PERSONAL C ARE PRODUC TS COUNCIL

TECHNIC AL GUIDELINES

Microbiology
Guidelines

Personal Care Products Council

Committed to Safety,
Quality & Innovation
ii | PCPC MICROBIOLOGY GUIDELINES
 P ER SONA L C A R E PRODUC TS CO U N CI L
T EC HNIC A L GUID EL INES

Microbiology
Guidelines

2016 EDITION

Editors
John F. Krowka, Ph.D.
Beth A. Jonas, Ph.D.
Production
Natasha Clover
Published by
Personal Care Products Council
1620 L Street, N.W., Suite 300
Washington, D.C. 20036
Phone: 202/331‑1770
Fax: 202/331‑1969
www.personalcarecouncil.org

PCPC MICROBIOLOGY GUIDELINES | iii

Copyright © 2016
The Personal Care Products Council
No portion of the PCPC Microbiology Guidelines may be reproduced in whole or in part, in any
form or by any electronic or mechanical means, including information exchange and retrieval sys‑
tems (except for the purpose of official, nonpublic use by the United States Government), without
prior written permission from The Personal Care Products Council, Inc., 1620 L Street, N.W.,
Washington, DC 20036
Library of Congress Control Number: 2013952022
ISBN 1-882621-51-4
Printed in the United States of America

iv | PCPC MICROBIOLOGY GUIDELINES

Foreword

In 1969, CTFA (now called the Personal Care Products Council (PCPC)) began publishing its
Technical Guidelines in the CTFA Cosmetic Journal. These guidelines were developed by the newly
organized CTFA Microbiology Committee and were concerned with microbiological issues. The
benefits of having the Guidelines available in a single volume, and presented in a standardized for‑
mat, were recognized, and in 1974, the first independent compilation of the Technical Guidelines
was published.
In 1993, after several major revisions and additions to the Guidelines, PCPC responded to requests
made by the users to split the Guidelines into separate volumes so that individuals might purchase
sets relating specifically to their areas of responsibility. The Guidelines are now published by PCPC
in three volumes: Microbiology, Quality Assurance, and Safety Evaluation.
The PCPC Technical Guidelines are dynamic documents that undergo extensive review and updat‑
ing to reflect best practices prior to publication by PCPC technical committees and staff, as well as
include input from PCPC members and nonmember companies, federal government agencies, and
scientific professional societies. Comments from individuals are welcome at any time. All guidelines
and methods were re-reviewed and updated for this edition of the Microbiology Guidelines.
While PCPC has sought to ensure that these Guidelines generally satisfy applicable U.S. federal stat‑
utory and regulatory requirements as of the date they were drafted, PCPC can assume no respon‑
sibility for their adequacy, nor does it purport to advise as to the necessity for their use in any par‑
ticular situation. In those Guidelines that address regulatory requirements, decisions such as when a
report must be filed and what information must be included in it can be made only by those indi‑
viduals responsible for making such submissions. With regard to all of the areas covered by PCPC
Guidelines, each company is responsible for making their own assessments and must independently
assume responsibility to ensure that their conduct is consistent with all current, applicable federal,
state and local laws and regulations.
It must be emphasized to the user that these Guidelines are intended only to aid manufacturers in
developing programs that meet their individual needs. The Guidelines must not be considered either
minimum or maximum requirements of effective programs. Alternative ways to reach the goals of
the Guidelines may well exist and may be equally useful. Guidelines on any topic must, of course,
be adapted to the particular operations of the manufacturer using them. We hope it proves to be an
especially valuable resource.

Lezlee J. Westine Beth A. Jonas, Ph.D.

President & CEO Chief Scientist &
Executive Vice President - Science
September 2016

PCPC MICROBIOLOGY GUIDELINES | v

vi | PCPC MICROBIOLOGY GUIDELINES
Acknowledgements

The Guidelines presented in this volume were developed by the PCPC Mi‑
crobiology Committee. As the development and updating effort has been an
iterative process, listing all of the experts involved from PCPC member com‑
panies would be beyond the capabilities of the current editors. Therefore, to
all who had a part, a very warm and sincere thank you. We are grateful to
Andress Johnson and Warren Holland-Recine who organized the efforts of the
Council’s Microbiology Committee to update these guidelines. We also wish
to thank Don English and Joyce Beauchamp for their invaluable assistance.
This edition is dedicated to the memory of Kimdra Smith-Webster and Scott
Sutton.
The editors also would like to thank Natasha Clover at PCPC for her assistance
in producing this volume.

