency of MPN Data .................................................... 230

IV-B-4 Comparison of Frequency of Log MPN Data ......... ,.............. :................ .. 230

IV-C-1 Raw Sample Data from the Analysis of Chlorinated Sewage ........ .
Treatment Plant Effluents.~ .................. .'... :........ ~·........... ~ ........................... :.: ..... . 237
IV-C-2 Logarithmic Transformation of the Data in Table IV-C-1 ................... .
: .
IV-C-3 Analysis of Difference Between Means ....................... :....... :............. :·....... . 241
V-B-1 Estimated Time Required·. fo.r Twenty MPN Analyses ................... ~ ..... .. 248
V-B-2 General Equipment and Supplies Minimum Program, Yearly .Basis 249
V-B-3 Equipment and Supplies for MF Analyses Minimum Program ... ,.....

V-B-4 Equipment and Supplies for MPN Analyses Minimum Program ...... 253

V-B-5 General Equipment and Supplies Full Program in Microbiology .... .

Weekly Basis ..................... .'.....................'.................... :........ :.................. :............ . 254

V-B-6 Equipment and Supplies for MF Analyses Full Program in ..............

Microbiology, Weekly Basis ... :...................................................:..... :......... :.... . 256

V-B-7 Equipment and Supplies for MPN Analyses Full Program in .......... .
Microbiology, We.ekly Basis ............ :................................................................ 257

V-B-8 Media for Full Program in · Microbio'logy Laboratory Usag~ for ...... .
each Week/ 100 Samples .............................................................;.......... :: .... .. 258
.. '

xi
V-C-1 Laboratory-acquired Infections Related to Personnel and Work ...... . 260

V-C-2 Sources of Laboratory-acquired Infections ................................................. 261

V-C-3 Normal Use Concentration of Disinfectants .................................................. 267

xii &EPA MICROBIOLOGICAL MANUAL 1978

 THE MICROBIOLOGY METHODS STEERING COMMITTEE
OF EPA

Cochairpersons: Robert Bordner and John Winter

Environmental Monitoring and Support Laboratory-Cincinnati

Members:

William Stang Harold Jeter (retired)

Edwin Geldreich Francis Brezenski
Kathleen Shimmin

xiii
 CONTRl.l~UTORS BY SECTION

Sampling ;,. Salmonella .

William Stang Kathleen Shimmin
NEIC-Denver Alameda Laboratory
Region IX
General Laboratory Equipment/Media
Robert Bordner and John Winter Donald Spino
EM SL-Cincinnati MERL-Cincinnati

Pasquale Scarpino Actinomycetes

UC Dept. of Environ. Engineering Robert Safferman
EMSL-Cincinnati
Isolation and Enumeration of Bacteria
Robert Bordner and John Winter Quality Control
EMSL-Cincinnati Robert Bordner
EMSL-Cincinnati ·
Pasquale Scarpino
UC Dept. of Environ. Engineering Development of a Quality Control Program
John Winter
Selection of Analytical Methods EMSL-Cincinnati
Robert Bordner and John Winter
EMSL-Cincinnati Manpower and Analytical Costs
Robert Bordner and John Winter
Standard Plate Count EMSL-Cincinnati
Raymond Taylor
MERL-Cincinnati Legal Considerations
Dave Shedroff
Total Coliforms Office of Enforcement
Harold Jeter (retired) Washington, DC
National Training Center
ERC-Cincinnati Carroll Wills
NEIC-Denver
Fecal Coliforms
Edwin Geldreich Safety
MERL-Cincinnati Robert Bordner and John Winter
EM SL-Cincinnati
Fecal Streptococci
Francis Brezenski Pasquale Scarpino
Region II UC Dept of Environ. Engineering

xt~ &EPA MICROBIOLOGICAL MANUAL 1978

 ACKNOWLEDGEMENTS

The Committee wishes to acknowledge the many EPA microbiologists and others who
participated in the development or review of the manual. These include, in regional and program
order:

Region
Howard Davis and Edward Gritsavage
Regional Laboratory
Needham Heights, MA

Victor Cabelli, Alfred Dufour . and Morris Levin

Environmental Research Laboratory
Narragansett, RI

Region II
Isidore Seidenberg (retired)
Edison Water Laboratory
Edison, NJ

Region Ill
Leonard Guarraia Don· Lear'
Office of Water & Hazardous Materials Annapolis Field Station
Washington, DC · Annapolis, MD

Region IV
Bobby Joe Carroll and Ralph Gentry Al Bourquin
S & A Division, SERL Pensacola Station
Athens, GA Pensacola , FL

Region V
James Adams
Central Regional Laboratory
Chicago, IL

Region VI
Harold Cumiford
Houston Facility
Houston, TX

Region VII
Carl Bailey
Regional Laboratory
Kansas City, MO

xv···
 Region VIII
John Manhart
Regional Laboratory, Denver Federal Center
Denver, CO

Region IX
Harold Scotten
Alameda Laboratory
Alameda, CA

Region X
George J. Vasconcelos and Richard Bauer
Regional Laboratory
Seattle, WA

Martin Knittel
Environmental Research Laboratory
Corvallis, OR

Ronald Gordon
Alaska Water Laboratory
College, AK

Cincinnati Environmental Research Center

Joseph Santner and Rocco Russomanno
National Training Center

Martin Allen, Harry Nash and Don Reasoner

Municipal Environmental Research Laboratory

Louis Resi
Division of Technical Support

Bernard Kenner (retired)

Municipal Environmental Reserach Laboratory (AWTRL)

Paul Britton, Terry Covert and Herbert Manning

Environmental Monitoring and Support Laboratory

Elmer Akin and Walter Jakubowski

Health Effects Research Laboratory

PREPARATION OF THIS VOLUME

The editors acknowledge gratefully the excellent technical skills of organiza~ion, prQofreading,
typing and computerized text editing performed by M. Mary Sullivan. Her contribution of hard work
and sacrifice of personal time to this manual cannot be overstated.

xvi &EPA MICROBIOLOGICAL MANUAL 1978

 PART I. INTRODUCTION

As the only direct measures of pollution by man and other warm-blooded animals,
microbiological parameters contribute unique information on water and wastewater quality and
public health risk from waterborne disease. Microbiological analyses are conducted to:

Monitor ambient water quality for recreational, industrial, agricultural and water supply uses,

Assure the safety of potable water,

Monitor municipal and industrial discharges,

Identify the sources of bacterial pollutants,

and evaluate water resources.

Role of the Aquatic Microbiologist

Although their primary role is to produce valid data for management decisions,
microbiologists should also participate in survey planning and evaluation, develop new microbial
parameters and methodology, consult on microbiological problems, establish and monitor criteria
and standards, testify in administrative hearings and court cases, train laboratory staffs and .
research special problems. Microbiologists should also go beyond sanitary microbiology to solve
taste and odor problems, to study microbiological transformations, and to apply other
measurements to the aquatic ecosystem.

Scope of the Microbiology Manual Series

This EPA manual provides uniform laboratory and field methods for microbiological analyses
of the environment. The analytical methods are standardized procedures recommended for use in
enforcement, monitoring and research. However, they are not intended to inhrbit or prevent
methods research and development. Exploratory and developmental methods are compiled sepa-
rately for evaluation but are part of the EPA Microbiology Manual Series.

The environmental areas covered will include:

• All waters - fresh, estuarine, marine, shellfish-growing, agricultural, ground, surface,

finished, recreational and industrial processing.

INTRODUCTION 1
 • All wastewaters of microbiological concern - domestic waste effluents, industrial
wastes such as food, dairy, meat, tanning, sugar, textile, pulp and paper, shellfish
processing and agricultural wastes such as feedlot and irrigation runoff.

• Other areas of the environment - air, sediments, soils, sludges, oils, leachates,
vegetation, etc.

Coverage of the First Edition ofthe Manual

Although the scope of the Manual Series is broad and inclusive of many parameters and
sample types, the first edition describes primarily the analytical methods that meetthe immediate
needs of the Agency. These are the key parameters that are accepted and used for water quality,
compliance monitoring and enforcement under Federal Water Pollution Control Act, PL 92-500,
Marine Protection, Research, and Sanctuaries Act, PL 92-532 and the Safe Drinking Water Act, PL
93-623. The necessary supportive sections include: sample collection, equipment and techniques,
cultural media, glassware preparation, quality control, data handling, safety, legal considerations
and selection of analytical methods.

Focus of the Manual

This Manual is intended for use by the supervisor or analyst who may be a professional
microbiologist, a technician, chemist, engineer or plant operator. Regardless of other skills, the
supervisor and analyst should have received at least two weeks training in each parameter from a
federal or state agency or from a university.

To assist the new analyst, Part II has been prepared as a basic discussion on laboratory operations
and for general guidance to permit use of the manual by those required to do microbiological
analyses. The trained analyst will be familiar and knowledgeable of most of these techniques.
The analytical procedures in Part Ill are written in a stepwise manner so that the manual can be
used both at bench level and as a reference book. Part IV emphasizes the important, but often neglect-
ed need for quality control in microbiological analyses, while Part V describes general considerations
for laboratory management.

Objectives

The objectives of This Manual are to:

• Select the best method currently available for use in the environmental monitoring,
compliance monitoring, enforcement and research activities of the Agency.

• Establish uniform application of microbiological methods so that only the best methods
are used and perpetuated, data from different laboratories or surveys can be fairly
compared and/or results can be stored in a common data bank, e.g., STORET, for later
use.
• Provide guidance on the use of these methods, their" advantages, limitations and
application to various types of water and wastes.

• Establish recognized procedures for method selection and evaluation that will form the
baseline against which other tests forthe same or new parameters can be measured.

• Emphasize the analytical quality control and management practices that should be
performed in the laboratory to assure valid data.

2 oEPA MICROBIOLOGICAL MANUAL 1978

Criteria

The first edition of This Manual describes the parameters of health and sanitary significance.
In the future, the criteria for addition of a method to the Manual are:

• The method is required to satisfy new or changing needs of the Agency.

• The method is practical for field and laboratory use. Equipment, supplies and' media are
available and the procedure provides results within reasonable time limits. •

• The method offers significant advantages over current methods.

• The method has been validated by the developer or by others according to the criteria
for Comparative Testing of Methodology and Method Characterization. (See IV-C-1 ).

• The method criteria and characterization have been reviewed and accepted by.the EPA
Steering Committee for Microbiology.

INTRODUCTION 3
 PART II. GENERAL OPERATIONS

This Part describes the general procedures which are applicable to the methods of analysis for
all parameters. The Sections provide the basic background information that must be understood
when the analytical procedures are carried out. The procedures are divided here into broad areas
offunction:

Section A Sample Collection, Preservation and Storage

Section B General Laboratory Equipment, Techniques and Media

Section C Isolation and Enumeration of Bacteria

Section D Selection of Analytical Methodology

4 oEPA MICROBIOLOGICAL MANUAL 1978

 PART II. GENERAL OPERATIONS

Section A . Sample Collection, Preservation and S,torage

Collection, preservation and storage 9f This Section is organized as follows:

water samples are critical to the results of
water quality analyses., The data are only as
valid as the water sample. 1. Sample Containers

A sampling program must be planned to 2. Sampling Techniq\JeS

satisfy the objectives of the study yet remain
within the limitations of available manpower, Composite Sampling
time and money. The survey should use the Surface Sampling by Hand
minimum number of samples that adequately Surface Sampling by
represent the effluent or body of water from Weighted Bottle Frame
which they are taken. The number of samples Depth Sampling
and location of sampling sites should be deter- Soil Sampling
mined prior to the survey and must satisfy the Sediment Sampling
requirements needed to establish water qual- Water Tap Sampling
ity standard or effluent permit violations.
3. Sample Identification and
The microbiologist should participate in Handling
the planning which specifies the microbiologi-
cal tests needed, the number of analyses to be 4. Chain of Custody Procedures
performed, and the equipment required. Con-
sideration should be given to the weather and 5. Selection of Sampling Sites and
other local conditions prior to the formulation Frequency
of a final plan. For example, seasonal varia-
tions in water temperature and flows would be Potable Water Supplies
important factors in deciding when to study Lakes and Impoundments
the effects of thermal pollution on bacteria. Stream Sampling
Sample collectors must know the exact loca- Marine and Estuarine
tion of the sampling sites and be fully trained Sampling
in the aseptic technique of sample collection Domestic and Industrial
as well as the use of any specialized sampling Waste Discharges
equipment. The sample collector is responsi- Recreational Waters
ble for the recording of all pertinent informa- Shellfish-Harvesting Waters
tion about the sample that might be significant Frequency of Sampling
in the evaluation and interpretation cif the
laboratory data or that might be necessary in 6. Preservation and Transit of
p9tential enforcement action. Samples

.· SAMPLING TECHNIQUES 5
1. Sample Containers 1.5 Wrapping Bottles: Protect the tops
and necks of glass stoppered bottles from
contamination by covering them before sterili-
1.1 Sample Bottles: Bottles must be resis- zation with aluminum foil or kraft paper.
tant to sterilizing conditions and the solvent
action of water. Wide-mouth borosilicate glass
bottles with screw-cap or ground-glass stopper 1.6 Sterilization of Bottles: Autoclave
or heat-resistant plastic bottles may be used glass or heat-resistant plastic bottles at 1 21 C
if they can be sterilized without producing toxic for 15 minutes. Alternatively, dry glassware
materials (see suggested sample containers in may be sterilized in a hot air oven at 170 C for
Figure 11-A-1 ). Screw-caps must not produce not less than two hours. Ethylene oxide gas
bacterlostatic or nutritive compounds upon sterilization is acceptable for plastic containers
sterilization. that are not heat-resistant. Sample bottles
sterilized by gas should be stored overnight
before being used to allow the last traces of
1.2 Selection and Cleansing of Bottles: gas to dissipate. See Part 11-B, 3 for steriliza-
Sample bottles should be at least 1 25 ml vol- tion procedures.
ume for adequate sampling and for good mixing.
Bottles of 250 ml, 500 ml and 1000 ml volume
are often used for multiple analyses. Discard 1.7 Plastic Bags: The commercially-
bottles which have chips, cracks, and etched available bags (Whirl-pak) are a practical sub-
surfaces. Bottle closures must be water-tight. stitute for plastic or glass sample bottles in
Before use, thoroughly cleanse bottles and sampling soil or sediment. See Figure 11-A-1.
closures with detergent and hot water, fol- The bags are sealed in manufacture and opened
lowed by a hot water rinse to remove all trace only at time of sampling. The manufacturer
of detergent. Then rinse them three times with states that such bags are sterilized.
laboratory-pure water (11-B,6). A test for the
biological examination of glassware where
bacteriostatic or inhibitory residues may 2; Sampling Techniques
be present, is described in Part IV-A, 5.1.

Samples are collected by hand or with a

1.3 Dechlorinating Agent: The agent sampling device if (1) depth samples are re-
must be placed in the bottle when water and quired or (2) the sampling site has difficult
wastewater samples containing residual chlo- access such as a manhole, dock, bridge or
rine are anticipated. Add sodium thiosulfate to bank adjacentto a surface water.
the bottle before sterilization at a concentration
of 0.1 ml of a 10 percent solution for each
125 ml (4 oz.) sample volume (1 ). This concen- 2.1 Chlorinated Samples: When samples,
tration will neutralize approximately 15 mg/I such as treated waters, ch.lorinated wastewa-
of residue chlorine. ters or recreational waters, are collected, the
sample bottle must contain a dechlorinating
agent (see this Section, 1.3).
1.4 Chelating Agent: A chelating agent
should be added to sample bottles used to
collect samples suspected of containing 2.2 Composite Sampling: In no case
>0.01 mg/liter concentrations of heavy metals should a composite sample be cOileCted for
such as copper, nickel or zinc, etc. Add 0.3 bacteriological examination. Data from indi-
ml of a 15 percent solution of ethylenedia- vidual samples show a range of values. A com-
minetetraacetic acid (EDTA) tetrasodium salt, posite sample will not display this range. Indi-
for each 125 ml (4 oz.) sample volume prior to vidual results will give information about in-
sterilization (2, 3). dustrial process variations in flow and compo-

6 oEPA MICROBIOLOGICAL MANUAL 1978

A B c

FIGURE 11-A-1. Suggested Sample Containers

A Screw-~ap Glass or Plastic Bottle.

B Plastic Bag (Whirl-pak).

C Glass Stoppered Bottle.

• I<•

SAMPLING TECHNIQUES 7
sition. Also, one or more portions that make up samples froni lakes, reservoirs, estuaries and
a composite sample may contain toxic or nutri- the oceans. These depth samplers require low-
tive materials and cause erroneous results. ering the sampling device and/or container to
the desired depth, then opening, filling, and
2.3 Surface Sampling by Hand: A grab clo·sing the container and returning the device
sample is obtained using a sample bottle pre- to the surface. Although depth measurements
pared as described in 1. above. Identify the are best made with a pre-marked steel cable,
sampling site on a chain of custody tag if the sample depths can be determined by pre-
required, or on the bottle label and on a field measuring and marking the nylon rope at inter-
log sheet (see 3). Remove the bottle covering vals with a non-smearing ink, paint, or finger-
and closure and protect from contamination. nail polish. The following list of depth sam-
Grasp the bottle at the base with one hand and plers is not inclusive but can serve as a guide:
plunge the bottle mouth down into the water to
avoid introducing surface scum. Position the 2.5.1 ZoBell J-Z Sampler: This sampler
mouth of the bottle into the current away from described by ZoBeITTn 1941 (4) was designed
the hand of the collector and away from the for deep sea sampling but is also used in fresh
side of the sampling platform or boat (see waters. Figure 11-A-4 shows its general ap-
Figure 11-A-2). The sampling depth should be pearance. It has a metal frame (A), a heavy
15 to 30 cm (6 to 12 inches) below the water metal messenger (8), a sealed glass tube (C)
surface. If the water body is static, an artificial attached to a rubber tube (D), and a sterile
current can be created, by moving the bottle 350 ml glass bottle (E) or a collapsible neo-
horizontally in the direction it is pointed and prene rubber bulb for shallow waters. The
away from the sampler. Tip the bottle slightly messenger (8) is released at the surface when
upwards to allow air to exit and the bottle to the sampler reaches the 'desired depth, and
breaks the glass tubing (C) at a file mark. The
fill. After removal of the bottle from the stream,
bent rubber tubing (D) then straightens out and
pour out a small portion of the sample to allow
the water is drawn in several inches from the
an air space of 2.5 to 5 cm (1 to 2 inches)
sampler. A partial vacuum created by auto-
above each sample for proper mixing of the
claving of the sealed unit draws the water into
sample before analyses. Tightly stopper and
the bottle.
label the bottle.
2.5.2 Niskin Sampler: This is sometimes
2.4 Surface Sampling by Weighted Bot- called a sterile-bag or "Book" sampler (see
tle Frame: When sampling from a bridge or Figure 11-A-5) (5). A messenger triggers the
other structure above a stream or body of opening of two plates (A) in V-fashion by spring
water, the sample collector places the bottle in power, and causes the sterile plastic bag (8) to
a weighted frame (see Figure 11-A-3) that holds inflate. At the same time a plastic filler tube (C)
the bottle securely. Remove cover and lower leading to the plastic container is cut by a
the device to the water. It is preferable to use guillotine knife (D) and the bag fills with water.
nylon rope which does not absorb water and The bag is then automatically sealed with a
will not rot. Face the bottle mouth upstream by clamp (E) and the apparatus is brought to the
swinging the sampling device first down- surface. Samplers are available that will hold
stream, and then allow it to drop into the water, 1, 2, 3, or 5 liters of water.
without slack in the rope. Pull the sample de-
vice rapidly upstream and out of the water, 2.5.3 New York Dept. of Health Depth
thus simulating the scooping motion of grab Sampler: This device (see FiQure 11-A-6) de-
sampling described in 2.3. Take care not to pends upon a vane (A) and lever (8) mechanism
dislodge dirt or other material that might fall to lift the glass stopper (C) as water inertia is
Into the open bottle from the sampling applied by a sharp upward tug on the line (D)
platform. attached to the apparatus. As the stopper is
lifted, the bottle fills before the detachment of
2.5 Depth Sampling: Several additional the stopper from the vane occurs and closes
devices are needed for collection of depth the sample bottle (6).

8 oEPA MICROBIOLOGICAL MANUAL 1978

 ~' ·.·. •' . ...

. . ...·.
~

.. ·.;'· .. .

' '

FIGURE 11-A-2. Demonstration of Technique Used in Grab Sampling of Surface Waters.

SAMPLING TECHNIQUES 9
 FIGURE 11-A-3. Weighted Bottle Frame and Sample Bottle for Grab Sampling.

10 &EPA MICROBIOLOGICAL MANUAL 1978

 B

FIGURE 11-A-4. Zobell J-Z Sampler. (A) metal frame, (B) messenger, (C) glass tube,
(D) rubber tube and (E) sterile sample bottle.

SAMPLING TECHNIQUES ·11

 A

FIGURE 11-A-5. Niskin Depth Sampler. (A) hinged plates, (B) plastic bag,
{C) plastic filler tube in sheath, (D) guillotine knife and (E)
closure clamp.

12 &EPA MICROBIOLOGICAL MANUAL 1978

 D

c2

c1

E
J
'
f

FIGURE 11-A-6. New York State Dept. of Health Depth Sampler. (A) vane, (B 1) lever
in closed position, (8 2) lever in open position, (C 1) glass stopper in
closed position, (C 2) glass stopper in open position, (D) suspension
line, and (E) metal frame.

SAMPLING TECHNIQUES 13
 2.5.4 Kemmerer Sampler (7): This depth finger, gloves or other materials. If the well
sampler (see Figure 11-A-7) has been used with- does not have pumping machinery, collect the
out sterilization to collect bacteriological sample using a weighted sterilized sample
water samples in high pollution areas. The bottle, such as described in 2.4 above, and
sampler consists of a cylindrical brass or plas- shown in Figure 11-A-3. Care must be taken to
tic tube (F} that contains a rubber stopper or avoid contaminating the sample with the sur-
valve at either end (D and G). The valves are face scum from the water surface.
connected to a rod (E) that passes through the
center of the cylinder. The device is lowered
Into the water in the open position, and a water 2.8 Soil Sampling
sample is trapped in the cylinder when the
valves are closed by a dropped messenger (8). 2.8. 1 Selection of the sampling site is
The Kemmerer sampler should not be used for based on knowledge of the area and the pur-
collecting bacteriological samples without ob- poses of the analyses, i.e., surface sampling for
taining data that support its use without natural background, surface contamination, or
sterilization. below surface sampling to monitor treatment
effect such as irrigation, or stormwater runoff.
2.6 Sediment Sampling with Van
The actual sites for sampling and the num-
Donsel-Geldreich Sampler (8): This device
ber of points to be sampled must be predeter-
(see Figure 11-A-8) collects sediment or mud in
mined by the survey objectives. Soil sampling
sterile "Whirl-Pak" plastic bags (A) down to 60
has the advantage of permitting the survey
foot depth. The bag mouth is wrapped over
planners to lay out a stable grid network for
a nosepiece (8), and the bag is kept closed
sampling and resampling over a given time
during descent to the bottom by a bag clamp
period.
bar (H). As the mud plate (D) contacts the bot-
tom, the nosepiece (8) is driven into the sedi-
2.8.2 If a surface sample is desired, scrape
ment by the weight (C) of the sampler. As the
the top one inch of soil from a square foot area
noseplece (8) moves downward, the bag (A)
using a sterile scoop or spoon.
slides through the bag clamp bar (H), opens,
and fills with sediment. The bag is sealed when
If a subsurface sample is desired, use a
the double noose (F) tied to the bottom of the
sterile scoop or spatula to remove the top
bag is pulled, before the apparatus is returned
surface of one inch or more from a one foot
to the surface.
square area. Use a second sterile scoop or
spoon to take the sample.
2. 7 Water Tap Sampling: Make certain
that samples are not collected from spigots Place samplings in a sterile one quart
that leak around their stems, or from spigots screw-cap bottle until it is full. Depending on
that contain aeration devices or screens within the amount of moisture, a one quart bottle
the faucet. For samples taken from direct water holds 300-800 grams of soil. Label and tag
main connections, the spigot should be flushed the bottle carefully and store at 4 C until
for 2-3 minutes to clear the service line. For analyzed.
wells equipped with hand or mechanical
pumps, pump the water to waste for five min-
utes befor.e the sample is collected. Remove 3. Sample Identification and Handling
the cap aseptically from the sample bottle. Hold
the sample bottle upright near the base while it 3. 1 Specific details on sample identifica-
Is being filled. Avoid splashing. Do not rinse the tion are entered on a permanent label. Take
bottle with the sample; fill it directly to within care in transcribing sampling information to
2.5 cm (1 inch) from the top. Replace bottle the label, because the enforcement action may
closure and hood covering. Caution must be depend upon evidence of primary labeling.
used to prevent contaminating the sample with See 4. in This Section. Labels must be clean,

14 oEPA MICROBIOLOGICAL MANUAL 1978

 Biif~__:._-D
11-------E

42cm n------F

11---------- E

FIGURE 11-A-7. Kemmerer Depth Sampler. (A) nylon line, (B) messenger, (C) catch set so
that the sampler_ is open, (D) top rubber valve, (E) con.nectin~ rod between
the valves, (F)tube body, (G) bottom rubber valve, (H) knot att~e bottom·of
the suspension line and (I) rubber tubing attached to the spring loaded
check valve.

SAMPLING TECHNIQUES 15
 FIGURE 11-A-8. Van bonsel-Geldreich Sediment Sampler. (A) sterile "Whirl-Pak" plastic
bag, (B) nose piece, (C) weight, (D) mud plate, (E) slide bar, (F) part of the.
double noose, (G) attachment for the suspension line and (H) bag clamp
bar;

16 .&EPA MICROBIOLOGICAL MANUAL 1978

waterproof, non-smearing and of sufficient (a) in actual physical possession, or
size for the necessary information. Label must
be securely attached to the sample bottle, but (b) in view after being in physical posses-
removable when necessary. Do not accept in- sion, or
sufficiently or improperly labeled samples for
examination. A sample label showing the mini: (c) in physical possession and locked up so
mum information required is pictured in Figure that no one could tamper with it.
11-A-9.
4. 1.2 Personnel should receive copies of
3.2 Field Data Record: A field record study plans and know the contents prior to the
should be completed on each sample to record study. A pre-study briefing shall be held to
the full details on sampling and other pertinent appraise participants of the objectives, sample
remarks such as flooding, rain or extreme tem- locations and chain of custody procedures.
perature which are relevant to interpretation of After chain of custody samples are collected, a
results. This record also provides a back-up de-briefing is held in the field to determine
record of sample identification. One example adherence to chain of custody procedures and
is shown in Figure ll-A-10. whether additional samples are required.

3.3 Marking Device: A marking pen or 4.2 Rules for Sample Collection
other device must be non-smearing if wetted,
and maintain a permanent legible mark. 4.2. 1 Handle the samples as little as
possible.
3.4 Transport Container: Insulated ice
containers in which the sample can be held, are 4.2.2 Obtain stream and effluent samples
recommended. using standard microbiological sampling
techniques.
3.5 Storage of Samples: A refrigerator is
necessary for storage of samples at the labora- 4.2.3 Attach sample tag or;label (Figure
tory. The temperature range of the refrigerator 11-A- 9 i to the sample container. The tag or
is 1-4 C. label should contain as· a minimum: serial
number of label, location, date andtime taken,
type of sample, sequence number (first sample
of the day - sequence No. 1, second sample,
4. Chain of Custody Procedures sequence No. 2, etc), analyses required and
sample collector. The tags must be filled out
4.1 General: An agency must demon- legibly in waterproof ink.
strate the reliability of its evidence in pollution
cases by proving the.chain of possession and 4.2.4 Use a bound notebook to record
custody of samples which are offered for evi- field measurements and other pertinent
dence or which form the basis of analytical information necessary to refresh the sampler's
results introduced into evidence. It is impera- memory if the person later becomes a witness
tive that the office and laboratory prepare writ- in an enforcment proceeding. A separate set of
ten procedures to be followed whenever evi- field notebooks should be maintajned for each
dence samples are collected, transferred, study and stored in a safe place where it can
stored, analyzed, or destroyed. be protected and accounted for. A sample log
sheet with a standard format should be
4. 1. 1 The primary objective of these pro- established to minimize field entries and
cedures is to create an accurate written record include the date, time, survey, type of samples,
which can be used to trace the possession of volume of each sample, type of analyses,
the sample from the moment of its collection label and sample numbers, sample location,
through its introduction into evidence. A sam- field measurements such as temperature,
ple is in custody if it is: conductivity, DO, pH, and any other pertinent

SAMPLING TECHNIQUES 17
 Station No. I
EPA, NATIONAL ENFORCEMENT INVESTIGATIONS CENTER
Date I
Time ISequence No.

Station Location Grab

I Comp.

c BOD
Solids
Metals
Oil and Grease
Remarks/Preservatives

:ii: COD D.O.

Cl
.Nutrients Bact•
...ct
..J Other
ct
a:w SamplerSa
Cl)

FIGURE 11-A-9. Example of a Sample Label.

SERIAL SHEET NO. - - - -

Samplers=------------

FIELD DATA RECORD

SAMPLE DATE OF TIME (HRS)

STATION SAMPLE SAMPLE pH TEMP OTHER REMARKS
NUMBER COUECTION TAICIN IKllYIO IY LAI 'C

US EPA, NEIC·Denver

FIGURE ll-A-10. Field Data Record.

18 &~ MICROBIOLOGICAL MANUAL 1978

information or observation (Figure ll-A-11 ). The lows transfer of custody of a group of samples
entries should be signed by the sample in the field, t0 the mobile laboratory or to the
collector. The responsibility for preparing and N EiC-Denver laboratory .. ' When a custodian
storing sample notebooks during and after a transfers a portion of the samples identified on
study should be assigned to a study the sheet to the field mobile laboratory, the
coordinator, or his designated representative. individual samples must be noted in the col-
umn with the signature .of the person relin-
4.2.5 A field collector is responsible for quishing the samples. The field laboratory per-
the samples collected until properly dis- son receiving the samples acknowledges re-
patched to the receiving laboratory or turned ceipt by signing in the appropriate column.
over to an assigned custodian. He must assure
that each container is in his physical posses- 4.3.2 If a custodian has . not been
sion or in his view at all times, or stored in a assigned, the field custodian or field sampler
locked place where no one can tamper with it. has the responsibility for packaging and
dispatching samples to the laboratory for
4.2.6 Color slides or photographs should analysis. The "Dispatch" portion of the Chain
be taken of the sample location and any visible of Custody Record must be filled out dated,
water pollution. The signature of the photo- and signed.
grapher, time, date, and site location must be
written on the back of the photo. Such photo- 4.3.3 Samples must be carefully packed in
graphs should be handled according to the shipment containers such as ice chests, to
established chain of custody procedures to avoid breakage. The shipping containers are
prevent alteration. padlocked for shipment to the receiving
laboratory.
4.3 Transfer of Custody and Shipment
4.3.4 Packages must be accompanied by
In transfer of -custody procedures, each the Chain of Custody Record showing
custodian of samples must sign, record and identification of the contents. The original
date the transfer. Most regulatory agencies must accompany the shipment. A copy is
develop chain of custody procedures tailored retained by the survey coordinator.
to their needs. These procedures may vary in
format and language but contain the same 4.3.5 If samples are delivered to the
essential elements. Historically, sample trans- laboratory when appropriate personnel are not
fer under chain of custody has been on a there to receive them, the samples must be
sample by sample basis which is awkward and locked in a designated area within the
time-consuming. However, EPA's National En- laboratory so that no one can tamper with
forcement Investigation Center (NEIC), Denver them. This same person must return to the
has set a precedent with its bulk transfer of laboratory, unlock the sarripies ·and deliver
samples. Bulk transfer is speedier, reduces custody to the appropriate custodian.
paperwork and the number of sample custodi-
ans. The following description of chain of cus- 4.4 Laboratory Custody Procedures
tody is essentially that of NEIC-Denver (9).
4.4.1 The laboratory shall designate a
4.3. 1 Samples must be accompanied by a ~·sample custodian" and an alternate to act in
Chain of Custody Record which includes the his absence. In addition, the laboratory shall
name of the study, collector's signature, sta- set aside as a "sample storage security area",
tion number, station location, date, time, type an isolated room with sufficient refrigerator
of sample, sequence number, number of con- space, which can be locked or just a locked
tainers and analyses required (Figure ll-A-12). refrigerator in smaller laboratories.
When turning over the possession of samples,
the transferor and transferee sign, date and 4.4.2 Samples should be handled by the
note time on the sheet. This record sheet al- minimum possible number of persons.

SAMPLING TECHNIQUES 19
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20 &EPA MICROBIOLOGICAL MANUAL 1978

SERIAL SHEET NO.

ENVIRONMENTAL PROTECTION AGEN.CY

Office Of Enforcement
NATIONAL ENFORCEMENT INVESTIGATIONS CENTER
Building 53, Box 25227, Denver Federal Center
Denver, Colorado 80225

CHAIN OF CUSTODY RECORD

SURVEY SAMPLERS: (Sl9noture)

SAMPLE TYPE
STATION
STATION LOCATION DATE TIME Water SEQ. NO. Of ...... .... Nf!~YSIS
NUMaER Ah NO. CONTAINERS REQUIRED
Comp. Grab.

Relinquished by: (Signature) Received by: (Signature} Date/Time

I
Relinquished by: (Signature) Received by: (Sl9naturoJ Date/Time
I
Relinquished by: (Signature) Received by: (Signature) Date/Time

I
Relinquished by: (Signature) Received by Mobile laboratory for field Date/Time
analysis: {Signature)
I
Dispatched by: (Slgnalur•) Date/Time Received for laboratory by: Dote/Time
I I I
Method of Shipment:

Di1tribution: Orig.- Accompany Shipment

1 Copy- Survey Coordinator Field Files GPO 854 - sog

FIGURE ll-A-12. Chain of Custody Record.

SAMPLING .TECHNIQUES 21
 4.4.3 Incoming samples shall be received received from the custodian until the tests
only by the custodian, who will indicate receipt were run.
by signing the Chain of Custody Record Sheet
accompanying the samples and retaining the 4.4. 10 Once the sample testing is com-
sheet as a permanent record. Couriers picking pleted, microbiological samples can be dis-
up samples at the airport, post office, etc. shall carded but identifying tags and laboratory
sign jointly with the laboratory custodian. record should be returned to the custodian.
Other documentation of work will also be
4.4.4 Immediately upon receipt, the custo- given to the custodian.
dian places samples in the sample room,
which will be locked at all times except when 4.4. 11 Tags and laboratory records of
samples are removed or replaced by the custo- tests may be destroyed only upon the order of
dian. To the maximum extent possible, only the the Laboratory Director, who will first confer
custodian should be permitted in the sample with the Chief, Enforcement Specialist Office,
room. to make certain that the information is no
longer required.
4.4.5 The custodian shall ensure that
microbiological samples are property stored
and maintained at 1-4 C. 5. Selection of Sampling Sites and
Frequency
4.4.6 Only the custodian will distribute
samples to personnel who are to perform tests. These will be described for potable and
recreational waters, streams, lakes, reservoirs,
4.4.7 The analyst records information in estuarine, and marine waters as well as do-
his laboratory notebook or analytical work- mestic and industrial wastewaters.
sheet, describing the sample, the procedures
performed and the results of the testing. The 5.1 Potable Water Supplies
notes shall be dated and indicate who per-
formed the tests. The notes shall be retained An expanded program to maintain the san-
es a permanent record in the laboratory and itary. quality Qf potable water supplies has
should include any abnormalities which oc- been recently established by the National In-
curred during the testing procedure. In the terim Primary Drinking Water Regulations (11).
event that the person who performed the tests The. sampling program includes examination
Is not available as a witness at time of trial, of water as it enters and flows throughout the
the government may be able to introduce the distribution system. For application of the EPA
notes in evidence under the Federal Business Drinking Water Standards, the frequency of
Records Act. sampling and the location of sampling points
are established jointly by the utility, the
4.4.8 Standard methods of laboratory Reporting Agency, and the Certifying
analyses shall be used as described in the Authority. Additionally, the laboratory, the
"Guidelines Establishing Test Procedures for methods of analyses, and the technical
Analysis of Pollutants," ( 10) and amendments. competence of personnel must be inspected
If laboratory personnel deviate from standard and approved by the Reporting Agency and
procedures, they should be prepared to justify the Certifying Authority.
their decision during cross-examination.
5. 1.1 Sampling Water Supplies: Figure ll-
4.4.9 Laboratory personnel are responsi- A-13 shows how reservoirs and lakes used as
ble forthe care and custody of a sample once it water supplies are sampled: (A) at inlets, (B) at
is handed over to them and should be pre- other possible sources of pollution, (C) at the
pared to testify that the sample was in their draw-off point, (D) at quarter point intervals
possession and view or secured in the labora- around the draw-off point at about the same
tory at all times from the moment it was depth and (E) atthe reservoir outlet.

22 &EPA MICROBIOLOGICAL MANUAL 1978

FIGURE ll-A-13. Sampling a Water Supply Reservoir. (A) influent stream, (8) possible
agricultural contamination, (C) water plant intake, (D) multi-point
sampling around intake and (E) reservoir outlet.

SAMPLING TECHNIQUES 23
 5. 1.2 Sampling Treatment Systems: Sam- 5.3. 1 Selection of Sampling Sites: A
pling should be representative of the distribu- typical stream sampling program includes
tion system and include sites such as munici- sampling locations upstream of the area of
pal buildings, public schools, airports and concern, upstream and downstream of waste
parks, hydrants, restaurants, theaters, gas sta- discharges, upstream and downstream from
tions, industrial plants and private residences. tributary entrances to the river and upstream
A systematic coverage of such points in the of the mouth of the tributary. For more
distribution system should insure the detec- complex situations, where several waste
tion of contamination from breaks in water- discharges are involved, sampling includes
lines, loss of pressure or crossconnections. sites upstream and downstream from the
The sampling program should also include combined discharge area and samples taken
special sampling locations such as dead-end directly from each industrial or municipal
distribution lines that are sources of bacterial waste discharge. Using available
contamination (12). bacteriological, chemical and discharge rate
data, the contribution of each pollution source
5.1.3 Sample Frequency: The minimum can be determined. See Figure ll-A-14 and
number of samples which must be collected ll-A-15.
and examined ea'ch month is based upon the
population density served by the distribution
system {Table 11-A-1). Samples should be col- 5.3.2 Small Streams: Small streams
lected at evenly spaced time intervals through- should be sampled at background stations
outthe month. In the event of an unsatisfactory upstream of the pollution sources and at
sample, repetitive samples must be collected stations downstream from pollution sources.
until two consecutive samples yield satisfac- Additional sampling sites should be located
tory quality water. Repetitive samples from any downstream tc delineate the zones of
single point or special purpose samples must pollution. Avoid sampling areas where
not be counted in the overall total of monthly stagnation may occur (backwater of a
samples. tributary) and areas located near the inside
bank of a curve in the stream which may not be
5.1.4 Standard Sample: The standards for representative of the main channel.
microbiological quality are based upon the
number of organisms allowable in a standard
sample. A standard sample for the membrane 5.3.3 Large Streams and Rivers: Large
filter technique is at least 100 ml. For the MPN streams are usually not well mixed laterally for
test, a standard sample consists of five stan- long distances downstream from the pollution
dard portions of either 10 ml or 100 ml. sources. Sampling sites below point source
pollution should be established to provide
desired downstream travel time and dispersal
5.2 Lakes and Impoundments as determined by flow rate measurements.
Particular care must be taken to establish the
Figure ll-A-14 shows the range of sam- proper sampling points as shown in Figure ll-A-
pling points in a recreational impoundment or 15: Sampling point (A) is the upper reach control
lake: (A) inlets, (B) source of pollution, (C} grid or station, (B) monitors a non-point source of
transect across the long axis of the water body, · pollution, (C) samples the waste discharge as it
(D) bathing beach and (E) outlet. enters the stream, (0) shows quarter-point
sampling below the pollution to detect chan-
neling, (D) also serves as an upstream monitor
5.3 Stream Sampling on the tributary measured as (E), and (F) moni-
tors the downstream effect of the tributary
The objectives of the initial survey dictate after mixing. Occasionally, depth samples
the location, frequency and number of sam- are necessary to determine vertical mixing
ples to be collected. patterns.

24 oEPA MICROBIOLOGICAL MANUAL 1978

 TABLE 11-A-1

Sampling Frequency for Drinking Waters Based on Population

Minimum number of Minimum number of

Population served: samples per month Population served: samples per month

25 to 1,000 ............................................. 1 90,001 to 96,000 .................................. 95

1,001 to 2,500 ....................................... 2 96,001 to 111,000 .............................. 100
2,501 to 3,300 ....................................... 3 111,001 to 130,000 ............................ 110
3,301 to 4, 100 ....................................... 4 130,001 to 160,000 ............................ 120
4, 101 to 4,900 ....................................... 5 160,001 to 190,000 ............................ 130
4,901 to 5,800 ....................................... 6 190,001 to 220,000 ............................ 140
5,801 to 6,700 ....................................... 7 220,001 to 250,000 ............................ 150
6,701 to 7,600 ....................................... 8 250,001 to 290,000 ............................ 160
7,601 to 8,500 ....................................... 9 290,001 to 320,000 ............................ 170
8,501 to 9,400 ....................................... 10 320,001 to 360,000............................ 180
9.401 to 10,300 ···································· 11 360,001 to 410,000 ............................ 190
10,301 to 11, 100 .................................. 12 410,001 to 450,000 ............................ 200
11,101 to 12,000 .................................. 13 450,001 to 500,000 ............................ 210
12,001 to 12,900 .................................. 14 500,001 to 550,000 ............................ 220
12,901 to 13,700 .................................. 15 550,001 to 600,000 ............................ 230
13,701 to 14,600 .................................. 16 600,001 to 660,000 ............................ 240
14,601 to 15,500 .................................. 17 660,001 to 720,000 ............................ 250
15,501 to 16,300 .................................. 18 720,001 to 780,000 ............................ 260
16,301 to 17,200 .................................. 19 780,001 to 840,000 ............................ 270
17,201 to 18, 100 .................................. 20 840,001 to 910,000 ............................ 280
18, 101 to 18,900 .................................. 21 910,001 to 970,000 ............................ 290
18,901 to 19,800 .................................. 22 970,001 to 1,050,000 ........................ 300
19,801 to 20,700 .................................. 23 1,050,001 to 1, 140,000 ···················· 310
20,701 to 21,500 .................................. 24 1,140,001 to 1,230,000 .................... 320
21,501 to 22,300 .................................. 25 1,230,001 to 1,320,000 ···················· 330
22,301 to 23,200 .................................. 26 1,320,001 to 1.420,000 .................... 340
23,201 to 24,000 .................................. 27 1.420,001 to 1,520,000 ···················· 350
24,001 to 24,900 .................................. 28 -
1,520,001 to 1,630,000 .................... 360
24,901 to 25,000 .................................. 29 1,630,001 to 1,730,000 .................... 370
25,001 to 28,000 .................................. 30 1,730,001 to 1,850,000 .................... 380
28,001 to 33,000 .................................. 35 1,850,001 to 1,970,000 .................... 390
33,001 to 37,000 .................................. 40 1,970,001 to 2,060,000 .................... 400
37,000 to 41,000 .................................. 45 2,060,001 to 2,270,000 .................... 410
41,001 to 46,000 .................................. 50 2,270,001 to 2,510,000 .................... 420
46,001 to 50,000 .................................. 55 2,510,001 to 2,750,000 .................... 430
50,001 to 54,000 .................................. 60 2,750,001 to 3,020,000 .................... 440
54,001 to 59,000 .................................. 65 3,020,001 to 3,320,000 .................... 450
59,001 to 64,000 .................................. 70 3,320,001 to 3,620,000 .................... 460
64,001 to 70,000 .................................. 75 3,620,001 to 3,960,000 .................... 470
70,001 to 76,000 .................................. 80 3,960,001 to 4,310,000 .................... 480
76,001 to 83,000 .................................. 85 4,310,001 to 4,690,000 .................... 490
83,001 to 90,000 .................................. 90 4,690,001 or more ............................... 500

SAMPLING TECHNIQUES 25
 FIGURE ll-A-14. Sampling a Lake or lmpoundment. (A). inlets, (8) potential source
of pollution, (8 1) village, (8 2) agricultural run-off, (8 3) home septic
tank, (C) multi-point transect, (D) bathing beach and (E) outlet above
and below dam.

26 oEPA MICROBIOLOGICAL MANUAL 1978

 /'

\\\\

F F
I

FIGURE ll-A-15. Sampling a Large Stream. (A) control station, (B) agricultural pollution,
(C) industrial discharge, (D) quarterpoint transect (E) tributary, and
(F) downstream monitoring.

. SAMPLING TECHNIQUES 27
 5.4 Marine and Estuarine Sampling waters. Despite the higher temperature, oxy-
gen concentrations are higher in shallow than
in deeper waters due to greater water move-
Sampling marine and estuarine waters
ment, surf action and photosynthetic activity
requires the consideration of other factors in
from macrophytes and the plankton.
addition to those usually recognized in fresh
water sampling. They include tidal cycles,
Moving from the shallow waters to the
current patterns, bottom currents and counter-
intermediate depths, one observes a modera-
currents, stratification, seasonal fluctuations,
tion of these shallow water characteristics. In
dispersion of discharges and multi-depth
the deeper waters, there is a marked stabliza-
samplings.
tion of conditions. Water temperatures are
The frequency of sampling varies with the lower and more stable. There is limited turbu-
objectives. When a sampling program is lence, little penetration of light, sparse vegeta-
started, it may be necessary to sample every tion and the ocean floor is covered with a layer
hour around the clock to establish pollutional of silts and sediments.
loads and dispersion patterns. The sewage 5.4.2 Estuarine Sampling: When a survey
discharges may occur continuously or is made on an estuary, samples are often taken
Intermittently. from a boat, usually making an end to end
traverse of the estuary. Another method in-
When the sampling strategy for a survey is volves taking samples throughout a ti'dal cycle,
planned, data may be available from previous every hour or two hours from a bridge or from
hydrological studies done by Coast Guard, an anchored boat at a number of fixed points.
Corps of Engineers, National Oceanic and
Atmospheric Administration (NOAA), U.S. In a large bay or estuary where many
Geological Survey, or university and private square miles of area are involved, a grid or
research investigations. In a survey, float series of stations may be necessary. Two sets
studies and dye studies are often carried out to of samples are usually taken from an area on a
determine surface and undercurrents. Initially given day, one at ebb or flood slack water, and
depth samples are taken on the bottom and at the other three hours earlier, or later, at the half
five feet increments between surface and tidal interval. Sampling is scheduled so that
bottom. A random grid pattern for selecting the mid-sampling time of each run coincides
sampling sites is established statistically. with the calculated occurrence of the tidal
condition.
5.4.1 Marine Sampling: In ocean studies,
the environmental conditions are most diverse In locating sampling sites, one must con-
along the coast where shore, atmosphere and sider points at which tributary waters enter the
the surf are strong influences. The shallow main stream or estuary, location of shellfish
coastal waters are particularly susceptible to beds and bathing beaches. The sampling sta-
dally fluctuations in temperature and seasonal tions can be adjusted as data accumulate. For
changes. example, if a series of stations half mile apart
consistently show similar values, some of
Sampling during the entire tidal cycle or these stations may be dropped and other sta-
during a half cycle may be required. Many tions added in areas where data shows more
ocean studies such as sampling over the conti- variability.
nental shelf involve huge areas and no two
areas of water are the same. Considerable stratification can occur be-
tween the salt water from the sea and the fresh
Selection of sampling sites and depths are water supplied by a river. It is essential when
most critical in marine waters. In winter, cool- starting a survey of an unknown estuary to find
ing of coastal waters can result in water layers out whether there is any marked stratification.
which approach 0 C. In summer, the shallow This can be done by chloride determinations at
waters warm much faster than the deeper different locations and depths. It is possible for

28 &EPA MICROBIOLOGICAL MANUAL 1978

stratification to occur in one part of an estuary marinas, or garbage collection areas (12).
and not in another. Samples of bathing beach water should be
collected at locations and times of heaviest
On a.flood tide, the more dense salt water use. Daily sampling, preferably in the after-
pushing up into the less dense fresh river noon, is the optimum frequency during the
water will cause ·an overlapping with the fresh season. Weekends and holidays which are pe-
water flowing on top. A phenomenon called a riods of highest use must be included in the
salt water wedge can form. As a result, stratifi- sampling program. Samples of estuarine bath-
cation occurs. If the discharge of pollution is in ing waters should be obtained at high tide, ebb
the salt water layer, the contamination will be tide and low tide in order to determine the
concentrated near the bottom at the flood tide. cyclic water quality and deterioration that
The flow or velocity of the fresh water will must be monitored during the swimming
influence the degree of stratification which season.
occurs. If one is sampling only at the surface, it
is possible that the data will not show the 5.6.2 Swimming Pools: Swimming pool
polluted underflowing water which was con- water should be monitored at least daily dur-
taminated at a point below the fresh water ing maximum use periods, preferably at the
river. Therefore, where stratification is sus- overflow. It is important to test swimming pool
pected, samples at different depths will be samples for neutralization of residual chlorine
needed to measure vertical distribution. at pool side to assure that the dechlorinating
agent was effective.
5.5 Domestic and Industrial Waste
Discharges 5.7 Shellfish-Harvesting Waters

It is often necessary to sample secondary Water overlying shellfish-harvesting areas

and tertiary wastes from municipal waste should be sampled during periods of most
tr.eatment plants and various industrial waste unfavorable hydrographic conditions, usually
treatment operations. In situations where the at low tide after heavy precipitation. However,
plant treatment efficiency varies considerably, shellfish beds are sometimes exposed during
grab samples are collected around the clock at low tide and must be sampled during other
selected intervals for a three to five day period. tidal conditions. Procedures for sampling of
If it is known that the process displays little shellfish and water in shellfish growing areas
variation, fewer samples are needed. In no are governed by the National Shellfish Sanita-
case should a. composite sample be collected tion Program's Manual of Operations (13).
for bacteriological examination. The NPDES
has established wastewater treatment plant 5.8 Frequency of Sampling
effluent limits for all dischargers. These are
often based on maximum and mean values. A The frequency of sampling depends upon
sufficient number of samples must be col- the type of pollution that is to be measured.
lected to satisfy the permit and/or to provide Cyclic pollution and its duration are measured
statistically sound data and give a fair.repre- as frequently as practical immediately down-
sentation of the bacteriological quality of the stream from the source. Uniform pollution
discharge. loads are measured at greater distances down-
stream from the source and at less frequent
5.6 Recreational Waters time intervals than cyclic pollution. Climatic
and tidal conditions must be considered. jn
5.6. 1 Bathing Beaches: Sampling sites at marine and estuarine sampling. A common
bathing beaches or other recreational areas approach for short-term studies is to collect
should include upstream or peripheral areas samples from each site daily and advance the
and locations adjacent to natural drains that sampling intervals one hour during each 24-
would discharge stormwater, or run-off areas hour period to obtain data for a 7-10 day
draining septic wastes from restaurants, boat study.

SAMPLING TECHNIQUES 29
 Often the numbers of samples to be not be held longer than six hours between
collected are specified by NPDES permits, collection and initiation of analyses (14). This
drinking water regulations, or by state limit is applied to fresh waters, seawaters and
requirements. Some standards require a shellfish-bed waters. The exception is water
minimum number of samples to be collected supply sam·ples mailed in from water treat-
each month. Other standards are less explicit ment systems. Current regulations permit
and simply indicate that the geometric mean these samples to be held up to 30 hours.
coliform density shall not exceed a certain
level each month, with no more than 10%, 6.2.1 Despite the establishment of a six
20%, etc. of samples exceeding a certain hour limit, sewage samples, organically-rich
value. Where the number of samples required wastes and marine waters are particularly
is undetermined, a sufficient number should susceptible to rapid increases or die-away and
be collected to measure the variations in hence should be held for the shortest time
stream conditions. possible to minimize change.

6.2.2 Temporary Field Laboratories: In sit-

6. Preservation and Transit of Samples uations where it is impossible to meet the six
hour maximum holding time between collec-
The adherence to sample preservation and tion and processing of samples, the use of
holding time limits is critical to the production temporary field laboratories located near the
of valid data. Samples exceeding these limits collection site should be considered.
should not be analyzed. The following rules
must be observed. 6.2.3 Delayed Incubation Procedure: If
6.1 Storage Temperature and Handling sampling and transit conditions require more
Conditions tha·n six hours, and the use of field laboratories
is impossible, the delayed incubation proce-
Bacteriological samples should be iced or dure for total and fecal coliforms and fecal
refrigerated at a temperature of 1-4 C during streptococci should be considered.
transit to the laboratory. Insulated containers
are preferable to assure proper maintenance 6.2.4 Public Transportation: Occasionally,
of storage temperature. Care should be taken commercial forms of transit such as airlines,
that sample bottles are not totally immersed in buslines or couriers can be used to transport
water during transit or storage. samples contained in ice chests to the labora-
6.2 Holding Time Limitations tory. These should be considered only when
storage time and temperature requirements
Although samples should be examined as and the proper disposition of the samples can
soon as possible after collection, they should be assured.

30 &EPA MICROBIOLOGICAL MANUAL 1978

 REFERENCES

1. Public Health Laboratory Service Water Subcommittee, 1953. The effect of sodium thiosulfate
on the coliform and Bacterium coli counts of non-chlorinated water samples. J. Hyg. ~:5 72.

2. Shipe, E. L. and A. Fields, 1954. Comparison of the molecular filter technique with agar plate
counts for enumeration of Escherichia coliin various aqueous concentrations of zinc and copper
sulfate. Appl. Microbiol. 2:382.

3. Shipe, E. L. and A. Fields, 1956. Chelation as a method for maintaining the coliform index in water
supplies. Public Health Rep. I._!_:97 4.

4. Zobell, C. E., 1941. Apparatus for collecting water samples from different depths for bacteriologi-
cal analysis. J. Marine Research 4: 173.

5. Niskin, S., 1962. Water sampler for microbiological study. Deep Sea Research 9:501.

6. Fuhs, G. Wolfgang, 1977. Personal Communication: Director of the Environmental Health Center,
Division of Lab and Research, New York State Health Department, Albany, New York.

7. Welch, P.S. 1948. Limnological Methods. Blakiston Company, Philadelphia, PA.

8. Van Donsel, D. J. and E. E. Geldreich, 1972. Relationships of Salmonella to fecal coliforms in

bottom sediments. Water Research 5: 1079.

9. Wills, Carroll, 1975 (June). Chain of Custody Procedures, National Enforcement Investigation
Center-Denver, Colorado, U.S. EPA.

10. Guidelines Establishing Test Procedures for the Analysis of Pollutants. 40 CFR Part 136, 52780,
as amended, December 1, 1976.

11. National Interim Primary Drinking Water Regulations, 40 Code of Federal Regulations, Amend-
ments to Part 141, December 24, 1975.

12. Geldreich, E. E., 1975. Handbook for Evaluating Bacteriological Water Laboratories, (2nd ed.)
U.S. Environmental Protection Agency, Municipal Environmental Research Laboratory, Cincin-
nati, Ohio. EPA-670/9-75-006.

13. Hauser, L. S. (ed.), 1965. National Shellfish Sanitation Program. Manual of Operations, Part I:
Sanitation of shellfish growing areas. U.S. Public Health Service, Washington, D.C. -- -

14. Public Health Laboratory Service Water Subcommittee, 1953. The effect of storage on the
coliform and Bacterium coli counts of water samples. Storage for six hours at room and
refrigerator temperatures.~ 61 :659.

SAMPLING TECHNIQUES 31
 PART II. GENERAL OPERATIONS

Section B Laboratory Equipment, Techniques and Media

Most equipment and supplies described in 1. Equipment and Supplies

This Manual are available in weM-equipped
bacteriology laboratories. Other items specific 1.1 Inoculating Needles and Loops:
for the membrane filter or multiple-tube dilu- Needles and loops of nichrome, platinum or
tion methods are used only in water laborator- platinum-iridium wire are used to transfer
ies. This Section describes the required equip- microbes aseptically from one growth medium
ment, media and preparation techniques used to another. A 24-26 gauge nichrome wire is
in the laboratory or in the field. The contents recommended. The loop diameter should be at
include: _least 3 mm. They are sterilized by heating to
redness in a gas flame or in an electric
incinerator. Resterilization is not required for
replicate transfers of the same bacteria or
1. Equipment and Supplies bacteria-containing materials to a sterile
medium. Sterile, disposable hardwood
applicator sticks or plastic loops can be used
2. Cleaning Glassware to inoculate fermentation tubes.

1.2 Plastic petri dishes (50 x 12mm) with

3. Sterilization tight-fitting lids are preferred for MF proce-
dures because they retain humidity and are
more practical for use in plastic bags sub-
4. Preparation and Use of mersed in a water bath incubator. Petri dishes
Culture Media (60 x 15 mm) with loose-fitting lids can be used
in incubators with controlled humidity or in
plastic boxes with tight covers, containing
6. Media Composition
moist towels.
General Use Media
1.3 Incubators: Incubators are constant
MF Media for Coliforms
temperature air chambers or water baths
MPN Media for Coliforms
which provide controlled temperature environ-
Media for Fecal Streptococci
ment for microbiological tests. The incubator
Media for Ss/monells and
must control temperature within specified
Other Enterics tolerance limits of the tests. The air type
Medium for Actinomycetes
incubator can be water-jacketed, a dry air unit
or aluminum block equipped with thermostat-
6. Laboratory Pure Water controlled heating units that maintain 35 C ±
0.5 C. Overcrowding of incubators with plates
arid tubes must be avoided, because this will
7. Dilution Water interfere with the constancy of the desired

32 oEPA MICROBIOLOGICAL MANUAL 1978

temperature. Water bath or aluminum block (b) To Deliver (TD) Pipets: Pipets designed
type incubators used in fecal coliform tests to release the exact calibrated amount when
must control the incubation temperature of the pipet tip is held vertically against the re-
44.5 C ± 0.2 C. Covers are required. An accurate ceiving vessel wall until draining stops. Exam-
thermometer in the water bath monitors tem- ples: bacteriological, Mohr, serological, and
perature. Recording thermometers are recom- volumetric pipets.
mended for use in the water baths.
To Contain and To Deliver pipets are indic-
1.4 Colony Counter: A standard counting atedby the letters TC or TD marked respec-
device equivalent to the Quebec Colony Counter tively on the neck with other calibration
that provides good visibility and magnification information.
of at least 1.5 diameters on a non-glare ruled
guide plate is recommended. (c) Blow-Out Pipets: These pipets are in-
tended for rapid use in serology and are emp-
1.5 pH Equipment: Electro metric pH meter tied by forceful blow-out. Because the blow-
must be accurate to at least 0.1 pH unit. out action always produces an aerosol such
pipets should never be used in the microbiol-
1.6 Balances: For routine weighing of ogy laboratory. The pipets do not deliver the
media and reagents, use a single pan top loader calibrated amount until they are completely
balance having a sensitivity of 0.1 gram at a emptied.
load of 150 grams. For weighings of less than 2
grams, use a four place analytical balance Blow-Out pipets are marked with a double
having a sensitivity of 1 mg at a load of 10 band etching or fired-in marking on the neck.
grams.
(d) Dual Purpose Pi pets: A new pi pet which
1. 7 Media Preparation Utensils: Use suit- combines three calibrations. The pipet has an
able non-corrosive utensils of plastic, glass, upper graduation mark which is the To Deliver
stainless steel, or non-chipped enamel. Utensils and Blow-Out line, a lower graduationwhich is
must be chemically clean before use to prevent the To Contain line, and carries the double
contamination of media with chemicals such as band for Blow-Out.
chlorine, copper, zinc, chromium and de-
tergents. For microbiological analyses, only bacteri-
ologTCal, serological or Mohr pi pets are recom-
1.8 Pipets and Graduated Cylinders mended for use in the To Derrver=-(TD) mode.
Dual purpose or serological pi pets which must
1.8.1 Pi pets: Because transfer pi pets are an be used as blow-out pipets, are not recom-
important element in any microbiological mended for microbiology because of the dan-
method, they must be properly used. If mouth- ger of infection through aerosol and the possi-
pipetting is practiced for non-polluted waters, ble mix-up of TD, TC and blow-out type pi pets
pipets should be cotton-plugged for safety. with subsequent misuse and improper
Blow-out pipets are not to be used. delivery.

The major types of transfer pipets (non- (e) Pipet Standards/Specifications: There
volunietric) used in the laboratory are based are several tolerance specifications used to
on the method of draining: characterize measuring pipets. The analyst
should be aware of these limits. When accu-
(a) To Contain (TC) Pipets: Pipets calibrated racy of measurements is critical, use only pi-
to holdor contain the exact amount specified pets within the following Class A limits:
by the calibration. Pipet must be completely
emptied to provide the stated volume. Ex- NBS Specification for Mohr Measuring Pi-
amples: Transfer Micro and Dual Purpose pets, Class A Volume Tolerances Circular 602
Pi pets. and Federal Specification NNN-P-350

EQUIPMENT, TECHNIQUES AND MEDIA 33

Capacity/Grad (ml) Tolerance (ml) cumbersome. A finger-mounted safety pipet-
tor which can be fabricated in the laboratory
1 / 10 in 1 / 100 ±0.0025 ( 1, 2) is recommended as the most efficient. It
2/10 in 1/100 ±0.004
1 in 1/100 can be fitted with a mouthpiece, hand-held
±0.01
1 in 1/10 ±0.01 bulb or attached to a vacuum pump. Figure 11-B-
2 in 1/10 ±0.01 1 shows the finger-held model and Figure 11-B-
5 in 1 /10 ±0.02 2 shows an enlargement of the plastic ring
10 in 1/10 ±0.03 suction device.
2.6 tn 1/10 ±0.05
1.8.3 Graduates: For normal laboratory
operations, graduates are used for measuring
For routine use, the following tolerances volumes greater than 10 ml. When extreme
are acceptable: accuracy is required above 10 ml, volumetric
pi pets up to 200 ml and volumetric flasks from
APHA Specification, Bacteriological Pi pets 10 ml to 6 liters are available. The tolerances
given below are acceptable for graduates.
Capacity/Grad (ml) Tolerance (ml)

1 in 1 ±0.025 1.9 Pipet Container: Only aluminum,

1.1 in 1.1, 1.0 ±0.025 stainless steel, pyrex glass or other non-
2.2 in 2.2, 2. 1 ±0.040
2.0, 1.0 corrosive heat-resistant containers, either cyl-
11 In 11 ±0.10 indrical or rectangular in shape, should be
used. Pipets may be also wrapped in kraft
paper. Copper or copper alloy containers must
USPHS Specification, Bacteriological Pipets not be used.
for Water Analyses
1.10 Dilution (Milk Dilution) Bottles or
Capacity/Grad (ml) Tolerance (ml) Tubes: Borosilicate or other non-corrosive
glass bottles with screw-caps and inert liners.
11 in 11, 10, 1 ±0.06
1.11 Fermentation Tubes and Vials: The
fermentation tubes should be large enough to
1.8.2 Pipetting Devices: Mechanical pipet- contain the media and inocula in no more than
ting devices are recommended for all purposes. half of the tube depth. There should be suffi-
Although several devices have recently be- cient media in the outer fermentation tube to
come available, some are not practical for fill the enclosed inverted vial after sterilization
water analyses because they are too slow and and partially submerge it.

NBS Specification, Tolerance of Graduates, in ml

NNN-C-940
Volume Demarcations NBS Class A Type 1 Style

2000 20 ±10.0
1000 10 ±5.0
500 5 +1.3 ±2.6
250 2 ±0.8 +1.4
100 1 ±0.4 ±0.6
50 1 ±0.26

34 oEPA MICROBIOLOGICAL MANUAL 1978

f \GU RE \\-B· \. f inge<-Moonted P\pettin9 Oe"\ce.

f\GURE \\'B-2· Enwgement of p;pettln9

oevice iiP·

36
 1.12 Sample Bottles: Sample bottles tion power and coagulates microbial proto-
should be borosilicate glass or plastic, resis- plasm. The temperature in the autoclave should
tant to sterilizing conditions and the solvent be monitored. The relationship between the
action of water and which do not produce toxic pressure of steam and the temperature is
substances upon sterilization. Wide-mouth shown in Table 11-8-1. The autoclave is used to
ground-glass stopper or screw-cap bottles are sterilize solid and liquid media, contaminated
acceptable. Bottles equipped with screw-caps materials, discarded cultures, glassware of all
are acceptable provided that bacteriostatic or types. filtering units, etc. Pressure cookers and
nutritive compounds are not produced from vertical autoclaves are not recommended.
caps or liners. Bottles should be at least 125
ml volume. 3.2 Dry Heat: The hot-air oven is used to
sterilize glassware such as petri dishes, pipets,
2. Cleaning Glassware sample bottles and flasks, hardwood applicator
sticks, and other articles, but not liquids or
In microbiology, clean glassware is crucial materials which ·will evaporate or deteriorate.
to insure valid results. Previously used or new Since moisture is not present in the oven, a
glassware must be thoroughly cleaned with a temperature of 165-170 C (329-338 F) is re-
phosphate-free laboratory detergent and hot quired for a 2 hour period.
water, then rinsed repeatedly with hot water,
followed by at least three rinses with labora- 3.3 Incineration: Contaminated materials
tory pure water. To determine whether the that are combustible may be disposed of by
detergent used contains inhibitory residues, burning. This method is also used for steriliz-
the test procedure for detergent suitability ing inoculating needles and loops, and flaming
should be performed each time a new type the lips of test tubes and flasks.
detergent is purchased. See Part IV-A, 5. 1.
3.4 Filtration. Filtration is used to sterilize
3. Sterilization liquids that are heat-sensitive. Filters include
those composed of: asbestos-cellulose (Seitz
Sterilization is the process that eliminates filter), cellulose esters (0.22 µm MF or molecu-
living organisms from treated substances or lar filter), unglazed porcelain (Chamberland-
objects. Disinfection is the destruction or Pasteur filter), or diatomaceous earth (Berkfeld
removal of the infectious agents by chemical filter). Although filters are normally used
or physical means. Usually chemical agents to remove bacteria, porosities small enough
are used as disinfectants (germicide and bac- .for the removal of viruses are available.
tericide are synonymous with the term, disin-
fectant). Pasteurization is a form of disinfection 3.5 Ultraviolet Radiation: Ultraviolet light
used for materials which may be altered or includes radiations between 150 and 4000
damaged by excessive heat. Low heat is applied Angstrom units (A.U.), but radiations less than
once or repeatedly to sensitive liquids to destroy 1800 A.U. are absorbed by atmospheric oxy-
vegetative cells. Sterilization can be accom- gen. The greatest killing effect on microorga-
plished by moist heat, dry heat, incineration, nisms occurs at 2600 A.U. Commercial germi-
filtration, radiation or by the use of the chemical cidal ultraviolet lamps emit primarily 2537
agent, ethylene oxide. Bottles should have loos- A.U. which has 85 percent of the germicidal
ened caps for penetration of steam or gas. ability of 2600 A.U. Ultraviolet radiation at the
germicidal wavelength is used to sterilize labo-
3.1 Moist Heat: The autoclave is used in ratory equipment such as membrane filter
the laboratory for moist heat sterilization. It is units, inoculating rooms, bacteriological
normally operated at 15 lbs. per sq. in. steam hoods and glove boxes. See Part IV-A, 4.2, for
pressure for 15 minutes, producing a tempera- monitoring procedures.
ture inside the autoclave of 121.6 C (250F) at
sea level. Steam under pressure provides 3.6 Ethylene Oxide Chemical
effective sterilization since it has good penetra- Sterilization: Low temperature ethylene oxide

36 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE 11-B-1

Relationship of Steam Pressure to Temperature in the Autoclave

Pounds of pressure of steam Corresponding Temperature

per square inch In Degrees•

Celsius Fahrenheit

0 100.0 212

5 108.3 227

10 115.5 240

15 121.6 250

20 126.6 260

25 130.5 267

30 134.4 274

*Correct at sea level and only if all air is evacuated from the sterilizing chamber since a mixture
of steam and air at a given pressure gives a temperature that is less than that of pure steam only.

EQUIPMENT, TECHNIQUES AND MEDIA 37

gas sterilization is used to sterilize plastics, used selectively according to the type and
rubber goods, delicate instruments and other volume of medium as described below:
materials that would be damaged by the high
temperature of the steam pressure autoclave. 4.2.1 MF Broths and Agars: Small volumes
Vent sterilized materials according to operating of broth are rehydrated in a boiling water bath
instructions. for 5 minutes. Agars and larger volumes of
broth require direct heating to the first bubble
4. Preparation and Use of Culture Media of boil. Such heating must be applied with
stirring and constant attention until agar is
The preparation of culture media and solu- dissolved.
tions is a critical aspect of water quality testing.
In 'many laboratories media are prepared by 4.2.2 Other Broths: Some heat may be
nonprofessional personnel. If such personnel required to dissolve the ingredients prior to
are properly trained and guided, they can autoclaving. Heat can be applied by flame or
perform the required tasks efficiently and waterbath.
reliably. However, the supervisor should
monitor media preparation to maintain quality 4.2.3 Other Agars: Heat is required for
control. Use commercial dehydrated media complete solution. For large volumes, direct
which require only weighing and dissolving of heat will effect solution more rapidly but must
the powder in laboratory pure water for prepa- be applied with stirring and constant attention
ration and are much more likely to have uniform to prevent scorching.
high quality than media compounded in the
laboratory. See IV-A, 7.3-7.5 for quality control 4.3 Sterilization: Media must be dissolved
on preparation of media. before autoclaving, to insure timing for com-
plete sterilization. The specific recommenda-
4.1 Supplies of Dehydrated Media: The tions for sterilization are described in subsec-
laboratory worker should keep a record of the tion 5, Composition of Media, in This Section.
lot numbers of commercial media in use. The
data of receipt of media and the date of open- The following general recommendations
ing should be recorded in the quality control can be made:
log. It is suggested that' supplies of dehydrated
media be purchased for anticipated use over Sterilization
the next year. Whenever practical, one-quarter
pound bottles should be purchased to insure
minimal exposure of contents to atmosphere. MF and Salmonella
Heat to boiling only
When a new lot number of medium is used, the Media
contents should be tested for expected per-
formance characteristics (see Part IV-A for Media Containing Autoclave, cool,
details). Stocks of dehydrated media should be Blood then add blood
stored in a cool, dry place away from sunlight.
Litmus Milk and 115 C for 20
4.2 Rehydration of Media: In this Manual, Other Milk-con- minutes
dry ingredients are added to 1 liter (1000 ml) taining Media (10 lbs. pressure)
of laboratory pure water. However, liquids
such as ethanol are added to graduate and
Most Carbohydrate- 118 C for 15
br9ught up to volume with laboratory pure Containing Media minutes
water. Care must be taken to completely dis- for Fermentation (12 lbs. pressure)
solve and mix the Ingredients before dispensing Reactions such as or
the medium into bottles, tubes or flasks. If Phenol Red Broth, 121 C for 12 minutes
heat is necessary, it must be applied with cau- Triple Sugar Iron
tion and for the shortest possible time. Direct Agar
heat, boiling water bath and flowing steam are

38 oEPA MICROBIOLOGICAL MANUAL 1978

 Culture media used for the examination of
MPN, some Salmonella 121 C for water and wastewaters are described sepa-
Media and General 12-15 minutes rately for:
Use Media such as (15 lbs. pressure)
Trypticase Soy Broth 5.1 General Use Media
and Agar, Nutrient 5.2 MF Media for Coliforms
Broth and Agar 5.3 MPN Media for Coliforms
5.4 Media for Fecal Streptococci
5.5 Media for Salmonella and
Other Enterics
As soon as the autoclave pressure has 5.6 Medium for Actinomycetes
fallen to zero, the sterilized media should be
removed from the autoclave for cooling before 5.1 General Use Media
use or storage. Refrigerated media should be
allowed to come to room temperature before 5.1. 1 Nutrient Agar (Difeo 0001-02, BBL
use. Incubate MPN tubed media overnight and 11471)
discard tubes showing bubbles. Media that
have been poured into petri dishes should be
used on the day of preparation or refrigerated. Use: This medium is used to cultivate pure
Water loss from evaporation can be prevented culture isolates for subsequent gram stain and
by storing plates in plastic bags. Plates should other examinations and for general cultivation
be stored inverted. See Part IV-A, 7.9 for stor- of microorganisms that are not fastidious.
age limits.
Composition:
Autoclaves should be inspected routinely
to insure proper functioning of pressure Peptone 5.0 g
gauges and .thermometers. The use of com- Beef Extract 3.0 g
mercially available temperature indicator de- Agar 15.0 g
vices within the sterilizer (heat-resistant spore
preparations, chemically-impregnated tapes,
vials containing chemicals, etc.) is recom- Final pH: 6.8 ± .1
mended to insure sterility (see Part IV-A for
details). Preparation: Add ·23 grams of nutrient
agar per liter of laboratory pure water and
mix well. Heat in boiling water bath to dissolve
5. Composition of Media the agar completely. Dispense in screw-cap
tubes, bottles or flasks and sterilize for 15
The formulas for media used in this Man- minutes at 121 C (15 lbs. pressure). Remove
ual and for other more commonly-used media tubes and slant.
are given in detail in this Section. Normally,
these media should not be prepared from basic 5.1.2 Nutrient Broth (Difeo 0003-02, BBL
ingredients when suitable commercial media 11478) --
are available.
Use: General laboratory use for the cultiva-
The catalogue numbers cited are those for tion of non-fastidious microorganisms.
1A lb. size if available, from the two U.S. manu-
facturers whose media were used in most of Composition:
the method development work. This listing is
not restrictive; other sources can be utilized if Peptone 5.0 g
the user confirms that the formulas are the Beef Extract 3.0 g
same as those cited here and that the media
produce comparable results. Final pH: 6.8 ± .1

EQUIPMENT, TECHNIQUES AND MEDIA 39

 Preparation: Add 8 grams of nutrient broth Dextrose 2.5 g
per liter of laboratory pure water and warm to Dipotassium Phosphate 2.5 g
dissolve the medium completely. Dispense in
containers and sterilize for 15 minutes at 1 21
C (16 lbs. pressure).
Final pH: 7.3 + .2

5.1.3 Trypticase Soy Agar (BBL 11042) Preparation: Add 30 grams of TS broth per
Tryptic Soy Agar(Difeo 036902) liter of laboratory pure water. Warm the broth
and mix gently to dissolve the medium com-
Use: A general purpose medium for the pletely. Dispense and sterilize for 15 minutes
cultivation of fastidious microorganisms. An to 121 C (15 lbs. pressure).
excellent blood agar is prepared by adding
sheep blood.
5. 1.5 Standard Methods Agar (BBL
Composition: 11637) Plate Count Agar (Difeo 04 79-02)
(Tryptone Glucose Yeast Agar)
Trypticase Peptone or
Tryptone 15.0 g Use: Standard Plate Counts in water and in
Phytone Peptone or general pour plate procedures.
Soytone 5.0 g
Sodium Chloride 5.o· g
Agar 15.0 g Composition

Final pH: 7 .3 ± .2
Tryptone or Trypticase
Preparation: Add 40 grams of TS agar per Peptone 5.0 g
liter of laboratory pure water and mix well. Yeast Extract 2.5 g
Heat In a boiling water bath to dissolve agar Dextrose 1.0 g
completely. Dispense into tubes, bottles or Agar 15.0 g
flasks and sterilize for 1 5 minutes at 12 1 C ( 15
lbs. pressure). Final pH: 7 .0 ± .2
For blood agar, cool the sterile, melted
agar to 45-46 C and add aseptically 5 ml of Preparation: Add 23.5 grams of tryptone
sterile defibrinated sheep blood for each 100 glucose yeast agar per liter of laboratory pure
ml of agar. Mix flask of agar by swirling and water. Mix well and heat in boiling water bath
dispense into petri dishes. Blood from other to dissolve agar completely. Dispense· into
species may be used for particular purposes. screw-cap tubes, flasks or bottles and sterilize
for 15 minutes at 121 C (15 lbs. pressure).
5.1.4 Tryptic Soy Broth (Difeo 0370-02
Trypticase Soy Broth (BBL 11767) 5. 1.6 Phenol Red Broth Base (Difeo
0092-02, BBL 11505)
Use: A general purpose medium for the
cultivation of fastidious microorganisms.
Use: Phenol red broth base with the addi-
Composition: tion of carbohydrates is used in fermentation
studies of microorganisms because its pH
Tryptone or Trypticase range of 6.9-8.5 indicates slight changes
Peptone 17.0 g toward acidity. Although 0.5-1.0%
Soytone or Phytone carbohydrates have been used, the 1.0 per-
Peptone 3.0 g cent level is recommended to prevent reversal
Sodium Chloride 5.0 g of the reactions.

40 &EPA MICROBIOLOGICAL MANUAL 1978

Composition: Preparation: Add 15-16 grams (depending
on manufacturer) of purple broth base per liter
Difeo (0092-02)
of laboratory pure water and mix to dissolve.
Beef Extract 1.0 g Add 10 grams of the test carbohydrate and dis-
Protease Peptone No. 3 10.0 g solve. Dispense in screw-cap fermentation
Sodium Chloride 5.0 g tubes and sterilize for not more than 15 minutes
Phenol Red 0.018 g at 118 C (12 lbs. pressure).

BBL (11505) Ten percent solutions of the following car-

bohydrates are prepared for differentiation of
Trypticase Peptone 10.0 g fecal streptococci.
Sodium Chloride 5.0 g
L-Arabinose (Difeo 0159, BBL 11960)
Phenol Red 0.018 g
Raffinose (Difeo 017 4, BBL 12060)
D-Sorbitol (Difeo 0179)
Final pH: 7 .4 + .2 Glycerol (Difeo 0282)
Lactose (Difeo 0156, BBL 11881)
Preparation: Add 15-16 grams (de-
Inositol (Difeo 0164)
pending on manufacturer) of phenol red broth
L-Sorbose (Merck 7760)
base per liter of laboratory pure water. Mix
well to dissolve. To this solution, add 10 grams Sterilize heat-labile carbohydrates and
of test carbohydrate if heat-stable. Mix to com- alcohols by passage through a sterile 0.22 µm
plete solution. Distribute the medium in fer- membrane filtration unit. The carbohydrates
mentation tubes and sterilize not more than 1 5 made up as 10% solutions are sterilized and
minutes at 118 C (12 lbs. pressure). Heat- added to the sterile base medium at a 1 %
sensitive carbohydrates oralcohols are filter- concentration, wt/unit volume. Check pH and
sterilized and added to the sterile medium adjust if necessary with 0.1 N NaOH after
tubes. Check pH and adjust if necessary with addition of carbohydrate. Careful use of asep-
0.1 N NaOH after addition and solution of the tic techniques is necessary to prevent contami-
carbohydrate. nation. As a QC check for contamination, incu-
bate the prepared tubes for 24 hours at 35 C.
5.1.7 Purple Broth Base (Difeo 0227-02,
BBL 11558_)__ 5. 1.8 Purple Broth Base with Sorbose, pH
10
Use: For preparation of carbohydrate broths (Medium may not be commercially
in fermentation studies, for the cultural identi- available).
fication of pure cultures of microorganisms,
particularly the fecal streptococci. Although Use: To determine the presence of Group
0.5-1.0 percent carbohydrate has been used, Q Streptococci.
1.0 percent is recommended rather than 0.5
p'ercent to prevent reversal of the reaction. Composition:
BBL product does not contain beef extract.
Same as purple broth base but add 1 %
sorbose and adjust pH to 10.0.
Composition:
Final pH: 10.0 + .2
Protease Peptone No. 3
or Peptone 10.0 g Preparation: Prepare and sterilize purple
Beef Extract 1.0 g broth base as in 5.1.7. Add sufficient volume
Sodium Chloride 5.0 g of a 10% filter-sterilized solution of sorbose to
Brom Cresol Purple 0.015 g produce a 1 % final concentration of sorbose.
Adjust pH to 10.0 with sterile 38% sodium
Final pH: 6.8 ± .2 phosphate solution (Na3P04·12 H20).

EQUIPMENT, TECHNIQUES AND MEDIA 41

 5. 1.9 IMViC Test Media (c) Simmons Citrate Agar (BBL 11619,
Difeo 0091-02)
Use: Differentiation of the coliform group
based on indole production from tryptophane Use: Differentiation of gram-negative en-
broth, acid production in a glucose broth indic- teric bacteria on the basis of citrate utilization.
ated by methyl red color change, formation of
acetoin (actylmethylcarbinol) in salt peptone Composition:
glucose broth and use of citrate as the sole
carbon source. Magnesium Sulfate 0.2 g
Monoammonium Phosphate 1:0 g
(a) Tryptone 1 % (Difeo 01 23-02) Trypto- Dipotassium Phosphate 1.0 g
phane Broth (BBL 11920) Sodium Citrate 2.0 g
Sodium Chloride 5.0 g
Use: For the detection of indole as a by- Brom Thymol Blue 0.08 g
product of the metabolism of tryptophane and Agar 15.0 g
forthe identification of bacteria.
Final pH: 6.8 + .2
Composition:
Preparation: Add 24.2 grams of Simmons
Tryptone or Trypticase Citrate agar per liter of laboratory pure water.
Peptone 10.0 g Heat in boiling water bath with mixing for
complete solution. Dispense into screw-cap
Final pH: 7.2 ± .2 tubes and sterilize for 15 minutes at 121 C
(15 lbs. pressure). Cool tubes as slants.
Preparation: Add 10 grams of Tryptone or
Trypticase to 900 ml of laboratory pure water 5.1.10 Motility Test Medium (Edwards and
and heat with mixing until dissolved. Bring Ewing)(BBL 11435) -
solution to 1000 ml in a graduate or flask.
Dispense in five ml volume tubes and sterilize Use: Detection of motility of gram-negative
for 16 minutes at 121 C (15 lbs. pressure). enteric bacteria.

(b) MR-VP Broth (Buffered Glucose) (BBL Composition:

11382, Difeo 0016-02)
Beef Extract 3.0 g
Use: For the performance of the Methyl Peptone 10.0 g
Red and Voges-Proskauer Tests in differentia- Sodium Chloride 5.0 g
tion of the coliform group. Agar 4.0 g

Composition: Final pH: 7 .3 ± .2

Buffered Peptone or Preparation: Add 22 grams of dry medium
Polypeptone 7.0 g to 1 liter of laboratory pure water. Add 0.05
Dextrose 5.0 g grams of triphenyl tetrazolium chloride/liter.
Dipotassium Phosphate 5.0 g Heat with mixing to boil for 1 minute. Dispense
10 ml volumes into tubes and sterilize for 15
Final pH: 6.9 ± .2 minutes at 121 C (15 lbs. pressure).

Preparation: Add 17 grams of MR-VP me-

dium to 1 liter of laboratory pure water. Mix to 5.2 MF Media for Coliforms
dissolve. Dispense 10 ml volumes into tubes
and sterilize for 15 minutes at 121 C (15 lbs. Prepare heat-sensitive broths ~ sterile
pressure). flasks .

42 .$.EPA MICROBIOLOGICAL MANUAL 1978

 5.2.1 M-FC Broth Base (Difeo 0883-02)
M-FC Broth (BBL 11364)
Prepare by adding 15 grams of agar per
liter of M-FC broth. Heat in boiling water bath
Use: Detection and enumeration of fecal to solution then cool to about 45 C and add to
coliform microorganisms by the membrane fil- 50 mm diameter glass or plastic petri dishes to
ter procedure. a minimal agar depth of 2-3 mm. Allow to
solidify. Protect the prepared medium from
Composition: light. It can be stored at 4 C for up to 2 weeks.

Tryptose or Biosate Caution: Do not autoclave M-FC Broth or

- ---- -----
Peptone 10.0 g Agar.
Protease Peptone No. 3
· or Polypeptone 5.0 g
5.2.2 M-Coliform Broth (BBL 11119) M-
Endo Broth MF (Difeo 0749-02) -
Yeast Extract 3.0 g
Sodium Chloride 5.0 g Use: A selective and differential medium
Lactose 12.5 g for enumeration of members of the coliform
Bile Salts No. 3 or group by the membrane filter technique.
Bile Salts Mixture 1.5 g
Aniline Blue 0.1 g Composition:

Final pH: 7.4 ± .2 Tryptose or Polypeptone 10.0 g

Thiopeptone or Thiotone 5.0 g
Preparation: Casitone or Trypticase 5.0 g
Yeast Extract 1.5 g
Lactose 12.5 g
Rosalie Acid Sodium Chloride 5.0 g
Dipotassium Hydrogen
Dissolve 1 gram of rosolic acid in 100 ml Phosphate 4.375 g
of 0.2 N sodium hydroxide to make a rosolic Potassium Dihydrogen
acid solution. Note: The quality of present sup- Phosphate 1.375 g
plies is quite variable. Performance of new Sodium Lauryl Sulfate 0.050 g
batches should be compared against previous Sodium Desoxycholate 0.1 g
batch before it is exhausted. Sodium Sulfite 2.1 g
Basic Fuchsin 1.05 g
Autoclaving will decompose rosolic acid
reagent. Stock solutions should be stored .in
Final pH: 7.2 + .2
the dark at 4 C. Discard after 2 weeks or sooner Preparation: Add 48 grams of M-Endo
if the color changes from dark red to muddy medium to 1 liter of laboratory pure water
brown. The rosolic acid may be omitted from containing 20 ml of 95% ethanol. Denatured
testing samples with stressed organisms and alcohol should not be used. Heat in boiling
low background count. water bath for solution. Store prepared medium
in the dark at 4 C. Discard the unused medium
after 96 hours.
M-FC
--- Broth
-
Prepare M-Endo agar by adding 15 grams
Add 37 grams of M-FC medium to 1 liter of of agar per liter of M-Endo medium. Heat to
laboratory pure water containing 10 ml of the boiling, cool to about 45 C and dispense into
rosolic acid solution. Heat in a boiling water glass or plastic petri dishes to provide minimal
bath to dissolve before use. Store the prepared agar depth of 2-3 mm and allow to solidify.
medium at 4 Cina refrigerator. Discard unused Protect prepared medium from light. It can be
medium after 96 hours. stored at 4 C for up to 2 weeks.

EQUIPMENT, TECHNIQUES AND MEDIA 43

 5.2.3 M-Endo Holding Medium Composition:

Use: Holding Medium in the delayed incu- Yeast Extract 1.2 g

bation total coliform procedure. Casitone or Trypticase Peptone 3.7 g
Thiopeptone or Thiotone 3.7 g
Composition: Tryptose or Biosate Peptone 7.5 g
Lactose 9.4 g
Same as M-Endo Broth but add 0.384 Dipotassium Hydrogen
grams of sodium benzoate per 100 ml. Phosphate 3.3 g
Potassium Dihydrogen
Preparation: Phosphate 1.0 g
Sodium Chloride 3.7 g
Sodium Desoxycholate 0.1 g
Prepare M-Endo Broth as described in
Sodium Lauryl Sulfate 0.05 g
5.2.2 and add 3.2 ml of a 12 % solution of Sodium Sulfite 1.6 g
sodium benzoate per 100 ml of medium. Add
Basic Fuchsin 0.8 g
cycloheximide if needed.
Agar 14-15.0 g
i

Sodium Benzoate Solution Final pH: 7.2 ± .2

Final pH: 7 .2 ± .2 Preparation: Add 50 or 51 grams, depend-
ing on manufacturer, of agar per liter of labora-
Dissolve 12 grams of sodium benzoate in
tory pure water to which has been added 20 ml
about 85 ml of laboratory pure water, then
of 95% ethanol. Heat in· boiling water bath
bring to 100 ml final volume. Sterilize by auto-
to dissolve completely. Cool to about 45 C
claving or filtration. Discard the solution after
and dispense into 60 mm glass or plastic petri
6 months.
dishes to provide a minimal agar depth of 2-3
mm. Allow to solidify. If larger dishes are used,
Cycloheximide Solution (Optional) dispense sufficient agar to give equivalent
depth. Protect prepared medium from light. It
can be stored at 4 C for up to 2 weeks.
Cycloheximide is used for samples that
have shown problems of overgrowth with
fungi. Prepare an aqueous solution containing Caution: Do not autoclave.
1.26 grams of cycloheximide/100 ml of labora-
tory pure water. Store solution in refrigerator. 5.2.5 M-Coliform Holding Broth (Difeo
Discard and remake after 6 months. Add 4 ml 084 2-02) (LES Holding Medium)
of the aqueous solution of cycloheximide per
100 ml of M-Endo Holding Medium. Use: Holding medium in the delayed-
incubation total coliform procedure.
Caution: Cycloheximide is a powerful skin
Irritant that should be handled with care. See Composition:
Manufacturer's Directions (Actidione, Upjohn,
Kalamazoo, Ml). Tryptone or Trypticase
Peptone 3.0 g
5.2.4 M-Endo Agar LES (Difeo 0736-02, M-Endo Broth MF 3.0 g
BBL 11203) - - Di potassium Hydrogen
Phosphate 3.0 g
Use: Determination of total coliforms us- Sodium Benzoate 1.0 g
ing a two-step membrane filter technique with Sulfanilamide 1.0 g
lauryl tryptose broth as the preliminary Paraminobenzoic Acid 1.2 g
enrichment. Cycloheximide 0.5 g

44 &EPA MICROBIOLOGICAL MANUAL 1978

Final pH: 7.1 ± .2 mentation tubes (150 X 20 mm tubes contain-
ing 75 X 10 mm tubes) for testing 1 ml or less
Preparation: Add 12.7 grams per liter of of samples. For testing 10 ml volumes of sam-
laboratory pure water and mix to dissolve. Do ples, add 71.2 grams of the medium per liter of
not heat to dissolve medium. - laboratory pure water and mix to dissolve. Dis-
pense in 10 ml amounts in fermentation tubes
5.2.6 M-VFC Holding Medium (3) (150 X 25 mm tubes containing 75 X 10 mm
tubes). Sterilize for 15 minutes at 121 C (15
Use: Holding medium in the delayed incu- lbs. pressure). The concentration of the me-
bation test for fecal coliform microorganisms. dium should vary with the size of the sample
according to the table below.
Composition:

Casitone, Vitamin Free 0.2 g Compensation in Lauryl Tryptose Broth

Sodium Benzoate 4.0 g for Diluting Effects of Samples
Sulfanilamide 0.5 g

Final pH: 6. 7 ± .2 Sample Size Medium Dehydrated

LTB Medium /Dilution Concen- LTB in
Preparation: Add 4.7 grams of medium /Tube in ml in ml tration grams/liter
per liter of· laboratory pure water containing
10 ml of 9 5 % ethanol. Denatured alcohol
should not be used. Heat slightly to dissolve
10 0.1 to 1.0 1x 35.6
ingredients, then sterilize by membrane filtra- 10 10 2x 71.2
tion (0.22 um). Store prepared medium at 4 C. 20 10 1.5x 53.4
Discard after 1 month.

5.3 MPN Media for Coliforms

5.3.1 Lauryl Sulfate Broth (BBL 11338) 5.3.2 Brilliant Green Bile 2% (Difeo
Lauryl Tryptose Broth (Difeo 0241-02) 0007-02) Brilliant Green Bile Broth 2% (BBL
11079) -------
Use: Primary medium for the Presumptive
Testforthetotal coliform group. Use: Recommended for the confirmation
of MPN Presumptive Tests for members of the
Composition: coliform group.

