Appendix C1

APPENDIX C1
OHIO WATER MICROBIOLOGY LABORATORY

M-ENDO METHOD FOR TOTAL COLIFORMS
Updated March 2013
The M-Endo method is a membrane-filtration method that determines concentrations of total
coliforms. M-Endo can be used in a two-step method for total coliforms and Escherichia coli (E.
coli) with NA-MUG (nutrient agar with MUG) as the second step; however, the Ohio Water
Microbiology Laboratory (OWML) no longer uses NA-MUG and it will not be described below.
The M-Endo method can be used in the field or the laboratory.
THEORY: The M-Endo plates are incubated at 35C for 22-24 hours. The M-Endo agar
contains selective and differential agents. Sodium lauryl sulfate and sodium desoxycholate
inhibit Gram-positive cocci and endospore-forming bacteria. Basic fuchsin and sodium sulfite
form a green metallic sheen on colonies able to ferment
lactose to acetaldehydethese colonies are counted as
total coliforms.
USE: The M-Endo method is suitable for use with
drinking water or groundwater. The method is not
recommended for use with raw surface waters because
non-target background growth may make plates difficult
to read.
MEDIA SOURCES: The M-Endo agar dehydrated
medium is available from Fisher Scientific (800/7667000, Cat DF0736154 (100 g) or DF0736172 (500g)).
Ethanol (95% denatured) needs to be purchased also
(Fisher Scientific, Cat A405P-4 (4 L)).
Prepoured plates can be purchased from Hardy
Diagnostics (800/266-2222, m-Endo Cat G128). If prepoured plates are purchased, 95% ethanol
is not needed.
Use phosphate buffered dilution water and 0.45 m membrane filters. Buffer can be purchased
from Hardy Diagnostics (Cat D699 (99mL) or Cat U193 (500mL)). See buffer preparation
(Appendix A2).
MEDIA PREPARATION:
Instructions for preparation of M-Endo medium (modified from American Public Health
Association and others, 2006)
NOTE: It is not recommended that this medium be made up in large quantities and stored in the
refrigerator; therefore, the instructions to prepare only 100 mL of media are given. Plates should
be poured immediately and used within 5 days.
1. Prepare 100 mL of a 2% ethanol solution by combining 2 mL of 95% denatured ethanol with

98 mL of deionized or distilled water. Mix well.
2. Combine 5.1 g of dehydrated m-Endo medium with 100 mL of the 2% ethanol solution in a
250-mL flask.
3. Stir the mixture well for several minutes to break up clumps and prevent medium from
adhering to the flask.
4. Place the flask in a heated water bath or on a hot plate and heat slowly to boiling. If
using a hot plate, stir the mixture constantly or use a stir bar and magnetic stirring hot
plate to prevent scorching. Do not autoclave.
5. When the medium approaches the boiling point, promptly remove from heat. Do not
boil.
6. Cool the medium to a temperature of about 45-50C and pour 6 to 7 mL in 50-mm Petridish
bottoms. Quickly place Petri-dish tops loosely on Petri-dish bottoms to allow condensation to
escape.
7. When the medium has solidified (about 10 minutes), close the Petri dishes by pressing firmly
on the tops. These plates are suitable for use after the medium has solidified.
8. About 15 to 20 plates can be prepared from 100 mL of medium. Label and date.
9. Prepared Petri dishes sealed in small plastic bags to prevent drying and stored in the darkness
of a refrigerator, can be used for a maximum of 5 days.
ANALYZING SAMPLES:
Refer to the USGS National Field Manual (Myers and others, 2007) for membrane filtration
procedures. Record results on a bench sheet form (an example is attached).
REFERENCES:
Britton, L.J., and Greeson, P.E., eds., 1989, Methods for collection and analysis of aquatic
biological and microbiological samples: U.S. Geological Survey Techniques of Water-Resources
Investigations, book 5, chap. A4, p. 13-16.
American Public Health Association, American Water Works Association, and Water
Environment Federation, 2006, Standard methods for the analysis of water and wastewater
Section 9222B. Standard total coliform membrane filter procedure: Washington, D.C., American
Public Health Association.
Myers, D.N., Stoeckel, D.M., Bushon, R.N., Francy, D.S., and Brady, A.M.G., 2007, Fecal
indicator bacteria (ver. 2.0): U.S. Geological Survey Techniques of Water-Resources
Investigations, book 9, chap. A7, section 7.1.3.C. accessed December 2012 from
http://pubs.water.usgs.gov/twri9A/.
NWIS CODES:
Total coliforms, M-Endo membrane-filtration method, colony-forming units per 100 mL
Parameter code: 31501
Method code: BAC51
E. coli, NA-MUG membrane-filtration method, colony-forming units per 100 mL
Parameter code: 50278

Method code: BAC24
Total coliforms on mENDO Water

To be filled out at time of collection:
Site Name:
_________________________
Date: ____/____/___
MM / DD /YY
Time: ________
(military)
To be filled out by laboratory analyst:

Analyzed by (initials): _____________
Analysis Date: ____________
Date mENDO poured: _____________
Time in 35C (2224hr): ____________
Filter (0.45 m) lot number: ____________
Time out of incubator: ____________
Read by: ____________
Date plates read: ____________
Colony counts:
Suggested sample volumes: 100, 30, 10, 3, 1, 0.3 (30mL of 10-2 dilution), and 0.1 (10mL of 10-2
dilution)
Sample size (volume - mL)
Colony count
Filter Blank
(before plating series)
Process Blank
(after plating series)
RESULTS
Total coliform colonies/100 mL
100 x
Count
Colonies
100 mL
Volume plated
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OHIO WATER MICROBIOLOGY LABORATORY

1. Prepare 100 mL of a 2% ethanol solution by combining 2 mL of 95% denatured ethanol with

Parameter code: 50278

Total coliforms on mENDO Water

To be filled out by laboratory analyst:

Analysis Date: ____________

Date mENDO poured: _____________

Time in 35C (2224hr): ____________

Filter (0.45 m) lot number: ____________

Time out of incubator: ____________

Read by: ____________

Date plates read: ____________

Sample size (volume - mL)

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