PCPC MICROBIOLOGY GUIDELINES | vii

viii | PCPC MICROBIOLOGY GUIDELINES
Table of Contents
Foreword v
Acknowledgements vii
Introduction 1
1. Microbiological Quality Assurance for the Personal Care Products Industry 3
2. Microbiological Evaluation of the Plant Environment 11
3. Cleaning and Sanitization 31
4. Microbiology Staff Training 53
5. Handling, Storage and Analysis of Raw Materials 67
6. Microbiological Sampling 71
7. Microbiological Quality for Process Water 79
8. Microbiology Laboratory Audit 91
9. Microbiological Validation and Documentation 105
10. Maintenance and Preservation of Test Organisms 127
11. Raw Material Microbial Content 141
12. Establishing Microbiological Quality of Personal Care Products 145
13. Determination of Preservation Efficacy in Water-Miscible Personal Care Products 151
14. Preservation Efficacy Testing of Eye-Area Personal Care Products 159
15. Microbiological Assessment of Product Quality After Use 163
16. Microbiological Risk Factor Assessment of Atypical Personal Care Products 171
17. Determination of Preservation Efficacy in Nonwoven Substrate Products 181
18. M-1 Determination of the Microbial Content of Personal Care Products 187
19. M-2 Examination for and Identification of Staphylococcus aureus, Escherichia coli, 195
Pseudomonas aeruginosa, and Candida albicans
20. M-3 Method for Preservation Efficacy Testing of Water‑Miscible Personal Care Products 209
21. M-4 Method for Preservation Efficacy Testing of Eye Area Personal Care Products 217
22. M-5 Methods for Preservation Efficacy Testing of Nonwoven Substrate 227
Personal Care Products
23. M-6 A Method for Preservation Efficacy Testing of Atypical Personal Care Products 237
24. M-7 A Screening Method for Preservation Testing of Water-Miscible 249
Personal Care Products
25. Glossary of Microbiological Terms 257

PCPC MICROBIOLOGY GUIDELINES | ix

x | PCPC MICROBIOLOGY GUIDELINES
 INTRODUCTION
Introduction

The production of quality personal care products requires a commitment from the manufacturer to
establish and maintain a total quality program. The microbiological component of such a program
is designed to ensure: (1) that the product that reaches the consumer is free of microorganisms that
could affect the product quality and consumer health, and (2) that during normal product use, the
quality of the product will not be compromised by microbial activity.
The Personal Care Products Council Microbiology Guidelines are intended to provide manufacturers
with guidance regarding best practices as well as establishing and maintaining a microbiological
quality program within their companies. The Guidelines are also recommended for contract manu‑
facturers and packagers as well as suppliers of raw materials. Sections of the Guidelines will vary as
to applicability for different sectors of the industry and for individual companies.
The Guidelines are organized into separate sections. The major provisions for an effective micro‑
biological quality program are outlined in the first guideline “Microbiological Quality Assurance
for the Personal Care Products Industry.” More detailed information on cleaning and sanitization,
staff training, raw materials, sampling, process water and other topics is presented in subsequent
sections. “Cosmetic products are not expected to be aseptic; however, they must be completely free
of high-virulence microbial pathogens, and the total number of microorganisms per gram must be
low.”1 In addition, cosmetics should remain in this condition when used by consumers.
As an alternative to manufacturing sterile products, the consideration of limits to microbiological
content is based on the best available information. Microbiological limits for finished products as
well as raw materials are covered in separate guidelines. Sections 16-21 provide methods for micro‑
bial content and preservation effectiveness testing of a variety of product types. A glossary of mi‑
crobiological terms is provided in Section 25. All sections of the 2016 edition of the Microbiology
Guidelines were reviewed and revised.
These Guidelines are not intended to establish minimum industry standards for all personal care
products. Also, the Guidelines do not cover all areas that might be addressed under a specific cate‑
gory. The Personal Care Products Council intends to include additional topics in future updates to
the Guidelines. In the interim, cosmetic companies are encouraged to refer to other microbiology
resources. Also, while these Guidelines can help ensure that products are microbiologically accept‑
able, they cannot substitute for day‑to‑day familiarity with the principles of microbial control. The
Guidelines must never be taken to restrict additional activities when circumstances dictate.
1
US Food and Drug Administration, Compliance Program Guidance Manual, Chapter 29 – Colors and Cosmetics Technology
–Part V Page 1, available at: http://www.fda.gov/downloads/Cosmetics/GuidanceRegulation/GuidanceDocuments/
UCM208412.pdf (Accesssed June 1, 2016)

PCPC MICROBIOLOGY GUIDELINES | 1

2 | PCPC MICROBIOLOGY GUIDELINES
 SECTION 1

Microbiological Quality
Assurance for the Personal
Care Products Industry

1
QUALITY ASSURANCE
MICROBIOLOGICAL
INTRODUCTION
Adequate control of the microbiological quality of finished personal care products depends upon
the implementation of an effective microbiological quality assurance program. Although the appli‑
cability of some aspects of such a program will vary for different types of products, processes, and
facilities, the major areas described below should be reviewed.
The reader is directed to review the “Glossary of Microbiological Terms” that follows these guide‑
lines to ensure a proper understanding of the guidelines.
Note: These guidelines do not apply to Over-the-Counter (OTC) products and/or drugs as defined
by regulatory agencies. Food and Drug Administration (FDA) Current Good Manufacturing Pro‑
cedures (cGMPs) for Finished Pharmaceuticals should be consulted for the manufacture of drug
products.1

QUALITY ASSURANCE
Quality assurance is defined as the activity of providing the evidence needed to establish confidence
that the quality function is being performed adequately.2 The goal of an effective microbiological
quality assurance program is to assure that the finished product consistently meets established mi‑
crobiological standards.
The microbiological quality assurance program can be viewed as having several major components:

• Personnel including: qualifications; functions; and training;

• Physical environment including: plant; grounds; equipment and sanitary procedures;
• Materials including: storage; raw materials; packaging; and finished goods; and
• Procedures including: sampling; testing; laboratory practices; and auditing.