Tryptose or Trypticase Composition:

Peptone 20.0 g
Lactose 5.0 g Peptone 10.0 g
Di potassium Hydrogen Lactose 10.0 g
Phosphate 2.i5 g Oxgall or Bile 20.0 g
Potassium Dihydrogen Brilliant Green 0.33 g
Phosphate 2.75 g
Sodium Chloride 5.0 g Final Ph: 7 .2 + .2
Sodium Lauryl Sulfate 0.1 g
Preparation: Add 40 grams of brilliant
Final pH: 6.8 + .2 green bile broth to 1 liter of laboratory pure
water. Dispense 10 ml volumes of the broth in
Preparation: Add 35.6 grams of the me- fermentation tubes (150 X 20 mm tubes con-
dium to 1 liter of laboratory pure water and mix taining 75 X 10 mm tubes). Sterilize for 15
to dissolve. Dispense 10 ml volumes in fer- minutes at 121 C ( 15 lbs. pressure).

EQUIPMENT, TECHNIQUES AND MEDIA 45

 5.3.3 Levine's Eosin Methylene Blue Agar 5.4 Media for Fecal Streptococci
{Difeo 0005-02, BBL 11220) - --
5.4.1 KF Streptococcus Agar (Difeo
Use: Isolation of coliform-like colonies as a 0496-02) KF Streptococcal Agar (BBL 11313)
preliminary to total coliform Completed Test
Procedure. Use: Selective cultivation and enumera-
tion of fecal streptococci by direct plating or
Composition: the membrane filter technique.

Peptone 10.0 g Composition:

Lactose 10.0 g
DI potassium Phosphate 2.0 g Protease Peptone No. 3
Agar 15.0 g or Polypeptone 10.0 g
Eosln Y 0.4 g Yeast· Extract' 10.0 g
Methylene Blue 0.065 g Sodium Chloride 5.0 g
Sodium Glycerophosphate 10.0 g
Final pH: 7.1 ± .2 Maltose 20.0 g
Lactose 1.0 g
Preparation: Add 37.5 grams of Levine's Sodium Azide 0.4 g
E.M.B. agar to 1 liter of laboratory pure water Brom Cresci Purple
and heat in a boiling water bath until dissolved (in Difeo medium only) 0.015 g
completely. Dispense into tubes, flasks or bot- Agar 20.0 g
tles and sterilize for 15 minutes at 121 C (15
lbs. pressure). A flocculant precipitate may Final pH: 7 .2 ± .2
form after autoclaving. Resuspend the precipi-
tate by gently shaking the flask prior to pour- Preparation: Add 76.4 grams of the me-
ing the medium into sterile petri dishes. dium per liter of laboratory pure water. Dis-
solve by heating in a boiling water bath with
6.3.4 EC Medium (Difeo 0314-02) EC Broth agitation. Heat in boiling water bath for 5
(BBL 11187) --- minutes after solution is complete. Caution: Do
not autoclave. Cool to 60. C and add 1 ml ofa
Use: Detection and enumeration of fecal filter-sterilized 1 % aqueous solution of 2, 3, 5-
coliform bacteria. triphenyl tetrazolium chloride per 100 ml of
agar. If necessary, adjust pH to 7.2 with 10%
Composition: Na 2 C0 3 . Do not hold the completed medium
(with indicator) at 44-46 C for more than 4
Tryptose or Trypticase hours before use. Store prepared medium
Peptone 20.0 g (without indicator) in the dark for up to 30 days
Lactose 5.0 g at 4 C. TTC solution is light-sensitive. It should
Bile Salts No. 3 or be stored in the refriger~tor and protected
Bile Salts Mixture 1.5 g from light.
Dlpotassium Phosphate 4.0 g
Monopotassium Phosphate 1.5 g 5.4.2 Azide Dextrose Broth (Difeo 0837-02,
Sodium Chloride 5.0 g BBL 11000)

Final pH: 6.9 ± .2 Use: Primary inoculation medium for Fecal

.. Preparation: Add 37 grams of EC medium Streptococci Pres~mptive T~st.

to 1 liter of laboratory pure water. Dispense Composition:

into fermentation tubes (150 X 20 mm tubes
containing 75 X 10 mm tubes), Sterilize for 15 Beef Extract 4.5 g
minutes at 121 C (15 lbs. pressure). Tryptose or Polypeptone 15.0 g

46 oEPA MICROBIOLOGICAL.MANUAL 1978

Dextrose 7.5 g Composition:
Sodium Chloride 7.5 g
Sodium Azide 0.2 g Pfizer Peptone c 17.0 g
Pfizer Peptone B 3.0 g
Pfizer Yeast Extract 5.0 g
Final pH: 7.2 + .2 Pfizer Bile 10.0 g
Sodium Chloride 5.2 g
Preparation; Add 34. 7 grams of azide dex- Esculin 1.0 g
trose broth to 1 liter of laboratory pure water. Sodium Citrate 1.0 g
Dissolve and dispense into tubes without inner Ferric Ammonium Citrate 0.5 g
vials. Note: Azide dextrose broth should be Sodium Azide 0.25 g
sterilizedat111fC for 15 iTiTrlUtes (12 lbs. Agar 15.0 g
pressure). Prepare-the medium in multiple
strength for larger inocula to preserve the cor-
Final pH: 7.1 + .2
rect concentration of ingredients. For exam- Preparation: Add 58 grams of PSE agar to
ple, if 10 ml of inoculum is to be added to 10 1 liter of laboratory pure water. Heat in a boil-
ml of medium, the medium should be prepared ing water bath to complete solution. Dispense
double strength. into tubes or flasks and sterilize for 15 minutes
at 121 C (15 lbs. pressure).
5.4.3 Ethyl Violet Azide Broth (BBL 11226)
EVA Broth (Difeo 0606-02) - - 5.4.5 Brain Heart Infusion Broth (BHI)
(Difeo 0037-02, BBL 11058)
Use: Confirmed Test for fecal
streptococci. Use: For separation of enterococcus group
organisms from S. bovis and S. equinus by
Composition: testing for growth at 10 C and 45 C. For
general cultivation of fastidious micro.
Tryptose or Biosate Peptone 20.0 g organisms.
Dextrose 5.0 g
Dipotassium Phosphate 2.7 g Composition:
Monopotassium Phosphate 2.7 g
Sodium Chloride 5.0 g Calf Brain Infusion 200.0 g
Sodium Azide 0.4 g Beef Heart Infusion 250.0 g
Ethyl Violet 0.83 mg Peptone 10.0 g
Sodium Chloride 5.0 g
Disodium Phosphate 2.5 g
Final pH: 7 .0 + .2 Dextrose 2.0 g

Preparation: Add 35.8 grams of the me-

Final pH: 7.4 + .2
dium to 1 liter of laboratory pure water. Dis- Preparation: Dissolve 37 grams of brain
solve and dispense in 10 ml amounts into heart infusion broth in 1 liter of laboratory pure
tubes. Sterilize for 15 minutes at 121 C ( 1 5 lbs. water. Dispense in 8-10 ml volumes in screw-
pressure). cap tubes and sterilize for 15 minutes at 121 C
(15 lbs. pressure). If the medium is not used the
5.4.4 PSE Agar (Pfizer Selective Entero- same day as prepared and sterilized, heat at
coccus Agar) 2248, formerly from Pfizer Diag- 100 C for several minutes to remove absorbed
nostics Division. Now available from Grand oxygen, and cool quickly without agitation,
Island Biological Company (GIBCO), 3175 Sta- just prior to inoculation.
ley Road, Grand Island, NY 14072.
5.4.6 Brain Heart Infusion Agar (Difeo
Use: Isolation offecal streptococci. 0418-02,BBL11064)

EQUIPMENT, TECHNIQUES AND MEDIA 47

 Use: Cultivation of streptococci isolates or Preparation of Medium: Add 37 grams of
other fastidious bacteria. BHI broth to 1 liter of laboratory pure water
and heat gently with agitation to dissolve. Dis-
Composition: Brain heart infusion agar pense 60 ml amounts of the medium into
contains the same components as BHI broth screw-cap flasks. Sterilize for 1 5 minutes at
(see 5.4.5 above) with the addition of 15.0 121 C (15 lbs. pressure).
grams agar.
Preparation of 10% oxgall: Add 10 grams
Preparation: Heat in boiling water bath of oxgall per 100 ml of laboratory pure water.
until dissolved. Dispense 10-12 ml of medium After dissolving and mixing, filter-sterilize the
in screw-cap test tubes and slant after steriliza- solution.
tion. Sterilize for 15 minutes at 121 C (15 lbs.
pressure). Preparation of Final Medium: Cool the BHI
broth and add 40 ml of the sterile 10% oxgall
Final pH: 7 .4 ± .2 solution to each 60 ml of sterile, cool BHI
broth, resulting in a 40% bile concentration.
5.4. 7 Brain Heart Infusion (BHI) Broth with Dispense as needed aseptically in 10 ml vol-
6.5%NaC-I- - - umes into sterile culture tubes.

Use: Identification of fecal streptococci. 5.4. 10 Starch Agar

Composition: Brain heart infusion broth (Medium may not be commercially

with 6.5% NaCl is the same as BHI broth in available).
5.4.5 above with addition of 60.0 grams NaCl
per liter of medium. Use: Starch hydrolysis tests for separation
and confirmation of fecal streptoccal species.
Since most commercially available dehy-
drated media already contain sodium chloride, Composition:
this amount is taken into consideration for
determining the final NaCl percentage above. Peptone 10.0 g
Yeast Extract 5.0 g
5.4.8 Brain Heart Infusion Broth (BHI), pH Sodium Chloride 5.0 g
9.6 ---- -- Starch (Soluble) 2.0 g
Agar 15.0 g
Use: Identification offecal streptococci.
Final pH: 6.8 ± .2
Composition: Same as for BHI broth above
in 5.4.5 with addition of sterile 38% sodium Preparation: Add 37 grams of starch agar
phosphate solution (Na 3 P0 4 •12 H2 0) to in 1 liter of cold laboratory pure water. Heat to
produce a final pH of 9.6. boiling to dissolve and dispense into tubes,
flasks or bottles. Sterilize for 1 5 minutes at
5.4.9 Brain Heart Infusion Broth with 40% 121 C (15 lbs. pressure). Cool medium after
Bile
---- ---- sterilization and pour into petri dishes. Allow to
solidify.
Use: Verification of fecal streptococci.
5.4. 11 Starch Liquid Medium
Composition: Same as for BHI broth above
in 5.4.5 with addition of 40 ml of sterile 10% (Medium may not be commercially
oxgall to 60 ml of basic medium or 668 ml to available).
each liter of medium.
Use: Starch hydrolysis for speciation of
Final pH: 7.4 ± .2 fecal streptococci.

48 &EPA MICROBIOLOGICAL MANUAL 1978

Composition: Preparation: Add 100 grams of litmus milk
to 1 liter of laboratory pure water and warm to
Tryptone or Trypticase about 50 C to dissolve the medium. Dispense
Peptone 10.0 g 10 ml volumes into screw-cap tubes and steril-
Yeast 'Extract 3.0 g ize for 20 minutes at 115 C (10 lbs. pressure).
Dipotassium Phosphate 2.0 g Do not overheat. Control pressure and time
Glucose 0.5 g carefully since overheating or prolonged heat-
Starch (Soluble) 5.0 g ing during sterilization can caramelize the milk
sugar.
Final pH: 6.8 ± .2
5.4. 14 Skim Milk with 0. 1 % Methylene
Preparation: Add 30.5 grams of dry ingre- Blue
dients to 1 liter of laboratory pure water and
heat to boiling. Dispense into tubes and steril- Use: Identification of fecal streptococci by
ize for 15 minutes at 121 C (15 lbs. pressure). reduction of methylene blue.

5.4.12 Nutrient Gelatin (BBL 11481, Difeo Composition:

0011-02)
Skim Milk Powder 100.0 g
Use: Detection of gelatin liquefaction for Methylene Blue Powder 1.0 g
identification of the fecal streptococci and
other microorganisms. Final pH: 6.4 + .2
Composition: Preparation: Add 100 grams of skim milk
powder and 1 gram of methylene blue to 1 liter
Peptone 5.0 g of distilled water, and warm to 50 C to dissolve
Beef Extract 3.0 g the medium. Dispense 10 ml volumes into
Gelatin 120.0 g screw-cap tubes and sterilize for 20 minutes
at 115 C (10 lbs. pressure). Do not overheat.
Final pH: 6.8 + .2 Prolonged heating or overheating during sterili-
zation results in caramelization of the milk
Preparation: Add 128 grams of nutrient sugar.
gelatin to 1 liter of cold laboratory pure water
and warm to. about 50 C to dissolve the me- 5.4. 15 Brain Heart Infusion Agar with
dium. Dispense 5 ml in screw-cap test tubes 0.04% PotaSSTUiTITeITUrfte
and sterilize for 15 minutes at 121 C (15 lbs.
pressure). Store tubes in refrigerator until use. Use: Identification of fecal streptococci by
tellurite reduction.
5.4.13 Litmus Milk (Difeo 0107-02, BBL
11343)
Composition:
Use: To separate and identify fecal strep-
tococci and generally to determine the action Calf Brain Infusion 200.0 g
of bacteria on milk. Beef Heart Infusion 250.0 g
Protease Peptone 10.0 g
Composition: Dextrose 2.0 g
Sodium Chloride 5.0 g
Skim Milk 100.0 g Disodium Phosphate 2.5 g
Litmus 0.75 g Agar 15.0 g

Final pH: 6.8 ± .2 Final pH: 7.4 ± .2

EQUIPMENT, TECHNIQUES AND MEDIA 49

 Preparation: Add 52 grams of brain heart Sodium Chloride 5.0 g
Infusion agar to 1 liter of cold laboratory pure Agar 14.0 g
water. Heat in a boiling water bath to dissolve
the agar and dispense 100 ml volumes in Final pH: 7.0-7.3
screw-cap flasks and sterilize for 15 minutes at Preparation of 50% Glucose Solution:
121 C (15 lbs. pressure). Cool to 50 C and add Weigh out 50 grams of reagent grade glucose.
1 ml of sterile warm (50 C) 4 % potassium Add to 50 ml laboratory pure water in a 100 ml
tellurite to each 100 ml flask of brain heart volumetric flask. Dissolve glucose and bring
Infusion agar. This should produce a final po- up to volume. Filter-sterilize solution and store
tassium tellurite concentration of 0.04%. Dis- in a screw-cap flask.
pense melted sterile medium into sterile petri
dishes. Preparation of 1 % TTC Solution: Weigh
5.4.16 Blood Agar with 0.04 % Potassium out 1 gram of 2, 3, 5 triphenyl tetrazolium
Tellurite - ----- chloride. Add to 50 ml laboratory pure water in
a 100 ml volumetric flask. Dissolve and bring
Use: Identification of fecal streptococci by up to volume. Filter-sterilize solution and store
tellurite reduction. in a screw-cap flask.

Composition: Preparation of Final Medium: Add 39

grams of TG agar to 1 liter of cold laboratory
Evans Peptone 10.0 g pure water and heat in a boiling water bath to
Sodium Chloride 5.0 g dissolve the agar. Sterilize for 15 minutes at
Meat Extract (Lab Lemco) 10.0 g 121 C (15 lbs. pressure) and cool to about 50
Yeast Extract 3.0 g C. To 970 ml of liquid TG agar, aseptically add
Agar 15.0 g 20 ml of 50% glucose solution and 10 ml of
1 % TTC solution. Mix well and pour into sterile
Final pH: 7 .2 ± .2 petri dishes.
Preparation: Add 43 grams of blood agar
5.4. 18 Blood Agar Base (Optional - 10%
base to 1 liter of laboratory pure water. Heat
· Blood) {Difeo 0045-02, BBL 11036)
in a boiling water bath to dissolve the agar and
dispense 100 ml volumes in screw-cap flasks, Use: Identification of hemolytic properties
sterilize for 15 minutes at 121 C ( 15 lbs. pres- of fecal streptococci.
sure) and cool to 44-46 C. Aseptically add 10%
sterile defibrinated horse blood to the medium. Composition:
The mixture is heated at 70 C for 10 minutes,
then cooled to 45 C. A filter-sterilized 4.0% BBL 11036
solution of potassium tellurite is added asep-
tically to give a final concentration of 0.04%. Beef Heart Infusion 375.0 g
Dispense the completed medium into petri Tryptose or Thiotone
dishes. (Peptone) 10.0 g
Sodium Chloride 5.0 g
6.4.17 Tetrazolium Glucose {TG) Agar or Agar 15.0 g
2, 3, 6-Triphenyl Tetrazolium Ci1i'Qi="ide {TTC
Agar) (Medium may not be commercially Difeo 0045-02
available).
Beef Heart Infusion 500.0 g
Use: Identification of fecal streptococci by
Tryptose or Thiotone
tetrazolium reduction.
(Peptone) 10.0 g
Composition: Sodium Chloride 5.0 g
Agar 15.0 g
Peptone 10.0 g
Beef Extract 10.0 g Final pH: 7.4 ± .2

60 &EPA MICROBIOLOGICAL MANUAL 1978

 Preparation: Add 40 grams of blood agar Preparation: Add 46 grams of tetrathion-
base to 1 liter of cold laboratory pure water. ate broth base to 1 liter of laboratory pure
Heat in a boiling water bath to dissolve the water and heat to boiling. Cool to less than 45
agar. Dispense in 90 ml volumes into screw- C and add 20 ml of iodine solution.* Mix and
cap flask. Sterilize for 15 minutes at 121 C (15 dispense in 10 ml volumes into screw-cap
lbs. pressure). Store at 4 C for later use. When tubes. Do not heat after the addition of the
ready to use, heat/cool the ·blood agar base ~o iodine. Do not autoclave. The tetrathionate
45-50 C and add 10% by volume of fresh broth base without iodine may be stored for
sterile defibrinated horse blood. Mix and pour later use. The complete medium (with iodine)
into sterile petri dishes. should be used on the day it is prepared.

5.5 Media for Sslmonells *The iodine-iodide solution is prepared by dis-

solving 6 grams iodine crystals and 5 grams
5.5.1 Selenite F Broth (BBL 11607, Difeo potassium iodide in 20 ml of distilled wateL
0275-02)
5.5.3 Dulcitol Selenite Broth
Use: Primary enrichment of salmonellae.

(Medium may not be commercially

Composition:
available).
Tryptone or Polypeptone 5.0 g
Lactose 4.0 g Use: Primary enrichment of salmonellae.
Disodium Hydrogen Phosphate 10.0 g
Sodium Selenite 4.0 g Composition:

Final pH: 7 .0 ± .2 Protease Peptone 4.0 g

Yeast Extract 1.5 g
Preparation: Add 23 grams of selenite Dulcitol 4.0 g
broth to 1 liter of laboratory pure water. Mix Sodium Selenite 5.0 g
and warm gently until dissolved. Dispense in Disodium Hydrogen
tubes to a depth of 6 cm and expose to flowing Phosphate 1.25 g
steam for 15 minutes. Avoid excessive heat- Potassium Dihydrogen
ing. Do not autoclave. Sterilization is unneces- Phosphate 1.25 g
sa..Y if broth is used immediately.
Final pH: 6.9 ± .2
5.5.2 Tetrathionate Broth Base (Difeo
0104-02, BBL 11706) Preparation: Add 16.5 grams of dulcitol
selenite broth to 1 liter of laboratory pure
water and heat carefully to dissolve ingredi-·
Use: Primary enrichment of salmonellae.
ents. Do not boil. The prepared medium should
be buff-colored. Dispense into screw-cap
Composition: tubes to a depth of 6 cm. Do not autoclave.

Protease Peptone or 5.5.4 Tetrathionate Brilliant Green Broth

Polypeptone 5.0 g
Bile Salts 1.0 g Use: Primary enrichment for salmonellae.
Calcium Carbonate 10.0 g
Sodium Thiosulfate 30.0 g Composition: Same as tetrathionate broth
base (5.5.2) with addition of 0.01 gram of
Final pH: 7.8 ± .2 brilliant green per liter.

EQUIPMENT, TECHNIQUES AND MEDIA 51

 5.5.5 Brilliant Green Agar (BBL 11072, Phenol Red 0.08 g
Difeo 0285-02) -- -- Agar 15.0 g
Use: As a primary plating medium for the Final pH: 7.4 ± .2
isolation of salmonellae.
Composition: Preparation: Add 45 or 4 7 grams of XL
agar base to 1 liter of cold laboratory pure
Yeast Extract 3.0 g water. Heat in a boiling water bath to dissolve
Polypeptone or Protease the agar. Prior to sterilization, add 1.25 ml of
Peptone 10.0 g 1 % aqueous brilliant green. Sterilize for 15
Sodium Chloride 5.0 g minutes at 121 C (15 lbs. pressure). Cool the
Lactose 10.0 g sterilized medium to about 45-50 C and add
Saccharose (Sucrose) 10.0 g 20 ml of a solution containing 34% sodium
Phenol Red 0.08 g thiosulfate and 4% ferric ammonium citrate.
Brilliant Green 0.0125 g Pour into sterile petri dishes.
Agar 20.0 g
5.5.7 Xylose Lysine Desoxycholate (XLD)
Final pH: 6.9 ± .2 Agar -- --

Preparation: Add 58 grams of brilliant

Use: Differentiation of Salmonella.
green agar to 1 liter of cold laboratory pure
water and heat to boiling. Dispense in screw-
Composition:
cap flasks and sterilize for 15 minutes at 1 21 C
(15 lbs. pressure). Pour into sterile petri dishes. BBL 11837
Warning: A longer period of sterilization
will reduce the selectivity of the medium. Xylose 3.5 g
L-Lysine 5.0 g
5.5.6 Xylose Lysine Brilliant Green (XLBG) Lactose 7.5 g
Agar Saccharose (Sucrose) 7.5 g
Sodium Chloride 5.0 g
Use: Salmonella Differentiation. Yeast Extract 3.0 g
Phenol Red 0.08 g
Composition of XL Agar Base: Agar 13.5 g
Sodium Desoxycholate 2.5 g
BBL 11836
Sodium Thiosulfate 6.8 g
Xylose 3.5 g Ferric Ammonium Citrate 0.8 g
L-Lysine 5.0 g
Lactose Difeo 0788-02
7.5 g
Saccharose (Sucrose) 7.5 g
Sodium Chloride 5.0 g Xylose 3.75 g
Yeast Extract 3.0 g
L-Lysine 5.0 g
Phenol Red 0.08 g Lactose 7.5 g
Agar 13.5 g
Saccharose (Sucrose) 7.5 g
Sodium Chloride 5.0 g
Difeo 0555-02 Yeast Extract 3.0 g
Phenpl Red 0.08 g
Xylose 3.75 g Agar 15.0 g
L-Lysine 5.0 g Sodium Desoxycholate 2.5 g
Lactose 7.5 g Sodium Thiosulfate 6.8 g
Saccharose (Sucrose) 7.5 g Ferric Ammonium Citrate 0.8 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g Final pH: 7 .4 ± .2
52 AEPA
- MICROBIOLOGICAL MA.NUAL 1978
 Preparation: Add 55 or 57 grams of XLD Yeast Extract 3.0 g
agar in 1 liter of cold laboratory pure water, Peptone 15.0 g
heat to boiling with mixing. Do not overheat Protease Peptone 5.0 g
and do not autoclave. Pour into sterile petri Lactose 10.0 g
disheS. - Saccharose (Sucrose) 10.0 g
Dextrose 1.0 g
Note: Taylor and Schelhart report better Ferrous Sulfate 0.2 g
recoveries by using XL Agar Base (BBL 11835 Sodium Chloride 5.0 g
or Difeo 9555); see 5.5.6, and adding sepa- Sodium Thiosulfate 0.3 g
rately, sterile solutions of the last three ingredi- Agar 12.0 g
ents (4). Phenol Red 0.024 g

5.5.8 Bismuth Sulfite Agar (Difeo BBL 11748

0073-02, BBL 11030)
Peptone 20.0 g
Use: Differentiation of salmonellae, espe- Lactose 10.0 g
cially S. typhosa. Saccharose (Sucrose) 10.0 g
Dextrose 1.0 g
Composition: .Ferrous Sulfate 0.2 g
Sodium Chloride 5.0 g
Polypeptone or Protease Sodium Thiosulfate 0.2 g
Peptone 10.0 g Agar 13.0 g
Beef Extract 5.0 g Phenol Red 0.025 g
Dextrose 5.0 g
Disodium Hydrogen Final pH: 7 .3 + .2
Phosphate 4.0 g
Ferrous Sulfate 0.3 g Preparation: Add 65 grams or 59.4 grams,
Bismuth Sulfite Indicator 8.0 g depending on manufacturer, of triple sugar
Brilliant Green 0.025 g iron agar to 1 liter of cold laboratory pure
Agar 20.0 g water and heat in a boiling water bath to dis-
solve the agar. Dispense into screw-cap tubes
Final pH: 7 .6 + .2 and sterilize for 15 minutes at 118 C (12 lbs.
pressure). Slant tubes for a generous butt.
Preparation: Add 52 grams of bismuth sul- Inoculated TSI slants must be incubated with
fite agar to. 1 liter of cold laboratory pure water loosened caps to prevent complete blackening
and heat to boiling. Do not autoclave or over- of the medium from H 2 S.
heat. Twirl the flask prior to pouring plates to
evenly dispense the characteristic precipitate.
Use the plated medium on the day prepared.
5.5. 10 Lysine Iron Agar

5.5.9 Triple Sugar Iron (TSl)Agar Use: Differentiation of Proteus, Citrobacter

and Shigella from S.almonella based on deami-
Use: Differentiation of gram negative en- nation of lysine and hydrogen sulfide produc-
terics by their differing ability to ferment dex- tion. Salmonella cultures produce large
trose, lactose and sucrose and ability to amounts of hydrogen sulfide and lysine decar-
produce hydrogen sulfide. boxylase.

Composition: Composition:

Difeo 0265-02 Difeo 0849-02

Beef Extract 3.0 g Peptone 5.0 g

EQUIPMENT, TECHNIQUES AND MEDIA 53

Yeast Extract 3.0 g to 900 ml laboratory pure water and boil to
Dextrose 1.0 g dissolve. Sterilize for 15 minutes at 121 C (15
L-Lysine 10.0 g lbs.pressure). Cool to 50-55 C and add asepti-
Ferric Ammonium Citrate 0.5 g cally 100 ml of filter-sterilized urea agar base.
Sodium Thiosulfate 0.04 g Mix and dispense in sterile tubes. Slant tubes
Brom Cresol Purple 0.02 g to form a 2 cm butt and 3 cm slant and cool.
Agar 15.0 g
Urea Agar Base 1OX Concentrate (Difeo
BBL 11362 0284-60)
Peptone 5.0 g
Yeast Extract 3.0 g Use: Same as urea agar base, for prepara-
Dextrose 1.0 g tion of small volumes of urea agar.
L-Lysine 10.0 g
Ferric Ammonium Citrate 0.5 g Composition: A filter-sterilized 1OX solu-
Sodium Thiosulfate 0.04 g tion of urea agar base, 10 ml volumes in tubes.
Brom Cresol Purple 0.02 g Refrigerate to store.
Agar 13.5 g
Preparation: Add 1.5 grams of agar to 90
Final pH: 6. 7 ± .2 ml of laboratory pure water and dissolve by
boiling. SteriJize for 15 minutes at 121 C (15
Preparation: Add 34.5 or 33 grams, de- lbs. pressure). Cool the agar to 50-55 C and
pending on manufacturer, of lysine iron agar to
aseptically add a 10 ml tube urea agar base
1 liter of laboratory pure water. Heat in a concentrate. Mix agar and concentrate. Dis-
boiling water bath to dissolve the agar. Dis-
pense aseptically into sterile tubes and slant.
pense in 4 ml amounts in screw-cap tubes and
sterilize for 12 minutes at 121 C (15 lbs.
pressure). Cool to give a deep butt and short 5.5.12 Phenylalanine Agar (BBL 11536,
slant. Inoculated LIA slants must be incubated Difco0745-02) --
with loosened caps.
Use: Differential tube medium fo~ the sep-
6.6.11 Urea Agar Base (BBL 11794, Difeo aration of members of the Proteus and Provi-
0283-02) - - - - - - dencia genera from other members of the En-
terobacteriaceae based on deaminase activity.
Use: To differentiate enteric microorga-
nisms, especially Proteus sp. on basis of Composition:
urease activity.
Yeast Extract 3.0 g
Composition: DL-Phenylalanine 2.0 g
Disodium Phosphate 1.0 g
Peptone 1.0 g Sodium Chloride 5.0 g
Dextrose 1.0 g Agar 12.0 g
Sodium Chloride 5.0 g
Monopotassium Phosphate 2.0 g
Urea 20.0 g Final pH: 7.3 + .2
Phenol Red 0.012 g
Preparation: Add 23 grams of phenylala-
Final pH: 6.8 ± .2 nine agar to 1 liter of cold laboratory pure
water. Heat in a boiling water bath to dissolve
Preparation: Add 29 grams of urea agar the agar. Dispense in screw-cap tubes and
base to 100 ml of laboratory pure water. Dis- sterilize in the autoclave for 15 minutes at 121
solve and filter-sterilize. Add 15 grams of agar C (15 Jbs. pressure). Slant and cool tubes.

54 &EPA MICROBIOLOGICAL MANUAL 1978

 5.5.13 Malonate Broth (Modified) (BBL 15 minutes at 121 C (15 lbs. pressure). The
11398, Difeo 0569-02-)- proper pH for the complete medium (6.5) is
indicated by purple color of broth.
Use: Differentiation of enteric organisms
based on utilization of malonate. Described by 5.5.15 Motility Test Medium (BBL 11435)
Leifson and modified by Ewing, the medium is
used in differentiation of Salmonella. Use. Recommended for detection of motil-
ity of gram-negative enteric bacilli.
Composition:
Composition:
Yeast Extract 1.0 g
Ammonium Sulfate 2.0 g
Beef Extract 3.0 g
Dipotassium Phosphate 0.6 g Peptone 10.0 g
Monopotassium Phosphate 0.4 g Sodium Chloride 5.0 g
Sodium Chloride 2.0 g
Agar 4.0 g
Sodium Malonate 3.0 g
Dextrose 0.25 g
Brom Thymol Blue 0.025 g
Final pH: 7 .3 + .2
Final pH: 6. 7 + .2 Preparation: Add 22 grams of motility test
medium to 1 liter of cold laboratory pure water
Preparation: Dissolve 9.3 grams in 1 liter and heat in a boiling water bath to dissolve the
of laboratory pure water. Dispense into tubes agar. Dispense in tubes and sterilize for 15
and sterilize for 15 minutes at 121 C (15 lbs. minutes at 121 C (15 lbs. pressure).
pressure).
To aid in recognizing motility, add 0.05
5.5.14 Decarboxylase Medium Base (Difeo grams of triphenyl tetrazolium chloride/liter
0872-02, BBL 11429) after sterilization.
Use: Differentiation of microorganisms
based on decarboxylase activity in presence of 5.5.16 Motility Sulfide Medium (Difeo
L-lysine HCI, L-arginine HCI, L-ornithine HCI, 0450-17)
glutamic acid or other amino acids.
Use: Determination of motility and the
Composition: production of hydrogen sulfide from 1-cystine.
Peptone 5.0 g
Composition:
Yeast Extract 3.0 g
Dextrose 1.0 g
Beef Extract 3.0 g
Brom Cresol Purple 0.02 g
Peptone No. 3 10.0 g
Final pH: 6.5 + .2 L-Cystine 0.2 g
Ferrous Ammonium Citrate 0.2 g
Preparation: Add 9 grams of base to 1 liter Sodium Citrate 2.0 g
of cold laboratory pure water and warm to Sodium Chloride 5.0 g
dissolve completely. Add 5 grams L-lysine, Gelatin 80.0 g
L-ornithine, L-arginine or other L-amino acids Agar 4.0 g
as desired per liter of medium and warm to
dissolve completely. When D/L amino acids are Final pH: 7 .3 ± .2
used add 10 g rather than 5 g. If ornithine HCI
is used, adjust pH with 10 N NaOH. (~bout 2.1 Preparation: Add 104 grams of Motility
ml required for 1 liter of medium containing 5 Sulfide Medium to 1 liter of cold laboratory
grams of ornithine HCI). Lysine or arginine do pure water. After wetting powder, heat care-
not require pH adjustment. Dispense in 5 ml fully to boiling on a hot plate to dissolve com-
volumes into screw-cap tubes and sterilize for pletely. Dispense in 4 ml amounts in tubes, cap

EQUIPMENT, TECHNIQUES AND MEDIA 55

loosely and sterilize for 15 minutes at 11 7 C Sodium Chloride 2.0 g
( 10 lbs. pressure). Dipotassium Phosphate 2.0 g
Magnesium Sulfate•7 :H 20 0.05 g
Calcium Carbonate 0.02 g
5.5.17 !:! Broth (Difeo 0451-02) Iron Sulfate• 7 H20 0.01 g
Agar 15.0 g
Use: Preparation of H agglutinating anti-
gens of members of genus, Salmonella. Final pH: 7.0-7.2

Composition: Preparation: Weigh out ingredients and

add in turn to 1 liter of laboratory pure water in
BBL 11288 a two liter flask. Dissolve ingredients using
gentle heat. Add the agar last and place in a
Thlotone (peptone) 10.0 g boiling water bath. Heat and stir occasionally
Beef Extract 3.0 g until dissolved. Dispense 250 ml volumes in
Dextrose 1.0 g 500 ml screw-cap flasks and 17 ml volumes in
Sodium Chloride 5.0 g screw-cap tubes. Sterilize for 15 minutes at
Dipotassium Phosphate 2.5 g 121 C (15 lbs. pressure).

Difeo 0451-02
6. Laboratory Pure Water
Thiotone (Peptone) 5.0 g
Tryptone 5.0 g Distilled or deionized water free of nutri-
Beef Extract 3.0 g tive and toxic materials is required for prepa-
Dextrose 1.0 g ration of media and dilution/rinse water.
Sodium Chloride 5.0 g
Dipotassium Phosphate 2.5 g 6.1 Distilled Water Systems: Water dis-
tillation units can produce good grades of pure
Final pH: 7.2 ± .2 water. Stills are dependable and long-lived if
maintained and cleaned properly. Use of sof-
Preparation: Add 21.5 grams of H broth to tened water as the source water increases the
1 liter of laboratory pure water, mix well and interval between cleanings of the still. Stills
dissolve by warming. Dispense 5 ml amounts characteristically produce a good grade of
in screw-cap test tubes. Sterilize for 1 5 water which gradually deteriorates as corro-
minutes at 121 C (15 lbs. pressure). sion, leaching and fouling set in. There is no
sudden loss of water quality unless a structural
5.6 Medium for Actinomycetes failure occurs. Stills are efficier:it in removing
dissolved chemicals but not dissolved gases.
Starch-Casein Agar Fresh laboratory pure water may contain chlo-
rine and ammonia. On storage, ammonia will
(Medium may not be commercially increase and C02 will appear from air contami-
available). nation. Distilled water systems should be
monitored continuously for conductance and
Use: Isolation of actinomycetes from analyzed monthly for chlorine, ammonia and
water or soil. standard plate count and at least annually for
trace metals. See Table IV-A-3.
Composition:
6.2 Deionized Water Systems: Deioniza-
Soluble Starch (or tion systems produce a good grade of pure
Glycerol) 10.0 g water and can produce an ultrapure water
Casein (Vitamin-free) 0.3 g when combined with filtration and activated
Sodium Nitrate 2.0 g carbon in a recirculating system.

56 oEPA MICROBIOLOGICAL MANUAL 1978

 In contrast to distilled water systems, diluents, but these are non-standard. Other
deionization systems do not gradually deterio- workers have used tap waters with the same
rate. Rather, they continue to produce the lack of uniformity and an added potential for
same quality water for a long period of time toxicity. Certainly for comparability of microbi-
until the resins and/ or carbon are exhausted, ological data the dilution water must be uni-
whereupon the quality deteriorates quickly. form between laboratories. The chemical ele-
Deionized water systems should be monitored ments and compounds required in natural con-
continually with a conductance meter and ditions to insure a balance of cell solutes and
analyzed monthly for ammonia/amines, total maintain cell turgidity must be reproduced in
organic carbon, specific organic pollutants and the laboratory.
Standard Plate Count and at least annually for
metals. Amines may elute from the resin. Inorganic constituents such as sodium,
Organic carbon results from organic chemicals potassium, magnesium, phosphate, chloride
in the water or from bacterial growth in the and sulfate, and soluble organics such as pep-
columns. Use of a 0.22 µm final filter is recom- tone are used in synthetic dilution waters. pH
mended to remove bacterial contamination. is usually held to a near-neutral reaction. Two
See Table IV-A-3. standard dilution waters are:

Avoid the sudden loss of good quality 7.1 Phosphate Buffered Dilution Water
water by continuously monitoring perfor-
mance of the system, anticipating the remain- 7. 1. 1 Stock Phosphate Buffer Solution
ing life of cartridges and replacing them be-
fore failure occurs. Potassium Dihydrogen
Phosphate (KH 2 P0 4 ) 34.0 g
6.3 Quality Control Labora~ory Pure Water 500 ml

Pure water systems should be monitored Adjust the pH of the solution to 7 .2 with 1
carefully as a part of the intralaboratory QC N Na OH and bring volume to 1000 ml with
program. The water quality should meet the laboratory pure water. Sterilize by filtration or
standards set in this Manual, Part IV-A, 5.2-5.4. autoclave for 15 minutes at 121 C (15 lbs.
pressure).
7. Dilution Water
7. 1.2 Storage of Stock Solution: After
Dilution water is used to reduce the num- sterilization of the stock solution store in the
ber of microbial cells/unit volume of sample so refrigerator until used. Handle aseptically. If
that the density of cells is low enough to permit evidence of mold or other contamination ap-
enumeration or manipulation by the technique pears, the stock solution should be discarded
of choice: pour-plate, spread plate, MF or MPN. and a fresh solution prepared.

The ideal dilution water is neutral in effect. 7. 1.3 Working Solution

It maintains bacterial populations without
stimulating cell growth and reproduction, Stoel< Phosphate Buffer 1.25 ml
damaging cells or reducing their ability to sur- MgCl2 Solution (38 g/liter) 5 ml
vive, grow or reproduce. Its basic purpose is to Laboratory Pure Water 1000 ml
simulate the chemical conditions of the natural
environment which are favorable to cell Final pH: 7.2 t0.1
stability.
7.2 Peptone Dilution Water
Microbiologists have tried different ap-
proaches to obtain an ideal diluent. Some Composition:
workers have copied the natural environment
by use of sterile fresh or marine waters as Peptone 1.0 g

EQUIPMENT, TECHNIQUES AND MEDIA 57

 Laboratory Pure Water 1000 ml pressure). Final volumes after sterilization
should be 99 ± 2 ml. Cool and separate bottles
Final pH: 7.0 ± 0.1 outside of the 99 ml± 2 ml li.mit. Tighten screw-
caps after sterilization and store in a cool
7.3 Preparation of Dilution and Rinse place.
Water

Dispense 102 ml volumes of phosphate Prepare dilution water for rinsing in 500
buffer or peptone dilution water into borosili- ml or larger volumes and autoclave for 30
cate glass, screw-cap dilution bottles scribed minutes or more. Bottles or flasks must be
at 99 ml. Loosen screw-caps on bottles and separated sufficiently in the autoclave to per-
sterilize at 121 C for 15 minutes (15 lbs. mit easy access for steam.

REFERENCES

1. Songer, J. R.• J. F. Sullivan and J. W. Monroe, 1971. Safe, convenient pipetting device. Appl.
Microbiol. 21: 109 7. · --

2. Songer, J. R., D. T. Brayrnen and R. G. Mathis, 1975. Safe, convenient pipetting station. Appl.
Microbiol.30:887. --

3. Taylor, R., R. Bordner and P. Scarpino, 1973. Delayed incubation membrane-filter test for fecal
coliforrns. Appl. Microbiol. 25:363.

4. Taylor, W. I., 1965. Isolation of Shigellae. I. Xylose lysine agars; new media for isolation of enteric
pathogens. Arn. J. Clin. Path . ..!_i:4 71.

GENERAL REFERENCES

Difeo Laboratories. General Conditions Pertaining to the Cultivation of Microorganisms; and, Preparation
of Media from Dehydrated Culture Media. Difeo Manual. 9th Edition. Difeo Laboratories, Detroit, Ml. p. 16
(1953).

Baltimore Biological Laboratories. General Suggestions for Use of Media: Dehydrated-Prepared. BBL
Manual of Products and Laboratory Procedures. 5th Edition, BBL, Division of Becton-Dickinson and Co.,
Cockeysvllle, MD. p. 88 (1968).

Geldreich, E. E.• 1975. Handbook for Evaluating Water Laboratories (2nd Edition). EPA-670/9-75-006,
U.S. Environmental Protection Agency, Cincinnati, OH p. 83.

American Public Health Association, 1976. Standard Methods for the Examination of Water and
Wastewater (14th Edition). American Public Health Association, Inc. p. 892.

68 oEPA MICROBIOLOGICAL MANUAL 1978

 PART II. GENERAL OPERATIONS

Section C Techniques for Isolation and Enumeration of Bacteria of

Sanitary Significance

This . Section describes the fundamental 1. Preparation for Analyses

laboratory procedures needed for
microbiological analyses of water and 1.1 Preparation of Data Sheets
wastewater. Although experienced
microbiologists and technicians may not Select a standard format for bench sheet
require the depth of information and the . or card and use to r~cord pertinent data:
degree of detail given in this Section, it is sample identification, sampling conditions,
provided to serve the technical personnel who chlorine residual, temperature, pH, sampling
are new to environmental microbiology. The site, station number, date and time of
procedures included are: collection, sample collector, required chain of
custody information, data and time received
and analyzed, time elapsed from sample
1. Preparation for Analyses
collection, analyses performed, sample
volume(s), analyst, and laboratory
2. Streak Plate, Pour Plate and identification numbers. (See Figures 11-C-1, 2
Spread Plate Methods and 3).

1.2 Disinfection of Work Area

3. Membrane Filtration Method
Wipe the work area before and after use
4. Most Probable Number Method with laboratory-strength disinfectant and allow
surface to dry before use. See Table V-C-3.

5. Staining Procedures Keep a covered container of iodophor or

quaternary ammonium disinfectant available
for emergency use. Phenolics are acceptable if
6. Shipment of Cultures analyses for these compounds are not per-
formed as part of laboratory work.

The specific details unique to tests are 1.3 Pretreatment of Samples

described in separate Sections of Part Ill:
Standard Plate Count, Total Coliform, Fecal Prior to dilution of samples for analyses,
Coliform, Fecal Streptococci, Salmonella and the analyst should examine the sample for free
Actinomycetes. Refer to Part IV, A & C for de- chlorine residual and possible uneven distribu-
tails of quality control on these analyses. tion of microorganisms.

ISOLATION AND ENUMERATION 59

 ltllCROBIOLOGY LABORATORY RECORD
UEUBRANE FILTER ANALYSIS
PROJECT: NO:
SAMPLE SOUllCE:
SAMPLING DATE: TIME: FILTRATION DATE: TIME:
COLLECTOR: TEMP: pH: ANALYST:

VOL. COUNT COUNT/100 ml VOL. COUNT COUNT/100 ml VOL. COUNT COUNT /100 ml

llemark•: Remerks: Remnrks:

El'A•20t (Cini
(ll•V·•Mlll

FIGURE 11-C-1. Microbiological Bench Card for MF Analyses.

MICROBIOLOGY LABORATORY RECORD

COLIFORM MPN ANALYSIS

Sample • - - - - - - - D a l e _ _ _ _ _ Time - - - fiH River------------

Location-----------------------------------

T'd
I e R.
Qin pal124 Hrs. De p th WalerTe "'.
p "C Secchi Disc

Dilution MPN/IOOml
-

Coli!OC'm Pres. LST 24Hrs.

48Hrs.
Coliform Con!. BGLB 48Hra.
f'ecal ColifOC'm EC 24Hrs.

I
I
AFO, Region Ill, USEPA

FIGURE 11-C-2. Bench Card for MPN Analyses.

60 &EPA MICROBIOLOGICAL MANUAL 1978

 BACTERIAL INDICATOR ORGANISMS OF POLLUTION

STATION _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ BENCH NO

LOCATION-----------------------------

DATE HOURS SAD NO.-------

COLLECTED ... ANALYST

EXAMINED .............. .

MPN
PRESUMPTIVE CONFIRMED
Oil. EC
LTB B.G.L.B. MEMBRANE FILTER
ml. 44.5•C
35'C 35'C
24 hrs. · 48 hrs. 24 hrs. 48 hrs. 24 hrs. VOLUME COLONY
-
- FILTERED COUNT

-- TOTAL
10 -- COLI FORMS

--
-
--
-- FECAL
10- -- COLI FORMS
--
--
--
--
10· --
--
--
--
-- ORGANISMS PER 100 ML.

10-
--
-- TOTALS

-- FE CA LS

--
--
10· --
-- SALMONELLA

-- ISOLATION
CONFIRMED MPN ........... . . . . . PER 100 ML.
SE RO TYPE (S)
FECAL COLIFORM MPN . . . . . ....... PER 100 ML.

REMARKS'------------~-~~--~--~------~

SERL, Region IV. US EPA

FIGURE 11-C-3. Combined Microbiological Bench Card

/SOLA TION AND ENUMERATION 61

 1.3.1 Water Samples with High Solids 1.4 Dilution of Samples

(a) Blending of sediments, primary efflu- 1.4. 1 Necessity for Dilutions: Dilutions of
ents, sludge and highly turbid waters is essen- the original sample Of water, wastewater or
tial for representative subsampling. other material are often necessary to reduce
the number of bacterial cells to measureable
(b) Blend the entire water sample contain-
levels or to isolate single cells for purification
ing particulates in a Waring-type blender. Use
and identification (see Part 11-B, 7 for details on
only autoclavable pyrex glass, stainless steel
dilution water).
or plastic blender containers with safety screw
covers to prevent release of aerosols.
1.4.2 Serial Dilutions: A known quantity of
(c) Limit blending to no more than 30 sec- the sample (usually 1.0 ml, but other volumes
onds at about 5000 RPM to avoid overheating can be used) is transferred through a series of
or shearing damage. known volumes (e.g., 9 or 99 ml) of dilution
water. This procedure is repeated until the
(d) Dilute sediments or soils containing desired bacterial density is reached. After dilu-
limited amounts of water at a 1:1, 1:2 ratio or tion of the sample, the bacteria are enumer-
more with dilution water to ensure good blend- ated using the membrane filtration, pour plate,
ing action and to reduce heat generation. Use streak plate, or the most probable number
of a large blender container rather than smaller technique.
units also reduces heat.
For ease of calculation and preparation,
1.3.2 Dry Solid Samples serial dilutions are usually prepared in suc-
ceeding ten-fold volumes called decimal dilu-
(a) Mix sample thoroughly and weigh 50 tions. T'1e decimal dilution procedure is shown
grams aliquot in a tared weighing pan. Dry at in Figure 11-C-4.
105-110 C to constant weight. The final weight
is used in calculating numbers of organisms/ 1.4.3 Special Dilutions for Membrane Fil-
gram dry solids. tration Procedures: The normally accepted lim-
(b) Prepare the initial dilution by weighing its for colonies per plate in membrane filtration
methods (20-60, 20-80 or 20-100) require
out a second aliquot of 11 grams of original
that decimal dilution series be modified to
sample. Add to a 99 ml volume of buffered dilu-
tion water for a 1:10 dilution and blend sample assume an MF plate count within the accepted
limit.
aseptically in a Waring-type blender at 5000
rpm for 30 seconds. Use only a pyrex glass,
stainless steel or plastic blender container with The recommended method for obtaining
safety screw lid to prevent release of aerosols. counts within these limits is to filter dilution
volumes of the decimal series which have a
(c) Transfer an 11 ml sample of the 1:10 factor of 3, 4 or 5 among them (see Table 11-C-1
dilution to a second dilution bottle containing for details).
99 ml buffered dilution water and shake vigor-
ously about 25 times. Repeat this process until 1.4.4 Filtration Volumes for Membrane
the desired dilution is reached. Filter Analyses: For sample volumes of 1-10 ml,
add 20 ml of dilution water to the funnel before
1.3.3 Analytical Method adding sample, to evenly disperse cells.

Although high solids samples with low 1.4.5 Preparation of Dilutions: Shake
microbial densities may require MPN or pour sample bottle vigorously(about 25 times in 7
plate procedures, high density samples such as seconds) to evenly distribute the bacteria.
polluted soils, sludges and feces are diluted so Take care to secure the screw-cap and prevent
that the MF method is applicable. leakage during shaking.

62 &EPA MICROBIOLOGICAL MANUAL 1978

 . DILUTION A DILUTION B
10:....2 10-4

WATER
LJ
99 ml 99 ml
~ ----------1 ml----------.... IBLANKI - - - - - - - 1 ml---_,.-.... IBLANK

c---

·.~ ~ ~
Ci)·
0
,....
:ti.
::!
o·
:z:
:ti.
I
DELIVERY VOLUME: 10 ml
.
1 ml 0.1 ml
- 1 ml

1
0.1 ml 1 ml 0.1 ml

:z:ti
~
~-
:ti..
::!
0
:z:
VOLUME OF.
ORIGINAL SAMPLE
IN TUBE/PLATE 10 ml 1 ml 10-1 ml 10-3 ml 10....:s ml.

) (_ ) ( _) ( ) ( ) (_ J.
FIGURE 11-C-4. Preparation of Decimal Dilution.
m
w
 TABLE 11-C-1

Recommended Filtration Volumes of Samples in MF Analyses

MF Count Range 20-60 MF Count Range 20-80 MF Count Range 20-100

Sample vol., ml added as: Sample vol., ml added as: Sample vol., ml added as:

.01 ml of 10-2 .04 4 ml of 10-2 .01 ml of 10-2

.03 3 ml of 10-2 .15 1.5 ml of 10-1 .05 5 ml of 10-2

.1 ml of 10-1

.3 3 ml of 10-1 .5 5 ml of 10-1 .25 2.5 of 10-1

1.0 ml sample

3 3 ml sample 2.0 2 ml sample 1.25 1.25 ml sample

10 10 ml sample 8.0 8 ml sample 6 6 ml sample

30 30 ml sample 30 30 ml sample 30 30 ml sample

64 oEPA MICROBIOLOGICAL MANUAL 1978

 (a) Withdraw 1.0 ml or 0. 1 ml of original to a 98.0 ml dilution blank, the final dilution
sample to test samples directly for a 1 x 1o0 ratio is calculated as follows:
ml and 1 x 10- 1 ml volumes, respectively.
2.0 ml 2 1
(b) Transfer a second 1.0 ml volume to a 98.0 + 2.0 ml 100 50
99 ml dilution water bottle (Dilution A). Shake
sample vigorously about 25 times and with- (f) If 0.5 ml is added to a dilution blank
draw 1.0 ml or 0. 1 ml of diluted sample for containing 99.5 ml of buffer, the dilution ratio
testing of 1 x 10-2 and11 x 110-3 1 ml sample is calculated as follows:
volumes. The;resultant dilution is calculated as
follows: 0.5 ml 0.5

Dilution
99.5 + 0.5 ml 100 200
Volume of Sample
Volume of Dilution Blank Ratio (g) Varying the final volume tested will also
+ Volume of Sample permit modification of dilutions without
increasing the number of dilution bottles as
or, follows:

1.0 ml A 1 :200 dilution can be obtained by test-

99.0 + 1.0 ml
ing 0.5 ml of a 1: 100 dilution.
100

or 1o- 2 (Dilution A) A 1 :500 dilution can be obtained by test-

ing 2 ml of a 1: 1000 dilution.
(c) When 1.0 ml is transferred from dilu-
tion bottle A to a second dilution bottle (B), the 1.4.6 Prompt Use of Dilutions: The poten-
dilution ratio for bottle B dilution shown in tial toxicity of phosphate dilution water and
Figure 11-C-4 is the product of the individual the stimulatory effect of peptone dilution
dilutions as follows: water increase rapidly with time. Therefore,
dilutions of samples should be tested as soon
A x B = Final or Total Dilution Ratio as possible after make-up and should be held
no longer than 30 minutes after preparation.

or 2. Streak, Pour and Spread Plate Methods

2.1 Summary: There are three plate dilu-

x tion techniques commonly used for isolating
100 100 10,000
and/or enumerating single colonies of bacteria:
Volumes of 0.1 ml can be tested directly from the streak plate, pour plate and spread plate
each serial dilution to provide intermediate proceduri;is. These techniques described herein
dilutions. use solid or melted agar plating media to dilute
out the microorganisms so that individual
(d) Alternatively, if an initial sample vol- species or cells can be selected or counted from
ume of 11 ml is transferred into the first 99 ml mixed cuftures. Because colonies can originate
volume dilution blank, an intermediate dilution from more than one cell, results may be report-
can be obtained with the added precision re- ed as colony-forming-units (CFUs).
sulting from measurements of 1 ml volumes
serially as opposed to the measurement of 0. 1 2. 1. 1 Streak Plate Method: To obtain a
ml volumes in (c). pure culture the analyst dilutes and isolates
bacteria from a mixed culture by drawing a
(e) Different dilutions can be obtained by small amount of the bacterial growth lightly
varying sample and dilution preparations. For across the surface of an agar plate in a pattern
example, if 2.0 ml of the sample is transferred with an inoculating needle or loop.

ISOLATION AND ENUMERATION 65

 In one suggested pattern, the plate is Plate Count Method (see Part Ill-A). The spread
streaked in parallel lines over half of the sur- plate method provides a quantitative method
face, rotated a quarter turn, streaked again, for aerobic surface growth of cultures against
rotated another quarter turn and streaked which other surface growth methods such as
once more. The inoculum is progressively di- the MF technique can be compared.
luted with each successive streak, and eventu-
ally single cells are deposited on the agar 2.2.2 Because the volumes tested with the
surface. After suitable incubation, single iso- spread plate technique are limited to
lated colonies develop in the path of the 0.1-0.5 ml, the sample must be diluted to con-
streak. (see Figure 11-C-5). tain at least 40-2000 cells/ml in order to have
a counting range of 20-200.
2.1.2 Pour Plate Method: The analyst di-
lutes apd isolatescells in a bacterial suspen-
2.2.3 In the pour plate technique, test vol-
umes are limited to 0.1-2 ml per 100 mi petri
sion by consecutively transferring a portion of
dish so the sample must be diluted to contain
the original sample through a series of dilution
water blanks. After an appropriate series of
60-3000 cells/ml to have a 30-300 counting
range.
dilutions, the original bacterial population is
diluted out to a countable level, as described in
2.3 Apparatus and Materials
1.4. Aliquots of the diluted sample are added
to sterile petri dishes and mixed with melted
agar. After the agar solidifies, the plates are
2.3.1 Incubator set at 35 ± 0.5 C.
inverted and incubated for a predetermined
time. Surface or subsurface colonies will de-
2.3.2 Water bath set at 44-46 C for tem-
pering agar.
velop in some of the agar plates. These colo-
nies can be counted to provide a quantitative
value for the bacterial density of the original 2.3.3 Colony Counter, Quebec darkfield
model or equivalent.
sample, or they can be picked for further quali-
tative study.
2.3.4 Hand tally or electronic counting
device.
2. 1.3 Spread Plate Method: The analyst
Isolates bacterial cells by delivering a small - 2.3.5 Thermometer which has been
volume of a diluted sample onto a solid agar checked against a National Bureau of
plate and spreading the inoculum by use of a Standards-Certified Thermometer or one of
sterile glass rod bent at an angle of about 120°. equivalent accuracy.
The inoculum is spread uniformly by holding
the stick at a set angle on the agar and rotating 2.3.6 Inoculating needle and loop.
the agar plate or rotating the stick until the
lnoculum is distributed evenly. 2.3. 7 Pi pet containers of stainless steel,
aluminum or pyrex for glass pipets.
2.2 Scope and Application
2.3.8 Petri dish containers of stainless
2.2.1 Streak and pour plate methods pro- steel, aluminum or pyrex glass for glass petri
vide the means to separate individual bacteria dishes.
so that each cell will develop into an isolated
colony In or on a solid medium. The methods 2.3.9 Glass spreader rods.
can Isolate specific bacteria by the use of se-
lective or differential media. 2.3. 1.0 Sterile T.D. bacteriological or Mohr
pipets, glass or plastic of appropriate volume.
The streak plate is only qualitative but the
pour plate procedure can be used to quantitate 2.3. 11 Sterile 100 mm X 1 5 mm petri
bacteria present in a sample as in the Standard dishes, glass or plastic.

66 oEPA MICROBIOLOGICAL MANUAL 1978

STEP 1 STEP 2 STEP 3

FIGURE 11-C-5. Suggested Pattern for Preparing a Streak Plate.

ISOLATION AND ENUMERATION· 67

 2.3.12 Dilution (milk dilution) bottles, py- separated pure colonies of bacteria, each th.eo-
rex, marked at 99 ml volume, screw-cap with retically arising from a single bacterium.
neoprene rubber liner.
2.5.6 After streaking, incubate the petri
2.3. 13 Bunsen/Fisher type burner or elec- dishes at 35 C for 24 hours (or other appropri-
tric incinerator. ate conditions) in an inverted position to pre-
vent condensation of water on the agar sur-
2.4Media face. Moisture interferes with development of
isolated colonies by spreading bacterial
growth over the agar surface.
2.4.1 Sterile agar medium dispensed in
bulk quantities in screwcapped bottles or
2.5. 7 For further purification, examine
flasks.
plates after incubation for single, well-isolated
colonies. Pick typical colonies using a sterile
2.4.2 Sterile dilution water in bottles con-
inoculating needle, suspend cells in dilution
taining 99 ± 2 ml volumes. water, and restreak on an agar plate, repeating
steps 2.5.2-2.5.6. Isolated, single coloni.es
2.6 Streak Plate Procedures from a plate containing like colonies may be
considered to be pure.
2.5.1 Melt the nonselective agar, such as
nutrient agar or Trypticase soy agar, temper at 2.5.8 Streaking may also be done on se-
44-46 C, and add about 15 ml agar to each lective media, such as Endo or EMB agars or on
sterile petri dish. Allow to harden and dry for selective/differential media e.g.) in Salmonella
best results in streaking. testing.

2.6.2 Bend an inoculation needle or loop 2.6 Pour Plate Procedure

at an angle about 1 cm from the needle tip to
prevent cutting of the agar during streaking. 2.6. 1 Shake the sample bottle vigorously
Sterilize the needle by heating it to redness in about 25 times to disperse ~he bacteria. Dur-
a flame, and air-cool. ing shaking, close cap tightly to prevent leak-
age of sample and the danger of
2.6.3 Remove screw cap and pick a small contamination.
amount of growth from an isolated colony or
from a mass of growth. 2.6.2 Using a 1.1 ml bacteriological pipet,
prepare the initial dilution by pipeting 1 ml of
2.6.4 Draw the needle containing the the sample into a 99 ml dilution water blank.
bacteria back and forth across the surface of a This initial dilution represents a 10-2 dilution
previously-poured and hardened agar plate in (see this Section, 1.4, Dilution of Samples).
a specific pattern, such as shown in Figure 11-C-
5. Streak 1/3-1/2 of the agar surface. Flame 2.6.3 Using the same pipet transfer 0. 1
and cool needle after each step and inoculate and 1.0 ml from the undiluted sample to two
plate further by drawing the needle across the separa.te petri dishes. ·
area previously streaked.
2.6.4 Shake the 1: 100 dilution bottle
2.6.6 Rotate the plate one quarter turn vigorously again and pipet 0. 1 and 1.0 ml of
clockwise as each step is completed to make the 1: 100 dilution into two petri dishes using
the streaking easier. Streaking patterns other another sterile 1. 1 ml pi pet.
than the model shown in Figure 11-C-5 can be
used; the objective is simply to deposit fewer 2.6.5 Pour aseptically 12-1 5 ml of the
and fewer cells along the streak until single melted agar medium cooled to 44-46 C into
cells are deposited on the agar surface. After each petri dish. Mix agar and inoculum by
Incubation, these cells will develop into well- rotation, being careful to prevent spillover of

68 &EPA MICROBIOLOGICAL MANUAL 1978

agar. One recommended technique uses a se- 2. 7 .4 Place cool glass spreader on agar
quence of five rotations to the left, five to the surface next to inoculum. Position spreader so
right and five forward and backward. Allow the that the tip forms a radius from the center to
agar to solidify on a level surface. the plate edge. Holding spreader motionless,
rotate plate several revolutions, or hold plate
2.6.6 Invert the dishes and incubate at the and move the spreader in a series of sweeping
specific temperature and time. After incuba- arcs. The purpose is to spread the inoculum
tion, well-isolated surface and subsurface colo- uniformly overthe entire surface of the agar.
nies should develop in some of the plates.
2. 7 .5 Lift the glass spreader from the agar
2.6.7 When the pour plate technique is and place in alcohol solution. Cover plate par-
used quantitatively, count plates containing tially, leaving open slightly to evaporate excess
between 30 and 300 colonies. This is the moisture for 15-30 minutes.
technique used in the Standard Plate Count
Method described in Part Ill-A. Pour plate 2.7.6 When agar surfaces are dry, close
counts are reported as the count per ml. dishes, invert them and incubate as required
for the specific test.
Count/ml
2.7.7 After incubation at the proper time
Sum of Plate Counts and temperature, isolated surface colonies
Total Volume of Sample in ml should develop in one or more dilutions within
the acceptable counting range of 20-200 col-
lonies. The maximum recommended number of
2.7 Spread Plate Procedure colonies/spread plate is fewer than for other
plate techniques because surface colonies are
2. 7. 1 Prepare the appropriate melted larger than subsurface colonies and crowding
agar. Pour about 15 ml of the melted agar into can result at lower count levels.
each 100 mm petri dish. Keep covers opened
slightly until agars have hardened and mois- 2.7.8 Count the colonies by normal tech-
ture or condensation have evaporated. Close niques and report on a count/ml or count/100
dishes and store in refrigerator. Warm at room ml basis dependent on the use of the data.
temperature before use.
2.8 Reporting Results
2.7.2 Prepare a series of dilutions based
upon the estimated concentration of bacteria 2.8. 1 Significant Figures: To prevent false
so that 0.1-0.5 ml of inoculum will give a precision in the reporting of counts, the plate
20~200 count (equivalent to a 40-2000 counts must be limited to the digit(s) known
count/ml in the diluted sample). The dilutions definitely plus one digit which is in doubt.
should bracket the. estimated density of These combined digits are termed the Signifi-
bacteria. The analyst must remember that if cant Figures (S.F.).
only 0.1 ml volume is tested, it must be plated
on the agar plate marked with the next higher (a) For example, if an analyst reports a
dilution; for example, 0.1 ml of the 10- 1 plate count of 124 to three significant figures
dilution onto the surface of the plate marked he is indicating that he is certain of the first
1_o-'2. Inoculate agar plates. two digits, 1 and 2, but is uncertain whether
the last digit is 3, 4 or 5. If the analyst were
2. 7 .3 Remove the glass spreader from al- reporting that same number to two significant
cohol and flame. Cool for 1 5 seconds. Test figures, he would report the first figures, 1, as
glass rod on edge of agar to verify safe temper- certain, the second figure, 2, as uncertain, and
ature before use. This step can be simplified by the third figure, 4, as unknown. Hence he
making and sterilizing a number of glass would report it as 120, inserting the zero only
spreaders. · as a spacer. Large counts of 1200, 12,000 and

ISOLATION AND ENUMERATION 69

12,000,000 only contain two significant fig- 3. Membrane Filtration Method
ures. Of course, zeros can be significant in
counts of 10, 60, 105, etc. 3.1 Summary: The membrane filter
method provides a direct count of bacterial
(b) In plate count and MF methods, the colonies on the surface of the filter. The
number of significant digits which can be re- sample is filtered as soon as possible after
ported are dictated by the' method itself as collection. After the sample is filtered, the
follows: within the acceptable counting range membrane filter is placed on a nutrient
of the method itself, i.e., 20-60, 20-80, medium formulated to encourage growth of
20-100 or 30-300 the actual number of colo- the bacteria for which the.test is designed and
nies observed is the best estimate of the true to ;;uppressthe growth of other microorganisms.
density. The number of significant figures are After incubation under the specified
equal to the number of colonies. conditions, the bacteria retained on the
surface of the membrane develop into visible
TABLE 11-C-2 colonies. The medium and the temperature of
incubation influence the kinds and
Number of Significant appearance of bacteria that develop. Two-step
enrichment and delayed incubation MF
Figures (S.F.) Reported
procedures can also be used. The two-step·
procedure involves an acclimation period on
Actual Pour Plate/ Membrane another medium before the selective growth
Colony Spread Plate Filtration step.
Count Method Mt;ithod
3.2 Scope and Application
1 - 9 S.F. S.F.
Membrane filter methods are preferred
10 - 99 2 S.F. 2 S.F. over MPN or other techniques, where applica-
ble because of the following advantages.
100 - 300 3 S.F.
3.2. 1 Advantages

(a) One of the primary advantages of this

2.8.2 Rounding Off Counts: Since plate method is its speed. Definitive results for total
counts must be limited to the number of signifi- and fecal coliforms can be obtained in 22-26
cant figures obtainable by the method, the hours, whereas 48-96 hours are required for
non-zero number which is not significant the multiple-tube fermentation method.
should be treated by the standard scientific
convention: (b) Considerably larger, more representa-
tive water samples can be examined than with
(a) If the insignificant digit is less than five, the. MPN. With waters of low bacterial densi-
replace it with a zero, e.g., 3530 becomes ties such as finished waters, larger sample
3500. aliquots can be used to enhance the reliability
of the results.
(b) If the insignificant digit is five, round
the preceding significant digit to the nearest (c) The precision is greater with the MF than
even number, e.g., with two S.F., 3450 be- the multiple-tube technique because the former
comes 3400, and 3550 becomes '3600. makes a direct count of colonies/unit volume.

(d) The method represents savings in time,

(c) If the insignificant digit is greater than labor, space, supplies and equipment.
five, drop the digit and increase the preceding
significant number by one, e.g., 3480 be- (e) Because ·of its portability, this proce-
comes 3500. dure is also very practical for field studies.

70 &EPA 'MICROBIOLOGICAL MANUAL 1978

 3.2.2 Limitations: Although the majority of 3.3.2 Water bath or other type of incuba-
water samples can be tested by membrane tors such as the aluminum heat sink incubator,
filtration, there are limitations with certain or equivalent, set at 44.5 + 0.2 C for fecal
samples and some problems with membrane coliform test (Part 111-C).
filters themselves.
3.3.3 Stereoscopic microscope, with mag-
nification of 10-15 X, wide-field type.
(a) Some samples contain large quantities
of colloidal materials or suspended solids such
3.3.4 A microscope lamp producing dif-
as iron, manganese or alum floes or clay (1).
fuse light from cool, white fluorescent lamps
Other samples may contain algae. These
adjusted to give maximum color or sheen
substances cari clog the filter pores and
appearance.
prevent filtration or can cause the
development of spreading bacterial colonies.
3.3.5 Hand tally.
When the bacterial counts of such samples are
high, a smaller volume or a higher sample
3.3.6 Pipet container of stainless steel,
dilution can be used to minimize the effect of
aluminum or pyrex glass for glass pi pets.
sample turbidity. The membrane filter method
may be used with samples containing turbidity
3.3.7 Graduated cylinders covered with
by filtration of several smaller replicate sample
aluminum foil or kraft paper and sterilized.
volumes and compositing the results.
However, with waters of high turbidity and low
3.3.8 Membrane filtration units (filter base
bacterial count, the membrane filter method
and funnel), glass, plastic or stainless steel, see
may not be applicable. In the latter situation
Figure 11-C-6. These are wrapped with alumi-
the multiple-tube procedure should be used.
num foil or kraft paper and sterilized. See Fig-
ure 11-C-7 for an exploded view of a stainless
(b) Large non-specific populations may
steel MF assembly and filter. ·
mask the appearance of indicators on selective
media such as M-Endo MF medium (4).
3.3.9 Ultraviolet sterilizer for the filter fun-
nel is optional (6).
(c) Industrial wastewaters may contain
zinc, copper, or other heavy metallic com-
3.3. 10 Line vacuum, electric vacuum
pounds (2) which adsorb onto the membrane
pump or aspirator is used as a vacuum source.
surface and interfere with subsequent bacterial
In an emergency or in the field, a hand pump or
development ( 1 , 2, 3).
a syringe can be used. Such vacuum-
producing devices should be equipped with a
(d) MF analyses require preparation of
check valve to prevent the return flow of air.
MPN tubed media for verification.
3.3. 11 Vacuum filter flask, usually 1 liter,
· (e) Inhibition may result in seawater or
with appropriate tubing. Filter manifolds to
from toxic materials such as chlorine or
hold a numbe.r of filter bases are optional.
phenols.
3.3. 12 Safety trap flask placed between·
(f) Indicator organisms stressed in the en-
the filter flask and the vacuum source.
vironment may be poorly recovered (5).
3.3.13 Forceps, straight or curved, with
smooth tips to permit easy handling of filters
3.3 Apparatus and Materials
without damage.

3.3.1 Incubator set at 35 ± 0.5 C for total 3.3. 14 Alcohol, ethanol or methanol, in.
coliform ·test (Part 111-B) and fecal streptococci small wide mouthed vials, for sterilizing
test (Part 111-D). forceps.

ISOLATION AND ENUMERATION 71

 )

FIGURE 11-C-6. Membrane Filtration Units Made by Various Manufacturers

for Detection of Bacteria in Aqueous Suspensions.

72 &EPA MICROBIOLOGICAL MANUAL 1978

FIGURE 11-C-7. Exploded View of a Stainless Steel Membrane Filtration Unit.

ISOLATION AND ENUMERATION 73

 3.3.15 Bunsen or Fisher type burner or to room temperature. Use sterile forceps for
electric incinerator unit. manipulation of absorbent pads and mem-
brane filters, contacting the outer edges only,
3.3. 16 Sterile T.D. bacteriological or Mohr to avoid touching the filtering area or damag-
plpets, glass or plastic, of appropriate volume. ing the membrane filter surface. Sterilize for-
ceps by immersing the tips in ethanol and
3.3. 17 Sterile petri dishes, 50 X 1 2 mm, flaming. Place absorbent pad in bottom of 50
with tight-fitting lids and 60 x 15 mm, with or 60 mm petri dish. Add 1.8-2.0 ml broth to
loose-fitting lids glass or plastic. the sterile absorbent pad. Saturate but do not
flood the pad. Tip the petri dish to drain off
3.3. 18 Dilution bottles (milk dilution) excess. If agar medium is used, add about 5-6
pyrex, marked at 99 ml, screw-cap with ml (to a depth of 2-3 mm) in the petri dish.
neoprene liners.
3.4.2 Arrange petri dishes in rows accord-
3.3. 19 Membrane filters, white, grid ing to the dilution series. Mark each dish to
marked, 4 7 mm diameter, with 0.45 µm .02± identify the sample, volume or dilution to be
um pore size or other pore size recommended filtered. ·
bythe manufacturerforwateranalyses.
3.4.3 Using sterile forceps, place a mem-
3.3.20 Absorbent pads of cellulosic brane filter, grid-side up, on the porous plate of
paper, 4 7 mm diameter. The paper should be the filter base.
of high quality and free of sulfites or other
substances that could inhibit bacterial growth. 3.4.4 Attach the funnel to the base of the
filter unit, taking care not to damage or dis-
3.3.21 Waterproof plastic bags. lodge the filter, The membrane filter is now
fitted between the funnel and the base. ·
3.3.22 Inoculation loops, at least 3 mm
diameter, or needles, nichrome or platinum 3.4.5 Shake the sample container vigor-
wire, 26 B&S gauge, in suitable holder. Dispos- ously about 25 times.
able applicator sticks or plastic loops as alter-
natives to inoculation loops. 3.4.6 Prepare at least three sample incre-
ments according to 1 .4.3, in this Section.
3.3.23 Media: Media required for a spe- Measure the desired volume of sample into the
cific test should be prepared in pre-sterilized funnel with the vacuum turned off. To measure
erlenmeyer flasks with metal caps, aluminum the sample accurately and to obtain good distri-
foil covers, or screw caps. bution of colonies on the filter surface, the
following methods are recommended:
3.3.24 Dilution Water (a) Sample volumes of 20 ml or 111ore:
Measure the sample in a sterile graduated cyl-
(a) Sterile buffered or peptone dilution inder and pour it into the funnel. Rinse the
water dispensed in 99 ±2 ml amounts in graduate twice with sterile dilution water, and
screw-capped dilution bottles. add the rinse water 'to the funnel. For potable
waters, 100 ml volumes may be measured
(b) Sterile dilution water prepared in larger directly i.n a precali_brated funnel. .
volumes for wetting membranes before addi-
(b) Sample volumes of J0-20 ml: Me.asure
tion of the sample and for rinsing the funnel
the sample with a sterile 10 ml or 20 ml pipet
after sample filtration.
into the funnel.
3.4 Procedure (c) Sample volumes of< 10 ml: Pour about
10 ml of sterile dilution water into the funnel
3.4.1 Prepare the required media as out- and add the sample to the sterile water using"
lined In Part 11-B. If the medium is an agar, cool appropriate sterile pipet.

74 oEPA MICROBIOLOGICAL MANUAL 1978

 (d) Sample volumes of less than 0. 1 ml: learn to recognize the difference between two
Prepare appropriate dilutions in sterile dilution or more colonies which have grown into con-
water and proceed as applicable in steps (b) or tact with each other and single, irregularly
(c) above. shaped colonies which sometimes develop on
membrane filters. The latter colonies are usu-
(e) The time elapsing between preparation ally associated with a fiber or particulate ma-
of sample dilutions and filtration should be terial and the colonies conform to the shape
minimal and never more than 30 minutes. and size of the fiber or particulates. Colonies
which have grown together almost invariably
3.4.7 Turn on the vacuum to filter the show a very fine line of contact.
sample. Leave the vacuum on and rinse down
the funnel walls at least twice with 20-30 ml of
sterile dilution water. Turn off vacuum. 3.6 Calculation of Results: Select the
membrane filter with the number of colonies in
the acceptable range and calculate count per
3.4.8 Remove the funnel from the base of
100 ml according to the general formula:
the filter unit. An ultraviolet sterilizer unit can
be used to hold and sterilize the funnel be-
count per 100 ml=
tween filtrations. At least 2 minutes exposure
time is required for funnel decontamination
(6). Protect eyes from UV irradiation with
glasses, goggles, or an enclosed UV chamber No. colonies counted
(7).
x 100
Volume of sample filtered, in ml

3:4.9 Holding the membrane filter at its

edge with a sterilized forceps gently lift and
place the filter grid-side up in the culture dish. 3.6. 1 Counts Within the Acceptable Limits
Slide the filter onto the absorbent pad or agar,
using a rolling action to avoid trapping air
bubbles between the membrane filter and the (a) The acceptable range of colonies which
underlying pad or agar. Reseat the membrane is countable on a membrane is a functfon of
if non-wetted areas occur due to air bubbles. the parameter as shown in Table 11-C-3.

3.4. 10 Invert the petri dishes and incubate (b) Assume that filtration of volumes of 50,
at the appropriate temperature in an atmo- 15, 5, 1.5, and 0.5 ml produced colony counts
sphere with close to 100% relative humidity of 200, 110, 40, 10, 5, respectively.
forthe required time.
(c) An analyst would not actually count the
3.5 Counting Colonies: The grid lines are colonies on all filters. By inspection he would
used in counting the colonies. select the membrane filter(s) with 20-80 coli-
form colonies and then limit his actual count-
3.5. 1 Count the colonies for the parameter ing to such membranes.
of interest following a preset plan such as
shown in Figure 11-C-8. Some colonies will be in
(d) After selecting the best membrane
contact with grid lines. A suggested procedure
filter for counting, in this case the MF with a 40
to reduce error in counting these colonies is
colony count, the analyst counts colonies
shown in Figure 11-C-9. ·count the colonies in
according to the counting procedures in 3.6·
the squares indicated by the arrows.
and applies the general formula as follows:
3.5.2 . The fluorescent lamp should be
nearly perpendicular to the membrane filter,
Count colonies individually, even if they are in 40
Colonies per 100 ml x 100 800
contact with each other. The technician must 5

ISOLATION AND ENUMERATION 75

 ~~
p--

~
-- -
-- ...........
~

-.... _::: '-..

... ....-::_ - - -~ I "I

-
J
"\
~ - - - -- - - - -
I•r LJ - - - _,_ -
.....
- --
""
!~ ~ I- - I- -

T - -~ \ ... ... I- -

t: { - - -- - 1-- - - I- - -

.... I-
- \w ~

l
\ \ I j
\ I I

"-' . "'- ,,,

~
.. i\. / ~
/
"-..... ...........
!'....
--- -~
~
/ ..oil
i;r

FIGURE 11-C-8. Colony-Counting Pathway. (The inner circle indicates the

effective filtering area, dashed line indicates the pathway.)

e
' i> ·v- -

• t -t>
•
• v
.. ~

FIGURE 11-C-9. Enlarged Portion of Grid-Marked Square of Filter.

(Colonies are counted in squares indicated by the arrow.)

76 &EPA MICROBIOLOGICAL MANUAL 1918

 TABLE 11-C-3
Acceptable Limits*

Parameters Lower Upper Remarks

Total coliform bacteria 20 (0 for potable waters) 80 Limit, 200 colonies

Fecal coliform bacteria 20 60 of all types
Fecal streptococci 20 100

• Colony counts < or > the limits cited above must be identified as outside of this range.

3.6.2 More Than One Acceptable Count Then calculate the arithmetic mean of these
counts to obtain the final reported value:
(a) If there are acceptable counts on repli- 75,000 + 100,000
87,500
cate plates, carry counts independently to final 2
reporting units, then calculate the arithmetic
mean of these counts to obtain the final re- Report as: 88,000/100 ml.
ported value.
3.6.3 !! All MF Counts are Below the Lower
Limit, Select the Most Nearly Acceptable
For example, 1 ml volumes produce coli- Count (for non-potable waters)
form counts of 26 and 36 or counts of 2600
and 3600/100 ml: For example, assume a count in which
sample volumes of 1, 0.3 and .01 ml produced
colony counts of 14, 3, and 0, respectively.
2600 + 3600
2
= 3100
Here, no colony count falls within recom-
mended limits. Calculate on the basis of the
and value = 3100/100 ml most nearly acceptable plate count, 14, and
report with aqualifying remark:
(b) If more than one dilution, indepen-
14
dently carry counts to final reporting units, - x 100 = 1400
then average for final reported value. 1.0

Report as: Estimated Count, 1400 per 100

For example, assume that volumes of 0.3, ml.
0.1, 0.03 and 0.01 ml produced coliform colony
counts of TNTC (Too Numerous To Count), 75, 3.6.4 If Counts from All. Membranes are
30 and 8, respectively. In this example, two Zero, Calculate Using Count from Largest Fil-
volumes, 0.1 and 0.03 produce colonies in tration Volume - - - - - - --
the acceptable counting range.
For example, sample volumes of 25, 10,
and 2 ml produced colony counts of 0, 0, and
Independently carry each MF count to a 0, respectively, and no actual calculation is
count per 100 ml: possible, even as an estimated report. Calcu-
late the number of colonies per 100 ml that
would have been reported if there had been
75 one colony on the filter representing the larg-
x 100 75,000/100 ml
0.1 est filtration volume, thus:. --
30 1
x 100 =100,000/100 ml x 100 4
0.03 25

ISOLATION AND ENUMERATION 77

 Report as:< (Less than) ~ colonies per 3.7.1 A percent verification can be deter-
mined for any colony validation test:
--
100 ml.

3.6.5 If All Membrane Counts are Above

the Upper Limit, Calculate Count withSmallest No. of colonies meeting verification test
VOiU'iTi0FT1terea No. of colonies subjected to verification

For example, assume that the volumes 1,

0.3, and 0.01 ml produced colony counts of x 100 Percent verification
TNTC, 150, and 110 colonies. Since all colony
counts are above the recommended limit, use
the colony count from the smallest sample 3.7.2 Verification is required for all posi-
volume filtered and estimate the count as: · tive samples from potable waters.
110
x 100 = 1, 100,000 3. 7 .3 Verification is also recommended
0.01 for establishing quality control in research for
Report as: Estimated Count use with new test waters, new procedures or
new technicians, for identifying unusual col-
1, 100,000 per 100 ml.
ony types and as support for data used in legal
3.6.6 If Colonies are Too Numerous To actions.
Count, UseUpper Limit Count with Smallest
Fiiii1itioi1VoiUiTie - - - - - - - 3.7.4 The worker is cautioned not to apply
the percentage of verification determined for
Assume in Example 3.6.5 that the volumes one sample to other samples.
1.0, 0.3, and 0.01 ml, all produced too many
colonies to show separated colonies, and that 3. 7 .5 The careful worker may also pick
the laboratory bench record showed TNTC (Too non-typical colonies and follow the verification
Numerous to Count). procedure to determine that false negative col-
onies do not occur.
Use 80 colonies (Upper Limit Count for
Total Coliform) as the basis of calculation with 3.8 Significant Figures: See 2.8. 1.
the smallest filtration volume, th!Js:
3.9 Repeatability and Reproducibility of
80 Counts: Analysts should be able to duplicate
0.01 x 100 = 800,000 their own colony counts on the same mem-
brane within 5% and the counts of other ana-
Report as:> (Greater Than) 8000,000 per lysts within 10%. Failure to agree within these
100 ml. -- -- limits should trigger a review of procedures.

3.6. 7 If there is no result because of con-

fluency, lab accident, etc. Report as: No Result 4. Most Probable Number (MPN) Method
and specify reason. - ---
4.1 Summary: The Most Probable Num.
3.6.8 Reporting Results: Report bacterial ber procedure estimates the number of spe-
densities per 100 ml of sample. See Figure 11- cific organisms in water and wastewater by
C-1 and 11-C-3 for examples of forms for the use of probability tables.
reporting results.
Decimal dilutions of samples are inocu-
3.7 Verification: A verified membrane fil- lated in series into liquid tube media. Positive
ter test establishes the validity of colony differ- tests are indicated by growth and/or fermenta-
entiation on a selective medium and provides tive gas production. Bacterial densities are
support evidence of colony interpretation. based on combinations of positive and nega~

78 &EPA MICROBIOLOGICAL MANUAL 1978

tive tube results read from the MPN table. The (d) The MPN may be the only method appli-
MPN procedure may be carried to three stages cable to problem sample materials such as ·
of completion: bottom sludges, muds, soils and sediments
(with blending).
4. 1. 1 The Presumptive Test provides a
preliminaryestimate of bacterial density 4.2.2 Limitations: The MPN procedure has
based on enrichment in minimally-restrictive disadvantages:
tube media. The results of this test are never
used without further analyses.

4. 1.2 The Confirmed Test, the second (a) This method is ordinarily limited to a
stage of the M PN, is the usual extent of testing. maximum sample volume of 10 ml per tube, but
Growth from each positive Presumptive Test 100 ml portions are used in shellfish waters.
tube is inoculated into a more selective inhibi-
tory medium. The tubes are incubated at the
prescribed temperature and time, the positive (b) The time required for the test may be as
reactions noted and counts calculated from long as 96 hours for a Confirmed Test result.
the MPN table.
(c) The MPN tables are probability calcula-
4. 1.3 The Completed Test is the third tions and inherently have poor precision and
stage of the MPN used for total coliform contain a 23% bias at the 5 tube, three dilution
analyses only. Positive tubes from the levels normally used.
Confirmed Test are submitted to additional
tests to verify the identification of the isolated (d) The man-hour requirements to prepare
microorganisms. Although the Completed Test glassware and media and to perform the tests
provides the greatest reliability, the amount of are significant.
time and the workload restrict its use to
periodic substantiation of Confirmed Test
results, to other QC checks on methodology (e) Relatively large amounts of bench space,
and analysts, and to research. incubator space and tube/rack storage space
are required.
4.2 Scope and Application
(f) The procedure does not lend itself to
4.2. 1 Advantages: The MPN procedure · field work. As compliance monitoring of water
has the advantages inherent in liquid nutrient quality and effluent standards becomes a major
media. legal requirement, the time, precision and
equipment limitations cited in (b), (c), (d) and (e)
(a) The Presumptive and Confirmed Tests above are more serious for the large number of
require only observing and recording of field analyses which will be required.
gas/no gas for coliforms and growth/no
growth for fecal streptococci. The tests require
minimal experience, training or interpretation (g) Background organisms or toxic con-
by the analyst. stituents in 10 ml volumes of marine water can
interfere and be undetected.
(b) Water samples with high turbidity or
large numbers of algae have no apparent dele-
terious effect on the tube reactions.
4.2.3 The minimum MPN test that is ac-
(c) If a toxic substance is present in the ceptable for water and wastewater analyses is.
sample, the resultant 1: 10 or 1: 100 dilution of the Confirmed Test because of the high proba-
that sample in the liquid broth may reduce the bility of false positive reactions in the Pre-
toxicity to the point of no effect. sumptive Test.

ISOLATION AND ENUMERATION 79

 4.3 Apparatus and Materials · 4.4 Media: Appropriate media dispensed
in test tubes or in fermentation test tubes. See
4.3. 1 Water bath or air incubator set at 35 Part 118. for specific media.
± 0.6 C for total coliform and fecal strepto-
cocci tests. Water bath at 44.5 ± 0.2 for fecal
coliform test. 4.5 Dilution Water: Sterile 99 ± 2 ml
volumes in screw-cap pyrex glass bottles. See
4.3.2 Pipet containers of stainless steel, Part 11-B, 7.
aluminum or pyrex glass for glass pi pets.

4.3.3 Inoculation loops, 3 mm diameter, 4.6 Presumptive Test

and needle of nichrome or platinum wire, 26
B&S gauge, in suitable holder.
4.6. 1 Shake the sample or dilution con-
4.3.4 Disposable applicator sticks or plas- tainer vigorously about 25 times.
tic loops as alternatives to inoculation loops in
4.3.3 above. 4.6.2 To perform the Presumptive Test,
arrange a series of three or more rows of
4.3.6 Compound microscope, oil immer- culture tubes containing the test medium in a
sion, 1000 X. rack, providing for five replicates in each row.
Use five rows for samples of unknown density.
4.3.6 Culture tube racks, 10 x 5 open- Inoculate each successive row with decreas-
ings; each opening to accept 25 mm diameter ing decimal dilutions of the sample. For exam-
tubes. ple, in testing polluted waters for total coli-
forms, the initial sample inoculations might be
4.3.7 Gas burner, Bunsen/Fisher types or 0. 1, 0.01, 0.001, 0.0001, 0.00001 ml of origi-
electric incinerator unit. nal sample into successive rows each contain-
ing five replicate volumes. This series of sam-
4.3.8 Sterile T.D. bacteriological or Mohr ple volumes would yield determinate results
pipets, glass or plastic, of appropriate sizes. from test waters containing up to 16,000,000
organisms per 100 ml by use of the MPN
4.3.9 Dilution bottles {milk dilution), pyrex tables.
glass, marked at 99 ml volume, screw cap with
neoprene liner. When removing sample aliquots or dilu-
tions for further inoculations, do not insert the
4.3.10 Pyrex culture test tubes 150 x 25 pipet tip more than 2.5 cm (1 inch) balow the
or 160 X 20 mm containing inverted fermen- surface of the sample.
tation vials,,75 x 10 mm an'd proper closures.
4.6.3 Incubate tubes for 24±2 hours at 35 C.
4.3.11 Gram stain solutions {optional). A positive presumptive test is gas production
See This Section 5.3. for the coliforms or growth for fecal strepto-
cocci. After 24 hours incubation, examine the
(a) Hucker's Crystal Violet Solution (stain). tubes for gas formation and/or growth. Inocu-
late positive tubes into Confirmed Test media.
(b) Lugol's Iodine Solution {mordant). tf there is no gas or growth reincubate these
negative tubes for an additional 24 hours.
(c) Acetone Alcohol (decolorizer).
4.6.4 If the Presumptive tubes are negative
(d) Safranin Solution (counterstain). after 48 ± 3 hours, discard tubes. If the Pre-
sumptive tubes are positive, the cultures are
4.3.12 Glass microscope slides, 2.5 x 7 .6 verified in the Confirmed Test. Record the
cm (1 X 3 inches). negative and positive results.

80 oEPA MICROBIOLOGICAL MANUAL 1978

 4.7 Confirmed Test 35 C for 24 hours. Pick typical coliform colonies
(or atypical colonies if no typical colonies are
4.7.1 The Confirmed Test is performed by present), inoculate into lauryl tryptose fermen-
verifying positive tubes from the Presumptive. tation tubes and incubate at 35 C for 24-48
Test at 24 and 48 hours. If Presumptive tubes h~urs. The formation of gas in any amount in
are positive at 24 hours, confirm them at that the fermentation tubes constitutes a positive
time. <::;ompleted Test for total coliforms.