The PCPC Quality Assurance Guidelines offer general guidance in establishing quality assurance
programs within personal care manufacturing facilities.3 In addition, the “Personal Care Good Man‑
ufacturing Practices” offers guidance in establishing the control systems designed to assure product
quality and consumer safety.4

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 SECTION 1 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY

PERSONNEL AND TRAINING

All personnel should have the necessary training or experience to perform their assigned functions
in manufacturing and quality control.5

A. Quality Assurance Microbiology Laboratory

QUALITY ASSURANCE

The personnel responsible for microbiological quality control should be of adequate number
MICROBIOLOGICAL

and have the necessary training and/or experience to ensure that personal care products meet
established specifications.
1. Microbiologist - Should have acquired by education and/or experience the expertise needed to
supervise operations and should be capable of:

• Sampling raw materials, process water, intermediates, and finished products;

• Developing, validating and performing test methods;
1

• Performing sanitation inspection of plant facilities;

• Performing environmental studies;
• Performing documentation and record keeping;
• Interpreting and reporting test results;
• Developing and implementing hygiene action plans; and
• Participating in investigation of out of specification (OOS) microbiological results.

2. Technician - Should be qualified by education and/or experience in microbiological technique.

B. Manufacturing/Operations
1. Supervisor - Should be qualified by training and/or experience to properly ensure maintenance
of the microbiological integrity of the product being manufactured.
2. Compounders, Filling Line Operators, etc. - Should have an understanding of causes of mi‑
crobiological contamination, common contamination sources, and their prevention.

C. Education Program
In order to maintain microbiological quality it is important to instill general microbiological
awareness and to train operating employees in hygienic practices. Examples of microbiological,
and hygiene training to be emphasized are listed below.
1. Potential sources for product contamination by the following avenues:

• Physical contact with manufacturing equipment, and formulation ingredients, especially

following poor personal hygiene;
• Gross contamination from process and/or rinse water; condensation on standing; dust and
particulate matter laden with microorganisms, including airborne spores and vegetative cells;

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 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY SECTION 1

• Unsanitary or dirty equipment; and

• Contaminated raw materials.

2. Training on proper cleaning and sanitizing procedures (See Section 3 – Cleaning and Sanitiza‑
tion).
3. Encouraging employees to report plant conditions that could affect product integrity.

1
4. Personal Hygiene:

QUALITY ASSURANCE
MICROBIOLOGICAL
• No person with any health condition that could adversely affect products should have direct
contact with raw materials, packaging products, or product contact surfaces.
• Personnel should store personal belongings, eat, drink, or use tobacco only in designated
areas.
• Personnel should be required to wear appropriate Personal Protective Equipment (PPE) and
clothing (hair net, booties, etc).

PHYSICAL ENVIRONMENT
A. Plant and Grounds
Buildings and equipment should be designed for ease of cleaning and sanitization. They should
be clean and maintained in an orderly manner.
The manufacturing area should be designed to minimize the risk of contaminating raw materials,
packaging components, or products. These areas should have walls and floors that are easy to
clean and sanitize. Overhead repositories for dust, such as piping and ductwork, should be kept to
a minimum and cleaned when necessary. Floor drains in manufacturing areas should be routinely
sanitized and properly maintained.
Building openings, including doors, should be designed, operated, and maintained to protect the
manufacturing areas and to minimize environmental contamination. Windows should be prop‑
erly screened and each manufacturing facility should have an effective rodent and insect control
system.
Ventilation systems should include, where appropriate, changeable filters properly maintained
to restrict entry of particulate matter, insects, microorganisms, and other contaminants. Positive
air pressure should be available in areas containing easily contaminated materials. Water used for
humidifying should be of acceptable microbiological quality.
Hand-washing and/or hand sanitizing facilities should be provided near the production area.
Signs reminding personnel to wash hands should be prominently displayed at the washing facili‑
ties. Hand cleansers and disposable towels should be available.
Eating and smoking should not be permitted in the manufacturing areas.

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 SECTION 1 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY

Clean containers, utensils and microbiologically acceptable water used with a disinfectant-type
cleaner should be available for general environmental cleaning. Clean containers appropriately
labeled should be provided for collecting waste and scrap materials.
Designated areas should be provided for storing raw materials and finished goods.

B. Machinery and Equipment

QUALITY ASSURANCE

It is desirable that equipment be constructed for effective cleaning and sanitization and that it
MICROBIOLOGICAL

be designed to protect products from contamination. The PCPC Quality Assurance Guidelines
provide important information on construction, cleanability, and related items.6
Possible Sources of Contamination:
• Pipes - May contain crevices, pits, sharp turns, dead ends, connections, unsanitary welded
joints.
• Equipment - May contain pits, crevices, poorly sealed lids, leaking pump shaft seals, defective
1

sight glasses.
• Utensils - Plastic is difficult to clean. Wood is not acceptable for most personal care manufac‑
turing applications.
• Personnel - The human body is a reservoir of large numbers of microorganisms. Protective
apparel should be worn whenever appropriate. Infected cuts or abrasions on the hands should
be covered.
• Atmosphere - Dust is laden with airborne microorganisms.
• Other - General condensation, standing water, reused filter pads, cleaning rags, and com‑
pressed air can be sources of microbial contamination.

Recommendations:
• Pipes - Stainless steel, glass and plastic hose are the best materials; sanitary snap joint fittings
are preferred. Pipes should be graded to drain with no dead legs.
• Equipment - Lids on compounding tanks should be tight fitting and vented to minimize con‑
densate formation. Drains should be at the lowest point. Equipment should be designed to
minimize backwash contamination potential. Hard to clean equipment should be dismantled
and cleaned out of place between product changeovers.
• Utensils - Should be made of stainless steel; should be thoroughly cleaned, rinsed, air dried,
and properly protected from contamination between uses.
• Personnel - Should be properly trained in personal hygiene (e.g., Proper hand hygiene after re‑
stroom use, contact with food, use of proper clothing, etc., and prior to contact with product)
and the sanitary use of equipment.
• Atmosphere - Introduction of clean air and the exclusion of particulate matter.
• Other - Single-service towels should be used where possible. Compressed air lines associated
with product contact equipment should be protected with appropriate point of use filters.

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 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY SECTION 1

SANITARY PROCEDURES
Cleaning and sanitization procedures are essential to ensure good microbial quality in the manu‑
facture of personal care products. Written cleaning and sanitizing procedures should be established,
distributed and implemented by responsible personnel. These procedures should be validated in
order to consistently meet hygienic manufacturing requirements. Refer to Section 3 (Cleaning and
Sanitization) for additional details.

1
QUALITY ASSURANCE
MICROBIOLOGICAL
STORAGE
Care should be taken to prevent introducing microbes when storing all materials. The following are
desirable conditions of storage: dry; protected from airborne contaminants; maintained within rea‑
sonable temperature limits; located within low traffic areas; and large enough to segregate incoming
materials from material already received and approved. Materials should be stored in a manner that
allows for sufficient cleaning and inspection.
Raw material containers, and storage areas should be protected from contamination by air, dust,
water, and personnel. Storage areas, and raw material containers should be cleaned on a schedule.
For more specific guidance for storing raw materials consult “Handling, Storage and Analysis of
Raw Materials” (Section 5).
Bulk storage of raw materials, process intermediates, and finished products should be protected
from microbial contamination. Bulk material should be properly labeled. Bulk subjected to extend‑
ed storage should be sampled and retested before use in accordance with established procedures.
A program should be established for cleaning and sanitizing bulk storage containers (Section 3 –
cleaning and Sanitization).

RAW MATERIALS
A. Specifications
Personal care manufacturers should evaluate the microbiological quality of their raw materials,
and establish appropriate specifications based on the best available scientific information. Micro‑
biological specifications should be established for all raw materials susceptible to contamination
(See Section 5 – Handling and Storage of Raw Materials and Section 11 – Raw Material Micro‑
bial Content). The microbiological quality assurance program should include provisions that:

• The material should be sampled immediately upon receipt from manufacturer.

• Material should be held in a clean quarantine area until testing is completed.
• Rejected materials should be clearly marked for prompt disposal.
• Accepted material should be so marked.
• Procedures are in place to re-sample and test susceptible raw materials stored for prolonged
periods prior to use.

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 SECTION 1 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY

B. Identifying Materials
All raw materials, intermediate, and finished product, should be clearly marked as to their iden‑
tity.7

C. Sampling Plan
For sampling plan see Section 6 (Microbiological Sampling) and the PCPC Quality Assurance
QUALITY ASSURANCE

Guidelines.8
MICROBIOLOGICAL

PACKAGING MATERIALS AND OTHER COMPONENTS

Packaging materials (tubes, jars, bottles, caps, brushes, applicators and other components) should
be properly controlled (e.g., handling, storage, testing and proper documentation of results) to min‑
imize contamination and to maintain microbiological standards and specifications.
1

A program should be established to ensure that appropriate packing materials and product contain‑
ers conform with in-house microbiological specifications.

FINISHED GOODS
Finished goods should be sampled and tested to assure that products meet established microbiolog‑
ical specifications and should not be released for distribution until the satisfactory completion of
the testing. See Section 6 (Microbiological Sampling) and Section 11 (Establishing Microbiological
Quality of Personal Care Products) for guidance.