4.7.2 A positive test is indicated by gas Typical colonies show a golden green
production for the coliform bacteria or growth metallic sheen or reddish purple color with
for fecal streptococci. After 24 ± 2 hours. incu- nucleation.
bation, examine the tubes for gas formation
and/or growth. If there is no gas/growth, rein- Atypical colonies are red, pink or colorless,
cubate these negative tubes for a second 24 unnucleated and mucoid.
hours.
4.~.2 Optional Gram Stain Procedure
4.7.3 After 48 hours ± 3 hours examine
tubes for gas and/or growth, record positive The gram stain test has been used in the
and negative results. Discard negative tubes. Completed MPN Test for demonstrating gram
Retain positive tubes if the test is to be carried negative, nonsporeforming rods from isolated
to completion for total coliform tests. colonies. Although the gram stain procedure is
proposed for revision of the 15th edition of
4.7.4 The fecal coliform MPN test is per- Standard Methods, it provides a final check on
formed by inoculating EC Broth tubes with results and remains useful for evaluating ques-
growth from all positive Presumptive tubes and tionable colony types.
incubating them at the elevated temperature of
44.5 C for 24 hours. Gas production is the posi- After incubation of the EMB agar plates for
tive reaction. 24 hours at 35 C (in 4.8.1) pick at least two
typical colonies (or atypicals if no typical colo-
4.7.5 Passage of positive Presumptive cul- nies are present) and inoculate onto nutrient
tures through the Confirmed Test completes the agar slants. Incubate for 24 hours at 35 C, and
MPN series for fecal streptococci and fecal proceed as in 5, this Section.
coliform bacteria.
4.9 Calculation of MPN Value
4.7.6 In routine practice, most sample ex-
aminations for total coliform are terminated at The calculated density of the Confirmed or
the end of the Confirmed Test. However, for Completed Test may be obtained from the MPN
quality control, at least five percent of the Con- table based on the number of positive tubes
firmed Test sampieS(and a minimumof one and reactions in each dilution.
sample per .test run) should be carried through
the_Completed Test. 4.9.1 Table 11-C-4 illustrates the MPN indi-
ces and 95% Confidence Limits for general use.
4.8 Completed Test for Total Coliform
MPN 4.9.2 Table 11-C-5 shows the MPN indices
and limits for potable water testing.
Positive Confirmed Test cultures may be
subjected to final Completed Test identification 4.9.3 Three dilutions are necessary to
through application of further culture tests, formulate the MPN code. For example in Table
as follows: 11-C-4 if five 10 ml, five 1.0 ml, and five 0.1 ml
portions are used as inocula and positive results
. 4.8.1 Streak Levine's EMB agar plates fro.m are observed in five of the 10 ml inocula,
each positive confirmatory tube and incubate at three of the 1.0 inocula, and none of the 0.1

ISOLATION AND ENUMERATION 81

,_,
O>
tABLEll.c.4

Most Probable Number Index and 95 % Confidence Limits for Five Tube, Three Dilution Series (8, 9)

No. or Tubes Giving 950/o Confidence No. or Tubes Giving 9510 Confidence
Positive Reaction out of MPN Limits Positive Reaction out of_ MPN Limits
Index Index
5 or 10 5 or 1 5 or 0.1 per Lower Upper 5 of 10 5 of 1 5 of O. 1 per Lower Upper
ml Each ml Each ml Each 100 ml ml Each ml Eact ml Each 100 ml
O:>
~ 0
0
0
0
0
1
<2
2 <0.5 7 4 2 1 26 9 78
0 1 0 2 <0.5 7 4 3 0 27 9 80
0 2 0 4 <0.5 11 4 3 1 33 11 93
E!
g 1 0 0 2 <0.5 7
4 4 0 34 12 93
0 1 1 4 <0.5 11 5 0 0 23 7 70;
0
~
0 1 1 0 4 <0.5 11 5 0 1 31 11 89
r- 1 1 1 6 <0.5 15 5 0 2 43 15 110
0
G') 1 2 0 6 <0.5 15 5 1 0 33 11 93
~r-
5 1 1 46 16 120
2 0 0 5 <0.5 13 5 1 2 63 21 150
2 0 1 7 1 17
~ 2 1 0 7 1 . 17 5 2 0 49 17 130
::z: 2 1 1 9 2 21 5 2 1 70 23 170
t
r-
2 2 0
0
9 .
12
2
3
21
28
5
5
2
3
2
0
94
79
28
25
220
190
2 3
..... 1 110 31
<o 5 3 250
(iii 3 0 0 8 1 19 5 3 2 140 37 340
3 0 1 11 2 25 5 3 3 180 44 500
3 1 0 11 2 25 5 4 0 130 35 300
3 1 1 14 4 34 5 4 1 170 43 490
3 2 0 14 4 34 5 4 2 220 57 700
3 - 2 1 17 5 46 5 4 3 280 90 850
3 3 0 17 5 46 5 4 4 350 120 1, 000
4 0 0 13 3 31 5 5 0 240 68 750
4 0 1 17 5 46 5 5 1 350 120 1, 000
4 1 0 17 5 46 5 5 2 540 180 1, 400
4 1 1 21 7 63 5 5 3 920 300 3, 200
4 1 2 26 9 78 5 5 4 1600 640 5, 800
4 2 0 22 7 67 5 5 5 > 2400
-· ·-· - ...
 TABLE · 11-C-5

Most Probable Number Index and 95% Confidence Limits for Testing Potable Waters

Number of Positive Tubes MPN

from five 10 ml Portions lndex/100 ml 95% Confidence Limits
Lower Upper

0 <2.2 0 6.0

2.2 0.1 . 12.6

2 5.1 0.5 19.2

3 9.2 1.6 29.4

4 16. 3.3 52.9

5 > 16. 8.0 Infinite

ISOLATION AND ENUMERATION 83

ml inocula, the coded results of the test are sample volume to increase the positive tubes
5-3-0. The code is located in the MPN Table, in the highest dilution selected. See Table 11-C-
and the MPN index of 79 per 100 ml is 6, Test4.
recorded.
(e) There should be no negative results in
4.9.4 When the series of decimal dilutions higher sample volumes than those chosen.
is other than 10, 1.0 and 0. 1 ml, select the However, if negative tubes are present, e.g.,
MPN value from Table 11-C-4 and calculate 4/5, 5/5, 3/5 and 0/5 the highest sample vol-
according to the following formula: ume with all positive tubes must be used along
with the next two lower sample volumes. See
10 Table 11-C-6, Test 5.
MPN (From Table) x
Largest Quantity Tested
(f) If all tubes are positive, choose the three
MPN/100 ml highest dilutions. See Table 11-C-6, Test 6.

(g) If all tubes are negative, choose the

As an example, five out of five 0.01 ml three lowest dilutions. See Table 11-C-6, Test 7.
portions, two out of five 0.001 ml portions,
and zero out of five 0.0001 ml portions from a (h) If positive tubes skip a dilution, select
sample of water, gave positive reactions. From the highest dilution with positive tubes and the
the code 5-2-0 in MPN Table (Table 11-C-4), the two lower dilutions. See Table 11-C-6, Test 8.
MPN index 49 is adjusted for dilutions:
(i) If only the middle dilution is positive,
select this dilution and one higher and
10 lower dilution. See Table 11-C-6, Test 9.
49 (From Table) X 49,000
0.01
4.9.6 A number of theoretically possible
The final corrected MPN Value combinations of positive tube results are omit-
4s,00011 oom1. ted in Table 11-C-4 because the probability of
their occurrence is less than 1 %. If such un-
4.9.5 If more than the above three sample likely tube combinations occur in more than
volumes are inoculated, the three significant 1 % of samples, review the laboratory proce-
dilutions must be determined. The significant dures for errors and note sample types. Collect
dilutions are selected using the following fresh samples for analyses.
rules:
4.9.7 The MPN can also be computed for
(a) Only three dilutions are used in the each sample based upon the number of
code for calculating an MPN value. positive and negative Presumptive, Confirmed
or Completed Tests, and the total number of
(b) To obtain the proper three dilutions, milliliters tested ( 10). MPN/100 ml =
\
select the smallest sample volume giving all
positive results and the two succeeding lesser
sample volumes. See Table 11-C-6, Test 1 and 2.
No. of Positive Tubes X 100
(c) If less than three dilutions show posi- (No. of ml in Negative Tubes) x
tive tubes, select the three highest sample (No. of ml in All Tubes)
volumes which will include the dilutions with
the positive tubes. See Table 11-C-6, Test 3.
Example: From a sample of water, five out
(d) If there are positive tubes in the dilu- of five 10 ml portions, two out of five 1.0 ml
tions higher than these dilutions selected, pos- portions, and zero out of five 0. 1 ml portions
itive results are moved up from these dilutions

84 oEPA MICROBIOLOGICAL MANUAL 1978

 TABLE 11-C-6

Selection of Coded Results, Five Tube Series

Positive TuQes/ml Sample Volume

Test 10 1.0 0.1 0.01 0.001 0.0001 Code

5• 3 0 0 0 0 5-3-0

2 5 5 4 0 0 0 5-4-0

3 4 0 0 0 4-1-0

4 5 5 4 0 5-4-2

5 4 5 3 0 0 5-3-0

6 5 5 5 5 5 5-5-5

7 0 0 0 0 0 0-0-0

8 4 0 2 0 0 4-0-2

9 0 1 0 0 0 0-1-0

*Underlines indicate positive tube series selected for code.

ISOLATION AND ENUMERATION 85 /

/
I.

I ---
gave positive results. Therefore, MPN/100 ml 5. 1.3 Air-dry the smear and fix by quickly
passing the slide several times through a por-
tion of the flame.
7 x 100
50.22
~ (3.5) x (55.5)
5.2 Gram Stain
or

MPN/100 ml = 50
Gram staining is a general test for charac-
4.10 Reporting Results: Report the MPN terization of bacteria and for examination of
value for water samples on the basis of 100 ml
culture purity.
of sample. Report the MPN values of solid type
samples on the basis of 1 gram of dry weight
sample.
5.2.1 Prepare and fix a bacterial smear as
Examples of bench forms are shown in in 5.1. For quality control, prepare a separate
Figures 11-C-1, 2 and 3. smear of known gram positive cocci and gram
negative rods.
4.11 Precision and Accuracy

4. 11. 1 The precision of the MPN value

Increases proportionately with the number of 5.2.2 Flood the smear with ammonium
replicates tested. oxalate-crystal violet stain for one minute.

4.11.2 Multiple-tube values are generally

high because MPN tables include a 23% posi-
tive bias. 5.2.3 Wash the slide in a gentle stream of
tap or pure water and flood with Lugol's iodine
4.11.3 MPN numbers represent only a sta- solution (mordant). Allow it to remain for one
tistical estimate of the true bacterial density in minute.
the sample. The 95% Confidence Limits for
each MPN value included in the Tables 11-C-4
and 5 show the limited precision of these
estimates. 5.2.4 Wash the slide in water and blot dry.

5. Staining Procedures

5.1 Preparation of Bacterial Smears 5.2.5 Decolorize with acetone alcohol,

either by adding it dropwise on the tilted slide
6.1.1 Place a small drop of laboratory pure until the blue color 'stops flowing from the
water on a clean slide. smear, or by gently agitating the slide up and
down in a beaker containing the alcohol wash
5.1.2 Using a sterilized inoculating nee- for about 30 seconds.
dle, pick a small amount of growth from the
agar slant. Mix the bacteria with the drop of
water on the slide and spread evenly over an
area the size of a quarter. Use loop for broth 5.2.6 Flood the smear with the safranln
cultures. counterstain for 10 seconds. Wash and air-dry .

. .
86 oEPA MICROBIOLOGICAL MANUAL 1978
 5.2.7 Examine under the oil immersion (b) Lugol's iodine: Dissolve 1 gram iodine
objective. Gram positive cells retain the crystal crystals and 2 grams potassium iodide in
violet stain and are blue in color. Gram negative about 5 ml of pure water. After crystals are in
cells are decolorized by the acetone alcohol so solution, add sufficient laboratory pure water
that they accept the safranin counterstain and to bring the final solution to a volume of 300
appear pink to red. ml.

5.3 Stain Solutions (c) Acetone-Alcohol: Combine fifty ml

volumes of acetone and 95% ethyl alcohol.
Only those stains and dyes which are certi-
fied by the National Biological Stain Commis- (d) Safranin: Dissolve 2.5 grams of safra-
sion should be used. nin in 100 ml of 95% ethyl alcohol. Store as a
stock solution. For working solution, add 10 ml
5.3.1 Loeffler's Methylene Blue of stock to 100 ml of laboratory pure water,
mix and store.
(a) Methylene Blue-ethyl alcohol solution.

(1) Dissolve 0.3 grams of methylene blue 6. Shipment of Cultures

(90% dye content) in 30 ml of 95% ethyl alcohol
(Solution A). 6. 1 Confirmation or further identification
to serotype may be required if the bacteriologi-
(2) Dissolve 0.01 grams of potassium cal data are to be used for specific needs such
hydroxide in 100 ml of laboratory pure water as enforcement cases, epidemiological stud-
(Solution B). ies, tracing sources of pollution or scientific
publication. The selected cultures should be
(3) Mix solution A and B. sent to an official typing center or state health
laboratory with pertinent information for the
5.3.2 Solutions for Gram Staining confirmatory identification. This service is usu-
ally available if the cultures are of public health
(a) Ammonium oxalate-crystal violet significance, but permission should be ob-
solution: tained from the reference laboratory before
sending cultures.
( 1) Dissolve 2 grams crystal violet (approxi-
mately 85% dye content) in 20 ml of 95% Observe the following instructions:
ethyl alcohol (Solution A).
6.1.1 Send only pure cultures.
(2) Dissolve 0.8 grams ammonium oxalate
in 80 ml laboratory pure water (Solution B). 6. 1.2 Provide a culture with discernable
growth on brain heart infusion agar, blood
(3) Mix Solutions A and B. agar base or nutrient agar stab in a screw-cap
tube or vial sealed with a cork soaked in hot
(4) Filter through cheesecloth or coarse paraffin. Triple sugar iron agar or other sugar-
filter paper. containing agars should not be used.

(5) Problems with the gram stain tech-

nique are frequently traceable to the 6.1.3 Complete the reference laboratory
ammonium oxalate-crystal violet solution. In form which requires information on the source
the event that decolorization is insufficient, the of the culture, tests completed, results
amount of crystal violet in the solution can be obtained and identification of the originating
reduced to as little as 10% of the laboratory. Include form with cultures, but on
recommended amount. outside of s~condary container.

/SOLA TION AND ENUMERATION 87

 6.2 Shipping ·Regulations for Cultures: SECONDARY CONTAINER provided that the
Transportation (DOT} regulations apply to sur- total aggregate volume does not exceed 50
face and air transportation, but shipment of ml. (NOTE: Multiple secondary containers of
cultures beyond 100-200 miles is only practi- cultures, which individually meet the
cal by air. Air freight service is limited in many packaging requirements for shipment of 50 ml
areas, hence passenger-carrying aircraft must or less, can be overpacked in a single outer
be used for safe and quick service. Strict ship- shipping container, provided that the total
ping regulations are imposed on such passen- aggregate volume does not exceed 4000 ml).
ger service shipments (11 ). Packaging and la-
beling of the cultures must conform with cur- 6.2.4 Data forms, letters and other infor-
rent federal shipping regulations for etiologi- mation identifying or describing the cultures
cal agents described in: 49 CFR 173.387 (12) should be placed around the outside of the
and 42 CFR 72.25 (c) (13). The requirements in SECONDARY CONTAINER. DO NOT ENCLOSE
6.2.1-6.2.8 that follow are also shown in Fig- WITHIN THE SECONDARY CONTAINER.
ure 11-C-10.
6.2.5 Place the SECONDARY CONTAINER
6.2.1 Place each culture in a securely and information form in an OUTER MAILING
closed, watertight PRIMARY CONTAINER TUBE OR BOX.
(screw-cap test tube or vial) and seal the cap
with tape. 6.2.6 Place an address label and
ETIOLOGIC AGENT/MICROBIOLOGICAL CUL-
6.2.2 Wrap the PRIMARY CONTAINER TURES label on the outer mailing tube or box.
with sufficient absorbent material (paper
towel, tissue, etc.) to absorb the entire con- 6.2. 7 Individual primary containers of
tents should breakage or leakage occur. greater than 50 ml of culture material require
special packaging and cannot be transported
6.2.3 Place the wrapped, sealed, on passenger-carrying aircraft.
PRIMARY CONTAINER in a durable, watertight
SECONDARY CONTAINER (screw-cap metal
mailing tube or sealed metal can). Screw-cap 6.2.8 International shipments must also
metal mailing tubes should be sealed with conform to the added regulations: US Post
tape. Several PRIMARY CONTAINERS of Office Publication 51, Air Cargo Restricted Art-
cultures, each individually wrapped in icles Tariff 6-D and DOT Regulations 49 CFR,
absorbent material, may be placed in the Section 173.

88 oEPA MICROBIOLOGICAL MANUAL 1978

 PART 1
PRIMARY
CONTAINER
CULTURE
(SCREW-CAP) PART 3
ABSORBENT
PACKING
PART 2
MATERIAL
CAP

c;;
0.....
),,
:::!
0
2:
),,

~ CAP MICROBIOLOGICAL MATERIALS

~ PACKAGING CONFORMS

~
WITH STANDARDS IN 49
CFR 173.387, AND
42CFR72.25
ABSORBENT
:::! PACKING
0
2: EA MATERIAL
LABEL

ADDRESS
LABEL

CROSS SECTION
OF PROPER PACKING

FIGURE 11-C-1 O. Packaging and Labelling of Microbiological Cultures for Shipment.

CX>
co
 REFERENCES

1. Clark, H.F., P. W. Kabler and E. E. Geldreich, 1957. Advantages and limitations of the membrane
filter procedures. Water Sewage Works 104:385.

2. Shipe, E. L and A. Fields, 1954. A comparison of the molecular filter technique with agar plate
count for enumeration of Escherichia coli. Appl. Microbiol. 2:382.

3. Shipe, E. L. and G. M. Cameron, 1954. A comparison of the membrane filter with the most
probable number method for coliform determinations from several waters. Appl. Microbial. 2:85.

4. Geldreich, E. E., H. L. Jeter and J. A. Winter, 1967. Technical considerations in applying the
membrane filter procedure. Health Laboratory Science 4: 113.

5. Proceedings of the Symposium on the Recovery of Indicator Organisms Employing Membrane

Filters. Jan 20-21, 1975. Environmental Monitoring and Support Laboratory, U.S. Environmental
Protection Agency, Cincinnati, OH 45268. (1977).

6. Rhines, C. E. and W. P. Cheevers, 1965. Decontamination of membrane filter holders by

ultraviolet light. J. Am. Water Works Association 5 7:500.

7. Manning, H., 1975. AQC Newsletter #26, July 1975. U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH 45268. p. 15.

8. Swaroop, S., 1938. Numerical estimation of B. coli by dilution method. Indian J. Med. Research
26:353. -- ----

9. Swaroop, S., 19 51. The range of variation of the most probable number of organisms estimated
by the dilution method. Indian J. Med. Research 39: 107.

10. Thomas, H. A., Jr., 1942. Bacterial densities from fermentation tubes. J. Am. Water Works
Association 34:572. - - -- --

11. Morbidity and Mortality Report, 1975. Center for Disease Control, Public Health Service,
USDHEW, Atlanta, GA 24:49.

12. Title 49, Code of Federal Regulations (CFR), Part 173.

13. Interstate Quarantine, regulations of the shipment of etiologic agents. Title 4 2, Code of Federal
Regulations, (CFR), Part 72, Section 25.

90 oEPA MICROBIOLOGICAL MANUAL 1978

 PART II. . GENERAL OPERATIONS

Section D Selection .of . Analytical Methqds

This Section discusses the selection of 3. .Recommendations for Methods

methods for monitoring water and wastewater in Waters and Wastewaters
in r~sponse :to the Laws, the microbi·ological
standards that have been established, and the
criteria that have been' recommended to en-· 1. _Methodology
force the laws. The major problems that have
. developed in. the application of .the methods .· Test procedures have .been specified and
·~re.. identified and solutions are given where published in Federal Register for drinking
they are available. water, wastewater discharges (NPDES) and
vessel discharges.
1. Methodology
1.1 National Interim Primary Drinking
1.1 National Interim Primary Water Regulations
Drinking Water Regu-
lations ' Although· the National Interim Primary
1.2 NPDES Guidelines Drinking Water Regulations (Title 40 CFR Part
1.3 Marine Sanitation R!Jgu- 141.) state that the total coliform analyses can
lations be performed by the membrane filter or MPN ·
1.4 Water Quality Standards·· procedures, the MF procedure is preferred
1.5 Water Quality Criteria because large volumes of samples can be
1.6 Alternate Test Procedures analyzed in a much shorter time, a critical
factor for potabl'e water. Samples containing
2. Problems in Application excessive noncoliform populations or turbidity
must be analyzed by the MPN technique.
2.1 Stressed Microorganisms These regulations specify the testing of
2.2 Incomplete Recovery/ sample sizes of 100 ml for the MF technique
Suppression and the testing of five replicate 10 or 100 ml
2.3 Interference by Turbidity volumes for the MPN procedure. The law
2.4 Analysis of Ground Water directs that the samples be taken at points
2.5 Field Problems representative of the distribution system.
2.6 Method Modifications The minimal schedules for the frequency
and Kits of sampllng are based on population and
2.7 Changes in Membrane the required response is given for positive
Filters and Methodology test results. A detailed description of the
2.8 K/ebsiellain Industrial proposed criteria for interim certifi-
Wastes cation of microbiology laboratories under

.. . .. ,
.· '. ~-: . . . ' .. ; .. :.·... -~ ·. ' .
91
METHOD SELECTION
the Safe Drinking Water Act is given in 2. Problems in Application
Appendix B.
Although the methods described in this
1.2 National Pollution Discharge Manual are judged the best available, there are
Elimination System (NP DES} Guidelines difficulties in the application of methods in
different geographical areas, in certain wastes
The NPDES established guidelines for and in some potable and surface waters. Addi-
analysis of pollutants under PL 92-500, tional problems can stem from the indiscrimi-
nate use of new and simplified equipment,
Section 304 (g). The parameters and methods
are described in 40 CFR Part 136, as amended supplies or media that have been proposed for
use in these procedures. ·
(40 Code of Federal Regulations, Protection of
the Environment, ch. 1 - Environmental
Protection Agency, Part 136, Guidelines 2.1 Stressed Microorganisms
Establishing Test Procedures for the Analysis
of Pollutants). The method must be specified · Some water and wastewater samples con-
and MPNs must be five tube, five dilution. See tain microorganisms which should reproduce
but do not under the conditions of test. These
Table 11-0-1.
organisms have been described as injured or
stressed cells. The stress may be caused by
1.3 Marine Sanitation Regulations
temperature changes or chemical treatment
such as chlorine or toxic wastes ( 1).
The regulations for marine sanitation
devices (40 CFR Part 140) established
Stressed organisms are particularly
performance standards and specified the
important in environmental measurements
analytical methods as those promulgated in
because tests for bacterial indicators or
40 CFR Part 136, cited in 1.2 above.
pathogens can give negative responses, then
recover later and multiply to produce
1.4 Water Quality Standards
dangerous conditions. Subsections 2.1.1 and
2.1 .2 describe efforts to recover stressed
Water quality standards (limits) have been
microorganisms.
established by law for drinking water and
certain sewage and industrial effluents. These
2.1.1 Ambient Temperature Effects
standards and the reference sources are listed
In Table 11-0-2. A standard must be specified in
Extreme ambient temperatures stress
the NPDES permit to be enforceable.
microorganisms and reduce recovery of
microbiological indicators. For example, in
1.5 Water Quality Criteria Alaska and other extremely cold areas, the
severe change from cold stream temperature
Water quality criteria have been to 44.5 C temperature of incubation reduces
recommended by the EPA for certain types of recovery of fecal coliforms. The two-step MF
water classified according to use. These test for fecal coliforms increases recoveries by
criteria are listed in Table 11-0-3. use of a 2-hour acclimation on an enrichment
medium at 35 C before normal incubation at
44.5 C.
1.6 Alternate Test Procedures
In contrast, water samples from natural
The amendments to 304 (g) also provide waters at high temperatures may include large
procedures for approval of alternate methods. numbers of non-coliform organisms which in-
National approval for test methods is obtained terfere with sheen production on MF's and
by application to EPA through EMSL- with positive gas production in MPN analyses.
Clnclnnati while case by case approval is An improved MF medium that provides greater
obtained by application through the EPA selectivity is desirable but may not be possible
Regional Offices (40 CFR 136.4). without sacrificing recovery.

92 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE 11-0-1

Approved Test Procedures for the Analysis of Pollutants (40 CFR 136)

Parameter per 100 ml Method Reference and Page No.

USGS 2 This Manual

Fecal Coliforms MPN 922 Part 111 C, 5

MF 937 45 Part 111 C, 2

Fecal Coliforms in MPN 922 Part Ill C, 5

presence of chlorine 3 MF 928, 937 Part Ill c.2

Total Coliforms MPN 916 Part Ill 8, 4

MF 928 35 Part Ill 8, 2

Total Coliforms in MPN 916 Part Ill 8, 4

presence of Chlorine MF 933 Part Ill B, 2

Fecal Streptococci MPN 943 Part 111 D, 4

MF 944 50 Part Ill D, 2
Plate Count 947 Part Ill D, 5

Standard Methods for the Examination of Water and Wastewater, 14th Edition, (1975).
2 Slack, K. V., et.al. Methods for Collection and Analysis of Aquatic Biological and
Microbiological Samples. USGS Techniques of Water Resources Inv., Book 5, ch. A4 (1973).
3 Since the MF technique usually yields low and variable recovery from chlorinated
wastewaters, ·the MPN method will be required to resolve any controversies.

METHOD SELECTION 93
 TABLE 11-D-2

Water Quality Standards

Microbiological
Standards Reference
Water or Coliforms/100 ml Source
Wastewater Total Fecal

Potable Water <5 PL 93-523

Chlorinated Effluents 200-400 PL 92-500

2• Treatment Wastes 200-400 40 CFR Part 133

Selected Industrial Wastes 200-400 PL 92-500

Leather and Tanning 400 40 CFR Part 425

Feed Lots 400 40 CFR Part 412

Meat Products 400 40 CFR Part 432

Beet Sugar 400 40 CFR Part 409

Canned Fruits and 400 40 CFR Part 407

Vegetables

Textiles 400 40 CFR Part 410

Effluents from Marine

Sanitation Devices with
Discharges Type I 1000 40 CFR Part 140
and Amendments
Type II 200 40 CFR Part 140
and Amendments

94 &EPA MICROBIOLOGICAL MANUAL 1918

 TABLE 11-0-3

Water Quality Criteria

· · Mfcrobiolog.icaf Criteria
Water or .Statistical Coliforms/ 100 ml Reference
Wastewater ,Measure Total Fecal Source

;.,) .... _: :.. ,·; . .. .

x
:

Public Water Supply log .. 20000 2000 A

·. ~ ,. - ..
Recreational Water:
Primary Contact log x130 days 200 B
maximum/30 days, 400 B
in 10% of Samples
..
General Contact log X/30 days 1060 B
maximum/30 days, 2000 B
in 10% of Samples
.. ..
Agricultural Water monthly x 5000 1000
'':' ~·

Shellfish-Raising Waters
Daily Median 70 14 c & D
Highest 10% of 230 43
Daily Values

A Water Quality Criteria, EPA. March, 1973. Superintendent of Documents, U.S. Governmen.t Printing
Office, Washington, DC 20402.

B Water Quality Criteria, FWPCA, April 1, 1968. Superintendent of Documents, U.S. Government Printing·
Office, Washington, DC 20402.

c National Shellfish Sanitation Program Manual of Operation. U.S. Dept. ou.f l:\11/, ·1965. Pu~lic. He.alth
Service Publ. No. 33. Superintendent of Documents, U.S. Government Printing Office, Washi.ngton, DC
20402. . , . . ·:'l

D Quality Criteria for Water, July 1976, O.W.H.M., US EPA.

95
 2, 1.2 Chlorinated Effluents and Toxic analyses of primary and secondary effluents or
Wastes industrial wastes containing toxic materials,
the MPN procedure is required. However, the
Although thiosulfate is added to all two-step MF procedure for total coliforms
samples suspected of containing chlorine, to described in this Manual and in Standard
neutralize its toxic effects, the membrane filter Methods is acceptable for toxic wastes.
procedure yields poor recovery of coliforms
from chlorinated effluents as compared to If the MF procedure is applied to
MPN recovery (1-6). A recent amendment to chlorinated or toxic samples, the laboratory
40 CFR 136 added Coliform bacteria (Fecal) in should require data from at least 10 samples
the presence of chlorine, as a specific. collected over 1 week of plant processing (but.
parameter and recommended analysis by the not less than 5 calendar days} to show ·
MF or MPN techniques (7). A qualifying state- comparability of the MF to the MPN technique.
ment appended to the method in 40 CFR Part See Part IV-C, 3 for details.
136 requires the five tube, five dilution MPN
and states: "Since the membrane filter 2.2 Incomplete Recovery/Suppression
technique usually yields low and variable
recovery from chlorinated wastewaters, the When coliforms are present in low num-
MPN method will be required to resolve any bers in drinking water, high levels of non-
controversies." Therefore, the MPN procedure coliforms can suppress growth or mask detec-
should be used in analySTS Ofchlorinated tion. This problem may appear as a mass of ·
effluentswhere thedata may bechallenged by confluent growth on a membrane filter or as
legal or enrc>rcementaetions. The MF may be spots of sheen in this confluent growth. In the
used currently for self-monitoring situations. MPN procedure, presumptive tubes may show
(See Table 11-D-1 ). heavy growth with no gas bubbles, dilution
skips or unusual tube combinations. Whe.n
Proposed changes in MF materials and these negative presumptive tubes are trans-
procedures include new membrane filter for- ferred to BGLB, they confirm in this more re-
mulations, an agar overlay technique, modified strictive medium, indicating that the coliform
media and twostep methods (1). Present modi- gas production in the Presumptive Test was
fications of the MF method have not produced suppressed by non-coliforms.
recoveries of fecal coliforms from chlorinated
effluents equivalent to MPN recoveries. Thor- 2.3 Interference by Turbidity
ough evaluation and approval of proposed pro-
cedures by EPA are required before changes The tendency of bacteria to clump and
will be acceptable. adhere to particles can produce inaccurate
results in the analysis of water samples. The
Certain types of wastes show recovery National Interim Primary Drinking Water Regu-
problemsfortotal and fecal coliforms: lations (NIPDWR) specify one turbidity unit as.
the primary maximum allowable level but per-
1. Primary and Chlorinated-Primary mit up to five turbidity units if this level does
Waste Effluents. not interfere with disinfection or microbiologi-
cal analyses. Turbidity can interfere with filtra-
2. Chlorinated-Secondary and Chlor- tion by causing a clumping of indicators or
inated-Tertiary Waste Effluents. clogging of pores. The turbidity as organic
solids can also provide nutrients for bacterial
3. Industrial wastes containing toxic growth and subsequently produce higher
metals or phenols. counts. The type of particles variably affects
the filtration rate; for example, clay, silt or
When turbidity and low recovery prevent organic debris clog more easily than sand.
the application of the MF technique to coliform Background organisms may also be imbedded

96 &EPA MICROBIOLOGICAL MANUAL 1978

in the particles and interfere with the coliform dure required for acceptance of an alternative
detection. procedure is described in 40 CFR Parts 136.4
and 136.5, as amended.
2.4 Analysis of Ground Water

Although total coliforms are a valid mea- 2. 7 Changes in Membrane Filters and
sure of pollution, their use as indicators in Methodology
analyzing ground waters and rural community
supplies may not sufficiently describe the There is an expected pattern of changes
water quality. For example, ground waters fre- in materials and methodology used in the
quently contain high total counts of bacteria manufacture of membrane filters. The changes
with no coliforms. Such waters pass Interim may or may not be announced by the manu-
Drinking Water Regulations but technical judg- facturer. Therefore, it is important for the
ment must conclude these are not acceptable laboratory to monitor membrane performance
as potable waters. as described in Section A of Quality Control
in this Manual.
2.5 Field Problems

Assurance of data validity demands sam- These changes include modification of

ple analyses within the shortest time interval formulations and the. replacement of the
after collection. This need requires field ana- 0.45 µm pore MF by a 0.7 µm retention pore
lyses using either a mobile laboratory or field MF for improved recovery. Tests by independ-
kit equipment. Since a mobile laboratory may ent investigators show that several MF's give
not be available for a survey, it is likely that at comparable recovery (5, 6, 8, 9), however,
leas.t a part of the analyses will need to be enrichment or two-temperature incubations
completed in an onsite facility. If the analyses are needed before recoveries approach the
can be done using membrane filtration tech- MPN values (See 2.1.2 in this Section).
niques, field kits such as Millipore's Water
Laboratory and MF Portable Incubator (heat
sink)· are particularly helpful for rapid set-up This discussion of problems with new
and analyses of limited samples. However, if methodology and membrane materials should
large numbers of samples are tested per day or not be interpreted as indicating that EPA dis-
the survey covers more than a few days, the courages new developments. Rather EPA en-
heat-sink incubator is impractical because of courages _the MF supply industry to test and
limited capacity and high cost. In such surveys, examine procedures, to innovate and to re-
a mobile laboratory utilizing water-jacketed in- search. The membrane filter manufacturers
cubators is more practical. should be commended and encouraged to
continue their efforts toward solving problems
2.6 Method Modifications and Kits and improving materials and techniques in
water microbiology.
Commercial manufacturers continue to of-
fer proprietary kits and method modifications
to speed or simplify the proced.ures used in 2.8 Klebsiella in Industrial Wastes
coliform and fecal coliform analyses, primarily
for field use. Most of these units have not been Klebsiel/a bacteria (part of the coliform
demonstrated to produce results comparable group) multiply in certain industrial W!'lStes, are
to the official procedures. If not tested to the not differentiated from fecal coliforms by MF
satisfaction of EPA, such method modifica- and MPN procedures and consequently are
tions and kits cannot be used for establishing included in the results. These recoveries have
total or fecal coliform numbers for permits been reported in textile, paper and pulp mills
under NPDES or for total coliform numbers and other wastE!S. Objections have been raised
under the Safe Drinking Water Act. The proce- to the application of fecal coliform standards

METHOD SELECTION 97
 TABLE 11-D-4

Selection of· Methods for Problem Samples

Problem Area 'Parameter Chosen Method of Choice

Shellfish-harvesting waters Total coliform MPN*

Fecal Coliform MPN*

Marine & Estuarine Waters Total Coliform MF/MPN

Fecal Coliform MF/MPN

Treated Industrial Wastes Fecal Coliform MF

(non-chlorinated, non-toxic)
Low Solids Wastes

Toxic Industrial Wastes Fecal Coliform MPN or alternate

(metals, phenolics) and procedure, tested
High Solids Wastes and approved ..

Primary and Chlorinated- Total Coliform MPN· or alternate

Primary Municipal/Industrial procedure, tested
Effluent , and .51pproved**

Fecal Coliform MPN o'r· a'lternate

procedure, tested
and· approved ..

Chlorinated-Secondary Effluent Total Coliform Two-Step MF

... Fecal Coliform MPN or . alternate

procedure, tested
and approved**
... • , ..... •''!> •• ' ... ,
•MPN recommended to conform with the MPN method specified for examination of
shellfish. ' ' ·'

•"Requires proof of comparability under. EPA's specified test regime that the alternate
procedure (MF, streak plate. etc.) is valid. See This Manual. IV-C. 3.

98 &EPA MICROBIOLOGICAL' MANUAL 1978

to these wastes because Klebsiella originate Control Act, the Marine Protection, Research
from other than sanitary sources. However, and Sanctuaries Act and the Safe Drinking
EPA does consider large numbers of Kleb- Water Act require recommendations on analyt-
siel/a, Aeromonas and other noncoliforms as ical methodology. Generally, the membrane
indicators of organic pollution. Further, these filter methods are preferred over MPN and
organisms do occur in low densities in human othe~ techniques, where proven applicable.
and animal wastes.

3. Recommendations for Methods in Waters In Table 11-0-4, problem ·samples are

and Wastewaters
identified and the analytical method recom-
The amended Federal Water Pollution mended for parameters of choice.

REFERENCES
1. Bordner, R. H.,. C. F. Frith and J. A. Winter, eds., 1977. Proceedings of the Symposium on
Recovery of Indicator Organisms Employing Membrane Filters, U.S. Environmental Protection
Agency, EPA-600.19-77-024, EMSL-Cincinnati, Cincinnati, OH 45268.

2. Lin, S. D., 1973. Evaluation of coliform tests for chlorinated secondary effluents. JWPCF, 45:3:498.

3. Greene, R. A., R. H. Bordner and P. V. Scarpino, 1974. Applicability of the membrane filter and
most probable number coliform procedures to chlorinated wastewaters. Paper .G87 given at
74th Annual Meeting of the American Society for Microbiology, May 12-17, 1974, Chicago, IL.

4. Rose, R~ E., E. E. Geldreich and W. Litsky, 1975. Improved membrane filter method for fecal
coliform analysis. Appl. Microbiol. 29:4:532.

5. Lin, S. D., 1976. Evaluation of Millipore HA and HC membrane filters for the enumeration of
indicator bacteria. Appl. Environ. Microbiol. 32:300.

6. Green, B. L., E. Clausen and W. Litsky, 1975. Comparison of the new Millipore HC with
conventional membrane filters for the enumeration of fecal coliform bacteria. Appl. Microbiol.
30:697. --

7. Guidelines for Establishing Test Procedures, 40 Code of Federal Regulations (CFR) Part 136,
Published in Federal Register, 4Q, 52180, Dec. 1, 1976.

8. Tobin, R. S. and B. J. Dutka, 1977. Comparison of the surface structure, metal binding, and fecal
coliform recoveries of nine membrane filters. Appl. Environ. Microbiol. 34:69.

9. Lin, S. D., 1977. Comparison of membranes for fecal coliform recovery in chlorinated effluents.
JWPCF, 49:2255.

.METHOD SELECTION 99
 PART Ill. ANALYTICAL METHODOLOGY

Part Ill of the manual describes the specific analytical procedures selected in response to the
parameters required under PL 92-500 and 93-523 (see Part V-D, Legal Considerations) and to
related parameters for indicators and pathogens which.supplement the required information. New
parameters and new methodology will be added as proven in actual usage. The methods are
presented in Sections as follows:

Section A Standard Plate Count

Section B Total Coliforms

Section c Fecal Coliforms

Section D Fecal Streptococci

Section E Sslmonells

Section F Actinomycetes

100 oEPA MICROBIOLOGICAL MANUAL 1978

 PART Ill. ANALYTICAL METHODOLOGY

Section A Standard Plate Count

1. Summary of Method tion of quantitative tests for coliforms (1-3).

The procedure may also be used to monitor
The Standard Plate Count (SPC) Method is quality changes in bottled water or emergency
a direct quantitative measurement of the via- water supplies.
ble aerobic and facultative anaerobic bacteria
in a water environment, capable of growth on
the selected plating medium. An aliquot of the 2. 1 Theoretically, each bacterium present
water sample or its dilution is pipetted into a in a sample multiplies into a visible colony of
sterile glass or plastic petri dish and a liquified, millions of bacteria. However, no standard
tempered agar medium added. The plate is plate count or any other total count procedure
rotated to evenly distribute the bacteria. Each yields the true number because not all viable
colony that develops on or in the agar medium bacterial cells in the water sample can repro-
originates theoretically from one bacterial cell. duce under a single set of cultural conditions
Although no one set of plate count conditions imposed in the test. The number and types of
can enumerate all organisms present, the bacteria that develop are influenced by the
Standard Plate Count Method provides the uni- time and temperature of incubation, the pH of
form technique required for comparative test- the medium, the level of oxygen, the presence
ing and for monitoring water quality in se- of specific nutrients in the growth medium,
lected situations. competition among cells for nutrients, anti-
biosis, predation, etc.

2. Scope and Application ( 1-6)

This simple technique is a useful tool for 2.2 This procedure does not allow· the
determining the bacterial density of potable more fastidious aerobes or obligate anaerobes
waters and for quality control studies of water to develop. Also, bacteria of possible impor-
treatment processes. The Standard Plate tance in water such as Crenothrix, Sphaeroti-
Count provides a method for monitoring /us, and the actinomycetes will not develop
changes in the bacteriological quality of fin- within the incubation period specified for pota-
ished water throughout a distribution system, ble water analysis.
thus giving an indication of the effectiveness
of chlorine in the system as well as the possi-
ble existence of cross-connections, sediment 2.3 Clumps of organisms in the water sam-
accumulations and other problems within the ple which are not broken up by shaking result
distribution lines. Tote:: bacterial densities in underestimates of bacterial density, since
greater than 500-1000 organisms per ml may an aggregate of cells will appear as one colony
indicate coliform suppression or desensitiza- on the growth medium.

STANDARD PLATE COUNT 101

3. Apparatus and Materials 5. Procedure

3.1 Incubator that maintains a stable 35 5.1 Dilution of Sample (See Part 11-C, 1.4
± 0.5 C. Temperature is checked against an for details)
NBS certified thermometer or one of equivalent
accuracy. 5.1.1 The sample is diluted to obtain final
pl~te counts of 30-300 colonies. In this range,
3.2 Water bath for tempering agar set at the plate counts are the most accurate and
44-46 c. precise possible. Sir....,~ the microbial popula-
tion in the original wate.r sample is not known
3.3 Colony Counter, Quebec darkfield beforehand, a series of dilutions must be pre-
model or equivalent. pared and plated to obtain a plate count within
this range.
3.4 Hand tally or electronic counting de- 5.1.2 For most potable water samples,
vice (optional). countable plates can be obtainep by plating 1
and 0. 1 ml of the undiluted sample, and 1 ml of
3.5 Pipet containers of stainless steel, alu- the 1:100 sample dilution (see Figure 111-A-1).
minum or pyrex glass for glass pi pets. Higher dilutions may be necessary with some
potable waters. ·
3.6 Petri dish containers of stainless steel
or aluminum for glass petri dishes. 5. 1.3 Shake the sample vigorously about
25 times.
3.7 Thermometer certified by National
Bureau of Standards or one of equivalent 5.1 :4 Prepare an. initial 1: 100 dilution by
accuracy, with calibration chart. pipetting 1 ml of the sample into a 99 ml
dilution water blank using a sterile 1 ml. pipet
3.8 Sterile TD (To Deliver) bacteriological (see Figure 111-A-1 ).
or Mohr pipets, glass or plastic of appropriate
5. 1.5 The 1: 100 dilution bottle is vigor-
volumes, see Part 11-B, 1.8. 1.
ously shaken and further dilutions made by
pipetting aliquots (usualli1 ,ml) into additional
3.9 Sterile 100 mm x 15 mm petri dishes, dilution blanks. A new sterile pipet must be
glass or plastic. 1,1sed for each transfer and each dilution must
be thoroughly shaken befor~ removing an ali-
3.10 Dilution bottles (milk dilution), pyrex quot for subsequent dilution.
glass, marked at 99 ml volume, screw cap with
neoprene rubber liner. 5.1.6 When an aliquot is removed, the
pipet tip should not be inserted more than 2.5
3. 11 Bunsen/Fisher gas burner or electric cm (1 inch) below the surface ofthe liquid.
incinerator.
5.2 Preparation of Agar ·
5.2.1 Melt prepared plate count agar (tryp-
4.Medla
tone glucose yeast agar) by heating in boiling
water or by flowing steam in an autoclave at
4.1 Sterile Plate Count Agar (Tryptone Glu-
100 C. Do not allow the medium to remain at
cose Yeast Agar) dispensed in tubes (15 to 20
these high temperatures beyond the time nec-
ml per tube) or in bulk quantities in screw cap
essary to melt it. Prepared agar should be
flasks or dilution bottles. See Part 11-B, 5.1.5.
melted once only.
4.2 Sterile buffered dilution water, 99 ± 2 5.2.2 Place melted agar in a tempering
mt volumes, in screwcapped dilution bottles. water bath maintained at a temperature of
See Part 11-B, 7. , 44-46 C. Do not hold agar at.this temperature

102 aEPA MICROBIOLOGICAL MANUAL 1978

 /ML
A

A
SAMPLE
O.IML

0
e

FIGURE 111-A-1. Typical Dilution Series for Standard Plate Count

STANDARD PLATE COUNT 103

 longer than three hours because precipitates plate will check the sterility of pipets, agar,
may form which confuse the counting of colo- dilution water and petri dishes. See Part IV-C,
nies. Maintain a thermometer immersed in a 1.3.
separate bottle or flask in the water bath to
monitor the temperature. 5.5 Incubation of Plated Samples

5.3 Preparation for Plating 5.5.1 After agar plates have hardened on a
level surface (usually within 10 minutes), invert
5.3. 1 Prepare at least duplicate plates for the plates and immediately incubate at 35 C.
each sample or dilution tested. Mark and ar-
range plates in a reasonable order for use. 5.5.2 Incubate te!)ts on all water samples
Prepare a bench sheet or card, including sam- except bottled water at 35 ± 0.5 C for 48 ± 3
ple identity, dilutions, date and other relevant hours. Incubate the tests on bottled water at
Information. 35 ·± 0.5 C for 72 -f 4 hours. The longer
incubation is required to recover organisms in
5.3.2 Aseptically pipet an aliquot from the bottled water with longer generation times.
. appropriate dilution into the bottom of each
petri dish. Use a separate sterile pi pet to trans- 5.5.3 Stacks of plates should be at least
fer an aliquot to each set of petri dishes for 2.5 cm from adjacent stacks, the top or sides
each sample or sample dilution used. Vigor- of the incubator. Do not stack plates more than
ously shake the undiluted sample and dilution four high. These precautions allow proper
containers before each transfer is made. circulation of air to maintain uniform tempera-
ture throughout the incubator and speed
5.3.3 Pipet sample or sample dilution into equilibration.
marked petri dish. After. delivery, touch the tip
once to a dry spot in the dish~
5.6 Counting and Recording Colonies:
5.3.4 To minimize bacterial density
changes In the samples, do not prepare any After the required incubation period, examine
more samples than can be diluted and plated plates and select those with 30-300 colonies.
within 20-25 minutes. Count these plates immediately. A Quebec-
type colony counter equipped with a guide
5.4 Pouring Agar Plates plate, appropriate magnification and light is
recommended for use with a hand tally.
6.4.1 Use the thermometer in the control
bottle In the tempering bath to check the tem- 5.6. 1 Electronic-assist devices are avail-
perature of the plating medium before able which register colony counts with a sens-
pouring. ing probe and automatically tabulate the total
plate count.
6.~.2 Add not less than 12 ml (usually
12-15 ml) of the melted and cooled (44-46 C) Fully-automatic colony counters are avail-
agar medium to each petri dish containing an able which count all colonies (particles) larger
aliquot of the sample or its dilution. Mix the than a preset threshold-size. These counters
Inoculated medium carefully to prevent spill- scan and provide digital register and a visual
ing. Avoid splashing the inside of the cover. image of the plate for further examination and
One recommended technique rotates plate recounting with different threshold if so
five times to left, five times to the right and five desired.
times in a back and forth motion.
Because the accuracy of automatic can-
5.4.3 Pipet a one ml volume of sterile ters varies with the size and number of colo-
dilution water into a petri dish, add agar, mix nies per plate, the analyst should periodically
and Incubate with test plates. This control compare its results with manual counts.

104 &EPA MICROBIOLOGICAL MANUAL 1978

 5.6.2 The following rules should be used Count reported: Estimated Standard Plate
to report the Standard Plate Count: Count, 220/ml.

(a) Plates with 30 to 300 Colonies: Count (c) If 1 ml volumes of original sample
all colonies and divide by the volume tested (in produce counts < 30, actual counts are
ml). If replicate plates from one dilution are reported.
countable (30-300), sum the counts of colo-
nies on all plates and divide by the volumes (d) Plate with No Colonies: If all plates from
tested (in ml) as follows: dilutions tested show no colonies, report the
count as < 1 times the lowest dilution plated.
Sum of Colonies For example, if 0. 1, 0.01 and 0.001 ml vol-
= S.P. Count/ml umes of sample were tested with no visible
Sum of Volumes Tested, ml
colonies developing, the lowest dilution, 0. 1
ml would be used to calculate a less than ( <)
count as follows:
Record the dilutions used, the number of
colonies on each plate and report as the Stan-
dard Plate Count per milliliter.
= < 10
Volume Tested 0. 1
If two or more consecutive dilutions are
countable, independently carry each calcula-
tion of plate count to a final count per ml, then Count reported: Standard Plate Count,
calculate the mean of these counts/ml for the > 10/ml.
·reported value.
(e) All Plates Greater than 300 Colonies:
For example, if 280 and 34 colonies are
When counts per plate in the highest dilution
counted in the 1: 100 and 1: 1000 dilutions of a exceed 300 colonies, compute the count by
water sample, the calculation is:
multiplying the mean count by the dilution
280 used and report as a greater than ( >),Standard
= 28,000/ml Plate Count per milliliter. For example, if
.01
duplicate 1.0, D.1 and 0.01 volumes of sample
were tested with average counts of > 500,
34 = 34,000/ml > 500 and 340 developing in the dilutions, the
.001
count would be· calculated as follows:
28000 + 34000
Reporting Value =
2
31000 SPC/ml Plate Count 340
= 34,000
(b) All Plates with Fewer than 30 Colonies: Volume Tested 0.01
If thereare less than 30 colonies on all plates,
record the actual number of colonies on the or count reported as: Standard Plate Count,
lowest dilution plated and report the count as: > 34,000/ml.
Estimated Standard Plate Count per milliliter.
For example, if volumes of 0. 1, 0.01 and 0.001
5.6.3 Count Estimations on Crowded
ml were plated and produced counts of 22, 2
Plates: The square divisions of thegrid on the
and 0 colonies respectively, the colony count
Quebec or similar colony counter can be used
of 22 from the largest sample volume (0. 1 ml)
to estimate the numbers of bacteria per plate.
would be selected, calculated and reported as
With less than 10 colonies per sq cm count the
follows:
colonies in 13 squares with representative
Plate Count 22 distribution of colonies. Select 7 consecutive
= 220
Volume Plate 0.1 horizontal squares and 6 consecutive vertical

STANDARD PLATE COUNT 105

squares for counting. Sum the colonies in 5.6.5 Remarks on Data Sheet: Any
these 13 sq cm, and multiply by 5 to estimate unusual occurrences such as missed dilutions,
the colonies per plate for glass plates (area of loss of plates through breakage,
65 sq cm) or multiply by 4.32 for plastic plates contamination of equipment, materials, media,
(area of 5 7 sq cm). With more than 10 colonies or the laboratory environment, as shown by
per sq cm, count 4 representative squares, sterility control plates, must be noted on the
average the count per sq cm, multiply by the data sheet. Report as: Lab Accident, etc.
number of sq cm/plate (usually 65 for glass
plates and 57 for plastic plates) to estimate the
colonies per plate. Then multiply by the
6. Reporting Results
reciprocal of the dilution to determine the
count/ml. When bacterial counts on crowded
Report Standard Plate Count or Estimated
plates are greater than 100 colonies per sq
Standard Plate Count as colonies per ml, not
cm, report the result as Estimated Standard per 100 ml. - - --
Plate Count greater than ( >) 6,500 times the
highest dilution plated.
Standard Plate Counts should be rounded
to the number of significant figures (S.F.)
5.6.4 Spreaders: Plates containing
obtainable in the procedure: 1 S.F. for 0-9
spreading colonies must be so reported on the
actual plate counts, 2 S.F. for 10-99 actual
data sheet. If spreaders exceed one-half of the
plate counts and 3 S.F. for 100-300 actual
total plate area, the plate is not used. Report as:
plate counts. See Part 11-C, 2.8. 1 of this
No results, spreaders.
manual.
Colonies can be counted on
representative portions of plates if spreading
colonies constitute less than one-half of the 7. Precision and Accuracy
total plate area, and the colonies are well-
distributed in the remaining portion of the 7. 1 Prescott et al (7) reported that the
plate. standard deviationofindividual counts from
30-300 will vary from 0-30 percent. This
(a) Count each chain of colonies as a single plating error was 10% for plate counts within
colony. the 100-300 range. A dilution error of about
3% for each dilution stage is incurred in
(b) Count each spreader colony that addition to the plating error. Large variations
develops as a film of growth between the agar can be expected from high density samples
and the petri dish bottom as one colony. such as sewage for which several dilutions are
necessary.
(c) Countthe growth that develops in a film
of water at the edge or over the surface of the 7 .2 Laboratory personnel should be able
agar as one colony. to duplicate their plate count values on the ·
same plate within 5%, and the counts of others
(d) Adjust count for entire plate and report within 10%. If analysts' counts do not agree,
as: Estimated Standard Plate Count/ml. review counting procedures for analyst error.

106 oEPA MICROBIOLOGICAL MANUAL 1978

 REFERENCES

1. Geldreich, E. E., H. D. Nash, D. J. Reasoner and R. H. Taylor, 1972. The necessity of controlling
bacterial populations in potable waters: Community Water Supply. J. Amer. Water Works Assoc.
64:596. ---. - - - - .- -

2. Geldreich, E. E., H. D. Nash, D. J. Reasoner and R.H. Taylor, 1975. The necessity of controlling
bacterial populations in potable waters: Bottled Water and Emergency Water Supplies. J. Amer.
Waterworks Assoc. 67:117. ---

3. Geldreich, E. E., 1973. Is the total count necessary? 1st AWWA Technology Conference
Proceedings, Amer. Water Works Assoc. Vll-1, Cincinnati, Ohio,

4. Clark, D. S., 1971. Studies on the surface plate method of counting bacteria .. Can. J. Microbial.
·17:943 -

5. Klein, D. A. and S. Wu, 197 4. Stress: a factor to be considered in heterotrophic microorganism

enumeration from aquatic environments. Appl. Microbial. 27:429:

6. Van Soestbergen, A. A. and C: H. Lee, 1969. Pour plates or streak plates? Appl. Microbial.
18:1092. --

7. Prescott, S. C,. C-E. A. Winslow, and M. H. Mccrady, 1946. Water Bacteriology. (6th ed.) John
Wiley and Sons, Inc., p. 46-50. --

STANDARD. PLATE
. .
'
COUNT
.· . 107
 PART Ill. ANALYTICAL METHODOLOGY

Section B Total Coliform Methods

This section describes the enumerative 2. Single-Step, Two-Step and Delayed-

techniques for total coliform bacteria in water Incubation Membrane Filter Methods
and wastewater. The method chosen depends
upon the characteristics of the sample. The
Section Is divided as follows: 2.1 Summary: An appropriate volume of a
water sample or its dilution is passed through
a membrane filter that retains the bacteria
1. Definition of the Coliform present in the sample.
Group
In the single-step procedure the filter retaining
the microorganisms is placed on M-Endo agar,
2. Single·Step, Two-Step
LES M-Endo agar or on an absorbent pad satu-
and Delayed-Incubation
rated with M-Endo broth ~n a petri dish. The
Membrane Filter (MF)
test is incubated at 35 C for 24 hours.
Methods

In the two-step enrichment procedure the filter

3. Verification retaining the microorganisms is placed on an
absorbent pad saturated with lauryl tryptose
(lauryl sulfate) broth. After incubation for 2
4. Most Probable Number
hours at 35 C, the filter is ·transferred to an
(MPN) Method
absorbent pad saturated with M-Endo broth,
M-Endo agar, or LES M-Endo agar, and incu-
5. Differentiation of the bated for an additional 20-22 hours at 35 C.
Coliform Group by Further The sheen colonies are counted under low
Biochemical Tests magnification and the numbers of total coli-
forms are reported per 100 ml of original
sample.

1. Definition of the Coliform Group

In the delayed-incubation procedure, the
filter retaining the microorganisms is placed
The coliform or total coliform group on an absorbent pad saturated with M-Endo
includes all of the aerobic and facultative an- preservative medium in a tight-lidded petri
aerobic, gram-negative, nonspore-forming, dish and transported from field site to the
rod-shaped bacteria that ferment lactose in laboratory. In the laboratory, the filter is trans-
24-48 hours at 35 C. The definition includes ferred to M-Endo growth medium and incu-
the genera: Escherichia, Citrobacter, Entero- bated at 35 C for 24 hours. Sheen colonies are
bacter, and K/ebsiel/e. counted as total coliforms per 100 ml.

108 oEPA MICROBIOLOGICAL MANUAL 1~78

 2.2 Scope and Application: The total coli- is checked against an NBS certified thermom-
form test can be used for any type of water or eter or equivalent. Incubator must have
wastewater, but since the development of the humidity control if loose-lidded pertri dishes
fecal coliform procedure there has been in- are used. See Part 11-B, 1 .2.
creasing use of this more specific test as an
indicator of fecal pollution. However, the total 2.3.2 A binocular (dissection) microscope,
coliform test remains the primary indicator of with magnification of 10 or 1 !? x, and a day-.
bacteriological quality for potable water, distri- light type fluorescent lamp angled to give max-
bution system waters, and public water sup- imum sheen appearance.
plies because a broader measure of pollution
is desired for these waters. It is also a useful 2.3.3 Hand tally.
measure in shellfish-raising waters.
2.3.4 Pipet container of stainless' steel,
Although the majority of water and waste- aluminum or pyrex glass for glass pi pets.
water samples can be examined for total coli-
forms by the single-step MF procedure, 2.3.5 Sterile 50-100 ml graduated cylin-
coliforms may be suppressed by high back- ders covered with aluminum foil or kraft paper,
ground organisms, and potable water samples
may require the two-step method. 2.3.6 Sterile, unassembled membrane fil-
tration units (filter base and funnel), glass, plas-
If the membrane filtration method is used tic or stainless steel, wrapped with aluminum
to measure total coliforms in chlorinated foil or kraft paper. Portable field filtration units
secondary or tertiary sewage effluents the are available.
two-step enrichment procedure is required.
However, it may be necessary to use the MPN 2.3. 7 Vacuum source.
method because of high solids in the wastes or
toxicity from an industrial waste (see Part 11-D, 2.3.8 Vacuum filter flask with appropriate
this Manual). tubing. Filter manifolds which hold a number
of filter bases can also be used.
The delayed-incubation MF method is
useful in survey monitoring or emergency 2.3.9 Ultraviolet sterilizer for MF filtration
situations when the single step coliform test units (optional).
cannot be performed at the sample site, or
when time and temperature limits for sample 2.3. 10 Safety trap flask between the filter
storage cannot be met. The method eliminates flask and the vacuum source.
field processing and equipment needs. Also,
examination at a central laboratory permits 2.3.11 Forceps with smooth tips.
confirmation and biochemical identification of
the organisms as necessary. Consistent results
2.3.12 Methanol or ethanol, 95%, in small
have been obtained with this method using ·
vial, for flaming forceps.
water samples from a variety of sources (1, 2).
The applicability of this method for a specific
water source must be determined in 2.3. 13 Bunsen/Fisher burner or electric
preliminary studies .by comparison with the incinerator.
standard MF method.
2.3. 14 Sterile TD bacteriological or Mohr
pi pets, glass or plastic, of appropriate size.
2.3 Apparatus and Materials
2.3. 15 Sterile petri dishes with tight-
2°.3.1 Water jacket, air, or heat sink incu- fitting lids, 50 X 12 mm or loose-fitting lids 60
bator that maintains 35 + 0.5 C. Temperature X 15 mm, glass or plastic.

TOTAL COL/FORMS 109

 2.3. 16 Dilution bottles (milk dilution), py- 2.5 Dilution Water (See Part ll-B, 7 for
rex, marked at 99 ml volume, screw cap with preparation).
neoprene rubber liner.
2.5.1 Sterile dilution water dispensed in
2.3.17 Membrane filters, white, grid- 99 + 2 ml amounts in screw-capped dilution
marked, 4 7 mm diameter, with 0.45 µm ± bottles.
0.02 µm pore size, or other pore size, as recom-
mended by manufacturer for water analyses. 2.5.2 Sterile dilution water prepared in
liter or larger volumes for wetting membranes
2.3. 1 8 Absorbent pads. before addition of small sample volumes and
for rinsing the funnel after sample filtration.
2.3.19 Inoculation loops, at least 3 mm
diameter, or needles, nichrome or platinum 2.6 Procedure: Refer to the general proce-
wire, 26 B&S gauge, in suitable holder. dure in Part 11-C for more complete details.

2.3.20 Disposable applicator sticks or 2.6. 1 Single-Step Procedure

plastic loops as alternatives to inoculation
loops. (a) Prepare the M-En'do broth, M-Endo agar
or LES M-Endo agar as.directed in Part 11-B.
2.3.21 Shipping tubes, labels, and packing
materials for mailing delayed incubation (b) Place one sterile absorbent pad in the
pistes. bottom half of each petri dish. Pi pet 1.8-2.0 ml
M-Endo broth onto the pad to saturate it. Pour
off excess broth. Alternatively, pipet 5-6 ml of
melted agar into each dish (2-3 mm) and allow
2.4 Media: Media are prepared in pre- to harden before use. Mark dishes and bench
sterillzed erlenmeyer flasks with metal caps, forms with sample identities and volumes.
aluminum foil covers, or screw caps.
(c) Place a sterile membrane filter on the
2.4.1 M-Endo broth or agar (See Part 11-B, filter base, grid-side up and attach the funnel to
5.2.2). the base of the filter unit; the membrane filter
is now held between the funnel and the base.
2.4.2 LES M-Endo agar (See Part 11-B,
5.2.4). (d) Shake the sample bottle vigorously
about 25 times and measure the desired vol-
2.4.3 Laury! tryptose broth (See Part 11-B, ume of sample into the funnel. Select sample
5.3.1). volumes based on previous knowledge to
produce membrane filters with 20-80 coli-
2.4.4 Brilliant green lactose bile broth form colonies. See Table 11-C-1. If sample vol-
(See Part 11-8, 5.3.2). ume is < 10 ml, add 10 ml of sterile dilution
water to the filter before adding sample.
2.4.5 M-Endo holding medium (See Part 11-
B, 5.2.3). It is desirable to filter the largest possible
sample volumes for greatest accuracy. How-
2.4.6 Sodium benzoate, U.S.P., for use in ever, if past analyses of specific samples have
the delayed incubation procedure (See Part 11- resulted in confluent growth, "too numerous to
8, 5.2.3). count" membranes, or lack of sheen from
excessive turbidity, additional samples should
2.4.7 Cycloheximide (Actidione - Upjohn, be collected and filtration volumes adjusted to
Kalamazoo, Ml) for use as antifungal agent in provide isolated colonies from smaller volumes.
delayed Incubation procedure (See Part 11-B, See 2. 7.2 in this Section for details on adjusting
5.2.3). sample volumes for potable waters.

110 oEPA MICROBIOLOGICAL MANUAL 1978

 The suggested method for measuring filter is now held between the funnel and the
sample volumes is described in Part 11-C, 3.4.6. base.

(e) Filter sample and rinse the sides of the (d) Shake the sample bottle vigorously
funnel at least twice with 20-30 ml of sterile about 25 times to obtain uniform distribution
dilution water. Turn off the vacuum and re- . of bacteria. Select sample volumes based on
move the funnel from the filter base. Asepti- previous knowledge to produce membrane fil-
cally remove the membrane filter from the ters with 20-80 coliform colonies. See Table
filter base and place grid-side up on the agar or 11-C-1. If sample volume is < 10 ml, add 10 ml
pad. of sterile dilution water to filter before adding
sample.
(f) Filter samples in order of increasing
sample volume, filter potable waters first.
(e) Filter samples in order of increasing
(g) If M-Endo broth is us~d, place the filter sample volume, rinsing with sterile buffered
on an absorbent pad saturated with the broth. dilution water between filtrations. The me-
Reseat the membrane, if air bubbles occur, as thods of measurement and dispensation of the
evidenced by non-wetted areas on the mem- sample into the funnel are given in Part 11-C,
brane. Invert dish and incubate for 24 ± 2 3.4.6.
hours at 35 + 0.5 C in an atmosphere with
near saturated humidity. (f) Turn on the vacuum to filter the sample
through the membrane, rinse the sides of the
(h) If M-Endo agar or LES M-Endo agar is funnel at least twice with 20-30 ml of sterile
used, place the inoculated filter directly on the dilution water. Turn off vacuum and remove
agar surface. Reseat the membrane if bubbles funnel from base.
occur. lnvart the dish and incubate for 24 ± 2
hours at 35 + 0.5 C in an atmosphere with (g) Remove the membrane filter asepti-
near saturated humidity. cally from the filter base and place grid-side up
on the pad in the top of the petri dish. Reseat
(i) If tight-lidded dishes are used, there is MF if air bubbles are observed.
no requirement for near-saturated humidity.
(h) Incubate the filter in the petri dish with-
U) After incubation remove the dishes from out inverting for 1 1/2 - 2 hours at 35 + 0.5 C
the incubator and examine for sheen colonies. in an atmosphere of near saturated humidity.
This completes the first step in the Two-Step
(k) Proceed to 2.7 for Counting and Enrichment Procedure.
Recording Colonies.
(i) Prepare M-Endo broth, M-Endo agar, or
2.6.2 Two-Step Enrichment Procedure LES M-Endo agar as directed in Part 11-B.

(a) Place a sterile absorbent pad in the top If M-Endo broth is used, place a new sterile
of each petri dish. absorbent pad in the bottom half of the dish
and saturate with 1.8-2.0 ml of the M-Endo
(b) Prepare lauryl tryptose broth as di- broth. Transfer the filter to the new pad. Reseat
rected in Part 11-B. Pipet 1.8-2.0 ml lauryl tryp- MF if air bubbles are observed. Remove the
tose broth onto the pad to saturate it. Pour off used pad and discard.
excess broth.
If M-Endo or LES M-Endo agar is used,
(c) Place a sterile membrane filter on the pour 5-6 ml of agar into the bottom of each
filter holder, grid-side up and attach the funnel petri dish and allow to solidfy. The agar me-
to the ba·se of the filter unit; the membrane. dium can be refrigerated for up to two weeks.

TOTAL COLIFOR.MS 111

 (j) Transfer the filter from the lauryl tryp- an absorbent pad saturated with M-Endo Hold-
tose broth onto the Endo medium. Reseat if air ing Medium or LES Holding Medium.
bubbles are observed.
(h) Place the culture dish in shipping con-
(k) Incubate dishes in an inverted position tainer and send to the examining laboratory.
for an additional 20-22 hours at 35 + 0.5 C. Coliform bacteria can be held on the holding
This completes the second step in the Two- medium for up to 72 hours with little effect on
Step Enrichment Procedure. the final counts. The holding period should be
kept to a minimum.
(I·) Proceed to 2. 7 Counting and
Recording. (i) At the examining laboratory remove the
membrane from the holding medium, place it
2.6.3 Delayed Incubation Procedure in another dish containing M-Endo broth or
agar medium, and complete testing for coli-
(a) Prepare the M-Endo Holding Medium or forms as described above under 2.6. 1.
LES Holding Medium as outlined in Part 11-B,
5.2.3 or 5.2.5. Saturate the sterile absorbent 2.7 Counting and Recording Colonies:
pads with about 2.0 ml of holding broth. Pour After incubation, count colonies on those
off excess broth. Mark dishes and bench forms membrane filters containing 20-80 golden-
with sample identity and volumes. green metallic surface sheen colonies and less
than 200 total bacterial colonies. A binocular
(b) Using sterile forceps place a mem- (dissection) microscope with a magnification
brane filter on the filter base grid side up. of 10 or 15 x is recommended. Count the
colonies according to the general directions
(c) Attach the funnel to the base of the given in Part 11-C, 3.5.
filter unit; the membrane filter is now held
between the funnel and base. 2. 7. 1 The following general rules are used
in calculating the total coliform count per 100
(d) Shake the sample vigorously about 25 ml of sample. Specific rules for analysis and
times and measure into the funnel with the counting of water supply samples are given in
vacuum off. If the sample is < 10 ml, add 10 2.7.2.
ml of sterile dilution water to the membrane
filter before adding the sample. (a) Countable Membranes with 20-80
Sheen Colonies, and Less Than 200 Total Bac-
(1) Select sample volumes based on previ- teria.I Colonies: Select the plate counts to be
ous knowledge to produce counts of 20-80 used according to the rules given in Part 11-C,
coliform colonies. See Table 11-C-1. 3.6, and calculate the final value using the
formula.
(2) Follow the methods for sample mea-
surement and dispensation given in Part 11-C, Total Coliforms/100 ml:
3.4.6

(e) Filter the sample through the mem- No. of Total Coliform Colonies Counted
brane and rinse the sides of the funnel walls at
Volume in ml of Sample Filtered
least twice with 20-30 ml of sterile dilution
water.
x 100
(f) Turn off the vacuum and remove the
funnel from the base of the filter unit. (b) Counts Greater Than the Upper Limit of
80 Colonies: All colony counts are above the
(g) Aseptically remove the membrane filter recommended limits. For example, sample vol-
from the filter base and place grid side up on umes of 1, 0.3, and 0.01 ml are filtered to

112 &EPA MICROBIOLOGICAL MANUAL 1978

produce total coliform colony counts of TNC, If filtration of multiple volumes of less than
150, and 110 colonies. 100 ml still results in confluency or high back-
ground count, the coliforms may be present
Use the count from the smallest filtration but suppressed. These samples should be ana-
volume and report as a greater than lyzed by the MPN Test. This MPN check should
count/100 ml. In the example above: be made on at least one sample for each prob-
lem water once every three months.
110
0.01
x 100 = 1, 100,000
(c) Membranes with Confluent Growth
or> 1, 100,000 coliforms/100 ml.
For potable water samples, confluence
(c) Membranes with More Than 200 Total requires resampling and retesting.
Colonies (Coliforms plus NOrl-COITTOrrTISf"" - -
(d) Verification. Because unsatisfactory
(1) Estimate sheen colonies if possible, samples from public water supplies containing
calculate total coliform density as in (a) above. 5 or more coliform colonies must be verified,
at least 5 colonies need to be verified for each
Report as: Estimated Count/100 ml. positive sample. Reported counts are adjusted
based on verification.
(2) If estimate of sheen colonies is not
possible, report count as Too Numerous to (e) Quality control procedures are speci-
Count (TNTC). fied by EPA under the law, and described in
Appendix C in this Manual.
(d) Membranes with Confluent Growth
2.7.3 Reporting Results: Report total coli-
form densities per 100 ml of sample. See
Report as: Confluent Growth and specify
Figure 11-C-3 for an example of a bench form for
the presence or absence of sheen.
reporting results. A discussion on significant
figures is given in Part 11-C, 2.8.
2.7.2 Special Rules for Potable Waters
2.8 Precision and Accuracy: There are
(a) Countable Membranes with 0-80 Sheen
no established precision and accuracy data
Colonies, and Less than 200 Total Colonies available at this time.
Count the sheen colonies per volume filter-
3. Verification
ed. Calculate and report the number of Total
Coliforms/100 ml. Verification of total coliform colonies from
M-Endo type media validates sheen as evi-
(b) Uncountable Membranes for Potable dence of coliforms. Verification of repr~senta­
Water Samples · tive numbers of colonies may be required in
evidence gathering or for quality control pro-
If 100 ml portions of potable water sam- cedures. The verification procedure follows:
ples cannot be tested because of high back-
ground counts or confluency, multiple volumes 3.1 Using a sterile inoculating needle, pick
of less than 100 ml can be filtered. For example, growth from the centers of at least 10 well-
if 60 colonies appear on the surface of one isolated sheen colonies (5 sheen colonies per
m.embrane through which a 50 ml portion of plate for potable waters). Inoculate each into a
the sample was passed, and 50 colonies on tube of lauryl tryptose broth and incubate 24-48
a second membrane through which a second hours at 35 C + 0.5 C. Do not transfer exclu-
50 ml portion of the sample was passed, the sively into brilliant green bile lactose broth.
colonies are totaled and reported as 110 total However, colonies may be transferred to LTB
coliforms per 100 ml. and BGLB simultaneously.

TOTAL COL/FORMS 113

 3.2 At the 24 and 48 hour readings, con- 4.2 Scope and Application
firm gas-positive lauryl tryptose broth tubes by
inoculating a loopful of growth into brilliant 4.2.1 Advantages: The MPN procedure is
green lactose bile broth and incubate for a tube-dilution method using a nutrient-rich
24-48 hours at 35 ± 0.5 C. Cultures that are medium, which is less sensitive to toxicity and
positive in BGLB are interpreted as verified supports the growth of environmentally-
coliform colonies (see Figure 111-B-1 ). stressed organisms. The method is applicable
to the examination of total coliforms in chlori-
nated primary effluents and under other
3.3 If questionable sheen occurs, the stressed conditions. The multiple-tube proce-
worker should also verify these colonies. dure is also better suited for the examination of
turbid samples, muds, sediments, or sludges
because particulates do not interfere visibly
with the test.

4. Most Probable Number (MPN) Method 4.2.2 Limitations: Certain non-coliform

bacteria may suppress coliforms or act syner-
gistically to ferment lauryl tryptose broth and
4.1 Summary: This method detects and yield false positive results. A significant num-
estimates the total coliforms in water samples ber of false positive results can also occur in
by the multiple fermentation tube technique. the brilliant green bile broth when chlorinated
The method has three stages: the Presumptive, primary effluents are te"sted, especially when
the Confirmed, and the Completed Tests. In stormwater is mixed with the sewage (3). False
the Presumptive Test, a series of lauryl tryp- negatives may occur with waters containing
tose broth fermentation tubes are inoculated nitrates (4). False positives are more common
with decimal dilutions of the sample. The for- in sediments.
mation of gas at 35 C within 48 hours consti-
tutes a positive Presumptive Test for members 4.3 Apparatus and Materials
of the total coliform group. However, the MPN
must be carried through the Confirmed Test 4.3. 1 Water bath or air incubator set at 35
for valid results. In this test, inocula from posi- +0.5C.
tive Presumptive tubes are transferred to tubes
of brilliant green lactose bile (BGLB) broth. The 4.3.2 Pipet containers of stainless steel,
BGLB medium contains selective and inhib- aluminum, or pyrex glass for glass pi pets.
itive agents to suppress the growth of all non-
coliform organisms. Gas production after incu- 4.3.3 Inoculation loops, at least 3 mm
bation for 24 or 48 hours cit 35 C constitutes a diameter and needles of nichrome or platinum
positive Confirmed Test and is the point at wire, 26 B & S gauge, in suitable holders.
which most MPN tests are terminated. The
Completed Test begins with streaking inocu- 4.3.4 Disposable sterile applicator sticks
lum from the positive BGLB tubes onto EMB or plastic loops as alternatives to inoculating
plates and incubating the plates for 24 hours loops.
at 35 C. Typical and atypical colonies are
transferred into lauryl tryptose broth fermenta- 4.3.5 Compound microscope, oil
tion tubes and onto nutrient agar slants. Gas immersion.
formation in the fermentation tubes and pres-
ence of gram-negative rods constitute a posi- 4.3.6 Bunsen/Fisher burner or electric in-
tive Completed Test for total coiiforms. See cinerator unit.
Figure 111-B-2. The MPN per 100 ml is calculated
from the MPN table based upon the Con- 4.3.7 Sterile TD Mohr or bacteriological
firmed or Completed test results. pi pets, glass or plastic, of appropriate size.

114 &EPA MICROBIOLOGICAL MANUAL 1978

 Pick 10 sheen colonies·
from each sample

I
Lauryl Tryptose Broth
24 hours at 35 C

~
Gas+ Gas-

1
Reincubate
24 hours at 35 C

Gas-
Negative
Test

Brilliant Green Lactose Bile Broth

24 hours at 35 C

Gas+ Gas-

Verified Re incubate
Coliform 24 hours at 35 C
Colony

Verified· Negative
Coliform Test
Colony

FIGURE 111-B-1. Verification of Total Coliform Colonies on the Membrane Filter

TOTAL COL/FORMS 115

 Sample

I
Lauryl Tryptose Broth
35 ± 0.5 c

Gas + Gas -
24 hr 24 hr

Reincubate
24 hr

~
L Gas -
48 hr
Negative Test

I
~ Gas +
24 hr
Brilliant Green Lactose Bile Broth
35 ±0.5 c

Gas -
24 hr
0
w
~
a:
u:::
z
0
u Reincubate
24 hr

L ~ Gas -
Negative Test

I
I-
t/)
w
l-
Nutrient Agar Slant
24 hrat35 ± 0.5 C
Eosin Methylene Blue Agar
24 hrat35 ± 0.5 C

Lauryl Tryptose Broth Tube

24-48 hr at 35 ± 0.5 C
o
~
w
~
11.
:I! Gram + Gram Gas + Gas -
0 Sporeformers Nonspore-forming
u

L Negative
Test
Coliforms
Present
Coliforms
Present

FIGURE 111-B-2. Flow Chart for the Total Coliform MPN Test
Negative
Test

116 oEPA MICROBIOLOGICAL MANUAL 1978

 4.3.8 Pyrex culture tubes, 150 X 25 mm 100, 10, 1, 0. 1 and 0.01 ml, respectively; the
or 150 x 20 mm, containing inverted fermen- latter two volumes delivered as dilutions of
tation vials, 75 X 10 mm with caps. original sample.

4.3.9 Culture tube racks to hold fifty, 25 (c) For known polluted waters the initial
mm diameter tubes. sample inoculations might be 0. 1, 0.01,
0.001, 0.0001, and 0.00001 ml of original
4.3. 10 Dilution bottles (milk dilution) py- sample delivered as dilutions into successive
rex glass, 99 ml volume, screw cap with neo- rows each containing five replicate volumes.
prene rubber liners. This series of sample volumes will yield deter-
minate results from a low of 200 to a high of
4.4Media 16,000,000 organisms per 100 ml.
4.4.1 Presumptive Test: Lauryl tryptose
broth. See Part 11-B, 5.3.1 . Lactose broth is not (d) Shake the sample and dilutions vigor-
used because of false positive reactions. ously about 25 times. Inoculate each 5-tube
row with replicate sample volumes in increas-
4.4.2 Confirmed Test: Brilliant green bile ing decimal dilutions and incubate at 35 C ±
broth. (See Part 11-B, 5.3.2). 0.5 c.

4.4.3 Completed Test: (e) After 24 + 2 hours incubation at 35 C,

gently agitate the tubes in the rack and exam-
(a) Eosin methylene blue agar (see Part 11-B; ine the tubes for gas. Any amount of gas
5.3.3). constitutes a positive test. If there is no gas
production in the tubes, reincubate for an
(b) Nutrient agar or plate count agar slants additional 24 hours and reexamine for gas.
(see Part 11-B, 5. 1. 1 and 5. 1.5). Positive Presumption tubes are submitted
directly to the Confirmed Test. Results are
4.5 Dilution Water: Sterile dilution water recorded on laboratory bench forms.
dispensed in 99 + 2 ml amounts preferably in
screw-capped bottles. (See Part 11-B, 7). (f) If a laboratory using the MPN test on
water supplies finds frequent numbers of Pre-
4.6 Procedure: Part 11-C describes the gen- sumptive test tubes with heavy growth but no
eral MPN procedure in detail. gas, these negative tubes should be submitted
to the Confirmed Test to check for suppression
4.6. 1 Prepare the media for Presumptive, of coliforms.
Confirmed or Completed Tests selected. (See
Part 11-B, 5.3). (g) If The Presumptive Test tubes are gas-
negative after 48 · + 3 hours, they are dis-
4.6.2 Presumptive Test (See Figure 111-B-2): carded and the results recorded as negative
To begin the Presumptive Test, arrange fer- Presumptive Tests. Positive Presumptive tubes
mentation tubes of lauryl tryptose broth in are verified by the Confirmed Test.
rows cf 5 tubes each in the tube rack. Select
sample volumes and clearly label each bank of (h) If the fecal colifrom test is to be· run,
tubes to identify the sample and volume (Part 111-C), the analyst can inoculate growth
inoculated. from positive Presumptive Test tubes into EC
medium at the same time as he inoculates the
(a) For potable waters, five portions of 10 Confirmed Test Medium.
ml each or five portions of 100 ml each are .
used. 4.6.3 Confirmed Test (See Figure 111-B-2)

(b) For relatively-unpolluted waters the (a) Carefully shake each positive Presump-
sample volumes for the five rows might be tive tube. With a sterile 3 mm loop or a sterile

TOTAL COL/FORMS 117

applicator stick, transfer growth from each into lauryl tryptose fermentation tubes and
tube to BGLB. Gently agitate the tubes to mix incubate tubes for up to 48 + 3 hours.
the inoculum and incubate at 35 ±
0.5 C. (c) The formation of gas in any amount in
the fermentation tubes and presence of gram
(b) After 24 ± 2 hours incubation at 35 C negative 'rods constitute a positive Completed
examine the tubes for gas. Any amount of gas in Test for total coliforms.
BGLB constitutes a positive Confirmed Test.
If there is no gas production in the tubes
(negative test) reincubate tubes for an addi- 4.6.5 Special Considerations for Potable
tional 24 hours. Record the gas-positive and Waters
gas-negative tubes. Hold the positive tubes for Sample Size - For potable waters the stan-
the Completed Test if required for quality con- dard sample shall be five times the standard
trol or for checks on questionable reactions. portion which is either 10 milliliters or 100
milliliters as described in 40 CFR 141 (5).
(c) After 48 ± 3 hours reexamine the
Confirmed Test Tubes. Record the positive and . Confirmation - If a laboratory using the
negative tube results. Discard the negative MPN test on water supplies finds frequent
tubes and hold the positive tubes for the Com- numbers of Presumptive test tubes with heavy
pleted Test if required as in (b) above. growth but no gas, these negative tubes
should be submitted to the Confirmed Test to
(d) In routine practice most sample ana- check for suppression of coliforms.
lyses are terminated at the end of the Con-
firmed Test. However, the Confirmed Test data Completion - In water supply laboratories,
should be verified by carrying 5% of Confirmed 10% of all samples and at least one sample
Tests with a minimum of one sample per test quarterly must be carried to completion but
run through the Completed Test. no gram stain of cultures is required.

(e) For certification of water supply labora- 4.7 Calculations: The results of the Con-
tories, the MPN test is carried to completion firmed or Completed Test may be obtained
(except for gram stain) on 10 percent of positive from the MPN table based on the number of
confirmed samples and at least one sample positive tubes in each dilution. See Part 11-C,
quarterly. 4.9 for details on calculation of MPN results.

4.6.4 Completed Test (See Figure 111-B-2) 4. 7 .1 Table 11-C-4 illustrates the MPN in-
dex and 95% Confidence Limits for combina-
Positive Confirmed Test cultures may be tions of positive and negative results when five
subjected to final Completed Test identifica- 10 ml, five 1.0 ml, and five 0. 1 ml volumes of
tion through application of further biochemical sample are tested.
and culture tests, as follows:
4.7.2 Table 11-C-5 provides the MPN indi-
(a) Streak one or more EMB agar plates ces and limits for the five tube, single volumes
from each positive BGLB tube. Incubate the used for potable water supplies.
plates at 35 ±
0.5 C for 24 + 2 hours.
4.7.3 When the series of decimal dilutions
(b) Transfer one or more well-isolated typi- is other than those in the tables select the MPN
cal colonies (nucleated with or without a metal- value from Table 11-C-4 and calculate accord-
lic sheen) to lcluryl tryptose broth fermentation ing to the following formula:
tubes and to nutrient or plate count agar
slants. Incubate the slants for 24 ± 2 or 48 ± MPN (From Table) X 10
3 hours at 35 ± 0.5 C. If no typical colonies are Largest Volume Tested
present, pick and inoculate at least two atypi-
cal (pink, mucoid and unnucleated) colonies = MPN/100 ml

118 &EPA MICROBIOLOGICAL MANUAL 1978

 4.8 Reporting Results: Report the MPN Some brands are not satisfactory and oth-
values per 100 ml of sample. See an example ers become unsatisfactory after aging. Both
of a report form in Figures 111-D-·2 and 111-D-3. amyl alcohol and benzaldehyde compound
should be purchased in as small amounts as
4.9 Precision and Accuracy: The preci- will be consistent with the volume of work
sion of the MPN value increases . with in- anticipated. Store the reagent in the dark in a
creased numbers of replicates tested. A five brown bottle with a glass stopper.
tube, five dilution MPN is recommended for
natural and waste waters. Only a five tube, (b) Methyl Red Test Reagent: Dissolve 0.1
single volume series is required for potable gram methyl red in 300 ml of 95% ethyl alco-
waters. hol and dilute to 500 ml with distilled water.

(c) Voges-ProskauerTest Reagents

5. Differentiation of the Coliform Group by
by Further Biochemical Tests ( 1) Naphthol solution: Dissolve 5 grams
purified alphanaphthol (melting point 92.5 C
5.1 Summary: The differentiation of the or higher) in 100 ml absolute ethyl alcohol.
members of the coliform group into genera and This solution. must be freshly prepared each
species is based on additional biochemical and day.
cultural tests (see Table 111-B-1 ). These tests
require specific training for valid results. (2) Potassium hydroxide solution: Dissolve
40 grams KOH in 100 ml distilled water.
5.2 Apparatus and Materials
(d) OxidaseTest Reagents
5.2.1 Incubator set at 35 ± 0.5 C.

5.2.2 Pipet containers of stainless steel, ( 1) Reagent A: Weigh out gram alpha-
aluminum or pyrex glass for glass pipets. napthol and dissolve in 100. ml of 95%
ethanol.
5.2.3 Inoculation loop, 3 mm diameter
and needle. (2) Reagent B: Weigh out 1 gram para-
aminodimethylaniline HCI (or oxylate) and
5.2.4 Bunsen/Fisher type burner or elec- dissolve in 100 ml of distilled water. Prepare
tric incinerator. frequently and store in refrigerator.

5.2.5 Sterile TD Mohr and bacteriological

pipets, glass or plastic, of appropriate volumes. 5.3 Media

5.2.6 Graduates, 25 - 500 ml.

5.3.1 Tryptophane broth for demonstrat-
5.2.7 Test tubes, 100 x 13 mm or 150 x ing indole production in the lndole Test. (See
20 mm with caps, in racks. Part 11-B, 5. 1.9 (a) for preparation).

5.2.8 Reagents 5.3.2 MR-VP broth (buffered glucose) to

demonstrate acid production by methyl red
color change in the Methyl Red Test and to
(a) lndole Test Reagent: Dissolve 5 grams demonstrate acetyl methyl carbinol
para-dimethylamino benzaldehyde in 75 ml production in the Voges-Proskauer test. (See
isoamyl (or normal amyl) alcohol, ACS grade, Part 11-B, 5. 1.9 (b) for preparation).
and slowly add 25 ml cone HCI. The reagent
should be yellow and have a pH below 6.0. If 5.3.3 Simmon's Citrate Agar to demon-
the final reagent is dark in color it should be strate utilization of citrate as a sole source of
discarded. carbon. (See Part 11-B, 5. 1.9 (c) for preparation).

TOTAL COL/FORMS 119

....
"'
0

TABLE 111-B-1

Differentiation of the Coliform and Related Organisms Based

Upon Biochemical Reactions

~)
m
~
Tests
~ Bacte.rium
(') Methyl Voges- Cytochrome Ornithine Lysine Arginine
::J:J lndole Citrate Motility
Red Proskauer Oxidase Decarboxylase Decarboxylase Dehydrolase

-...
0
l1J
0
0
Escherichia coli + + - - - v v v ±

-~...
C>

Citrobacter freundii - + - + - v v v +

~
::c: Enterobacter aerogenes - - + + - + + - +
§
.......
co Klebsiella :t - + + - - +
~

Pseudomonas - - - + + - - -(+)1 +

Aeromonas + - + + + + - + +

V = variable
( ) 1 = reaction of P. aeruginosa
 5.3.4 Nutrient agar slant for oxidase test. (d) A dark red color in the amyl alcohol
(See Part 11-B, 5. 1. 1 for preparation). layer on top of the culture is a positive indole
test; the original color of the reagent, a nega-
5.3.5 Decarboxylase medium base con- tive test. An orange color may indicate the
taining lysine HCI, arginine HCI or ornithine presence of skatole and is reported as a +
HCI to demonstrate utilization of the specific reaction.
amino acids. (See Part 11-B, 5.5.14 for
preparation).

5.3.6 Motility test medium (Edwards and 5.4.3 Methyl Red Test
Ewing). (See Section 11-B, 5. 1. 10 for
preparation). (a) Inoculate a pure culture into 10 ml of
buffered glucose broth.
5.3.7 Multitest Systems (optional to Sin-
gle Test Series) - -- (b) Incubate for 5 days at 35 C.

(a) API Enteric 20 (Analytab Products, Inc.). (c) To 5 ml of the five day culture, add 5
drops of methyl red indicator.
(b) Enterotube (Roche Diagnostics).
(d) A distinct red color is positive and dis-
(c) lnolex (lnolex Biomedical Division of tinct yellow, negative. Orange color is dubious,
Wilson Pharmaceutical and Chemical Corp.). may indicate a mixed culture and should be
repeated.
(d) Minitek (Baltimore Biological Labora-
tories, Bioquest). 5.4.4 Voges Proskauer Test: This procedure
detects the production of acetyl methyl carbinol
(e) Pathotec Test Strips (General Diagnos- which in the presence of alphanapthol and
tics Division of Warner-Lambert Company). potassium hydroxide develops a reddish color.

(f) r/b Enteric Differential System (Diagn- (a) Use a pure culture to inoculate 10 ml of
ostic Research, Inc.). buffered glucose broth or 5 ml of salt peptone
glucose broth or use the previously inoculated
5.4 Procedure buffered glucose broth from the Methyl Red
Test.
5.4.1 Biochemical tests should always be
performed along with positive and negative (b) Incubate the inoculated salt peptone
controls. See Table IV-A-5. glucose broth or the buffered glucose broth at
35 ± 0.5 C for 48 hours.
5.4.2 lndole Test
(c) Add 0.6 ml naphthol solution and 0.2
(a) Inoculate a pure culture into 5 ml of ml KOH solution to 1 ml of the 48 hour salt
tryptophane broth. peptone or buffered glucose broth culture in a
separate clean test tube. Shake vigorously for
(b) Incubate the tryptophane broth at 35+ 10 seconds and allow the mixture to stand for
0.5 C for 24+2 hours and mix well. 2-4 hours.

(c) Add 0.2-0.3 ml test reagent to the 24 (d) Observe the results and record. A pink
hour culture, shake. and allow the mixture to to crimson color is a positive test. Do not read
stand for 10 minutes. Observe and record the after 4 hours. A negative test may develop a
results. copper or faint brown color.

TOTAL COL/FORMS 121

 5.4.5 Citrate Test for the reactions of the other tubes to be valid.
Positive purple tubes must have growth as
(a) Lightly inoculate a pure culture into a evidenced by turbidity because uninoculated
tube of Simmon's Citrate Agar, using a needle tubes are also purple; nonfermenters may
to stab, then streak the medium. Be careful not remain alkaline throughout incubation.
to carry over any nutrient material.

(b) Incubate at 35 C for 48 hours. 5.4.8 Motility Test

(c) Examine agar tube for growth and color (a) Stab-inoculate the center of the tube of
change. A distinct Prussian blue color in the Motility Test Medium to at least half depth.
presence of growth indicates a positive test;
no color change is a negative test. (b) Incubate tubes 24-48 hours at 35 C.

5.4.6 Cytochrome Oxidase Test (c) Examine tubes for growth. If negative,
(lndophenol): The cytochrome oxidase test can reincubate at room temperature for 5 more
be done with commercially-prepared paper days.
strips or on a nutrient agar slant as follows:
(d) Non-motile organisms grow only along
(a} Inoculate nutrient agar slant and incu- the line of inoculation. Motile organisms grow
bate at 35 C for 18-24 hours. Older cultures outward from the line of inoculation and
should not be used. spread throughout the medium producing a
cloudy appearance.
(b) Add 2-3. drops of reagent A and reag-
ent B to the slant, tilt to mix and read reaction (e) Addition of 2, 3, 5 triphenyl tetrazolium
within 2 minutes. chloride (TTC) will aid recognition of motility.
Growth of microorganisms reduces TTC and
(c) Strong positive reaction (blue color produces red color along the line of growth.
slant or paper strip) occurs in 30 seconds.
Ignore weak reactions that occur after 2 5.4.9 Additional Biochemical Tests: If
minutes. other biochemical tests are necessary to fur-
ther identify enteric bacteria, for example spe-
5.4. 7 Decarboxylase Tests (lysine, argi- cific carbohydrate fermentation, see the Table
nine and ornithine) 111-E-5, Biochemical Characteristics of Entero-
bacteriaceae.
(a) The complete decarboxylase test series
requires tubes of each of the amino acids and a 5.4. 10 Multitest Systems: Multitest sys-
control tube containing no amino acids. tems are available which use tubes containing
agar media that provide numerous biochemi-
(b) Inoculate each tube lightly. cal tests, plastic units containing a series of
dehydrated media, media-impregnated discs
(c) Add sufficient sterile mineral oil to the and reagent-impregnated paper strips. Some
broths to make 3-4 mm layers on the surface of the systems use numerical codes to aid
and tighten the screw caps. identification. Others provide computerized
identification of bacteria. A number of inde-
(d} Incubate for 18-24 hours at 35 C and pendent investigators have compared one or
read. Negative reactions should be re- more multitest systems with conventional or
lncubated up to 4 days. traditional biochemical tests. Some of the ear-
lier systems have been improved. Most of the
(e) Positive reactions are purple and recent studies report the correct identification
negative reactions are yellow. Read the control of high percentages of isolates. The systems
tube without amino acid first; it must be yellow are described in Part 111-E, 5.6.

122 &EPA MICROBIOLOGICAL MANUAL 1978

 REFERENCES

1. Geldreich, E. E., P. W. Kabler, H. L. Jeter and H. F. Clark, 1955. A delayed incubation membrane
filter test for coliform bacteria in water. Amer. Jour. Public Health 45: 1462.

2. Brezenski, F. T. and J. A. Winter, 1969. Use of the delayed incubation membrane filter test for
determining coliform bacteria in sea water. Water Res. 3:583. ·

3. Geldreich, E. E., 1975. Handbook for Evaluating Water Bacteriological Laboratories ·(2nd ed.),
EPA-670/9-75-006. U.S. Environmental Protection Agency, Cincinnati, Ohio.

4. Tubiash, H., 1951. The Anaerogenic Effect of Nitrates and Nitrites on Gram-negative Enteric
Bacteria. Amer. Jour. Public Health 41 :833. ·
-~ -- -------
5. National Interim Primary Drii1king Water Regulations, 40 Code of Federal Regulations (CFR) Part
141.14 (b) and (c), Published in Federal Register, 40, 59566, December 24, 1975.