LABORATORY PRACTICES
A. Microbiological Quality Assurance Laboratory
Several key functions of the Microbiological Quality Assurance Laboratory are:

• Analyze raw materials for microbial content, and determine if microbiological specifications
are met;
• Check to ensure that plant hygiene procedures are implemented, and effective;
• Ensure that the microbial status of finished product meets established specifications;
• Investigate, and resolve contamination problems;
• Establish a program to routinely monitor critical control points, including cleaning, sanitiza‑
tion, and storage of processing and filling equipment;
• Establish appropriate documentation, and record-keeping procedures for laboratory testing;

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 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY SECTION 1

• Establish, and manage an environmental monitoring (EM) program in the manufacturing

facility.

Microbiological testing should be conducted in a laboratory specifically designed for this pur‑
pose. Alternatively, microbiological quality assurance may be subcontracted. For additional guid‑
ance see the Section 8 (Microbiology Laboratory Audit) guideline.

1
B. Procedures

QUALITY ASSURANCE
MICROBIOLOGICAL
The following are the main suggested procedures for the microbiological laboratory:

• Sampling - It is recommended that the procedure as outlined in “Microbiological Sampling”

be followed.
• Testing - It is recommended that raw materials, bulk in-process, and finished goods be tested
for microbial content. See Section 11 (Establishing Microbiological Quality of Personal Care
Products) and Section 10 (Raw Material Microbial Content)
• Water - Particular attention should be given to water, as it is the most important raw material
as well as a solvent for cleaning, disinfecting and rinsing. See Section 7 (Microbiological Qual‑
ity for Process Water).
• Preservation - The inability of microorganisms to survive in packaged products should be ver‑
ified during product development. See Section 12 (Determination of Preservation Efficacy in
Water-Miscible Personal Care Products).
• Monitoring - Control and monitor sanitization procedures by the use of swabs, direct contact
plates, air samplers, and other means. Refer to Section 2 (Microbiological Evaluation of the
Plant Environment).
• Documentation/Record Keeping - Maintain accurate, detailed records providing the history
of a material. A central file should be maintained for periodic review by a microbiologist and
should be the prime record for all testing performed. See Section 9 (Microbiological Validation
and Documentation).

It is recommended that completed records be maintained for at least two years after distribution
of the batch of manufactured product.4

MONITORING
The PCPC Quality Assurance Guidelines recommends periodic self-audits.4 Periodic surveillance or
inspection of facilities, operations, practices, housekeeping and sanitation is an excellent adjunct to
a microbiological quality assurance program. Such monitoring helps to verify consistent compliance
with established procedures, to confirm that the systems continue to be adequate for provision of
safe and effective products, and to identify areas that may require improvement. Appropriate mea‑
sures should be taken where undesirable trends become evident or when conditions are noted that
may cast doubt on product or process integrity.

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 SECTION 1 MICROBIOLOGICAL QUALITY ASSURANCE FOR THE PERSONAL CARE PRODUCTS INDUSTRY

REFERENCES
1. “Current Good Manufacturing Procedures (cGMP’s) for Finished Pharmaceuticals,” Title 21,
Code of Federal Regulations, Part 211 (21 CFR 211).
2. Nikitakis, J.M. (ed.) 2014. “Glossary of Terms,” PCPC Quality Assurance Guidelines, The
Personal Care Products Association, Washington, DC 20036 (December 1992).
3. Nikitakis, J.M. (ed.) 2014. PCPC Quality Assurance Guidelines, The Personal Care, Products
Association, Washington, DC 20036 (December 1992).
QUALITY ASSURANCE
MICROBIOLOGICAL

4. Nikitakis, J.M. (ed.) 2014. ISO 22716 Cosmetic Good Manufacturing Practices. In PCPC
Quality Assurance Guidelines, The Personal Care Products Association, Washington, DC 20036.
5. Nikitakis, J.M. (ed.) 2014. Annex 1 Personnel and Training. In PCPC Quality Assurance
Guidelines, The Personal Care Products Association, Washington, DC 20036, pp (December
1992).
6. Nikitakis, J.M. (ed.) 2014. Annex 3 Equipment – Part 2 – Processing. In PCPC Quality
1

Assurance Guidelines, The Personal Care Products Association, Washington, DC 20036, pp. 21-31.
7. Nikitakis, J.M. (ed.) 2014. Annex 6 Finished Products/Lot Identification & Control. In PCPC
Quality Assurance Guidelines, The Personal Care Products Association, Washington, DC 20036, pp
55-58.
8. Nikitakis, J.M. (ed.) 2014. Annex 17 Sampling: Part I - General Provisions Sampling Plan. In
PCPC Quality Assurance Guidelines, The Personal Care Products Association, Washington, DC
20036, pp 103-106.

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 SECTION 2

Microbiological
Evaluation of the
Plant Environment
INTRODUCTION
The personal care product manufacturing plant environment may directly or indirectly affect the
microbiological quality of personal care finished products. Environmental assessment of the plant
primarily employs air and surface monitoring techniques. Evaluation of monitoring results takes
into consideration the intrinsic factors that affect the microbial environmental quality within the
facility. Changes in environmental data (i.e., trend analysis) can serve as useful indicators of the need

2
for investigation and possible corrective actions.

PLANT ENVIRONMENT
EVALUATION OF THE
For facilities that manufacture products that are regulated as over-the-counter (OTC) drug products
in the United States, refer to the United States Pharmacopeia (USP),1 Parenteral Drug Association
(PDA),2 and Current Good Manufacturing Practices (cGMPs)3-5 for guidance in how to conduct
environmental monitoring for a manufacturing plant.
Manufacturers have the responsibility to determine what type of program is most suitable for their
facilities, since each manufacturing plant is unique. This guideline sets forth general and specific
information to aid manufacturers in designing environmental monitoring programs that are suited
to their own needs. Some information offered may not be directly applicable to the operations of
every facility. However, an established environmental monitoring program can provide the data,
tools, and procedures needed to maintain a well-functioning facility. An effective program can pro‑
vide information about areas of the plant that potentially may affect the microbial quality of the
finished product.
The Food and Drug Administration (FDA) has another approach that could be considered, which is
Hazard Analysis Critical Control Point (HACCP). HACCP is a systematic approach for evaluating
hazards and risks of various parts of a process and places controls and systems at critical points.6
Good communication between microbiologists and facility engineers is essential in maintaining
awareness of changing environmental factors that could alter the microbiological quality of the
plant environment.

MANUFACTURING ENVIRONMENT
The quality of the manufacturing plant environment is largely influenced by five basic factors: facili‑
ties, equipment, personnel, housekeeping, and cleaning and sanitization. Understanding these factors

PCPC MICROBIOLOGY GUIDELINES | 11

 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

is essential when developing an environmental control program. Additional guidance on these topics
is given in Section 3 (Cleaning and Sanitization) and the PCPC Quality Assurance Guidelines.7

A. Manufacturing Facility
1. Design
A well-designed, well-constructed manufacturing facility can contribute to a high-quality fin‑
ished product. Proper design can minimize cross-contamination and contamination from the
surrounding environment. Contamination of the plant environment by microorganisms, dust,
and dirt can be controlled by the use of vent filters, drain traps, and tight-fitting doors and
windows. Airborne dust contamination can be minimized by filtered air-handling systems that
provide adequate ventilation, temperature, and humidity controls to prevent cross-contamina‑
tion. Materials used for building interiors should be durable, designed to be easily cleaned, and
adequately maintained. Overhead utilities (pipe work) can be designed so as not to adversely
affect the manufacturing environments. Ductwork for these utility systems should be com‑
posed of nonporous and non-flaking material.
General building design includes suitable barriers for separating manufacturing and packaging
areas from warehouses, offices, locker rooms, and washrooms. In particular, a good building
PLANT ENVIRONMENT

design provides separate areas for material receipt, storage, weighing, compounding, filling,
EVALUATION OF THE

packaging etc. Traffic flow of both personnel and materials (e.g., raw ingredients, packaging
component, and finished stock) can be minimized in processing and packaging areas.
When considering building design concepts, some decisions on the desired level of control
may be based on present and anticipated requirements of products and manufacturing
2. Operational Influences
Both internal and external conditions are important factors that can affect the microbiological
2

quality of the plant environment. These diverse factors are taken into consideration when de‑
termining facility design as well as the frequency of microbiological monitoring.
Some examples of internal influences are:

• Start-up of air conditioning or heating systems

• Construction
• Duct and vent cleaning
• Modifications to equipment
• Plant alterations
• Equipment maintenance
• Change in activity level

Some examples of external influences are:

• Construction
• Surrounding environment (farm lands, wet lands)

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 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT SECTION 2

• Climate (temperature, humidity)

• Seasonal changes

B. Manufacturing and Filling Equipment

Equipment that is constructed for effective cleaning and sanitization and is designed to protect
products from contamination is recommended. When designing or purchasing new manufacturing
or filling equipment, microbiology, quality, engineering, and manufacturing personnel should eval‑
uate this equipment for the ease of cleaning and sanitization in addition to other factors such as cost
and efficiency. Some useful factors that influence sanitary maintenance of this equipment include:

• Equipment drawings and/or schematics, which can provide helpful guidance for cleaning and
sanitization protocols (Refer to Section 3 - Cleaning and Sanitization).
• Equipment manufactured from smooth, nonporous materials, for example 316L stainless steel
• Valves and gauges that are easily disassembled for cleaning
• Equipment made from materials that are compatible with products and cleaning and sanitiza‑
tion solutions

2
In the PCPC Quality Assurance Guidelines, Annex 3 which covers packaging and processing

PLANT ENVIRONMENT
EVALUATION OF THE
equipment gives important direction on construction, cleanability, and related items.8,9

C. Personnel
Personnel are encouraged to practice good personal hygiene. Wearing clean uniforms and appro‑
priate clothing like, head covers, beard covers, clean gloves or finger cots will help prevent con‑
tamination. Adequate locker room facilities, washrooms, and eating areas should be physically
separated from the manufacturing, filling, and packaging areas of the plant.
It is recommended that employees responsible for sanitation and housekeeping be thorough‑
ly trained in all pertinent procedures as part of an ongoing training program. All employees
involved with manufacturing and packaging should be trained to follow Personal Care Good
Manufacturing Practices through a regular training program.10 Training programs are most effec‑
tive if documented and conducted periodically according to a pre-planned schedule. For more
information, refer to Section 3(Cleaning and Sanitization).