TOTAL COL/FORMS 123

 PART Ill. ANALYTICAL METHODOLOGY

Section C Fecal Coliform Methods

The direct membrane filter (MF), the 2. Direct Membrane Filter (MF) Method
delayed-Incubation MF and the multiple-tube,
most probable number (MPN) methods can be 2.1 Summary: An appropriate volume of a
used to enumerate fecal coliforms in water and water sample or its dilution is passed through
wastewater. For a general description of the a· membrane filter that retains the bacteria
fundamental laboratory techniques refer to present in the sample. The filter containing the
Part 11-C. The method chosen depends upon microorganisms is placed on an absorbent pad
the characteristics of the sample. The Section saturated with M-FC broth or on M-FC agar in a
Is divided as follows: petri dish. The dish is incubated at 44.5 C for
24 hours. After incubation, the typical blue
1. Definition of the Fecal Coliform colonies are counted under low magnification
Group and the number of fecal coliforms is reported
per 100 ml of original sample.
2. Direct Membrane Filter (MF)
Method
2.2 Scope and Application
3. Delayed-Incubation Membrane •
Filter Method 2.2. 1 Advantages: The results of the MF
test are obtained in 24 hours. Up to 72 hours
4. Verification are required for the multiple-tube fermentation
method. The M-FC method provides direct enu-
5. Most Probable Number (MPN) meration of the fecal coliform group without
Method enrichment or subsequent testing. Over 93%
of the blue colonies that· develop in this test
using M-FC medium at the elevated tempera-
1. Definition of the Fecal Coliform Group ture of 44.5 C ± 0.2 C are reported to be fecal
coliforms (1). The test is applicable to the ex-
1.1 The fecal coliforms are part of the total amination of lakes and reservoirs, wells and
coliform group. They are defined as gram- springs, public water supplies, natural bathihg
negative nonspore-forming rods that ferment waters, secondary non-chlorinated effluents
lactose In 24 ± 2 hours at 44.5 ± 0.2 C with from sewage treatment plants, farm ponds,
the production of gas in a multiple-tube proce- stormwater runoff, raw municipal sewage, and
dure or produce acidity with blue co.lonies in a feedlot runoff. The MF test has been used with
membrane filter procedure. varied success in marine waters.

1.2 The major species in the fecal coliform 2.2.2 Limitations: Recent data (2, 3)
group is Escherichia coli, a species indicative indicate that the single-step MF fecal coliform
of fecal pollution and the possible presence of procedure may produce lower results than
enteric pathogens. those obtained with the fecal coliform

124 oEPA MICROBIOLOGICAL MANUAL 1978

multiple-tube procedure, particularly for 2.3.9 Safety trap flask between the filter
chlorinated effluents. Since chlorination flask and the vacuum source.
stresses fecal coliforms and significantly
reduces recovery, this method should not be 2.3. 10 Forceps with smooth tips.
used with chlorinated wastewater.
Disinfection and toxic materials such as 2.3:11 Ethanol, 95% or methanol, in small
metals, phenols~ acids or caustics also affect vial, for sterilizing forceps.
recovery of fecal coliforms on the membrane
filter. Any decision to use this test for stressed 2.3. 12 Bunsen/Fisher burner or electric
microorganisms requires parallel MF/MPN incinerator.
evaluation based on the procedure described
in Part IV-C, 3. 2.3. 13 Sterile TD bacteriological or Mohr
pi pets, glass or plastic, of appropriate size.
Recently-proposed solutions to problems
of lower recovery (2, 4, 5, 6) include the use of 2..3. 14 Sterile petri dishes, 50 x 12 mm
two-step incubation, two-step incubation over- plastic with tight-fitting lids.
lay and/or enrichment techniques and modifi-
cation of membrane filter structures. 2.3. 1 5 Dilution bottles (milk dilution), py-
rex glass, marked at 99 ml volume, screw-cap
2.3 Apparatus and Materials with neoprene rubber liner.

2.3. 1 Water bath, aluminum heat sink, or 2.3.16 Membrane filters, white, grid
other incubator that maintains a stable 44.5 + marked, 47 mm diameter, 0.45+0.02 µm pore
0.2 C. Temperature is checked against an NBS size or other pore size recommended by manu-
certified thermometer or one of equivalent facturer for water analyses. The Millipore
accuracy. HC MF, not the HA, is recommended.

2.3.2 Binocular (dissecting type) micro- 2.3.17 Absorb.ent pads.

scope, with magnification of 10-15 x and
daylight-type fluorescent lamp. 2.3.18 Water-proof plastic bags.

2.3.3 Hand tally. 2.3.19 Inoculation loops, 3 mm diameter,

or needle of nichrome or platinum wire, 26
2.3.4 Pipet containers of stainless steel, B&S gauge, in suitable holder.
aluminum or pyrex glass for glass pi pets.
2.3.20 Disposable applicator sticks or
2.3.5 Graduated cylinders, covered with plastic loops as alternatives to inoculation
qluminum foil or kraft paper before loops.
sterilization.
2.3.21 Ultraviolet sterilizer for MF filtra-
2.3.6 Sterile, unassembled membrane fil- tion units (optional).
tration units (filter base and funnel), glass, plas-
tic or stainless steel, wrapped with aluminum 2.4 Media
foil and kraft paper.
2.4.1 M-FC broth or agar prepared in pre-
2.3. 7 Vacuum source. sterilized erlenmeyer flasks (See Part 11-B, 5.2.1 ).

2.3.8 Vacuum filter flask, with appropriate 2.4.2 Lauryl tryptose broth prepared in 10
tubing. Filter manifolds which hold a number ml volumes in fermentation tubes (see Part 11-B,
of filter bases can also be used. 5.3.1) for verification.

FECAL COL/FORMS 125

 2.4.3 EC medium prepared in 10 ml vol- 2.6.5 Do not filter less than 1.0 ml of
umes in fermentation tubes (see Part 11-8, undiluted sample.
5.3.4) for verification.
2.6.6 Filter the sample and rinse the sides
2.5 Dilution Water (See Part 11,8, 7 for of the funnel walls at least twice with 20-30 ml
preparation). of sterile dilution water.

2.5.1 Sterile buffered dilution water or 2.6. 7 Turn off the vacuum and remove the
peptone water dispensed in 99+2 ml amounts · funnel from the filter base.
in screw-capped dilution bottles.
2.6.8 Aseptically remove the membrane
2.5.2 Sterile buffered water or peptone filter from the filter base. Place the filter, grid
water prepared in 500 ml or larger volumes for side up, on the absorbent pad saturated with
wetting membranes before addition of the M-FC Broth or on M-FC agar, using a rolling
sample, and for rinsing the funnel after sample motion to prevent air bubbles.
filtration.
2.6.9 Incubate the petri dishes for 24 + 2
hours at 44.5 ± 0.2 C in sealed waterproof
2.6 Procedure: The general membrane fil- plastic bags submerged (with the petri dishes
ter procedure is described in detail in Part 11-C. inverted) in a waterbath, or without plastic bag
in a heat-sink incubator. MF cultures should be
placed in incubator within 30 minutes of
2.6. 1 Prepare the M-FC broth or agar me- filtration.
dium as outlined in Part 11-8, 5.2. 1. Saturate the
sterile absorbent pads with about 2.0 ml of 2.6.10 After 24 hours remove dishes from
broth or add 5-6 ml of M-FC agar to the bottom the incubator and examine for blue colonies.
of each 50 x 12 mm petri dish (to a depth of
2-3 mm). Pour off excess liquid from broth- 2. 7 Counting and Recording Colonies:
saturated pads. Mark dishes and bench forms Select those plates with 20-60 blue (some-
with sample identity and sample volumes. times greenish-blue) colonies. Non-fecal
colonies are gray, buff or colorless and are
2.6.2 Using a sterile forceps place a sterile not counted. Pinpoint blue colonies should be
membrane filter on the filter base, grid side up. counted and confirmed. The colonies are
counted using a microscope of 10-15x and
Attach the funnel to the base of the· filter unit; a fluorescent lamp. Use of hand lens or other
• the membrane filter is now held between the simple optical devices of lower magnification
funnel and base. make difficult the identification and differentia-
tion of typical and atypical blue colonies.
2.6.3 Shake the samp!e vigorously about
2.7.1 The general counting rules are given
25 times and measure the sample into the
in Part 11-C, 3.5. The following rules are used
funnel with the vacuum off. If sample volume is
in calculating the fecal coliform count per
< 10 ml, add 10 ml of sterile dilution water to
100 ml of sample:
the filter before adding the sample.

2.6.4 Sample volumes for fecal coliform

enumeration in different waters and wastewa- (a) Countable Membranes with 20-60
ters are suggested in Table 111-C-1. These vol- Blue Colonies. Count all blue colonies using
umes should provide the recommended count the formula:
of 20-60 colonies on a membrane filter. Fecal No. of Fecal Coliform Colonies Counted
coliform levels are generally lower than total Volume in ml of Sample Filtered
coliform densities in the ~ame sample; there- fecal coliform
fore larger volumes are sampled.
x 100
count/100 ml

126 oEPA MICROBIOLOGICAL MANUAL 1978

 TABLE 111-C-1

Suggested Range of Sample Volumes for Fecal Coliform Tests

Using the Membrane Filter Method

Sample Source 100 30 10 3 1 0.3 0.1 0.03 0.01 0.003 0.001 0.0003 0.0001

;;:
...~ Swimming Pools x
0 Wells, Springs x x
...0
-
~
::0
Lakes, Reservoirs x x x
~
(I) Water Supply Intakes x x x x x
Bathing Beaches x x x x x
River Water x x x x x
Chlorinated Sewage Effluent x x x x x
Raw Sewage x x x x· x

.....
r-.>"
......
 For example, if 40 colonies are counted 2.8.2 Laboratory personnel should be able
after the filtration of 50 ml of sample, the to duplicate their own colony counts on the
calculation is: same plate within 5%, and the counts of other
analysts on the same plate within 10%.
40
50 X 100 = 80 fecal coliforms/100 ml.

3. Delayed-Incubation Membrane Filter (MF)

(b) Countable Membranes With Less Than Method
20 Blue Colonies. Report as: EStimate'd
COuntTiOO ml and specify the reason. 3.1 Summary: Bacteria are retained on
0.45 µm filters after passage of selected sam-
ple volumes through the filters. The filters are
{c) Membranes With No Colonies. Report placed on M-VFC broth (a minimum growth
the count as: Less than (calculated value)/100 medium) and transported from field sites to the
ml, based upon the largest single volume laboratory. In the laboratory, the filters are
filtered. transferred to the M-FC medium and incubated
at 44.5 C for 24 hours. Blue colonies are coun-
For example, if 10, 3 and 1 ml are filtered ted as fecal coliforms.
and all plates show zero counts, select the
largest volume, apply the general formula and 3.2 Scope and Application
report the count as a <(less than) value:
3.2. 1 Advantages: The delayed incubation

w 1
x 100 = 10
MF method is useful in survey monitoring or
emergency situations when the standa.rd fecal
coliform test cannot be performed at the sam-
or < 10 fecal coliforms/100 ml. ple site, or when time and temperature limits
for sample storage cannot be met. The method
(d) Countable Membr~nes With More Than eliminates field processing and equipment
60 Blue Colonies. Calculate count from high- needs. Also, examination at a central labora-
est dilution and report as a > value. tory permits confirmation and biochemical
identification of the organisms as necessary.
(e) Uncountable Membranes With More Consistent results have been obtained with
Than 60 Colonies. Use 60 colonies as the this method using water samples from a vari-
basis of calculation with the smallest filtration ety of sources (7).
volume, e.g., 0.01 ml:
3.2.2 Limitations: The applicability of this
_§Q_ method for a specific water source must be
0.01
x 100 600,000
determined in preliminary studies by compari-
son with the standard MF method. For exam-
Report as: > 600,000 fecal ple, limited testing has indicated that the
coliforms/100 ml. delayed-incubation method is not as effective
in saline waters (7).
2.7.2 Reporting Results~ Report fecal coli-
form densities per 100 ml. See discussion on 3.3 Apparatus and Materials
significant figures in Part 11-C, 2.8.1.
3.3. 1 Water bath, aluminum heat sink, or
2.8 Precision and Accuracy equivalent incubator that maintains a 44.5 ±
0.2 C temperature.
2.8.1 Ninety-three percent of the blue col-
onies that develop on M-FC medium at the 3.3.2 Binocular (dissection) microscope,
elevated temperature of 44.5 ±
0.2 C were with magnification 10 or 15 x, - binocular,
verified as fecal coliform ( 1). wide-field type. A microscope lamp producing

128 &EPA MICROBIOLOGICAL MANUAL 1978

diffuse daylight from cool white fluorescent 3.3. 18 Ultraviolet sterilizer for MF filtra-
lamps. tion units (optional).

3.3.3 Hand tally. 3.4 Media: The following media are pre-
pared in pre-sterilized erlenmeyer flasks with
3.3.4 Pipet containers of stainless steel, metal caps, aluminum foil covers, or screw-
aluminum or pyrex glass for glass pi pets. caps:

3.3.5 Graduated cylinders, covered with 3.4. 1 M-VFC holding media (see Part 11-B,
aluminum foil or kraft paper before 5.2.6).
sterilization.
3.4.2 M-FC broth or agar (see Part 11-B,
3.3.6 Sterile unassembled membrane fil- 5.2.1).
tration units (filter base and funnel), glass, plas-
tic or stainless steel wrapped with aluminum 3.5 Dilution Water
foil or kraft paper.
3.5. 1 Sterile dilution water dispensed in
3.3. 7 Vacuum source. 99 + 2 ml volumes in screw-capped bottles.
3.3.8 Filter flask to hold filter base, with 3.5.2 Sterile dilution water prepared in
appropriate tubing. Filter manifold to hold a large volumes for wetting membranes before
number of filter bases can also be used. In the the addition of the sample, and for rinsing the
field, portable field kits are also used. funnel after sample filtration.

3.3.9 Safety trap flask between the filter- 3.6 Procedure: The general membrane fil-
ing flask and the vacuum source. ter procedure is described in detail in Part 11-C.

3.3. 10 Forceps with smooth tip. 3.6.1 Prepare the M-VFC holding medium
as outlined in Part 11-B, 5.2.6. Saturate the
3.3.11 Ethanol, 95% or methanol, in small sterile absorbent pads with about 2.0 ml of M-
vial, for sterilizing forceps. VFC broth. Pour off excess broth. Mark dishes
and bench forms with sample identity and
3.3. 1 2 Bunsen/Fisher type burner. volumes.

3.3.13 Sterile TD bacteriological or Mohr 3.6.2 Using sterile forceps place a mem-
pi pets, glass or plastic, in appropriate volumes. brane filter on the filter base grid side up.

3.3. 1 4 Sterile petri dishes, 50 X 12 mm 3.6.3 Attach the funnel to the base of the
plastic with tight-fitting lids. filter unit; the membrane filter is now held
between the funnel and base.
3.3. 15 Dilution bottles (milk dilution), py-
rex glass, 99 ml volume, screw-caps with neo- 3.6.4 Shake the sample vigorously about
prene rubber liners. 25 times and measure into the funnel with the
vacuum off. If the sample is < 10 ml, add 10
3.3.16 Membrane filters, white, grid mark- ml of sterile dilution water to the membrane
ed, 47 mm in diameter,0.45±0.02µm pore size, filter before adding the sample.
or other pore size recommended by the manu-
facturer for water analyses. The Millipore HC (a) Sample volumes for fecal coliform enu-
MF, not t,he HA is recommended. meration in different waters and wastewaters
are suggested in Table 111-C-1. These volumes
3.3.17 Shipping tubes, labels, and packing should produce membrane filters with a re-
materials for mailing delayed incubation plates. commended count of 20-60 colonies.

FECAL COL/FORMS 129

 (b) Follow the methods for sample mea- 4. Verification
surement and dispensation given in Part 11-C,
3.4.6. Verification of the membrane filter test for
fecal coliforms establishes the validity of col-
3.6.5 Filter the sample through the mem- ony differentiation by blue color and provides
brane and rinse the sides of the funnel walls at supporting evidence of colony interpretation.
least twice with 20-30 ml of sterile dilution The verification procedure corresponds to the
water. fecal coliform MPN (EC Medium) test.

3.6.6 Turn off the vacuum and remove the 4.1 Pick from the centers of at least 10
funnel from the base of the filter unit. well-isolated blue colonies. Inoculate into lauryl
tryptose broth and incubate 24-48 hours at
3.6.7 Aseptically remove the membrane 35+0.5 c.
filter from the filter base and place grid side up
on an absorbent pad saturated with VFC 4.2 Confirm gas-positive lauryl tryptose
medium. broth tubes at 24 and 48 hours by inoculating
a loopful of growth into EC tubes and incubat-
3.6.8 Place the culture dish in shipping ing for 24 hours at 44.5 ± 0.2 C. Cultures that
container and send to the examining labora- produce gas in EC tubes are interpreted as
tory. Fecal coliform bacteria can be held on the verified fecal coliform colonies (see Figure 111-
VFC holding medium for up to 72 hours with C-1 ).
little effect on the final counts. The holding
period should be kept to a minimum. 4.3 A percent verification can be deter-
mined for any colony-validation test:
3.6.9 At the examining laboratory remove
No. of colonies meeting verification test
the membrane from the holding medium,
place it in another dish containing M-FC broth No. of colonies subjected to verification
or agar medium, and complete testing for fecal
coliforms as described above under 2.6.
x 100 Percent verification

3.7 Counting and Recording Colonies: Example: Twenty blue colonies on M-FC
After the required incubation select those medium were subjected to verification studies
plates with 20-60 blue (sometimes greenish- shown in Figure 111-C-1. Eighteen of these colo-
blue) colonies. Gray to cream colored colonies nies proved to be fecal coliforms according to
are not counted. Pin-point blue colonies are provisions of the test:
not counted unless confirmed. The colonies
are enumerated using a binocular microscope 18
with a magnification of 10 or 1 5x.
Percent verification = _ X 100 = 90%
20

Refer to 2.7.1, for rules used in reporting 4.4 A percent verification figure can be
the fecal coliform MF counts. applied to the direct test results to determine
the verified fecal coliform count per 100 ml:

3.8 Reporting Results: Record densities Percent verification count per

as fecal coliforms per 100 ml. Refer to Part 11-C, 100 x 100 ml
2.8, for discussions on the use of significant
figures and rounding off values. Verified fecal
coliform count

3.9 Precision and Accuracy: As reported Example: For a given sample, by the M-FC
In 2.8, this Section. test, the fecal coliform count was found to be

130 oEPA MICROBIOLOGICAL MANUAL 1978

 Pick 10 blue colonies

Laury! Tryptose Broth

24 hours at 35 C

Gas+ Gas-

Reincubate
24 hours at 35 C
I

Gas+ Gas-

Negative
EC Broth Test
24 hours at 44.5 C
I

I
Gas+ Gas-
I
1
Verified Negative
Fecal Coliform Test
Colony

FIGURE 111-C-1. Verification of Fecal Coliform Colonies on the Membrane Filter

FECAL COL/FORMS 131

42,000 organisms per 100 ml. Supplemental 5.2.8 Pyrex test tubes, 150 X 20 mm,
studies on selected colonies showed 92% containing inverted fermentation vials, 75 x
verification. 10 mm, with caps.

Verified fecal 92 5.2.9 Culture tube racks to hold fifty, 25

coliform count = =-
1 oo x 42,000 38,640 mm diameter tubes.

5.3Media
Rounding off = 39,000 fecal coliforms
per100ml 5.3. 1 Lauryl tryptose broth (same as total
coliform Presumptive Test medium) prepared
The worker is cautioned not to apply per- in 10 ml volumes in appropriate concentration
centage of verification determined on one for sample volumes used. (Part 11-B, 5.3. 1).
sample to other samples.
5.3.2 EC medium prepared in 10 ml vol-
umes in fermentation tubes (Part 11-B, 5.3.4).
5. Most Probable Number (MPN) Method
5.4 Dilution Water: Sterile buffered or
peptone dilution water dispensed in 99 ±
2 ml
5.1 Summary: Culture from positive tubes volumes in screw-capped bottles.
of the lauryl tryptose broth (same as presump-
tive MPN Method, Part 111-B) is inoculated into 5.5 Procedure: Part 11-C describes in detail
EC Broth and incubated at 44.5 C for 24 hours the general MPN procedure. See Figure 111-C-2.
(see Figure 111-C-2). Formation of gas in any
quantity In the inverted vial is a positive reac- 5.5. 1 Prepare the total coliform Presump-
tion confirming fecal coliforms. Fecal coliform tive Test medium, (lauryl tryptose broth) and
densities are calculated from the MPN table on EC medium. Clearly mark each bank of tubes,
the basis of the positive EC tubes (8). identifying the sample and the volume
inoculated.
5.2 Apparatus and Materials
5.5.2 Inoculate the Presumptive Test me-
6.2.1 Incubator th at maintains 35 ± 0.5 C. dium with appropriate quantities of sample
following the Presumptive Test total coliform
6.2.2 Water bath or equivalent incubator procedure, (Part 111-B).
that maintains a 44.5 ±
0.2 Ctemperature.
5.5.3 Gently shake the Presumptive tube.
6.2.3 Pipet containers of stainless steel, Using a sterile inoculating loop or a sterile
aluminum or pyrex glass for glass pipets. wooden applicator, transfer inocula from posi-
tive Presumptive Test tubes at 24 and 48
5.2.4 Inoculation loop, 3 mm diameter hours to EC confirmatory tubes. Gently shake
and needle of nichrome or platinum wire, 26 B the rack of inoculated EC tubes to insure mix-
& S gauge, in suitable holder. Sterile applica- ing of inoculum with medium.
tor sticks are a suitable alternative.
5.5.4 Incubate inoculated EC tubes at
6.2.6 Sterile pipets T.D., Mohr or bacterio- 44.5 ± 0.2 C for 24 ± 2 hours. Tubes must be
logical, glass or plastic, of appropriate size. placed in the incubator within 30 minutes after
inoculation. The water depth in the water bath
5.2.6 Dilution bottles (milk dilution), pyrex, incubator must come to the top. level of the
99 ml volume, screw-cap with neoprene liners. culture medium in the tubes.

6.2. 7 Bunsen or Fisher-type burner or elec- 5.5.5 The presence of gas in any quantity
tric Incinerator unit. in the EC confirmatory fermentation tubes af-

132 oEPA MICROBIOLOGICAL MANUAL 1978

 Sample

Laury! Tryptose Broth

35 ±0.5 c

I-
C/)
w Gas+ Gas-
1- 24 hr 24 hr
w
>
~
~
:::>
Cl) Reincubate
w
a: 24 hr
0...
I

Gas+ Gas-
Negative
Test

Elevated Temperature Test

EC Medium at 44.5 ± 0.2 C

I-
C/)
w Gas+ Gas-
I-
C 24 hr 24 hr
w
~
a:
u:
z
0 Fecal Coliforms Present Negative
u
Test

L Calculate
Fecal Coliform
MPN

FIGURE 111-C-2. Flow Chart for the Fecal Coliform MPN Tests.

FECAL COL/FORMS 133

ter 24 ± 2 hours constitutes a positive test for from five of the 10 ml portions, three of the 1.0
fecal coliforms. ml portions, and none of the 0.1 ml portions,
the coded result of the test is 5-3-0. The code
5.6 Calculations is located in the MPN Table 11-C-4, and the MPN
per 100 ml is recorded. See Part 11-C, 4.9 for
5.6.1 Calculate fecal coliform densities on rules on selection of significant dilutions.
the basis of the number of positive EC fermen-
tation tubes, using the table of most probable
5.7 Reporting Results: Report the fecal
numbers (MPN).
coliform MPN values per 100 ml of sample.
5.6.2 The MPN results are computed from
three dilutions that include the highest dilution 5.8 Precision and Accuracy: The preci-
with all positive tubes and the next two higher sions of the MPN counts are given as confi-
dilutions. For example, if five 10 ml, five 1.0 ml, dence limits in the MPN tables. Note that the
and five 0.1 ml sample portions are inoculated precision of the MPN value increases with
initially into Presumptive Test medium, and increased numbers of replicates per sample
positive EC confirmatory results are obtained tested.

REFERENCES

1. Geldreich, E. E., H.F. Clark, C. B. Huff, and L. C. Best, 1965. Fecal-coliform-organism medium for
the membrane filter technique. J. Amer. Water Works Assoc. 57.:208.

2. Bordner, R. H., C. F. Frith and J. A. Winter, (Editors), 1977. Proceedings of the Symposium on the
Recovery of Indicator Organisms Employing Membrane Filters. U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH. (EPA-
600/9-77-024).

3. Lin, S. 0., 1973. Evaluation of coliform tests for chlorinated secondary effluents. J. Water
Pollution Control Federation 45:498. · - --

4. Lin, S. D., 1976. Evaluation of Millipore HA and HC membrane filters for the enumeration of
indicator bacteria. Appl. Environ. Microbiol. 32:300.

5. Lin, S. D., 1976. Membrane filter method for recovery of fecal coliforms in chlorinated sewage
effluents. Appl. Environ. Microbiol. 32:547.

6. Green, B. L., E. M. Clausen, and W. Litsky, 1975. Two-temperature membrane filter method
for enumeration of fecal coliform bacteria from chlorinated effluents. Appl. Environ.
Microbiol. 33: 1259. --

7. Taylor, R.H., R.H. Bordner, and P. V. Scarpino, 1973. Delayed-incubation membrane-filter test for
for fecal coliforms. Appl. Microbiol. 25:363:

8. Geldreich, E. E., 1966. Sanitary Significance of Fecal Coliforms in the Environment, U.S. Dept. of
the Interior, FWPCA, WP-20-3, 1 22 pp. -- - -

134 .S,EPA ·MICROBIOLOGICAL MANUAL 1978

 PART Ill. ANALYTICAL METHODOLOGY

Section D Fecal Streptococci

The membrane filter (MF), most probable 1.2 Fecal Streptococcal Intermediates
number (MPN), and direct pour plate proce- and Biotypes: Current information indicates
dures can be used to enumerate and identify that other streptococci belonging to Lance-
fecal streptococci in water and wastewater. field's serological Group Q occur in the feces
For a general description of these fundamental of humans and other warm-blooded animals,
techniques refer to Part 11-C. The method se- especially chickens. S. avium is characteristi-
lected depends upon the characteristics of the cally found in the feces of chickens and occa-
sample. The Section is divided as follows: sionally in the feces of man, dogs and pigs. The
Group Q antigen is found in the cell wall of
1. The Fecal Streptococcus Group these organisms, and in addition, the Group D
antigen is located between the cell wall and
2. Membrane Filter Method the cytoplasmic membrane where it occurs
naturally in the established Group D species.
3. Verification These common antigens indicate a relation-
ship between Group D and Group Q orga-
4. Most Probable Number Method nisms. The Group Q Streptococcci may ac-
count for the occurrence of the "intermediate
5. Pour Plate Method strains or biotypes" of Group D Streptococci
(1). Group Q organisms grow on media com-·
6. Determination of FC/FS, Ratios manly used for the isolation and enumeration
of enterococci. Kenner et al. (2) observed that
7. Identification to Species enterococcus biotypes occur more commonly
in the feces of fowl than of pigs, sheep, cows
and humans. Forty percent of the streptococci
1. The Fecal Streptococcus Group isolated from the feces of fowl by Kenner et al.
were enterococcus biotypes with the restbe-
1.1 Fecal Streptococci and Lancefield's ing the enterococcus group. It is probable that
Group D Streptococcus: The terms "Fecal some of the biotypes they described were
Streptococcus" and "Lancefield's Group D Group Q Streptococci. The Group Q Strepto-
Streptococcus" have been use synonymously. cocci, with a type species, should be consid-
When used as indicators of fecal contamina- ered in the Fecal Streptococcus Group.
tion the following species and varieties are
implied: S. faecalis, S. faecalis subsp. liquefa-
ciens, S. faecalis subsp. zymogenes, S. fae- 1.3 Definition of the Fecal Streptococ-
cium, S. bovis and S. equinus. For sanitary cus Group: The term, fecal streptococci, will
analyses, media and methodology for quantifi- be used to describe the streptococci which
cation are selective for these organisms. indicate the sanitary quality of water and

FECAL STREPTOCOCCI 135

wastewater. The fecal streptococci group in- animals. High numbers of these organisms are
cludes the serological groups D and 0. associated with pollution from meat process-
ing plants, dairy wastes, and run-off from feed-
Fecal Streptococci
lots and farmlands. Because of limited survival
time outside the animal intestinal tract their
S. faecalis
(/)
::s presence indicates very recent contamination
0 from farm animals.
0 0. S. faecalissubsp. /iquefaciens
8 ::s
o e
...c:
Qi (!) S. faecalissubsp. zymogenes
2. Membrane Filter (MF) Method
w
S.faecium
2.1 Summary: A suitable volume of sam-
S. bovis ple is passed through the 0.45 ]:lm membrane
filter which retains the bacteria. The filter is
S. equinus placed on KF Streptococcus agar and incu-
c. bated at 35 C for 48 hours. Red and pink
::s
S.avium ea
(!)
colonies are counted as streptococci ( 1, 2).

1.4 Viridans Streptococci: The viridans 2.2 Scope and Application: The mem-
streptococci, primarily S. salivarius and S. mi- brane filter technique is recommended as the
tis are not considered as part of the fecal standard method for assaying fecal strepto-
streptococci as defined in 1.~ and 1.3. These cocci in fresh and marine waters and in non-
inhabitants of the nasopharyngeal tract have chlorinated sewage. Wastewaters from food
been reported by a few workers in feces and processing plants, slaughter houses, canner-
do grow on some fecal streptococci media. ies, sugar processing plants, dairy plants, feed-
However, their low numbers when present, the lot and farmland run-off may be analyzed by
low frequency of occurrence and the limited this procedure. Colonies on a membrane filter
data available at this time concerning their can be transferred to biochemical media for
presence, have resulted in their exclusion from identification and speciation to provide infor-
the classification of fecal streptococci. mation on the source of contamination. The
general advantages and limitations of the MF
1.5 Scope and-Application: Fecal strepto- method 3re given in Part 11-C.
cocci data verify fecal pollution and may pro-
vide additional information concerning the re-
cency and probable origin of pollution. In com- 2.3 Apparatus and Materials
bination with data on coliform bacteria, fecal
streptococci are used in sanitary evaluation as 2.3.1 Water bath, aluminum heat sink or
a supplement to fecal coliforms when a more air incubator set at 35 ±0.5 C. Temperature
precise determination of sources of contami- checked with an NBS thermometer or one of
nation is necessary. The occurrence of fecal equivalent accuracy.
streptococci in water indicates fecal contami-
nation by warm-blooded animals. They are not 2.3.2 Stereoscopic (dissection) micro-
known to multiply in the environment. Further scope, with magnification of 10 to 15 x, pref-
identification of streptococcal types present in erably wide field type. A microscope lamp with
the sample may be obtained by biochemical diffuse light from cool, white fluorescent tubes.
characterization. (See Figure 111-D-2 "Isolation is recommended.
and Identification of Fecal Streptococci").
Such information is useful for source investi- 2.3.3 Hand tally.
gations. For example, S. bovis and S. equinus
are host specific and are associated with the 2.3.4 Pipet containers of stainless steel,
fecal excrement of non-human warm-blooded aluminum or pyrex glass for glass pi pets.

136 oEPA MICROBIOLOGICAL MANUAL 1978

 2.3.5 Sterile graduated cylinders, covered 2.5.2 Sterile buffered water or peptone
with aluminum foil or kraft paper. water prepared as described in Part 11-B, 7, in
large volumes for wetting membranes before
2.3.6 Sterile, unassembled membrane fil- the addition of the sample, and for rinsing the
tration units (filter base and funnel), glass, plas- funnel after filtration.
tic or stainless steel, wrapped with aluminum
foil or kraft paper.
2.6 Immediate MF Procedure: The gen-
eral membrane filter procedure is described in
2.3.7 Vacuum source.
detail in Part 11-C, 3.
2.3.8 Filter suction flask to hold filter base,
with appropriate tubing. Filter manifolds that 2.6. 1 Clearly mark each petri dish and
hold a number of filter bases can also be used. aseptically add 5-6 ml of the liquified agar
medium (to each dish to a depth of 2-3 mm).
2.3.9 Safety vacuum flask.
2.6.2 Place a sterile membrane filter on
2.3. 10 Forceps, with smooth tips. the filter base, grid-side up and attach the
funnel to the base of the filter unit; the mem-
2.3.11 Ethanol, 95%, or methanol, in brane filter is now held securely between the
small vial for sterilizing forceps. funnel and base.

, 2.3. 12 Bunsen/Fisher burner or electric 2.6.3 Shake the sample vigorously about
incinerator. 25 times and measure the sample into the
funnel with the vacuum off. ·
2.3, 13 Sterile T.D. bacteriological or Mohr
pi pets, glass or plastic, of appropriate size. 2.6.4 Filter appropriate volumes of water
sample through the sterile membrane to ob-
2.3. 14 Sterile petri dishes, 50 x 12 mm tain 20-100 colonies on the membrane
plastic or 60 X 15 mm glass or plastic. surface.

2.3.15 Dilution bottles (milk dilution), py- At least 3 sample increments should be
rex glass, marked at 99 ml volumes, screw-cap filtered in order of increasing volumes. Where
with neoprene rubber liner. no background information is available, more
may be necessary. The methods of measure-
2.3. 16 Membrane filters, manufactured ment and dispensation of the sample into the
from cellulose ester materials, white, grid funnel are given in Part 11-C, 3.4.6.
marked, 4 7 mm in diameter, 0.45 + 0.02 µm
pore size or other pore size as recommended 2.6.5 Filter the sample and rinse the sides
by the manufacturer for fecal streptococci of the funnel at least twice with 20-30 ml of
analyses. sterile buffered dilution water. Turn off the
vacuum and remove the funnel from the filter
2.3.17 Ultraviolet sterilizer for MF filtra- base.
tion units (optional).
2.6.6 Aseptically remove the membrane
2.4 Media: KF Streptococcus agar pre- from the filter base and place grid-side up on
pared as described in Part 11-B, 5.4.1. the agar.

2.5 Dilution Water 2.6.7 Incubate the petri dishes in the in-
verted position at 35 + 0.5 C for 48 hours.
2.5. 1 Sterile buffered dilution water or
peptone water dispensed in 99 + 2 ml vol- 2.6.8 After incubation, remove dishes and
umes in screw-capped dilution bottles. examine for red to pink colonies and count.

FECAL STREPTOCOCCI 137

 2.7 Counting and Recording Colonies: to filter water samples at a field site, place
Select those plates with 20-100 pink to dark membranes on the KF agar medium in tight-
red colonies. These may range in size from lidded petri dishes and hold these plates for up
barely visible to about 2 mm in diameter. Colo- to 3 days. After the holding period, plates are
nies of other colors are not counted. Count the incubated for 48 hours at 35 C and counted in
colonies as described in Part 11-C, 3.5, using the normal manner. This 72 hour holding time
low-power (10-15 X) microscope equipped can be used to air mail the membranes on KF
with overhead illumination. agar to a central laboratory for incubation and
counting. (National Pollution Surveillance Sys-
Fecal streptococcal density is reported as or- tem FWPCA, data collected from geographical
ganisms per 100 ml. Use the general formula locations around the Nation; and Kenner et al.,
to calculate fecal streptococci densities: Kansas City data.) --

Fecal Streptococci/100 ml =
3. Verification
No. of Fecal Streptococcus Colonies Counted
Volume of Sample Filtered, ml Periodically, typical colonies growing on
the membrane filter should be verified. When
x 100 a survey is initiated or a new body of water is
being sampled, it is recommended that at
For example, if 40 colonies are counted least 10 typical colonies from the membrane
after the filtration of 50 ml of sample the or agar plate used in computing the final
calculation is: density be picked and transferred into BHI

Fecal Streptococci/100 ml = :g x 100 = 80. ·

broth or onto BHI agar slants. After 24-48
hours incubation, subject the . cultures to a
catalase t~st: Catalase activity indicates the
See Part 11-C, 3.6 for calculation for results. nonstreptococci. Atypical colonies should also
be verified to determine false negative reac-
Reporting Results: Report fecal strepto- tions on the membrane filter. Final confirma-
coccal densities per 100 ml of sample. See tion of fecal streptococci is achieved by
discussion on significant figures in Part 11-C determining growth of catalase negative
2.8. isolates in BHI broth at 45 C and in 40% bile
within two days (see Figure 111-D-1 ).
2.8 Precision and Accuracy

2.8. 1 Extensive precision and accuracy 3.1 Apparatus and Materials

data are not available, however, KF Strepto-
coccus agar has been reported to be highly
efficient in the recovery of fecal streptococci
3. 1.1 Incubators set at 35 ± 0.5 C and 45
(2, 3, 4). In the analyses of feces, sewage and +O.?C.
foods, KF yielded a high recovery of fecal
streptococci with a low percent (18.6) of non- 3.1.2 Inoculating needle and loop .
.fecal streptococci. ·

2.8.2 Laboratory personnel should be able 3.1.3 Bunsen/Fisher burner or electric

to duplicate their colony counts on the same incinerator.
plates within 5%, and the counts of others
within 10%. 3.1.4 Solution of 3% hydrogen peroxide.
2.9 Delayed MF Procedure: Because of
the stability of the KF agar and its extreme 3.1.5 Glass microscope slides, 2.5 X 7.6
selectivity for fecal streptococci, it is possible cm ( 1 X 3 inches).

138 oEPA MICROBIOLOGICAL MANUAL 1978

 3.1.6 Media (turbidity) in azide dextrose broth after incuba,.
tion at 35 C for 24-48 hours. To confirm the
(a) Brain heart infusion (BHI) agar. (See Part presence of fecal streptococci, a portion of the
11-B, 5.4.6). growth from each positive azide dextrose
broth tube is streaked onto PSE or equivalent
(b) Brain heart infusion (BHI) broth. (See esculin-azide agar and incubated at 35 C for
Part 11-B, 5.4.5). 24 hours (5). The presence of brownish-black
colonies with brown halos confirms fecal
3.2 Procedure streptococci. The MPN is computed on the
basis of the Confirmed Test results read from
3.2.1 Plates for verification should con- and MPN table.
tain 20-100 colonies. Pick at least 10 typica I
colonies from the selected membrane or agar 4.2 Scope and Application: This method
plate and inoculate into a BHI agar slant and can be used for detection of fecal streptococci
into a BHI broth tube. in water, sewage or feces, but is more time-
consuming, less convenient and less direct
3.2.2 After 24-48 hours incubation at 35 than the other procedures. The MPN must be
+0.5 C, transfer a loopful of growth from the used for samples which cannot be examined
BHI slant to a clean glass slide and add a few by the MF or direct plating techniques because
drops of freshly tested 3% hydrogen peroxide of turbidity, high numbers of background bac-
(H 2 0 2 ) to the smear. If the catalase enzyme is teria, metallic compounds, the presence of co-
present, it cleaves the H2 0 2 to water and visible agulants, the chlorination of sewage effluents
oxygen gas. Bubbles constitute a positive cata- or sample volume limitations of the plating
lase test and indicate non-streptococcal spe- technique.
cies. Confirmation need not be continued. Use
a platinum loop, not nichrome, to avoid false 4.3 Apparatus and Materials
positive reactions.
4.3. 1 Water bath or air incubator set at 35
3.2.3 If a negative catalase reaction occurs, + 0.5 C. Temperature checked with an NBS
transfer a loopful of growth from the BHI broth thermometer or one of equivalent accuracy.
to fresh BHI broth and BHI broth+ 40% bile and
incubate at 45 C and at 35 C. Growth within two 4.3.2 Pipet containers of stainless steel,
days indicates fecal streptococcal species (see aluminum or pyrex glass for pi pets.
Figure 111-D-1 and 111-D-2).
4.3.3 Culture tube racks to hold fifty 25
mm diameter tubes.
3.2.4 Further identification of streptococ-
cal types present in the sample may be ob-
4.3.4 Sterile T.D. bacteriological or Mohr
tained by biochemical characterization. (See
pi pets, of appropriate sizes.
Figure 111-D-2 to 111-D-4 for identification offecal
streptococci. Such information is useful for
4.3.5 Dilution bottles (milk dilution), pyrex
investigating sources of pollution. See Part 11-B glass, 99 ml volume, screw-capped, with neo-
for preparation of media used in the schematic prene rubber liner.
outlines).
4~3.6 Test tubes, pyrex, culture 150 x 25
4. Most Probable Number Method or 150 X 20 mm, with caps.

4.1 Summary: The multiple-tube proce- 4.3.7 Inoculating loop, 3 mm diameter, in

dure estimates the number of fecal strepto- holder or disposable applicator sticks or loops.
cocci by inoculating decimal dilutions of the
sample into broth tube media. Positive tubes in 4.3.8 Bunsen/Fisher burners or electric
the Presumptive Test are indicated by growth incinerator.

FECAL STREPTOCOCCI 139

 10 Colonies Typical in Appearance of
Fecal Streptococci on Isolation Media

Brain Heart Infusion Broth and Agar Slant

(24-48 Hours at35 C)

Catalase Negative

Growth at 45 C Growth in 40% Bile

Verification of Fecal Streptococci

FIGURE 111-0-1. Verification Procedure for Fecal Streptococci.

140 &EPA MICROBIOLOGICAL MANUAL 1978.

 Sample

PSE Agar KF Agar

(Brownish-black colony
with brown halo) (pink-red colony)

Catalase Negative

Growth at 45 c and 1o c Growth at 45 C only

Positive Starch Hydrolysis

Growth in 6.5 % NaCl Lactose

and pH 9.6 BHI Broth Fermentation

I
0.1 % Methylene Blue in Milk Acid only No change

~
Reduction No Reaction

I
Enterococcus
. I.
Tentative S. bovis S. equinus
Group Group Q (Livestock and
Poultry Sources)

See Figures 111-0-3

and 111-0-4

.FIGURE 111-0-2. Isolation and Identification of Fecal Streptococci, General Scheme

FECAL STREPTOCOCCI 141

4.4 Media 4.6.5 Shake the rack of inoculated culture
tubes to mix well and incubate them at 35 +
4.4.1 Azide dextrose broth prepared in 10 0.5 C for 48 hours + 3 hours. Examine tubes
ml volumes in test tubes without fermentation for turbidity after 24 ± 2 hours and 48 ± 3
vial (see Part 11-8, 5.4.2 for preparation). Ethyl hours.
violet azide broth is not used because of false
positive reactions. 4.6.6 Read and record the results from
each tube. A positive Presumptive Test shows
4.4.1 Azide dextrose broth prepared in 10 growth consisting of turbidity in the medium or
ml volumes in test tubes without fermentation a button of sediment at the bottom of the
vial {see Part 11-8, 5.4.2 for preparation). culture tube, or both.

4.4.2 Pfizer Selective Enterococcus (PSE) 4.6. 7 · For the Confirmed Test, streak
or equivalent esc1,.1lin-azide agar in pour plates growth from each positive azide dextrose
(see Part 11-B, 5.4.4 for preparation). · broth tube onto PSE Agar plates, making cer-
tain that the label on the plate corresponds to
4.5 Dilution Water: Sterile dilution water the positive azide dextrose tube used.
dispensed in 99 ml ± 2 ml amounts in screw-
capped bottles. 4.6.8 Incubate the PSE Agar plates at 35
+ 0.5 C for 24 hours.
4.6 Procedure: The general MPN proce-
dure is described in detail in Part 11-C, 4. 4.6.9 Read and record the results of each
plate corresponding to the positive azide dex-
4.6. 1 Prepare the media for the .Presump- trose tube. A positive Confirmed Test is evi-
tive Test. (azide dextrose broth) and the Con- denced by the presence of brownish-black col-
firmed Test, (PSE Agar plates). (see Part 11-8, onies with brown halos. The number of posi-
5.4.2 and 5.4.4 respectively). tive confirmed azide tubes in each dilution is
used to compute the density from an MPN
4.6.2 Mark culture tubes to identify sam- table.
ples and sample volumes.
4. 7 Calculations
4.6.3 Shake the sample vigorously about
25times. 4.7.1 Calculate fecal streptococci densi-
ties on the basis of the number of positive
4.6.4 Inoculate the azide dextrose broth Confirmed Tests from the PSE agar plates,
with appropriate sample volumes for the Pre- using the Table of Most Probable Numbers
sumptive Test. The number of fecal strepto- (MPN) in Table 11-C-1.
cocci In a water polluted with n:rnnicipal
wastes is generally lower than the number of 4.7.2 The MPN results are computed from
coliforms. Therefore, larger sample volumes 3 usable Confirmed Test dilutions. For exam-
must be used to inoculate the MPN tubes for ple, if positive Confirmatory Test results are
fecal streptococci than for coliforms. For ex- obtained from 5 of the 10 ml portions, three of
ample, if sample volumes of 1.0, 0. 1, 0.01, and the 1.0 ml portions, and none of the 0. 1 ml
0.001 ml are used for the coliform test, a portions, the coded results of the test is 5-3-0.
series of 10, 1.0, 0. 1, and 0.01 ml volumes are The code is located in the MPN Table 11-C-4,
Inoculated for the fecal streptococci test. Use and the MPN per 100 ml is recorded. See Part
single-strength broth, 10 ml tubes for inocula 11-C, 4 for details.
of 1.0 ml or less, and double-strength broth, 10
ml tubes for inocula of 10 ml. Sample volumes 4.8 Precision and Accuracy: The precision
from feedlots, meat packing plants, and storm- of the MPN value increases with increased
water run-off with more fecal streptococci numbers of replicates tested. Five tubes are
than coliforms must be adjusted accordingly. recommended for each dilution.

142 oEPA MICROBIOLOGICAL MANUAL 1978

5. Pour Plate Method 5.3.6 Sterile T.D. bacteriological or Mohr
pi pets of appropriate sizes.
5.1 Summary: Aliquots of the water sam~
pie or diluted sample are delivered to the bot- 5.3. 7 Sterile 100 mm X 15 mm petri
tom of a petri dish, and liquified Pfizer Selec- dishes, glass or plastic.
tive Enterococcus (PSE) agar, equivalent
esculin-azide agar or KF agar is added and 5.3.8 Dilution bottles (milk dilution), pyrex,
thoroughly mixed with the water sample. Fecal 99 ml volume, screw-capped, with neoprene
streptococci on PSE agar are 1 mm in diameter rubber liners.
and brownish-black with brown halos after
18-24 hours at 35 C. On KF agar fecal strepto- 5.3.9 Bunsen/Fisher burner or electric
cocci are red or pink after 48 hours at 35 C. incinerator.

5.3. 10 Hand tally.

5.2 Scope and Application: The pour
plate method is recommended as an alternate 5.4 Media: Sterile Pfizer Selective Entero-
procedure to the MF technique when chlori- coccus agar (PSE), equivalent esculin-azide
nated sewage effluent and water samples with agar, (Part 11-B, 5.4.4) or KF Streptococcus agar
high turbidity are encountered. PSE agar, the (Part 11-B, 5.4.1) are prepared in pre-sterilized
medium of choice, has several advantages: ( 1) erlenmeyer flasks or bottles with metal foil
it requires only 24 hours incubation compared covers, or screw-caps.
to 48 hours for other media, and (2) it exhibits
consistent recovery, regardless of. sources. 5.5 Dilution Water: Sterile dilution water
dispensed in 99 ± 2 ml amounts preferably in
With the pour plate technique, only small screw-capped dilution bottles (see Part 11-B, 7).
volumes of sample may be analyzed. This is a
disadvantage when the fecal streptococcal 5.6 Procedure
density is low and a large volume of sample
would be required for an accurate density de- 5.6.1 Shake the sample bottle vigorously
termination. Consequently, the M.F technique about 25 times to disperse the bacteria. Take
should be used unless the water is so turbid care that the closure is tight to prevent leakage
thatfiltration is impossible. of sample during shaking.

5.6.2. Dilute the sample to obtain final

5.3 Apparatus and Materials plate counts between 30-300 colonies. The
number of colonies within this range gives the
5.3.1 Air incubator set at 35 + 0.5 C. most accurate estimation of the microbial pop-
Temperature is checked against a ·National ulation. Because the magnitude of the micro-
Bureau of Standards thermometer or one of bial population in the original water sample is
equivalent accuracy. not known beforehand, a range of dilutions
must be prepared and plated to obtain a plate
5.3.2 Water bath for tempering agars set within this range of colony counts.
at 44-46 C.
5.6.3 Transfer 0. 1 and 1.0 ml from the
5.3.3 Colony Counter, Quebec. darkfield undiluted sample to each of 2 separate petri
model or equivalent. dishes.

5.3.4 Pipet containers of stainless steel, 5.6.4 Prepare the initial 1: 100 or 1o-2
aluminum or pyrex glass for glass pi pets. 9ilution by pipetting 1 ml of the sample into a
99 ml dilution water blank using a sterile 1. 1
5.3.5 Petri dish containers for glass or ml pipet (see Part 11-C, 1.4 "Preparation of
plastic petri dishes. Dilutions," and Figure 11-C-1 ).

FECAL STREPTOCOCCI 143

 5.6.5 Vigorously shake the 1: 100 dilution (a) Plates with 30-300 Fecal Streptococ-
bottle to obtain uniform distribution of cal ColOrlieS:Count all colonies for each plate
bacteria. within the 30-300 range. Calculate the aver-
age count for these plates correcting for the
5.6.6 Pi pet 0. 1 and 1.0 ml of this 1: 100 dilution as follows:
dilution into each of 2 separate petri dishes
using another sterile pi pet. Sum of Colonies
- - - - - - - - - - - x 100 = FS/100 ml
Sum of Volumes tested, ml
5.6.7 Make additional dilutions as re-
quired for raw wastes or stormwater run-off
and prepare pour plates containing the dilu- (b) All Plates Greater than 300 Colonies:
tion aliquots. When counts for all dilutions contain more
than 300 colonies, e.g., > 500 for 1.0 ml,
5.6.8 Prepare duplicate petri dishes for > 500 for 0. 1 ml, and 340 for 0.01 ml; com-
each sample increment (Figure Ill-A~). Mark pute the density by counting the plate having
each petri dish with the number of the sample, nearest to 300 colonies. In this case use the
the dilution, the date, and any other necessary 0:01 ml plate.
information. Deliver the liquid into the dish,
and touch the tip once against a dry area in the 340
petri dish bottom while holding the pipet 0.01 x 100 = 3,400,000
vertically.
Report as: Estimated Fecal Streptococcal
5.6.9 Pour 12-15 ml of liquified cooled Count, 3,400,000/100 ml
agar medium into each petri dish containing
the sample or its dilution. Mix the medium and (c) All Plates with Fewer than 30 Colonies:
the sample thoroughly by gently rotating and If all plates are less than 30 colonies, record
tilting the petri dish. Not more than 20 minutes the actual number of colonies on the lowest
should elapse between dilution, plating, and dilution plated and report the count as the
addition of the medium. Refer to Part 11-C, 2.6, Estimated Fecal Streptococcal Plate Count
for further information. per 100 ml.

5.6.10 Allow agar to solidify as rapidly as (d) Plate with No Colonies: If plates from all
possible after pouring, and place the inverted dilutions show nocolonies, assume a count of
PSE plates at 35 ± 0.5 C for 18-24 hours and one ( 1) colony; then divide 1 by the largest
KF plates at 48 ± 3 hours. volume filtered and report the value as a less
than ( <) count. For example, if 0.1, 0.01 and
5.7 Counting and Recording Colonies 0.001 ml were filtered with no reported colo-
nies, the count would be:
5.7.1 After the specified incubation period,
select those plates with 30-300 fecal strepto- 1
- x100=<1000
coccal colonies. Fecal streptococci on PSE agar 0.1
are brownish-black colonies, about 1 mm in
diameter with brown halos. On KF agar, fecal Report the co.unt as: < 10/100 ml
streptococci are pink to red and of varying
sizes. (e) When all plates are crowded, it is possi-
ble to use the square divisions of the grid on
5.7.2 Count colonies in the plates with the the Quebec or similar counter to estimate the
aid of a colony counter (10-15x magnification) numbers of colonies on the plate. See Part 111-
equipped with a grid. A, 5.6.3 for details.

5.7.3 Observe the following rules for re- 5.8 Precision and Accuracy: Replicate
porting the fecal streptococcal plate counts. plate counts from the same sample deviate

144 oEPA MICROBIOLOGICAL MANUAL 1978

because of errors introduced from a variety of source information. There are several precau-
sources. tions to be observed when ratios are being
used.
Prescott et al. (6) reported for the Standard
Plate Count (butapplicable here) that the stan- (a) Bacterial densities can be altered dras-
dard deviation of individual counts from tically wheh the pH of the sample is below 4.0
30-300 will vary from 0-20%. This plating or above 9.0.
error was 10% for higher plate counts within
the 100-300 range. The authors pointed out (b) Due to limited survival capability of
that a dilution error of about 3% for each some of the fecal streptococci, it is essential to
dilution stage is incurred in addition to the sample close to the pollution source to obtain
plating e'rror. Therefore, large variations can reliable ratios. This is especially true for the
be expected from high density samples such highly sensitive S. bovis and S. equinus
as sewage from which several dilutions are species.
made.
(c) It is difficult to use ratios effectively
Laboratory personnel should be .able to when mixed pollution sources are present.
duplicate their plate count values for the same
plate within 5%, and the counts of others (d) In marine waters, bays, estuaries, and
within 10%. irrigation returns, FC/FS ratios have been of
limited value in accurately defining major pol-
lutional source·s.
6. Determination of Fecal Coliform/F~cal
Streptococcus Ratios (FC/FS) (e) If fecal streptococcal counts are
<; 100/100 ml, ratios should not be applied.
The relationship of fecal coliform to fecal
streptococcus density may provide informa-
tion on the potential source(s) of contamina- 7. Identification of Fecal Streptococci to
tion. Estimated per capita contributions of in- Species
dicator bacteria for animals were used to de~
velop FC/FS ratios (7, 8). These ratios are as 7.1 Summary: Although the fecal st. pto-
follows: cocci are enumerated as described in the lre-
vious sections and are verified with simple
FC/FS Ratios biochemical tests in Part 111-D, 3 above, it is
important at times to identify the fecal strepto-
Man 4.4 cocci to species to further verify animal and
Duck 0.6 human sources of pollution and to determine
Sheep 0.4 the sanitary significance of isolates. This iden-
Chicken 0.4 tification to speeies is performed using the
Pig 0.4 additional biochemical tests described to dif-
Cow 0.2 ferentiate an~ confirm the Group Q strepto-
Turkey 0.1 cocci, the bovis-equinusGroup and the entero-
cocci. The enterococci can be separated as S.
From the data, it was reasoned that ratios faecium and S. faecalisvarieties or into groups
greater than 4:1 were indicative of pollution according to original source.
derived from domestic wastes composed of
man's body wastes. Ratios of less than 0.7 7.2 Scope and Application: The initial
suggested that contamination originated from biochemical test confirms that the isolates are
livestock and poultry wastes, milk and food fecal streptococci by negative catalase reac-
processing wastes o.r from stormwater run-off tion. Group Q streptococci and enterococci are
(non-human source). Further speciation of the separated from the bovis-equinus group by
fecal streptococci provides more specific positive growth at 10 and 45 C and are then

FECAL STREPTOCOCCI 145

verified by growth in 6.5% NaCl and at pH 9.6. (e) Brain heart infusion (BHI) broth
The enterococci are separated from the Group with 40% bile. (Part 11-B, 5.4.9).
a streptococci by reduction of methylene blue
milk. Subsequently, the enterococci are either (f) Starch agar plates. (Part 11-B, 5.4.10).
speciated or separated by origin using addi-
tional biochemical tests. The Streptococcus {g) Starch liquid medium. (Part 11-B,
bovis and equinus are verified by hydrolysis 5.4.11).
of starch and separated by lactose fermenta-
tion. These tests require specific training for (h) Nutrient gelatin. {Part 11-B, 5.4. 12).
valid results.
{i) Litmus milk. {Part 11-B, 5.4. 13).

7.3 Apparatus and Materials 0) Skim milk with 0. 1 % methylene blue.

{Part 11-B, 5.4. 14).
7.3.1 Incubators set at 10 ± 0.5 C, 35 +
0.5 C, and 45 ± 0.5 C (water baths recom- (k) Potassium tellurite in brain heart infu-
mended for 10 and 45 C). Temperatures sion. {Part 11-B, 5.4. 15).
checked with a National Bureau of Standards
thermometer or one of equivalent accuracy. (1) Potassium tellurite in blood agar
{optional). {Part 11-B, 5.4.16).
7.3.2 Pipet containers of stainless steel,
aluminum or pyrex glass for glass pi pets. {m) Tetrazolium glucose (TG) agar or 2, 3,
5-triphenyl tetrazolium chloride (TTC) agar.
7.3.3 Sterile T.D. bacteriological or Mohr (Part 11-B, 5.4. 17).
plpets, of appropriate sizes.
{n) Blood agar with 10% blood. (Part 11-B,
7.3.4 Sterile petri dishes 100 x 15 mm or 5.4.18).
60 X 15 mm, glass or plastic.
{o) 1 % D-sorbitol solution in purple broth
7 .3.5 Dilution bottles (milk dilution), pyrex, base. {Part 11-B, 5. 1. 7).
99 ml volume, screw cap, with neoprene rub-
ber liners. (p) 1 % glycerol in purple broth base. (Part
11-B, 5. 1. 7).
7 .3.6 Inoculation loop, 3 mm diameter, or
needle. {q) 1 % L-arabinose solution in purple broth
base. {Part 11-B, 5.1. 7).
7.3.7 Bunsen/Fisher burner or electric
incinerator. (r) 1 % lactose solution in purple broth
base. (Part 11-B, 5. 1.8).
7.3.8 Media
{s) 1 % sorbose solution in purple broth
(a) Brain heart infusion (BHI) broth. (Part 11- base. (Part 11-B, 5. 1.8).
B, 5.4.5).
{t) 1 % sorbose solution in purple broth
(b) Brain heart infusion (BHI) agar. (Part 11-B, base at pH 10. (Part 11-B, 5. 1.8).
5.4.6).
7.4 Procedure: Follow the schematic out-
(c) Brain heart infusion (BHI) broth with lines in Figures 111-D-2 to 4 for the identification
6.5% NaCl. (Part 11-B, 5.4.7). of fecal streptococcal species.

(d) Brain heart infusion (BHI) broth, pH 9.6. 7.4.1 Isolation and Confirmation of Fecal
(Part 11-B, 5.4.8). Streptococci {Figure 111-D-2) - --

146 &EPA MICROBIOLOGICAL MANUAL 1978

 (a) Pick .colonies typical of fecal strepto- reactions in 6.5% NaCl and pH 9.6 BHI broths
cocci from the membranes or the pour plates, and no reaction in 0.1 % methylene blue indicate
and inoculate them onto BHI agar slants and the tentative identification of group Q.
into BHI broth tubes.
(a) Growth Test in 6.5% NaCl-BHI Broth:
(b) After 24-48 hours incubation at 35 + Transfer 1 drop of 24hour BHI broth culture to
0.5 C, transfer a loopful of growth from the BHI a tube of BHI broth containing 6.5% sodium
slant to a clean glass slide, and add a few chloride. Incubate at 35 + 0.5 C, and check for
drops of freshly-tested 3% hydrogen peroxide growth as evidenced by turbidity within a 3-7
to the smear. A positive control such as staphy- day period. Growth is a positive test.
lococcus and a negative control such as S.
faecalis should be used for testing the 3% (b) Growth Test in BHI Broth of pH 9.6:
Hz Oz. Transfer 1 drop of the24 hour BHfbroth cul-
ture to a tube of BHI broth adjusted to pH 9.6.
(c) If the catalase enzyme is present, it Incubate at 35 + 0.5 C, and check for growth
cleaves the H 2 0 2 to produce water and visible as evidenced by turbidity at 1, 2, 3, and 7 days.
oxygen gas bubbles. The presence of bubbles Growth is a positive test.
constitutes a positive catalase test that indica-
tes non-streptococcal species. Verification (c) Reduction of 0. 1 % Methylene Blue in
need not be continued. Skim Milk: Transfer1 drop of the 24 hour BRT
broth culture to a tube of sterile skim milk
(d) If the catalase test is negative, a separa- containing 0.1 % methylene blue. Positive re-
tion of the enterococcus and Q group orga- duction of methylene blue is evidenced by the
nisms from S. bovis, and S. equinus can be color change from blue to white.
made by testing for growth at 10 and 45 C.

7.4.2 Separation of Enterococci and

Group Q Streptococci (Figure 111-D-2) 7.4.4 Separation of Enterococcus Group
by Species (Figure 111-D-3) ---
(a) Transfer 1 drop of the growth from the
BHI broth tube from 7.4.1 to each of 2 BHI The enterococci can be separated into
broth tubes. species as described in 7.4.4 or into groups by
original source as described in 7.4.5. The
(b) Place 1 tube in a 45±0.5 C water bath enterococcus group can be separated into
and observe for growth (as evidenced by tur- species by observing the reduction of potassium
bidity) within 2 days. Place the other tube in a tellurite and 2, 3, 5-triphenyl tetrazolium chlo-
10 + 0.5 C water bath and check for growth ride and the fermentation of D-sorbitol and
within 5 days. Growth at 10 and 45 C indicates glycerol.
that the culture is a potential member of the
enterococcus or Q groups. On the other hand, (a) Stre.ak the 24 hour BHI broth culture
S. equinus and S. bovis exhibit growth at 45 C onto an agar plate containing a final concen-
but not at 10 C. (See 7.4.7 for these tration of 0.4% potassium tellurite. Invert the
speciations). plates, incubate at 35 ± 0.5 C, and observe
the plates each day for 7 days. Colonies reduc-
7.4.3 Confirmation of Enterococcus Group ing the potassium tellurite will appear black on
(Figure 111-D-2) - -- this medium.

This is done by testing for growth in 6.5% (b) Streak the 24 hour BHI broth culture
NaCl and at pH 9.6 in SHI broth and observing onto tetrazolium glucose agar (TG). Reduction
for reduction of 0.1 % methylene blue in milk. of tetrazolium to formazin is observed after 48
Positive reactions in all cases confirm the hours at 35 + 0.5. C. The degrees of reduction
presence of the enterococcus group. Positive are indicated as follows:

FECAL STREPTOCOCCI 147

 Enterococcus Group

Enterococcus. by Species Enterococci by Origin

Reduction of K2Te03. Tetrazolium Separation of Vegetation

and Fermentation of D-sorbitol Insect and Animal
and Glycerol Source$

Positive Negative

I
Hydrolysis
I
Fermentation (See Figure 111-0-4)
of Gelatin of L-Arabinose

~
Positive Negative Positive

I
S.fa.c•lis
Beta-Hemolysls S. faeclum
Subap. liqu11f•cit1ns

~
Posltlvo Negative

S.f•11c•lis S. faecalis
Subsp. zymog11n11s

FIGURE 111-0-3. Identification of Fecal Streptococci, Separation of Enterococcus

Group by Species and by Original Source of Culture.

148 oEPA MICROBIOLOGICAL MANUAL 1918

 Strong Reduction (+4 to +3) = Red cen- Alpha-Hemolysis- Some streptococci par-
tered colony. tially lyse red blood cells and reduce hemoglo-
Moderate Reduction ( + 2) = Pink centered bin to methemoglobin producing a discolor-
colony. ation of the red blood cells. This appears as
Weak Reduction ( + 1) = Pale pink cen- greenish zones around the colonies.
tered colony.
No reduction (0) =White colony. Beta Hemolysis- Enzymes of fecal strepto-
cocci completely lyse red blood cells produc-
(c) Transfer a small amount of growth from ing yellowish hue, or clear, colorless zone in
each 24 hour BHI agar slant culture to sepa- the blood agar surrounding the colony. S. fae-
rate purple broth base tubes containing 1 % D- calissubsp. zymogenesdemonstrates beta he-
sorbitol and 1 % glycerol. Be careful not to molysis of the blood.
penetrate the agar slant and carry over agar to
the carbohydrate medium. Incubate the inocu- Gamma-Hemolysis - Some streptococci
lated carbohydrate media at 35 + 0.5 C, and produce no hemolysis which is designated as
note acid production over a 4 day incubation gamma hemolysis, S. faecalis is alpha or
period. A negative reaction in D-sorbitol or gamma hemolytic.
glycerol shows that S. faecium is present. The
indicator will be unchanged (purple in color). It is important to note that upon serial
transfer of the culture in the laboratory, hemol-
(d) Reduction of potassium tellurite (black ysis may be lost. This is especially true for S.
colonies), reduction of tetrazolium to formazan faecalis subsp. zymogenes where beta hemol-
on rrc· agar and the fermentation of glycerol ysis may not occur after serial transfer.
and D-sorbitol indicate S. faecalisand its varie-
ties. The determination of S. faecalis subspe- (e) Negative reactions in potassium tellur-
cies is performed as follows: ite and tetrazolium media and failure to fer-
ment D-sorbitol and glycerol indicate S.
faecium.
(1) .Stab-inoculate gelatin with a small
amount of growth from a 24 hour BHI agar (f) Transfer a small amount of growth from
slant. Incubate the culture at 35 + 0.5 C for the 24 hour BHI agar slant to a purple broth
2-14 days, according to the rate of growth. base tube containing 1 % L-arabinose. Be care-
After incubation, place the tube in a cold water ful not to penetrate the agar slant surface and
bath or refrigerator 1 5-30 minutes to deter- carry over agar to the carbohydrate medium.
mine whether or not the gelatin will still solid- Incubate the inoculated carbohydrate medium
ify. Uninoculated controls must be run in paral- at 35 ± 0.5 C, al"!d note acid production over a
lel, especially when prolonged incubation peri- 4 day incubation period. If S. faecium is
ods are encountered. Liquefaction dictates S. present, the L-arabinose is fermented, acid will
faecalis subsp. liquefaciens, whereas solidifi- be produced and the medium will turn yellow.
cation indicates S. faeca/is or S. faeca/is
subsp. zymogenes. The latter 2 strains are 7.4.5 Separating Enterococci by Origin
separated by their hemolysis reactions. (Vegetation, Insect and Animal Sources) - - -

(2) The hemolytic properties of the fecal In contrast to the separation of entero-
streptococci are determined by streak or pour cocci by species described in 7.4.4, the mem-
plates. Melt blood agar base, cool and add bers of the enterococcus group can be sepa-
10% sheep blood. Inoculate streak plates or rated according to original source of culture.
prepare pour plates and incubate for 48 hours. The starch hydrolysis tests separate the enter-
After incubation, read plates. Overnight refrig- ococci originating on vegetation from those
eration may enhance the hemolytic reactions. typically found in insects and animals; the pep-
Hemolysis is classified as 3 types: tonization of litmus milk test separates the

FECAL STREPTOCOCCI 149

Insect-origin from warm-blooded animal- (c) Peptonization of Milk: Peptonization in
source enterococci, as shown in Figure 111-D-4. litmus milk is used to separate S. faeca/is
subsp. /iquefaciens (from insect sources) from
S. faeca/is from vegetation hydrolyzes enterococci derived from warm-blooded ani-
starch while S. faeca/is subsp. liquefaciens mal sources.
from insects and enterococci from warm-
blooded animals do not. The starch hydrolysis (1) Tra·nsfer 1 drop of the isolate growing
test can be performed satisfactorily with in a 24 hour BHI broth culture to a tube of
starch agar plates or in a liquid starch tube sterile litmus milk.
medium(9).
(2) Incubate the tube at 35 ± 0.5 C, and
(a) Starch Hydrolysis Plate Test observe at 1, 2, 3 and 7 days.

(3) .A positive peptonization includes liqui-

(1) Pour the starch agar medium into petri faction and clearing of the milk with the devel-
dishes and allow to solidify. With a wax pencil opment of a brownish color. Peptonization in-
divide the bottoms of the petri dishes into 6 dicates S. faeca/is subsp. liquefaciens. Color
individual areas to allow the testing of 6 changes without clearing, or coagulation are
isolates. negative reactions and indicate enterococci
from warm-blooded animal sources are
(2) Streak each isolate from a 24 hour BHI present.
agar slant onto one of the areas and incubate
at 35 ± 0.5 C for 24 hours. 7 .4.6 Identification of Group 0
Streptococci
(3) After incubation, flood the agar me-
dium with Lugol's iodine solution (Part 11-C, The group 0 Streptococci are initially sep-
5.3). Streaks showing clear hydrolytic zones arated from the Enterococcus Group and tenta-
with absence of the usual blue-black color of tively identified by growth in 6.5% NaCl in BHI
the starch-iodine complex in the zones sur- and in BHI broth at pH 9,6, but no growth in
rounding the colonies are considered positive. 0. 1 % methylene blue in milk. See Figure 111-D-
2.
(b) Starch Hydrolysis Tube Test
Other pertinent physiological characteris-
tics of Group 0 Streptococci are: fermentation
(1) The starch test may also be performed of sorbose, growth in sorbose medium at pH
using a liquid medium instead of starch agar 10, but no growth in tellurite medium and
plates. Inoculate tubes of sterile liquid medium negative hydrolysis of starch and gelatin. The
with the test organisms. After 1 8 hours of tests may be carried out as follows:
incubation at 35 ± 0.5 C, test the tubes for
starch hydrolysis using a modification of the (a) Inoculate a sorbose fermentation tube
iodine test (8). For this modification, 0.2 ml of with a small amount of growth from an isolate
2% FeCI solution and 0.2 ml of Lugol's iodine on a 24 hour BHI slant. Be careful not to
solution are added to 5 ml of the inoculated penetrate the agar slant surface and carry over
medium and to 5 ml of the uninoculated me- agar to the carbohydrate medium. Incubate
dium (control). the carbohydrate medium at 35 ± 0.5 C and
note acid production over a 4 day incubation
(2) Hold the tubes for 3 hours at room period. Sorbose fermentation is indicated by
temperature. Compare the inoculated tubes to acid production and change in the medium
the control tube. The control tube (negative color from purple to yellow.
test) maintains a violet color, but positive test
cultures hydrolyze the starch and decolorize (b) Transfer a small amount of growth from
the medium to a reddish-violet hue. the 24 hour BHI slant to the 1 % sorbose me-

160 &EPA MICROBIOLOGICAL MANUAL 1978

 Separation of
Enterococcus Group
by Original Source,
Vegetation, Insect
and Animal Sources

Starch Hydrolysis

Positive Negative

Peptonization of
Litmus Milk

Positive Negative

Atypical S. faecalis Enterococci

S. faecalis Subsp. /iquefaciens (Warm-blooded
(Vegetation sources) (Insect Sources) Animal Sources)

FIGURE 111-D-4. Identification of Fecal Streptococci, Separation of Enterococci from

Vegetation, Insect and Animal Sources.

FECAL STREPTOCOCCI 151

dium {described in Part 11-8, 5. 1.8 above) which therefore, identification in those cases will de-
has been adjusted to pH 10 with sterile 38% pend solely on physiological characteristics.
sodium phosphate solution (see Part 11-8,
5.4.8). Incubate the inoculated medium at 35 7.4.7 Separation and Speciation of S. bo-
± 0.5 C for at least 4 days. Growth indicates a visand S. equinus -- ---
positive test.
(a) S. bovis and S. equinuswere separated
(c) Inoculate the growth from a 24 hour from the Enterococci and Group Q Strepto-
BHI slant into BHI broth containing 0.04 % cocci by growth at 45 C but no growth at 10 C
potassium tellurite and incubate at 35 ±
0.5 C (see Figure 111-0-2). S. bovis and S. equinus can
for 7 days. Group Q organisms do not grow on be separated by the lactose fermentation test
this medium. in which S. bovisproduces acid and S. equinus
produces no change,(c)lbelow.
(d) Stab-inoculate gelatin with a small
amount of growth from the 24 hour 8HI slant. (b) Starch Hydrolysis Test: Perform starch
Incubate the culture at 35 ± 0.5 C for 2-14 hydrolysis test as in 7.4.5 (a) or (b).
days, according to the rate of growth. After
Incubation, place the tube in a cold water bath
Positive starch hydrolysis test confirms S.
or refrigerator for 15-30 minutes to determine
whether or not the gelatin will solidify. Unino- bovisand S. equinus.
culated control must be done in parallel, espe-
cially when prolonged incubation periods are (c) Lactose Fermentation Test: To differen-
used. tiate between S. bovis and S. equinus by lac-
tose fermentation, transfer a small amount of
{e) Group Q organisms do not hydrolyze growth from the 24 hour BHI agar slant to a
starch. Perform starch hydrolysis test as in purple broth base tube containing 1 % lactose
7 .4.5 {a) or {b) above. and an inverted fermentation tube. Do not pen-
etrate the agar slant surface and carry over
{f) Streptococcus avium sp. constitute agar to the carbohydrate medium. Incubate
Lancefield's Group Q. Consequently, Group Q the inoculated carbohydrate medium at 35 ±
antiserum may be used in the precipitin test to 0.5 C and observe the reaction over a 4 day
provide further identification. However, the Q incubation period. S. bovis gives an acid reac-
antigen is not demonstrable in all strains, tion only; S. equinusshows no change.

152 oEPA MICROBIOLOGICAL MANUAL 1978

 REFERENCES

1. Nowlan, Sandra and R. H. Diebel, 1967. Group Q streptococci I. Ecology, serology, physiology
and relationship to established enterococci. J. Bacterial. 94, (2):291.

2. Kenner, B. A., H. F. Clark and P. W. Kabler, 1960. Fecal streptococci. II. Quantification of
streptococci in feces. Am. J. Public Health. 50: 1553.

3. Kenner, B. A., H. F. Clark and P. W. Kabler, 1961. Fecal streptococci. I. Cultivation and
enumeration of streptococci in surface water. Appl. Microbial. 9: 15.

4. Pavlova, M. T., F. T. Brezenski and W. Litsky, 1972. Evaluation of various media for isolation,
enumeration and identification of fecal streptococci from natural sources. Health Lab. Sci. 9:289.

5. Clausen, E. M., B. L. Green and W. Litsky, 1977. Fecal Streptococci: Indicators of Pollution, pp.
24 7-264. In: A. W. Hoadley and B. J. Dutka, Eds., Bacterial Indicators/Health Hazards Associated
with Water, ASTM STP635, American Society for Testing and Materials, Philadelphia, PA.

6. Prescott, S. C., C-E. A. Winslow and M. H. Mccrady, 1946. Water Bacteriology. (6th ed.) John
Wiley and Sons, Inc., p. 46-50. --

7. Geldreich, E. E., H. F. Clark and C. B. Huff, 1964. A study of pollution indicators in a waste
stabilization pond. J. WPCF, 36 ( 11 ): 1372.

8. Geldreich, E. E., 1976. Fecal coliform and fecal streptococcus density relationships in waste
discharges and receiving waters. In: CRC Critical Reviews in Environmental Control. p. 349.

9. Pavlova, M. T., W. Litsky and F. J. Francis, 1971. A comparative study of starch hydrolysis by fecal
streptococci employing plate and tube techniques. Health Lab. Sci. 8:67.

FECAL STREPTOCOCCI 153

 PART Ill. ANALYTICAL METHODOLOGY

Section E Salmonella

Recommended methods are presented for 1. The Genus, Salmonella

recovery of Salmonella from water and waste-
water and their subsequent identification. The 1 .1 Definition
methods are particularly useful for recreational
and shellfish-harvesting waters. No single The genus Salmonella is comprised of a
method of recovery and identification of these large number of serologically related, gram-
organisms from waters and wastewaters is negative, nonspore-forming bacilli that are
appropriate for all sampling situations. The 0.4-0.6 µm in width x 1-3 µm in length, and
method selected depends on the character- which occasionally form short filaments: They
istics of the sample and the microbiologist's are motile with peritrichous flagella or are non-
motile. Ordinarily salmonellae do not ferment
experience with the techniques. Multiple
lactose, sucrose, malonate or salicin but do
option techniques are described for sample
ferment numerous carbohydrates including
concentration, enrichment, isolation and
glucose, inositol and dulcitol. These bacteria
Identification. The Section is divided as are positive for lysine and ornithine decarboxy-
follows: lase and negative for urease and phenylalanine
deaminase. Usually they produce hydrogen
sulfide and do not liquify gelatin. All of the
1. The Gen_l!S, Salmonella known Salmonella species are pathogenic for
warm-blooded animals, including man. They
2. Methods for Concentration of cause enteritis, (via contaminated water, food
Salmonella or food products) enteric fevers and are found
in reptiles, amphibians and mammals. Edwards
3. Primary Enrichment for and Ewing have puplished an authoritative
Salmonella .work on the isolation and characterization of
Salmonella (1 ).
4. lsolat,on of Salmonella
In Bergey's 8th edition (2), the salmonellae
have been reclassified tentatively into 4 sub-
5. Biochemical Identification of
genera containing II subdivisions. However,
Salmonella
the problem of Kaufman's listing of many sero-
types, and tre lack of agreement as to what
6. Serological Test for Salmonella constitutes the genus Salmonella or its spe-
cies, leaves the taxonomy in a fluid state.
7. Quantitative Techniques
1.2 Identification Schemes
8. Optional Fluorescent Antibody A comprehensive scheme of the recom-
Screening Technique mended isolation, detection and identification

154 oEPA MICROBIOLOGICAL MANUAL 1978

methods is outlined in sequence in Figure 111-E- (b) Limitations: This is not a quantitative
1. procedure, since some salmonellae may pass
through the swab, others may desorb from the
When space and equipment are limited, swab during the exposure period, and the
the number of options at each stage depicted water volume sampled is unknown. It is not
in Figure 111-E-1 can be reduced and salmonel- possible to predict the salmonellae concentra-
lae isolated successfully. One such simplified tion in .the water· or ·wastewater from the con-
scheme is outlined in Figure 111-E-2. centration in the swab nor does the procedure
reflect changes or cycling of salmonellae con-
The procedures outlined in these schema centrations at the sample site.
are described in the following subsections
2-8. 2. 1.3 Apparatus and Materials

(a) Cheesecloth roll, 23 cm wide.

2. Methods for Concentration of Salmonella
(b) Paper cutter or large pair of shears.
The initial steps for detection of salmonel-
lae in water and wastewater require concen- (c) Length of 1 6 gauge wire.
tration of the organisms by one of several
methods: the gauze swab, diatomaceous (d) Sterile 250 ml flasks or jars'., screw-cap,
earth, the cartridge filter, or the membrane containing enrichment media, Part 111-E, 3.3. 10.
filter technique. The volume of sample tested
is directly related to the level of pollution. (e) Sterile plastic bags (e.g., Whirl-pak or
heavy-duty food freezer bags).
2.1 Swab Technique Modified After
Moore's Method (3) (f) Insulated container with ice (optional).

2. 1. 1 Summary: In this method sterile 2. 1.4 Procedure

gauze swabs are immersed for about 5 days
just below the surface of a water or wastewa- (a) Prepare a swab from a length of cheese-
ter. After the exposure period during which cloth 180 cm long x ·23 cm .wide by folding
bacterial concentration occurs, the gauze the length 5 times to form a pad 36 cm x 23 cm.
swabs are retrieved, placed in sterile bags, Cut the folas at one end. From this end, cut
iced and returned to the laboratory for exami- the pad into 5. parallel strips; 4.5 cm wide and
nation. The swab, portions of the swab, or the 2~ cm long, leaving an uncut top section of
expressed liquid from the swab are added to 10 cm (see Figure· 111-E-3).
enrichment media for selective growth of sal-
monellae and suppression of coliforms and (b) Bind the top of the gauze swab with 16
other non-salmonellae. gauge wire to fotm a mop-head shape, with the
strips hanging free (see Figure 111-E-4).
2. 1.2 Scope and Application
(c) Wrap swab in kraft paper and
(a) Advantages: The gauze swab technique autoclave.
is superior to grab sampling, because salmo-
nellae concentration occurs in the swab (d) Place the swab just below the surface
permitting improved detection. Although this of the water or wastewater to be examined and
technique is not quantitative,· it has proved secure with the wire to a solid support.
effective in detection of low numbers of salmo-
neilae in waters and wastewater. The tech- (e) Leave the swab in place for about 5
'nique is simple and inexpensive. days.

SALMONELLA 156
 SAMPLE

I
CONCENTRATION OF SAMPLE

I
Gauze Diatomaceous Cartridge Membrane
Swab Earth Plug Filter Filter

I
ENRICHMENT
I
Dulcitol Tetrathionate
Selenite Selenite Tetrathionate Brilliant
Broth Broth Broth Green Broth

FA Screening Option

ISOLATION PLATING
I
Xylose Lysine Xylose Lysine Bismuth
Desoxycholate Brilliant Brilliant
Sulfite
Agar Green Agar Green Agar
Agar
I
BIOCHEMICAL IDENTIFICATION
(Primary Screening)
I
Triple Sugar Urea Lysine Cytochrome
Iron Agar · Agar Iron Agar Oxidase Test
I
'ADDITIONAL BIOCHEMICAL IDENTIFICATION
(Minimal Biochemical Set by Single Tube or Multitest Systems)
I
Lysine Tryptophane Phenylalanine Phenol Red
Malonate
Decarboxylase Deaminase Dulcitol
Broth Broth
Broth Broth Broth

I
SEROLOGICAL VERIFICATION

Polyvalent 0 : Vo I --- -1 Polyvalent H

Confirmation by a
Reference Laboratory

FIGURE 111-E-1. Scheme for the Concentration, Isolation and Identification of Salmonella.

166 &EPA MICROBIOLOGICAf MANUAL 1978

 SAMPLE

CONCENTRATION OF SAMPLE

Membrane
Filter

.ENRICHMENT

I Tetrathionate
Dulcitol
Selenite Broth
Broth

I
ISOLATION PLATING

I
Xylose Lysine Brilliant
Desoxycholate Green
Agar Agar

I
BIOCHEMICAL IDENTIFICATION
(Primary Screening)

I
Triple Sugar Urea
Iron Agar Agar

I
ADDITIONAL BIOCHEMICAL IDENTIFICATION
(Multitest System)

SEROLOGICAL VERIFICATION

Polyvalent 0

FIGURE 111-E-2. Simplified ~cheme for Concentration, Isolation and Identification

of Salmonella.

SALMONELLA 157
 23 cm
.i1

. ~~~ ~ :... ! ": .: . .

...
E . .. . . . -- ...

-
, ... 1 .
(.)

0 .. ::;: ·i:: . ::. ~ .-·

..: : .": ! ~. i ~~ ::· : ·~:. : ..

l ...
•· ····:
.
I •'•
_. ...
.··.·.:.:.: -

..... . ......
. .. . . ...
. ·: .:
.. .. ... -.. .........·-..
-......' . . ' .. -· ...
... - . : ..
~
·•·I••••
-~ .. : ...
. . . : . ::.:·:: i ..
.. •• ·1 • • •
; .. : . J.;
..
'
• -: : - ::: : I. • -- ........t
•;, • • o.l •

.... ...
:~:: ·;:~~~~:7'~~:-·
,,,, ·-. ... .: ~.::. ·:: . .... .....,,_.
••

.
''"T'

' -
.....

,....
·- - ·;• •.. ·-..
~
.! ":'' .

•·
......
......
1·. 7 . . . •
1
:;

·:°·-·:·-··
..... ......... 9#

~ . .: i
• ;: .l!' ...
;
....
.·: ·:::.-:···-
. .......... '· - ..
.'
....
-· .

.... .',."':
,. .
. : ~ ..: . .._., ~ ·: '
..... . -· -
·- r.
: ~ ::=-
::.·
·- .... -. .. : .. ·- ·: ~~-.;

IE-4.5cm~I

FIGURE 111-E-3. Dimensions of the Gauze Swabs.

158 &EPA MICROBIOLOGICAL MANUAL 1978

 WIRE

·.
;.·• ~ ...
•,

.•.·, .
:.,;..·~··~"=t---=-F=-1N~GERS
·,
.·,,· OF SWAB

.... '·.· .
. ..~:·· 5:· ·~:~·-~·
........ -
..~·;.:-_::
.·.·· .·:... ··.·

........ ·.·

. .. .. .. . ...:. ~

. . ~·. ·: . . .
• • >.· ••
.·.. :" ..:......
'".:
. '
·.· . ·• '~ ~ .. ·:
.
. . ".:'•.

FIGURE 111-E-4. The Gauze·Swab in Position.

SALMONELLA 159
 (f) After the exposure period, retrieve the (e) Safety trap flask between the fil_tering
swab and place it directly into a sterile plastic flask and the vacuum source.
bag (Whirl-Pak), ice and return to the labora-
tory for examination. See enrichment step (f) Sterile spatula.
3.4.1. Alternatively, the swabs may be placed
In enrichment media and incubated on-site, (g) Forceps.
then iced and returned to the central
laboratory. (h) Sterile graduated cylinders, 1000 ml
size, covered with aluminum foil or kraft paper.
2.2 Diatomaceous Earth Technique
(i) Containers for glass pi pets.
2.2.1 Summary: The filtering action of dia-
0) Sterile absorbent pads of cellulosic pa-
tomaceous earth is used to concentrate the
per, 4 7 mm in diameter.
organisms (4). Diatomaceous earth is loosely
packed on top of an absorbent pad in the
(k) Sterile T.D., 10 ml Mohr pi pets, glass or
funnel of an assembled membrane filtration
pl-astic.
unit. One to two liters of a water sample are
passed through the diatomaceous earth using
( 1) Rinse water, sterile phosphate buffered
vacuum and portions of the diatomaceous
water, prepared in large volumes, See Part 11-B,
earth plug are added aseptically to enrichment
broth.
7 for preparation.

2.2.2 Scope and Application 2.2.4 Procedure

(a) Advantages: Although it is not enumer- (a) Assemble the membrane filter unit, sub-
ative, this method is quantitative in the sense stituting an absorbent pad for the membrane
that known volumes of water or wastewater filter.
are filtered through the diatomaceous earth.
(b) With the funnel in place, loosely pack
(b) Limitations: The diatomaceous earth approximately 2.5 cm thickness of diatoma-
filter Is easily clogged with the suspended ceous earth on top of the absorbent pad.
material found in turbid waters. This slows the
filtering process but may not prevent its use. (c) Add enough sterile buffered water with
Also salmonellae ·may pass through due to a 10 ml pi pet to saturate the diatomaceous
improper formation of the diatomaceous filter. earth. Draw water through filter under vacuum
(15 lb.).
2.2.3 Apparatus and Materials
(d) Filter 1 liter or more of the sample
under vacuum. The volume of sample filtered
(a) Diatomaceous earth (Johns-Manville's
will depend upon the estimated amount of
"Celite" or equivalent).
pollution in the water or wastewater.
(b) Sterile membrane filter unit (filter base
and funnel), plastic, glass or stainless steel, (e) Rinse the funnel at least twice after
wrapped with aluminum foil or kraft paper. sample filtration with 20-30 ml of the rinse
water.
(c) Vacuum source.
(f) Remove the funnel. Add the absorbent
(d) Vacuum flask, 2-liter, to hold the filter pad, the diatomaceous earth plug or halves
base, with appropriate tubing. An alternative is of each to flasks of selected enrichment broths.
a commercially-available manifold. See the enrichment step 3.4.2.

160 oEPA MICROBIOLOGICAL MANUAL 1978

 2.3 Membrane Filtration of Samples (g) Membrane filter, white, grid-marked,
142 mm in diameter with 0.45 +
0.02 µm
2.3. 1 Summary: The water or wastewater pore size.
sample is passed under positive pressure
through a 0.45 µm pore, 142 mm diameter (h) Prefilter (Millipore AP15-142-50 or
filter in a pressure filtration unit. After filtra- equivalent).
tion, the membrane filter is aseptically divided
into portions that are added ·to enrich.ment 2.3.4 Procedure
broths.
(a) Aseptically add the sterile membrane
2.3.2 Scope and Application filter to the sterile filter holder.

(a) Advantages: Membrane filtration is (b) Add a 2-20 liter sample to the dispens-
used when the sample volume desired is larger ing pressure vessel. The sample size is limited
than the 1-2 liters employed with tlw diatoma- by the level of solid~ or turbidity in the sample.
ceous earth technique, when the 5 day sample
period for the swab technique is not feasible, (c) If sample is turbid use a prefilter ahead
or when the sample is not too turbid to permit of the membrane filter.
passage of the desired volume. The method is
useful for water of very low organic and partic- (d) Using positive pressure, force the sam-
ulate matter content. It is quantitative in that it ple through the membrane filter.
retains all cells from the filtration of a known
volume of water or wastewater. (e) After filtration, the membrane and pre-
filters are added to enrichment media. See
(b) Limitations: Use of this technique is enrichment step 3.4.3.
somewhat limited because the sample may
clog pores and prevent filtration. Also, the limi- 2.4 Cartridge Filter Technique (5)
tations of membrane filtration cited in Part 11-C,
3.2.2, are applicable. 2.4. 1 Summary: The water or wastewater
sample is drawn under negative pressure
2.3.3 Apparatus and Materials through a filter of borosilicate glass microfi-
bers bonded with epoxyl!resin. The volume
(a) Membrane filter holder, stainless steel, filtered may be measured. After filtration, the
142 mm, autoclavable for use in pressure fil- filter is placed aseptically into enrichment
tration. (Gelman Disc, Filter Holder, 11872, broth.
Millipore SS Filter Holder, YY22 14200, or
equivalent). 2.4.2 Scope and Application

(b) Dispensing pressure vessel, 10/12 liter (a) Advantages: Because this technique
size. (Gelman 15108, Millipore XX67 00003, can be used to filter 20 liters or more of sam-
or equivalent). ple, it is applicable to waters with low concen-
trations of organisms. It can be combined with
(c) Pressure pump, capable of maintaining an MPN procedure for a quantitative estimate
high pressure necessary for pressure filtration of bacterial density.
(7 kg/cm 2 maximum pressure).
(b) Limitations: The presence of high num-
(d) Forceps, with smooth tips. bers of background organisms may make re-
covery of Salmonella very difficult by this tech-
(e) Sterile shears to divide membrane. nique. The autoclave must be sufficiently large
to hold sample reservoir containers. As with
(f) Dilution water, phosphate buffered, pre- other filtration procedures, high turbidity in the
pared in large volumes. sample lengthens filtration time.

SALMONELLA 161
 2.4.3 Apparatus and Materials (g) When a new sample is to be filtered,
replace glass tubing, filter, filter holder and
(a) Vacuum pump, capable of operation at sample container with new sterile units.
a pressure differential up to 69 kN/m 2 .

(b) Vacuum gauge. 3. Primary Enrichment for Salmonella

(c) Water trap (heavy-walled flask or bottle, Selenite and tetrathionate broths are used
with at least a 5-liter capacity) closed by a 2- for primary enrichment. The selenite may be
holed rubber stopper, with 2 short pieces of combined with dulcitol to improve the selectiv-
glass tubing inserted. ity for salmonellae. The tetrathionate may be
used as a broth base alone or combined with
(d) Glass and rubber vacuum tubing, with brilliant green dye which enhances its selectiv-
shut-off valve. ity for salmonellae other than S. typhi.

(e) Optional for MPN technique: manifold,

capable of 5 simultaneous filtrations. 3.1 Summary: After concentration 9f sal-
monellae, the gauze swabs, diatomaceous
(f) Sterile, 20 liter, heavy-walled pyrex earth plugs, membrane filters or cartridge fil-
glass carboy, calibrated from 1-20 liters (Corn- ters are placed in flasks of selenite, tetrathion-
ing CWG No. 434490). ate or other selected broth. The broth encour-
ages salmonellae to grow while inhibiting
(g) Balston (Lexington, MA) type AA car- other bacteria. Multiple flasks of the enrich-
tridge filter, 2.5 X 6.4 cm or equivalent and a ment broth are incubated for 3-4 days at se-
type 90 filter holder. Filters, filter holders and lected temperatures. After incubation, cultures
tubing are wrapped and sterilized by autoclave are streaked onto differential plating media for
at 121 C for 15 minutes. salmonellae isolation. These differential plates
should be streaked from the enrichment media
2.4.4 Procedure every 24 hours over a 3-4 day period.