D. H ousekeeping
The general plant environment should be maintained in a clean and orderly state. For example,
cleaning and /or sanitization of floors, walls, ceilings, vents, pipes, fixtures and equipment exteri‑
ors should be conducted on a regular schedule according to written operating procedures. Equip‑
ment, hoses, tools, and other items not in use should be stored in a clean state and protected from
contamination. Personal Care facilities should have effective programs for control of rodents and
other pests, and for proper refuse disposal. For additional guidance, refer to Annex 2 - Premises
(Facility) in the PCPC Quality Assurance Guidelines.11

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

E. Cleaning and Sanitization

Equipment cleaning and sanitization is carried out on an established schedule, usually between
batches of different products, according to written procedures. It is important to assure that
cleaning and sanitization is documented and validated, that equipment is identified as to sanitary
status, and that cleaned and / or sanitized equipment is kept dry and covered. Refer to Section 3
(Cleaning and Sanitization) for information on appropriate procedures, frequencies, expiration
times, and validation processes.
See the PCPC Quality Assurance Guidelines8 and Section 9 (Microbiological Validation and
Documentation) for additional guidance.

ENVIRONMENTAL MONITORING (EM) PROGRAM

The objective of an environmental monitoring program is to obtain microbiological data that can
serve as indicators of change in the environment as well as to document the state of control of the
facility. An effective EM program should promptly ascertain the potential areas of concern for intro‑
duction of contamination and/inactive of change in the environment. Monitoring these indicators
allows for a preventative action to be taken before any microbiological contamination occurs. Cri‑
PLANT ENVIRONMENT

teria are determined based on in-house needs.

EVALUATION OF THE

The following are among the elements to be considered when establishing an environmental mon‑
itoring (EM) plan.

A. Training
Personnel involved with environmental sampling should be properly trained according to a writ‑
2

ten procedure applicable to such testing, which includes at least the following:

• Methods and materials for collecting and processing samples

• Appropriate areas for monitoring
• Frequency of monitoring
• Interpretation of test results
• Determination of alert and action levels
• Proper documentation and communication of results
• Corrective action procedures

B. Documentation
Documentation provides an organized record of the microbiological evaluation. It is recommend‑
ed that the following information be included for proper documentation of an environmental
monitoring program:

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 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT SECTION 2

1. Procedural information

• Physical location (manufacturing area, warehouse, etc.)

• Sampling site
• Sampling method
• Collection / recovery medium used
• Incubation time and temperature
• Sampling frequency

2. Reported Data

• Specific site sampled

• Media quality control information
• Date and time of day sample collected
• Weather conditions
• Activities occurring near the sampling site at time of sampling
• Results

2
• Date and time
• Signature of investigator (a microbiologist or suitably trained individual)

PLANT ENVIRONMENT
EVALUATION OF THE
Additional information may be included based on in house needs or company policies.12

C. Baseline Data
Periodic microbiological monitoring of physical surfaces should be performed to determine the
baseline levels of the microbial flora within the different areas of the manufacturing environment.
Baseline variations in the microbial levels may be variable depending on internal and external
conditions. Operational influences are summarized previously in this section in the discussion of
the “Manufacturing Environment.”
Statistical analysis of environmental historical microbiological test data may be used to set the
alert and action level criteria for deciding when to investigate a shift in the trend. It is common
practice to periodically reevaluate the alert and action levels. There are several statistical methods
for evaluating the data.12,13

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

SURFACE SAMPLING
A. Surfaces14,15
The microbiological quality of physical surfaces within a manufacturing environment can direct‑
ly or indirectly affect the microbial quality of finished personal care products. Physical surfaces
coming into direct contact with finished product may include:

• Bulk raw material storage vessels or containers

• Intermediate and finished product storage vessels
• Processing equipment
• Filling equipment
• Transfer pumps and lines
• Pumps
• Valves
• Utensils
• Ancillary equipment and other working contact surfaces

Surfaces not coming in direct contact with the product that could affect microbiological quality
PLANT ENVIRONMENT

may include:
EVALUATION OF THE

• Walls
• Floors
• Ceilings
• Overhead lighting and piping
• Vertical and horizontal support beams
2

• Overhead walkways
• External processing and filling equipment surfaces
• Pallets and forklift trucks
• External packaging materials
• Air handling systems

B. Monitoring Frequency
The type and frequency of microbiological monitoring of physical surfaces depend on the sus‑
ceptibility of the finished product to microbial contamination as well as other factors. Additional
factors to be considered are the scale of manufacturing, condition and design of plant, type of
process, local environmental factors, and company policies. Areas that are in direct contact with
finished product are usually monitored more frequently than areas that are not.
In areas directly contacting product, any increase from predetermined microbial alert and ac‑
tion levels may indicate a potential microbiological problem that could affect product quality.
Increased frequency of testing, adjustments to cleaning and sanitization protocols, or changes in

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 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT SECTION 2

other processes may be indicated if a potential microbiological problem is found to potentially

affect product quality. Supervisory personnel should routinely review the microbial test data gen‑
erated and, if required, adjust the frequency of environmental monitoring.