(a) Connect the parts of the filtration appa- 3.2 Scope and Application
ratus as depicted in Figure 111-E-5.
3.2.1 Advantages: The enrichment .me-
(b) Collect or transfer water sample to ster- thods described provide an optimal environ-
ile, calibrated container. ment for salmonellae and other enteric patho-
gens, and to some extent suppress the other
(c) Insert filter and filter holder into sample organisms that are present.
container. Protect container from contamina-
tion by sealing glass tubing in neck of con- 3.2.2 Limitations: Although enrichment
tainerwith sterile cotton and aluminum foil. broths provide an optimal environment for sal-
monellae development, recovery is not quanti-
(d) Start vacuum pump and filter desired tative.
volume of water. To avoid rupture of filter, the
pressure differential should not exceed 69 3.3 Apparatus and Materials
kN/m 2 •
(e) After filtering sample, aseptically re-· 3.3.1 Incubators set at 35 ± 0.5 C, 41.5 ±
move filter and filter holder. Separate filter and 0.5 C, and optionally at 37 ±
0.5 and 43 ±
0.5
place in appropriate enrichment medium. See c.
3.4.4.
3.3.2 Sterile shears and spatula.
(f) Place another sterile filter in the filter
holder and repeat filtration procedure. 3.3.3 Sterile forceps.

162 oEPA MICROBIOLOGICAL MANUAL 1978

 3.3.4 Sterile beakers, 500 ml size covered 3.4.1 The Gauze Swab: Squeeze the gauze
with aluminum foil or kraft paper. swab in the plastic bag to express the liquid
from the swab into a sterile 500 ml beaker.
3.3.5 Sterile erlenmeyer flasks, 125 ml After this, the entire swab or portions of it.may .
size, to hold 50 ml of enrichment broth. be placed directly into the enrichment broth
using sterile forceps and shears to divide the
3.3.6 Bunsen, Fisher burner or electric swab if necessary into the number of portions
incinerator. required for the enrichment media to be used.
For example, 2 enrichment media incubated at
3.3.7 Sterile petri dish. 2 temperatures, 35 C and 43 C, would require
4 portions. The pieces of swab and measured
3.3.8 Sterile aluminum foil. volumes of the liquid can be apportioned into
125 ml flasks containing 50 ml of enrichment
3.3.9 Alcohol, 95% ethanol, in a vial. broth made double strength to compensate for
the dilution effect of liquid.
3.3. 10 Media
3.4.2 The Diatomaceous Earth Plug: Re-
(a) Selenite broth (Difeo 0275, BBL move the diatomaceous earth plug from the
11608, or equivalent). (See Part 11-B, 5.5. 1). membrane filtration funnel by carefully tap-
ping the funnel on a sheet of sterile metal foil.
(b) Tetrathionate broth (Difeo 0104, BBL Tap the sides of the funnel and shake it gently
11706, or equivalent). (See Part 11-B, 5.5.2). to dislodge the plug. Transfer the entire plug to
an enrichment broth or divide the plug into
(c) Dulcitol selenite broth (see Part 11-B, halves using a sterile spatula. Add one half of
5.5.3). the plug to each 125 ml flask of medium used.
Inoculate at least 2 media in flasks containing
(d) Tetrathionate brilliant green broth · 50 ml of sterile single-strength broth. Incubate
(same as (b) above with the addition of 0.01 at 2 temperatures. Halve the absorbent pad
gram of brilliant green per liter of medium). with sterile shears, and add a portion to each
(See Part 11-B, 5.5.4). of the previously inoculated flasks.

3.4.3 The Membrane' Filter: Remove the

3.4 Procedures for Enrichment membrane filter aseptically from the filter base
with sterile forceps, and hold over the bottom
Select at least two enrichment media, of a sterile petri dish. Cut the membrane in ·
preferably one selenite and one tetrathionate quarters with the sterile shears, and let the
type, for each sample, The actual choice of quarters fall grid-side up into the petri dish
medium is based on the experience of the bottom.· Insert aseptically each quarter of a
analyst. membrane filter into a 125 ml flask containing
50 ml of single-strength medium.
Selenite and tetrathionate enrichment
broths are useful for all Salmonella including 3.4.4 The Cartridge Filter: Remove the car-
S. typhi. Although the addition of brilliant tridge filter from the holder aseptically and
green to tetrathionate broth base increases its place in the selected enrichment medium.
selectivity for salmonellae, it inhibits the recov- More than I filter may be used in succession on
ery of S. typhi. It is reported that tetrathionate a single sample and placed in the same culture
broth is toxic to salmonellae at a temperature medium. Repeat for the second enrichment
of 43 C (6). Dulcitol selenite medium may not medium.
completely recover S. typhi, S. cho/erae-suis,
S. enteritidis bioserotypes Paratyphi A and 3.4.5 After inoculation in 3.4. 1, 3.4.2,
Pullorum, from some samples because these 3.4.3 or 3.4.4, incubate enrichment flasks at
species ferment dulcitol slowly (7). 35 C, 41.5 C and other selected temperatures

SALMONELLA 163
for at least 24 hours. However, some salmonel- tion and isolation of Salmonella from water
lae are slow-growing and recovery may be ( 11, 1 2, 1 3).
increased by incubating for successive 24-
hour periods up to 96 hours before streaking 3.5.5 Although investigators have used
on isolation agars. different elevated temperatures, the results
are inconclusive for a single incubation tem-
3.4.6 If a sample must be collected in an perature. The general conclusion of most stud-
area some distance fr"om the central laboratory ies on comparison of recoveries of salmonel-
responsible for Salmonella identifications, the lae at different incubation temperatures is that
scheme can be interrupted at different points. a greater number of isolates and more species
First, the samples can be concentrated, iced are isolated at 41-43 C than at 35-37 C (9,
and transported back to the central laboratory. 14-19). This manual recommends two temper-
Second, the samples can be concentrated, atures in enrichment and isolation procedures
placed in enrichment media, incubated at the for Salmonella, 35 C and 41.5 C.
selected temperature for 18-24 hours at a field
laboratory then iced and transported to the Optional Fluorescent Antibody Screening
central laboratory. Technique

3.5 Incubation Temperature as a Selec- The FA technique is a rapid screening me-

tion Technique for Salmonellae thod that can be used after primary enrich-
ment but it is tentative and optional. The de-
3.5.1 Historically 37 C has been used for tailed procedure is described in Part 111-E, 8
the Isolation of enteric microorganisms be- following the conventional methods for detec-
cause it is representative of the gut tempera- tion and identification of Salmonella.
ture of humans and other warm-blooded
animals. This temperature was considered
optimal for the detection and isolation of 4. Isolation of Salmonella
these enterics.
4.1 Summary: The organisms that de-
3.5.2 Some workers have used 35 C in velop in the primary enrichment broth media
place of 37 C because the latter temperature is are isolated and differentiated on solid media.
close to the upper tolerance limit for the group The enriched cultures are streaked every 24
and might prevent the growth of some desired hours for 3-4 days onto the surface of XLD,
species. This inhibitory effect would be most BG, XLBG and bismuth sulfite media. After
significant if the 37 C temperature were not incubation, the plates are examined for typical
well-controlled and might be exceeded by 2-3 C. colonies of salmonellae wl,iich are picked and
Consequently, double temperatures of 35 C and characterized biochemically and serologically.
37 C came into common usage (1, 8) for isola-
tion and identification of Enterobacteriaceae. 4.2 Scope and Application

3.6.3 Because the large populations of 4.2. 1 Advantages: Pure cultures of Salmo-
normal gut microorganisms interfere with the nella can be isolated by the careful selection
Isolation of pathogens such 'as Salmonella and use of plating media and incubation tem-
from humans and other warm-blooded ani- peratures. Brilliant green agar is favored for
mals, temperatures above 37 C were proposed the development and identification of salmo-
to reduce the background microorganisms. nellae except for S. typhi and a few other
species whereas bismuth sulfite agar allows
3.6.4 The 43 C temperature was proposed the growth of most Salmonella including S.
as an aid to the isolation of Salmonella, partic- typhi.
ularly from various heavily contaminated ma-
terials (9, 10). The 41.5 C temperature has 4.2.2 Limitations: Bacteria other than sal-
been recommended by others for the detec- monellae may compete with the salmonellae

164 oEPA MICROBIOLOGICAL MANUAL 1978

on the secondary differential media, thus inter- 4.4.4 Bismuth sulfite agar (see Part 11-B,
fering with their isolation and identification. 5.5.8).
The use of brilliant green agar at an elevated
temperature of 41.5 C reduces the number of 4.5 Procedure
interfering organisms, but also inhibits devel-
opment of some serotypes of Salmonella. 4.5. 1 Prepare two selected media (XLD,
BG, XLBG or bismuth sulfite agars) in petri
4.3 Apparatus and Materials dishes. As a minimum xylose lysine desoxy-
cholate (XLD) and brilliant green (BG) or xylose
4.3. 1 Incubators set at 35 ± 0.5 C, 41.5 ± lysine brilliant green (XLBG) agars are recom-
0.5 C, and optionally at 37 + 0.5 C and 43 ± mended. Bismuth sulfite agar permits the pre-
0.5C. sumptive detection of S. typhi and/or S.
enteriditis.
4.3.2 Water bath set at 44-46 C for tem-
pering agar. 4.5.2 Streak the surface of a previously
poured and solidified agar with a loopful of the
4.3.3 Petri dish canisters for glass petri enrichment culture.
dishes.
4.5.3 Duplicate plates should be streaked
4.3.4 Thermometer certified by National from each enrichment culture every 24 hours
Bureau of Standards or one ·of equivalent for 3-4 days.
accuracy.
4.5.4 Inoculate duplicate plates from each
4.3.5 Inoculating needle and 3 mm loop.· enrichment culture and incubate, one each, at
35 C and 41.5 C (and optionally at 37 C and 43
4.3.6 Colony Counter, Quebec darkfield C). Incubate the XLD and XLBG plates for 24
model or equivalent. hours, and the BG agar and bismuth sulfite
agar plates for 48 hours.
4.3. 7 Bunsen/Fisher burner, or electric
incinerator. 4.5.5 After incubation, examine plates for
colony appearance. Table 111-E-1 describes the
4.3.8 Sterile 100 mm x 15 mm petri appearance of colonies on XLD, XLBG, BG and
dishes, glass or plastic. bismuth sulfite agars. The salmonellae colo-
nies on BG agar are pinkish white with a red
4.3.9 Sterile phosphate buffered or background. Lactose fermenters will form
peptone dilution water in bottles, 99 + 2 ml greenish colonies or other colorations. Occa-
volumes (see Part 11-B, 7 for preparation). sionally, slow lactose fermenters such as Pro-
teus, Citrobacter, Pseudomonas and Aeromo-
nas mimic Salmonella.
4.4 Media: The following agar media are
dispensed in bulk quantities in screw-capped
4.5.6 Pick growth from the centers of well-
bottles .or flasks. (See Part 11-B, 4 for
isolated colonies that have the characteristic
preparation).
appearance of salmonellae, and streak onto
the screening media described in This Section,
4.4.1 Xylose lysine desoxycholate (XLD) 5.4.1 and 5.5. 1. Isolated, single colonies from
agar (see Part 11-B, 5.5. 7). a plate where all colonies appear alike may be
assumed to be pure. At least 2 colonies of each
4.4.2 Brilliant green (BG) agar (see Part 11- type suspected to be Salmonella should be
B, 5.5.5). . picked.

4.4.3 Xylose lysine brilliant green (XLBG) 4.5.7 The suspected colonies of Salmo-
agar (see Part 11-B, 5.5.6). nella should now be characterized by the sin-

SALMONELLA 165
 TABLE 111-E-1

Colonial Appearance of Salmonella and Other Enterics on Isolation Media

Colony Appearance Salmonella Other Enterics

1. Bismuth Sulfite Agar

Round jet black colonies s. typhi

with or without sheen

S. enteritidis ser
Enteritidis

S. enteritidis ser
Schottmuelleri
------------------
Flat or slightly raised S. enteritidis ser Proteus spp.
green colonies Typhimurium

S. enteritidis bioser
Paratyphyi

S. cholerae-suis

2. Brilliant Green Agar

Slightly pink-white, Salmonella spp

opaque colonies surrounded
by brilliant red medium
-Yellow-green ---- ---------
. - - --- - - - - - - - - -. - - - - - - - - - - - - - - - - - - - - - - Escherichia,
colonies Klebsie/la,
surrounded by yellow-green Proteus spp. (lactose or
zone sucrose fermenters)

3. XLD or XLBG Agar

Red, black centered colonies Salmonella spp.

Red colonies Shigella spp.

Yellow colonies Escherichia spp.

and biotypes

Citrobacter spp.

Klebsiella spp.

Enterobacter spp.

Proteus spp.

166 &EPA MICROBIOLOGICAL MANUAL 1978

gle biochemical tests or multitests in Part 111-E, tories that do not routinely perform these iden-
5.6. An 0-1 bacteriophage screening test may tification tests.
also be used on the isolate for a rapid (4-5
hours) determination of the tentative identifi- (d) The systems offer these same advan-
cation of Salmonella. Results must be verified tages for the identification and differentation of
(20, 21). other enteric bacteria such as E. coli, K/ebsiel/a,
Enterobacter, Citrobacter and Shigella.
5. Biochemical Identification of Salmonella 5.2.2 Limitations: Whether accomplished
by individual tube or multitest methods, bio-
5.1 Summary: Salmonellae can be identi- chemical identification of large numbers of
fied to genus by determining their reactions in a cultures is expensive and time-consuming. It
series of biochemical tests. These tests can be should not be attempted independently with-
made in single tube media or in commercial out previous training and experience in read-
multitest systems. This Section offers the mini- ing reactions and interpreting results.
mal required set of tests, a series of additional,
optional tests and a brief description of available 5.3 Apparatus and Materials
multitest systems. These tests require specific
training for valid results. 5.3. 1 Incubator set at 35 ± 0.5 C.
5.2 Scope and Application 5.3.2 Bunsen/Fisher burner, or electric
incinerator.
5.2. 1 Advantages: The biochemical reac-
tions characterize the Salmonella-like isolate, 5.3.3 Inoculating needle and loop.
permit a separation from closely related bacte-
ria, and provide presumptive identification as 5.3.4 Culture tubes, 100 x 13 or 150 x
Salmonella. Confirmed identification requires 20 mm.
additional serological tests.
5.3.5 Fermentation tubes, 75 x 10 mm.
The multitest systems have a number of
advantages over single tube media prepared 5.3.6 Closures to fit culture tubes.
in the laboratory:
5.3.7 Thermometer certified by National
(a) The different,designs of these systems Bureau of Standards, or one of equivalent
permit the user to select tests to fit his needs, accuracy.
facilities and budget.
5.4Media
(b) The multitest systems may be used with
confidence in the laboratory or in field situa- 5.4. 1 Screening Media
tions as rapid and convenient screening me-
thods for cultures suspected to be Salmonella. (a) Triple Sugar Iron Agar (TSI) (Difeo
Large numbers of cultures can be examined in 0265, BBL 117 49). See Part 11-B, 5.5.9.
a relatively short time; and those tentatively
identified as Salmonella can then be held for (b) Lysine Iron Agar (LIA) (Difeo 0849, BBL
later serological confirmation. 11363). See Part 11-B, 5.5. 10.

(c) These systems offer the advantages of (c) Motility Sulfide Medium (Difeo 0450).
minimal space requirements, immediate avail- See Part 11-B, 5.5. 16.
ability, and economy when compared with the
preparation and use of tubed media. They are (d) Urea Agar Base Concentrate (Difeo
ideally suited to field work and to small labora- 0284). See Part 11-B, 5.5. 11.

SALMONELLA 167
 (e) Cytochrome Oxidase Test Reagents 5.4.3 Optional Biochemical Tests

Reagent A. Weigh out 1 gram alpha- (a) Carbohydrate Utilization

napthol and dissolve in 100 ml of 95%
ethanol. Purple Broth Base (Difeo 0092, BBL
11 506). See Part 11-B, 5. 1. 7 containing:
Reagent B. Weigh out 1 gram of para-
Dulcitol (Difeo 0162),
amlnodimethylaniline HCI (or oxylate) and dis-
Lactose (Difeo 0156), or
solve in 100 ml of laboratory pure water. Pre-
Inositol (Difeo 0164)
pare frequently and store in refrigerator.
(b) Decarboxylase Activity
5.4.2 Minimal Biochemical Set
Decarboxylase Medium Base (Difeo
(a) Phenylalanine Agar (Difeo 0745, BBL 0872). See Part 11-B, 5.5.14 containing:
11537). See Part 11-B, 5.5. 12.
Lysine HCI (Difeo 0705),
(b) Ferric chloride reagent: Prepare a 10% Arginine HCI (Difeo 0583), or
solution (w/v) of ferric chloride in laboratory Ornithine HCI (Difeo 0293)
pure water. Store in a brown bottle in the
refrigerator. (c) ONPG Reagents

( 1) Monosodium Phosphate Solution

(c) lndole test (tryptophane broth). See Part
11-B, 5.1.9.
Dissolve 6.9 grams NaH 2 Po 4 •HOH in 45
ml of laboratory pure water. Add 3 ml of 30%
( 1) Tryptone (Difeo 01 23). NaOH and adjust pH to 7 .0. Bring to 50 ml with
laboratory pure water and store in refrigerator.
(2)Trypticase (BBL 11921).
(2) ONPG Solution
(3) lndole Test Reagent: Dissolve 5 grams
paradlmethylamino benzaldehyde in 75 ml iso- Dissolve 80 grams of o-nitrophenyl-B·D-
amyl or normal amyl alcohol, ACS grade. galactopyranoside (ONPG) in 15 ml pure water
Slowly add 25 ml cone HCI. The reagent at 37 C. Add 5 ml of 1 M NaH 2 P0 4 from (1)
should be yellow and have a pH below 6.0; if above. This 0.75 M solution of ONPG should
the final reagent is dark in color it should be be colorless. Store in a refrigerator. A portion
discarded. Examine the reagent carefully dur- of the buffered solution sufficient for the num-
ing preparation because some brands are not ber of tests to be done should be warmed to 37
C before use.
Slltisfactory after aging. Both amyl alcohol and
benzaldehyde should be purchased in a small
amount consistent with the volume of work 5.4.4 Multitest Systems (optional to Sin-
gle Test Series) - --
anticipated. Refrigerate the reagent in a glass-
stoppered bottle.
(a) API Enteric 20 (Analytab Products, Inc.).

There has been some difficulty in obtain- (b) Enterotube (Roche Diagnostics).
ing amyl alcohol. If this problem occurs, Gillies
describes an alternate paper strip test for in- (c) lnolex (lnolex Biomedical Division of
dole production (22). Wilson Pharmaceutical and Chemical Corp.)

(d)Malonate Broth, Modified (Difeo 0569, (d) Minitek (Baltimore Biological Labora-
BBL 11436). See Part 11-B, 5.5. 13. tories, Bioquest).

168 ~EPA
" MICROBIOLOGICAL MANUAL 1978
 (e) Pathotec Test Strips (General Diagnos- (4) Typical reactions:
tics Division of Warner-Lambert Company).
Salmonella: Kl Ag with H 2S, 1 + to 4 +.
(f) r/b Enteric Differential System S. typhi: KlA with H2S, trace to 1 +.
(Diagnostic Research, Inc.) Citrobacter: K/Ag or A/Ag with H 2S, 1 +
to3+.
5.5 Procedures: Since cultures are found
which react atypically, an isolate should not be (5) Atypical reactions: TSI tubes showing
eliminated because of a single anomalous re- alkaline slants and acid butts without H 2S
action, rather the biochemical reactions production should be inoculated into Motility
should be considered as a group. For example, Sulfide Medium to verify the negative H2S
LIA tubes not showing H 2S production, but reaction. If still H 2S negative, perform serolog-
having alkaline slants and alkaline butts may ical testing to confirm an atypical Salmonella.
be atypical S. typhi. These tubes should be
retained for further characterization. • (b) Lysine Iron Agar

Further, to insure that the media are yield- (1) Inoculate by stabbing the butt twice
ing proper reactions, the analyst is urged to and streaking the slant.
incorporate both positive and negative control
cultures into Single Test and Multitest (2) Incubate for 18-24 hours, and if nega-
Procedures. tive for an additional 24 hours, at 35 C.

5.5. 1 Screening Tests: Pick growth from (3) Read and record reactions. The slant or
the center of a single isolated colony on a butt is yellow from an acid reaction and
selective plating medium and inoculate into blue/purple for an alkaline reading. Gas
the primary screening medium. production is evidenced by bubbles in the me-
dium and H2S production by blackening of the
Fermentation tube reaction code for TSI medium along the stab line. Proteus has a
and LIA Agars: distinctive red slant caused by oxidative deam-
ination and a yellow butt.
Report slant/butt where K, A and N
indicate alkaline, acid and neutral reactions (4) Typical reactions:
respectively; G, g indicate large and small
amounts of gas production, respectively; and Salmonella: K/K or K/N with H2S +(-).
H 2S 1 + to 4 + indicate levels of blackening S. typhi: K/K with H2S -( + ).
due to hydrogen sulfide production. For Citrobacter:K/Awith H 2S-or +.
example." K/Ag is a·n alkaline slant and an acid Proteus: R(red)/A with H2S-( + ).
butt with a small amount of gas.
• (c) Urea Agar (Christensen)
• · (a) Triple Sugar Iron Agar
( 1) Inoculate slant only, using a heavy
( 1) Inoculate by stabbing the butt and inoculum.
streaking the slant.
(2) Incubate for 18-24 hours at 35 C with
(2) Incubate at 35 C for 18-24 hours with cap loose. A positive reaction for Proteus may
cap loose. · be recorded in 2-4 hours, but all negative tests
at 2-4 hours must be held for 18-24 hours.
(3) Read arid record reactions. Color of
slant or butt is yellow for an acid reaction or (3) Reactions are red for urease positive
red for an alkaline reading. Gas production is and yellow for urease negative. Salmonella
evidenced by bubbles in the medium, and H 2S give negative urease reactions. Cultures of
production by blackening of the medium. ·

SALMONELLA 169
Citrobacter may yield weak delayed positive · • (b) lndole Test
reactions at 18-24 hours.
(1) Inoculate the tryptophane broth lightly
• (d) Cytochrome Oxidase Test from the TSI agar slant culture.

The cytochrome (indophenol) oxidase test (2) Incubate the broth at 35 + 0.5 C for 24
can be done with prepared paper strips or the + 2 hours, with cap loose. -
following test on a nutrient agar slant:
(3) Add 0.2-0.3 ml indole test reagent to
(1) Inoculate nutrient agar slant and incu- the culture, shake and allow the mixture to
bate at 35 C for 18-24 hours. Older cultures stand for 10 minutes.
should not be used.
(4) Observe and record the results.
(2) Prepare reagents as in 5.4. 1 (e).
(5) A dark red color in the amyl alcohol
(3) Add 2-3 drops of reagent A and reag- layer on top of the culture is a positive indole
ent B to the slant, tilt to mix and read reaction test; the original yellow color of the reagent is
within two minutes. a negative test.

(4) A strong positive reaction (blue color (6) With rare exceptions, Salmonella and
slant or paper strip) occurs in 30 seconds. Citrobacterare indole-negative.
Ignore weak reactions that occur after two
minutes. Pseudomonads, aeromonads and • (c) Malonate Broth Test
vibrlos are positive. Salmonella i.s negative.
(1) Inoculate from the 18-24 hours TSI
agar slant culture.
5.5.2 Minimal Biochemical Set: After read-
ing the TSI reaction, use growth from the slant (2) Incubate for 48 hours at 35 C. Observe
to inoculate the minimal biochemical set (in tubes after 24 and 48 hours. Positive reactions
5.4.3 above) with a straight wire needle. Suffi- are indicated by a change in color of the me-
cient culture to inoculate all of the minimal dium from green to a deep blue. Lots of malo-
biochemical media is provided by one applica- nate medium should be checked with positive
tion of the tip of the needle to the TSI growth. and negative cultures.

• (a) Phenylalanine Agar (3) Salmonella arizonae and some strains

of Citrobacter utilize malonate. Other Salmo-
( 1) Inoculate surface of slant heavily. nella do not.

(2) Incubate for 18-24 hours ·at 35 C with • (d) Fermentation of Dulcitol in Phenol Red
cap loose. A positive reaction for Proteus may Broth Base
be recorded in 4 hours but negative tests must
be held for 18-24 hours. ( 1) Inoculate the Dulcitol broth lightly us-
ing a 24-hour culture. ·
(3) Test for phenylalanine deaminase by
allowing 4-5 drops of a 10% solution of ferric (2) Incubate at 35 C and examine daily for
chloride to run down over the growth on the 7 days.
slant.
(3) A positive reaction is production of acid
(4) A dark green color on the agar slant with yellow color.
and in the fluid is a positive reaction. A yellow
or brown color is negative. Salmonella and (4) Most salmonellae and some Citrobac-
Cltrobactergive negative reactions. ter utilize dulcitol. Some that do not use it or

170 &EPA MICROBIOLOGICAL MANUAL 1978

use it slowly include: S. typhi, S. cholerae-suis, menters may remain alkaline throughout
S. enteritidis bioser Paratyphi A and Pullorum, incubation.
and S. entertidisserTyphimurum.
(6) Salmonella are positive for lysine; posi-
5.5.3 Optional Biochemical Tests: If the tive or delayed positive for arginine; and posi-
minimal set of biochemical tests has not satis- tive for ornithine. S. typhi and bioser Gallina-
factorily identified cultures as Salmonella, or rum are negative for ornithine. Citrobacterand
variable reactions have been observed, pro- bioser Paratyphi A are negative for lysine.
ceed with the following optional tests. Strains are variable for arginine and ornithine.

• (a) Fermentation of Lactose in Phenol Red • (c) Gelatin Liquefaction

Broth and Inositol in Phenol Red Broth
(1) Inoculate nutrient gelatin tube by
(1) Inoculate the broth lightly using a 24- stabbing.
hour culture.
(2) Incubate at 35 C for 5 days.
(2) Incubate at 35 C and examine daily for
7 days. (3) Cool tubes to 20 C and inspect. Failure
to solidify is a positive reaction.
(3) A positive reaction is production of acid
and yellow color with or without gas (4) All Group I Enterobacteriaceae are
production. negative, except for S. arizonae which shows a
delayed positive reaction (see Table 111-E-5).
(4) Salmonella do not utilize lactose, but
some strains of Citrobacterdo. • (d) ONPG Test, o-nitrophenyl-f3-D-
galoctopyranoside (23)
Some Salmonella do utilize inositol.
( 1) Emulsify a large loopful of growth from
• . (b). Decarboxylase tests (lysine, arginine each culture in 0.25 ml of physiological saline
and ornithine) in a 10 X 75 mm fermentation tube.

( 1) The complete decarboxylase test series (2) Add one drop of toluene to each tube
requires tubes of each of the amino acids and a and shake well. Let tubes stand for 5 minutes
control tube containing no amino acids. in 35 C water bath.

(2) Inoculate each tube lightly. (3) Add 0.25 ml of buffered 0.75 M ONPG
solution to each tube (see 5'.4.3, (c) ( 1)) and
(3) Add sufficient sterile mineral oil to the incubate again in 35 C water bath.
broth to make 3-4 mm layer on the surface
and tighten the screw cap. (4) Read tubes at 1/2, 1 and 24 hours. A
positive result is development of yellow color.
(4) Incubate for 18-24 hours at 35 C. Lactose fermenters have f3 galactosidase so
Negative reactions should be reincubated up Citrobacter are positive and most Salmonella
to 4 days. negative.

(5) Positive reactions are deep purple and • (e) Motility Test: A test for motility is used
negative reactions. remain yellow. Read the in serology but is not recommended in the
control tube without amino acid first; it must biochemical reactions because:
be yellow for the reactions of the other tubes to
be valid. Positive purple tubes must have ( 1) Most members of the family Enterobac-
growth as evidence by turbidity because teriaceae are motile, so the test would not add
uninoculated tubes are also purple, nonfer- much to a characterization of the isolate.

SALMONELLA 171
 (2) A negative test cannot be considered reagent-filled capillary chambers. A single iso-
conclusive until the culture is passed once or lated colony is picked into broth and cultured
twice through a motility or broth medium and for 3 112 hours at 35 C. After incubation, the
the isolate retested. broth tube is centrifuged, the cells resus-
pended in water and inoculated into each cap-
5.6 Multitest Systems: Multitest systems illary chamber on the card. Each card is incu-
are available which use tubes containing pre- bated for 3 hours at 35 C in its own plastic
pared agar media, plastic units containing de- container. Isolates can be identified in 7 hours
hydrated media, media-impregnated discs and unless additional tests are required. A numeri-
reagent-impregnated paper strips. Some of cal binary code 1Jamed Var-ident is part of the
the systems use numerical codes to aid identi- system. ·
fication of bacteria. Others provide computer-
ized Identification of bacteria. A number of (d) The r/b Enteric Differential System con-
Independent investigators have compared one sists of4Beckford tubes, 2 basic and 2 ex-
or more multitest systems with conventional pander tubes. The 4 tubes contain agar media
biochemical tests. Some of the earlier systems and are constricted to form upper and lower
have been improved. Most of the recent stud- compartments .which provide 14 biochemical
ies report the correct identification of high reactions. The tubes are stabbed, streaked and
percentages of isolates (24-29). incubated at 35 C for 24 hours. A color chart of
typical tube reactions is provided for
5.6. 1 The following systems for the identi- identification.
fication of Enterobacteriaceae are commer-
cially available. These have been quality tested (e) The Pathotec Test Strips are individual
by the manufacturers and others and can be biochemical paper strips imp-regnated with
used with confidence. reagents that test for enzymes or end products
characteristic of certain bacteria. A cell sus-
(a) API Enteric 20 consists of 20 small pension prepared from isolated colonies is
chambers (called cupules) in a plastic strip, used as the inoculum to demonstrate the bio-
each containing dehydrated medium. An iso- chemical reactions. An incubation period of 4
lated colony is used to prepare a cell suspen- hours at 35 C may be required for the cell
sion to inoculate the media. The inoculated suspension. The earlier Rapid 1-D System has
media are incubated for 18 hours at 35 C in a been discontinued. A new identification sys-
special plastic chamber. A numerical identifi- tem for enterics is planned for marketing in
cation system based on thousands of reaction 1978-1979.
combinations is available. The identification
system is updated periodically. Computer serv- (f) The Minitek Microorganism Differentia-
ices may be obtained which are more compre- tion SyStem consists of 10 impregnated discs
hensive and accurate than the manual system that test for 12 biochemical reactions .. A single
(27, 28). colony suspended in special broth serves as
the inoculum. The 10 basic discs are part of 34
(b) Improved Enterotube with 8 compart- discs and 37 reactions offered to identify aero-
ments of agar media in a single plastic tube bic and anaerobic bacteria. The inoculated
provides tests for 11 biochemical reactions. discs are contained in a plastic tray within a
The media are inoculated by touching one end humidor and incubated at 35 C for 18-24
of a wire to an isolated colony and drawing the hours. Accessory equipment required to proc-
wire containing the inoculum through the me- ess the discs includes plastic inoculum plates,
dia. The Enterotube is incubated for 18-24 a 10-disc dispenser, a special pi petter, pi pet
hours at 35 C. A manual numerical identifica- tips, a pi pet tips organizer, an incubation humi-
tion aid, ENCISE, is part of the system. dor, a color comparator card set and inoculum
broth. The system stresses the biochemical
(c) The lnolex system (formerly Auxotab) is options and flexibility of the system. A flow
comprised of a test card unit containing 10 diagram is provided for identifications.

172 &EPA MICROBIOLOGICAL MANUAL 1978

 5.6.2 Factors for Selection: The 6 multi- proportional to the amount and number of
test systems briefly described above were exposures to pathogens. Some multitest sys-
designed for the biochemical identification of tems are more dangerous to handle than oth-
members of the family, Enterobacteriaceae. ers because they require more opening, clos-
Most of the isolates suspected as Salmonella ing and manipulating of the test container
could be identified by any of the multitest which may pose added hazards to the worker.
systems. Some of the factors that should be Some systems such as Enterotube and r/b use
considered in selecting a multitest system are: direct colony picks for reactions. API, lnolex,
Pathotec and Minitek require culturing and
(a) Biochemical Reactions in the Multitest additional handling of cell suspensions which
System: Since analysts working with Salmo- are greater hazards.
nella develop a series of tests that yield good
results for them, they should consider a system (g) Cost and Source of Multitest Systems:
which fits their preferred test pattern. The per unit cost of the multitests varies with
the system, as shown in Table 111-E-4.
(b) Need to Identify Atypical Salmonella:
Because it is important for the laboratory to 5.7 Biochemical Characteristics of
identify typical and atypical Salmonella from a Enterobacteriaceae: The analyst may wish to.
series of samples, systems that use numerical differentiate salmonellae from the other
identification should be selected (API, Entero- Enterobacteriaceae by applying additional
tube, and lnolex). Further, systems containing biochemical tests. Table 111-E-5 is a chart of
the most tests have a better chance for identifi- these biochemical reactions.
cation of typical and atypical Salmonella. Only
one system, API, requires no additional tests. 5.8 Serological Verification: The analyst
should understand that completion of the bio-
(c) Multitests: The production rate and the chemical tests does not yield identification of
time span required to identify typical Salmonella. The cultures that have been bio-
Salmonella varies with the system as shown in chemically confirmed should be verified
Table 111-E-2. serologically.

(d) Refrigerated Storage: Refrigerated

space is required for some systems. This can 6. Serological Testing for Salmonella
be a problem in purchase of a large supply.
Table 111-E-3 shows the reported shelf-life of the 6.1 Summary: Serological typing of Sal-
multitest systems, with and without monella strains is done by using slide-
refrigeration. agglutination for somatic (0) antigens and tube
testing for flagellar (H) antigens.
(e) Purchase of Special Equipment: Some
of the test systems contain all materials and 6.2 Scope and Application: Serological
equipment necessary to do the analyses. testing completes the identification of Salmo-
Others require . purchase of special items of nella. It is the only testing which identifies to
equipment for full use. For example, lnolex serotype and bioserotype levels. However, se- ·
requires a small centrifuge; API can be sight- rological testing is an expensive, complex pro-
read or can utilize a profile register for easy, cedure that should be carried out only by
rapid identification of atypical and typical trained personnel.
strains; Minitek uses a starter kit which
includes special plates, broth, pipetter, pipet 6.3 Apparatus and Materials
tips, color comparator card set, incubation
humidor, pipet tips, organizer and dispenser. 6.3.1 Small inverted fluorescent lamp.

(f) Safety Considerations: The probability 6.3.2 Incubator set at 35 + 0.5 C, and a
of laboratory-acquired infections is directly water bath set at 50 C.

SALMONELLA 173
 TABLE 111-E-2

Production Rate and Time Requirements of Multitest Systems*

Multltest Analyst's Time Time Span of Cultures

System Per Culture in Min. Test in Hours Per Day Per Analyst

API 10 18-22 80

Enterotube 6 18-24 100

tnolex 30 7 10-15

r/b Diff. 8 24 80

Pathotec 15 4 20-30

Minitek 10 18-24 80

•eased on experience in EMSL-Cincinnati.

TABLE 111-E-3

Reported Shelf-Life of Multitest Systems With or Without Refrigeration

Refrigeration No
System Required Ref~igeration •

API 12 months

lnohtx 12 months

Pathotec 3 years .. 12 months

Enterotube 7 months

r/b 6-12 months

Minitek 2 years

•Store in cool, dark place, ambient temperature.

••Refrigeration not required, but will extend the shelf-life.

174 &EPA MICROBIOLOGICAL MANUAL .1918

 TABLE 111-E-4

Cost and Source of M"11titest Systems 1

Multitest Cost Cost Address of

System per Unit per Box Manufacturer

API Enteric 20 $2.05 2 $ 51.25 (25/box) Analytab Products, Inc.

200 Express Street
Plainview, NY 11803

Improved Enterotube $2.16 $54.00 (GSA) (25/box) Roche Diagnostics

Div. of Hoffmann-La Roche, Inc.
Nutley, NJ 07110

Ino lex $0.91 ~22.80 (25/box) lnolex Biomedical Division

lnolex Corporation
3 Science Road
Glenwood, IL 60425

r/b Enteric Differ- $1.90 .$38.60 (20 sets/box) Diagnostic Research, Inc.
ential System 3 tubes/ 25 Lumber Road
set Roslyn, Long Island, NY 11576

Pathotec Test $0.20 $20.00 (100 tests/ Warner-Lambert Company

Strips box) General Diagnostics Division
Morris Plains, NJ 07950

Minitek $1.80 3 $90.00 (50 tests/kit) Baltimore Biological Laboratories

Cockeysville, MD 21030

1. As of October, 1977.

2. Plus $99.00/year, if the numerical identification system, Analytical Profile

lnolex Service, is used.

3. Requires one time purchase of accessories for $94. 10.

SALMONELLA 175
 TABLE 111-E-5

Biochemical Characteristics of the Enterobacteriaceae

Group I Group II Group Ill Group IV

....
~ ....
.
Q)

..,"'
.;:
"' .
u"' ~
. ~
0
·;::
"'
..,
.c:
~. .0
Q)
c:
0

.
.§
::::
Q)
-~
"'
.0
~ .!!!
.
.!!!
iii ~
"'::i .!!!
·~
~
:t .!<> .0 Q)
.E t::
'tl .c: .!!! c: 0
~
Raacllon in ""= :r: "' &::
Ill
"' "' "' "'
Catalaso + + + + oa + + + + + +
OxidHO
B·Galaclosldaso
-
+
-- -
+
-
D
-
d
-
+
-
+
-
+
-
+
-- -
+
Gn from Glucose
al 35C + + + + - d + + d D -
KCN (growth on) -+ - + D - + + + + + -
Muc:alo (acid)
N1trato roduclion +
-
+
+
+
D
+
-
+
d
+
d
+
-
+
-
+ + +

C1tbohydra1os:
(acid produclion)
Adonitol - - - - - d + - d D D
Arabinoso + - + + + + + - - +
Dulcilol d - d D
-
d d· - - -
-
- -
Esculin d - d D - d D
lnosilol - - - d - + D - d D -
Lactoso
MaUoso
+or x
+ +
- +or x
+
D
+
D D
+
+
+
-
+
-
+
-
D
-
+
Mannilol
Sallcln
+
d
-- +
d
+
-
D
-
+
+
+
+
+
-
+
+
D
d
+
D
Sorbitol + - + + + + - - D
Sucroso d - d - D + + - or x + D D
Trehaloso + - + + + + + + c1 +
Xyloso d - + + D + + + d D D

Rolatcid C sources;
C11r1to - - + + - d + + + -
Gluconato - - - + + + +
Malonato
d·Ta11r110
-
d
-
-+
d
+
D
D
- D
d
+
- -
- -
D
M.R + + + + D - - - + +
VP, - - - - - D + + D d -

Prot111n roactions,
Argintna d - d + - - D - - - -
Gelalin hydrolysis - -+ -D D - (d) (+) - + D -
H2S from TSI
Jndolo
-
+ + D
+
-
-
D
-
d
-
-
-
-
-
-
D
D
D
D
Lyslno
docarboxylase + + - + - d D + + d -
Otnilhlna d + d + d - + + + D D
Uroa hydrolysis - - (+) - - d (d) - - D b
Glutamlc acid - - -- - - - +
Phonylalanina - - - - - - - - + -
10 • d1llorenl roactions given by different species of a genus; d = different reactions given by different strains of a species or serotype;
X • late end irregularly positive (mutative).

From Bergey's Manual of Determinative Bacteriology, Eighth Ed. (2)

176 &EPA MICROBIOLOGICAL MANUAL 1978

 6.3.3 Bunsen/Fisher burner or electric 6.4.5 Blood agar base (without blood)
incinerator. · (Difeo 0045-02, BBL 11036, or equivalent)
(Part 11-B, 5.4. 18).
6.3.4 Inoculating loop (3 mm) and needle.
6.4.6 Nutrient agar (Difeo 0001-02, BBL
6.3.5 McFarland Barium Sulfate Standard 114 7.1, or equivalent) (Part 11-D, 5.1.1 ).
# 10 (Difeo 0691 ).
6.4.7 Phenolized saline (0.6 grams phenol
6.3.6 Test tubes, 150 x 25 or 150 .x 20 in 100 ml of0.5-0.85% NaCl solution).
mm and 100 x 13 mm.
6.4.8 Formalinized saline (0.6 ml formalin
6.3.7 Glass microscope slides, 5.0 X 7.6 in 100 ml of0.5-0.85% NaCl solution).
cm (2" x 3") cleaned to remove grease and oil. ·
6.5 Procedure: as described in Figure 111-E-
6.3.8 Salmonella 0 Antiserum Poly (Difeo 1, there are serological procedures for the 3
2264, BBL 40707, Sylvana 27-1 OBA or antigen groups, 0, Vi and H. However, it is
equivalent). usually necessary only to test for the 0 and Vi
antigens for verification of the Salmonella
6.3.9 Salmonella Vi Antiserum (Sylvana identification.
27-106A, BBL 40708, Difeo 2827, or
equivalent). 6.5. 1 Slide Agglutination Test for 0
Grouping (3--or-
6.3. 10 Salmonella 0 Antisera Set A-I
(Difeo 2892) includes 1 vial each of Groups, A, (a) Prepare a dense suspension of organ-
B, C 1, C2 , D, E1, E1, E2 , E4 , F, G, Hand I; Poly A-I isms from a fresh 24 hour BHI slant in 0.5 ml
and Vi. of phenolized saline solution. The suspension
should be homogeneous and at least as
Salmonella Grouping Serum Kit (BBL concentrated as that of McFarland Barium
40709) includes one vial each of Groups A, B, Sulfate Standard #10, which corresponds to
C 1• Cz, D and E Polyvalent and Vi. 3 x 109 cells/ml.

6.3.11 Salmonella H Antiserum (BBL (b) Mark rectangular areas on an alcohol-

407-99, Difeo 2406-4 7 or equivalent). cleaned glass slide with a wax pencil. Mark
heavy continuous lines to prevent flow of sus-
6.3.12 Capillary pipets with rubber bulb. pension from one section to another as shown
in the example below. For safety, it is recom-
6.4Media mended that the outside perimeter be in-
scribed with a wax line to prevent flow off the
6.4. 1 Brain heart infusion (BHI) broth (BBL edge of the slide. Note that slide sections 1, 2
11058, Difeo 0027 or .equivalent) (Part 11-B, and 3 are controls and section 4 is the one
5.4.5). complete test:

6.4.2 Brain heart infusion (BHI) agar slant 1 2 3 4

(BBL 11064, Difeo 0418 or equivalent) (Part 11- ,.
B, 5.4.6).
011
6.4.3 Motility medium (Difeo 0869, BBL ,, Q,.
01I
11
Q
,,
I 1
,'•,
11436, or equivalent) (Part 11-B, 5.5.16). I' •• I I
••
(j 0 0 0
6.4.4 H-broth (Difeo 0451, BBL 11289, or
equivalent) (Part 11-B, 5.5. 17).

SALMONELLA 177
 Section 1 -Add antiserum alone. (g) Mix the antigen and antiserum further
by tilting the slide back and forth until aggluti-
Section 2 - Combine antiserum and nation (or clumping) is apparent. If agglutina-
0.85% NaCl. tion is not evident or if it is weak at the end of 1
minute, consider the reaction negative. Com-
Section 3 - Combine bacterial suspension pare reactions with controls. As a QC function,
and 0.85% NaCl. · test antisera against cultures of known reac-
tions, monthly or as indicated.
Section 4 - Combine bacterial suspen-
sion, 0.85 % NaCl and antiserum. (h) If the polyvalent test is positive, test the
culture with Salmonella 0 groups A, 8, C, D
For large numbers of cultures, the 2 x 3 and E antisera. Kits. containing additional 0
inch (50 X 75 cm) glass slide can be used to group antisera are described in 6.3.
accommodate 12 or more sections per slide in
2 'rows. It is recommended that only one cul-
ture be tested at one time against the series of 6.5.2 Slide Agglutination Test for Vi Anti-
sera to avoid premature drying. As in the fol- gen (S. typh~ (30): Occasionally S. typhiwill be
lowing example: isolated without the capsule-like Vi antigen.
The procedure described in 6.5. 1 will identify
it as Salmonella, 0 group D. If the biochemical
reactions are characteristic of S. typhi, record
A 8 the results presumptively as S. typhi, no Vi
antigen detected and in this case, check other

0 9,. 0 0,. .... q..

0 colonies from same plate for Vi antigen. If Vi

z ..••
:•
•• II
II
II
.,
'I
I
'I
,1
,1
",. '•.. ••..
antigen is present, it will block 0 agglutination
and the procedure described in 6.5. 1 will not
identify the serogroup. If S. typhiis suspected,
wO
a:-
::> z
0 0 0 0 0 0 proceed as follows:
(/)
~w
:::> a.. (a) To three rectangles on a glass slide,
()~ add respectively:
Cl)

(1)Polyvalent Salmonella antiserum.

(2) Group D Salmonel/aO antiserum.

(c) Place a drop of Salmonella polyvalent 0 (3) Vi antiserum.

antiserum near the top of a rectangle and a
drop of saline nearthe bottom. (b) Mix with antigen as in procedure 6.5.1.

(d) Using a wire loop, suspend a sufficient (c) Reactions and interpretation:
amount of growth from an isolated colony or a
SHI slant into the drop of saline to produce a (1) No agglutination in any rectangle - not
milky suspension. a common Salmonella.

(e) Mix the suspension with the antiserum, (2) Agglutination in polyvalent antiserum
using the wire loop. Make a long, narrow track only- possibly a Salmonella other than Group
rather than a circular pool. D. Check antigen in other 0 groups.

(f) Flame loop well between uses to pre- (3) Agglutination in Vi antiserum only or in
vent cross-reactions from contamination of Vi and weakiy in polyvalent - presumptive S.
sera. typhi.

178 SEPA MICROBIOLOGICAL MANUAL 1978

 (d) Follow-up on reactions (3) immediately (a) Inoculate H-broth from an 18-24 ho1.4r
above: pure culture and incubate 18 hours at 30 C or
with heavy inoculum in 35 C water bath for 4
(1) Make a heavy suspension of antigen in hours.
0.5 ml of phenolized saline.
(b) Dilute the 18 hour culture 1: 1 with
(2) Heat the suspension for 15 minutes in a formalinized saline and mix. Allow to stand at
boiling water bath. room temperature for 1 hour.

(3) Cool and retest suspension in the (c) Pipet 0.5 ml of the formalinized culture
above three antisera. Include the three con- to small test tubes ( 13 x 100 mm), 1
trols as in 6.5. 1 (b). tube/antiserum.

(4) Compare results of slide agglutination (d) Prepare dilution of polyvalent H antis-
obtained with live and heated antigens. S. ty- era according to directions of manufacturer
phiwill give the following reactions: (usually 1: 1000). Add 0.5 ml to each tube
using a fresh pi pet for each tube. Mix by pipet-
Antigen ting the solution up and down several times.
Antiserum Live Heated
(e) Incubate tubes for 1 hour in 50 C water
bath. If the H-antigen is present,
Polyvalent Salmonella + or - + flocculation/agglutination may occur in 5-10
0 group D +
minutes but will occur within 1 hour. The posi.
Vi +
tive test is indicated by a diffused, fluffy sedi-
ment in the bottom of the tube. A negative
reaction gives a tight "button-like" group of
6.5.3 Alternative Procedure for Salmo- cells in the bottom of the tube.
nella, Including S. typhi: Edwards & Ewing and
Douglas & Washington describe this alternate
6.5.5 Confirmation of Salmonellato sero-
procedure as a more rapid serological method
type by an official typing center or state health
(1, 30).
laboratory is recommended when required for
tracing sources of contamination, for enforce-
(a) Slide test the organism for agglutina-
ment or for other Agency actions. This service
tion in polyvalent 0, Vi, and all of the common
is usually available if cultures are of public
0 group antisera at the same time. When indic-
health significance. See 11-C, 6 for details 011
ated, heat antigen as described in 6.5.2 (d).
proper shipment procedures.
(b) Interpret results as outlined in 6.5. 1
and 6.5.2.
7. Quantitative Techniques
6.5.4 Tube Test for H Antigen (1): Before
tests are made for H (flagellar) antigen, test for 7.1 Summary: These quantitative me-
·motility by inoculating the pure cultures into thods are time-consuming, but necessary
Motility Test Medium (5.5. 1 6), incubating and when it is desirable to determine salmonellae
reading. If the tests are negative, transfer the densities in recreational waters.. shellfish-
cultures again through the motility medium raising waters, stormwater run-off, wastewa-
before performing the flagellar antigen test. ters and sludges.
Motility medium in large diameter tubes or
small petri dishes may be inoculated on one
side and motile descendants picked on the 7.1.1 Samples are concentrated if neces-
other side after 24 hours. The test procedure sary by the standard techniques described in
follows: this Section.

SALMONELLA 179
 7.1.2 Samples or sample concentrates are 7.4 Concentration by Cartridge Filter (5)
inoculated into enrichment media in the stan-
dard 5-tube, 3-dilution sequence of the MPN. The cartridge filter is particularly useful for
See Part 11-C. concentrating large sample volumes (5). The
sample is placed in a sterile, calibrated
7.1.3 After incubation, each tube culture container and measured volumes (e.g. 10, 1
is streaked on selective plating media. and 0. 1 liters) are passed through separate
filters as described in this Section, 2.4. To
7. 1.4 Colonies reacting as Salmonella are speed the analyses, 5 replicate portions are
picked and confirmed biochemically or filtered simultaneously through 5 cartridge
serologically. filters using a manifold. If turbidity is a
problem, successive filters may be used. The
7.1.5 Based on the confirmation above, filters are removed aseptically and placed in
the number of tubes confirmed as positive for the selected enrichment media. The
Salmonella are tabulated and the MPN index concentration procedure is repeated for each
calculated from the regular MPN table. sample volume. Verification tests are done on
each enrichment culture and the MPN is
7.2 Concentration by MPN (15) calculated from the positive verification tests.

The need for concentration and sample

volumes required vary with the anticipated 8. Optional Fluorescent Antibody Screening
salmonellae densities. High density samples Technique
such as sewage may be inoculated directly as
10 ml portions into double-strength, or as 1 ml The fluorescent antibody (FA) technique
or less portions into single strength may be used as a rapid screening method for
enrichment media in MPN tubes (15). Low the detection of salmonellae particularly from
density samples may be concentrated by MF, large numbers of samples, after primary
diatomaceous earth, or cartridge filters before enrichment cultures (18-24 hours). Because
analysis by the MPN procedure. this technique does not require pure cultures
or serological typing, positive FA results should
7.3 Concentration by Membrane Filter be confirmed by the isolation procedures
or Dlatomaceous Earth Filter (12, 31, 32, 33) described in this Section, 4.5. If the FA test
is negative, the salmonellae detection pro-
Another quantitative technique uses either cedure may be terminated.
a large MF (142 mm diameter, 0.45 µm pore)
with filter aid (12) or diatomaceous earth with 8.1 Summary: An optional FA screening
a support pad. One liter or larger sample is technique concentrates bacteria from a water
either mixed with 1 % Celite and filtered through sample in diatomaceous earth ( Celite' or
an MF or is filtered through the diatomaceous equivalent). See this Section, 2.2. The diato-
earth and pad. After filtering, the Celite/MF maceous earth with the entrapped bacteria is
or diatomaceous plug and pad are placed in added to enrichment broth. After incubation
a sterile 1 pint (473 ml) blender jar containing of the broth, loopfuls of enrichment culture
100 ml sterile 0.1 % peptone water and blended are transferred to selective plating media
at about 5000 RPM for 1 minute on a Waring to produce spot cultures on the agar sur-
blender (see Part 11-C, 1.3). Beginning with faces. After the plates are incubated for 3
10 ml of the homogenate, serial tenfold hours, slide impression smears are prepared
dilutions (5 replicates per dilution) are inocu- from the micro-colonies and stained with a
lated Into enrichment media. Verification tests Salmonella polyvalent antiserum labeled with
are done on each positive enrichment tube fluorescent dye. Fluorescent cells indicate a
and the MPN is calculated from the number of positive reaction and the possible presence of
tubes which verify. Salmonella.

180 ~EPA MICROBIOLOGICAL MANUAL 1978

 8.2 Scope and Application (b) Fluorescent antibody pre-cleaned
micro slides, 2.5 X 7.6 cm.
8.2. 1 Advantages: Rapid screening of
large numbers of samples for Salmonella (c) Cover glasses for FA slides,. 16-. 19 mm
eliminates negative samples from further thick.
testing.
(d) FA Kirkpatrick fixative (Difeo 3188, or
8.2.2 Limitations: Careful interpretation of equivalent).
fluorescence is critical for this technique but
difficult to attain. Positive FA results must be · (e) Petri dishes, 100 x 15 mm, pyrex
confirmed by the conventional cultural glass.
techniques described above (see Part 111-E, 4, 5
and 6). The cost of equipment for fluorescence (f) Nail polish.
microscopy is approximately $5,000.
(g) Phosphate buffered saline, pH 8.0. Add
10 grams of dry FA buffer (Difeo 2314-33, or
8.3 Apparatus and Materials equivalent) to 1 liter of fresh laboratory pure
water. Dissolve completely and adjust pH to
8.3. 1 Equipment 8.0 with Na OH.

(a) Light microscope and low (h) FA mounting fluid (Difeo 2329-57, or
autofluorescence optics suitable for UV equivalent), reagent grade glycerine adjusted
microscopy. to pH of 9.0-9.6.

(b) Condenser, Cardioid Dark-Field, oil (i) Laboratory pure water.

immersion objective 95 x, with iris diaphragm
and with a numerical aperture (N.A.) at least U) Methanol or ethanol, 95%, for sterilizing
0.05 less than N.A. of objective lens. forceps.

(c) Filter Systems (k) Staining assembly consisting of jar,

cover and slide rack with handle. At least 5 are
Heat Filters - BG22, KG-1, KG-2 or needed: I each for fixative, ethanol and
equivalent. laboratory pure water and 2 for buffered saline
rinses.
Excitation Light Filters - UG 1 and BG 12 or
equivalent. ( I) FA Salmonella polyvalent conjugate is
a fluorescein conjugated anti-salmonellae
Barrier Filters - GG-9 and OG 1 or globulin (Difeo 3187, 3185; Sylvana
equivalent. 27-1 OOA, or equivalent).

(d) Intense Light System: Fluorescence When the conjugate is rehydrated, pre-
illuminator with power source, voltage pare 0.2-0.3 ml aliquots and freeze for future
regulator and mercury arc, quartz iodide or use. Rehydrated conjugates stored in the re-
tungsten light source. frigerator are not stable. (See 8.4.5 for titration
of conjugates).
(e) Incubator set at 35 C + 0.5 C.
(m) Moist chamber used to hold slides
containing conjugated-stained smears. A sim-
8.3.2 Materials ple chamber consists of a culture dish bottom
(150 X 20 mm) placed over wet toweling. A
(a) Non-drying low fluorescence immer- 'larger dish for this purpose may be prepared
sion oil. by placing moist towels onto a flat tray, then

SALMONELLA 181
placing the slides face-up on this surface and later placed over 2 of the drops, both inocula-
covering the tray with an inverted glass baking tion points will be included on the slide. This is
pan or similar metal pan. essential, since glass slide impression smears
of the inoculated points will be made after
8.3.3 Media incubation of the plates.

(a) Brain heart infusion (BHI) broth (Difeo (c) After the spots have been placed on the
0038, BBL 11059, or equivalent). See Part 11-B, agar surface of the differential plates, incubate
5.4.5 .. the plates at 35 C for 3 hours.

(b) Brilliant green agar (BG) (BBL 11073, (d) Remove the plates after incubation and
Difeo 0285-01 ). See Part 11-B, 5.5.5. make impression smears.

(c) Xylose lysine desoxycholate agar (XLD) 8.4.3 Impression Slides: Place a clean FA
(BBL 11838, Difeo 0788-01 ). See Part 11-B, X 3 inch glass slide (nonfluorescent) over 2
6.5.7. of the inoculated points on the agar. Press
down lightly, without moving the glass slide to
(d) Xylose lysine brilliant green agar either side. ·Too much pressure will cause
(XLBG) XL agar base (BBL 11836, Difeo 0555). movement of the slide and accumulation of
See Part 11-B, 5.5.6. agar on the slide. Repeat the process for the
remaining 2 inoculation points with another
8.4 FA Staining Techniques (13, 34, 35) clean slide. Make impression slides for each
differential plate.
8.4. 1 Preparation

Collect samples and concentrate them by

cartridge filter, membrane filter or diato- (a) Air dry the smears.
msceous earth technique as described in this
Section, 2.2, 2.3 and 2.4. (b) Fix for 2 minutes in Kirkpartick's Fixa-
tive (mix 60 ml absolute ~thanol, 30 ml chloro-
After concentration, place whole or half form, and 10 ml formalin).
plugs and pads, cartridge filters or membrane
filters into flasks of selenite broth, tetrathionate (c) Rinse the slides briefly in 95% ethanol.
broth, dulcitol selenite broth or tetrathionate
brilliant green broth. Incubate flasks for (d) Air dry. Do not blot.
18-24 hours at 35 and 41.5 C. See this
Section, 3. 8.4.5 Titration of Conjugates

8.4.2 Spot Culture Plates: After the primary

enrichment has been incubated 18-24 hours, After the slides have been fixed, stain with
prepare spot culture plates on the differential Salmonella FA conjugate. However, each lot
media: brilliant green {BG) agar, xylose lysine number of conjugate must be titered before
desoxycholate (XLD) agar, and xylose lysine use.
brilliant green (XLBG) agar as follows:
Based on the previous experience of the
(a) Mark the bottom of the plate to locate analyst or other workers, the conjugate and
the drops of inoculum then place 1 drop of test components must have been proven with
enrichment culture at 4 separate points on the water samples. Sources of polyvalent conju-
surface of each solid medium. gates known to be acceptable (CDC Approved
List) are: Burroughs Wellcome & Company,
(b) Space the drops 6-7 cm apart on the Clinical Sciences Inc., Difeo Laboratories and
piste so that when an FA microscope slide is Sylvana Company.

182 &EPA MICROBIOLOGICAL MANUAL 1978

 The Difeo Bacto-FA Poly Conjugate con- nella culture as a procedure control. Place the
tains antibodies against the major 0, Vi and H slides in a moist chamber to prevent evapora-
antigens. The Sylvana Salmonella Polyvalent tion of the staining reagent. Tap off excess
OH Globulin represents the somatic antigens reagent onto paper towel.
of Salmonella 0 groups A through S. The Difeo
Panvalent conjugate is not recommended be- (b) After 30 minutes, wash away the ex-
cause of excessive cross-reactions. cess reagent by dipping the slides into a bath
of saline buffered at pH 7 .2.
Titer the conjugate as follows:
(c) Transfer the slides to a second bath of
(a) Inoculate a known Salmonella culture buffered saline for 10 minutes.
into BHI broth. Incubate at 35 C for 18-24
hours. (d) Replace the rinse solutions in (b) and (c)
above, after each use.
(b) From this broth culture, make smears
on clean FA glass slides. Prepare enough (e) Rem'ove slides, rinse in a laboratory
smears for each conjugate dilution, and con- pure wat~r bath and allow to drain dry. Do not
trols. Include a known 4 + Salmonella control blot.
to be used as a reading standard. Air dry the
smears and follow the instructions for fixing (f) Place a small drop of FA mounting fluid
slides, Part 111-E, 8.4.4. (Difeo 2329-5 7) onto the smear and cover
with a coverslip. Seal the edges of the cover-
(c) Prepare the following dilutions of the slip with nail polish. Such slides may be stored
conjugate in buffered saline: 1 :2, 1 :4, 1 :6, 1 :8 for up to 1 year in a freezer at -70 C with
and 1: 10. Most batches are effective at the 1 :4 minimal loss of fluorescence.
dilution.
(g) Examine the slides under a
(d) Cover a smear with one of the conju- fluorescence microscope (900-1000 X) fitted
gate dilutions or the undiluted conjugate. with the proper filters. Use a known 4 + slide
Place slides in moist chamber and proceed as for comparison. Read slides within 2 1h hours if
in Part 111-E, 8.4.6 (b)-(f) and 8.4. 7 (a)-(b). Use the slides have not been sealed and stored in a
known 4 + Salmonella control as the standard. deep-freeze.

(e) The titer of conjugate to be used is the 8.4.7 Interpretation of Fluorescence, Re-
second highest dilution which gives 4+ porting Results
fluorescence. For example, if the conjugate
dilutions outlined above in 8.4.5 (c) above gave (a) Fluoresence results are recorded as
fluorescence intensity ratings (in order) 4+, shown in the following table. The number of
4+, 4+, 2+, 1 +,the conjugate dilution to be fluorescing cells and the degree of
employed would be 1 :4. This value should be fluorescence/cell are the criteria on which
marked on the conjugate bottle. Prepare an positive results are based. Smears with large
amount of chosen conjugate dilution sufficient numbers of strongly fluorescent cells (3 + or
for each day's run. 4+) are positve; weakly fluorescent cells are
negative. Smears with few fluorescent cells
8.4.6 Staining Impression Slides: After the should be examined carefully.
slides have been fixed, they are stained with
Salmonella FA conjugate. (b) Cultures giving positive FA reactions
(4+ or 3+) must be isolated on differential
(a) Cover each smear with one drop of the plating media, confirmed by biochemical iden-
predetermined dilutions of conjugate (Difeo tification, and verified serologically. Cultures
FA Salmonella Polyvalent, or Sylvana Polyva- displaying negative FA results may be
lent OH Globulin). Include a known 4 + Sa/mo- discarded.

SALMONELLA 183
 Rating Intensity Description
4+ Brilliant yellow-green fluorescence, cell sharply-outlined
Positive
3+ Bright yellow-green fluorescence, cell sharply outlined with
dark centers

2+ Dull yellow-green fluorescence, cells not sharply outlined

Negative
1+ Faint green discernible in dense ar·eas, cells not outlined
0 No fluorescence

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28. Robertson, E. A. and J. D. MacLowry, 1974. Mathematical analysis of the API enteric 20 profile
register using a computer diagnostic model. Appl. Microbiol. 28:691.

29. Robertson, E. A. and J. D. MacLowry, 1975. Construction of an interpretive pattern directory for
the API 10 Skit and analysis of its diagnotstic accuracy. J. of Clin. Microbial. .!.:_6:515.

30. ·Douglas, G. W. and J. A Washington, 1973. Identification of Enterobacteriaceae in the clinical

laboratory, Center for Disease Control, Public Health Service, USDHEW, Atlanta, GA.

31. Presnell, M. N. and W. H. Andrews, 1976. Use of the membrane filter and a filter aid for concen-
trating and enumerating indicator bacteria and Salmonella from estuarine waters. Water
Research 10:549.
32. Kaper, J. B., G. S. Sayler, M. M. Baldini and R. R. Colwell, 1977. Ambient-Temperature primary
nonselective enrichment for isolation of Salmonella spp. from an estuarine environment. Appl. &
Environ. Microbial. 33:4:829. -- -

33. Olivieri, V. P. and S. C. Riggio, 1976. Experience on the assay of microorganisms in urban runoff,
in proceeding of workshop on microorganisms in urban stormwater, EPA-60012-76-244,
Municipal Environmental Research Laboratory, EPA, Cincinnati, OH.

34. Thomason, B. M., 1971. Rapid detection of Salmonella microcolonies by fluorescent antibody.
Appl. Microbial. 22: 1064.

35. Thomason, B. M. and J. G. Wells, 1971. Preparation and testing of polyvalent conjugates for
fluorescent-antibody detection of salmonellae. Appl. Microbiol. 22:876.

SALMONELLA 185
 PART Ill. ANALYTICAL METHODOLOGY

Section F Actinomycetes

1. Summary of Method
3. Scope and Application
The pour-plate technique is the prinicipal
method for measuring actinomycete popula- 3.1 Actinomycetes are of interest in water
tions. Selective media are employed that favor treatment and waste treatment facilities be-
actlnomycete development over fungi and cause of the taste and odor problems they
other bacteria. The mass of branching fila- cause in potable waters and fish, and the foam-
ments characteristic of this bacterial group ing problems they can cause in waste treat-
offers distinctive features for identification. To ment plants.
facilitate identification and counting of the ac-
tinomycete colonies, plates are prepared by 3.2 The taste and odor problems result
the two-layer agar technique. Since only the from volatile products characterized by an in-
upper layer is inoculated, the method assures a tense earthy-musty odor (2, 3). Evidence points
predominance of surface colonies. to 2 highly odoriferous metabolites, geosmin
and 2-methylisoborneol, as the sources of the
2. Definition problem (4, 5, 6). It appears that the relative
abundance of these 2 metabolites in natural
2.1 The actinomycetes are a group of waters is linked to an ecological factor not yet
microorganisms with cells ranging from resolved. Traces of these products can impart
0.6-2.0 µm diameter but normally less than a disagreeable persistent odor to a municipal
1.0 µm diameter, which usually develop as water supply, which is extremely difficult to
non-septate hyphae in branching mycelial treat. These natural odorants, prevalent in
masses. The actinomycetes are generally many parts of the world, can also affect com-
saprophytic but some are parasitic or patho- mercial fishing.
genic to plants, animals and man.
3.3 Actinomycete distribution in waters
2.2 The actinomycetes are fungal in mor- with an earthy-musty odor shows a correlation
phology and in spore formation, but lack a between actinomycete counts and odor levels,
membrane around nuclear materials. They indicating such enumeration to be a useful
have a sensitivity to bacterial antibiotics, are parameter in measuring quality of water. The
susceptible to specific phage and have other genus of most interest is Streptomyces.
biochemical characteristics which class them
as filamentuous, branching bacteria ( 1). Al- 3.4 Actinomycetes have also been recog-
though actinomycetes are found in water and nized as the cause of disturbances in the oper-
sediments, the greatest natural reservoir for ation of activated sludge wastewater treat-
these organisms is the soil. ment plants where massive growths of these

186 &EPA MICROBIOLOGICAL MANUAL 1978

organisms can produce thick foams (7, 8). The 4. 11 Screw-cap culture tubes, borosilicate
genus involved is Nocardia. glass, 25 X 150 mm.

3.5 Because of diverse nutrient require- 4. 12 Dilution bottles (milk dilution), pyrex,
ments, no single medium has been devised marked at 99 ml, screw-cap with neoprene
that will support the growth of all actinomy- rubber liner.
cetes. Moreover, the culture media that have
proven useful in their isolation are not neces-
sarily the preferred media for encouraging 5. Media
abundant growth. The isolation media are re-
strictive and nutritionally poor. They act by 5.1 Sterile starch-casein agar or equiva-
depressing growth of other microorganisms lent agar prepared in 17 ml volumes in screw-
and by favoring a higher proportion of actino- cap tubes and in 250-300 ml volumes in 500
mycete colony development. ml, screw-cap bottles or flasks. See Part 11-B,
5.6.
3.6 The pour-plate method does not indic-
ate whether the isolated colony originated 5.2 Sterile buffered dilution water in bot-
from individual spores, spore aggregates, tles containing 99 + 2. ml volumes. See P~rt 11-
small mycelial fragments or a mycelial mat. B, 7.

5.3 Cycloheximide Stock Solution. Weigh

4. Apparatus and Materials out 100 mg cycloheximide (antifungal
antibiotic), bring to 100 ml with distilled water,
4.1 Incubator set at 28 + 0.5 C. pour int() a screw-cap flask and.mix until dis-
solved. Sterilize for 15 minutes. at 121 C ( 15
4.2 Water bath set at 44-46 C for temper- lbs. pressure). Cycloheximide is available as
ing agar. Actidionefrom the Up john Company.

4.3 Electric oven set at 105-110 C.

6. Sample Preparation
4.4 Hand tally or electronic counting de-
vice (optional). 6.1 Water Samples

4.5 Thermometer· which has been 6.1.1 F'ili sa.mple bottle only 3/4 f~ll so
checked against a National Bureau of that ample airspace is left in the bottle for
.Standards-Certified Thermometer. thorough mjxing of the water.sample .

4.6 Pipet containers of stainless steel, alu- 6. 1.2 Mix water sample by shaking vigor-
minum or pyrex glass for glass pi pets. ously about 25 times. Using a pipet, transfer
11 ml immediately after mixing to a 99 ml
4. 7 Petri dish canister of stainless steel or water blank.
aluminum for glass dishes.
6. 1.3 Repeat for. desired dilutions. A dilu-
4.8 Erlenmeyer flasks, pyrex, screw-cap, tion of 1o-3 is usually sufficient for plating
250 and 500 ml volume. water samples.

4.9 Sterile T.D., bacteriological or Mohr 6.2 Soil Samples

pi pets, glass or plastic, of appropriate size.
6.'2.1 Mix so'il . sample thoroughly and
4. 10 Sterile 100 mm X 1 5 mm petri weigh out a 50 gram sample in a tared weigh-
dishes, glass or plastic. ing pan. Dry at 105-110 C to constant weight.

· ACTINOMYCETES 187
The final weight is used in calculating numbers 8.2 Rules for making plate cou'nts are
of organisms/gram dry soil. given in Part Ill-A, 5.6.2.

6.2.2 Prepare the initial dilution by weigh- 8.3 Examine plates macroscopically hold-
ing out 11 grams of soil and adding to a 99 ml ing toward a light source. Actinomycete colo-
volume of buffered dilution water for a 1: 10 nies appear dull or chalky when covered with
dilution. Shake dilution bottle vigorously for 1 aerial mycelium. The edges of the colonies are
minute. less dense producing a halo effect. Colonies
adhere strongly to the agar and have a tough,
6.2.3 Transfer a 11 ml sample of the 1: 10 leathery texture. In contrast, bacterial colonies
dilution to a second dilution bottle containing ·appear shiny or opalescent with a soft texture,
99 ml buffered dilution water and shake vigor- adhere weakly to the agar, and show no gen-
ously about 25 times. Repeat this process until eral distinction between the edge and the col-
the desired dilution is reached. Dilution of ony as a whole (see Figure 111-F-1 ).
1 o-3 to 1 o- 6are usually necessary for enumer-
ation of soil samples. 8.4 Actinomycete colonies can be verified
at a magnification of 100 x. Because of their
filamentous growth, they typically have fuzzy
7. Plating Procedures borders which contrast sharply to the smooth
borders characteristic of bacteria. (See Figures
Prepare triplicate plates for each test dilu- 111-F-2 and 3).
tion using the two-layer agar technique as
follows: 8.5 Addition of cycloheximide to the isola-
tion medium suppresses development of fun-
7.1 Cool flask of starch casein agar to gal colonies. If fungi do develop, they can be
44-46 C and pour 15 ml layer in each petri differentiated from actinomycetes by their
dish. Allow to harden. woolly appearance and much larger cell diam-
eter. With a little experience in examining fun-
7.2 Melt starch-casein agar in tubes and gal and actinomycete colonies, it is fairly easy
cool to 44-46 C. Add 2 ml of sample or sample to distinguish them macroscopically.
dilution and 1 ml of cycloheximide solution.
Mix tube contents well.
9. Reporting Results
7.3 Pipet immediately 5 ml of the inocu-
lated agar over the solid agar base layer in 9. 1 Calculate the actinomycete density in
each plate with gentle swirling to evenly dis- water samples in counts/ml according to the
tribute the inoculated agar. Each 5 ml contains following equation:
0.5 ml of the particular dilution used. This 0.5 Sum of Replicate Plate Counts
factor must be taken into consideration in cal-
Total Volume of Original
culating the final dilution.
Sample Tested, in ml
7 .4 After plates are solidified, invert and
Incubate at 28 C for approximately 7 days and
count. Sum of Replicate Plate Counts
No of
Replicates X
Sample Dilution
Tested x Agar Plate
Dilution
8. Counting of Colonies Factor (See 7 .3)

8.1 Select plates for counting with Actinomycete

30-300 colonies. =
Count/ml

188 oEPA MICROBIOLOGICAL MANUAL 1978

FIGURE 111-F-1. A plate containing bacterial and actinomycete colonies.

A- The dull, powdery appearance of a sporulating actinomycete colony.

B- The smooth, mucoid appearance of a bacterial colony.

ACT/NOMYCETES 189
 FIGURE lll-F-2. An actinomycete colony showing the branching filaments
that cause the fuzzy appearance of its border. X 225

FIGURE lll-F-3. A bacterial colony with its relatively-distinct, smooth

border. x 225

190 oEPA MICROBIOLOGICAL MANUAL 1978

 For example, if the triplicate sample vol- 6.2, this Section. Calculate the counts as
umes tested at a 1: 10 dilution yielded 45, 39 follows:
and 42 actinomycete colonies, the calculation
would be: wt. of collected soil
wt. of dried soil conversion factor
126

3(rep) x 0.1 (dil) X 0.5 (A.P.D. factor)

conversion factor x count/gm collected
soil = count/gm dry soil
126 840
Determine count/gm collected soil by the
0.15 Actinomycetes/ml
equation given in 9.1.
9.2 Correct the actinomycete counts from '
soil and mud samples for water contents. See

REFERENCES

1. Lechevalier, H. A. and M. P. Lechevalier, 1967. Biology of Actinomycetes. Am. Rev. Microbial.

21:71. - -

2. Adams, B. A., 1929. C/adothrixdichotomaand allied organisms as a cause of an "indeterminate"

taste in chlorinated water. Water & Water Eng. ~:327.

3. Thaysen, A. C., 1936. The origin of an earthy or muddy taint in fish. Ann. Appl. Biol. 23:99.

4. Rosen, A. S., C. I. Mashni and R. S. Safferman, 1970. Recent developments in the chemistry of
odour in water: the cause of earthy/musty odour. Water Treatment Exam.~: 106.

5. Piet, G. J., B. C. J. Zoeteman and A. J. A. Kraayeveld, 1972. Earthy-smelling substance in surface

waters of the Netherlands. Water Treatment Exam. 21 :281.

6. Yurkowski, M. and J. L. Tabachek, 1974. Identification, analysis and removal of geosmin from
muddy-flavored trout. J. Fish. Res. Board Can. 31 :1851.

7. Lechevalier, H. A., 1975. Actinomycetes of sewage-treatment plants. U.S. Environmental Protec-

tion Agency, Environmental Protection_Technology Series, EPA-600/2-75-031, Cincinnati, OH.

8. Lechevalier, M. P. and H. A. Lechevalier, 1974. Nocardia amarae, sp. nov., an actinomycete

common in foaming activated sludge. Int. J. Syst. Bacterial. 24:278.

ACTINOMYCETES 191
 PART IV. QUALITY ASSURANCE

Regulatory agencies making decisions on water quality standards and wastewater discharge
limits require formal analytical quality control programs for their laboratories and program
participants to assure validity of their data. For example, water quality regulations include
provisions for quality control and testing procedures (the Safe Drinking Water Act, National Interim
Primary Drinking Water Regulations published in Federal Register, Title 40 Part 141, 59566,
December 24, 1975). Further, the Act states that analyses for maximum contaminant levels must
be conducted by laboratories approved in a formal certification program. The quality control
procedures specified in the Manual for the Interim Certification of Laboratories Involved in
Analyzing Public Drinking Water Supplies (Appendix B) are a recommended minimal program and
are not equivalent to the comprehensive system described in Part IV of this Manual.

A laboratory quality control program is the orderly application of the practices necessary to
remove or reduce the errors that occur in any laboratory operation due to personnel, equipment,
supplies, sampling procedures and the analytical methodology in use.

A quality control program must be practical, integrated and require only a reasonable amount
of time or it will be by-passed. When properly administered, a balanced, conscientiously applied
quality control program will assure the production of uniformly high quality data without inter-
fering with the primary analytical functions of the laboratory. This within-laboratory program
should be supplemented by participation of the laboratory in an interlaboratory quality control
program such as that conducted by EPA and detailed in Part V. The major considerations for quality
control are discussed under three separate Sections:

Section A Laboratory Operations

1. Sample Collection and Handling

2. Laboratory Facilities
3. Laboratory Personnel
4. Laboratory Equipment and Instrumentation
5. General Laboratory Supplies
6. Membrane Filters
7. Culture Media

Section B Statistics for Microbiology

1. Measures of Control Tendency

2. Measures of Dispersion
3. Normal Distribution
4. Poisson Distribution
5. Measures of Performance

192 &EPA MICROBIOLOGICAL MANUAL 1978

Section C Analytical Quality Control Procedures

1. Quality Control on Routine Analyses

2. Quality Control in Compliance Monitoring
3. Comparative Testing of Methodologies

QA/LABORATORY OPERATIONS 193

 PART IV. QUALITY ASSURANCE

Section A Laboratory Operations

Section A describes the checks and moni- Sample sites and a sampling frequency
toring procedures that should be performed are selected to provide data representative of
on materials, supplies, instrumentation and the characteristics and the variability of the
the physical facility. These checks should be water quality at that station. The most important
documented completely and recorded as per- quality control factor in sampling is the
formed. See Part V-A, 1.2 for details on this immediate analysis of the sample. If the sample
documentation. cannot be analyzed at once, it should be
refrigerated and analyzed within six hours.
1. Sample Collection and Recommended procedures for collecting,
transporting and handling water and waste-
Handling water samples are described separately in
Part II-A of this Manual.
2. Laboratory Facilities

3. Laboratory Personnel 2. Labora~ory Facilities

4. Laboratory Equipment and 2.1 Ventilation: Laboratories should be

well-ventilated and free of dust, drafts and
Instrumentation extreme temperature changes. Central air con-
ditioning has advantages: 1) The incoming air
5. General Laboratory Supplies is filtered, reducing contamination of the labo-
ratory and culture work. 2) The uniform tem-
6. Membrane Filters perature control of air conditioning permits
stable operation of incubators. 3) With closed
7. Culture Media windows, drafts and air currents which can
cross-contaminate surroundings and work
areas during warm weather are minimized. 4)
1. Sample Collection and Handling Low humidity reduces moisture problems with
media, chemicals, analytical balances and
The acquisition of valid data begins with other instrumentation.
collection of a representative water sample or
other environmental material being tested. 2.2 Space Utilization: Ideally the areas
Samples must be maintained as closely as provided for the preparation and sterilization
possible to original condition by careful han- of media, glassware and equipment should be
dling and storage. separated from the laboratory working area
but located close enough for convenience. In
public health laboratories that analyze many

194 oEPA MICROBIOLOGICAL MANUAL 1978

different types of samples a separate work 2.5 Monitoring for Cleanliness in Work
area is desirable for water analysis. Special Areas: High standards of cleanliness should
work areas such as an absolute barrier or be maintained in work areas. The laboratory
vented laminar flow hood (see Part V-C) are can be monitored for cleanliness by one or
often used for dispensing and preparing sterile more of the procedures described below.
media and tissue cultures, for transferring Since these monitoring procedures cannot re-
microbial cultures or· for working with. cover all of the microorganism populations
pathogenic materials. In smaller laboratories it present, absolute limits are difficult to develop.
may be necessary to carry out these separate Rather, the tests should be used regularly on a
activities in different sections of the same .vyeekly or other basis to monitor counts in the
room. However, limited facilities and restricted same work areas over time or to make compar-
work space may seriously hamper the quality isons between different work areas.
of the work and influence the validity of
results. Visitors and through traffic should be 2.5. 1 RODAC Agar Plates ( 1)
discouraged in work. areas. Through traffic can
be prevented by laboratory design. Work areas and other surfacel) can be
checked by RODAC plates which contain gen-
· 2.3 Laboratory Bench Areas: Sufficient eral growth media for total counts or selective
clean bench space should be available for the media for coliforms, enteric pathogens, strep-
analyses to be performed efficiently. For rou- tococci, staphylococci, or other microorga-
tine work, 6 linear feet is the recommended nisms cultured in the laboratory. RODAC is an
minimum work area for each analyst. Research acronym for Reproducible Organism Detection
work or other analyses using specialized and Counting.-The RODACdish has atest area
equipment may require significantly more of about 25 cm with Quebec style grids em-
space per worker. These estimates of bench bossed in the plate. It is specifically designed
space are exclusive of work areas used for to enumerate the microbial population of fl<!t
preparatory and supporting activities. Labora- solid surfaces by contacttechniques.
tory lighting should be even, screened to re-
duce glare, and provide about 100 footcandle (a) Purchase RODAC plates prefilled with
light intensity at working surfaces. desired test medium or prepare plates by fill-
ing the center well with about 1 6 ml of appro-
Bench tops should be set at heights of priate agar. When preparing agar.in the labora-
35....:38 inches with a depth of 28-30 inches. tory, add 0.0.7% soy lecithin or 0.5% polysor-
This height is comfortable for work in a stand" bate (Tween 80) to the agar to neutralize the
ing or sitting position. Desk tops or sit-down effect of the disinfectant on test surfaces.
benches are set at 30-31 inches height to Cooling leaves a raised bed of agar about 1
accommodate microscopy, plate counting, cal- mm higher than the rim of the dish, allowing
culations and writing activities. Bench tops contact of the sterile agar to the test surface
should be stainless steel, epoxy plastic or for direct counts.
other smooth impervious material which is in-
ert, corrosion-resistant and .has minimum (b) To sample an area, remove the plastic
seams. cover and carefully press the agar to the solid
surface being sampled. It is important that the
2.4 Walls and Floors: Walls should be entire agar layer contacts the test surface. Use
covered with waterproof paint, enamel or a rolling motion with uniform pressure on the
other surface material that provides a smooth back of the plate to insure complete contact.
finish which is easily cleaned and disinfected. Replace the cover and incubate in an inverted
Floors should be covered with good quality position for the appropriate time and
tiles or other heavy duty material which can be temperature.
maintained with skid-proof wax. Bacteriostatic
agents contained in some wall or floor finishes (c) Count the colonies with a Quebec col-
increase the effectiveness of disinfection. ony counter and report as the number of cola-

oEPA MICROBIOLOGICAL MANUAL 1978 195

nles per RODAC plate or number of colonies vial and break or cut with a sterile scissors
per 25 sq. cm. leaving the swab head in the vial. Replace the
cap and store vial at 4 Coron i~e until analysis.
2.5.2 Swab Method (1)
When using alginate swabs, rinse solution
(a) The swab contact method can be used vials should contain 4.5 ml after sterilization.
to monitor the contamination of work areas Prepare a 10% solution of sodium
and especially those with cracks, corners, hexametaphosphate in appropriate vials and
crevices and rough surfaces. Dacron swabs steam sterilize. Follow the above swabbing
rinse out more easily than cotton ones. These procedures but after swabbing the fifth 8 sq.
may be purchased from Econ Microbiological in. area, deposit the swab head in the rinse vial
Laboratory, 2716 Humboldt Avenue, South, and add 0.5 ml of sterile hexametaphosphate
Minneapolis, MN. Rayon swabs are available solution.
from Fuller Pharmaceutical Company, Minne- To begin plating, shake the vial vigorously
apolis, MN, and Consolidated Laboratories to dislodge the bacteria from the swab. Pi pet 1
Inc., Chicago Heights, IL. Swabs made of cal- ml and 0.1 ml aliquots of the rinse solution.
cium alginate which is soluble in water, can The 1 ml portions represent a 1: 10 dilution
also be used. These are available from Consoli- and the 0. 1 ml portions represent a 1: 100
dated Laboratories, Inc., Chicago Heights, IL. dilution. Pour the plates with Standard Me-
thods Agar. Mix and cool to solidify and incu-
Rinse Solution Vials - Add 1.25 ml stock bate at 35 C for 48 hours.
phosphate buffer solution, 5 ml of 10% aque-
ous sodium thiosulfate, 4 grams Asolectin (As- (c) Count colonies and convert the value to
sociated Concentrates, 32-30 61 st Street, the number/ml which equals the count per 8
Woodside, Long Island, NY 11377) and 10 sq. in. of area.
grams Tween 20 or Tween 80 (Hilltop Re-
search, Inc., Miamiville, OH 4514 7) to 500 ml 2.5.3 Air Density Plates ( 1)
distilled water; heat to solution in boiling water
bath. Cool and make up to 1 liter. Dispense in The number of microorganisms in the lab-
screw-capped vials in 10 ml or other volumes oratory air is directly proportional to the
and sterilize for 15 minutes at 15 lb. pressure amount and kind of activity. These organisms
(121 C). Because Asolectin is hygroscopic, affect results when the suspended cells con-
store it in a desiccator and weigh quickly. tact materials and equipment or when they
settle on exposed test materials and surfaces.
(b) To sample an area, open a sterile swab
container, grasp the end of the stick and re- (a) The numbers and types of airborne
move aseptically. Open a 10 ml vial of neutral- microorganisms can be determined by expos-
izing buffer, moisten swab head, and press out ing petri plates for a specified time at points
excess solution against the inside of the vial. where inoculating, filtering, plating and trans-
Hold the swab handle at a 30° angle against fer work is done. This exposure method can be
the sampling area. Rub the swab head slowly used to monitor total bacteria or the specific
and throughly over approximately 8 sq. in. of organisms being tested such as coliforms, en-
surface area. Repeat procedure 3 times over teric pathogens, streptococci, staphylococci,
the same area, turning the swab and reversing yeasts or molds.
directions. Place the swab head in the neutral-
lziog buffer vial, rinse briefly in solution and (b) Pour the petri dishes with the appropri-
press out the excess liquid. ate agar and allow to harden. Store poured
plates in the refrigerator if they are not used on
Test four more 8 sq. in. areas of surface, the same day.
rinsing the swab in solution after each swab-
bing. After the fifth area has been swabbed, (c) Remove petri dish covers and place
remove the excess liquid, place swab head in dishes top side up on sterile towels. Expose the

196 QA/LABORATORY OPERATIONS

plates in selected work sites for 15 minutes ety and complexity of the tasks and tests per-
and mark them with sample site identification. formed determine the professional and on-the-
Replace covers. Incubate Standard Methods job-training required.
Agar at 35 C for 48 hours and other media for
the specified time periods. 3.1 Professional Microbiologist: The mi-
crobiologist directs and participates in the col-
(d) Count the colonies and report the num- lection and storage of samples, the prepara-
ber per square foot. The number of organisms tion of glassware, equipment and media, the
which settle in 15 minutes of exposure on a analysis of a variety of waters and wastewa-
petri dish is equivalent to that for 1 sq, ters and the evaluation of procedures as re- .
ft./minute because the area of a standard-size quired. He takes part in the quality control
petri dish is approximately 1/15 sq. ft. The program. He counts, tabulates and summa-
microbial density should not normally exceed rizes data and prepares or helps to prepare
15 colonies per sq. ft. Air density plates should reports from the results. He should· have at
be taken weekly during peak work periods for least a BS degree in microbiology or a BS/BA
routine monitoring. degree in biology with a minor in
microbiology.
2.6 Laboratory Maintenance: Laboratory
benches, shelves, floors and windows should The professional microbiologist initially
be cleaned on a scheduled basis. Proper main- carries out routine tasks and progresses to
tenance is evidenced by lack of dust and soil more difficult work as he gains experience.
build-up on shelves, in corners, etc. Floors When he reaches the senior level he performs
should be wet-mopped and treated with a dis- the most complex duties.
infectant solution to reduce contamination of
air in the laboratory. Sweeping or dry-mopping 3.2 Senior Grade and Supervisory Micro-
should not be permitted in a microbiology biologists: A senior grade microbiologist per-
laboratory. Work benches should be wiped forms the duties described in 3.1 but carries
down with disinfectant before and after each out a wider range of assignments and more
use and at the end of the day. difficult tests. He participates in program plan-
ning and laboratory management and solves
Every attempt should be made to maintain significant microbiological problems that re-
the laboratory areas free of clutter. Laboratory quire a higher than bench-level skill and knowl-
benches, space under benches, shelves and edge. The experienced microbiologist consults
drawers accumulate equipment, materials and and advises on procedural problems and trains
supplies at a steady rate. The disorder can personnel. He provides expert testimony,
interfere with laboratory operations. It can be interprets results, prepares reports and
controlled by making a directed effort to clean recommends microbiological standards or
up work areas immediately after each use and actions for regulatory programs.
by conducting a weekly clean-up of the labora-
tory. Discard unneeded materials and store The supervisory microbiologist performs
equipment and supplies not in current use. The administrative functions of: planning and
·most important step in maintaining an orderly directing water quality monitoring programs,
laboratory is to have ample storage space designing field surveys, advising administrative
near-by. officers on policy matters related to micro-
biology and inspecting laboratory facilities. He
is recognized for his authoritative scientific
3. Laboratory Personnel competence.

Microbiologists, technicians and support 3.3 Technicians: The technician performs

personnel in the microbiology laboratory must semi-professional and professional duties of
have training and experience appropriate to limited scope and complexity. Typically, he
the laboratory's analytical program. The vari- assists professionals by doing the routine tests.

QA/LABORATORY OPERATIONS 197

Under supervision, he performs tasks involving non-professional technician, an approved con-
a series of steps. Technicians learn through sultant microbiologist should be available for
on-the-job instruction and by performing guidance and assistance.
standard tasks repetitively.
Work assignments in the laboratory
Technicians' tasks begin at tpe simplest" should be clearly defined. The analyst should
level but can progress to the mor~ detailed be trained in basic laboratory procedures and
procedures performed by the professional mi- should perform well at a given level of respon-
crobiologist. However, these higher levels of sibility before new assignments are made. The
skill and knowledge are generally limited to supervisor should periodically review proce-
the specific areas in which they have received dures such as sample collecting and handling,
on-the-job-training. media and glassware preparation, sterilization·,
routine testing procedures, counting, data han-
dling, and quality control techniques. Problem
3.4 Support Personnel: Laboratory aides
areas should be identified and solved by the
and clerks provide the necessary support serv-
staff. Improved laboratory results will be the
ices to the laboratory. Aides prepare glass-
measure of effective personnel practice and
ware, make media and sterilize materials. One
training.
or. more aides are recommended for every
bench microbiologist. Clerical duties include
recording data, filing records, typing reports,
4. Laboratory Equipment and
ordering supplies, maintaining inventories,
Instrumentation
distributing mail and answering the telephone.
Quality control of laboratory apparatus in-
3.5 On-the-Job-Training and Experience: cludes servicing and monitoring the operation
In addition to the formal academic training, the of incubators, waterbaths, hot-air sterilizing
professional microbiologist should have ovens, autoclaves, water stills, refrigerators,
experience in aquatic or environmental freezers, etc. Each item of equipment should
microbiology. Technical or on-the-job-training be tested to verify that it meets the manufac-
for at least two weeks is required for each turer's claims and the user's needs for accu-
parameter tested in water microbiology. The racy and precision. Maintenance should be
formal training of the technician ranges from performed on a regular basis by a technician
high school to technical or academic training who is familiar with the equipment.
short of a degree in microbiology. Technical
and scientific experience is often substituted 4.1 A summary of recommended monitor-
for advanced formal training. ing procedures for laboratory equipment is
given in Table IV-A-1.
The microbiologist and technician should
be encouraged to attend courses at centers of 4.2 Monitoring Procedures for UV
expertise such as universities, commercial Lamps
manufacturers, US EPA facilities, the Center
for Disease Control (CDC) of USPHS, FDA and 4.2. 1 Spread Plate Irradiation
other federal and state governmental agen-
cies. Courses might include fluorescent anti- This test should be run when a new UV
body techniques, isolation and identification lamp is installed and rerun quarterly to mea-
of the Enterobacteriaceae and laboratory sure continuing effectiveness of the UV
safety. irradiation.

3.6 Laboratory Supervison: Ideally, the (a) Prepare 100 ml or more of plate count
laboratory should be directed by a profes- agar. Pour 10-1 5 ml of the melted agar into
sional microbiologist. However, in a small each 100 mm petri dish as needed. Keep cov-
laboratory where the staff consists of a single ers opened slightly until agar has hardened·

198 oEPA MICROBIOLOGICAL MANUAL 1978

and moisture and condensation have evapo- 4.2.2 Measurements of UV Light Intensity
rated. Close dishes and store in refrigerator
until use. To monitor performance of UV lamps, it is
necessary to measure UV light intensity using
(b) Prepare a series of dilutions of a coli- the UV light meter manufactured for this pur-
form culture so that 0.5 ml of inoculum will pose. The shortwave UV meter Model J-225 is
give a 200-250 colony count (see Geldreich available from UV Products, Inc., San Gabriel,
and Clark). CA 91778 for about $125. This instrument or
equivalent is recommended.
(c) Pipet 0.5 ml of the selected dilution to
each agar plate.
(a) Measure and average light intensities
of a new UV lamp at proper distances of use.
(d) Remove a glass spreader-rod from the
Record results in QC log, noting readings and
alcohol container and ignite by passing
dates of installation.
through flame. Let burn completely and cool
for 15 seconds. Test glass rod on edge of agar
to verify safe temperature before use. Glass (b) Monitor lamp intensity quarterly there-
spreaders may also be autoclaved. after. Replace bulb when light is down to 80%
of original intensity.
(e) Place sterile, cool glass spreader on
agar surface next to inoculum. Position (c) Measure and average light intensities
spreader so that the tip forms a radius from the of replacement lamp at proper distances.
center to the plate edge. Holding stick motion- Record readings and date of installation.
less, rotate plate several revolutions, or hold
the plate and move the stick in a series of
sweeping arcs to spread the inoculum uni- 5. ~neral Laboratory Supplies
forml"y overthe entire surface ofthe ag'ar.
5.1 Laboratory Glassware
(f) Lift the glass spreader from the agar and
place in disinfectant solution.
5 .. 1. 1 A summary of procedures for main-
tenance of glassware is given in Table IV-A-2).
(g) Repeat the spread plate procedure with
other petri plates.
5. 1.2 Glassware pH Check: Traces of
(h) With cover removed, place agar spread some cleaning solutions are difficult to remove
plates under UV lamp at points where sterility completely. Before using a batch of clean
is desired. glassware, test several pieces for an alkaline
or acid residue by adding and dispersing a few
(i) Place one inoculated plate under ordi- drops of bromthymol blue or other pH indica-
nary laboratory lighting as a control. tor and observing the color reactions. Brom-
thymol blue is particularly advantageous for
0) Expose plates for two minutes. this check because it shows color changes of
yellow to blue green to blue, in the pH range
(k) Close plates and incubate at 35 C for 6.5 to 7.3.
24 hours. Remove and examine for growth.
To prepare bromthymol blue indicator,
( 1) Count all plates and record results. add 16 ml of 0.01 N NaOH to 0. 1 gram of
Control plate should contain 200-250 colo- bromthymol blue. Dilute to 250 ml with dis-
nies. UV-irradiated plates should show 99% tilled water (0.04 % solution).
reduction in the count of the control plate. If
count reduction is less than 80%, replace 5. 1.3 Test Procedure for Suitability of De-
lamp. . tergents Used in Washing (2) - - ·

QA/LABORATORY OPERATIONS 199

 (a} Wash and rinse six, 100 mm diameter water; however, the potential quality of the
petri dishes in the usual manner. These are water will vary with the type of system used.
Group A.
The quality of water obtainable from a
(b) After normal washing, rinse a second pure water system differs with the system used
group of 6 petri dishes 12 times with succes- and its maintenance. The acceptable limits are
sive portions of non-toxic distilled water. given in Table IV-A-3.
These are Group B.
5.3 Water Suitability Test
(c) Wash 6 petri dishes with the detergent
wash water using detergent concentrations 5.3.1 Summary: The water suitability
normally employed, and dry without rinsing. procedure of Geldreich and Clark (3) is a
These are Group C. sensitive test for determination of toxic or
stimulatory effects of distilled or deionized
(d) Sterilize dishes in the usual manner. water on bacteria. It is based on the growth of
Enterobacter aero genes in a chemically defined
(e} Add the proper dilution (usually two medium. A.eduction of 20% or more in the
different dilutions are used) of a water sample bacterial population compared to a control, is
yielding 30-300 colonies to triplicate petri ju'dged toxic. Increased growth greater than
dishes from each Group (A, B and C). Proceed 300% is called stimulatory.
according to the Standard Plate Count Proce- 5.3.2 Scope and Application: The test
dure in Part Ill-A. is recommended for periodic use and as a
special measure of water suitability. This
(f) The results are interpreted as follows: test called the Test for Bacteriological
Properties of Distilled water, in the 14th
(1) Differences in bacterial counts of less edi~ion of Standard Methods is required
than 15 % among all Groups indicate the .deter- annually of laboratories in the Interim Certi-
gent has no toxicity or inhibitory effect. fication Program for Drinking Water Supplies.

(2) Differences in bacterial counts of 15% However, the test is not easily done on
or more between Group A and B demonstrate an infrequent basis because it requires: work
that inhibitory residues are left on glassware over a four day period to complete, an ultra-
after the normal washing procedure used. pure control water and very pure reagents,
and absolute cleanliness of culture flasks,
(3) Disagreement in averages of less than petri dishes, test tubes and pipets, etc. It is
15% between Groups A and B, and greater a complex method that requires skill and
experience, is very sensitive to toxicants and
than:15% between Groups A and C indicates
cannot be related directly to routine analytical
that detergent used has inhibitory
results.
properties which are eliminated° during routine
washing. 5.3.3 Apparatus and Materials
5. 1.4 Sterility Checks on Glassware (a) Incubator set at 35 ± 0.5 C.
(b)Water bath for tempering agar set at
After sterilization of a load of bottles, 44-46 c.
flasks or tubes, test items of each type for (c) Colony counter, Quebec dark field
sterility by adding to one of each aerobic or model or equivalent.
anaerobic broth medium (e.g., lauryl tryptose (d) Hand tally or electronic counting de-
or fluid thioglycollate broth). Incubate and vice (optional).
check for growth. (e) Pipet containers of stainless steel,
aluminum or pyrex glass for glass pipets.
5.2 Pure Water Quality: Pure water sys-
(f) Petri dish containers of stainless steel
tems are meant to produce the best possible
or aluminum for glass petri dishes.

200 oEPA MICROBIOLOGICAL MANUAL 1978

 (g) Thermometer certified by National (b) Ammonium sulfate solution: Dissolve
Bureau of Standards or one of equivalent 0.60 g ammonium sulfate, (NH4)2S04, in
accuracy, with calibration chart. 500 ml redistilled water.
(h) Sterile TD (To Deliver) bacteriological (c) Salt mixture solution: Dissolve 0.26 g
or Mohr pipets, glass or plastic of appropriate magnesium sulfate, MgS04 ·7H20; 0.17 g
volumes, see Part 11-B, 1.8.1. calcium chloride, CaCl2·2H 20; 0.23 g ferrous
(i) Sterile 100 mm x 15 mm petri dishes, sulfate, FeS04 · 7H20; and 2.50 g sodium
glass or plastic. chloride, NaCl, in 500 ml redistilled water.
(j) Sterile pyrex glass flasks, 100 ml. (d) Phosphate buffer solution: Stock
500 ml, and 1000 ml volume. phosphate buffer solution, diluted 1 :25 in
(k) Dilution bottles (milk dilution), .pyrex redistilled water, see Part 11-B, 7 .1 in this
glass, marked at 99 ml volume, screw cap Manual.
with neoprene rubber liner.
Boil reagent solution 1-2 min.to kill vege-
(I) Bunsen/Fisher gas burner or electric' tative cells. Store in sterilized glass-stoppered
incinerator. bottles in the dark at 5 C for several months but
(m) Inoculation loops, at least 3 mm test for sterility before each period of use.
diameter, or needles, nichrome or platinum Prepare the salt-mixture solution without the
wire, 26 B&S gauge, in suitable holder. ferrous sulfate for long-term storage. To use
the mixture, add an appropriate amount of the
5.3.4 M~dia
freshly prepared and freshly boiled iron salt.
(a) Sterile Plate Count Agar (Tryptone Solutions with a heavy turbidity should be
Glucose Yeast Agar) dispensed in tubes (15 discarded and a new solution prepared. Bac-
to 20 ml per tube) or in bulk quantities in terial contamination may cause turbidity in the
screw cap flasks or dilution bottles. See phosphate buffer solution. Discard if this
Part 11-B, 5.1.5. occurs.
(b) Sterile buffered dilution water, 99 ± 2
ml volumes, in screw capped dilution bottles. 5.3.6 Culture: Isolate a pure culture of
See Part 11-B, 7.
Enterobacter aerogenes (IMViC type --++)
from a polluted stream or sewage sample.
(c) Nutrient agar slants with slopes approx-
imately 6.3 cm (2% in) length in 125 x 16 mm 5.3.7 Procedure
screw cap tubes. (a) Collect 200 ml each, of the unknown
5.3.5 Reagents: Sensitivity of the test Test Water and the Control Water (laboratory
depends on the purity of the reagents used. pure reference water) in sterile 500 ml screw
Use reagents of the highest purity and cap flasks. Boil for 2 minutes and cool to
prepare them in water freshly redistilled from room temperature.
a glass still. (b) Label five, sterile. 200 ml screw cap
(a) · Sodium citrate solution: Dissolve flasks as A, B, C, D, and E. Add the Test
0.29 g sodium citrate, Na 3 C6 Hs0r2H20. in Water, Control Water, and· reagents to eai;h
500 ml redistilled water. flask as described in the following table:

Control Test (ml) Optional Tests (ml)

Unknown
Reagents Control Test Nutrient Nitrogen Carbon
Water Water Available Source Source
A B c D E
Sodium citrate solution 2.5 2.5 2.5
Ammonium sulfate solution 2.5 2.5 2.5
Salt-mixture solution 2.5 2.5 2.5 2.5 2.5
Phosphate buffer (7 .3 ± 0.1) 1.5 1.5 1.5 1.5 1.5
Test Water 21.0 21.0 21.0 21.0
Control Water 21.0 5.0 . 2.5 2.5
Total volume 30.0 30.0 30.0 30.0 30.0

QA/LABORATORY OPERATIONS 201

 (c) Perform a Standard Plate Count on consistent, while densities above 100
prepared reagents, Control Water, and Test cells/ml decrease sensitivity to nutrients in
Water as a check on contamination. the Test Water. Make an initial bacterial
count by plating triplicate 1 ml portions from
(d) On the day before performing the
each culture flask in plate count agar. Mix
distilled-water suitability test, inoculate a
well and incubate Test A through E at 35 C
strain of £. aerogenes onto a nutrient agar
slant. Streak the entire agar surface to
for 24 + 2 hours. Prepare final plate counts
from each flask, using dilutions of 1, 0.1,
develop a continuous-growth film and
0.01, 0.001, and 0.0001 ml.
incubate 18-24 hours at 35 C. To harvest
viable cells, pipet 1-2 ml sterile dilution water
5.3.8 Calculations
from a 99 ml water blank onto the 18-24
hour culture. Emulsify the growth on the (a) For growth-inhibiting substances:
slant by gently rubbing the bacterial film
with the pipet, being careful not to tear the . colony count/ml Flask B
Rat10 = --~-----­
agar; then pipet the suspension back into the colony count/ml Flask A
original 99 ml water blank.
A ratio of 0.8 to 1.2 (inclusive) shows no
(e) Make a 1:100 dilution of the original toxic substances; a ratio of less than 0.8
bottle into a second water blank, a further shows growth-inhibiting substances in the
1:100 dilution of the second bottle into a water sample.
third water blank, then 10 ml of the third
(b) For nitrogen and carbon sources that
bottle into a fourth water blank, shaking
promote growth:
vigorously after each transfer. Pipet 1.0 ml of
the fourth dilution (1 :1 o- 6 ) into each of Flasks . colony count/ml Flask C
R at10 = - - " - - - - - - - -
A, 8, C, D, and E. This procedure should colony count/ml Flask A
result in a final dilution of the organisms to
a range of 30-80 viable cells from each ml (c) For nitrogen sources that promote
of test solution. growth:

(f) Variations among strains of the same . colony count/ml Flask D

Rat10 =---'--------
colony count/ml Flask A
organism, different organisms, media, and
surface area of agar slopes will possibly
(d) For carbon sources that promote
necessitate adjustment of the dilution pro-
bacterial growth:
cedure in order to arrive at a specific density
range between 30 and 80 viable cells. To . colony count/ml Flask E
establish the growth range numerically for a R at10 = .
colony count/ml Flask A
specific organism and medium, make a series
of plate counts from the third dilution to (e) Do not calculate ratios b, c, or d when
determine the bacterial density. Then choose ratio a indicates a toxic reaction. Ratios b, c,
the proper volume from this third dilution, or d, in excess of 1.2 indicate an available
which, when diluted by the 30 ml in Flasks A, source for bacterial .growth.
8, C, D, and E, will contain 30-80 viable
cells/ml. If the procedures are standardized 5.3.9 Interpretation of Results
as to surface area of the slant and laboratory
technic, it is possible to reproduce results on (a) The colony count from Flask A after
repeated experiments with the same strain of 20-24 hours at 35 C, will depend on the
microorganism. number of organisms initially planted in
Flask A and on the strain of E. aerogenes
(g) Add a suspension of Enterobacter used in the test procedures. This is the
serogenes (IMViC type --++) of such density reason the control, Flask A. must be run for
that each flask will contain 30-80 cells/ml, each individual series of tests. However, for
prepared as directed above. Cell densities a given strain of E. aerogenes under identical
below this range result in ratios that are not environmental conditions, the terminal count

202 oEPA MICROBIOLOGICAL MANUAL 1978

should be reasonably constant when the 5.4.1 Use a single batch of pure water,
initial plant is the same. The difference between glassware, membrane filters, or other needed
30 and 80 cells in the initial plant in Flask A materials as specified to control all other vari-
will produce a three-fold difference in the final ables except the one under study.
counts, providing the growth rate remains
constant. Thus, it is essential that the initial 5.4.2 Use the reference lot and the test lot,
colony counts on Flask A and Flask B be approx- to conduct parallel pour plate or MF plate count
imately equal to secure accurate data. tests on five natural or treated water samples
according to standard procedures in this
(b) When the ratio exceeds 1.2, it may be Manual.·
assumed that growth-stimulating substances
are present. However, this procedure is ex- When comparing sources of pure· water,
tremely sensitive and ratios up to 3.0 would conduct the tests in parallel using the reference
have little significance in actual practice. water and test water separately for all water
Therefore, when the ratio is between 1.2 and purposes in the tests (dilution, rinse, media
3.0, Tests C, D, and E do not appear to be preparation).
necessary except in special circumstances.
5.4.3 After incubation, compare the
(c) Usually Flask C will be very low and bacterial colonies from the two lots for size
Flasks D and E will have a ratio of less than 1.2 and appearance. If the colonies on the test lot
when the ratio of Flask B to Flask A is between are atypical or noticeably smaller than colonies
0.8 and 1.2. The limiting factors of growth in on the reference lot plates, record the evidence
Flask A are the nitrogen and organic carbon of inhibition or other problem, regardless of
present. An extremely large amount of count differences.
ammonia nitrogen with no organic carbon
could increase the ratio in Flask D above 1.2, or 5.4.4 Count plates and calculate the indi-
the absence of nitrogen with high carbon con- vidual counts per ml or per 100 ml, as required
centration could give ratios above 1.2 in Flask E, for final reporting values.
with a B:A ratio between 0.8 and 1.2.
5.4.5 Transform the final reporting values
(d) A ratio below 0.8 indicates that the to logarithms.
water contains toxic substances, and this ratio
includes all allowable tolerances. As indicated 5.4.6 Compile the log-transformed results
in the proceding paragraph, the ratio could go for the two lots in parallel column.sand calculate
as high as 3.0 from 1 .2 without any undesirable the+ or - difference, d, between the two trans-
consequences. formed results for each sample.
5.4 Use Tests for Media, Membranes and
5.4.7 Calculate the mean, d, and the
Laboratory Pure Water
standard deviation sd of these differences.
The Use Test, a pragmatic approach to
evaluation of materials and supplies, uses the 5.4.8 Calculate the Student's t statistic,
routine methods of analysis to compare current using the number of samples as n:
and new batches or lots. Such tests operate
on the theory that if a stimulatory or toxic effect
cannot be demonstrated in actual test use,
there is no effect.
5.4.9 Use the critical value, 2.78, selected
When a new shipment or lot of culture from a Student's t table at the .05 significance
medium, membrane filters or a new source of level for five sa mp Ies (4 degrees of freedom), for
laboratory pure water is to be used, or at annual comparison against the calculated value.
testing period for water, conduct comparison
tests of the current lot in use (reference lot) 5.4.10 ·If the calculated t value does not·
against the new lot (test lot) as follows: exceed 2. 78, the lots do not produce signifi-

QA/LABORATORY OPERATIONS 203

cantly different results and the test lot is knowledge in manufacturing processes. Con-
acceptable. tinuity of reagent quality depends on com-
pliance of .each new reagent with minimum
5.4.11 If the calculated t value exceeds specifications.
2.78, the lots produce significantly different
results. If the test lot results exceed reference 5.6.2 Repeat quality control procedures
lot results, the test lot is more stimulatory. If the each time that reagent batches are prepared,
test lot results are less than the reference lot, regardless of the expiration date given by the
the test lot is less stimulatory. manufacturer.

5.4.12 If condition 5.4.3 or 5.4.11 occurs, 5.6.3 Discard sera or antigens if contami-
review test conditions, rerun the test and/or nation is discovered.
obtain different lots for testing and use.
5.6.4 Select another working dilution if the
5.6 Reagents in General: The quality of level of activity has dropped.
test reagents must be assured. They must be
correctly prepared and properly stored. The 5. 7 Fluorescent Antibody Reagents
following general rules should be followed:
Highly specific reagents, antigens of high
5.5.1 Use ACS or AR grade chemicals that purity and very specific potent antibodies are
meet ACS specifications for preparing reagents. required for fluorescent antibody techniques.
Impurities in uncertified or lesser grades of The antisera must exhibit high staining intensi-
chemicals may inhibit bacterial growth, provide ties. Some sera may have high titers in one type
nutrients or fail to produce the desired reaction. of serological test, but demonstrate poor
staining titer and vice versa.
5.5.2 Date chemicals and reagents when
received and when opened for use. 5. 7.1 $tore desiccated fluorescent anti-
body (FA) reagents at 4 C. Prepare aliquots of
5.5.3 make reagents up to volume in volu- the .rehydrated conjugate in screw-cap glass
metric flasks. For storage, transfer to good vials, freeze and store at -20 C until working
quality plastic (polyethylene, polypropylene or dilutions are prepared.
tetrafluoroethylene) or borosilicate glass bot-
tles with polyethylene or other inert plastic 5. 7.2 Test FA reagents for correct reactions
stoppers or caps. with positive and negative controls before each
use. Results of positive controls should be
5.5.4 Identify prepared reagents with the within one dilution of the average titer.
generic name, the concentration, the date pre-
pared and the initials of preparer. 5.8 Dyes/Stains: Organic chemicals are
used as selective agents (e.g., brilliant green in
5.5.5 Store reagents under the conditions brilliant green lactose bile broth), as indicators
recommended by the manufacturer. in bacteriological media (phenol red lactose),
and as bacteriological stains (gram stain). Dyes
6.6.6 Run positive and negative controls from any commercial supplier vary from lot to
with each series of cultural or biochemical lot in percent dye, dye complex, insolubles and
tests. inert materials present. Because dyes for micro-
biological uses must be of proper strength and
6.6 Serological Reagents stability to produce the correct reactions, pur-
chase only dyes which have been certified by
5.6.1 Evaluate serological antisera against the Biological Stain Commission for biological
known antigens and compare with antisera that use.
have demonstrated acceptable reactivity. The
quality of commercial serological reagents is 5.8.1 Fluorescent Dyes: An important fac-
subject to methodology changes and new tor in the preparation of antisera-dye conju-

204 oEPA MICROBIOLOGICAL MANUAL 1978

gates is the purity of the fluorescein dye. 6.2.1 Some manufacturers certify that
Infrared studies reveal that the purity of fluor- their membranes meet stated specifications
escein isothiocyanate (FITC) commonly used on sterility, retention, recovery, pore size, flow
in FA tests, ranges from 30 to 100% in com- rate, pH, total acidity, phosphate and other
mercial products. Ideally 100% pure FITC extractables.
preparations are used. Dyes of less purity may
be satisfactory if the weight used to label the 6.2.2 Most manufacturers sell sterile
protein component of the serum allows for the membranes and those packaged for steriliza-
impurities, and if the impurities do not increase tion by the user. Membranes may be sterilized
non-specific staining. by autoclaving, ethylene oxide or irradiation.

5.8.2 Check bacteriological stains before

use with at least one positive and one negative 6.3 User's Quality Assurance
control culture.
6.3.1 In addition to the quality assurance
provided by the manufacturer, the user should
6. Membrane Filters determine that the membranes perform satis-
factorily by inspecting and testing each lot he
6.1 Government Specifications orders. The membranes should not be mis-
shapen or the gridlines distorted after
6.1.1 The quality and performance of autoclaving.
membrane filters vary with the manufacturer,
type, brand, lot number and storage conditions. 6.3.2 A membrane should allow good dif-
Membranes considered for purchase by federal fusibility of medium and be completely wetted
agencies must confo'rm to the government within 1 5 seconds after it is placed on the
specifications published in 1974 Military medium substrate with no dry areas indicated
Specifications (4). The detailed specifications by colorless or lighter-colored areas.
required of the manufacturer by the federal
government are an important advantage to 6.3.3 After incubation, the colonies
other users of. membrane filters because by should be of the expected size, with defined
meeting federal requirements, manufacturers shape and clearly delineated edges. The color
realize it is most efficient to make all mem- and morphology of the colonies must be typi-
branes to these specifications. Hence, mem- cal of those defined by the test procedure.
brane filters purchased from companies selling
to federal agencies meet the federal specifi- 6.3.4 The analyst should observe that the
cations. gridline ink does not "bleed" across the sur-
face, or restrict colony development on or ad-
6. 1.2 The specifications list requirements jacent to the cross-hatching. Colonies should
for pore size, porosity, flow rate, diffusibility, be evenly distributed across the membrane
autoclavability, sterility, diameter, thickness, surface. Membranes containing sizable areas
bacterial retention and bacterial cultivation. with no colony development are questionable.
The grid lines must be easily visible, permanent
and not imprinted into the surface so as to 6.4 ASTM Test Procedures
cause channelling of growth. The grid ink must
not stimulate or inhibit growth. Because of variable recoveries of micro-
organisms on different membrane filters, there
6. 1.3 The pads must have a specified di- is a critical need for standard procedures for
ameter and thickness, limited acidity, no evaluating membrane filters. Procedures are
waxes, sulfites, and other stimulating or inhib- being developed by Committee D-19.08.04 of
itive materials, and must absorb uniformly a ASTM for physical, chemical and microbiologi-
specified volume of medium. cal characteristics of membrane filters. The
procedures are intended for the larger labora-
6.2 Manufacturer's Quality Assurance tory and the manufacturer to test new batches

QA/LABORATORY OPERATIONS 205

or lots of MF's. Brief descriptions of these tests 6.4.2 Inhibitory Effects: The test for inhibi-
for bacterial retention, inhibitory effects, re- tory effects compares membrane filter counts
covery, extractables and flow rate follow. on one or more test lots of membrane filters,
with spread plate counts on Plate Count Agar
6.4.1 Bacterial Retention Test using a specific pure culture of Escherichia coli
(IMViC + + - -).
The test is based on filtration of a standard
culture through a 0.45 µm MF into a broth. ' (a) Prepare a dilution of the stabilized 24-
Sterile equipment and aseptic techniques must hour culture in 0. 1 % peptone dilution water so
be used. Five randomly-selected membranes that 0.2 ml contains 30-150 viable cells. Add
from five randomly-selected packages should about 20 ml of sterile dilution water to the
be tested. Control membranes should be taken funnel before filtration.
from the same package as the test mem-
branes. (b) Filter five replicate sample volumes of
0.2 ml through five replicate membrane filters
(a) Add 140 ml of double strength Trypti- of each MF test lot and place each on a plate of
case soy or Tryptic soy broth to a liter vacuum Tryptic soy agar.
flask and attach vacuum tubing. Wrap flask in
kraft paper and sterilize. (c) · Prepare the corresponding spread
plates in random fashion to avoid a time effect,
(b) Using an 18 hour broth culture of following a randomization table.
Serratia marc~scens, prepare a final dilution in
0.1 % peptone, containing 1000 cells per (d) With the same pipet, deliver five addi-
100 ml. tional 0.2 ml volumes to the surface of five
plates of Tryptic soy agar. The samples are
(c) Assemble a membrane filtration appa- distributed evenly over the surfaces of the
ratus using a sterile vacuum flask containing plates with sterile glass rods.
the broth. Insert the test filter, turn on vacuum
and pour in 20 ml of sterile rinse water to set (e) Incubate the membrane filters at 44.5 C
the membrane. Filter 100 ml of the culture and agar plates at 35 C for 24 hours. Count the
suspension. Rinse funnel twice with 20 ml of colonies. Compare recoveries on spr~ad plates
peptone water. and membrane filters. Acceptable filters should
recover some set percentage of the spread
(d) After the filtration remove funnel and plate count. This percentage should be estab-
transfer the membrane filter to a Trypticase lished by each laboratory based on previous
soy or Tryptic soy agar plate. Then aseptically performance by a known acceptable lot.
remove membrane filteration base from the
flask and insert a sterile stopper. Repeat 6.4.3 Recovery (5): The procedure for re-
filtrations four more times with sterile MF covery compares the fecal coliform counts on
assemblies, culture flasks and membranes. test membranes to the counts on spread plates
using M-FC agar substrate. Four' polluted
(e) Incubate agar plates and flasks for 48 waters and one raw sewage sample are
hours at 35 C. Examine membranes for growth analyzed.
outside of filtering areas which indicates a
leaky filtration assembly that could cause a (a) To determine sample test volumes, pre-
false positive test. Turbidity in the culture flask pare serial dilutions of each sample in 0. 1 %
indicates bacterial growth and failure of the peptone water to produce a suspension con-
membrane to retain the 0.2-0.3 µm sized bac- taining approximately 20-60 fecal coliforms
teria, but does not indicate the numbers of per 0. 1 ml. Hold original samples at 4 C. Deter-
organisms passing the filter. mine the fecal coliform density of each of the
samples or dilutions by membrane filter or
(f} Turbidity indicates filter failure. Repeat spread plate test. Read the results after 22-24
the test and control procedures. · hours incubation at 44.5 ± 0.2 C.

206 &EPA MICROBIOLOGICAL MANUAL 1978

 (b) If the fecal coliform density in the raw or suspected to have a high concentration of
water samples is less than 10 per 0.1 ml, seed pseudomonads or aeromonads, perform the
the sample with raw sewage, allow to stabilize cytochrome oxidase test. As in the verification
at 4 C for 24 hours and test as in (a). Then procedure, a minimum negative· oxidase test
proceed with the standard test procedure. confirmation of 80% should be achieved for
the membranes to be considered acceptable.
(c) Use the optimum dilution~ from (a) or (b)
for the test membranes and the spread plate
6.4.4 Extractables
controls.
(a) Total Extractables
(d) Perform the membrane filter tests ac-
cording to the procedure described in Part 11-C,
3 of This Manual. (1) Dry filters for 15 minutes at 70 C then
bring to room temperature in a desiccator.
(e) Prior to beginning the spread plate Weigh to constant weight on a four-place ana-
tests,airdrythesurfaceof the M-FC agar con- lytical balance.
tained in 100 ml petri dishes. Aseptically de-
liver 0.1 ml of the selected sample dilution to (2) Boil filters in 100 ml of distilled water
the agar surface and spread with a sterile bent for 20 minutes. Remove the filters and dry at
glass rod. Allow the sample aliquot to be com- 70 C for 60 minutes. Bring filters to room
pletely absorbed before inverting the dish. temperature in a desiccator and reweigh to
constant weight. Weighings shall be to the
(f) Alternate MF tests with spread plate nearest 0.1 milligram.
controls to randomize systematic errors.
Original. Weight - Weight After Extraction
(g) Insert petri dishes into waterproof bags Original Weight
or seal with waterproof tape, and submerge in
the waterbath incubator. Incubate for 22-24
Percent
hours at 44.5 + 0.2 C. Record the temperature x 100 Extractables
continuously during the incubation period .. Af-
ter incubation, remove plates and examine.
(b) Specific Extractables
(h) Count the blue colonies. If more than
one dilution was prepared, select the plates
with between 10 and 100 colonies, but prefer- (1) Immerse filter in ASTM Type 1 reagent
ably with 20-60 colonies. Calculate the arith- grade water for 24 hours (6).
metic mean of the five replicate fecal coliform
counts and the five replicate spread plates. (2) Remove filter and assay the extract for
Determine percent recovery: metals, total organics, phosphorus and
ammonia.

MF X Count 6.4.5 Flow Rate

--------- x 100 % Recovery
Spread Plate X Count Water flow rate shall be determined by
timing the passage of 500 ml of particle-free
(i) Verification: Pick 20 blue colonies from distilled water through a filter at 25 C and at a
each of 2 randomly-selected filters and 2 differential pressure of 70 centimeters of mer-
spread plates. If plates contain less than 20 cury. Particle-free water for this test is produced
colonies, pick all blue colonies. Verify the colo- by passage of a high purity water through a
nies in EC media as described in Part 111-C, 4 of 0.2 µm membrane filter, three times in succes-
This Manual. To be' acceptable, 80% of the sion, at 25C and at a differential pressure of
colonies must verify. If the samples are known 177 cm of mercury.

QA/LABORATORY. OPERATIONS 207

 60 composition and sensitivities of different
The flow rate = t
X 500 ml media, it is impossible to establish universal
time limits for holding unopened bottles of
EFA cm 2 media. Therefore, a conservative and protec-
tive recommendation is to limit thestorageof
== ml/min/cm 2 unopened bottles of cufo.iral media to two
years. This limit shOuld insure good perfor-
Where: t = experimental time in minutes for mance-of media withpr:oper-s~
filtering 500 ml of particle-free dis- conditions.
tilled water
7.3 Preparation of Media
and
EFA = effective filtering area of a 47 mm diam-
7.3.1 In high humidity areas, store opened
bottles of media in a large hinged-door desic-
eter filter
cator. Open bottles as briefly as possible during
the weighing process and return to the desic-
An average flow rate reported by manufac-
cator immediately after use.
turers for 0.45 µm pore size, 47 mm diameter
filters is 65 ml/min/cm 2 •
7 .3.2 Discard opened bottles of media 6
7. Culture Media months after initial use.

Since even the best cultural procedure is 7.3.3 Weigh media to the nearest 0.1
ineffectual if the medium is not prepared cor- gram on a single pan, top loader balance as
rectly, it is important to train personnel to use quickiy as possible.
the best materials and techniques in media
preparation, storage and application. Some 7.3.4 Keep balance out of drafts and away
factors that must be considered follow: from high humidity. Use a plastic shield around
the balance to protect from drafts.
7.1 Ordering Media
7.3.5 Clean the balance and surrounding
7. 1. 1 Order media in quantities to last up area immediately after weighing media.·
to one year. Always use oldest stock first.
7.4 Solution of Media
7.1.2 Whenever possible and practical,
order media in 1/4 pound multiples rather 7.4.1 Check cleanliness of glassware. Use
than one pound bottles to insure sealed pro- bromthymol blue indicator to spot-check pH of
tection of the supply as long as possible. Most glassware (5.1.2 in this Section).
deterioration nf media occurs after bottles are
opened. 7.4.2 Prepare media in a deionized or dis-
tilled water of proven quality. Ultra-pure water
7 .1.3 Maintain an inventory record of me- from a recirculating deionization system is re-
dia: the dates received, sizes, number of units, commended. Measure volumes with the great-
etc. Review the inventory quarterly for neces- est accuracy possible using the proper pi pet or
sary reordering. Date each bottle when re- graduate (Part 11-8, 1.8).
ceived and when opened. Bottles should be
inspected for color changes, caking or other Check the pH of media after solution·and
indication of deterioration. Discard such bot- after st.erilization using a laboratory model pH
tles and reorder. meter. Enter results in the QC record book.
The reading should be within 0.2 units of the
7.2 Holding Time Limits for Media stated value. If not, discard batch and remake.
If the pH is still incorrect, use another bot-
Because of the myriad of environmental tle or batch. pH differences indicate a prob-
conditions affecting media, and the unique lem of distilled water quality, deterioration of

208 .$EPA MICROBIOLOGICAL MANUAL 1978

medium or improperly-prepared medium. The between loads. As soon as the autoclave is
problem must be identified and corrected. The loaded and closed, steam can be admitted to
problem should be carefully documented in the chamber and in a relatively short time, full
the quality control record book and reported to pressure and temperature are developed in the
the manufaoturer if the data indicate the me- chamber. The total exposure time for media
dium as the source of error. sterilized 15 minutes at 121 C should not
exceed 45 minutes.
7.4.3 Note in the QC record any unusual
color development, darkening, or precipitation 7 .5.3 Avoid overcrowding in an autoclave
of media. Check the sterilization time and tem- which reduces its efficiency. Large volumes of
perature. If there is a drastic change in appear- media should be preheated to reduce the lag
. ance, discard medium and remake it. If the period before placing them in the autoclave .
problem still exists, remake the batch using a
different lot of medium. 7.5.4 Remove sterile media from the auto-
clave as soon as pressure is at zero.
7.4.4 Containers for preparation of
batches of broth or agar should be twice the 7.5.5 Media must be discarded if contami-
volume of the medium being prepared. nation is suspected. Reautoclaving is not
permitted.
7 .4.5 Media should be stirred continu-
ously while being heated to avoid burning. 7 .5.6 A preventive maintenance contract
Agar media are particularly susceptible to is recommended for autoclaves. It should in-
scorching and boilover. The only insurance clude checks on the accuracy of pressure and
against scorching or boilover is use of a boiling temperature gauges and recorders and opera-
water bath for small batches of media or con- bility of safety valve.
stant attention while heating larger batches on
a hot plate or burner. A combination hot plate 7 .5. 7 Check the effectiveness of steriliza-
and magnetic stirrer is recommended for solu- tion weekly, using strips or ampuls of Bacillus
tion of media. stearothermophilus spores. Commercial pack-
ages of these spores are available in ampuls of
7 .4.6 Bottles, tubes or plates of prepared growth-indicator media. Sterilization at 12 1 C
media are identified and dated. for 12-1 5 minutes will kill the spores. Incubate
the autoclaved cultures at 55-60 C and if
7.5 Sterilization by Autoclave growth occurs, sterilization is inadequate.

7 .5. 1 Media should be sterilized for the 7 .6 Sterilization by Filtration

minimal time specified by the man-ufacturer.
The amount of time required to sterilize a me- 7.6.1 Non-autoclavable solutions can be
dium in an autoclave will vary with the type sterilized by membrane filtration. With careful
and volume of medium and the size and shape preparation of the sterile filtration and receiving
of containers. See the table in Part 11-B, 4.3 apparatus, passage of a solution through a
on sterilization. 0.2 µm membrane filter will produce a sterile
solution.
7 .5.2 Since the potential for damage to
media increases with increased exposure to 7 .6.2 The filtration and subsequent sterile
heat, the amount of lag time before the auto- dispensations should be performed in a safety
clave is at full pressure and temperature can cabinet or bio-hazard hood.
a
be critical factor in whether media are dam-
aged. The danger from an extended heating 7.7 Gaseous Sterilization
period is reduced with use of a double-walled
autoclave which allows the operator to main- Equipment, supplies or other solid or dry
tain full pressure and temperature in the jacket materials which are sensitive to heat can be

QA/LABORATORY OPERATIONS 209

sterilized by long exposure to ethylene oxide (a) Bring the base medium to 44-46 C
gas (ETO) using available commercial equip- before addition of a labile constituent.
ment. Check sterility with culture of B. subti/us
var. nigerspores. (b) Warm enrichments such as blood or
serum to room temperature before adding to a
7.8 Use of Agars, Broths, and Enrich- base medium.
ment Media
(c) Once labile material is added to a me-
7.8.1 Agars dium, prepare plates or tubes as soon as possi-
ble. Do not hold the batch medium in a water
(a) Agar plates to be used for streaking or bath for more than 10 minutes.
spread plates are kept open slightly for 15
minutes after pouring or after taking out of 7.9 Storage of Media
refrigerated storage to evaporate free mois-
ture which would cause confluent growth on 7 .9. 1 The recommended time limits for
streak plates. holding prepared media in the laboratory are:

(b) Agar plates used for MF and spread MF broths in screw-cap 96

plate work must be free of lumps, uneven flasks at 4 C hours
surfaces, pock marks, bubbles or foam which (Work Week)
prevent good contact between the agar and
the membrane or uniform growth on spread MF agars in plates with Two
plates. tight-fitting covers Weeks

(c) Melted agars should be held in a tem- at 4 C

pering water bath at 44-46 C but no longer than
three hours. As a safety precaution against the Agar or broths in loose- One
use of agars which are too hot and might kill cap tubes, at 4 C Week
cells, place a bottle of agar in the same boiling
water or under the same autoclave conditions Agar or broths in screw- Three
as the agars to be used. After the agar is melted, cap tubes, tightly Months
transfer agar to a tempering bath. Insert close·d, at 4 C
thermometer in the agar bottle and use it to
determine when the temperature of the agars Agar plates (non-MF) with Two
is at approximately 44-46 C and safe for use loose-fitting covers, in Weeks
In pour plates. sealed plastic bags, at
4 c
7 .8.2 Broths
Large volumes of agar in Three
(a) Handle sterile MPN fermentation tubes screw-cap flasks or Months
of lauryl tryptose broth or brilliant green bile bottles tightly-closed,
broth carefully prior to use. Shaking can en- at 4 C
trap air in the inner tubes and produce a false
positive. Examine fresh tubes before use and 7 .9.2 Store fermentation tube media in
discard any with a bubble. the dark at room temperature or 4 C. If refriger-
ated, incubate overnight at room temperature
(b) Reduced media such as thioglycollate to detect false positive gas bubbles.
broth oxidize in storage. Before use, these
broths must be heated in boiling water for 7.9.3 Since loss of moisture is a major
20-30 minutes to reduce the medium. problem of storage, screw-capped tubes and
flasks are recommended. Prepoured agar
7.8.3 Enrichments

210 &EPA MICROBIOLOGICAL MANUAL 1978

plates can be sealed in plastic bags to retain Include the .date, the name of the medium
moisture and refrigerated. and lot number, amount of medium weighed
out and volume of medium prepared; the
7 .9.4 A simple check for loss of moisture record of sterilization, pH measurements,
in broth tubes can be made by marking the pH adjustments made, special handling or
original level in several tubes of each batch preparation techniques, e.g., use of heat-
and then observing the loss of moisture over sensitive compounds or components, and
time. If the estimated moisture loss exceeds name of preparer.
ten percent, discard the tubes of broth.
7. 10.2 Incubate five percent of each batch
7 .9.5 Protect media containing dyes from of medium for 2 days at 35 C and inspect for
light. If color changes are observed, discard growth.
the medium.
7. 10.3 Check each batch of medium when
7.9.6 Prepared sterile broths and agars used by inoculating 2 tubes or plates with pure
are available from commercial sources. Their cultures of species producing positive and
use may be advantageous when analyses are negative reactions for that medium.
done intermittently, when staff is not available
for such preparation work; or when cost of 7.10.4 Test new batches of differential
their use can be balanced against other factors media by inoculating with organisms of known
of laboratory operation. However, purchase of fermentative or other bioGhemical ability. Simi-
prepared media does not reduce the responsi- larly, enrichment and selective media are
bility of the laboratory for checking the pe:ior- tested for productivity of the desired microor-
mance of the media, regardless of the stated ganisms and inhibition of other microorga-
quality control practices of the manufacturer. nisms. Tables IV-A-4 and IV-A-5 list a group of
organisms with the broths, agars, biochemical
tests and reactions to which they can be
applied.
7.10 Quality Control of Prepared Media
7. 10.5 Record sterility and positive/ nega-
7.10.1 Maintain a book with a' complete tive performance checks in the media prepara-
record of each batch of medium prepared. tion portion of the quality control log.

QA/LABORATORY OPERATIONS 211

 TABLE IV-A-1

Monitoring Laboratory Equipment

Item Monitoring Procedure

1. Balance a. An analytical balance with a sensitivity of 1 mg or less at a

10 g load should be used for weighing 2 g or less. For larger
'quantities, a balance with accuracy of 50 mg at a 150 g load
should be used.

b. Check balance monthly with a set of certified class S weights.

c. Wipe balance and weights clean after each use.

d. Protect weights from laboratory atmosphere and corrosion.

e. Contract with a qualified expert for balance maintenance on an

annual basis.

2. pH Meter a. Compensate for temperature with each use.

b. Date standard buffer solutions when first opened and check

monthly with another pH meter. Discard the buffer solution If
the pH is more than ± 0. 1 pH unit from the manufacturer's
stated value or if it is contaminated with microorganisms.

c. Standardize with at least one standard buffer (pH 4.0, pH 7.0,

or pH 10.0) before each use.

d. Do not re-use buffer solution.

e. Have meters inspected at least yearly as part of a maintenance

contract.

3. Water Deionizer a. Monitor water for conductance daily. Monitor trace metals and
other toxic or nutritive compounds monthly. See Table IV-A-3.

b. Replace cartridges as indicated by manufacturer or as Indicated

by analytical results.

c. Monitor bacterial counts at exit point of unit. Replace the

cartridges when standard plate count exceeds 10,000/ml.

4. Water Still a. Drain and clean monthly according to instructions from the
manufacturer.
b. Drain and clean distilled water reservoir quarterly.

c. Check distilled water continuously or daily using a conductance

meter. See Table IV-A-3.

d. Conduct chemical tests on water to detect toxicity or stimulation

effect. See Table IV-A-3.

e. Conduct standard plate counts monthly on stored water and

clean out reservoir if count > 10,000/ml.

212 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-A-1
(continued)

Monitoring Laboratory Equipment

Item Monitoring Procedure

5. Dispensing Apparatus a. Check accuracy of dispensation with an NBS class A, ·graduated

cylinder at the start of each volume change and periodically
throughout extended runs.

b. Lubricate moving parts according to manufacturer's instructions

or at least once per month.

c. Correct immediately any leaks, loose connections or malfunctions.

d. After dispensing each type of medium, pass a large volume of

hot distilled water through dispenser to remove traces of agar
or medium.

e. At the end of the work day, break down unit Into parts, wash
well, rinse with distilled water and dry.
I
6. Ultraviolet a. Remove plug from outlet and clean ultraviolet lamps monthly by
Sterilizer wiping with a soft cloth moistened with ethanol.

b. Test ultraviolet lamps with light meter quarterly; if they emit

less than 80% of their rated initial output, replace them.

c. Perform spread plate Irradiation test quarterly. see This

Section, 4.2. 1.

7. Membrane Filter a. Check funnel support for leaks.

Equipment
b. Check funnel and funnel support to make certain they are
smooth. Discard funnel if inside surfaces are scratched.

c. Clean thoroughly after each work day.

8. Spectrophotometer a. Maintain quality control and calibration check as recommended

by the manufacturer.

b. Have Inspected yearly by a factory maintenance man.

9. Centrifuge a. Check brushes and bearings for wear every six months.

b. Check rheostat control against a tachometer at various loadings

every six months to ensure proper gravitational fields.

QA/LABORATORY OPERATIONS 213

 TABLE IV-A-1
(continued)

Monitoring Laboratory Equipment

Item Monitoring Procedure

10. Microscope a. Allow only trained technicians to use.

b. Appoint one laboratory worker to be responsible for the care

of the microsc.ope.

c. Clean optics and stage after every use. Use only lens paper
for cleaning.

d. Keep covere(j when not in use.

e. Establish annual maintenance on contract.

f. Maintain in one location if possible.

11. Microscope, a. Allow only trained technicians to use microscope and light
Fluorescence source.

b. Keep a log of lamp operation time.

c. Monitor lamp with meter. See Section 4.2.2. Replace the

lamp when < 80% of original fluorescence is observed.

d. Check lamp alignment, particularly if bulb has been changed.

Realign the fluorescent light source if necessary.

e.. Use known 4+ fluorescence slides as controls.

12. Safety Cabinet a. Check filters monthly for plugging or obvious dirt accumulation.
(Hood) Clean or replace filter as needed.

b. Check cabinet for leaks and for rate of air flow every three
months.

c. Expose blood agar plates to air flow for one hour once per
month to measure contamination.

d. Remove plug from the outlet and clean ultraviolet lamps every
two weeks by wiping with a soft cloth moistened with ethanol.

e. Test ultraviolet lamps quarterly with a light meter. If lamp

emits less then 80% of the rated output, replace lamp.

f. Perform maintenance as directed by the manufacturer.

g. Purchase and use a pressure monitor control device to measure

efficiency of air flow.

214 oEPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-A-1
(continued)

Monitoring Laboratory Equipment

Item Monitoring Procedure

13. Thermometers and a. Check the accuracy of thermometers and temperature recording
Recording Devices instruments, in the monitoring range, at least annually against a
certified thermometer or one of equivalent accuracy. Thermometer
graduations should not exceed the 0.2 or 0.5 C deviation permitted
in the analytical method. Check mercury columns for breaks.

b. Record calibration checks in quality control (QC) record. Mark

NBS calibration correction on each thermometer or on the outside
of the incubator, refrigerator or freezer containing the thermometer.

c. Record daily temperature checks on charts and keep for at least

three years. A simple, one year chart is shown in Figure IV-A-1.

14. Water Bath a. Check and record temperature daily. Bath must maintain the uniform
temperature needed for the test in use.

b. Maintain accurate thermometer completely immersed in water bath.

c. A recording thermometer and alarm system are recommended.

d. Clean monthly.

e. Use only stainless steel, rubber, plastic-coated, or other

corrosion-proof racks.

15. Refrigerator at 4 C a. Check and record temperature daily.

b. Clean monthly.

c. Require identification and dating of all material.

d. Defrost unit and discard outdated materials in refrigerator and

freezer compartments every three months.

16. Hot Air Oven a. Test performance with spore strips or suspensions quarterly.

b. Equip and monitor sterilization with a thermometer accurate in

160-180 C range.

1 7. Freezers a. Check temperature and record daily.

b. Use of recording thermometer and alarm system recommended.

c. Require identification and dating of all materials.

d. Clean and defrost freezer every six months. Discard outdated

materials.

QA/LABO.RATORY OPERATIONS 215

 TABLE IV-A-1
(continued)

Monitoring Laboratory Equipment

Item Monitoring Procedure

18. Autoclave a. Record temperature and pressure for each run. Recording
thermometer recommended.

b. Verify that autoclave maintains uniform operating temperature.

c. Check operating temperature with a min/max thermometer on a

weekly basis.

d. Test performance with spore strips or suspensions weekly. If

' evidence of contamination occurs, check until the cause is
identified and eliminated.

e. Procure semi-annual preventive maintenance inspections.

19. Incubator (Air /Water.Jacket) a. Check and record temperature daily.

b. If partially-submersible glass thermometer is used, bulb and stem

must be immersed in water to the mark on stem.

c. Measure temperatures daily on top and bottom shelves.

Periodically measure temperature on all shelves in use.

d. Expand test points proportionately for walk-in incubators.

e. Recording thermometer and alarm system are recommended.

f. Locate incubator where room temperature does not go outside

of the 16-27 C range.

216 oEPA MICROBIOLOGICAL MANUAL 1978

Instrument - - - - - - - - - - - - - - Temperature - - - - - Room

Read daily.
Record temperature in space provided.

Date Jan. Fe b . ·Mar. A,pr. Mav J une .Tu 1 . A ug. s ep. 0 ct. Nov. Dec. Date

1 1
2 2
3 3
4 4
5 I 5
6 6
7 l I
I 7
8 8

9 I 9
10 10
11 11
12 l I 12
13 I 13
'
14 14
15 I 15
16 16
17 ' 17
18 ' 18
19 19
20 20
21 21
22 22
23 23
24 24
25 25
26 26
27 27
28 28
29 29
30 30
31 31

FIGURE IV-A-1. Equipment Operation Temperature Record.

QA/LABORATORY OPERATIONS 217

 TABLE IV-A-2

Glassware Maintenance

Item Monitoring Procedure

1. Utensils and Use utensils and containers of non-corrosive and non-contaminating

Containers for materials such as pyrex glass, stainless, steel or
Medill Prep11ratlon aluminum.

2. Gl11Ssw11re (Reusable) a. With each use, examine glassware especially screw-capped dilution
bottles and flasks, for chipped or broken edges and etched
surfaces. Discard chipped or badly-etched glassware.

b. Inspect glassware after washing. If water beads excessively on the

cleaned surfaces, run the glassware through again.

c. Test for acid or alkaline residues by adding .bromthymol blue indicator

to representative glassware items (see 5. 1.2 in This Section).

d. Test for residual detergent by the test in 5. 1.3, This Section.

218 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-A-3

Laboratory Pure Water for Bacteriological Testing

Ideal
Monitoring
Parameter Frequency Limit

Chemical Tests

Conductivity Continl!ously 1-2 µmhos/crri

or with each at 25 C
use

pH With each use 5.5-7.5

Total Organic Monthly < 1.0 mg/liter

Carbon

Trace Metal, Single Monthly <0.05 mg/liter

Trace Metals, Total Monthly < 1.0 mg/liter

(Od, Cr, Cu Ni, Pb, Zn)

Ammonia/ Amines Monthly <0.1 mg/liter

Free chlorine With each use <0.1 mg/liter

Biological Tests

Standard Plate Count

Fresh Water Monthly < 1000 bacteria/ml
Stored Water Monthly < 10,000 bacteria/ml

Water Suit- Yearly and when Ratio: 0.8-3.Q

ability Test conditions change

Water Use-Test Yearly and when Calculated

conditions change t value
< 2.78

QA/LABORATORY OPERATIONS 219

 TABLE IV-A-4

Quality Control of Media

Medium Control Cultures Expected Results

M·Endo MF Broth Escherichia coli Golden green metallic sheen

or Agar Enterobecter eerogenes Golden green metallic sheen
Achromobecter sp Red colonies
Pseudomonas sp Red colonies
Salmonella sp Red colonies if medium overheated

M·FC Broth E. coli Blue colonies

or Agar K. pneumonlae Blue colonies
E. aerogenss No growth

Brllllant Green Bile E. coli Growth with gas

Lactose Broth E. aerogenes Growth with gas
C. frsundil Growth with gas
Staph. aureus No growth

Laury! Tryptose E. coli Growth with gas

Broth E. aerogenes Growth with gas
S. typhlmurlum Marked to complete Inhibition
S. aureus Marked to complete inhibition

Levine's Eosln E. coli Nucleated black colonies with

Mothyleno Blue Agar golden green metallic sheen
E. aerogenes Pink colonies with dark centers
C. freundli Colorless colonies
Salmonella sp Colorless colonies
Klebsiella sp Large brown mucoid colonies

Xylose Lysine Salmonella sp Red colonies, to red with black centers

Doaoxycholata Agar Klebsiella sp Yellow colonies
(XLD) E. coli Yellow colonies
E. aerogenes Yellow colonies

Bismuth Sulfite Agar Salm. typhosa Black colony with black or brownish·
black zone, with or without sheen
Other Salmonella sp Raised green colonies
Coliforms Green colonies

Brllllant Green Agar Salmonella sp Pink-white opaque colonies surrounded

by brilliant red zone
E. coli Inhibition or yellow green colonies
P. vulgarls Marked to complete inhibition or red
colonies

220 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-A-4
(continued)

Quality Control of Media

Medium Control Cultures Expected Results

KF Streptococcus Agar Strep. faecai;s Pink to red colonies

Strep. pyogt1nes No growth
S. aureus No growth
E. coll No growth

PSE Agar S. faeca/is Black colonies

f. qpl/ No growth
S. aureus No growth

QAl~ABORATORY OPERATIONS 221

 TABLE IV-A-5

Quality Control of Biochemical Tests

Test Control Culture Expected Results

BHI Broth at pH 9.6 Strep. faecalis Positive: growth

Strep. mitis-salivarius Negative: no growth

BHI Broth with 6.6% S. faecalis Positive: growth

NaC1 S. mitis-salivarius Negative: no growth

Arginine Dehydrolase Salm. typhlmurium Positive: alkaline reaction

(Moellor's medium) reddish violet color
Salm. flexneri Negative: yellow color

Lysine Decarboxylase S. typhlmurium Positive: alkaline reaction

(Mooller·s medium) reddish violet color
S. flexneri Negative: acid, yellow reaction

Ornlthlne Decarbox- S. typhimurium Positive: alkaline reaction

ylase (Moeller·s reddish violet color
medium) S. flexneri Negative: yellow color

lndolo Production Escherichia coli Positive: red color

(rryptophane Broth) Salmonella sp Negative: orange/yellow color
Enterobacter aerogenes ~egative: orange/yellow color

Methyl Red (Buffered E. coli Positive: red color

Peptone Glucose Broth) E. aerogenes Negative: no change

Vogos-Proskauer E. aerogenes Positive: pink color

(Buffered Peptone E. coli Negative: no color change
Glucose Broth)

Citrate Utllization E. aerogenes Positive: growth, change to

(Simmons Citrate Broth) blue color
E. coli Negative: no color change,
no growth

Urease Production Proteus mirabilis Positive: color change, pink

(Chrlstansen's Urea to red
Agu) Salmonella sp Negative: no color change

Cata lase S. aureus Positive: bubbles

(BHI agar slant) S. foecalis Negative: no bubbles

222 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-A-5
(continued)

Quality Control of Biochemical Tests

Test Control Culture ~xpected .Results

Gytochrome Oxidase Pseudomonas aeruginosa Positive: blue color

(~lpha-napthol and para- E. coli Negative: no change
!ll"l'llno-dimethylaniline S. aureus Negative: no ·change
0>1alate)

Phenylalanine P. mirabilis Positive: green color

Qeaminase Salmonella sp Negative: no color change
(P~enylalanine Agar) E. coli Negative: no color change

M19lonate Utilization K. pneumoniae ·Positive: blue color change

(M11lonate Broth) E. coli Negative: no growth or color
change

Milk, Methylene S. faecalis Positive: reduction of

~lue, 0.1% methylene blue
Group Q Streptococci Negative: no growth
Strep. salivarius Negative: no growth
· P. aeruginosa Peptonization and digestion
C. perfringens · Acid, coagulation and gas

~itrate Reduction E. coli Positive: red color change

(Potassium Nitrate P. aeruginosa Negative: no color change
BrQ.th)

2, 3, 5-Triphenyl S. faecalis Positive: reduction of ITC

T~tra~olium Chloride (red color)
11'1 TG Agar Strep. faecium Negative: no color change

Tellurlte Agar S. faecalis Growth

S. faecium No growth

Beta-Hemolysis S. faecalis Positive: lysis of red blood cells

In Blood Agar var. zymogenes
S. faecalis Negative: no lysis of red blood cells

Slant Butt H2S Production

Hvdrogen Sulfide £. coli A AG
(Triple Sugar P. vulgaris. A AG. +
Iron Agar) S. typhimurium K AG +

QA/LABORATORY OPERATIONS 223

 TABLE IV-A-5
(continued)

Quality Control of Biochemical Tests

Test Control Culture Expected Results

Slant Butt H2S Production

Lysine s. typhimurium K K +
(Lysine Iron Agar) s. flexneri K A

Gelatin Liquefaction S. marcescens Positive: liquefaction

at 20 C S. faeca/is Positive: liquefaction
(Nutrient Gelatin) var. liquefaciens
S. faecalis Negative: no liquefaction
C. perfringens Positive: liquefaction
E. coli Negative: no liquefaction

REFERENCES

1. American Public Health Association, 1972. Standard Methods for the Examination of Dairy
Products, (13 ed.) American Public Health Assoc1at1on, Inc., Washington, DC. RODAC:p. 192;
Swab: pp. 43-44; and Air: p. 44.

2. American Public Health Association, 1975. Standard Methods for the Examination of Water and
Wastewater (14 ed.) American Public Health Association, Inc., Washington, DC. p. SS~ -

3. Geldreich, E. E. and H. F. Clark, 1965. Distilled water suitability for microbiological applications.
J. Milk and Food Tech. 28:351.

4. Military specifications for Disk, Filtering, Microporous, Hydrosol Type, 47 mm diameter 100s.
MIL-D-37005 (DSA-DM) 5 September, 1973. Directorate of Medical Materiel, Defense Personnel
Supply Center, Dept. of Defense. Philadelphia, PA.

5. Tentative method for evaluating water testing membrane filters for fecal coliform recovery.
D3508-76T. 1978 Annual Book of ASTM Standards, Part 31, p. 1127.

6. Standard specification for reagent water. D1193-77. 1978 Annual Book of ASTM Standards,
Part 31, p. 20.

224 oEPA MICROBIOLOGICAL MANUAL 191lJ

 PART IV. QUALITY ASSURANCE

Section B Statistics for Microbiology

r:t

The Section is divided into five major An example of microbiological data is shown
areas of statistical measure: in Table IV-B-1.

1. Measures of Central Tendency TABLE IV-B-1

2. Measures of Dispersion Microbiological Results, Count/100 ml

3. Normal Distribution 79 220 330

110 230 490
4. Poisson Distribution
130 280 490
130 330 790
170 330 950
In this Section the computational formats 220 330 1100
for the more commonly used measures in sta-
tistics are described. In ~.he following discus-
sion let xi denote a typical observed result so Arithmetic Mean, X 372.7
that (x 1 , x 2 , .... , xn) represents a sample of n ob-
servations. A good reference book for further Geometric Mean, X9 287.8"
details on these parameters is Dixon and
Massey (1). Median 305
1. Measures of Central Tendency
Mode 330
1.1 The Arithmetic Mean: The most com-
monly used measure of central tendency is the Standard Deviation {S) 293.2
arithmetic mean which is often simply called
"the mean". Denote the sample mean by X and Range 1021
the population mean, of which X is an esti-
mate, byu. The computational formula is:

n The sample mean is calculated as follows:

. E 1 Xj
I=
n n
E x·
i= 1 I
n 6709
where l X· x --- 372.7
i=1 I n 18

QA/STATISTICS 225
 1.2 The Geometric Mean: A second mea- is tedious. It is relatively simple to show the
sure of central tendency that is preferred for following relationship:
certain distributions such as the Poisson. It will
be discussed later, in 4.3 but is defined as:
n 2 n 2
n1: X - (E Xi)
s - =
i=1 i=1
n(n - 1)

The derived formula is preferable because of

The geometric mean of the data in Table IV-B-1 its adaptability to the desk calculator. The sam-
is287.8. ple standard deviation of the microbiological
data in Table IV-B-1 is calculated as follows:
1.3 The Median: Another measure of cen-
tral tendency is the median. The median is the s (18) (3961541) - (6709) 2
value such that half of the other values are 18 (17)
greater and half are less. To find the sample
median, the data are arranged in ascending or
descending order and the middle value is 71307738 - 45010681
picked. When there is an even number of ob- 306
servations, the average of the two middle val-
ues ls taken. For the data in Table IV-B-1, the 2629i057
median is 305. 306

1.4 The Mode: The mode, one other mea-

sure of central tendency, is the most frequently
-v· 85939 = 293.2
occurring value. In Table IV-B-1 the mode is .
330, since this value occurs four times. The Confidence Limit (95% and 99%): The range
population mode is the value corresponding to ·of values within which a single analysis will be
the peak of the frequency distribution curve. included, 95% or 99% of the time.

95% C. L. = X ± 1.965
Frequency distributions with more than one
peak are called multimodal. In a symmetrical
frequency distribution, the mean, median, and 99% C. L. = X ± 2.585
mode are all equal.
2.2 The Variance: The sample value 5 2 is
referred to as the sample variance and is
2. Measures of Dispersion merely the square of the sample standard devi-
ation. Often it is more convenient in conversa-
2.1 The Standard Deviation: Of the sev- tion as well as computation to refer to the
eral measures of dispersion, the most common variance. This should not cause confusion if
ls the standard deviation. Denote the sample the above relationship is kept in.mind.
standard deviation by S and the population
value by cr(of which S is an estimate), when the The population variance is represented by
computational formula is: cr 2• Its formula is;

cr2 =
s = n

This is the same as the formula for 5 2

However, the computation using this formula except that the true population mean u is used

226 oEPA MICROBIOLOGICAL MANUAL 1978

rather than its estimate X and the numerator is 3. 1.2 Small deviations from the mean are
dl°t'ided by n instead of n - 1. extremely likely.

In calculating the sample variance the true 3. 1.3 Large deviations from the mean are
mean is not known and the estimate of the extremely unlikely.
mean from the data is used instead. Because
the sample mean is being used to calculate the 3.2 The normal distribution is completely
variance of the same data, only n - 1 of the defined by its mean,µ, and its standard devia-
squared difference terms are independent. It tion cr in the following manner:
can be shown that the estimate of the variance
must be based upon the sum of independent 3.2.1 The area under the normal curve
squared terms, thus indicating the division by betweenµ minus cr andµ plus cr is 68 percent of
n - 1, which is called the number of degrees of the total area, to the neare.st 1 percent.
freedom (d.f.) in the sample. As a rule, in any
calculation, for every parameter that must be 3.2.2 The area .under the normal curve
estimated! one degree of freedom is lost. betweenµ minus 2cr andµ plus 2cr is 95 per-:
cent of the area, to the nearest 1 percent.
2.3 The Range: The range is also used as a
measure of dispersion. It is the difference be- 3.2.3 The area under the normal curve
tween the highest and lowest values in a set of between µ minus 3cr and µ plus 3cr is 99.7
data. percent of the total area, to the nearest 0. 1
percent.

3.3 If the frequency distribution of a sam-

For the data in Table IV-B-1 the range is then: ple is a good approximation to the normal
curve, these characteristics of the normal
R= 1100-79= 1021 curve can be used to develop a great deal of
information about the underlying population.
A rough estimate of S can be made by dividing
the range of the sample by the square root of n,
the number of observations, when n ~ 10: 4.Non-symmetric Distribution

R
s - rn 4, 1 Asymmetry: In some investigations
one encounters distributions which are not
symmetric. For example, distributions of
bacterial counts are often characterized as
Use of the range as a measure of dispersion is having a skewed distribution because of the
generally limited to instances where the labor many low and a few extremely high counts. This
of computing the standard deviation is characteristic leads to an ari.thmetic mean
impractical. · which is considerably larger than the median
or the geometric mean. The frequency curves of
these d.istributions have a. long right tail, as
3. The Normal Distribution shown in Figure IV-B-2, and are said to display ·
positive skewness.
3. 1 The most important theoretical distri-
bution in statistics is the familiar bell-shaped 4.2 Logarithmic Transformation: For
normal distribution which is symmetric about practical and theoretical reasons, statisticians
its peak (see Figure IV-B-1). The following as- prefer to work with symmetric distributions
sumptions give rise to this distributional form: like the normal curve. Therefore, it is usually
necessary to convert skewed data so that a
3.1.1 Values above or below the mean are symmetric distribution resembling the normal
equally likely to occur. distribution results: An approximately normal

QA/STA TIS TICS 227

 >-
u
z
UJ
::::>
Cl
UJ
0::
u..

-3cr -2cr -lcr Jl +lcr +2a +3cr

QUANTITY MEASURED

FIGURE IV-B-1. Normal Distribution Curve.

>-
u
z
UJ
::::>
Cl
UJ
0::
u..

QUANTITY MEASURED

FIGURE IV-B-2. Positively-Skewed Distribution Curve.

228 &EPA MICROBIOLOGICAL MANUAL 1978

distribution can be derived from positively- log x9
skewed distributions by expressing the original
data as logarithms. An example of coliform
counts and their logarithms are shown in Therefore, the best measure of central tend-
Table IV-B-2. A comparison of the frequency ency for microbiological data is the log-
tables for the original data and their logs in transform.
Tables IV-B-3 and 4, shows that the logarithms
more closely approximate a symmetric
distribution.
The mean of tne log MPN data in Table IV-
B-2 is:

4.3 The Best Measure of Central Ten-

dency for Microbiological Data: Assuming 32.737
that the microbiological data has been normal- log X = 2.1825
n 15
ized through a logarithmic transformation, the
arithmetic mean is the best estimate of central
tendency. However, there is a direct relation-
ship between this mean and the geometric
mean of the original data: and the true mean of the MPN data is:

(log X) = antilog (2. 1825) 152

n

REFERENCES

1. Dixon, W. J. and F. J. Massey, Jr., 1969. Introduction to Statistical Analysis, 3rd Edition, McGraw-Hill,
Inc., New York, NY.

TABLE IV-B-2
Coliform Counts and Their Logarithms

Coliform
Count/100 ml log MPN
MPN

11 1.041
27 1.431
36 .1.556
48 1.681
80 1.903
85 1.929
120 2.080
130 2.114
136 2.134
161 2.207
317 2.501
601 2.779
760 2.881
1020 3.009
3100 3.491

QA/STATISTICS 229
 TABLE IV-B-3

Comparison of Frequency of MPN Data

Class Interval Frequency (MPN)

0 to 400 11
400 to 800 2
800 to 1200 1
1200 to 1600 0
1600 to 2000 0
2000 to 2400 0
2400 to 2800 0
2800 to 3200

TABLE IV-B-4

Comparison of Frequency of ·Log MPN Data

Class Interval Frequency (log MPN)

1.000 to 1.300 1
1.300 to 1.600 2
1.600 to 1.900
1.900 to 2.200 5
2.200 to 2.500 1
2.500 to 2.800 2
2.800 to 3.100 2
3.100 to 3.400 0
3.400 to 3.700

230 ~EPA
" MICROBIOLOGICAL MANUA_L 1978
 PART IV. QU~LITY ASSURANCE

Section C Analytical Quality Control Procedures

The Section on Analytical Quality Control pies using buffered water and the medium
is divided into three major areas of statistical batch at the start of the test series and follow-
usage: ing every tenth sample. When sterile controls
indicate contamination, data on samples af-
1. Quality Control on Routine fected should be rejected and a request made
Analyses for immediate resampling of those waters
involved.
2. Quality Control in Compliance
Monitoring
1.4 MF Colony Counting by More than
3. Comparative Testing of One Analyst: At least once per month, two or
Methodology more analysts should count the colonies on the
same membrane from a polluted water source.
4. Method Characterization Colonies on the membrane should be verified
and the analysts' counts compared to the veri-
fied count.

1. Quality Control on Routine Analyses 1.5 Check Analyses on Water Supply

Program by State Laboratories: "in a local
Each laboratory must establish quality laboratory, a minimum .r.umber of the water
control over the microbiological analyses in supply samples should be analyzed by the
use. Fifteen percent of total analyst time State laboratory. For example, · 1aboratories
should be spent on quality control practices that are required to test less than 100 samples
discussed in this Manual. per month should submit an additional 10% of
the number to the State laboratory for analysis.
1.1 Duplicate Analyses: Run duplicate Water systems with sample requirements
analyses on 10% of the known positive sam- above 100 per month should submit an addi-
ples analyzed and a minimum of one per tional 2% to the State Laboratory for analysis.
month. The duplicates may be run as split
samples by more than one analyst. 1.6 Reference Sample: Laboratories
should analyze reference samples quarterly
1.2 Positive Control Samples: Test a min- when available for the parameters measured.
imum of one pure culture of known positive
reaction per month for each parameter tested. 1. 7 Performance Sample: ·Laboratories
should analyze at least one unknown perfor-
1.3 Negative (Sterile) Control: Include mance sample per year when available, for
one negative control with each series of sam- parameters measured.

QA/ANALYTICAL PROCEDURES 231

 1.8 MF Verification: Five percent of the 1.10 Measurement of Analyst Precision:
analyses performed should be verified. If the routine work of the laboratory includes
samples from different wastewaters, surface
1.8.1 Total Coliforms: Pick at least 10 waters, water supplies or finished waters, the
isolated sheen colonies from each sample. following steps should be accomplished for
Transfer into lauryl tryptose broth. Incubate each type.
and read. Transfer positive tubes into brilliant
green bile broth for verification of coliforms.

Since samples from public water supplies Perform duplicate analyses on the first 15
with 5 or more sheen colonies must be verified, typical samples with positive responses.
at least 5 colonies are picked from each positive Although each set of duplicates must be
potable water sample. run by the same analyst, all analysts per-
forming routine analyses should contrib-
The laboratory should make every effort to ute a share of this initial data.
detect coliforms from samples with excessive
non-coliforms on the membrane filter. Any
sheen colonies appearing in mixed confluent
growth must be verified (see Part 111-B).
Calculate the logarithms of results. If ei-
1.8.2 Fecal Coliforms: Pick at least 10 ther of a set of duplicate results is zero,
isolated colonies containing blue to blue-green add 1 to both values before calculating the
pigment and transfer to lauryl tryptose broth. logarithms.
Incubate and read. Transfer positive tubes to
EC broth where gas production verifies fecal
coliform organisms (see Part 111-C).

1.8.3 Fecal Streptococci: Pick at least 10 Calculate the range (R) for each pair of
isolated pink to red colonies from MF or pour transformed duplicates and the mean (R)
plates. Transfer to BHI agar or broth. After of these ranges.
growth, perform catalase test. If negative (pos-
sible fecal streptococci) transfer growth to BHI
and 40% bile BHI broth tubes and incubate at
45 C and 35 C respectively. Growth at both tem-
Thereafter, run 10% of routine samples in
peratures verifies fecal streptococci (see
duplicate. Transform the duplicates as in
Part 111-D).
Step 2 and calculate their range. If this
range is greater than 3.2 7 R, analyst preci-
1.9 MPN Completion of Total Coliform Test
sion is out of control and all analytical
results since the last precision check must
1.9. 1 For routine analyses, complete the be discarded. The analytical problem must
MPN test on five percent of the positive con- be identified and resolved before doing
firmed samples and a minimum ·of one sample furtheranalyses (1).
pertest run.

1.9.2 For potable waters, complete the

MPN test once each quarter on 10 percent of
positive confirmed samples. If insufficient pos- In order that the criterion used in Step 4
itive tubes result from potable water samples, be kept up-to-date, periodically repeat
perform the completed test on positive source Steps 2 and 3 using the most recent sets
waters. of 15 duplicate results.

232 oEPA Ml<;ROBIOLOGICAL MANUAL 1978

2. Quality Control in Compliance Monitoring 2.3 Whenever the analytical standard de-
(National Poilution Discharge Elimination viation must be estimated from the study data
System)· the following procedure should be followed
for each major type of discharge.

2. 1 For any legal assurance of non- Step _l_

compliance within a permit, analytical results
must exceed the permit limit by a statistically Take 3 simultaneous grab samples at each
significant amount. This requires allowance of of K (at least 3) randomly or systematically
the analytical deviation known to occur at a selected sampling times which are
level equal to the permit limitation. expected to represent the range of
discharge throughout the study period.
2.2 It is apparently common for many
monitoring agencies to judge compliance with
microbiological permit limitations based upon
one analysis each for single grab samples Analyze the samples and convert the re-
taken at about the same time on 3 consecutive sults to logarithms.
days. The largest of these 3 results is then
compared to a maximum discharge limitation,
while· the arithmetic mean is compared to a
daily · average limitation. Judgment based Using these logarithms, calculate the fol-
upon such a sampling program is only valid lowing statistics:
under certain assumptions; namely that there
is no relationship between the discharge level
and the time of sampling, and that the variabil-
K 3 3
ity among analytical results for samples taken 1 2
2K ( l: ( E Xii - ( E
simultaneously is either inconsequential or s i=1 i=1 i=1
can be properly estimated from previous data.
3
Xi ( E . Xij)/3
The first assumption is usually invalid be- j=1
cause the discharge level is dependent upon K
production or processing operations which are X ( r. Xf)/K
not uniform, but rather follow some daily or i=1
weekly cycle. To keep such periodic cycles
from biasing the results, the grab samples
should be taken at randomly-selected times where: S = the pooled e~timate of the
during the study or, if there is some knowledge analytical standard deviation
of the cycle, at times selected systematically to
define its extremes. ·
Xii = the analytical result fort.he jth
The second assumption is important in grab sample taken at
order to make a compliance judgment. As sampling time i,
noted in 2. 1, this judgment must take into
account the estimated analytical standard de- xi = the mean result for the 3 grab
viation at a level equal to the permit limit. If an samples taken at
appropriate estimate of this' standard devia- sampling time i,
tion is not already available then it must be
estimated during the compliance study, either X the overall mean for the K
from multiple analyses of each grab sample or sampling times
from single analyses of multiple grab samples throughout the study
taken simultaneously. period.

. .
QA/ANALYTICAL PROCEDURES 233
 Step i_ 3. Comparative Testing of Methodologies

The maximum discharge limitation (Dmax)

has been violated if for any sampling time 3.1 Protocol for Testing: If a laboratory
i: proposes a new or modified method for accept-
ance certain minimal testing should be
Xi - t,95(2K){S/V3)" > Dmax completed.

where t. 95 (2K) is the Student's t value at a

3. 1. 1 Collect and test at least 10 samples
96 percent confidence level and 2K de-
from each sample source or type for which the
grees of freedom.
method is intended, i.e., fresh surface water,
sewage treatment plant influent or effluent,
industrial effluents, saline water, etc. Source
waters should be chosen which are represent-
An average limitation (D) over the study
ative and which span the relevant range of
period (daily, weekly, monthly, etc.) has
concentrations.
been violated if:

X- t,95(2K)(S/i/3K) > D 3.1.2 Collect the samples and perform the

analyses over 1 week of plant processing, but
not less than 5 calendar days.
2.4 If an estimate of the analytical stan-
dard deviation (S) is available that has at least
30 degrees of freedom, and if the estimate 3. 1.3 Take an even number of aliquots (at
applies to the same type of waste and was least 8) from each sample and simultaneously
generated from samples at a level near the analyze half of them using the accepted me-
discharge limitation of the permit under study, thod and the other half using the proposed
then Steps 1-5 can be modified as follows: alternative method. Throughout this protocol,
the analysis of aliquots from a single sample
using one method will be referred to as "repli-
cate analyses" and the resulting data will often
Take one grab sample at each of K (at least be referred to as "replicates." ·
3) sampling times selected as before.
3.2 Statistical Analyses: Compile the
data and perform the following steps for each
Analyze samples and convert results to sample type:
logarithms.
Step .1._: Transform the Data

K
Calculate: X = I E X1)/K
i=1 Calculate the logarithms of the basic data
and use these transformed data as the
basis for all subsequent statistical
analyses.
Dm 11 x has been violated if X 1 > Dmax + 2S
for any sampling time i. The . characteristics of microbiological
data have been discussed by many
Step§__ authors. Of particular importance to this
procedure is the effect that a logarithmic
5 has been violated if X > D + 2S/~ transformation has on such data (2, 3).

234 &EPA MICROBIOLOGICAL MANUAL 1978

Specifically, a logarithmic transformation This is done by applying Cochran's Test (4)
normalizes the distribution of results from as follows:
replicate analyses of the same sample and
equalizes the within-sample standard
deviations among samples with different
microbiological densities. These
conditions are required assumptions for K =the number of samples
many of the statistical procedures that
follow. Si = the standard deviation estimate from
the set of replicates for the ith
sample,
Step 2: Calculate the Basic Statistics.
Smax =the largest value for Si i =1, 2, ... , K
Calculate the arithmetic mean (X) and
Look up the critical C value for n-1 degrees
standard deviation (S) for each set of
of freedom on each of K standard devia-
replicates, and tabulate the data and their
tions and a .01 significance level, i.e., c. 99
basic statistics.
(n-1, K) in Dixon and Massey (5).

If C ~ C_ 9 9 (n-1, K), then pool the standard

Step 3: Test for Suspected Outliers deviations for the method (M) as follows:

For each set of n replicates, test for outli- K

ers by calculating the Extreme Studen- s~ ( l: Sf )/K
i= 1 .
tized Deviate as T = (XE - X)/S, where XE
= the maximum or minimum replicate,
S~· is· the pooled within-sample variance
whichever is farthest from X.
estimate forthe method.

Look up the critical T value for the number If C > C_ 99 (n-1, K), then the within-sample
of replicates on each sample and a .01 ( 1 standard deviation of this method is not a
percent) significance level in Standard constant over the concentration range
D2777-72, 1975 Book of ASTM Stan- represented in the data. To correct for this,
dards, Part 31, Water, page 1 5. If T is the data must be stratified, i.e., subdi-
greater than the. critical value, reject all vided, into sets of samples which have a
data for that sample with 99 percent confi- common standard deviation among the
dence that XE is an outlier. results of replicate analyses by this me-
thod. Care should be take.n to minimize the
number of strata to those that are required
This unfortunate loss of data is necessary in order to justify the development of
because the following statistical proce- pooled within-sample standard deviation
dures have been simplified and only apply estimates. If both methods require stratifi-
to the unique situation where, for each of cation, common strata should be devel-
K sample, there are exactly n replicates by oped which are jointly suitable. Step 4
each of the two methods. should be repeated to verify the proposed
stratification. Subsequently, Step 5
Step 4: Test for Equality Among the should be carried out for each stratum
Within-Sample Standard Deviation independently. If you have any problems
Estimates with stratifying the data, please seek the
assistance of a qualified statistician/data
For each met.hod, test for equality of the analyst before proceeding.
standard deviation estimates (S) caicu-
lated in Step 2 from the replicates for each Step 5: Test for Equality of the Pooled
sample. Within-Sample Variance Estimates

QA/ANAL YT/CAL PROCEDURES 235

 Having developed a pooled within-sample sewage treatment effluents. Recognized prob-
variance estimate for each method, within lems in Method 1 include: deviation among
common strata if necessary, test the replicate results is considered excessive, aver-
equality of these estirnates by performing ages show a positive bias of about 40 percent,
the following F test: and the procedure is difficult and time-
consuming.
2 2 2 2
F = SM /SM ,where SM > SM
1 2 1 2
Suppose that Method 2 is quicker and
easier than Method 1. Then, if the following
statistical analysis shows the performance of
Look up the critical F value for K(n-1) Method 2 to be at least equal to Method 1, EPA
degrees of freedom for each variance and may designate Method 2 as an alternative to
a .05 significance level, i.e., F. 95 (K(n-1 ), Method 1. If Method 2 offered no operational
K(n-1 }), in any standard statistical advantage over Method 1, the statistical analy-
reference. If F ~ F. 96 (K(n-1 ), K(n-1 )), then sis would have to show the performance of
the two methods produce results with Method 2 to be significantly superior to Me-
within-sample variances which are not thod 1.
significantly different. If F > F. 95 (K(n-1 ),
K(n-1)), then. there is a statistically Steps 1 and~
significant difference between the
variances. Transform the Data (Table IV-C-1) and Cal-
culate the Basicstatistie:s: -
Step 6: Test for Equality of the Method
Means The logarithmic transformation was found
most appropriate for the data in Table IV-
Finally, calculate the difference between C-1, see the results in Table IV-C-2.
the method means for each of the K sam-
ples as:

dj - XM - XM I i = 1, 2, ... ,K. Test for Suspected Outliers in Table IV-C-

1 2
2.
Using the mean (d) and standard deviation
(Sd) of these differences, calculate the Stu- IXe - Xt
T
dent's statistic as: s
t
d-
=--
Sd/VK
Sample 2, method 2, replicate 1:
Refer to a statistical reference for the crit-
ical Student's t value at the .05 signifi- Xe= 3.3711
cance level and K-1 degrees of freedom,
i.e, t.9 6(K-1 ). If t > t. 95 (K-1 ), then the means 3.3711 - 3.67731
for the two methods are significantly T 1.76
.1738
different.

3.3 Example of the Statistical Analysis Sample 4, method 2, replicate 1:

Procedure
Xe= 2.7497
Statement of the Problem

Method 1 is the accepted method for de- 12.7497 - 2.52991

T 1.69
termining fecal coliform levels in chlorinated .1301

236 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-C-1

Raw Sample Data from the Analysis of Chlorinated Sewage

Treatment Plant Effluents

Sample Replicates (XR)

No. Date Method in counts/ 100 ml ~XR XR SR

3/11 1 1850, 1350, 1050, 1800, 1150 7,200 1440 368.1

2 1200, 850, 650, 500, 1050 4,250 850 .285.0

2 3/11 1 7800, 8100, 5900, 5450, 7150 34,400 6880 1163.3

2 2350, 5300, 5650, 6350, 5450 25, 100 5020 1545.8

3 3/17 1 4000, 4400, 2650, 3000, 3650 17,700 3540 715.4

2 2400, 2150, 2050, 1600, 2200 10,400 2080 297.1

4 3/18 1 625, 512, 512, 462, 700 2,811 562 97.5

2 562, 350, 300, 275, 275 1,762 352 121.1

5 3/24 1 3870, 2330, 3330, 3670, 2530 15,730 3146 685.2

2 2730, 3930, 2530, 2200, 1730 13, 120 2624 822.3

6 3/25 1 7200, 4400, 7800, 6600, 5200 31,200 6240 1410.0

2 4400, 4000, 4400, 3600, 6200 22,600 4520 996.0

7 3/26 1 1200, 1000, 1000, 1200, 1600 6,000 1200 245.0

2 700, 600, 900, 900, 900 4,000 800 141.4

~ ..

QA/ANAL YT/CAL PROCEDURES 237

 . TABLE IV-C-2

Logarithmic Transformation of the Data in Table IV-C-1

Sample Transformed Replicates

No. Method (X = log XR) l:X x s

1 1 3.2672, 3.1303, 3.0212, 3.2553, 3.0607 15.7347 3.1469 .1115

2 3.0792, 2.9294, 2.8129, 2.6990, 3.0212 14.5417 3.9083 .1544

2 1 3.8921, 3.9085, 3.7709, 3.7364, 3.8543 19.1622 3.8324 .0756

2 3.3711, 3.7243, 3.7520, 3.8028, 3.7364 18.3866 3.6773 .1738

3 1 3.6021, 3.6435, 3.4232, 3.4771, 3.5623 17.7082 3.5416 .0903

2 3.3802, 3.3324. 3.3118, 3.2041, 3.3424 16.5709 3.3142 .0664

4 1 2.7959, 2.7093, 2.7093, 2.6646, 2.8451 13.7242 2.7448 .0735

2 2.7497, 2.5441. 2.4771, 2.4393, 2.4393 12.6496 2.5299 .1301

6 1 3.5877, 3.3674, 3.5224, 3.5647, 3.4031 17.4453 3.4891 .0984

2 3.4362, 3.5944. 3.4031. 3.3424, 3.2380 17.0141 3.4028 .1310

6 1 3.8573, 3.6435, 3.8921, 3.8195, 3.7160 18.9284 3.7857 .1033

2 3.6435, 3.6021, 3.6435, 3.5563, 3.7924 18.2377 3.6475 .. 0886

7 1 3.0792, 3.0000, 3.0000, 3.0792, 3.2041 15.3625 3.0725 .0836

2 2.8451, 2.7782, 2.9542, 2.9542. 2.9542 14.4860 2.8972 .0816

238 &EPA MICROBIOLOGICAL MANUAL 1978

Sample 5, method 2, replicate 2:

XE= 3.5944 Test for Equality of the Pooled Within-

SampleVariance EStlmates
13.5944 - 3.40281
T 1.46
.1310 2 2
F = s M2 /S M1 .0153/.0084 1.82

Sample 6, method 2, replicate 5:

The critical F value at .05 significance
XE= 3.7924 level, when both variance estimates have
7(4) or 28 degrees of freedom, equals
1.88. Since the critical value is not ex-
13.7924 - 3.64751
T 1.64 ceeded, these methods do not produce
.0886
significantly different within-sample vari-
ances. The same statement can be made
. . regarding within-sample standard
The critical T value for sets of 5 replicate deviations.
and a .01 significance level is 1.76. Since
none of the T values is greater than 1.76,
we must accept all of the suspected
values.
Test for Equality ofthe Method Means

First, for each sample, calculate the differ-

Test for Equality Am.ong the Within- ence between the means in Table IV-C-2.
Samp~Standard Deviation EStimates (S) These differences (di) are shown in Table
for Each Method in Table IV-C-2: - IV-C-3 along with their mean (CJ) and stan-
dard deviation (Sd). Then calculate:
2 K 2
Recall that C = Smaxl E Si
t = d/Sd/Viij = .1765/(.0547/Vl)
i=1

For Method 1: C = (.115) 2 /.0590 = .2106 .1765/.0207 8.54

For Method 2: C (.1738) 2 /.1071

The critical Student's t value at a .05 sig-
.2822 nificance level and 6 degrees of freedom
The critical Cochran statistic for 7 vari- is 2.45. Since the calculated value ex-
ances with 4 degrees of freedom each and ceeds the critical value, the methods do
a .01 significance level equals .5080. produce significantly different means.
Since both the calculated C values are less
then the critical value, the within-sample
variances are equal and we can proceed to To Summarize the Results
calculated pooled variance and standard
deviation estimates for each method: (a) The within"sample standard deviations
for these methods are not significantly differ-
s2
M1
.0590
7
.0084 ... SM
1
= .0918
ent at the .05 significance level.

(b) The mean for Method 1 is significantly

s2
M2
.1071
7
.0153 ... SM
2
.1237
higher than the mean for Method 2 at well over
the .05 significance level.

QAiANALYT/CAL PROCEDURES 239

 Discussion of the Results trary degree, for example, three orders of mag-
nitude, compared with growth and recovery on
Referring to Table IV-C-3, it can be easily .non-selective media.
seen that Method 1 produces the larger mean.
The difference can be calculated as:
4.3 Precision is a measure of the deviation
among multiple measur;ements of a single
(antilog(3.3733} - antilog(3.1967)\ quantity. The most widely used expression of
100
\:' antilog(3.1967} ) precision is the standard deviation (o-) which is
equal to the square root of the variance and
- (2362 - 1573\ 100 indicates the deviation of the values about the
- 1573 ) mean.

Like accuracy, the true precision of a me-

=- {_ 7 s 9 ) 100 = 50.2% thod must be generated in a collaborative
\1573 study among at least 15·1aboratories.

Since Method 1 results are known to be 4.4 Accuracy is a measure of the close-
about 40% greater than the true value, it can ness of observed values.to a known true value.
be estimated that Method 2 results are about 7 The lack of available standards for comparison
percent less than the true value. Therefore, of microbiological methodology has resulted
besides being quicker and easier, Method 2 in methods with known precision, but with
offers comparable precision with improved ac- limited accuracy information.
curacy. Such results would qualify Method 2
for approval as an alternative to Method 1. In microbiology, accuracy has been deter-
mined by applying the test method to a sus-
pension of a pure culture while independently
4. Method Characterization determining the cell number in the suspension
using a non-selective medium. Natural water
The choice of a method of analysis among samples are tested with and without the pure
several considered should be based on com- culture spike and the recovery determined by
parison of relative performance using these difference. The recovery from the spiked sam-
measureable characteristics. ple is compared with the count on the non-
selective medium (assumed to be true value)
4. 1 Specificity is the ability of a ·method to and expressed as a percent of this true value.
recover the desired organisms identified by a
selective or differential characteristic and veri- True accuracy of a method must be gener-
fied by additional tests. A method is judged ated in a collaborative study among at least 1 5
specific if the recovered microorganisms ver- laboratories using known levels of organisms.
ify as the desired organism, and the colonies
designated as "other organisms" do not verify
as the desired organism whe'n picked and
tested. The acceptable level of specificity for a 4.5 The Section on Sanitary and Health
method cannot be set absolutely, for example Effects Microbiology in the American Society
as 90%, but rather must be established for for Testing and Materials (ASTM) Committee
standard procedures or for new parameters on D-19:24 has also proposed a characteristic
a best judgment basis by comparison with the called Counting Range. The Counting Range is
accepted methods. described as the range from the lowest num-
ber to the highest number of colonies that can
4.2 Selectivity is the ability of a method to be measured on a single agar plate or mem-
encourage growth of the desired organism brane filter, without affecting the reliability of
while reducing other organisms by some arbi- the method.

240 oEPA MICROBIOLOGICAL MANUAL 1978

 TABLE IV-C-3

Analysis of Difference Between Means

Sample Mean from Table IV-C-2 for Difference Between

No. Method 1(XM ) Method 2 (XM2) Mean!:\ (ci1 = XM - XM l
1 1 2

3.1469 3,9083 .2386

2 3.8324 3.6773 .1551

3 3.5416 3.3142 .2274

4 2.7448 2.5299 .2149

5 3.4891 3.4028 ,0863

6 3.7857 3.6475 .1382

7 3.0725 2.8972 .1753

== =
XM
1
3.3733 XM2 3.1967 d = .1765 sd .0547

QA/ANALYTICAL PROCEDURES 241

 REFERENCES

1. Grant, E. L. and R. S. Leavenworth, 1972. Statistical Quality Control. Fourth Edition. McGraw-Hill, Inc.,
New York, NY. p. 87.

2. Eisenhart, C. and P. W. Wilson, 1943. Statistical Methods and Control in Bacteriology. Bacteriological
Reviews 7:57.

3. Velz, C. J., 1951. Graphical Approach to Statistics, Part 4: Evaluation of Bacterial Density. Water and
Sewage Works, 98:66. -- -

4. Dixon, W. J. and F. J. Massey, Jr., 1969. Introduction to Statistical Analyses, Third Edition. McGraw-Hill,
Inc., New York, NY. p. 310.

5. ibid. p. 537.

242 .::.EPA
"" MICROBIOLOGICAL MANUAL 1978
 PART V LABORATORY MANAGEMENT

Part V addresses those laboratory activities which supplement the .analytical methodology and
are primarily the responsibility of the laboratory manager.

Section A Development of a Quality Control Program

Section B Manpower and Analytical Costs

Section C Safety

Section D Legal Considerations

MANAGEMENT OF QUALITY CONTROL 243

 PART V. LABORATORY MANAGEMENT

Section A Development of a Quality Control Program

1. lntralaboratory Quality Control 2. 1 An Operating Manual is prepared

which describes the sampling techniques, ana-
1.1 To insure a viable quality assurance lytical methods, laboratory operations, mainte-
effort, management must recognize the need nance and quality control procedures. Specific
for a formal program and require its develop- details are given on all procedures and quality
ment. Management must commit itself to the control checks made on materials, supplies,
program by setting aside 15% of the labora- equipment, instrumentation and facilities. The
tory man-years for quality control activity. It frequency of the checks, the person responsi-
must meet with supervisors and staff to estab- ble for each check (with necesssary back-up
lish levels of responsibility for management, assignments), the review mechanism in the QC
supervisors and analysts. Laboratory person- program to be followed, the frequency of the
nel should participate in the planning and review ahd the corrective actions to be taken
structuring of the QA program. are specifed. A copy is provided to each
analyst.
1.2 Once the QA program is functioning,
supervisors review laboratory operations and 2.2 A Sample Log is maintained which
quality control with analysts on a frequent records chronologically information on sam-
(weekly) basis. Supervisors use the results of ple identification and origin, the necessary
the regular meetings with laboratory person- chain of custody information, and analyses
nel to inform management on a regular performed.
(monthly) basis of the status of the QA pro-
gram. These meetings are important to identify 2.3 A Written Record is also maintained of
the problems at the laboratory level and to get all analytical QC checks: positive and negative
the backing of management in the actions culture controls, sterility checks, replicate ana-
necessary to correct problems. lyses by an analyst, comparative data between
analysts, use-test results of media, membrane
filters and laboratory pure water, replicate ana-
lyses done to establish precision of analysts, or
of methodology used to determine non-
2. Documentation of the lntralaboratory
compliance with bacterial limits and water
Quality Control Program
quality standards.
Unless a record is made of the quality
control checks and procedures described in
Part IV of this Manual, there is no proof of
performance, no evidence for future reference 3. lnterlaboratory Quality Control
and for practical purposes, no quality control
program in operation. The following documen- An intei"laboratory quality control program
tation should be made and maintained. consists of: 1) formal collaborative method

244 ~EPA
""' MICROBIOLOGICAL MANUAL 1978
studies to establish precision and accuracy of unknown samples, 5) follow up on problems
selected methodology, 2) specific minimal detected in onsite inspections and perfor-
standards for personnel, sampling and sample mance evaluations.
preservation procedures, analytical methodol-
ogy, equipment, instrumentation, facilities and
within-laboratory quality control programs, 3) EPA has established such a interlabora-
verification of acceptable standards through tory quality control program in response to
annual on-site inspections, 4) periodic perfor- the FWPCA Amendments of 1972 and the Safe
mance tests of analytical capabilities using Drinking Water Act of 1974.

MANAGEMENT OF QUALITY CONTROL 245

 PART V. LABORATORY MANAGEMENT

Section B Ma,npower and Analytical Costs

Laboratories planning to begin or increase lyst on day one. Two hours are required to
microbiological activities have difficulty in de- complete the counting, calculation of results
termining their added manpower, equipment and verification , on day ~wo.
and supply needs. This Section provides esti-
mates of added costs: If fecal and. total coliform tests are per-
formed using 3 dilutions, twenty (20) samples
1. Time Expenditures can be analyzed in. one day by one analyst. On
the second day, counting of plates takes an
2. Specialized Equipment and estimated . 2 1 /2 hours. An estimated four
Supplies hours are needed for preparation of. media,
dilution water, dishes and pipettes for the test
period if analyses are performed over five
days. The time estimates include 10% devoted
to quality control procedures.
1. Time Expenditures for Microbiological
Analyses 1.2 MPN Analyses

The following estimates of time required After preparation of tubed media, one
for membrane filter (MF) and most probable worker can process 15 samples for MPN anal-
number (MPN) tests were prepared in re- yses for total and fecal coliforms in an 8-hour
sponse to Information requests from Regional day. The procedures include 5 tube x 5 or more
Offices and States planning new or additional dilutions to assure the positive and negative
microbiological work. The estimates are tubes result in 3 significant dilutions needed to
presented~ guidelines only. obtain the optimum MPN index. If 15 samples
are analyzed each day for a 5 day week, prepar-
The Microbiological Methods Section, ation and clean-up of tubed media and supplies
BMB and the Quality Assurance Branch, both for the test period would require an estimated
of EMSL-Cincinnati prepared the estimates 8 hours. Estimates include 10% of time devoted
based on average performance of one techni- to quality control procedures (Part IV of this
cian qualified by short-course or on-the-job Manual).
training and experience in the specific tech-
niques. The procedures are those described in Since the MPN requires reading and trans-
This Manual. fer of growth from positive tubes of lauryl
tryptose broth to brilliant green bile broth and
1.1.MFAnalyses EC broth at 24 and 48 hours, the time span for
one day's samples may cover four days. These
If only fecal coliform bacteria are being times are indicated as 8, 4, 2 and 1 hours on
tested, thirty (30) samples (estimating 3 the four successive days. New samples tested
dilutions/sample) can be prepared by one ana- on the 2, 3, 4th day, etc., add to each day's

246 &EPA MICROBIOLOGICAL MANUAL 1978

workload. This add-on effect results in 8 hours The laboratory will do membrane filtrations
+ 4 hours + 2 hours + 1 hour for a workload as single analyses at three dilution levels or
of 15 hours from the 4th day onward. Either MPN's as five tube, five dilution tests.
the sample load must be reduced or one must
provide a second technician. The accumulative
load is shown in Table V-B-1. The amounts of supplies or numbers of
items are based on needs, then adjusted to
2. Specialized Equipment and Supplies take advantage of quantity discounts by the
dozen, box, 1OO's, etc. For both laboratory
The following tables of . materials and efforts, the expendable items are based on 2
equipment are provided as guidance to labora- weeks usage, while consumable media are
tories. Tables V-B-2, 3 and 4 are designed for a based on usage/week in the high effort labora-
minimal program of 2 samples/day, 7 days a tory and usage/year in the low effort laboratory.
week. Estimates of expendable supplies are
based on those used in 1 year. Tables V-B-5, 6,
7 and 8 are designed for laboratories planning Laboratories doing primarily drinking
a program of 15 samples per day, 5 days per water analyses may require fewer items; other
week. Estimates for expendable supplies are laboratories analyzing only polluted waters may
based on those used in 1 week. require more items.

The following assumptions are made:

Costs are current figures provided to give
The basic laboratory fa~ility and equipment rough estimates only. The necessary chara,cter-
are available (space, benches, lighting, utilities, istics of equipment and materials are cited to
chemicals and standard glassware). assist the purchaser in his choices. ·

ANAL YT/CAL COSTS 247

I')
~
CX>

TABLE V-B-1

Estimated Time Required for Fifteen MPN Analyses/Day*

Day l_ Day~ Day~ Day !_ Day~ Day~ Day ?_ Day !_

First Series
Samples Start
15 MPNS 4 hours 2 hours 1 hour
0) 8 hours

~ Second Series
15 MPNs
~ 8 hours 4 hours 2 hours 1 hour
0
::0
0
QJ
0
r- Third Series
0 15 MPNs
-g
(i)

r-
8 hours 4 hours 2 hours 1 hour

~ Fourth Series
:i!:: 15 MPNs
§; 8 hours 4 hours 2 hours 1 hour
r-
~
(0

~
Fifth Series
15 MPNs
8 hours 4 hours 2 hours 1 hour

*The time required per MPN analysis is not 1 /15 of the time estimated because there is a time savings
in preparing larger numbers of samples.
 TABLE V-B-2

General Equipment and Supplies

Minimum Program

Cost
of
Item Quantity Quantity*

Incubator, gravity convection, 35 ± 0.5 C, $360.00

DWH, 18 X 19 x 28", (46 X 48 X 71 cm)

Incubator, waterbath, 44.5 ± 0.2 C for fecal coliforms, 380.00

LWH 18 X 12 X 7112'', (46 X 31 X 19 cm)

Sterilizer, steam, bench top, electric heat with temperature 1500.00

and pressure controls and gauges l.D.: 9" (23 cm)
Qiameter chamber, 16" (41 cm) deep

pH meter, analog, 0-14 pH range, accuracy ± 0. 1 pH 225.00

units, temperature compensated, with electrode

Oven, double wall, gravity connection RT to 225 C, 575.00

automatic temperature control, l.D.: LHD
19 X 18 X 16", (48 x 46 x 41 cm)

Refrigerator, 3 cubic ft. (. 19 M 3 ) with freezer 425.00

compartment

Thermometer, mercury, range 0-50 C graduated in 0. 1 C. 2 40.00

Meets NBS specs.

Cylinder, graduated, 100 ml, 1 ml graduations 12 66.00

Cylinder, graduated, 50 ml, 1 ml graduations 18 85.00

Bottles, dilution, 99 ml mark, screw-cap, Pyrex glass 48 50.00

Bottle, sample, polypropylene, wide mouth, screw-capped

autoclavable,
125 ml 12 5.90
250 ml 12 9.00
500 ml 12 13.00

Bottle, sample, glass, wide mouth, screw-cap, autoclavable

130 ml 12 6.80
210 ml 12 8.32

Beaker, stainless steel, 4 qt. waterbath for media 15.00

preparation

Pump, vacuum, polypropylene, water powered, 11.5 2.92

liters/min capacity

*1978 prices.

ANAL YT/CAL COSTS 249

 TABLE V-B-2

General Equipment and Supplies

Minimum Program (continued)

Cost
of
Item Quantity Quantity*

Burner, Bunsen, utility 5.25

Needle, inoculating, in holder 2 2.50

Tongs, flask . 1 pr. 5.25

Pen, ink, felt-tip, waterproof 12 . 11.00

Balance, torsion, two pan, 200 g capacity, accurate 310.00

to 10 mg, with 100 g weight

Hot Plate/Magnetic Stirrer, variable speed and heat, 125.00

6 X 6", (15 X 15 cm) top

Plpottor, automatic, volume of 5-50 ml, speed· of 485.00

10-60 deliveries/minute with glass syringe

* 1978 prices.

250 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE V-B-3

Equipment and Supplies for MF Analyses

Minimum Program

Cost
of
Item Quantity Quantity*

Filters, membrane, white, gridded 4 7 mm, 0.45 JJm 2100 MFs $400.00
or equivalent pore size in pkgs of 100

Pads absorbent, 4 7 mm (optional) 2100 MFs pads 32.00

in pkgs of 100

Dishes, petri, plastic, tight-lid, 50 mm X 12 mm 2100 ·dishes ,.· 210.00

in boxes of 500

Bag, plastic, waterproof for 44.5 C waterbath 1000 in boxes 60,QO

incubator of 500

Pi pet, glass Mohr or bacteriological, 10 ml 24 45.00

in 0.1 ml increments

Pipet, glass Mohr or bacteriological, 2.0 or ta 28.00

2.2 ml, in 0.1 increments

Pi pet, glass Mohr or bacteriological, i .0 or 18' 25.00

1.2 ml, in 0. 1 increments

Can, pipet, stainless steel, 2.5 X 2.5 x 16" 3 42.00

6.5 x 6.5 x 41 cm

Jar, polypropylene, for disinfection of pipets, 20.00

6.5 X 20" (16.5 X 50 cm)

Flask, vacuum, pyrex glass, 1 liter 6.00

Water Trap, glass bottle, stoppered with glass tube 1.00

in let/ outlet

Tubing, rubber, vacuum, 3/6" O.D. & 3/32" l.D. 48 9.00

(1.3 cm 0.D. & .24 cm l.D.)

Pinchclamp, flat jaw 2 2.00

Forceps, blunt with smooth tip 2 3.00

Microscope, dissecting, binocular, 15 power 700.00

llluminator, microscope, fluorescent, fits round tube 62.00

microscope

*1978 prices.

· ANAL YT/CAL COSTS . 251

 TABLE V-B-3

Equipment and Supplies for MF Analyses

Minimum Program
(continued)

Item Quantity Cost 1

M Endo MF broth (optional) a x 1/4#/yr $60.00

LES Endo agar (optional) a x 1/4#/yr 72.00

M-FC broth a x 1/4#/yr 64.00

Rosollc Acid (for M-FC broth) 54 x 1 g/yr 90.00

Mathenol, 96%, in small vial for forceps disinfection 1 pt/yr 1.00

Ethanol, 96%, unadulterated, for M-Endo, MF broth 500 ml/yr 5.00

end LES Endo agar

Equipment and Supplies for Verification of MF Analyses

Minimum Program

Item Quantity Cost 1

Tubes, Culture, w.o. lip, borosilicate glass, a x 24 $30.00

160 x 20 mm, reusable. 2

Tube, fermentation, borosilicate, w.o. lip, a x 24 15.00

76 x 10 mm, reusable. 2

Reck, wire, for 1O x 4 culture tubes 6 48.00

Basket, wire, rectangular galvanized, 6 35.00

1O x 6 x 6", (26 x 16 x 15 cm)

Caps, culture tube, aluminum 22 mm l.D. 16 x 12 40.00

Leuryl Tryptose broth 1 #/yr 13.80

Brilliant Green Bile broth 1 #/yr 17.90

EC broth 1 #/yr 16.45

11979 prices.
2Aveilable as disposables.

262 &EPA MICROBIOLOGICAL MANUAL 197~

 TABLE V-B-4

Equipment and Supplies for MPN Analyses

Minimum Program

Item Amount Cost 1

Tubes, Culture, w.o. lip, borosilicate glass, 2 x 576 $170.00

150 x 20 mm, reusable.2

Tubes, Culture, w.o. lip, borosilicate glass, 288 70.00

150 x 25 mm, reusable.2

Tube, fermentation, borosilicate, w.o. lip; 2 x 720 105.00

75 X 10 mm, reusable. 2

Rack, wire, for 10 x 5 culture tubes 15 x 225.00

Basket, wire, rectangular galvanized, 10 x 80.00

10 x 6 x 6", (25 x 15 x 15 cm)

Caps, culture tube, aluminum 22 mm l.D. 60 x 12 150.00

Caps, culture tube, aluminum 27 mm l.D. 24 x 12 60.00

Laury! Tryptose broth 14 x 1#/yr 200.00

Brilliant Green Bile broth 16 x 1#/yr 300.00

EC broth 15 x 1#/yr 250.00

i
1 978 prices.
2Available as disposables.

ANAL YT/CAL COSTS 253

 TABLE V-B-5

General Equipment and Supplies

Full Program in. Microbiology

Cost
of
Item Quantity Quantity*

Pl pets, glass, Mohr or bacteriological T.D., 10 ml 96 70.00

in 0.1 ml graduations

Pi pets, glass, Mohr or bacteriological T.D., 2 or 2.2 ml 96 60.00

in 0.1 ml graduations

Pi pets, glass, Mohr or bacteriological T.D., 1 ml 96 55.00

In 0.1 ml graduations

Can, pipet, stainless steel, 2.5 x 2.5 x 16" 15 210.00

(6.6 X 6.6 X 41 cm)

Jar, polypropylene, for disinfection of pipets, 2 40.00

6.6 X 20" (16.6 X 50 cm)

Cylinder, graduated, 100 ml 24 132.00

Cylinder, graduated, 50 ml 36 170.00

Bottle, dilution, pyrex glass, marked at 99 ml, 96 100.00

screw-cap

Bottle, sample, glass, wide mouth, screw-cap,

autoclavable
125 ml 72 42.00
250 ml 72 50.00
Bottle, sample, polypropylene, wide mouth,
screw.cap, autoclavable
125 ml 96 72.00
260 ml 96 108.00
500 ml 48 52.00
1000 ml 24 38.00
Thermometer, mercury, 0-50 C, graduated in 0. 1 C, 2 40.00
meets NBS specifications

Beaker, stainless c;teel, 4 quart, waterbath for 15.00

media preparation
Flask, vacuum, aspirator, pyrex glass, 1 liter 3 18.00
Water-trap, glass (glass bottle, stoppered, with glass 1.00
tube inlet and outlet)

• 1978 prices.

254 oEPA MICROBIOLOGICAL MANUAL 1978

 TABLE V-B-5

General Equipment and Supplies

Full Program in Microbiology (continued)

Cost
of
Item Quanti~y Quantity*

Tubing, rubber, vacuum, 3/ 16' l.D. x 3/32' O.D. 48' 9.00

(1.3 cm l.D. x 2.4 cm O.D.)

Needle, inoculating, with holder 4 6.00

Tongs, flask 1 pr 5.25

Pen, ink, felt tip, waterproof 12 11.00

Balance, single pan, electric, top loader, 200 g capacity, 700.00

sensitivity 0.02 g for weighings of 2-200 grams
'.
pH meter, analog, 0-14 pH range accuracy ± 0.1 pH 225.00
units, temperature compensated line voltage, with
comb. electrode
or
pH meter, same as above, with accuracy ± 0.05 500.00
pH units

Burner, gas, Bunsen 2 10.50

Hot plate-magnetic stirrer combination, variable 125.00

speed and heat, 15 X 1 5 cm. top

± 0.5 C, water jacketed radiant heat,

Incubator, 35 C 2 3380.00
18 X 38 X 27", (46 X 96 X 68 cm)
or
Incubator, 35 C ± 0.5 C, forced air, large, single chamber 2 2300.00
l.D.: DWH 18 X 36 X 27'', (46 X 96 X 68 cm)

Incubator, 44.5 C ± 0.2 C water bath circulating, 720.00

l.D., LWD 36 X 18 X 91/2'', (91 X 46 X 24 cm)

Sterilizer, large rectangular, double-wall steam, electric 14000.00

automatic, l.D., LWH 38 X 20 X 20"
(97 X 51 X 51 cm)

Oven, double wall, gravity convection RT to 225 C 750.00

l.D., LWH 24 X 20 X 20" (61 X 51 x 51 cm)

Refrigerator, 13 cubic foot, with freezer compartment, 500.00

autpmatic defrost

* 1978 prices.
ANALYTICAL COSTS ' 255
 TABLE V-B-6

Equipment and Supplies for MF Analyses

Full Program in Microbiology

Cost
of
Item Quantity Quantity*

Membrane filtration assembly, stainless steel, for 3 $360.00

4 7 mm filters

Membrane filtration assembly, plastic, autoclavable, 3 24.00

for 4 7 mm filters

Membrane filtration assembly, pyrex, for 4 7 mm filters 3 120.00

UV sterilizer unit for sterilizing MF filtration assemblies 550.00

Manifold, PVC, 3 place, for multiple filtrations 200.00

Microscope, binocular, dissecting type, 15 power 200.00

Lamp, fluorescent, microscope illuminator, fits round 62.00

tube microscope

Filters, membrane, white, 4 7 mm 0.45 ~m or equiv. 300 57.00

pore size, gridded

Pads, absorbent, 4 7 mm 300 4.50

Dishes, petri, plastic, 50 x 12 mm, tight lid 500 50.00

Bags, plastic, waterproof for submersion of M-FC dishes 500 30.00

In waterbath

Bottle. rinse water. round, wide-mouth, screw-cap, 24 38.00

polypropylene, 1000 ml

Forceps, blunt with smooth tip 3 4.00

Counter, mechanical, hand type 9.00

Pump, vacuum, polypropylene, water powered, 11.5 3.00

liters/min capacity
or
Pump, vacuum/pressure, electric, portable, produces 130.00
20" mercury vacuum

Plnchclamp, flatjaw 12 6.00

• 1979 prices.

256 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE V-B-7

Equipment and Supplies for MPN Analyses

Full Program in Microbiology

Cost
of
Item Quantity Quantity 1

Basket, wire, rectangular galvanized, 190 $1520.00

1o· x 6 x 6" (25.4 x 15 x 15 cm)

Rack, wire, for 10 X 5 culture tubes, 150 2250.00

150 X 25 mm

Tubes, culture w.o. lip, borosilicate glass, 30 x 576 2560.00

150 x 20 mm, reusable.2

Tubes, culture w.o. lip, borosilicate glass, 17 x 288 1200.00

150 x 25 mm, reusable. 2

Tubes, fermentation w.o. lip, borosilicate glass, 21 x 720 1100.00

75 X 10 mm, reusable~ 2

Caps, culture tube, aluminum, 22 mm l.D. 1250 x 12 3125.00

Caps, culture, tube, aluminum, 27 mm l.D. 408 x 12 1020.00

1 1978 prices.
2Available as disposables.

ANAL YT/CAL COSTS 257

 TABLE V-B-8

Media for Full Program in Microbiology Laboratory

Usage for each Week/100 Samples

Medium Usage/Week Cost/lb 1

MF Analyses:

M Endo MF Broth 0.1 lb/week $17.90

M-FC Broth 0.07 lb/week 24.85

LES Endo Agar 0. 17 lb/week 20.95

Rosollc Acid (for M-FC Broth) 1 g/week 1.70/g

MPN Analyses:

Lauryl Tryptose Broth · 1.96 lb/wk 2 13.80

Brilliant Green Bile Broth 2.2 lb/wk 17.90

EC Broth 2.04 lb/wk 16.45

EMB Agar variable 18.70

MF Verification Costs on 5 Samples/Week

20 Colonies/Sample

Lauryl Tryptose Broth .07 lb/week 13.80

Brilliant Green Bile Broth .07 lb/week 17.90

EC Broth .07 lb/week 16.45

11978 prices.
2For single strength medium.

258 &EPA MICROBIOLOGICAL MANUAL 1978

 PART V. LABORATORY MANAGEMENT

Section C Laboratory and Field Safety

Introduction 1. Administrative Considerations

This Section has been compiled from the 1.1 Development of a Safety Program
best available sources. The procedures given
for general laboratory safety follow the OSHA 1. 1. 1 If a laboratory safety program is to
regulations ( 1, 2). Specific recommendations be effective, management ·must· know the
for microbiology were selected from literatu~e causes of infections in order to develop and
of the Center for Disease Control, the National upgrade safety procedures, equipment a~d
Institute for Occupational Safety and Health of rules, and to reduce incidence of infection. The
the Public Health Sel"'{ice, the U.S. Environ- lack of information on sources of laboratory
mental Protection Agency and other sources infection (Table V-C-1 and 2) prevents improve-
(3-12). ment in safety programs and emphasizes the
need for reporting all accidents. A preponder-
Safety procedures should be performed ance of evidence in the literature indicates that
as an integral part of the analytical methods if the known causes of lab9ratory infections
and should be included in program planning· were eliminated, the remaining infections
on a day to day basis. could be considered to be caused by airborne
transmission.
The objectives of a laboratory and field
safety program are 1) to protect the laboratory
worker, the laboratory environment and the Some common microbiological proce-
surrounding community from microqial agents dures shown to produce aerosols include pi-
studied and 2) to protect the integrity of the petting into a petri plates or flasks, opening
microbiological studies. The program is dis- lyophilized culture ampuls, opening culture
cussed as follows: containers, inserting a hot loop into a culture
container and removing the cover from stan-
1. Administrative Considerations dard blender after mixing a sample.

2. Sources of Hazard
1. 1.2 Job attitudes can be the cause of
3. Field Guidelines laboratory accidents: overly rigid work habits,
failure to recognize dangerous situations,
4. Laboratory Guidelines work at excessive speed and deliberate viola-
tions of rules. These attitudes can only be
5. Biohazard Control overcome by the development of safe work
habits through continued education and
6. Safety Check List training.

SAFETY 259'
 TABLE V-C-1

Laboratory-acquired Infections Related to Personnel and Work*

Type of Infective Agent

Total # of
Distribution Infections Bacteria Viruses Rickettsia Parasites

No. of cases 2262 1303 519 293 62 85

No. of deaths 96 53 31 8 2 2

!Ye! of Personnel (where known)

Trained Scientific
Personnel .. 1534 856 358 206 57 57

Students 82 80

Animal Caretakers 221 137 39 40 4

Clerks, occasional
visitors, maintenance 87 48 15 23

Types of Work {where known)

Diagnostic 525 393 96 3 16 17

Research 726 289 230 155 31 21

Biological Reagent
Production 51 19 31

Classwork 37 37

Combination of
Activities 599 335 81 133 11 39

•Reference 3.

**Includes research assistants, professional and technical workers and graduate students.

260 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE V-C-2

Sources of Laboratory-acquired Infections*

Type of Infective Agent

Source of
Infection Bacteria Parasites

Work Situation but No Known. Incident

Clinical specimens 103 17

Autopsy, including known

accidents 95

Aerosols 135 39

Handled infected animals

and ectoparasites 126 5

Work with agent 412 16 27

Discarded glassware 21

Known accidents

Needle and syringe 66 3 2

Pipetting 66

Spilling and splattering 41 5 2

Injury with broken glass,

etc. 44

Bite of animal or
ectoparasite 28 8 2

Centrifuge 2 3

•Reference 3.

SAFETY 261
 1. 1.3 Safety training of laboratory and It is recommended that the safety officer
field personnel, formulation of safety regula- at each installation conduct a quarterly safety
tions, and the establishment of mechanisms inspection of the facilities to identify and cor-
for reporting and investigating accidents are rect dangerous conditions or procedures. He
prime responsibilities of the laboratory and should make full use of the safety check lists
field supervisors and higher management. developed for the laboratory. An example of a
First aid courses should be provided to the safety check list is given at the end of this
laboratory supervisor and at least one other Section.
permanent employee.

Each employee should have a copy of the 2. Sources of Hazard

sa'fety program. Joint supervisor-employee
safety committees should identify potential 2. 1 Causative Agents
laboratory hazards and formulate workable
safety regulations. Laboratory safety regula- Only a small percentage of microorgan-
tions should stress the protection of the labora- isms are capable of producing disease in
tory personnel, janitorial and maintenance man; they include bacteria, fungi, yeasts,
staff and others who might come in contact protozoa, actinomycetes, animal and human
with the laboratory and its personnel. viruses, and rickettsiae (3, 4). Because
innocuous and infectious microorganisms
cannot be differentiated in natural materials
1.2 Reporting Laboratory Infections and and because microorganisms considered
Accidents ' harmless may produce disease in man under
favorable conditions, it is good practice to
1.2. 1 Because many laboratory accidents treat all microorganisms and materials as if
are not reported, most laboratory infections they are pathogens or disease carriers.
are never traced to a specific cause. To im-
prove safety, it is important to maintain good 2.2 Sources in Sampling
records of laboratory accidents and infections.
It Is necessary to know the pathogens involved Laboratory and field personnel collecting
and the circumstances under which the infec- samples and isolating cultures from natural
tion or accident occurred. It is recommended sources must be made aware that pathogens
that each laboratory set up a formal system for are present in environmental samples.
reporting accidents as they occur so that ap- Disease-causing organisms are found in natu-
propriate prophylatic measures may be insti- ral waters, municipal effluents and sludges,
tuted. A record of accidents with after effects, industrial wastewaters from packing plants, in
particularly those resulting in infection, can be soils and runoff from feedlots and from septic
of considerable value. tank systems. These pathogens have also been
found in inadequately-treated finished water
1.2.2 In EPA, laboratory-acquired systems and in ground water supplies. Before
Infections o'r accidents must be reported to the . working with environmental samples, field
immediate supervisor. EPA Form 1440, personnel should have thorough training in
Supervisor's Report of Accident, and Form CA aseptic technique and handling pathogens.
1, "Federal Employee's Notice of Traumatic
Injury and Cl3im for Continuation of 2.3 Sources in the Laboratory
Pay/Compensation," must be completed by
the employee and the supervisor and filed Table V-C-1 and 2 present data on
within two working days. Form CA 2, "Federal laboratory-acquired infections in the United
Employee's Notice of Occupational Disease States which were collected in a survey of
and Claim for Compensation" is to be 5000 laboratories over a 20-year period (3).
completed by the employee and submitted Table V-C-1 shows the distribution of
within 30 days. laboratory-acquired infections according to

262 &EPA MICROBIOLOGICAL MANUAL 1978

personnel and their work. It is significant that 3.1 Automotive Safety
trained scientific personnel had 1534 of the
1924 infections identified by type of person- 3. 1. 1 The driver should make certain that
nel. Among laboratory workers, researchers he has valid state and agency driver licenses
led with 726 of 1938 infections. These data on his person before operating a vehicle.
suggest that the majority of infections occur
because of carelessness of trained and knowl- 3. 1.2 If the driver observes a questionable
edgeable workers and not because of igno- or unsafe condition when first operating a ve-
rance. However, new technicians should be hicle, he should return it directly to the carpool
made aware that culturing microorganisms regardless of work demands.
from natural sources develops extremely large
numbers of cells which could cause disease if 3.1.3 Continuous driving in excess of ten
not properly handled. hours, in any 24-hour period, is not
recommended.
Table V-C-2 presents data on the sources
of laboratory-acquired infections. This table 3.1.4 Occupants of vehicles should wear
shows that the five most common accidents seat belts and shoulder harnesses, where pro-
causing laboratory infection were: vided, whenever vehicles are in motion. The
driver should carry a kit provided by the labor-
1. Accidental inoculation with syringes atory that includes fire extinguisher, flares,
and needles. reflectors, and a first-aid kit.

2. Accidental oral aspiration of infectious 3. 1.5 Safety screens should be installed in

material through a pi pet. carryall and van-type vehicles to separate the
cargo and passenger compartments. If safety
3. Cuts or scratches from contaminated screens are not installed, cargo should not be
glassware. stacked higher than the back of the seat.

4. Spilling or splattering of pathogenic 3. 1.6 Employees required to tow a trailer

cultures on floors, table tops, and other should be instructed in the proper handling of
surfaces. the equipment involved.

5. Bites of animals or ectoparasites. 3.1.7 Vehicles used to tow any kind of

trailer should be equipped with west-coast
It is significant that in about 80% of the type mirrors and the necessary connections
laboratary infections studied in Table V-C-2 for trailer signal, tail lights, brakes, and safety
the mechanism was not known. It was only chains.
known that the individual had worked with the
agent or had tended infected animals. 3.1.8 If a boat is transported in a pickup
truck, it should not obstruct the vision of the
driver or extend over the vehicle cab.
3. Field Safety Guidelines

The sample collector or investigator must 3. 1.9 A driver backing a vehicle with a
also consider safety in his work. The potential trailer in tow should have someone outside the
for accidents in field work is much greater than vehicle to direct him.
in the laboratory. The following rules on field
safety were extracted from a comprehensive 3.2 Boat Safety
safety manual developed by EPA's National
Enforcement and Investigations Center at 3.2. 1 Only qualified employees should op-
Denver (5). They are intended as guidelines to erate watercraft. Boat operators must have
assist the laboratory in developing its own completed advanced emergency first-aid
protocol. training.

SAFETY 263
 3.2.2 The boat operator must not operate 3.3.3 Wear fluorescent vests or jackets
the boat without a second person on board. while sampling from roadways and bridges.
During sampling, post a yellow flasher on ap-
3.2.3 The boat operator is responsible for proaches at each end of the bridge. On heavily
the safety of persons and equipment on board. traveled roads post flagmen or warning de-
He should provide a a boat safety briefing to vices at each end of bridge that lacks 24-inch
occupants before embarking. walkways. Such sampling points should be
avoided where possible.
3.2.4 Occupants must wear life jackets
onboard. No exceptions are permitted. Soft- 3.3.4 Wear rubber gloves while handling
soled, non-skid shoes are recommended. samples that might be toxic or corrosive. Wear
work gloves while handling sampling equip- .
3.2.5 Flare gun, fire extinguishers and first ment. During collection and transport, the field
aid kit should be kept on each boat. worker should store containers to prevent
spilling or splashing of samples. Disinfect
3.2.6 Boats operated in estuaries or open hands immediately after handling sewage
seas should be equipped with depth-finding samples and equipment for sampling sewage.
instruments, navigational aids and two-way ra-
dios adequate to communicate with at least 3.3.5 Do not sample from railroad bridges
one shore station. Boats with marine radios unless there is an adequate walkway or the
should monitor distress frequency except railroad dispatcher has been contacted and it
when transmitting. has been positively determined that no trains
will run during sampling period.
3.2. 7 Boats should not be operated in high
winds, storms, heavy rain, fog, etc. Boats 3.3.6 Equip vehicles for sampling and as-
should not be operated more than one half sociated work with rotary amber caution
mile from launch point on estuaries, large lights. Operate such lights whenever vehicles
lakes, and large rivers until acquiring reliable are driven slowly on roadways or are parked
weather forecasts. near roadways. Do not park vehicles on
bridges.
3.2.8 The operator should attach a red
pennant to the radio mast when operating at 3.3. 7 Inform employees of the safety rules
slow speeds (e.g., sampling, dredging, towing). in force within industrial sites. Employees
must conform to rules promulgated by the
industry while on-site.
3.2.9 The operator must install and use
lights according to established practice for
night operations. 3.3.8 Properly ground electrical apparatus
employed in field operations and use battery
straps to handle or move wet-cell batteries.
3.3 General Rules for Sampling
3.4 Sampling from Manholes
3.3.1 Require two people on each gauging
or night sampling crew or on other hazardous 3.4. 1 Erect barricades around manholes
projects. where samples are being collected. Do not
leave manholes uncovered while unattended
or unbarricaded.
3.3.2 Wear safety glasses, safety shoes,
hard hats, respirators, gas masks, and ear-
protection devices, as appropriate in hazard- 3.4.2 Do not enter a manhole until it is
ous areas. cleared by using a blower for at least five

264 oEPA MICROBIOLOGICAL MANUAL 1978

minutes. Following the five-minute ventilation, 4. Laboratory Safety Guidelines
use a lead acetate swab to check for H 2 S. If
H 2 S is present, wear a respirator when enter- The following safety rules are intended as
ing manhole. Substitute a respirator for blower . guidelines. They were deve,oped from the
ventilation where explosive gases are present available safety literature (3-9) and have con-
(i.e., in storm sewer or domestic sewer). sidered the Occupational Safety and Health
Administration (OSHA) regulations (1, 2). Us-
3.4.3 A sample collector entering man- ing such source materials, the laboratory direc-
holes must wear safety lines handled by two tor, laboratory supervisor or senior profes-
persons outside of manholes. Keep safety lines sional should develop rules that are specific
taut at all times. Fifteen minutes is the maxi- for the laboratory program and the organisms
mum time allowed in manhole. Keep a vehicle involved.
at hand in case of emergency.

3.4.4 Do not enter sewer lines for any 4.1 Personal Conduct and Clothing
reason.
· 4. 1. 1 Store coats, hats, jackets, and other
items of personal clothing outside of the mi-
3.5 Sampling Channels and Streams crobiology laboratory. Do not mix laboratory
and street clothes in the same locker.
3.5. 1 Sample collector must work from
behind a barricade or wear a safety line at- 4. 1.2 Wear a non-flammable laboratory
tached to a secure object when sampling fast- gown or coat in the laboratory. If clothing
moving channels or streams from shore, walk- becomes contaminated, autoclave before
way, etc. The same rule applies to any open laundering. Laboratory clothing should not be
channel when footing is questionable, i.e., worn in clean areas or outside the building.
snow, steep bank, etc. Open-toed shoes, or extreme shoe styles
should not be. worn, since they provide little
3.5.2 Wade only to knee-depth in swift protection or are unstable.
waterstreams, or to hip-depth in placid water.
When wading in fast moving water secure 4.1.3 Wear goggles or safety glasses to
safety lines to shore and have at reast two protect eyes from UV irradition.
other persons in attendance.
4. 1.4 Wash hands carefully after labora-
3.5.3 Attach lines from sampling devices tory and field duties, using a germicidal .soap.
to a secure object but never to sampling
personnel.
4.1.5 Use forceps or rubber gloves when
there is a significant danger of contamination
such as during the clean-up of pathogenic
3.6 Sampling Under Ice
material.
3.6.1 Two people are a minimum crew for
operations involving ice cover. One person 4. 1.6 Do not touch one's face, lick labels
must remain on solid footing until thickness of or put pencils and other materials in one's
ice is known. mouth.

3.6.2 Do not sample on ice if the ice thick-

ness is less than four inches. 4.1.7 Don't smoke, eat, drink or chew gum
in the laboratory or while sampling. Do not
3.6.3 Wear life preservers and secure keep food or drinks in the lab refrigerator or
safety lines to an object on shore when sam- cold room. Do not brew coffee or tea in the
pling·on ice-covered water. laboratory area.

SAFETY 265
 4. 1.8 Keep conversation to an absolute 4.2. 10 Lyophilization procedures can be a
minimum during bench work to prevent self- source of laboratory infection. When vacuum
Infection or loss of analytical data. is applied during lyophilization, the contami-
nated air is withdrawn from the ampuls
4.1.9 Keep reading matter, surplus materi- through the pump and into the room. Use bio-
als and equipment out of the laboratory area. logical air filters or air decontamination proce-
dures to reduce hazard. Aerosols are also often
4.1.10 Laboratory and field personnel created by opening lyophilized ampuls. Re-
handling polluted samples should be vacci- duce this hazard by wrapping the ampul in a
nated against typhoid, tetanus and polio. disinfectant-soaked pledget of cotton before
breaking.

4.2 Laboratory Equipment 4.2.11 Read 11-C-6 for instructions on

proper packing of cultures for mail shipment
4.2.1 Limit traffic through the work areas. before sending any isolates to a central labora-
tory for confirmation.
4.2.2 Treat all cultures and samples as if
they are potentially pathogenic. The degree of
risk Is Increased greatly in culture work be- 4.2. 12 Periodically clean out freezers, ice
cause the microorganisms are produced in chests and refrigerators to remove any broken
very large numbers. ampuls, tubes, etc., containing infectious ma-
terials. If units contain pa~hogenic cultures,
4.2.3 Do not mouth-pipet polluted water, use rubber gloves during this cleaning. Use
wastewater or other potentially infectious or respiratory protection if actinomycetes, fungi
toxic fluids; use a bulb or other mechanical · or other easily disseminated agents are
device. See Part 11-B, 1.8.2. involved.

4.2.4 For potable waters, plug pipets with

non-absorbent cotton. Do not use pipets with 4.3 Disinfection/Sterilization
wet plugs.
4.3. 1 Disinfect table tops and work carts
4.2.5 Use a hooded bunsen burner or before and after laboratory work. A bottle of
shi~ded electric incinerator to protect against disinfectant and gauze squares or towelling for
splattering during culture work. washing and wiping purposes should be avail-
able in laboratory for routine and emergency
4.2.6 Maintain benches in a clear and un- use.
cluttered condition for maximum efficiency
and safety. 4.3.2 Use a disinfectant which specifies
germicidal activity against the organisms most
4.2.7 Perform all culture work in a often encountered in the laboratory. Organo-
blohazard hood to protect cultures and iodine complexes, quaternary ammonium
workers. compounds, phenolics and alcohols which are
effective against vegetative bacteria and
4.2.8 Do not use the kitchen type blender viruses are recommended for general use.
for mixing materials containing infectious However, these disinfectants are not sporo-
agents. Safety blenders are available in which cidal. If spore-forming bacteria are encounter-
Infectious materials may be mixed without dis- ed, formaldehyde or formaldehyde/alcohol
semination of infectious aerosols. solution is recommended. See Table V-C-3.
j

4.2.9 When a vacuum line is used, inter- Mercury salts, chlorine-containing com-
pose suitable traps or filters to insure that pounds or home-use products are not recom-
Infectious agents do not enterthe system. mended for the laboratory.

266 &EPA MICROBIOLOGICAL MANUAL 1978

 TABLE V-C-3 4.3.8 Check autoclaves with the use of
spore strips or spore suspensions of B. stear-
Normal . Use Concentration of Disinfectants othermophilus and maximum-minimum
recording thermometers. Ideally autoclaves
are equipped with temperature recording de-
Use vices so that a permanent record may be
Compound Concentration maintained.
mg/liter

Check hot air ovens and gas sterilizers

Organo-lodine Complexes 100-150 periodically with spore strips or the indicator,
8. subtilisvar. niger.
Quaternary Ammonium
Compounds 700-800
4.3.9 Wet-mop floors weekly, using water
Phenolics containing a disinfectant. Dry or wet pickup
vacuum cleaners with high-efficiency exhaust
Alcohol, 70% w/v water air filters are recommended. Wax floors with
solution bacteriostatic floor waxes i{available ..
Formaldehyde 8%
4.4 Chemicals and Gases
Formaldehyde in
70% Alcohol Solution 8% 4.4. 1 Label containers plainly and perma-
nently. Dispose of material in unlabelled con-
tainers carefully. Wipe or rinse residual ma-
terial from the external surfaces of. rea.gent
.4.3.3 If a culture or infective material is containers after use .
spilled, notify the laboratory supervisor at
once, then disinfect and clean up the area. 4.4.2 Store flammable solvents in an ap-
proved solvent storage cabinet or a well-
4.3.4 Never pour viable cultures or con- ventilated area.
taminated materials in the sink. Never leave
infectious material or equipment unattended 4.4.3 When opening bottles which may be
during use. under pressure i.e., hydrochloric acid, ammo-
nium hydroxide, cover the bottle with a towel
4.3.5 Immediately after use, place con- to divert chemical spray.
taminated pipets in a disinfectant container
which allows complete immersion; place cul- 4.4.4 Use bottle carriers to transport bot-
tures and contaminated materials in color- tles containing hazardous chemicals (acids,
coded biohazard bags and seal. Disinfectant corrosives, flammable liquids). Large cylinders
containers of pi pets and sealed bags of materi- are transported only by means of a wheeled
als are autoclaved as units. cart to which the cylinder is secured. Store and
' . transport compressed gas cylinders with sh.ip-
4.3.6 Place used glassware in special cans ping caps on, in an upright position, always
marked for autoclaving. Keep broken glass-· securely clamped or chained to a firm support
ware in a .separate container. Place plastic and away from heat. ·
items in separate cans to prevent fusing of
plastic around glass items. 4.4.5 Reagents a.nd chemical which might .
react in water drains or be dangerous to the
4.3.7 Mark contaminated items as Con- environment must be disposed of in 0th.er
taminated before removal from the laboratory ways. Examples are 1) sodium azide which
for autoclaving. Use temperature-sensitive reacts with metal.drains to produce very explo-
tapes which indicate exposure to heat. Pre- sive lead or copper azides and 2) mercury and
printed tapes or tags simplify this task. its salts which should not be returned to the

SAFETY 267
environment. Consult reference texts to deter- 5. Biohazard Control
mine the proper disposal procedure for each
chemical (8, 9). 5.1 Safety Cabinets

4.5 Handling Glassware 5. 1. 1 The safety cabinet is the most impor-

tant primary barrier available to the microbiol-
4.5. 1 Discard broken, chipped or badly ogist for isolation and containment of microor-
scratched glassware. Use gloves or sweep up ganisms and for protecting the laboratory envi-
broken glass, do not use bare hands. Pick up ronment, and the surrounding area from con-
fine glass particles with wet paper towelling. tamination. Transfers of cultures especially
pathogenic fungi, actinomycetes and yeasts
4.5.2 Fire polish tubing and rods. should be conducted in the safety cabinets.

4.5.3 Protect hands with gloves, towel, or 5. 1.2 UV lamps are commonly used in
tubing holder when inserting tubing into stop- biohazard hoods to maintain sterility of the
pers. Lubricate the tubing with water or glycer- work area. Goggles should be worn to protect
ine. Handle tubing close to the stopper and out the worker and cultures should be protected
ofline with end of the tube. from undesirable exposure (see Part IV-A, 4 in
this Manual).
4.5.4 Use asbestos-centered wire gauze
when heating glass vessels over a burner. 5. 1.3 There are several types of ventilated
cabinets available for use ( 10, 13):
4.5.5 Do not attempt to catch falling
glassware.
(a) Partial Barrier Cabinet

4.6 Electrical Equipment The open or closed front cabinet is usually

referred to as a partial barrier ventilated cabi-
4.6.1 Keep materials, tools and hands dry net. This cabinet can be used with the glove
while handling electrical equipment. panel removed, depending upon an inward
flow of air of at least 100 linear ft. per min. to
4.6.2 Use grounded outlets only. prevent escape of airborne particles. It can
also be used with the glove panel in place and
4.6.3 Do not use electrical equipment near arm-length gloves attached, in which case it
flammable solvents. will be maintained under a reduced air pres-
sure of about one inch of water gauge. When
4.6.4 Use only carbon dioxide or dry pow- operated closed, the partial barrier needs an
der fire extinguishers in case of fire in or near attached air lock for movement of materials. A
any electrical equipment. third mode of operation consists of using a
cabinet with glove panel attached, but with
4.7 Emergency Precautions gloves removed.

4.7.1 Install and maintain both foam and (b) Absolute Barrier Cabinet
carbon dioxide fire extinguishers within easy
access of the laboratory. The second type of ventilated cabinet is
the gas-tight cabinet system, referred to as an
4. 7 .2 Fire exits should be clearly marked absolute barrier cabinet. Absolute barrier cabi-
and accessible. nets are connected to form a modular cabinet
system with enclosed refrigerators, incuba-
4.7.3 Install and maintain a complete first tors, etc. Air is drawn into the cabinet system
aid kit and an oxygen respiration unit in the through ultrahigh efficiency filters and is ex-
laboratory. hausted through ultrahigh efficiency filters.

268 3EPA MICROBIOLOGICAL MANUAL 1978'

 (c) Vented Laminar Flow Cabinet microorganisms, a gas-tight absolute barrier
cabinet with glove ports or laminar flow cabi-
This third type of safety cabinet is not gas- net is recommended. These provide protection
tight. It relies on high efficiency filters to pro- of cultures as well as the worker and the work
tect the worker and the environment. In the environment. Because laminar flow cabinets
Figure V~C-1, the blower ( 1) set into the bottom do not require glove openings, yet do protect
of the cabinet pushes air up through a rear personnel and culture work and are easy to
duct into the top of the cabinet (2) and causes a use, they are recommended for all but the most
slight negative pressure in the work area (3). In hazardous microbiological operations.
the top of the cabinet (2), part of the air is
forced out the exhaust through a High Effi- 5.2 Biohazard Identification
ciency, Particulate, Air (HEPA) filter (4). How-
ever, themajor partof the air is forced down 5.2. 1 The revised Federal Occupational
through a large HEPA filter (5), in a vertical flow Safety and Health Act of 1972 requires biolog-
into the work area (6). This vertical flow (lam- ical hazard signs and tags to signify the actual
inar flow) combines with the negative pressure or potential biologic'al haza.rd ( 1, 10). The haz-
from the blower to draw in enough make-up ards are defined as infectious agents that
from the room to replace that exhausted present a risk to human well-being. These
above. The make-up air (7) is pulled through signs and tags are used to identify equipment,
vents at the edge of the hood opening and is containers, rooms, materials, experimental an-
drawn down to the blower withput contacting imals, or combinations of the above, which
the work area. It combines with the filtered air contain or are contaminated with viable haz-
and is recirculated through the HEPA filters. ardous agents.
The laminar flow draws off any contaminating
particles emanating from the work area. HEPA 5.2.2 The biological or biohazard symbol
filters are 99.99% efficient in removing parti- design is shown in Figure V-C-2. It has a fluo-
cles 0.3 µm diameter or larger, by the DOP test. rescent orange or orange-red color, and may
contain appropriate wording to indicate the
5. 1.4 Selection of a Cabinet nature or identity of the hazard, the name of
the individual responsible for its control, pre-
The partial barrier cabinet with open front cautionary information, etc. The wording must
gives some protection to the worker and the not be superimposed on the symbol. Back-
laboratory environment but does 'not protect ground color is optional, but enough contrast
the cultures. With the glove panel in place, it must be provided so that the symbol can be
protects the worker and adjacent laboratory clearly defined.
area. The absolute barrier and laminar flow
cabinets provide the greatest protection to the
worker and the cultures. 6. Safety Check List

For routine water bacteriology and limited The laboratory safety check list that fol-
work with pathogens, a partial barrier cabinet lows is provided as a guide for a laboratory to
with glove panel can be used. If a significant incorporate wholly or in part into its own safety
portion of the workload involves pathogenic program.

SAFETY
 FIGURE V-C-1. Laminar Flow Cabinet.

270 &EPA MICROBIOLOGICAL MANUAL 1978

CAUTION

FIGURE V-C-2. Example of Biohazard Sign.

SAFETY 271
 Safety Check List
for Microbiological Water Laboratories

Survey By:
Laboratory:
Location:
Date:
Code: S=Satisfactory, U=Unsatisfactory

1. Administrative Considerations

(a) Laboratory has a formal documented safety program.

(b) Each worker has a copy of the safety program.

(c) Employees are aware of procedures for reporting accidents

and unsafe conditions.

(d) New employees are instructed on laboratory safety.

(e) Joint supervisor-employee safety committee has been estab-

lished to identify potential laboratory hazards.

(f) Records are maintained of accidents and consequences.

(g) Name and phone number of the supervisor and an alternate are
posted at door of the laboratories so he may be contacted
in case of an emergency.

(h) Laboratory supervisor and at least one other permanent

employee have attended appropriate first aid courses. If
so, when:
(date)

(i) Emergency telephone numbers for fire, ambulance, health

centers, and poison control center are placed in a conspic-
uous location near the telephone.

(j) Employees know the location of first aid supplies.

(k) Emergency first aid charts, and hazardous agents charts

are posted in the laboratory.

(1) Fire evacuation plan is established for the laboratory

and is posted in a conspicuous location.

2. Personal Conduct

(a) Personal clothing is stored outside of the microbiology

laboratory.

(b) Lab coats and street clothes are kept in separate

lockers.

272 oEPA MICROBIOLOGICAL MANUAL 197B

 (c) Laboratory coats are worn at all times in the laboratory.

(d) Germicidal soap or medicated surgical sponges are available

for employees' use.

(e) Preparing, eating or drinking food and beverages are not

permitted in the laboratory.

(f) Smoking or chewing gum are not permitted in the laboratory.

(g) Food or drink are not stored in laboratory refrigerators.

(h) Reading materials are not kept in the laboratory.

(i) Laboratory coats are not worn outside the lab.

U) Employees who have cuts, abrasions, etc. on face, hands,

arms, etc. do not work with infectious agents.

3. Laboratory Equipment

(a) ·Bulb or mechanical device is used to pi pet polluted water,

wastewater or other potentially infectious or toxic fluids.

(b) Pipets are immersed in disinfectant after use.

(c) Benches are maintained in clear and uncluttered condition.

(d) Centrifuge cups and rubber cushions are in good condition.

(e) A suitable disinfectant is available for immediate use.

(f) Blender is used with sealed container assembly.

(g) Microscopes, colony counters, etc. are kept out of the work
area.

(h) Water baths are clean and free of growth and deposits.

(i) Employees are instructed in the operation of the autoclave

and operating instructions are posted near the· autoclave.

(j) Autoclaves, hot air sterilizing ovens, water distilling

equipment, and centrifuges are checked routinely for safe
operation.

Give frequency and last date

Autoclave

Water still

Centrifuge

Hot Air Oven

(k) No broken, chipped or scratched glassware are in use.

(1) Broken glass is discarded in designated containers.

SAFETY 273
 (m) Electrical circuits are protected against overload with
circuit breakers or ground-fault breakers.

(n) Power cords, control switches and thermostats are in good

working order.

(o) Water taps are protected against back-siphoning.

4. Disinfection/Sterilization

(a) Proper disinfectant is used routinely to disinfect table

tops and carts before and after laboratory work.

(b) Receptacles of contaminated items are marked.

(c) Performance checks of autoclaves, gas sterilizers and hot air

ovens are conducted with the use of spore strips, spore
ampuls, indicators, etc.

Item Frequency Last Date

(d) Safety glasses are provided to employees.

(e) Safety glasses are used with toxic or corrosive agents and
during exposure to UV irradiation.

5. Biohazard Control

(a) Biohazard tags or signs are posted in hazardous areas.

(b) Safety cabinets of the appropriate type and class are

provided.

(c) Lab personnel are vaccinated for typhoid fever, tetanus

and polio.

(d) Floors are wet-mopped weekly. with a disinfectant solution.

(e) Personnel are trained in the proper procedures for handling

lyophilized cultures where used.

6. General Handling and Storage of Chemicals and Gases

(a) Containers of reagents and chemicals are labelled properly.

(b) Flammable solvents are stored in an approved storage

cabinet or well-ventilated area away from oil burners,
hot plates , etc.

(c) Bottle carriers are provided for hazardous substances.

274 oEPA MICROBIOLOGICAL MANUAL 1978

 (d) Gas cylinders are securely clamped to a firm support.

(e) Toxic chemicals are clearly marked poison or toxic.

7. Emergency Precautions

(a) Foam and carbon dioxide fire extinguishers are installed

within easy access to laboratory and are properly
maintained. Frequency

(b) Eye wash stations , showers _ _ _ , oxygen

respirators , and fire blankets are available
within easy access.

(c) Fire exits are marked clearly.

(d) First aid kits are available and in good condition.

(e) At least one full-time employee is trained in first aid.

(f) Source of medical assistance is available and known to

employees.

8. Suggested Areas of Improvement:

9. General Comments:

{Signature of Installation Officer) {date)

. SAFETY 275
 REFERENCES

1. 29 Code of Federal Regulations (CFR) Part 1910, "Occupational Safety and Health Standards"
and Amendments.

2. Occupational Health and Safety Administration, 197 4. Provisions for Federal Worker Safety and
Health, OSHA (October, 197 4), Reference File 41 :6241

3. American Public Health Association, 1963. Diagnostic Procedures and Reagents, (4th ed.),
APHA, Inc. pp. 89. -

4. Pike, R. M., 1976. Laboratory-associated infections: summary and analysis of 3921 cases. Health
Laboratory Science,~: 105. --

6. National Field Investigation Center, 1973. NFIC-Denver Safety Manual, NFIC-Denver, EPA,
Denver, CO. EPA 330/9-7 4-002 (In revision).

6. Wedum, A.G., 1961. Control of laboratory airborne infection, Bacterial Reviews 25.

7. Reitman, M. and A.G. Wedum, 1971. Infectious hazards of common microbiological techniques.
In: Handbook of Laboratory Safety. (N. V. Steere, ed.), The Chemical Rubber Co., Cleveland, OH
pp.633. -- •

8. Shapton, D. A. and R. G. Board, Ed. 1972. /n:Safety in Microbiology. Academic Press, New York,
NY. --

9. Steere, N.V., editor, 1971. Handbook of Laboratory Safety. The Chemical Rubber Co., Cleveland,
OH.

10. Manufacturing Chemists Association, 1975. Laboratory Waste Disposal Manual, Washington,
DC. --

11. US Public Health Service, 197 4. National Institutes of Health, Biohazards Safety Guide, USPHS,
DHEW, GPO 17 40-00383.

12. U.S. Environmental Protection Agency, 1977. Occupational Health and Safety Manual, TN5
(9-12-77). ------

13. Runkle, K. S. and G. B. Phillips, Ed. 1969. Microbial Contamination Control Facilities. Van
Nostral)d Reinhold Co., New York, NY.

1:.l;

276 oEPA MICROBIOLOGICAL MANUAL 1978

 PART V. LABORATORY MANAGEMENT

SECTION D LEGAL CONSIDERATIONS

This Section is intended to guide practic- 1.4 The Marine Protection,

ing microbiologists in assessment of their re- Research and Sanctu-
sponsibilities and role under the three federal aries Act of 1972,
laws on water quality. It is not intended as Public Law 92-532
formal legal guidance or as representative of 1.5 The Safe Drinking Water
official legal position. It is based on the three Act of 1974, Public
Federal laws on water quality, (1, 2, and 3), on Law 93-523
A Primer on the Law, Evidence and Manage-
ment of Federii!Water Pollution control Cases, 2. Application of the Laws
Legal Support Division, US EPA May 1972 (4) to Microbiology
and on "Enforcement Activities," David I. She-
droff, in Proceedings of the First Microbiology 2.1 Gathering and Preserving
Seminar on Standard!Zat!on of Methods, US Evidence
EPA, March 1973 (5). Lawyers should be con- 2.2 Admissibility of Evidence
sulted for the exact interpretation of the laws 2.3 Preparation for
and their applications. Testimony
2.4 Testimony in Court
The Section describes the portions of the
Federal laws on water quality that are relevant
to microbiologists and relates analytical me-
thods and record-keeping to these laws. It also
shows how the analytical results become evi-
dence in administrative or court proceedings 1. Enabling Legislation
and will help the analyst to understand his role
as a witness. A brief outline of the contents of 1.1 Scope and Application: To under-
the Section follows: stand the role of microbiology in environmen-
tal and compliance monitoring, it is necessary
to briefly describe EPA's responsibilities for
1. Enabling Legislation {Federal development of methodology, assistance to
Laws Dealing with Water the States, pro~ulgation of criteria and guide-
Quality) lines, establishment of compliance with per-
mits and conduct of enforcement actions.
1. 1 Scope and Application Much of the work of the microbiologist in EPA
1.2 Federal Laws Dealing will involve generation of data to determine
with Water Quality compliance with the Federal laws on water
1.3 Federal Water Pollution quality. These laws with related regulations
Control Act Amend- limit the choice of analytical technique and
ments of 1972 Public sometimes require more documentation than
Law 92-500 the analyst might otherwise provide.

LEGAL ASPECTS 277

 1.2 Federal Laws Dealing with Water The law extends the Federal program to all
Quality(6) U.S. surface waters, not just interstate waters.
The States have submitted water quality stan-
Congress has passed three principal laws dards for intrastate waters to EPA for approval
on water quality which concern the microbiol- or revision. While the States retain primary
ogist: The Federal Water Pollution Control Act, responsibility to prevent, reduce, and elimi-
as amended; the Marine Protection, Research ·nate water pollution they must now do so
and Sanctuaries Act, commonly known as the within the framework of a new national pro-
Ocean Dumping Law; and the Safe Drinking gram. The law sets forth guidelines for the
Water Act. These acts have a common theme: control of industrial and municipal water pollu-
tion, expands water quality standards, estab-
1.2.1 Legislation is passed by Congress lishes a new system of permits for discharges
providing the general framework of Federal into the Nation's waters, and creates stringent
interest and control of an area of the environ- enforcement machinery and heavier penalties
ment. EPA promulgates general rules describ- for violations.
ing, requiring and/or limiting the qualities of
wastewater discharges or drinking water. 1.3.2 The Regulatory Scheme (6): Under
this act the States establish the minimum
1.2.2 EPA or appropriate state agencies water quality standards for streams and these
issue permits placing specific limitations on standards are approved by EPA. The Adminis-
discharges, establish Maximum Contaminant trator determines minimum acceptable efflu-
Levels in drinking and ambient water, and pro- ent limits for municipal treatment plants and
vide general rules controlling underground for specific industries based on current tech-
injections. nology'. These limits become more restrictive
over time. Using the more stringent water qual-
1.2.3 Permittees or others subject to the ity or treatment limitations, the Administrator
particular act may be required to self-monitor or State determines specific limits for a dis-
their discharges, and report findings to the charge. These limits set forth in a·permit for a
State and/or EPA. direct discharge are enforceable by civil pen-
alty, civil or criminal process, or revocation of
1.2.4 Provisions are made for enforce- permit.
ment actions when permit, variance, or abate-
ment schedules are violated. A microbiologist may be called as a wit-
ness to prove the violation of the permit by
direct discharge or to prove the violation of
1.3 Federal Water Pollution Control Act specific limitations placed on industrial firms
Amendments of 1972, Public Law 92-500 discharging to municipal plants. Generally
(1) these microbiological limitations will relate to
discharges from municipal treatment plants or
(See Appendix A for listing and summary from industries such as food processors.
of pertinent sections of the law, and the related
microbiological activities). Other enforcement activities for the micro-
biologist under the FWPCA include a permit
1.3. 1 Background and Summary (6): This program covering sewage sludge discharges
Is the most comprehensive program ever en- and standards for discharges from marine san-
acted to prevent, reduce, and eventually elimi- itation devices and their performance (40
nate water pollution. The two general goals of CFR/40 Amendments, 41 No. 20, January 29,
the Act are: To achieve wherever possible lly 1976). If such permits or standards contain
July 1, 1983, water that is clean enough for microbiological limitations, the microbiologist
recreational uses and for the protection of · may be called on to prove or disprove a viola-
aquatic life; and by 1985 to have no dis- tion (Section 405). Standards have been set
charges of pollutants into the Nation's waters. for discharges from marine sanitation devices

278 oEPA MICROBIOLOGICAL MANUAL 1978

 and their performance (Federal Register 41, be maintained under this Act. shall upon con-
January, 1976). viction, be punished by a fine of not more than
$10,000, or by imprisonment for not more
1.3.3 Analytical Guidelines and Criminal than six months, or by both."
Sanctions (6) -
. 1.3.4 Alternative test procedures are per-
Analytical guidelines and criminal sanc- mitted for use in NPDES, 40 CFR Part 136 and
tions for improper analysis or furnishing false in the Drinking Water regulations under 40
results are included in the Act. Section 304 (g) CFR Part 141. Information on application for
calls for the Administrator to promulgate use of alternative test procedures is given in
" ... guidelines establishing test procedures for these issues of CFR. The details of the compar-
- the analysis of pollutants ... ". The initial guide- ative testing which may be required are given
lines for monitoring the National Pollution Dis- in this Manual, under Quality Control, Part IV-C.
charge Elimination System (NP DES) (7) contain
test procedures for total coliforms, fecal coli-
forms, and fecal streptococci using methods 1.4 The Marine Protection, Research
referenced from the 14th Edition of Standard and Sanctuaries Act of 1972, (Ocean-
Methods(B). The guidelines defining second- Dumping) Public Law 92-532 (2)
ary treatment originally included fecal coli-
form limitations, and permits issued to munici- (See Appendix A for listing and summary
pal plants until July, 1976 contain such limita- of pertinent sections of the law, and the related
tions. These regulations were amended in microbiological activities).
July, 1976 and no longer contain microbiolog-
ical limitations. Such limitations may still be 1.4. 1 The Regulatory Scheme: The Marine
required in permits issued after that date if Protection, Research and Sanctuaries Act of
required for compliance with water quality 1972 regulates the dumping into ocean
standards or if such parameters are required in waters of all types of materials which would
order to comply with State law. Guidelines-for adversely affect human health and welfare, the
certain food related industries include fecal marine environment, ecological systems or
coliforms as a limiting parameter.. The economic development.
amended guidelines for municipal and indus-
trial wastewaters place restrictions on the This Act bans dumping of radiological,
measurement of fecal coliforms in chlorinated chemical or biological warfare agents and
or. toxic wastewaters. For these wastewaters high-level radioactive wastes. With one excep-
the membrane filter or most probable number tion, permits are required for transporting ma-
(MPN) methods may be used, but the MPN is. terials for ocean dumping and for the dumping
the method of choice when the results may be itself. The Corps of Engineers issues permits
involved in controversy. (Refer to the amend- for dredge spoils; EPA issues permits for all
ments to 40 CFR Part 136 (9)). The methods other materials (see Appendix A). The excep-
are described in Part Ill, B, C and D. tion is fish-processing wastes. Since they are a
natural ocean waste product, no permit is re-
Microbiologists performing analyses re- quired unless harbors or other protected
quired under this Act should be aware of the waters are involved as the receiving waters, or
specialized enforcement procedures in Sec- unless the EPA Adminis.trator finds that such
tion 309 (c)of FWPCA relating to analyses and deposits in certain offshore areas could endan-
reports of results: "Any person who knowingly ger health, the envi_ronment or ecological
·makes any false statement, representation, or systems.
certification in any application, record, report,
plan, or other document filed or required to be In evaluating permit applications, EPA and
maintained under .this Act_ or who falsifies, the Corps of Engineers must consider:
tampers with, or knowingly renders inaccurate
any monitoring device or met~od required to The need forthe proposed dumping.

LEGAL ASPECTS 279

 The effect on human health and welfare, 1.5.1 National Objectives: This Act has as
including economic, aesthetic and recrea- its main objective the establishment and en-
tional values. forcement of primary drinking water stan-
dards. These standards, which are to be en-
The effect on fisheries, resources, plank- forced by the States, will apply ·to public water
ton, fish, shellfish, wildlife, shorelines, systems and specify maximum levels of: 1)
beacJies and marine ecosystems. Those contaminants which may have adverse
health effects (primary standards) and, 2) those
The effect of dumping particular volumes contaminants which should be limited to pro-
and concentrations of materials and the tect the public welfare (secondary standards).
persistence of the effect. The protection of underground drinking water
sources by regulation of State underground
The effect on other uses such as scientific injection control programs and the appoint-
study, fishing and "other resource exploitation. ment of a 15-member National Drinking Water
Advisory Council are also provided for under
Appropriate locations and methods of dis- this Law. '
posal or recycling, including land-based
alternatives and the probable impact of 1.5.2 The Regulatory Scheme
requiring the use of such alternate loca-
tions or methods. Interim Primary Drinking Water Regula-
tions: The Administrator of EPA proposed na-
The law charges the Secretary of Com- tional interim primary drinking water regula-
merce with responsibility for a comprehensive tions 90 days after enactment of the Law. The
and continuing research program involving interim regulations were promulgated in De-
the possible long-range effects of pollution, cember, 1975(10).
overfishing or man-induced changes in ocean
ecosystems. Research efforts are to be coordi- Secondary Drinking Water Regulations:
nated with EPA and the Coast Guard. Following the enactment date of this Law, pro-
posed secondary regulations will address the
The basic research objective of the law is to aesthetic characteristics of water such as
find ways to minimize or to end all ocean dumping taste, appearance, etc. Ninety days later these
within five years. It will cover the effects of dump- secondary regulations will be promulgated,
ing materials into ocean or coastal waters and but will not be enforceable.
into the Great Lakes or their connecting waters.
Study of Maximum Contaminant Levels in
1.4.2 Civil and Criminal Sanctions: The Drinking Water: The National Academy of Sci-
law provides for both civil and criminal penal- ences conducted a study to determine the
ties for violations but there is no penalty for Maximum Contaminant Levels which should
dumping materials from a vessel as emer- be recommended to protect human health.
gency action to safeguard life at sea. Any indi- The study investigated contaminants that
vidual may initiate a civil suit to enjoin any might have adverse health effects but the lev-
person, including Federal, State and local gov- els of which could not be determined in drink-
ernment or agency, who is alleged to be violat- ing water. The results of the above study are
ing any prohibition, limitations, criterion or reported to Congress. EPA then publishes pro-
permit established or issued under this law. posals in the Federal Register for recom-
mended maximum levels, which will subse-
1.5 The Safe Drinking Water Act of quently be promulgated.
1974, Public Law 93-523 (3)
Revised Primary Drinking Water Regula-
(See Appendix A for listing and summary tions: Following the National Academy of Sci-
of pertinent sections of the law, and the related ences study, EPA proposes revised primary
microbiological activities). drinking water regulations to be adopted in

280 &EPA MICROBIOLOGICAL MANUAL 1978

180 days and to be effective 18 months after termines that the State no longer meets its
promulgation. These primary drinking water requirements under the Act.
regulations specify a Maximum Contaminant
Level or require the use of treatment tech- (b) Federal Enforcement: If a State fails to
niques for each contaminant in lieu of assure enforcement of drinking water regula-
Maximum Contaminant Levels. tions, the EPA Administrator notifies the State
concerning the violation, and provides advice
Regulation of State Underground Injec- and technical assistance to the State and to
tion Control Programs: The Act provides for the public water system that is in non-
regulations which contain minimum require- compliance, to bring the system into
ments for effective (State) programs to prevent compliance.
underground injection which endangers drink-
ing sources. To be approved, a State program 1.5.5 Civil and Criminal Sanctions: The
must prohibit underground injection without a EPA Adiministrator may bring a civil action to
state permit within three years after the enact- require compliance with either the national
ment date of the Law. Applicants for under- primary drinking water regulations or with any
ground injection permits must satisfy the State requirement of an applicable underground in-
that the injection will not endanger drinking jection control program. A maximum penalty
water sources. No regulations are promul- of $5,000 per day may be imposed by the
gated that allow underground injection which court for each day in which a violation occurs.
endangers drinking water sources.
·2. Application of the Laws to Microbiology

1.5.3 Quality Control Requirement 2. 1 Gathering and Preserving Evidence

In the Safe Drinking Water Act, the Admin- 2.1. 1 Stream Standards: To establish a ·
istrator can specify analytical methodology standard violation, it is necessary to show the
which includes quality control and testing navigable waterway is below approved water
procedures. quality standards. Water quality criteria spec-
ify permissible levels of chemjcal and biologi-
cal constituents for receiving waters. It must
1.5.4 Enforcement
be demonstrated that the defendant's dis-
charge caused or contributed to a reduction in
(a) State Primary Enforcement Responsi- receiving water quality below one or more
bility: A State has the primary enforcement applicable standards. Samples should be col-
responsibility for protection of drinking water lected 1) upstream of the discharge, 2) at the
if it has a program acceptable to EPA. The EPA point of discharge and 3) downstream of the
Administrator must determine that the State discharge at a point after a reasonable mixing
has drinking water regulations no less strin- zone.
gent than Federal regulations. The State may
permit variances and exemptions as pre- Although all State water quality standards
scribed in the Law, and must have an adequate include criteria for the same basic parameters,
plan for providing safe drinking water under there are differences among th~ .states as to
emergency circumstances. In . addition, the the sampling and test procedures which must
State must have monitoring programs that be followed in order to establish a standards
comply with Federal requirements and must violation. It is thus imperative that only the
possess sufficient enforcement authority. testing method specified be used in order to
show that a particular state water quality stan-
Approval by the EPA Administrator of the dard has or has not been violated.
State's underground injection program gives
to the State the primary enforcement responsi- 2. 1.2 Efflu.ent Standards: The Agency has
bility until such time as the Administrator de- not provided specific guidance in sampling

LEGAL ASPECTS 281

and tes't protocol to microbiologists working idual or corporate) property without his con-
with the effluent standards and compliance sent cannot be introduced into evidence in
monitoring. A protocol must vary with the way either a civil or a criminal case 'because of the
in which the permits are written. The stan- Fourth Amendment guarantee against unrea-
dards may set maximum values and average sonable searches and seizures. Consent need
values by the day, week, month, and year with- not be obtained to take samples on the public
out defining the number and kinds of sample portions of a waterway, usually up to the ordi-
required to establish compliance with a given nary high water level (11). Almost all Fourth
standard. Specifications of numbers and types Amendment objections can be prevented by
of samples are needed to provide weekly, sending an advance, written notification of the
monthly or yearly maxima or averages. time, scope, and purpose of any proposed EPA
inspection, or sampling visit and by obtaining
Each Regional Office of EPA has devel- the written consent of the party to be in-
oped an approach to compliance monitoring. spected ( 12). If a search warrant has not been
Uniformity of sampling and testing schemes is obtained, unannounced investigatory inspec-
desirable for data validation and for compari- tions may be made only if the voluntary con-
son of data between different laboratories. sent of a person in authority is secured ( 13).
The need for uniformity is even more impor-
tant with the transfer of the responsibility to 2.2 Admissibility of Evidence
the states for issuance of permits, as legally
permitted. Some states are following EPA- 2.2. 1 Types of Legal Action: Violations of
Regional guidance while others are develop- Public Laws 92-500, 92-532, or 93-523 can
ing their own plans for compliance monitoring. result in civil penalties assessed by the Admin-
istrator, a hearing board, or a court; criminal
The sampling plan should select the sam- sanctions by a court; or a court order requiring
pling techniques, volumes, frequency, replica- a discharge source to take or cease a particular
tion, etc., to meet the standard being chal- action. Except for civil penalties imposed by
lenged. All sampling and analyses to verify the Administrator, formal hearing will be
compliance with a particular standard should required.
be performed in the same fashion and with the
same frequency. Three rules for a good sam- 2.2.2 Authentication of Testimony: Au-
pling plan are: 1) fit the design to meet the thentication as a conditionprecedent to ad-
effluent standard, i.e., use a reasonable means missibility may require testimony under oath
to obtain statistical validity 2) apply the plan (14). Witnesses are subject to cross-
uniformly and 3) document the plan, indicate examination. The form and admissibility of evi-
its source and record its use. dence presented in Federal courts are clearly
defined in the Federal Rules of Civil Procedure
2.1.3 Drinking Water Standards: The Safe .(15) and the Federal Rules of Evidence (14).
Drinking Act has established Maximum Contami- These formal rules of evidence may be par-·
nant Levels (MCL's) on an interim basis for com- tially annulled for administrative hearings. Lab-
munity and non-community systems. The MCL's oratory records may not be acceptable evi-
are based primarily ori the 1962 Public Health dence without proof of authenticity.
Service Standards. For microbiology, the minimal
sampll]lg frequency per month is specified in The hearsay rule is one of the most direc-
Title 40 Part 141. Samples shall be taken at tive statutes. It states that generally persons
regular time intervals and in numbers propor- may only testify to what they know personally
tional to the populations served. Samples shall and that they must be subject to cross-
also be taken at points representative of the examination. However, some exceptions to the
conditions in the distribution systems. hearsay rule are allowed. Evidence that would
normally be hearsay is admissible in the ad-
2. 1.4 Constitutional Protections: Sample ministrative hearing, if it has probative value in
evidence taken from the defendant's (indiv- the opinion of the hearing officer. Also, evi-

282 &EPA MICROBIOLOGICAL MANUAL 1978

dence may be presented in written form in a whether it was specifically done in anticipa-
hearing, but this is more likely when the pre- tion of litigation. This caution in admitting evi-
ceeding is not a full-fledged trial-type hearing. dence is an indication of distrust of the situa-
tion, not of the individuals involved.
Additional exceptions to the hearsay rule
are cited in the recently-enacted Federal Rules 2.2.5 Contacts with Parties to Adjudica-
of Evidence (14). These rules state that under tory or Adversary Proceedings: The following
certain circumstances a witness does not have statements are quoted directly from the May 5,
to be present for his statement to be admissi- 1975, memorandum of the Acting Assistant
ble. In addition, records of regularly-conducted Administrator for Enforcement, US EPA (16):
business activities, public reports, reports pre-
pared by law enforcement personnel and fac- As we are now becoming involved in more
tual findings resulting from legal investiga- and more adjudicatory hearings on
tions may be admitted without the testimony NPDES (National Pollution Discharge Elim-
of the person or persons involved. ination System) permits and in enforce-
ment actions, both through Administrative
2.2.3 Admissibility of Records: Under Rule Orders and in the Courts, it is very impor-
803 (6) of the Federal Rules of Evidence (14), tant that our staffs clearly understand that
written records made in the regular course of contacts and discussions with parties to
any business (i.e., laboratory operation) may these proceedings be carefully controlled.
also be introduced into evidence in civil ac-
tions without the testimony of the person(s) We have recently had inquiries about
who made the record. Prior to enactment of cases in which requests for adjudicatory
the Federal Rules of Evidence, this authority hearing had been granted and in which
was contained in the Federal Business EPA technical staff members, without the
Records Act, 29 U.S. Code, Section 1732A. knowledge of either the attorney assigned
or of the Enforcement Director, met with
Although preferable, it is not always possi- company representatives to discuss the
ble to have the individuals who collected, kept, pending case. In each case, the merits of
and analyzed samples testify in court. In addi- EPA's position as compared to that of the
tion, if the opposing party does not i11tend to company were discussed, as were possi-
contest the integrity of the sample or testing ble areas of compromise with respect to
evidence, admission under the Business EPA's position.
Records Act can save much trial time. For
these reasons, it is important that the proce- Case preparation and decisions on strat-
dures followed in evidence, sample collections egy for adjudicatory hearings are the re-
and analyses be standardized and described in sponsibility of our regional attorneys with
an instruction manual which can be offered as assistance from Headquarters Counsel for
evidence of the standard operating procedure Adjudicatory Hearings. Accordingly, I
followed by the laboratory. would appreciate your instructing your
staff members not to discuss permit ques-
2.2.4 Limitations on the Admissibility of tions or technical issues applicable to a
Records: Although the statutes do not specifi- particular industrial facility which is the
cally cover the point, it is clear from the exami- subject of an adjudicatory hearing or an
nation of cases that one of the requirements enforcement action until the appropriate
for admissibility is that the document has in- Enforcement Division attorney, either in
herent probability of trustworthiness. Thus, a· the Regional Office or in Headquarters is
trial judge has discretion in allowing or not - notified.
allowing a document into evidence.if there is
doubt as to its trustworthiness. One criterion The foregoing is not to be construed as
for the judge to consider is whether the partic- discouraging settlement discussions in
ular analysis was done as a routine matter or pending cases, but is only intended to

LEGAL ASPECTS 283

 provide for orderly resolution of matters preferably in a bound book or on bench cards,
which can be negotiated. that can be incorporated into a permanent
record log. Provision should be made for the
signatures of sample collectors, analysts and
2.3 Preparation for Testimony direct line supervisors in the sample log and in
the data log so that the laboratory data are
2.3.1 Gathering and Preserving Evidence authenticated. As described in Part V-A of this
in Water Pollution Entorcement Actions: In ev- manual, a quality control log book should be
ery water pollution suit, expert testimony will maintained on a day-to-day basis. It should
be of primary importance. To meet its burden record quality control checks on: media and
of proof, the Government may have to present supplies, equipment and instrumentation, the
expert testimony on sampling, laboratory ana- actual analyses, data handling and storage.
lyses, test results and the harmful effect attrib- Training of analysts should include familiariza-
utable to the defendant's discharge. If the Gov- tion with the quality control book and identifi-
ernment's expert witnesses do not testify ef- cation of their responsibilities in the program.
fectively, the lawsuit may be jeopardized. Each analyst should have a personal copy of
the manual as a guide.
2.3.2 Testimony on Sampling: In the order
of proof in a trial concerning pollution there 2.3.4 Pre-Trial Discovery: Whenever an
wlll be testimony by witnesses who have taken agency is a party to any federal court litigation,
samples. The samples may be effluents, receiv- it will be subject, under the Federal Rules of
ing waters, potable waters, sludges or sedi- Civil Procedure, to pre-trial discovery. The
ments. These witnesses will explain how, agency will be required to answer the oppos-
where and when the samples were taken. The ing party's questions and to produce re-
choice of sampling location and what to sam- quested documents. Technical personnel re-
ple depends to a large extent on the type of sponding to a motion to produc.e documents
legal action contemplated. should deliver related documents to the
agency attorney handling the case. Docu-
2.3.3 Documentation of Procedures: In an- ments should not· be withheld because they
ticipation of possible court presentation of evi- appear to be damaging to the government's
dence, laboratories must maintain an orderly, case. The responsible government attorneys
complete and permanent record-keeping and will determine, on the basis of the law of dis-
filing system. A laboratory operating manual covery, which documents must be submitted
should be used in all laboratories. The manual to the opposing party.
formalizes the operation of the laboratory by
describing in detail the sampling procedures, A sensible filing system should be set up
the line of technical responsibility, specific and followed. The objective of a filing system
analytic methods followed, data handling pro- is to store information so that it can be found
cedures, the continuous quality control pro- quickly. Information which is known but is not
gram established for daily operations, partici- reflected in the file is of no use and will not be
pation in interlaboratory and intralaboratory available when needed. However, the files
quality control programs and safety guide- should be examined regularly and outdated or
lines. Since routines change, personnel should superfluous information discarded to maintain
sign dated receipts that indicate they have manageability. Critical or outspoken com-
received the operating instructions and modifi- ments on notes, route slips or in margins,
cations when issued. should not be retained unless the originator
and recipient are prepared to defend them in
Complete records of samples received court.
must be kept in a separate log and official
chain of custody requirements must be ob- 2.3.5 Testimony on Methodology: A wit-
served. The laboratory data records, analytical ness may be required to provide testimony on
results and cQmputations should be written, methods of analyses and test results. For ac-

284 &EPA MICROBIOLOGICAL MANUAL 1976

ceptance in court, the witness must be able to 2.4 Testimony in Court
testify that the analytical method or procedure
employed has wide use in the microbiological 2.4. 1 General Instructions for a Witness:
community. For example, the procedures out- The following suggestions are made for pros-
lined in Standard Methods(B) and in This Man- pective witnesses to lessen the apprehensions
ual are recognized and accepted. In court everyone feels when first testifying before a
cases on record, results obtained using Stan- board, commission, hearing officer, or in court.
dard Methods have been admitted into evi- Even veteran ·witnesses often experience
dence while deviations from Standard Me- some anxiety. However, if a witness is properly
thods have had to be explained and justified. prepared on the subject matter of his testi-
mony and his conduct on the witness stand, he
It may be necessary to present testimony is much more confident about testifying. The
on parameters that are not included in these witness will be required to take an oath to tell
publications or on special types of samples to nothing but the truth. The important point is
which the methods described are not applica- that there are two ways to tell the truth-one is
ble. In such cases effective testimony may be in a halting hesitant manner, which makes the
based upon the best methodology currently board member, hearing officer, judge or jury
available, utilizing as substantiating evidence. doubt that the witness is telling all the facts in
published reports, other method manuals, etc. a truthful way, and the other is in a confident
to demonstrate that the methods do have rec- straight forward manner, which gives cre-
ognition in the scientific community. dence to the witness' words.

The specific test methods to be used in the If a scientist is a witness in a case involv-
application of the Federal Water Pollution Con- ing testimony concerning the appearance of
trol Laws may be identified in the Code of an object, place or condition, he should refresh
Federal Regulations (CFR). For example, the his recollection by inspecting the object, place
procedures required for Section 304 (g) of the or condition, etc., before the hearing or trial.
Federal Water Pollution Control Amendments Later he should try to picture the item and
of 1972 appear in 40 CFR, Part 136. These recall the important points of his testimony. He
guidelines establish the methodology to be should repeat this procedure until he has thor-
used for compliance monitoring and the me- oughly familiarized himself with the points that
thods become those that are acceptable as will be made in the testimony.
standards in court. Part 136 of 40 CFR also
provides a mechanism and rule for obtaining Before testifying, the witness should visit
approval for any alternate procedure that may a court trial or board hearing and listen to other
be proposed when the recommended method witnesses testifying. This will familiarize him
is not appropriate. with such surroundings and help him to under-
stand court protocol and the problem of testi-
mony. The scientist should arrive at the hear-
ing in time to listen to other witnesses testify
2.3.6 Testimony by Expert: A court may before taking the witness chair himself.
require that an expert witness' opinion be
based on studies and tests conducted or su- A good witness listens to the question and
pervised by him personally. However, experts then answers it calmly and directly in a sincere
are frequently permitted to offer testimony in manner. He knows the facts and can communi-
the form of an opinion in "the area of compe- cate them. He testifies in this manner on cross-
tence or based on someone else's work. Such examination as well as on direct examination.
testimony can be developed through the use of
hypothetical questions and objections tend to The witness should wear neat, clean
add weight to the expert's testimony rather clothes when he testifies and should dress
than to cast doubt on the witness' conservatively .. He should speak clearly and
competency. not chew gum while testifying.

LEGAL ASPECTS 285

 2.4.2 Direct Examination enough so that anyone in the hearing room or
courtroom can hear: him easily. The witness
(a) In a discussion on administrative proce- makes certain that the reporter taking the ver-
dures, E. Barrett Prettyman, Retired Chief batim record of his testimony is able to hear
Judge, U.S. Court of Appeals for the District of him and record what he says. The case will be
Columbia, gave the following advice (4): decided entirely on the words that are re-
ported as the testimony given at the hearing or
The best form of oral testimony is a series trial. The witness must give complete state-
of short, accurate, and complete state- ments in sentence form; half statements or
ments of fact. It is to be emphasized that incomplete sentences may convey the thought
the testimony will be read by the finder of in the context of the hearing, but be unintelligi-
the facts, and that he will draw his findings ble when read from the cold record months
from what he reads ... confused, discursive, later.
incomplete statements of fact do not yield
satisfactory findings.
2.4.3 Cross-Examination

(b) The witness should stand upright when (a) Concerning cross-examination, the fol-
taking the oath, pay attention, say "I do" lowing advice is given to prospective wit-
clearly, and not slouch in the witness chair. If nesses (4):
the witness has prepared answers to possible
questions, he should not memorize them. It is, Don't argue. Don't fence. Don't guess.
however, very important that he familiarize Don't make wisecracks. Don't take sides. Don't
himself as much as possible with the facts get irritated. Think first, then speak. If you do
about which he will be called to testify. not know the answer to a question, say so. If
you do not know the answer but have an opin-
(c) During direct examination, the witness ion or belief on the subject based on informa-
may elaborate and respond more fully than is tion, say exactly that and let the hearing officer
advisable on cross-examination. However, decide whether you shall or shall not give such
when volunteering information, he should not information as you have. If a 'yes or no' answer
ramble or stray from the main point raised in to a question is demanded but you think that a
his lawyer's question. Testimony is a dialogue, qualification should be made to any such an-
not a monologue. If testimony concerns a spe- swer, give the 'yes or no' and at once request
cialized technical area, the court or hearing permission to explain your answer. Don't
board will find it easier to understand if it is worry about the effect an answer may have.
presented in the form of short answers to a Don't worry about being bulldozed or embar-
logical progression of questions. In addition, rassed; counsel will protect you. If you know
by letting his lawyer control the direction of his the answer to a question, state it as precisely
testimony, the witness will avoid making re- and succinctly as you can. The best protection
marks which are legally objectionable or tacti- against extensive cross-examination is to be
cally unwise. brief, accurate and calm.

(d) The witness should be serious at all The hearing officer, board member or jury
times and avoid laughing or talking about the wants only the facts, not hearsay, conclusions,
case in the building where the hearing or trial or opinions. The witness usually will not be
Is being held. allowed to testify about what someone else
has told him.
(e) While testifying, the witness should talk
to the board member, hearing officer or jury, (b) The witness must be polite, even to the
looking at him or them most of the time, and attorney for the opposing part. He should not
speaking frankly and openly as if to a friend or be a cocky witness. This will lose him the
neighbor. He should speak clearly and loudly respect and objectivity of the trier of the facts

286 oEPA MICROBIOLOGICAL MANUAL 1978

in the case. He should not exaggerate or em- important things which he na~urally would re-
broider his testimony. member. If asked about little details which a
person naturally would not remember it is best
(c) The witness should stop instantly when to say that one does not remember, but he
the judge, hearing officer or board member must not let the cross-examiner place him in
interrupts, or when the other attorney objects the trap of answering question after question
to what is said. He must not try to sneak the with "I don't know."
answer in or nod his head for a "yes" or "no"
answer. The reporter has to hear an answer to (g) The witness must not act nervous. He
record it. If the question is about distances or should avoid mannerisms which will make him
time and the answer is only an estimate, he appear frightened, not telling the truth, or not
must say that it is only an estimate. telling all that he knows. Above all, it is most
important that the witness not lose his temper.
(c) The witness should listen carefully to Testifying at length is fatiguing. Fatigue will be
the questions asked. No matter how friendly recognized by crossness, nervousness, anger,
the other attorney may seem on cross- careless answers and a willingn·ess to say any-
exa mi nation, he may be trying to damage the thing or answer any questions in order to leave
testimony. ·He must understand the question the witness stand. When the witness feels
completely and should have it repeated if nec- these symptoms, he must recognize them and
essary, then give a thoughtful answer. He must strive to overcome these feelings. Some attor-
not give a snap answer. He cannot be rushed neys on cross-examination try to wear out the
into answering, yet taking too much time witness so he will lose his temper and say
would make the board member, hearing offi- things that are not correct, or that will hurt the
cer or jury think the witness is making up the testimony. The witness must not let this
answers. happen.

(d) The witness must answer the question (h) If the witness does not want to answer
that is asked-not the question that he thinks a question, he should not ask the judge, hear-
the examiner (particularly the cross-examiner) ing officer or board member whether he must
intended to ask. The printed record shows only answer it. If it is an improper question, his
the question asked, not what was in the exam- attorney will object for him. One must not ask
iner's mind and a non-responsive answer may the presiding officer, judge or board member
be very detrimental to the case. This situation for advice or help in answering a question. The
exists when the witness thinks "I know what witness is on his own. If the question is an
he is after but he hasn't asked for it." Answer improper one, his attorney will object. If the
only what is asked. The witness must explain judge, hearing officer, or board member then
his answers if necessary. directs the witness to answer it, he must do so.
He cannot hedge or argue with the opposing
(e) If by chance one's answer is wrong, attorney.
correct it immediately; if the answer was not
clear, clarify it immediately. The witness is (i) There are trick questions which may be
sworn to tell the truth. Every material truth asked and which, if answered, signify "yes" or
should be readily admitted, even if not to the "no", an~ will damage the credibility of the
advantage of the party for whom he is testify- testimony. Two examples follow:
ing. He must not stop to figure out whether the
answer will help or hurt his side.
( 1) "Have you talked to anybody about this
(f) The witness must give positive, definite matter?" If you say "no", the hearing officer or
answers when at all possible and avoid saying board member, or a seasoned jury, will know
"I think", "I believe", "in my opinion." If he that is not correct because good lawyers al-:
does not know, he must say so and not make ways talk to the witnesses before they testify.
up an answer. One can be positive about the If one says "yes", the lawyer may try to infer

LEGAL ASPECTS 287

that you were told what to say. The best thing you are being paid to say what your side wants
to say is that you have talked to Mr. , you to say. Your answer should be something
your lawyer, to the appellant, etc., and that you like "No, I am not getting paid to testify, I am
were just asked what the facts were. All that is only getting compensation for my time off
wanted is the truth. from work, and my expenses incurred in being
here." A witness should never be paid a contin-
(2) "Are you getting paid to testify in this gency fee as it indicates strongly that since his
appeal?" The lawyer asking this hopes your compensation depends upon the results, he
answer will be "yes", thereby inferring that may be inclined to overstate the case.

REFERENCES

1. Federal Water Pollution Control Act Amendments of 1972, Public Law 92-500, October 18,
1972, 86 Stat. 816, 33 United States Code (USC) Sec. 1151.

2. Marine Protection, Research and Sanctuaries Act of 1972, Public Law 92-532, October 23,
1972,86Stat.1052.

3. Safe Drinking Water Act, Public Law 93-523, December 16, 1974, 88 Stat. 1660, 42 United
States Code (USC) 300f.

4. U.S. Environmental Protection Agency, Legal Support Division, 1972. A Primer on the Law,
Evidence, and Management of Federal Water Pollution Control Cases, Washington, 0.C. pp.
43-52, 54=58. - -- --
5. Shedroff, D. I., 1973. Enforcement activities. In: Proceedings of the First Microbiology Seminar
on Standardization of Methods, EPA-R4-73-022, Office of"Research and Monitoring, U.S.
'Environmental Protection Agency, Washington, D.C., pp. 1-11.

6. Shedroff, D.1., 1976. Personal Communication. Office of Enforcement, US EPA, Washington, DC.

7. Guidelines Establishing Test Procedures for Analysis of Pollutants, 40 Code of Federal Regu,la-
tions (CFR) Part 136, Published in Federal Register, 38, p. 28758, October 16, 1973.

8. American Public Health Association. 1976. Standard Methods for the Examination of Water and
Wastewater(14th ed.) American Public Health Association, Inc., Washington, DC. p. 874

9. Guidelines for Establishing Test Procedures, 40 Code of Federal Regulations (CFR) Part 136,
Published in Federal Register, 40, 52780, Dec. 1, 1976.

10. National Interim Primary Drinking Water Regulations, 40 Code of Federal Regulations (CFR) Part
141, Published in Federal Register, 40, 59566, December 24, 1975.

11. Borough of Ford City vs. United States, 345 F. 2d 645 (3rd Cir. 1965).

12. Camara vs. Municipal Court, 387 U.S. 523 (1967); See vs. Seattle, 387 U.S. 54 7 (1967).

13. United States vs. Hammond Milling Co., 413 F. 2d 608 (5th Cir. 1969), cert. den. 396 U.S. 1002
(1970); and United States vs. Thriftimart, Inc. 429 F. 2d 1006 (9th Cir. 1970) cert. den. 400 U.S.
926 (1970).

14. The Federal Rules of Evidence, Public Law 93-595, January 2, 1975, 88 Stat. 1926, 28 United
States Code (USC) App.

15. Federal Rules of Civil Procedure, Rule 43, adopted by the U.S. Supreme Court pursuant to Title
28, U.S. Code (USC) Section 2072, as amended effected July, 1975.

16. Johnson, R. H., Acting Assistant Administrator for Enforcement, EPA Office of Enforcement. May
5, 1975. "Contacts with Parties to Adjudicatory or Adversary Proceeding", Memorandum to EPA
Assistant and Regional Administrators.

288 oEPA MICROBIOLOGICAL MANUAL 1978

 APPENDIX A

TABLE-1

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500
Microbiological Activities Under Relevant Sections of the Law

Sections of Law Summary of Sections Microbiological Activity

104(a)(S)
Water Quality Surveillance The Administrator is required to establish and maintain Conduct research, develop methodology
System a water quality surveillance system with States and and technology, complete necessary
other Federal Agencies. Agencies in the system will analyses, perform surveys, and provide
collect and disseminate basic data on the chemical, expertise in microbiology.
physical and biological effects of varying water quality.
They are to develop new methods for identifying and
measuring the effects. of pollution on the chemical,
physical and biological integrity of the water.

106 (c)
Grants for State Pollution EPA is to provide assistance and guidance to the States Provide necessary assistance in
Control. Program on the development and operation of procedures and microbiological expertise and
systems to monitor water quality, including biological consultation to the States.
monitoring.

108 (a)
Pollution Control in the EPA is to conduct projects in cooperation with other Analyze water samples to support nethod
Great Lakes agencies for demonstrating new methods and developing and development plans for Great Lakes
plans for their use in controlling pollution on the pollution control.
Great Lakes.

301 (b) and 402

Permits for Publicly Owned Municipal treatment plants must attain" . . . second- Analyses to determine compliance or
(Municipal) Treatment Works ary treatment", as defined by Administrator, or non-compliance with microbiological
treatment necessary to meet water quality portions of permit requirements.
standards, whichever is more stringent, by 1977,
and " . . . best practicable waste treatment tech-
nology over the life of the works" by 1983.

l\l
00
co
N
co TABLE-1
0
(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500
Microbiological Activities Under Relevant Sections of the Law

Sections of Law Summary of Sections ~licrobiological Activity

301 (b) and 402

Permits for Non-Publicly Owned (1) Existing Plants: Must attain "· .• best practicable Analyses to determine compliance or
(Industrial) Treatment Works control technology currently available", or water non-compliance with microbiological
~) quality standards, whichever is more stringent, by portions of permit requirements.

~
1977, and"· •• best available technology economi-
cally achievable" by 1983.

(2) New Plants: Must comply with " . . . National Indus-

~ trial Standards of Performance" which for a particular
C')
::0 industry reflect "· •. the greatest degree of effluent
0 reduction • . • achievable through the application of
!XI the best available control technology, processes,
0
r- operating methods, or other alternatives."
0
C> 301 (b) and 307 (b)
~
r-
Pretreatment Standards for Dis-
charges by Non-Publicly Owned
Private industry discharging into public treatment plants
must demonstrate compliance with pretreatment standards
Analyses to determine compliance or
non-compliance with microbiological
Enterprises into Publicly-Owned which are determined by the type of waste source and whether portion of the pretreatment standard.
~ Plants the plant is already existing or is new since the passage of
2:: the Act. Standards for both new and old plants are designed
~
r-
to prevent the discharge through publicly-owned treatment
works of pollutants which" . . . interfere with, pass
....
co through, or (are) otherwise incompatible with such works."
;:;: Existing sources must comply by three years after promulga-
tion of applicable pretreatment standards.

304(a) (b) and (g)

Information and Guidelines EPA must develop water quality criteria which reflect know- Develop microbial water quality
ledge of the effects on plankton, fish, shellfish, wildlife, criteria based on the analyses of
plant life, esthetics and recreation which may be expccte.d all navigable, ground and coastal
from presence of pollutants in any body of water or in waters and the ocean.
ground water. Information must be developed on what factors
are needed to restore and maintain the chemical, physical
and biological integrity of navigable waters, ground waters,
coastal waters and oceans. The Administrator is also re-
quired to issue guidelines for identifying and evaluating
the nature and extent of nonpoint sources of pollutants.
 TABLE-1
(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500
Microbiological Activities Under Relevant Sections of the Law

Sections of Law Summary of Sections Microbiological Activity

304 (b)
Publication of Effluent The Administrator shall publish regulations providing Provide advice, technical assistance
Limitation Guidelines guidelines for effluent limitations. and analyses required to establish
effluent limitations.

304 (g)
Guidelines for Test The Administrator shall promulgate guidelines estab- Provide advice, technical assistance
Procedures lishing test procedures for the analyses of and analyses required to establish
pollutants. microbiological procedures.

307 (a)
Toxic Pollutants Discharge limitations are established or materials are Identification and quantification of
prohibited that are designated by the Administrator as pollutants, including viruses,
toxic, taking into account " . . . toxicity . . . persist- designated as toxic by the Adminis-
ence . . . degradability . . . presence of the affected trator.
organisms and the nature and extent of the· effect of the
toxic pollutant· on such organisms."

308
Inspections, Monitoring and Owners and operators of pollution point sources shall Inspection of microbiological portions
Entry establish and maintain records; make reports; install, of records, microbiological equipment
use, and maintain monitoring equipment or methods, in- or methods used by owner or operator
cluding biological monitoring methods; and sample effluents of pollution point source; sampling
at locations, intervals, and with methods prescribed by and analysis of effluents required to
the Administrator. be sampled by the owner or operator
of the pollution point source.

309
Federal Enforcement On the basis of any information available that indicates Provide advice and technical assistance
non-compliance of the requirements of a permit issued by to the State and persons in non-compliance
a State, the Administrator may notify the person in to bring them into compliance; provide
alleged violation and the State of such findings. If analytical data, expertise and testimony
after the thirtieth day after notification the State has as required to establish EPA's case.
not commenced appropriate enforcement action, the Adminis-
trator may issue a compliance order or bring civil action
to enforce the permit conditions or limitations.
,..,
co
_.
 N
<D
N

TABLE-1
(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500
Microbiological Activities Under Relevant Sections of the Law
Sections of Law Summary of Sections Microbiological Activity

310
0) International Pollution The Administrator may call a hearing when he has reason Microbiological surveys to determine

~
Abatement to believe pollution is occurring from U. S. sources if domestic pollution is adversely
"· •• which endangers the health or welfare of affecting a foreign country; survey
persons in a foreign country." results provide the Administrator
with data to assist him in deciding
~ whether to call a hearing.
(')
::0
0 311
Ill Oil and Hazardous Substance This section bans the discharge of oil and any other Performance of degradability tests
0r- Liability "· .• elements and compounds which, when discharged and experiments under actual or
0 in any quantity into . • . waters • . . present an simulated conditions.
E? inuninent and substantial danger to the public health or
welfare, including, but not limited to, fish, shellfish,
R
r- wildlife, shorelines, and beaches." The ban applies
to any substance which fits this description and that
~ the Administrator designates as "hazardous."
~
~
r-
312 (b)
Marine Sanitation Devices Vessel sanitation devices must conform to performance Analyses to confirm com~liance with
...co standards issued by the Administrator. New vessels must microbiological portions of per-
comply within two years of promulgation; existing formance standards. Analyses to
~ vessels have five years in which to comply. determine if the device operates
in conformity with the standards;
the Coast Guard is responsible
for such testing of the devices.
403 (c)
Ocean Discharge Criteria The Administrator is required to promulgate guidelines Conduct required analyses to
for determining the degradation of territorial waters, establish guidelines for monitor-
coastal waters and oceans. ing· the degradation of territorial
and coastal waters and the
oceans.
 TABLE-1
(Continued)

FEDERAL WATER POLLUTION CONTROL ACT AMENDMENTS OF 1972, PUBLIC LAW 92-500
Microbiological Activities Under Relevant Sections of the Law

Sections of Law Summary of Sections Microbiological Activity

405
Permits for Disposal of Permits are required for disposal of sewage sludge Analysis of sludges at time of
Sewage Sludge (including removal of in-place sewage sludge from one transport and at disposal site to
location and its deposit in another iocation) where determine compliance or non-compliance
disposal " . . . would result in any pollutant . . . with permit requirements.
entering • • . waters."

504
Emergency Powers An injunction prohibiting discharge by a particular Detection of pathogens in water and
source may be issued on proof of" . . . iniminent and from their sources; and enumeration
substantial endangerment to the health of persons or of indicators authorizing closure
to the welfare of persons where such endangerment is of shellfish beds and identifica-
to the livelihood of such persons." tion of pollutant sources.

N
co
w
,_,,
(0
~
APPENDIX A

TABLE-2

MARINE PROTECTION, RESEARCH AND SANCTUARIES ACT OF 1972, PUBLIC LAW 92-532
Microbiological Activities Under Enforcement and Compliance Monitoring Sections

Sections of Law Summary of Sections Microbiological Activity

~) 102
m EPA Permits for Ocean Establishment of a program for the issuance of EPA permits Analyses to determine compliance or
Dumping based on criteria which consider the effects of ocean non-compliance with microbiological
~ dumping on human welfare, shellfish and fisheries portions of permit requirements.
resources, plant and animal life, shorelines, beaches, Possible conduct of microbiological
~ and marine ecosystems. analyses to determine that the
(') proposed dumping "· . . will not un-
::0
0 reasonably degrade or endanger human
OJ health, welfare, or amenities, or
0
,... the marine environment, ecological
systems, or economic potentialities."
0 (Regulations in 40 CFR 227 set forth
Ci)
criteria for evaluation of permit
~,... applications for materials containing
living organisms. See especially 227.36.)
~ 103
2:: Corps of Engineer Permits The responsibility for issuing permits (based on the Analyses to determine compliance
~
,... criteria in Section 102 above) for the ocean dumping or non-compliance with microbiolog-
of dredged materials is under the jurisdiction of the ical portions of permit requirements .
....
co Army Corps of Engineers.
Q:l
 APPENDIX A

TABLE-3

SAFE DRINKING WATER ACT OF 1974, PUBLIC LAW 93-523

Microbiological Activities Under Enforcement and Compliance Sections

Sections of Law Summary of Sections Microbiological Activity

1412 (a) Cl)

Establish Primary Interim EPA has the responsibility for establishing national interim Provide the expertise and advice to
Standards primary drinking water regulations which will protect human assist in the establishment of the
health by using the technology which is generally available. required safe interim primary standards
for drinking water.

1412 (c)
Proposed National Secondary The Agency promulgates National Secondary Drinking Water Provide the expertise and advice to
Drinking Water Regulations regulations which are mostly related to the aesthetic EPA for establishing National Second-
characteristics of drinking water. ary Standards relating to micro-
biology.

1412 (e)
Study by Independent The Administrator shall arrange studies with the National Provide input as needed for estab-
Organizations Academy of Sciences or other independent scientific lishment, performance and evaluation
organization to determine maximum contaminant levels of of studies by the National Academy
known or anticipated contaminants, and to identify those of Sciences or other independent
contaminants in drinking water which are at levels too scientific organization.
low to measure.

1413 (b)
Recommended Maximum The Agency must also establish for each contaminant a Conduct research and monitoring analyses
Contaminant Levels maximum contaminant level which will produce no known to establish acceptable levels and
adverse effects and allows an adequate margin of safety. maximum levels for bacterial indicators,
pathogens and viruses.
1414 (a) (1) (A)
Failure by State to Assure If the Administrator finds that a State with primary en- Provide advice and technical assistance
Enforcement of Standards forcement responsibility has not maintained compliance in to the State and water systems to
its public water systems, he shall so notify the State and bring them into compliance.
provide assistance in achieving compliance.

N
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TABLE-3
{Continued)

SAFE DRINKING WATER ACT OF 1974, PUBLIC LAW 93-523

Microbiological Activities Under Enforcement and Compliance Sections

Sections of Law Summary of Sections Microbiological Activity

1414 (a) (1) (B)

0) Civil Action If the Administrator determines that the State abused its Provide analytical data, expertise
discretion in carrying out its primary enforcement responsi- and testimony as required to establish
i bility, the Administrator may commence a civil action to
enforce the standards.
EPA's case.

~ 1414 (f)
~ Non-Compliance/Public Hearings If the Administrator finds non-compliance by a public Provide confirming analytical data,
0 water system in a State with primary enforcement responsi- recommendations and expert advice on
~ bility, he may hold hearings to gather information from microbiological aspects demonstrating
,...
0 technical and other experts and may issue recommendations non-compliance.
0 on actions which will achieve compliance.
C)

,...~
1421
Underground Injection Control The Administrator shall publish and promulgate regulations Determine the feasibility of micro-
Systems for State underground injection control systems after biological criteria and perform
~ public hearing. The States will issue permits for under- analyses of injected wastewaters and
<: ground injection which will not endanger drinking water of ground waters if required.
§,... sources; and will inspect, monitor and keep records of
the permitted underground injection wells .
...
co
~ 1431 (a)
Emergency Powers EPA may obtain an injunction against a non-complying water Proof of presence of bacterial indica-
system on proof of the presence of a contaminant which tors, pathogen~ or viruses in suffi-
presents an imminent and substantial endangerment to human cient numbers to pose a danger to
health and if the appropriate State or local authority has human health.
not acted.

410
Amendments to the Bottled After the promulgation of drinking water regulations, the Conduct analyses to determine if the
Drinking Water Standards Food and Drug Administration must either promulgate amend- quality of bottled drinking water
(Section 4. Chapter IV of the ments to the bottled drinking water standards or publish meets drinking water regulations.
Federal Food, Drug, and reasons for not making amendments.
Cosmetic Act)
 APPENDIX B

FROM: Manual for the Interim Certification of Laboratories Involved in Analyzing Public Drinking Water Supplies, EPA
600/8-78-008, May, 1978. OMTS, Office of REsearch and Development, U.S. Environmental Protection Agency,
Washington, DC 20460

Chapter V
MICROBIOLOGY: CRITERIA AND PROCEDURES FOR INTERIM
CERTIFICATION OF LABORATORIES INVOLVED
IN ANALYSIS OF PUBLIC WATER SUPPLIES

The criteria and procedures described herein, shown in bold, are minimum requirements consid-
ered essential for laboratories seeking certification for microbiological analysis of public water sup-
plies. The requirements include laboratory equipment and supplies, laboratory practices, methodology,
sample collection, and certain quality control measures. The other items, involving personnel, facili-
ties, additional quality control procedures, data reporting, and action response, are optional. For a
commercial laboratory to qualify for certification in microbiology, it must process a minimum of 20
potable water samples per month using either the multiple tube procedure or membrane filter test.
Until National Revised Primary Drinking Water Regulations require certification of water supply
laboratories, all specifications will be considered as guidelines to be used by certification officials.
At that time, minimal requirements will be essential to certification of laboratories involved in anal-
ysis of public water supplies.
The minimum requirements must be in compliance, or action must be taken to correct defi-
ciencies prior to certification. A laboratory that exceeds these minimum requirements is encouraged
to maintain and improve those higher standards for facilities, equipment, methodology, and quality
control, as well as to continue the upgrading of personnel through training efforts to ensure routine
production of reliable data. .
The required methods of analyses are referenced in "Standard Methods for the Examination of
Water and Wastewater," 13th edition; however, some criteria in this document are more specific and
permit fewer variations than "Standard Methods." ·
The guidelines for quality assurance procedures are those in EPA's quality assurance program as
cited in the EPA Manual, "Microbiological Methods for Monitoring the Environment" (EMSL EPA
Cincinnati). A valuable source of further detail and background information for the laboratory eval-
uator is available in EPA's "Handbook for Evaluating Water Bacteriological Laboratories" (EPA-
67 0/9-7 5-006, August 197 6).
Minimum requirements are shown throughout in bold.
PERSONNEL1 (OPTIONAL REQUIREMENTS)

Analyst
The analyst performs microbiological tests with minimal supervision in those specialties for
which he is qualified by education and/or training and experience.
1Exceptions wiH be made for those persons employed by the laboratory and currently doing the required analy-
ses prior to promulgation of the interim regulations provided that within 2 years after June 24, 1977, they receive a
minimum of 2 weeks of additional training in water microbiology.

297
 • Academic training: Minimum of high school diploma in academic or laboratory-oriented
vocational courses.
• Job training: Minimum of 30 days on-the-job training plus one week of supplementary train-
ing acceptable to the Federal and State regulatory agency or agency responsible for prim~cy.
Personnel should take advantage of courses available to Federal and State regulatory
agencies.
• Supervision: Supervision by an experienced professional scientist. In the small water plant
laboratory consisting of a single analyst, the services of a State-approved outside consultant
must be available.

S11pervlsor/Consulta11t

The supervisor directs technical personnel in the proper performance of laboratory procedures
and the reporting of results. If no technical supervisor is available, a consultant should be available.

• Academic training: Minimum of a bachelor's degree in microbiology, biology, chemistry, or

a closely related field. Exceptions will be made for employees of laboratories that serve
communities with populations of 50,000 or less if they receive at least 2 weeks of additional
training in water microbiology from a Federal agency, State agency, or university.
• Job training: Technical training in water microbiology for a minimum of 2 weeks from a
Federal agency, State agency, or university in the parameter to be tested. Consultant must
have 1 year of bench experience, approved by the State, in total coliform analysis. State
laboratory expertise would be the most desirable source of outside consultation.
• Experience: One year of bench experience in sanitary (water, milk, or food) microbiology.

LABORATORY FACILITIES (OPTIONAL REQUIREMENTS)

Laboratory space should be adequate (200 ft 2 and 6 linear ft of bench space per analyst) to
accommodate periods of peak work load. Working space requirements should include sufficient
bench-top area for processing samples; storage space for media, glassware, and portable equipment
items; floor space for stationary equipment (incubators, waterbaths, refrigerators, etc.); and associ-
ated area for cleaning glassware and sterilizing materials. The space required for both laboratory
work and materials preparation in small water plant laboratories may be consolidated into one
room, with the various functions allocated to different parts of the room.
Facilities should be clean, air-conditioned, and with adequate lighting at bench top (100 ft-
candles).
Laboratory safety, which must be an integral and conscious effort in laboratory operations,
should provide safeguards to avoid electric shock, prevent fire, prevent accidental chemical spills,
and minimize microbiological dangers, facility deficiencies, and equipment failures. While safety
is not an aspect of laboratory certification, the evaluation should point out on an informal basis,
potential safety problems observed during an on-site visit.

298 oEPA MICROBIOLOGICAL MANUAL 1978

LABORATORY EQUIPMENT, SUPPLIES, AND MATERIALS (MINIMUM REQUIREMENTS)

The laboratory must have available or access to the items required for the total coliform mem-
brane filter or most probable number procedures as listed below.

• pH Meter: Accuracy must be ±0. l units.

• Balances-top loader or pan: Balance must be clean, not corroded, and be provided witp ap-
propriate weights of good quality. Balance must tare out and detect 50-mg weight accurate-
ly: this sensitivity is required for use in general media preparation of 2g or larger quantities.
• Temperature-monitoring devices:

-Glass or metal thermometers must be graduated in 0.5°C increments.

-Continuous temperature recording devices must be sensitive to within 0.5°C.
-Liquid column of glass thermometers must have no separation.
-A certified thermometer or one 'of equivalent accuracy must be available.

• Air (or water jacketed) incubator/incubator rooms/waterbaths/aluminum block incubators:

-Unit must maintain internal temperature of 35.0° ± 0.5°C in area of use at maximum
loading.
-When aluminum block incubators are used, culture dishes and tubes must be snug-fitting in
block.

• Autoclave:

-Autoclave must be in good operating condition when observed during operational cycle or
when time-temperature charts are read. Vertical autoclaves are not recommended. For
most efficient operation, a double-walled autoclave constructed of stainless steel is sug-
gested (optional).
-Autoclave must have pressure and temperature gauges on exhaust side and an operating
safety valve.
-Autoclave must reach sterilization temperature (121°C) and be maintained during steriliza-
tion cycle: no more than 45 minutes is required for"a complete cycle.
-Depressurization must not produce air bubbles in fermentation media.

• Hot-air oven: Oven must be constructed to ensure a stable sterilization temperature . .Its use
is optional for sterilization of glass pipets, bottles, flasks, culture dishes, etc. (optional).
• Refrigerator: Refrigerator must hold temperature at 1° to 4.4°C (34° to 40°F).
• Optical/counting/lighting equipment: Low power magnification device (preferably binocular
microscope with 10 to 15x) with fluorescent light source must be available for counting MF
colonies. A mechanical hand tally can be used for counting colonies (optional).
• Inoculation equipment:

-Loop diameter must be at least 3 mm and of 22 to 24 gauge Nichrome, chromel, or

299
 platinum-iridium wire. Single-service metal loops, disposable dry heat-sterilized hardwood
applicator sticks, pre-sterilized plastic, or metal loops may be used (optional).

• fttembrane filtration equipment:

-Units must be made of stainless steel, glass, or autoclavable plastic. Equipment must not
leak and must be uncorroded.
-Field equipment is acceptable for coliform detection only when standard laboratory MF
procedures are followed.

• Membrane filters and pads:

-Membrane filters must be manufactured from cellulose ester materials, white, grid-marked,
47-mm diameter, 0.45 µm pore size. Another pore size may be used if the manufacturer
gives performance data equal to or better than the 0.45-µm membrane filter.
-Membranes and pads must be autoclavable or presterilized.

• Laboratory glassware, plastic ware, and metal utensils:

-Except for disposable plastic ware, items must be resistant to effects of corrosion, high
temperature, and vigorous cleaning operations. Metal utensils made of stainless steel are
preferred (optional).
-Flasks, beakers, pipets, dilution bottles, culture dishes, culture tubes, and other glassware
must be of borosilicate glass and free of chips, cracks, or excessive etching. Volumetric
glassware should be Class A, denoting that it meets Federal specifications and need not be
calibrated before use.
-Plastic items must be of clear, inert, nontoxic material arid must retain accurate calibration
marks after repeated autoclaving.

• Culture dishes:

-Sterile tight or loose-lid plastic culture dishes or loose-lid glass culture dishes must be used.
-For loose-lid culture dishes, relative humidity in the incubator must be at least 90 percent.
-Culture dish containers must be aluminum or stainless steel; or dishes may be wrapped in
heavy aluminum foil or char-resistant paper.
-Open packs of disposable sterile culture ~ishes must be resealed between uses.

• Q1lt11re tubes and closures:

-Culture tubes must be made of borosilicate glass or other corrosion resistant glass and
must be of a sufficient size to contain the culture medium, as well as the sample portions
employed, without being more than 3/4 full. It is desirable that the fermentation vial
extend above the medium (optional).
-Caps must be snug-fitting stainless steel or plastic; loose-fitting aluminum caps or screw
caps are also acceptable.

300 &EPA MICROBIOLOGICAL MANUAL 1978

 • Measuring equipment:
-Sterile, glass or plastic pipets must be used for measuring 10 ml or less.
-Pipets must deliver the required volume quickly and accurately within a 2.5 percent toler-
ance .
...:..Pipets must not be badly etched; mouthpiece or delivery tips must not be chipped; gradua-
tion marks must be legible. ·
-Open packs of disposable sterile pipets must be resealed between uses.
-Pipet containers must be aluminum or stainless steel.
-Graduated cylinders must be used for samples larger than 10 ml; calibrated membrane
filter funnel markings are permissible provided accuracy is within a 2.5 percent tolerance.

GENERAL LABORATORY PRACTICES (MINIMUM REQUIREMENTS)

Sterilization Procedures

• The following times and temperatures must be used for autoclaving materials:

Material Temperature/Minimum Time

Membrane filters and pads 121°C/10 min.

Carbohydrate-containing media· 121°C/12-15 min.
(lauryl tryptose, brilliant green
lactose bile broth, etc.)
Contaminated materials and discarded 121°C/30 min.
tests
Membrane filter assemblies (wrapped), 121°C/30 min.
sample collection bottles (empty),
individual glassware items
Rinse water volumes of 500 ml to 1,000 121°C/45 min.
ml
Rinse water in excess of 1,000 ml 121°C/time adjusted for volume; check
for sterility
Dilution water blank 121°C/30 min.

Membrane filter assembles must be sterilized between sample filtration series. A filtration series
ends when 30 minutes or longer elapse between sample filtrations. At least 2 minutes of UV lighfor
boiling water may be used on membrane filter assembly to prevent bacterial carry-over between
filtrations (optional).
Dried glassware must be sterilized at a minimum of 170°C for 2 hours.

Laboratory Pure Water (Distilled, Deionized, or OtJ:ier Processed Waters)

• An analyst must test the quality of the laboratory pure water or have it tested by the State
or by a State-authorized laboratory.

oEPA MICROBIOLOGICAL MANUAL 1978 301

 • Only water determined as laboratory pure water (see quality control section) can be used
for performing bacteriological analyses.
Although processed water may be acceptable for routine chemistry, there is a good chance that
it contains enough of some constituent to be toxic or stimulatory to microorganisms (optional).

Rinse and Dilution Water

Stock buffer solution ~ust be p~epared according to "Standard Methods" using laboratory pure
water adjusted to pH 7.2. Stock buffer must be autoclaved or filter-sterilized, labeled, dated, and
stored at 1° to 4.4°C. The stored buffer solution must be free of turbidity.
Rinse and dilution water must be prepared by adding 1.25 ml of stock buffer solution per liter
of laboratory pure water. Final pH must be 7 .2 ± 0.1.

Media Preparation and Storage

The following are minimum requirements for storing and preparing media:

• Laboratories must use commercial dehydrated media for routine bacteriological procedures
as quality control measures.
• Lnuryl tryptose and brilliant green lactose bile broths must be prepared according to
"Standard .Methods"; lactose broth is not permitted.
• Dehydrated media containers must be kept tightly closed and stored in a cool, dry location.
Discolored or caked dehydrated media cannot be used.
• Laboratory pure water must be used; dissolution of the media must be completed before
dispensing to culture tubes or bottles.
• The membrane fi~ter broth and agar media must be heated in a boiling water bath until com-
pletely dissolved.
• Membrane filter (MF) broths must be stored and refrigerated no longer than 96 hours. MF
agar media must be stored, refrigerated and used within 2 weeks.
• Most probable number (MPN) media prepared in tubes with loose-fitting caps must be used
within 1 week. If l\1PN media are refrigerated after sterilization, they must be incubated over-
night at 3S°C to confirm usability. Tubes showing growth or gas bubbles must be discarded.
• Media in screw cap containers may be held up to 3 months, provided the media are stored in
the dark and evaporation is not excessive (0.5 ml per 10 ml total volume). Commercially
prepared liquid and agar media supplies may be used (optional).
• Ampouled media must be stored at 1° to 4.4°C (34° to 40°F); time must be limited to man-
ufacturer's expiration date.

METHODOLOGY (MINIMUM REQUIREMENTS)

The required procedures, which are mandatory, are described

J
in the 13th edition of "Standard
Methods": standard coliform MPN tests (p. 664-668), single step or enrichment standard total
coliform membrane filter procedure (p. 679-683). Tentative methods are not acceptable. All other
procedures are considered alternative analytical techniques as described in section 141.27

302 oEPA MICROBIOLOGICAL MANUAL 1978

of the National Interim Primary Drinking Water Regulations. Application for the use of alternative
methods may require acceptable comparability data.
The membrane filter procedure is preferred because it permits analysis of large sample volumes
in reduced analysis time. The membranes should show good colony development over the entire
surface. The golden green metallic sheen colonies should be counted and recorded as the coliform
density per 100 ml of water sample. The following rules for reporting any problem with MF results
must be observed:

• Confluent growth: Growth (with or without discrete sheen colonies) covering the entire
filtration area of the membrane. Results are reported as "confluent growth per 100 ml, with
(or without) coliforms," and a new-sample requested.
• TNTC (Too numerous to count): The total number Of bacterial colonies on the membrane is
too numerous (usually greater than 200 total colonies), not sufficiently distinct, or both. An
accurate count can.not be made. Results are reported as "TNTC per 100 ml, with (or with-
out) coliforms," and a new sample requested.
• Confluent growth and TNTC: A new sample must be requested, and the sample volumes
filtered must be adjusted to apply the MF procedure; otherwise the MPN procedure must be
used.
• Confirmed MPN test on problem supplies: If the laboratory has elected to use the MPN test
on water supplies that have a continued history of confluent growth or TNTC with the MF
procedure, all presumptive tubes with heavy growth without gas production should be sub-
mitted to the confirmed MPN test to check for the suppression of coliforms. A count is
adjusted based upon confirmation and a new sample requested. This procedure should be
carried out on one sample from each problem water supply once every 3 months.

SAMPLE COLLECTION, HANDLING, AND PRESERVATION (MINIMUM REQUIREMENTS)

When the laboratory has been delegated responsibility for sample collecting, handling, and pres-
ervation, there must be strict adherence to correct sampling procedures, complete identification of
the sample, and prompt transfer of the sample to the laboratory as described in "Standard Meth-
ods," 13th edition, section 450, p. 657-660.
The sample must be representative of the potable water system. The sampling program must
include examination of the finished water at selected sites that systematically cover the distribution
network.
Minimum sample frequency must be that specified in the National Interim Primary Drinking
Water Regulations, 40 CFR 141.Zl.
The collector must be trained in sampling procedures and approved by the State regulatory
authority or its delegated representative.
The water tap must be sampled after maintaining a steady flow for 2 or 3 minutes to clear serv-
ice line. The tap is free of aerator, strainer, hose attachment, or water purification devices.
The sample volume must be a minimum of 100 ml. The sample bottle must be filled only to the
shoulder to provide space for mixing. ·
The sample report form must be completed immediately after collection with location, date· and
time of collection, chlorine residual, collector's name, and remarks.

303
 Sample bottles must be of at least 120 ml-capacity, sterile plastic or hard glass, wide mouthed
with stopper or plastic screw cap, and capable of withstanding repeated sterilization. Sodium thio-
sulfate ( 100 mg/I) is added to all sample bottles during preparation. As an example, 0.1 ml of a 10
percent solution is required in a 4-oz (120-ml) bottle.
Date and time of sample arrival must be added to the sample report form when sample is re-
ceived in the laboratory.
State regulations relating to chain-of-custody, if required, must be followed in the field and in
the laboratory.
Samples delivered by collectors to the laboratory must be analyzed on the day of collection.
Where it is necessary to send water samples by mail, bus, United Parcel Service, courier service,
or private shipping, holding/transit time between sampling .and analyses must not exceed 30 hours.
When possible, samples are refrigerated during transit and during storage in the laboratory (optional).
If the laboratory is required by State regulation to examine samples.after 30 hours and up to 48
hours, the laboratory must indicate that the data may be invalid because of excessive delay before
sample processing. Samples arriving after 48 hours shall be refused without exception and a new
sample requested. (The problem of holding time is under investigation by EPA.)

QUALITY CONTROL PROGRAM

Minimum Requirement
A written description for current laboratory quality control program must be available for
review. Management, supervisors, ahd analysts participate in setting up the quality control pro-
gram. Each participant should have a copy of the quality control program and a detailed guide
of his own portion. A record on analytical quality control tests and quality control checks on
media, materials, and equipment must be prepared and retained for 3 years.

Analytical Quality Control Tests for General Laboratory Practices and Methodology

Minimum and optional requirements for analytical quality control tests for general practices
and methodology are:
• Minimum requirements:
-At least five sheen or borderline sheen colonies must be verified from each membrane con-
taining five or more such colonies. Counts must be adjusted based on verification. The
verification procedure must be conducted by transferring growth from colonies into lauryl
tryptose broth (LTB) tubes and then transferring growth from gas-positive LTB cultures to
brilliant green lactose bile (BGLB) tubes. Colonies must not be transferred exclusively to
BGLB because of the lower recovery of stressed coliforms in this more selective medium.
However, colonies may be transferred to LTB and BGLB simultaneously. Negative LTB
tubes must be reincubated a second day and confirmed if gas is produced. It is desirable to
verify all sheen and borderline sheen colonies (optional).
-A start and finish 1\1F control test (rinse water, medium, and supplies) must be conducted
for each filtration series. If sterile controls indicate contamination, all data on samples
affected must be rejected and a request made for immediate resampling of those waters
involved in the laboratory error.

304 &EPA MICROBIOLOGICAL MANUAL 1978

 -The MPN test must be carried to completion, except for gram staining, on 10 percent of
positive confirmed samples. If no positive tubes result from potable water samples, the
completed test except for gram staining must be performed quarterly on at least one
positive source water.
-Laboratory pure water must be analyzed annually by the test for bactericidal properties
for distilled water ("Standard Methods," 13th edition, p. 646). Only satisfactorily tested
water is permissible in preparing media, reagents, rinse, and dilution water. If the tests do
not meet the requirements, corrective action must be taken and the water retested.
-Laboratory pure water must be analyzed monthly for conductance, pH, chlorine residual,
and standard plate count. If tests exceed requirements, corrective action must be taken
and the water retested.
-Laboratory pure water must not be in contact with heavy metals. It must be analyzed
initially and annually thereafter for trace metals (especially Pb, Cd, Cr, Cu, Ni, and Zn). If
tests do not meet the requirements, corrective action must be taken and the water re-
tested.
-Standard plate count procedure must be performed as described in "Standard Methods,"
13th edition, p. 660-662. Plates must be incubated at 35° ±"0.5°C for 48 hours.
-Requirements for laboratory pure water:

pH 5.5-1.5
Conductivity Greater than 0.2 megohm as resistivity or
less than 5.0 micromhos/cm at 25°C
Trace metals:
A single metal Not greater than 0.05 mg/I
Total metals Equal to or less than 1.0 mg/I
Test for bactericidal properties of dis-
tilled water ("Standard Methods;"
13th edition, p. 646) 0.8 - 3.0
Free chlorine residual 0.0
Standard plate count Less than 10,000/ ml

-Laboratory must analyze one quality control sample per year (when available) for param-
eter(s) measured.
-Laboratory must satisfactorily analyze one unknown performance sample per year (when
available) for parameter(s) measured.

• Optional requirements:

-Duplicate analyses should be run on known positive samples at a minimum frequency of

one per month. The duplicates may be run as a split sample by more than one analyst,
with each split being a 50-ml sample.
-Water plant laboratories should examine a minimum of one polluted water source per
month in addition to the required number of distribution samples.
-If there is more than one analyst in laboratory, at least once per month each analyst

305
 should count the sheen colonies on a membrane from a polluted water source. Colonies on
the membrane should be verified and the analysts' counts compared to the verified count.
-A minimum number of the official water supply samples required for each system should
be analyzed by the State laboratory. For example, systems that are required to have less
than 10 samples examined per month should submit one additional sample to a State
authorized laboratory. Water systems with sample requirements above 10 per month
would submit two additional samples to a State authorized laboratory.

Quality Control Checks of Laboratory Media, Equipment, and Supplies

Minimum and optional requirements for quality control checks of laboratory media, equipment,
and supplies are:

• Minimum requirements:

-pH meter must be clean and calibrated each use period with pH 7.0 standard buffer. Buf-
fer aliquot must be used only once. Commercial buffer solutions must ~e dated on initial
use.
-Balances (top loader or pan) must be calibrated annually.
-Glass thermometers or continuous recording devices for incubators must be checked year-
ly and metal thermometers quarterly (or at more frequent intervals when necessary)
against a certified thermometer or one of equivalent accuracy.
-Temperature in air (or water jacketed) incubator/incubator room/waterbaths/aluminum
block incubators must be recorded continuously or recorded daily from in-place thermom-
eter(s) immersed in liquid and placed on shelves in use.
-Date, time, and temperature must be recorded continuously or recorded for each steriliza-
tion cycle of the autoclave.
-HQ.t air oven must be equipped with a thermometer calibrated in the range of 170°C or
with a temperature recording device. Records must be maintained showing date, time, and
temperature of each sterilization cycle. It is desirable to place the thermometer bulb in
sand and to avoid overcrowding (optional).
-Membrane filters used must be those recommended by the manufacturer for water analy-
sis. The recommendation must be based on data relating to ink toxicity, recovery, reten-
tion, and absence of growth-promoting substances.
-Washing processes must provide clean glassware with no stains or spotting. With initial use
of a detergent or washing product and whenever a different washing product is used, the
rinsing process must demonstrate that it provides glassware free of toxic material by the
inhibitory residue test ("Standard Methods," 13th edition, p. 643).
-At least one bottle per batch of sterilized sample bottles must be checked by adding ap·
proximately 25 ml of sterile LTB broth to each bottle. It must be incubated at 35 ± 0.5°C
for 24 hours and checked for growth.
-Service contracts or approved internal protocols must be maintained on balances, auto-
clave, water still, etc., and the service records entered in a log book.

306 oEPA MICROBIOLOGICAL MANUAL 1978

 -Records must be available for inspection on batches of sterilized media showing lot num-
bers, date, sterilization time-temperature, final pH, and technician's name.

• Optional requirements:

-Positive and negative cultures should be used, and testing should be carried out to deter-
mine recovery and performance compa~ed to a previous acceptable lot of medium.
-Media should be ordered on a basis of 12-month needs. Bottles should be dated on receJpt
and when opened initially. Except for large volume uses, media should be purchased in 1I4-
lb bottles. Bottles of media should be used within 6 months after opening; however; in no
case should opened media be used after one year. Opened bottles should be stored in a des-
iccator to extend storage time beyond 6 months. Shelf life of unopened bottles is 2 years.
-Testing should be carried out in media and membranes to determirie recovery .an.d per-
formance compared to previous acceptable lot.
-Lot number of membrane filters and date of receipt should be recorded.
-Heat sensitive tapes and spore strips or ampoules should be used during sterilization. Maxi-
mum i:egistering thermometer is recommended.

DATA REPORTING (MINIMUM REQUIREMENTS)

Where the laboratory has the responsibility for sample collections, the sample collector should
complete a sample report form immediately after each sample is taken. The information on the
form includes sample identification number, sample collector's name, time and date of collection,
arrival time and date in the laboratory, direct count, MF verified count, MPN completed count,
analyst's name, and other special information.
Results should be calculated and entered on the sample report form to be forwarded. A careful
check should be made to verify that each result was entered correctly from the bench sheet and
initialed by the analyst.
All results should be reported immediately to the proper authority.
Positive results are reported as preliminary without waiting for MF verification orMPN comple-
tion. After MF verification and/or MPN completion, the adjusted counts should be reported.
A copy of the sample report form should be retained either by the laboratory or State program
for 3 years. If results are entered into a computer storage system, a printout of the data should be
returned to the laboratory for verification with bench sheets.

ACTION RESPONSE TO LABORATORY RESULTS (MINIMUM REQUIREMENT).

When action response is a designated laboratory responsibility, the proper authorities should be
promptly notified on unsatisfactory sample results, and a request should be made for resampling
from the same sampling point.

307
SAMPLE FORMS FOR ON-SITE EVALUATION OF LABORATORIES INVOLVED IN
ANALYSIS OF PUBLIC WATER SUPPLIES-MICROBIOLOGY

SURVEY BY:

AFFILIATION:-----------------------------

CODES FOR MARKING ON-SITE EVALUATION FORMS:

S-Satlsfactory
U- Unsatisfactory
NA-Not Applicable

308 &EPA MICROBIOLOGICAL MANUAL 1978

Laboratory _ _ _ _ _ _ _ _ _ _ _ _ _ __ Evaluator _ _ _ _ _ _ _ _ _ _ _ _ __

Location _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Date _ _ _ _ _ _ _ _ _ _ _ _ _ _ ~

PERSONNEL

The form dealing with personnel can be found on the following page.

LABORATORY FACILITIES

Space in laboratory and preparation room is adequate for needs during peak work periods
(200 ft 2 and 6 linear ft of usable bench space per analyst).
Facilities are clean, with adequate lighting (100 ft-candles) and air conditioning.

NOTE: Material on pages 53-65, except where indicated, are minimum requirements.

309
....w PERSONNEL
0 Laboratory _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

Location _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

Dat•t=-~~~~~-

Evaluator - - - - - ----

Academic training
Present Experience
Position/title Name
~:> specialty (years/area)
HS BA/BS MA/MS Ph.D.

J Laboratory director

~
0
~
0
DJ Supervisor/consultant
0,...
0
Ii)

,...~
~
~~ ~!P>!L~ W© ~~ ~ ~ILYr
:z:
c::
,...
); Professionals
...
co
(note discipline)
Ci!

Technician/analyst
Laboratory - - - - - - - - - - - - - - - - - - - - Evaluator - - - - - - - - -

·Location - - - - - - - - - - - - - - - - - - - - D a t e - - - - - - - - - - -

LABORATORY EQUIPMENT, SUPPLIES, AND MATERIALS

1. pH Meter
M a n u f a c t u r e r - - - - - - - - - - - - - - - - - - Model

Clean, calibrated to 0.1 pH units each use period; record maintained ............ .
Aliquot of standard pH 7 .0 buffer used only once .......................... .

2. Balance-Top Loader or Pan

Manufacturer------------------ Model

Clean. Detects a ?O-mg_ weight accurately (for a general media pr~paration of.>"2:g
quantities) ..........................................•..............
Good quality weights in clean condition ................................. .

- 3. Temperature-Monitoring Devices··
Accuracy checked annually against a certified thermometer or one of equivalent
·accuracy ....... : ....... ·.............. ; ; ......................... .
Legible graduations in 0.5°C-increments ................................. .
No separation.fa liquid.column ........................................ .

4. Incubator or Incubator Room

Manufacturer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Model _ _ _ _ _ _ _ __

Sufficient size for daily work load, ...................... ·................ .

Uniform temperature maintained on shelves in all areas used (35.0° ± 0.5°C) .. , .. .
Calibrated thermometer with bulb immersed in liquid and located on shelves in use ..
Temperature recorded daily or recording thermometer sensitive to± 0.5°C ....... .

5. Autoclave . -. •... • ~

Manufacturer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ M o d e l - - - - - - - - -

Reaches sterilization temperature (121°C), maintains 121°C during sterilization

cycle, and requires no more than 45 min for a complete cycle ................ .
Pressure and temperature gauges on exhaust side and an operating safety valve .... .
No air bubbles producedin fermentation vials during depressurization ........... .
Record maintained on time and temperature for each sterilization cycle ......... .

311
 6. Hot-Air Oven
M a n u f a c t u r e r - - - - - - - - - - - - - - - - - - Model - - - - - - - - -

Operates at a minimum of l 70°C ................. : ..................... .

Thermometer inserted or oven equipped with temperature-recording thermometer
device ........................................................... .
Time and temperature record maintained for each sterilization cycle ............ .
Thermometer bulb in sand (optional)

7. Refrigerator
Temperature maintained at 1° to 4.4.°C (34° to 40°F) ....................... .

8. Optical Equipment
Low power magnification device (preferably binocular microscope with 10 to I SX)
with fluorescent light source for counting MF colonies ..................... .
Colonies counted with a mechanical hand tally (optional}

9. /11oculation Equipment
Sterilized loops of at least 3-mm diameter, 22 to 24 gauge Nichrome, Chrome!, or
platinum-iridium wire ............................. ·.................. .
or
Disposable dry heat-sterilized hardwood applicator sticks or presterilized loops .....

I 0. Membrane Filtration Equipment

M a n u f a c t u r e r - - - - - - - - - - - - - - - - - - Model - - - - - - - - -

Made of stainless s'teel, glass, or autoclavable plastic ......................... .

Nonleaking and uncorroded ........................................... .

11. Membrane Filters and Pads

Manufacturer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ M o d e l - - - - - - - - -

Filters recommended by manufacturer for water analyses ..................... .

Filters and pads presterilized or autoclavable .............................. .

12. Glass, Plastic, and Metal Utensils for Media Preparation

Washing process provides glassware free of toxic residue as demonstrated by the
inhibitory residue test .............................................. .
Glass items of borosilicate, free of chips and cracks ................. : ....... .
Utensils clean and free from foreign residues or dried medium ................ .
Plastic items clear with visible graduations ................................ .

312 &EPA MICROBIOLOGICAL MANUAL 1978

13. Sample Bottles
Wide-mouth hard glas·s bottles; stoppered or plastic screw-capped; capaCity at least
120 ml .......................................................... .
Glass-stoppered bottles with tops covered with aluminum foil or kraft
paper ........................................................... .
Screw-caps have leakproof nontoxic liners that can withstand repeated sterilization
(30 min at 121°C) ................................................. .
Sterile sample bottles contain 10 mg of dechlorinating agent per 100 ml of sample ..

14. Pipets
Brand~~~~~~~~~~~~~~~~~~~~~Type~~~~~~~~~~

Sterile; glass or plastic; with a 2.5 percent tolerance ......................... .

Tips unbroken; graduations distinctly marked .............................. .

15. Pipet Containers

Aluminum, stainless steel ............................................. .
Pipets wrapped in quality kraft paper (char-resistant) ....................... .
_Open packs of disposable sterile pipets resealed between uses .................. .

16. Culture Dishes

Sterile plastic or glass ................................................ .

Open packs of disposable sterile plastic dishes resealed between uses ............ .
Dishes are in containers of aluminum or stainless-steel with covers or
are wrapped with heavy aluminum foil or char-resistant paper ................ .

17. Culture Tubes and Closures

Sufficient size to contain sterile medium and sample without danger of spillage .... .
Metal or plastic caps; plastic plugs ........................................ .
Borosilicate glass or other corrosion-resistant glass .......................... .

313
Laboratory _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Evaluator---------
Location _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Date _ _ _ _ _ _ _ _ _ __

GENERAL LABORATORY PRACTICES

1. Sterilization Procedures
Satisfactory sterilization procedures and/or records ......................... .
Tube.broth media and reagents sterilized at 121°C 12 to 15 min ............... .
Tubes and flasks packed loosely in baskets or racks for uniform heating and cooling.
MF presterilized or autoclaved at 121°C for I 0 min with fast exhaust ........... .
MF assemblies and empty sample bottles sterilized at 121°C for 30 min .......... .
MF assemblies sterilized between sample filtration series ..................... .
Rinse water volumes of 500 to 1,000 ml sterilized at 121 ° C
for 45 min ............................................ , .......... .
Dilution water blanks autoclaved at 121°C for 30 min ....................... .
Wire loops, needles, and forceps sterilized ................................. .
Total exposure of MPN media to heat not over 45 min ....................... .
Timing for sterilization begins when autoclave reaches 121° C.................. .
Individual glassware items autoclaved at 121°C for 30 min .................... .
Individual dry glassware items sterilized 2 hours at I 70°C (dry heat) ............ .
Pipets, culture dishes, and inoculating lo.ops in boxes sterilized at l 70°C for 2 hours.
MPN media removed and cooled as soon as possible after sterilization and stored in
cool dark place (optional)
UV light or boiling water for at least 2 min may be used on membrane filter assem-
blies to reduce bacterial carry-over between each filtration (optional)

2. Laboratory Pure Water

Only laboratory pure water used in preparing media, reagents, rinse water, and dilu-
tion \Yater........................................................ .
Laboratory pure water not in contact with heavy metals ..................... .

Source: Laboratory-prepared _ _ _ _ _ _ _ _ Purchased _ _ _ _ _ _ _ __

If laboratory-prepared:
Still m a n u f a c t u r e r - - - - - - - - - - - - - - - - - - - - - - -
Deionizer m a n u f a c t u r e r - - - - - - - - - - - - - - - - - - - - -
Record of recharge frequency _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

Production rate and quality adequate for laboratory needs .................... .

Inspected, repaired, cleaned by service contract or in-house service ............. .

a. Chemical quality control

Record of satisfactory annual analyses for trace metals
A single metal not greater than 0.05 mg/I .......................... .

314 .SEPA MICROBIOLOGICAL MANUAL 1978

 Total metals: equal to or less than 1.0 mg/l. ........................ .

Record of monthly analyses of laboratory pure water

Conductance: >0.2 megohm resistivity or <5.0 microhmos/cm .......... .,
pH: 5.5-7.5 ................................................. .
Standard plate count: <10,000/ml. ............................... .
Free chlorine residual: 0.0 ........................ : ............. .

b. Microbiological quality control

Test for bactericidal properties of distilled water (0.8-3.0) performed at least

annually ...................................................... .
Testing laboratory _ _ _ _ _ _ Date _ _ _ _ _ _ R a t i o - - - - - - -

3. Rinse and Dilution Water

Stock buffer solution prepared according to "Standard Methods," 13th edition .... .
Stock buffer solution adjustyd to pH 7 .2 ................................. .
Stock buffer autoclaved at 121°C, stored at 1° to 4.4°C (34° to 40°F) or filter
sterilized ......................................................... .
Stock buffer labeled and dated ......................................... .
Stock potassium phosphate buffer solution (1.25 ml) added per liter distilled water
for rinse and dilution water .......................................... .
Final pH 7.2 ± 0.1 ................................................... .

4. Media

Dehydrated media bottle kept tightly closed and protected from dust and excessive
humidity in storage areas ............................................ .
Dehydrated media not used if discolored or caked .......................... .
Laboratory pure water used in media preparation ........................... .
Dissolution Of media complete before dispensing to culture tubes or bottles ...... .
MPN tube media with loose-fitting caps used in less than 1 week ............... .
Tube media in screw-capped tubes held no longer than 3 months ............... .
Ampouled media stored at 1° to 4.4°C and time limited to manufacturer's expira-
tion date ...................................... ·............. ~ ..... .
Media stored at low temperatures are incubated overnight prior to use and
tubes with air bubbles discarded ...................................... .
Media protected from sunlight ......................................... .
MF media stored in refrigerator; broth medium used within 96 hours, agar within
two weeks if prepared in tight-fitting dishes .............................. .

';315
 5. 1A11ryl Tryptose Broth

Manufacturer-------------- Lot No. _ _ _ _ _ _ __

Single strength composition, 35.6 g per liter pure water ...................... .

Single strength pH 6.8 ± 0.2; double strength pH 6. 7 ± 0.2 .................... .
Not less than 10 ml per tube ............................................ · -
Media made to result in single strength after addition of sample portions ......... .

6. Brilliant Green lActose Bile Broth

Manufacturer~------------- Lot No.~-------

Medium composition 40 g per liter pure water ............................. .

Final pH 7.2 ± 0.2 .................................................. .

7. M-Endo Media

Manufacturer------------- Lot No.~-------

Medium composition 48.0 g per liter pure water optionally 15 g agar added/I ..... .
Reconstituted in laboratory pure water containing 2 percent ethanol (not
denatured) ....................................................... .
Final pH 7.2 ± 0.2 .................................................. .
Medium held in boiling water bath until completely dissolved ................. .

8. Standard Plate Count Agar

Correct composition, sterile and pH 7 .0 ± 0.2 ............................. .

Sterile medium not remelted a second time after sterilization .................. .
Culture dishes incubated 48 hours at 35° ± 0.5°C ........................... .
No more than 1.0 ml or less than 0.1 ml sample plated (sample or dilution) ....... .
Liquefied agar, 10 ml or more; medium temperature between 44° to 46°C ......... .
Melted medium stored no longer than 3 hours before use ..................... .
Only plates with between 30 to 300 colonies counted; when 1 ml of undiluted
sample is plated, colony density may be less than 30 ....................... .
Only two significant figures recorded and calculated as standard plate count/
1.0 ml I I I I I I I I I I I I I I I I I I I I I I I a I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

316 oEPA MICROBIOLOGICAL MANUAL 1978

Laboratory _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ___,._ _ Evaluator _ _ _ _ _ _ _ _ __

Location - - - - - - - - - - - - - - - - - - - . . . . . . - - D a t e - - - - - - - - - - -

METHODOLOGY
Methodology specified in "Standard Methods," 13th eclition, or EPA manual ........ .
M-Endo broth, M-Endo agar, or Les Endo agar used in a single step procedure ........ .
In two-step Les M-Endo procedure, MF incubated on lauryl-tryptose-broth-saturated
absorbent pad for 1.5 to 2 hours at 3.5° ± 0.5°C; then on M-Endo broth or Les
Endo agar for 20 to 22 hours at 35° ± 0.5°C ................................ .

1. Total Coliform Membrane Filter Procedure

Samples containing excessive bacterial populations (greater than 200), confluency,
or turbidity retested by the MPN procedure .............................. .
Filtration assembly sterile at start of each series .......... : ................. .
Absorbent pads saturated with medium, excess discarded; or 4.0 ml of agar medium
can be used per culture dish instead of a pad ............................. .
Sample shaken vigorously immediately before test .......................... .
.Test sample portions measured and not less than 100 ml ..................... .
Funnel rinsed at least twice with 20- to 30-ml portiqns of sterile buffered water ... .
MF removed with sterile forceps grasping area outside effective filtering area ...... .
MF rolled onto medium pad or agar so air bubples are not trapped .............. .

2. Incubation of Membrane Filter Cultures

Total incubation time 22 to 24 hours at 35° ± O.~cic ........................ .
Incubated in tight-fitting culture dishes or loose-fitting dishes incubated in high
relative humidity chambers .......................................... .

3. Membrane Filter Colony Counting

Samples repeated when coliforms are "TNTC" or colony growth is confluent, possi-
bly obscuring coliform development and/or detection ...................... .
Total coliform count calculated in density per 100 ml ....................... .
Samples containing five or more coliforms per I 00 ml are resampled and tested ... .
Low power magnification device with fluorescent light positioned for maximum
sheen visibility .................................................... .

4. Verification of Total Coliform Colonies

All typical coliform (sheen) colonies or at least five randomly selected sheen colo-
nies verified in lauryl tryptose broth and BGLB. . . . . . . . . . . . . . . . . . . . . . . . . . . . _ _,
Counts adjusted based on verification .................................... .
All atypical coliform (borderline sheen) colonies or at least five randomly-selected
colonies verified in LTB and BGLB .................................... .
Counts adjusted based on verification ......... , .•.........................

317
 5. MF Field Equipment
Manufacturer _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ M o d e l - - - - - - - - -

Only standard laboratory MF procedures adapted to field application ........... .

6. Total Coliform Most Probable Number Procedure
a. Presumptive Test
Five standard portions, either 1a or 1aa ml ............................ .
Sample shaken vigorously immediately before test ....................... .
Tubes incubated at 3S 0 ± a.S°C for 24 ± 2 hours ........................ .
Examined for gas (any gas bubble indicates positive test) .................. .
Tubes that are gas-positive within 24 hours submitted promptly to confirm test .
Negative tubes returned to incubator and examined for gas within 48 ± 3 hours;
positives submitted to confirm test ................................. .
Public water supply samples with heavy growth and no gas production con-
firmed for presence of suppressed coliforms ........................... .
Adjusted count reported based upon confirmation ....................... .
Adequate test labeling and tube dilution coding (optional)

b. Confrrmed Test
Presumptive positive tube gently shaken or mixed by rotating .............. .
One loopful or one dip of applicator transferred from presumptive tube to
BGLB ........................................................ .
Incubated at 3S°C ± a.S 0 ; checked within 24 hours ± 2 hours for gas production.
Positive confrnned tube results recorded; negative tubes reincubated and read
within 48 ± 3 hours ............................................. .
Unsatisfactory sample defined as three or more positive confirmed tubes ...... .

c. Completed Test
Applied to 1a percent of all positive samples each quarter ................. .
Applied to all positive confirmed tubes in each test completed .............. .
Positive confrnned tubes streaked on EMB plates for colony isolation ........ .
Plates adequately streaked to obtain discrete colonies ..................... .
Incubated at 3S 0 ± a.s 0 c for 24 ± 2 hours ............................. .
Typical nucleated colonies, with or without sheen, on EMB plates selected for
completed test identification ...................................... .
If typical colonies absent, atypical colonies selected for completed test
identification .................................................. .
If no colonies or only colorless colonies appear, confirmed test for that particu-
lar tube considered negative ....................................... .
An isolated typical colony or two atypical colonies transferred to lauryl tryptose
broth ......................................................... .
Incubated at 3S 0 ± a.S°C; checked for gas within 48 ± 3 hours .............. .
Cultures producing gas in lauryl tryptose broth within 48 ± 3 hours are consid-
ered coliforms ................................................. .

318 SEPA MICROBIOLOGICAL MANUAL 1978

SAMPLE COLLECTION, HANDLING, AND PRESERVATION
Representative samples of potable water distribution system ......... ~ ............ .
Minimal sampling frequency as specified in the Natiohal Interim Primary Drinking Water
Regulations .................................... ·.................. ; .. .
Sample collector trained and approved as required by State regulatory authority or its
. delegated representative ............................ ·.................... .

1. Sample Bottles
Sterile sample bottles of at least 120 ml; able to withstand repeated sterilization ... .
Ample air space remains after sample collected to allow for adequate mixing ...... .
Sodium thiosulfate, I 00 mg/I, added to sample bottle before sterilization ..... : .. .

2. Sampling
Sample collected after maintaining a steady flow for 2 to 3 min to clear service
line ............................ , ........... '· .............. , ...... .
Tap free of aerator, strainer, hose attachment, water purification, or other devices ..
Samples refrigerated when possible during transit and storage periods in the labora-
tory (optional)

3. Sample Identification
Sample identified immediately after collection ............................. .
Identification includes, water source, location, time and date of collection, and col-
lector's name; insufficiently identified samples discarded .................... .
Chlorine residual where applicable

4. Sample Transit Time

Transit time for potable water samples sent by mail or commercial transportation,
not in excess of 30 hours .......................... : .................. .
No sample processed after 48-hour transit/storage .......................... .
Samples delivered to laboratory by collectors examined the day of collection ..... .
Data marked as questionable on samples analyzed after 30 hours ............... .

5. Sample Receipt in Laboratory

Sample logged in when received in laboratory, including date and time of arrival and
analysis ......... ·............................................ : .... .
Chain-of-custody procedures required by State regulations followed ... ; ........ .

319
Laboratory_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Evaluator _ _ _ _ _ _ _ _ __

Location _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Date _ _ _ _ _ _ _ _ _ __

QUALITY CONTROL
A written laboratory quality control program is available for review ................ .

I. Analytical Quality Control

A record containing results of analytical control tests available for review ........ .

a. Verification of MF Colonies
At least five coliforms verified from each positive sample .................. .
Sheen colonies in mixed confluent growth reported and verified (optional)

b. Negative Coliform Controls

A start and finish MF control test (rinse water, medium, and supplies) run with
each filtration series ....................................... ·...... .
When controls indicate contamination occurred, afi data on affected samples
rejected and resampling requested .................................. .

c. Total Coliform Confirmed Test

Presumptive tubes with heavy growth but no gas production submitted to con-
firmed test to check for suppression of coliforms. Confirmation procedure
carried out every 3 months on one sample from each problem water supply ...

d. Duplicate analyses (optional)

Duplicate analyses run on positive p~lluted samples not to exceed 10 percent
but a minimum of one per month (optional) ......................... .

e. Positive Control Samples (optional)

One positive control sample (polluted water) run each month (optional)

f. Colony Counting (If More Than One Analyst in Laboratory) (optional)

Two or more analysts count sheen colonies; all colonies are verified; analysts'
counts compared to verified counts; procedure is carried out at least once per
month (optional)

g. Check Analyses by State Laboratories (optional)

A minimum of samples, proportional to the local laboratory work load, proc-
essed by State laboratory (see criteria for recommendations) (optional)

320 &EPA MICROBIOLOGICAL MANUAL 1978

2. Quality Control of Equipment, Supplies and Media
a. Records
Satisfactory records containing complete quality control checks on equipment,
supplies, and media available for inspection ........................... .

b. Equipment and Supplies

Service contracts or approved internal protocol maintained on balance, auto-
clave, water still, etc.; service records entered in a log book ............... .
Glass thermometers calibrated annually against a certified thermometer; metal
thermometers checked quarterly ................................... .
pH Meters standardized with pH 7 .0 buffer. ............... ; ............ .
Laboratory pure water analyzed as described in criteria ................... .
Lot numbers and dates of receipt of membrane filters recorded (optional)
Heat-sensitive tapes and/or spare strips/ampoules used during sterilization (optional)

c. Media Quality Control

Laboratory chemicals of Analytical Reagent Grade ...................... .
Dyes certified for bacteriological use .................................. .
pH checked and recorded on each batch of medium after preparation and
sterilization ........................................ ·........... .
Causes for deviations beyond ± 0.2 pH units specified .................... .
Media ordered on a basis of 12-month need; purchased in 1/4-lb. quantities,
except those used in large amounts (optional)
Bottles dated on receipt and when opened (optional)
Opened bottles of routinely used media discarded within 6 months (if stored in
desiccator storage may be extended) (qptional)
Shelf life of unopened bottles not in excess of 2 years (optional)
New lots of media quality tested against satisfactory lot using natural water
samples (optional) · .

321
Laboratory._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Evaluator _ _ _ _ _ _ _ __

L o c a t i o n - - - - - - - - - - - - - - - - - - - - Date _ _ _ _ _ _ _ _ _ __

DATA REPORTING
Sample information and laboratory data fully recorded .................... ·..... .
Direct MF counts and/or confirmed MPN results reported promptly· ... ~ ........... .
After MF verification and/or MPN completion, adjusted counts reported ............ .
One copy of report form retained in laboratory or by State program for 3 years ...... .
Test results assembled and available for inspection (optional) ·

322 &EPA MICROBIOLOGICAL MANUAL 1978

Laboratory_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Evaluator _ _ _ _ _ _ _ __

L o c a t i o n - - - - - - - - - - - - - - - - - - - - ' - - D a t e _ _ _ _ _ _ _ _ _ __

ACTION RESPONSE TO LABORATORY RESULTS

Unsatisfactory test results given action response. and resampled as defined in National
Interim Primary Drinking Water Regulations ..................... ·............ .
·state and responsible local authority notified within 48 hours after check.samples c;_9,n-
firm coliform occurrence ....... , ·... ·... .' ............................·... · . ." .. .
All data reported to State and local authorities within 40 days ...... '. ............. .

323 ·:
 APPEN'b1·x C

BIBLIOGRAPHY

1. Kittrell, R. W., 1969. A Practical Guide to Water Quality Standards of Streams, Publ. No.
CWR-5. U.S. Department of the Interior, FWPCA, U.S. Government Printing Office,
Washington, DC.

2. American Public Health Association, 1975. Standard Methods for the Examination of
Water and Wastewater, (14th ed.) Washington, DC. - - -

3. Society of American Bacteriologists, 1957. Manual of Microbiological Methods,

McGraw-Hill Book Company, Inc., New York, NY.

4. Geldreich, E. E., 1975. Handbook for Evaluating Water Bacteriological Laboratories,

(2nd ed.) U.S. Environmental Protection Agency, Cincinnati, OH. EPA-670/9-75-006.

5. Scarpino, P. V., 1971. Bacterial and viral analysis of water and wastewater. Chapter 12.
In: Water and Water Pollution Handbook. Volume 2. (L. L. Ciaccio, ed.) Marcel Dekker,
Inc., New York, NY. 2:639.

6. Mitchell, Ralph (ed.), 1972. Water Pollution Microbiology. John Wiley and Sons, Inc.,
New York, NY. --

7. Current Practices in Water Microbiology, 1973. U.S. Environmental Protec.tion Agency,

Office of Water Program Operations, National Training and Operational Technology
Center, Cincinnati, OH.

8. Greeson, P.E., T. A. Ehlke, G. A. Irwin, B. W. Lium, and K. V. Slack, 1977. Techniques

of Water Resources Investigations of the United States Geological Survey. Chapter A4,
Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples.
Book 5, Laboratory Analysis. U.S. Dept., of the Interior, Superintendent of Documents,
U.S. Government Printing Office, Washington, D.C. 20402.

9. Bordner, R.H., C. F. Frith and J. A. Winter, Eds., 1977. Proceedings of the Symposium
on Recovery of Indicator Organisms Employing Membrane Filters. U.S. Environmental
Protection Agency, EPA-60019-77-024, EMSL-Cincinnati, Cincinnati, OH 45268.

324 &EPA MICROBIOLOGICAL MANUAL 1978

 INDt:X

inhibitory effects, 206

Absorbent pads , 7 4 , 205 recovery, 206

Accidents, reporting , 262 Asymmetry of data, 227

Accuracy, method , 240 Autoclave, steam

quality control, 216
Actinomycetes, analytical method for, 186 specifications, 36, 37, 38
temperature control, 216
Agar, nutrient, 39
Automotive safety, 263
Agglutination test
for H antigen, 179 Azide dextrose broth, 46
for 0 grouping, 177
for 0 and Vi, alternate procedure, 179
for Vi antigen, 178 Balances
quality control , 212
Air density plates in quality control, 1 as specifications, 33

Alcohol Bacteria
for sterilization procedures, 71 , 7 4 see total coliforms, 108
in MF media, 43-45 fecal coliforms, 124
fecal streptococci, 135
Alternate test procedures, 92 Salmonella, 154
· standard plate count, 101
Analytical cost, 246
Bathing beaches, sampling, 29
Analytical quality control
comparative testing of methodologies, 236 BHI tests
in compliance monitoring , 23~ 10 C and 45 C, 146
in routine analyses, 231 6.5% NaCl and pH 9.6, 14 7
with 0.4 % potassium tellurite, 14 7
Antisera , for serological testinQ of
Salmonella, 177 Bench forms
for membran~ filter analyses, 60
Applicator sticks , 3 2 , 7 4 , 80 for MPN analyses", 60 .

Arabinose (L) fermentation, 41 _1 149 Biochemical characterization of the

Eilterobacteriaceae, 17 6
Arabinose, (L), Solution in purple broth
base, 41 Biochemical identification of Salmonella, 167
Arginine decarboxylase test, 1 Z2, 171
Biochemical tests
ASTM tests for membrane filters coliforms, 119
bacterial retention, 206 fecal streptococci, 146
extractables, 207 multitest systems, 172
flow rate , 207 Salmonella, 167

325
 Blohazard, control of, 268 Check list, safety, 269

Bismuth sulfite agar, 53 Chelating agent, 6

Blending, high solids samples, 62 Chemicals and gases, safe use of, 267

Blood agar base, with 10% blood, 50 Chlorinated effluents, 6, 96

,.
Blood agar with 0.4 % potassium tellurite, 50 Chlorination of wastes, 6, 96

Boat safety, 263 Chlorine, damage to cells, 6, 96

Bottles, sample, 36 Citrobabter, 108

Brain heart infusion Cleaning glassware, 36

agar, 47
agar with potassium tellurite, 49 Coliforms, differentiation of, 119
broth, 47
broth pH 9.6, 48 Coliforms, fecal
broth with 40% bile, 48 definition , 124
broth with 6.5% NaCl, 48 MF test, delayed incubation, 128
MF test, direct, 124
MF test, verification, 130
Brilliant green agar, 52 MPN test, 132

Brilliant green lactose bile broth, 45 Coliform test limitations

fecal coliforms, MF, 124
Brom thymol blue, pH check on glassware, total coliforms, MF, 108
199 total coliforms, MPN, 114

Buffered glucose medium, see MR-VP Broth, Coliforms, total

42 differentiation of, 119
MF test, immediate two-step, 111
Buffered water MF test, single step, 110
peptone, 57 MF test, delayed, 112
phosphate, 57 MF verification, 113
MPN test, 114

Calculating results Colony

MF analyses, 75 counting, pour plate, 69
MPN analyses, 81 counting, spread plate, 69
spread plates, 69 counting, membrane filter, 75
counting by more than one analyst, 231
Catalase test, 147 spreader colonies, 106

Celito (diatomaceous earth), 160 Colony counters , 33

Centrifuge, quality control of, 213 Conjugate

approved list, 182
Certification program, Appendix B, 297 fluorescent antibody, 182
titration of, 182
Citrate test, 122
Comparative testing of methodologies, 234
Chain of custody, 17
Completed MPN, 81
See also Total Coliform, 118
Characterization of Enterobacteriaceae, 17 6 Analytical Quality Control, 231

326 &EPA MICROBIOLOGICAL MANUAL 1978

Composite sampling, 6 Differentiation of coliforms, 119

Concentration techniques, for Salmonella, 155 Differentiation of Enterobacteriaceae, 17 6 .

Confirmed MPN, 81 Dilution

See Fecal Coliform, 132 bottles, 34, 7 4
See also Fecal Streptococci, 142 necessity for, 62
See also Total Coliform, 117 serial, 62
water, 57, 62
Costs of microbiological analyses, 246
Dilution water
Counting colonies, more than one analyst, 231 peptone dilution water, 57
phosphate buffer solution, stock, 57
Counting range, method characteristic, 240 phosphate buffer, 5 7
preparation of dilution and rinse water, 58
Cross-examination, in court, 286
Direct MF method, 124
Culture dishes, 32
Disinfectants, safe use, 266
Culture media
dehydrated, 38 Distilled water, 56
preparation and use, 38, 208
quality contro.I of, 211 Documentation, of quality control progam, 244
rehydration of, 38
specific media, 39 Drinking water
sterilization, 38 analyses of, 109
regulations, 280
Culture tubes, 34
Dry heat sterilization, 36
Cultures, shipment of, 89

Cytochrome oxidase test, 122, 170 Dulcitol fermentation test, 170

Dulcitol selenite broth, 51

Data transformation , 227
Duplicate analyses, 231
Decarboxylase medium, 55
EC medium (broth), 46
Decarboxylase tests, 122, 171
EDTA, chelating agent, 6
Dechlorinating agent, sodium thiosulfate, 6
Electrical equipment, safe use, 268
Decontamination of laboratory, 266
Enrichment procedures, Salmonella, 162
Deionized water, 56
Enterobacter, 108
Delayed incubation MF method
for fecal coliforms, 128
for total coliforms, 112 Enterobacteriaceae, differentiation of,
Table, 176
Depth samplers
Kemmerer, 1 4 Eosin methylene blue agar, 46
New York Dept. of Health, 8
Niskin, 8
Equipment and instrumentation
ZoBell J-Z, 8
costs of, 246
Detergent suitability test for glassware, 199 quality control of, 198

327
 Equlvalency program , 92
Fermentation tests
Escherichia, 108 arabinose (L), 149
glycerol, 14 7
Estuarine waters, sampling in, 28 inositol , 171
lactose, 152, 171
Ethanol, 43-45, 71, 74 malonate, 170
sorbose , pH 10, 150
Ethyl violet azide broth, 47 sorbitol (D), 14 7

Ethylene oxide sterilization, 36 Field kits, 97

Evidence, legal, 281 Field log sheets , 17

Field problems, 97
Facilities, requirements for, 194
Field safety guidelines , 263
Fecal coliform
Filter, cartridge , 161
definition 'of the group, 124
MF. delayed-Incubation method, 128
Filter, diatomaceous earth, 160
MF direct method, 124
MF verification, 130
Filter, membrane (MF) method
MPN, 132
concentrating enterics, 161
fecal coliforms, 124, 128
Fecal streptococci fecal streptococci, . 136
confirmation of enterococci, 14 7 general, 70
definition of, 135 total coliforms, 108
determination of fecal coliform/fecal
streptococcal ratios, 145 Filter funnel, 71-73
identification of Group Q streptococci, 150
Identification of species, 145 Filtration sterilization , 36
Isolation and confirmation, 146
membrane filter method, 136 Filtration techniques for concen~rating enterics
membrane filter verification, 138 cartridge (Balston), 161
MPN method, 139 diatomaceous earth, 160
pour plate method, 143 membrane filter (flat), 161
separation of enterococci and Group Q
streptococci , 14 7 Filtration volume, 62
separation of enterococcus group by species, Fluorescent antibody testing, 180
147
separation of enterococci by origin , 149 Fluorescent antibody reagents, 204
separation and speciation of S. bovis and
S. equinus, 152 Fluorescent dyes, 204
Fluorescent light, 71
Fecal streptococci test limitations Forceps, 71
MF method, 136
MPN procedure, 139 Freezer, 215
pour plate method , 143 Funnel, membrane filter, 71-73

Federal Water Pollution Control Amendments, Gelatin hydrolysis test, 149, 171
Act of 1972, 278
Glassware
cleaning, 36
Fermentation detergent suitability test for, 199
tubes, 34 dilution bottles, 34
vials, 34 fermentation tubes and vials, 34

328 &EPA MICROBIOLOGICAL MANUAL 1978

 graduates and pipets, 33 • lndole test, 121 , 170
pH check, 1 99
quality control, 199 Industrial wastes, Klebsie/la in, 97
sample bottles, 36
sterility checks, 200 Infections
sterilization, 36 laboratory acquired , 262
reporting , 262
Glucose broth, buffered (MR-VP broth), 42

Glycerol , fermentation test, 14 7 Injured cells, 92

Grab samples, 8 Inoculation loops, needles, applicator sticks,

32
Graduated cylinders, 34
Inoculation of cultures, methods for, 65
Gram stain, 86
Interference, by turbidity, 96
Ground water, analysis of, 97
lnterlaboratory quality control, 244
H antigens, test for, 179
Intra laboratory quality control, 192, 244
H broth, 56
Isolation of bacteria
pour plate method, 65
Hazards, laboratory. sources, 262
spread plate method, 65
streak plate method, 65
Heat, dry, sterilization by, 36

Isolation of Salmonella, plating methods, 164

Heat, moist, sterilization by, 36
Isolation techniques, 65
Hemolysis test, 149

Hoods (safety cabinets), 268

KF streptococcus agar, 46
Hot air sterilization, 36
Kemmerer sampler, . 14
Hydrogen ion concentration , 212
Klebsiella in industrial wa~tes, 97

IMViC test media, 42

Klebsie/la pneumoniae, 108

Incineration, sterilization by, 36

LES MF holding medium, 44
Incomplete recovery, suppression, 96 Laboratory check list for safety, 269

Identification of Enterobacteriaceae, 17 6 Laboratory equipment, quality control of, 198

Immediate incubation MF method, (direct MF), Laboratory facilities, quality control of, 194
124 Laboratory management
development of a QC program, 244
Impression slides, 182 legal considerations, 277
manpower and analytical costs, 246
Incubation temperatures for Salmonella, 1 64 safety, 259

Incubators Laboratory personnel, quality control of, 197 .

quality control , 21 6
specifications, 32 Laboratory pure water, 56, 200
temperature control , 216 use test for, 203

329
Laboratory records, 17 fecal coliform • 124 • 128
MF analyses, 59 fecal streptococci , 136
MPN analyses, 59 general MF techniques, 70
total coliform • 108
Laboratory supplies, quality control of, 199
M-Coliform broth, 43
Lactose, fermentation, 152, 171
M-Coliform holding broth. (LES holding medium),
Lactose in purple broth base, 41 44

Lakes and lmpoundment sampling , 24 MPN, (most probable number), 78

Lauryl tryptose sulfate broth, 45 MR-VP broth, 42

Laws, Federal Water M-VFC holding medium. 45

Federal Water Pollution Control Act
Amendments of 1972, Public Law Malonate broth , 55
92-500, 278
Marine Protection, Research and Marine water, sampling of, 28
Sanctuaries Act of 1972, Public
Law 92-532, 279 Malonate broth test. 170
Safe Drinking Water Act of 197 4,
Public Law 93-523, 280 Manpower and analytical costs, 246
sections relevant to microbiology, 289
Marine Protection Research and Sanctuaries Act
Levine's EMB agar, 46 of 1972, 92, 279

Limitations of, Marine Sanitation Regulations. 92. see Marine

coliform test (total), MF, 108 Protection • Research and Sanctuaries Act of
coliform test (total), MPN , 114 1972, 279
fecal coliform test, MF, 124
fecal coliform MPN method, 132 McFarland's barium sulfate standard, 177
fecal streptococci test
MF method, 136 Mean
MPN method, 139 arithmetic, 225
pour plate method, 143 geometric. 226
MF method, general, 70
MPN method, general, 78 Media. culture
azide dextrose broth, 46
Litmus milk, 49 bismuth sulfite agar. 53
blood agar base, with 10% blood , 50
Lysine decarboxylase test, 122 , 171 blood· agar with 0.4% potassium tellurite. 50
brain heart infusion agar, 4 7
Lysine iron agar (LIA) test. 53, 169 brain heart infusion agar with potassium
tellurite, 49
brain heart infusion broth, pH 9.6. 48
M·FC agar, 43 brain heart infusion broth with 40% bile,
M-FC broth, 43 48
brain heart infusion broth with 6.5% NaCl,
M·Endo holding medium. 44 48
M-Endo agar LES. 44 brilliant green agar, 52
M-Endo broth MF, 43 brilliant green bile broth 2%, 45
comparative testing, 2Q3
MF methods decarboxylase medium, 55
concentration of SB/monel/B, , 161 dulcitol selenite broth, 51
cost, 246 EC medium (broth), 46

330 &EPA MICROBIOLOGICAL MANUAL 1978

eosin methylene blue agar, see L.:evine's EMB triple sugar iron agar, 53
agar, 46 2, 3, 5-triphenyl tetrazolium chloride agar,
ethyl violet azide broth, 4 7 (tetrazolium Glucose Agar), 50
general use, 39 tryptic soy broth , 40
glucose broth, buffered (MR-VP broth), 42 trypticase soy agar,· 4.0
H broth, 56 tryptone glucose yeast agar (Standard
IMViC test media Methods Agar), 40 -
tryptone 1 % , 4 2 tryptophane broth, (tryptone, 1 %) , 42
MR-VP broth, 42 urea agar base, 54
Simmon's citrate agar, 42 urea agar base, 1OX, 54
KF streptococcus agar, 46 ' xylose lysine brilliant green ftgar, 52
lactose 10% in purple broth base, 41 xylose lysine desoxycholate agar, 52
lauryl tryptose broth, see lauryl sulfate
broth, 45 Media dispensing apparatus, 213
LES MF holding medium, coliform, s~e
M-coliform holding broth, 44 Media , quality control of, 208
litmus milk, 49 use test for, 203
lysine iron agar, 53
M-coliform broth, 43 Median, 226
M-coliform holding broth, see LES
holding medium, 44 Membrane filter apparatus, 71
M-Endo agar LES, 44
Membrane filter method
M-Endo holding medium, 44
concentrating Salmonella, 161
M-FC agar, 42
costs, 246 ·
M-FC broth, 42
fecal coliforms, 124, 128
M-VFC holding medium, 45
fecal streptococci, 136
malonate broth , 55
total coliforms, 108
media for fecal streptococci, 46
media for Salmonella and other enterics, 51 Membrane filters
medium for actinomycetes, 56 ASTM tests for, 205
MF media for coliforms, 42 changes in, 97
motility sulfide medium, 55 , comparative testing, 203
motility test medium, 42 government specification, 205
MPN media for coliforms, 45 quality control of, 211·
nutrient agar, 39 specifications and use, 74
nutrient broth, 39 use tests, 205
nutrient gelatin, 49
phenylalanine agar, 54 Measurement of analysts' precision, 232
plate count agar, (Standard Methqds Agar),
40 Meter, for UV light, 199
potassium tellurite in blood agar, 50
potassium tellurite in brain heart infusion Method characterization, 240
agar, 49
purple broth base , 41 Method modifications, 97
purple broth base with sorbose , pH 10, 41
PSE agar (Pfizer selective enterococcus), 4 7 Method selection , 91
selenite F broth , 51
Simmon's citrate agar, 42 Methyl red test, 12 1
skim milk with 0. 1 % methylene blue, 49
standard methods agar, 40 Methylene blue, reduction in milk tElSJ, 147
starch agar, 48
starch casein agar, 56 Microbiologists' responsibilities under water
starch liquid medium,· 48 laws, Appendix A, 289
TTC agar (Tetrazolium Glucose Agar), 50
tetrathionate brilliant green broth , 5 1 Microscope, compound
tetrathionate broth base, 51 quality control of, 214
tetrazolium glucose agar, (TTC agar), 50 use in' stain 'examination, 80, 87

331.
Microscope, fluorescence Oven, dry heat, 36
use in fluorescent antibody techniques, 180

Microscope, low power Parallel testing, 96, 234

quality control, 214
use In MF method, 71 Pathogens, 154
laboratory safety guidelines, 265
Miik, peptonization test, 150 shipment of cultures, 88
sources of hazard, 262
Mode, 226
Moist heat sterilization, 36 Peptone, dilution water, 57

Most probable number (MPN) methods Performance characteristics

costs, 246 in method development, 240
fecal coliforms, 132 in method evaluation and comparison, 240
fecal streptococci, 139
general technique, 78 Performance sample, 231
tables, 82
Salmone/Je, 180 Performance specifications, equipment and
total collforms, 114 n:iaterials
balance, 33
Motility test, 122, 171
dilution blanks, 58
Motility test medium, 42 general, 198
graduates, 33
Multi-test systems, (biochemical tests), 122, 172 incubators, 32
membrane filters, 205
pipets, 34
National Interim Primary Drinking Water
Regulations (NIPDWR), 91 Personal safety, 265

National Pollution Discharge Elimination Personnel, requirements, 197

System (NPDES) Guidelines, 92
Petri dishes, 32
Negative Controls, 231
membrane filters, 7 4
pour and streak, 66
Neutralization of toxic materials and metals, 6
pH measurements
glassware, 199
New York Dept. of Health depth sampler, 8
media, 208
Nlskin sampler, 8 pH meter
quality control, 205
Normal distribution, 227 specifications, 33
Nutrient agar, 39 Phenol red broth base, 40

Nutrient broth, 39 Phenylalanine agar, 54

Phenylalanine test, 170 ·
Nutrient gelatin, 49
Phosphate-buffered water, 5 7
0 and VI antigens, alternate test for, 179
Pipet containers, 34
0 Group test for Salmonella, 177 Pi pets
specifications, 34
ONPG test (0-nltrophenyl-B-D-galacto-
tolerances, 34
pyranoslde), 171

Ornlthlna decarboxylase test, 122, 171 Pipetting devices, 34

332 oEPA MICROBIOLOGICAL MANUAL 1978

. Plate count agar, (Standard Methods Agar), culture media , 208
40 development of a QA program, 244
equipment and instrumentation , 198
Plating methods general laboratory supplies, 199 .
pour, 65 laboratory facilities, 194
spread, 65 laboratory management, 244
streak, 65 laboratory operations, 194
membrane filters, 205
Plates, culture, 32 personnel, 197
routine analyses, 231
sampling collection and handling, 194
Plates, spot culture, 182 statistics for microbiology, 225

Portable equipment, see Field kits,. 97 Quality assurance of media

preparation , 208 ·
purchase, 208
Positive controls, 231 record maintenance, 211
sterilization, 209
Potable water· analyses storage recommendations. 210
MF procedures, 108 use of agars, broths and enrichment
MPN procedures, 114 media, 21,0
special rules for counting, 113
total coliforms, · 108 Quality 'assurance program
documentation , 244
Potable water, sampling, 22 interlaboratory, 244
intralaboratory. 244
Potassium tellurite
in blood agar, 50 Quality control records, 194, 244
in brain heart infusion agar, 49
Quantitation of Salmonella, 17 9
Pour plate method
Standard Plate Count, 101 Quebec colony counter, 66
Fecal Streptococci , 143

Precision, method , 240

f'ange, 227
Presumptive test
Total Coliform, 117
Reagents, quality control of
Isolation and Enumeration, 78
chemical, 204
dyes and stains, 204
Pretreatment of samples, 59
FA reagents, 204
serological , 204
PSE agar, 47
Records
Public water supplies, sampling, 22-24
field, 17
laboratory
Pure cultures, 65
MF, 19, 60-61
MPN, 19, 60
Purple broth base, 41
quality control, 244
sampling, 22
Purple broth base with sorbose, pH 10, 4·1
~ecovery
ambient temperature effect. 92
Quality assurance
incomplete recovery, 96
analytical quality control procedures, 231
interferences, 96
comparative testing of methodologies, 234
suppression. 96
compliance monitoring, 233

333
Recreational waters, sampling multitest systems, 172
bathing beaches, 29 optional biochemical tests, 171
swimming pools, 29 screening tests, 169
cartridge filter, 161
Reference sample, 231 concentration, 155
definition of genus, 154
Refrigerator diatomaceous earth, 160
quality control, 215 fluorescent antibody screening technique,
temperature control, 215 180
isolation of, 164
Repeat sampling, potable water supplies, 24 membrane filtration, 161
primary enrichment, 162
Reporting infections and accidents, 262 quantitative techniques, 179
cartridge filter, 180
Results, reporting MF/dia'tomaceous earth filter, 180
MF analyses, 75 serologic.al testing, 173
MPN analyses, 81 slide agglu.tination test for 0
spread plates, 69 grouping, 177
slide agglutination test for Vi
RODAC plates, agar, 195 antigen, 17·8
alternative slide agglutination test,
Rosollc acid , 43 179 .
tube test for H antigen, 179
Rounding off numbers, 70 swab, technique, 155

Sample
Safe Drinking Water Act of 1974, 280 containers, 6
dechlorination of, 6
Safety dilution of, 62
administrative considerations, 259 high solids, 62
biohazard control , 268 holding time limitations, 30
field guidelines, 263 identification and handling, 14
laboratory guidelines, 265 preservation and transit, 30
safety check list, 269 pretreatment, 59
sources of hazard , 262 report forms, 17-18, 20, 21
solid-type, 62
Safety cabinets (hoods) storage temperature, 30
description, 268 volume, 6
quality control, 198, 214
Sample Collection, Quality Control in, 194
Safety check list, 269
Sampling
Safety guidelines chain of custody, 17
automotive, 263 domestic and industrial wastes, 29
biohazard control, 268 equipment, 8-14
boat, 263 frequency of, 24
field, 263 general use lakes and impoundments, 24
laboratory, 265 marine & estuarine waters, 28
safety check list, 269 potable water supplies, 22
sampling rules, 264 recreational waters, 29
sampling under ice, 265 safety, 264
sediment, 14
Safety program , development, 259 shellfish-harvesting waters, 29
site selection, 22
Salmonella sludges, 14
biochemical identification procedures soil, 14
minimal biochemical set, 168 streams, 24

334 &EPA MICROBIOLOGICAL MANUAL 1978

Sampling techniques, 6 Spread plates
technique, 66
Screening procedures for Salmonella monitoring UV light effectiveness, 198
biochemical tests, 169
flourescent antibody technique, 180 Spreader colonies, 106

Sediments, 62 Stain
crystal violet, 87
Sediment sampler gram procedure, 86
Van Donsel-Geldreich , 14 Loeffler's methylene blue, 87
Lugol's iodine, 87
Selection of analytical methods, 91 safranin, 87
smears, preparation of, 86
Selectivity, method , 240
Staining procedures, 86
Selenite dulcitol broth, 51
Standard deviation, 226
Selenite F broth , 51
Standard methods agar, 40
Serological testing, Salmonella, 173
Serological reagents, 204
Standard plate count
apparatus and materials, 102
Serratia marescens, retention test for
counting and reporting results, 104
MFs, 206
dilution of sample, 102
media, 102
Sewage, 92
precision and accuracy, 106
Shellfish-harvesting waters, sampling, 29 procedure, 102
scope and application , 101
Shipment of cultures, 87
Starch agar, 48
Significant figures, 69
Starch casein agar, 56
Simmons Citrate agar, 42
Starch hydrolysis test, 150
Slides, impression, 182
Starch liquid medium, 48
Sodium thiosulfate, for dechlorinatio!'l
(neutralization), 6
Statistics for microbiology
measures of central tendency, 224
Soil sampling , 14
measures of dispersion , 226
normal distribution, 227
Sorbitol (D) fermentation test, 149

Sorbose pH 10 fermentation test, 152

Steam sterilization, 36
Specifications, performance and tolerance·
balances, 33 Sterilization procedures
dilution blanks, 58 alcohol, 74
graduates, 34 dry heat, 36
incubators, 32 ethylene oxide chemical, 36
MFs, 205 filtration, 36
pipets, . 33 incineration, 36
moist heat (steam), 36
ultraviolet irradiation , 36
Specificity, method, 240
Sterilizer, steam, 38
Spectrophotometer, quality control, 213
Storage Tetrathionate brilliant green broth enrichment,
dehydrated media, 208 for Salmonella, 163
Impression slides, 183
prepared media, 210 Tetrathionate broth base, 51
samples, 30
Tetrathionate broth enrichment for
Streak plate method, 65 Salmonella, 1 63
Streptococci, fecal Tetrazolium chloride, 2, 3, 5-triphenyl
definition, 135 reduction test, 14 7
determination of FC/FS ratios, 145
identification of species, 145 Tetrazolium glucose agar {TTC agar), 50
methods for enumeration and identification ,
136 Thermometer. 215
MF method, 136
MF verification, 138 Time expenditures for microbiological analyses,
MPN method, 139 246
pour plate method, 143
Titration of FA conjugate. 182
Streptomycetes {actinomycetes), 186
Tolerances
Stressed microorganisms, 92 balances, 33
dilution blanks, 58
Stream sampling, 24
graduates. 33
incubators. 32
Suitability test, detergent, 199 pipets, 33
for laboratory pure water, 200
Total Coliforms. Analyses for
Supplies differentiation of, 1 1 9
costs, 247 MF test, delayed, 1 1 2
quality control of, 199 MF test, single-step, 110
MF test, two-step, 111
Suppression, 96 MF test, verification , 1 1 3
MPN test, 114
Surface sampling
by hand, 8 Toxic metals, neutralization of, 6
by weighted frame, 8
Training for personnel, 198
Surface sampler, 8

Swab contact method, 196 Transit time, 30

Swab technique for concentrating enterics, 155 Triple sugar iron agar {TSI) test, 169

Swimming pools, sampling, 29 2, 3, 5-triphenyl tetrazolium chloride agar,

{tetrazolium glucose agar), 50
Tables, MPN, 82
Triple sugar iron agar, 53
Temperature of incubation for Salmonella, 164
Tryptic soy broth , 40
Temperature recording devices, 215
Trypticase soy agar, 40
Test, water suitability, 200
Tryptone glucose yeast agar ,(Standard Methods
Testimony, in court, 284 Agar), 40

Tetrathlonate brilliant green broth, 5 1 Tryptophane broth, (tryptone, 1 %) , 42

336 oEPA MICROBIOLOGICAL MANUAL 1978

TTC (2, 3, 5-triphenyl tetrazolium chloride), 147 Water, deionized, 56

TTC agar (tetrazolium glucose agar), 50 Water, deionizer, 212

Tubes, culture, 34 Water, dilution, 57, 62

Turbidity standard, (McFarland's barium sulfate), Water, distilled, 56

177
Water, laboratory pure, 56

Ultraviolet lamp sterilizer Water laws, Federal, 277

meter, 199
monitoring efficiency, 198 Water quality criteria, 92
use, 75
Water .quality standards, 92
Ultraviolet light meter, 212 , 199
Water quality tests
Ultraviolet sterilization, 36 use test, 203
water suitability· test, 200
Unsatisfactory samples, potable waters, 24

Urea agar base, 54 Water still, 56, 212

Urea agar base, 1OX, 54 Water suitability, test for, 200

•
Urease test, 169 Water tap sampling , 14

Use test for media, membranes, and . Waterbath, for tempering agar, 66, 68, 102
laboratory pure Vl(ater, 203
Waterbath, incubator
Van Donsel-Geldreich sediment sampler, 14· quality control , 21 5
specifications" 32
Variability of replicates (precision) 240 temperature control , 215

Variance, 226 Workload, guidelines for, 246

Verification
general.. 78
fecal coliforms, 130
fecal streptococci, 138 Xylose -lysine brilliant green agar, 52
membrane filter tests, 78
total coliforms, 11 3
Xylose lysine desoxycholate agar, 52
Vi antigen, test for, 178
ZoBell J-Z sampler, 8
Voges-Proskauer test, 121

337
 TECHNICAL REPORT DATA
(Please read lntJructions on the reverse before completing)
1 Rt PORT NO, 3. RECIPIENT'S ACCESSIOr..NO.
12.
EPA-600/8-78-017
4. TITLE AND SUBTITLE 5. REPORT DATE
December 1978
MICROBIOLOGICAL METHODS FOR MONITORING THE ENVIRONMENT 6. PERFORMING ORGANIZATION CODE
Water and Wastes
7. AUTHOR(SJ Editors: 8. PERFORMING ORGANIZATION REPORT NO.
Robert H. Bordner and John A. Winter, EMSL-Cincinnati;
Pasquale Scarpino, University of Cincinnati
g_ PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO.
lHD 621
11. CONTRACT/GRANT NO.
SAME AS BELOW
68-03-0431
12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED
Environmental Monitoring and Support Lab, - Cinn, OH Final
Office of Research and Development 14. SPONSORING AGENCY CODE
U.S. Environmental Protection Agency
Cincinnati, OH 45268 EPA/600/06
1!5, SUPPLEMENTARY NOTES
Project Officer: John Winter, EMSL, Cincinnati

16, ABSTRACT

This first EPA manual contains uniform laboratory and field methods for
microbiological analyses of water~ and wastewaters, and is recommended in
enforcement, monitoring and research activities. The procedures are prepared
in detailed, stepwise form for the bench worker. The manual covers coliform,
fecal coliform, fecal streptococci, Salmonella, actinomycetes and Standard
Plate Count organisms with the necessary support sections on sampling, equip-
ment, media, basic techniques, safety, and quality assurance.

17. KEY WORDS AND DOCUMENT ANALYSIS

.a. DESCRIPTQRS b.IDENTIFIERS/OPEN ENDED TERMS c. COSATI Field/Group

Aquatic microbiology Methodology Analytical procedures
Coliform bacteria Microbiology Standard plate count
Enterobacteriaceae Surface waters Total colif orms
Po table wa.ter Statistics Fecal coliforms 06/M
Public law Water analysis Pathogens
Quality assurance Water pollution Indicator organisms
Safety Water quality Fecal streptococci
T.T., c: t-.,. ... ., r ,,,.
19. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES

RELEASE TO PUBLIC Unclassified 354

20. SECURITY CLASS (This page) 22. PRICE
Unclassified
EPA Form 2220·1 (9·73)
338 'llU.S. GOVERNMENI' PRINTING OFFICE: l 9 91· s 4 B-18 7/ 4 o s 41