C. Methods
Sampling by means of swabs, direct contact devices, or contact plates are the most common
methods of monitoring surfaces for microbial contamination.16,17 Note that swabs and contact
plates will not recover total microbial bioburden from a surface.
Exit monitoring of rinse water can be used to evaluate interior surfaces of manufacturing and
filling equipment. However, rinse water testing may not be useful in detecting the presence of
biofilm bacteria.18 See Table 2-1 for different surface sampling methods.
1. Swabbing: Sterile swabs can be used to sample environmental surfaces for the presence of
microbial contamination. The sterile swab is wetted in sterile buffer, saline solution, or broth
and rubbed over a measured portion of the surface to be monitored. The swab is then either
streaked across an agar plate or placed into a sterile broth tube. The plate or tube is incubated
for the appropriate length of time.

2
a. Swabs: Swabs can be used on flat, irregular surfaces and on hard-to-reach areas. The three

PLANT ENVIRONMENT
EVALUATION OF THE
most common composition materials for swabs are dacron, cotton wool, and calcium al‑
ginate. Calcium alginate is a fibrous material that dissolves in sodium citrate or sodium
hexametaphosphate, a characteristic that facilitates the total release of microorganisms that
have been recovered on the swab from the surface. This allows for a quantitative analysis.19
Leachables from cotton wool swabs, such as fatty acids, may be inhibitory or detrimental
to microbial growth.20 Whichever type of swab is used, all on-going testing should be per‑
formed with the same type of swab and be processed as soon after collection as feasible.
In cases where there is a delay (e.g., swab samples need to be shipped to a laboratory for
processing and analysis), transport swabs may be used. Transport swabs are designed to
maintain the viability and numbers of microbes present at the time of sampling until the
time of processing. The swab manufacturer should be consulted for storage and temperature
conditions to determine how long after use a transport swab can maintain the viability of
microorganisms.21
b. Sampling: Using aseptic technique, the sample site is sampled by rubbing a pre-moistened
swab over the surface. Moistened swabs are essential for recovery of the highest possible
numbers of bacteria, molds or yeasts. Solutions used to wet the swab may be, but are not
limited to, the following: sterile buffer, saline solution, or broth.
If sanitizer or disinfectant residues are present on the surfaces being sampled, they may
interfere with the test results. All of the solutions used to wet swabs should contain a neu‑
tralizer if disinfectant or sanitizer residues are expected.
An appropriate cleaner such as 70% alcohol should be used after sampling to remove swab
residue. A sterile template (e.g., 2 x 2in. area) may be used to standardize the amount of
surface area sampled.

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

c. Processing Swabs: Three basic techniques are commonly used to process swabs after sampling
a surface.
i. Direct Swab Methods. After a swab has been used to sample a test surface, it can either be
streaked directly onto an agar surface in a Petri dish or it can be added to an enrichment
broth as described below. A variety of media both general and selective, may be used, e.g.,
Trypticase Soy Agar, Pseudomonas Isolation Agar, MacConkey, Sabouraud Dextrose, etc.
If general and selective media are used, inoculate the general media first. If the selective
media is inoculated first, inhibitory ingredients may be carried over to the general media
and prevent growth. Include a neutralizer in the media if the sanitizer/disinfectant resi‑
dues on the sampled surface may interfere with the test results.
This technique may be used for testing those surface areas on which low numbers of mi‑
croorganisms are expected and for which a quantifiable result is needed. Streaked Petri
dishes or enrichment broth are incubated for the appropriate period and temperature.
Note: The use of selective agars when directly plating swabs can be inhibitory to injured
microorganisms. The results, either microbial growth or no growth, may not be repre‑
sentative of the types of microorganisms that are actually present on the surface. Unless
looking for specific types of microorganisms, the use of general microbial growth media
or enrichment techniques may give more useful information in an environmental moni‑
PLANT ENVIRONMENT

toring plan.
EVALUATION OF THE

Test results may be recorded as:

• Growth or no growth per swab

• Growth or no growth per unit area (e.g., per square inch or square centimeter)
• If low numbers are recovered, individual colonies may be counted and recorded as the
number of microorganisms per swab or unit area
2

ii. Swab Enrichment Methods. This technique may be used in areas where low numbers of
microorganisms are expected. After sampling a test surface, aseptically transfer the used
swab directly into a test tube of enrichment broth. Include a neutralizer in the enrich‑
ment broth if there is a concern that sanitizer/disinfectant residues on the sampled surface
may interfere with the test results. Incubate the test tube with swab for the appropriate
period and temperature.
Test results may be recorded as:

• Growth or no growth per swab, based on the presence or absence of turbidity in a gen‑
eral enrichment broth or
• Growth or no growth per unit area (e.g., per square inch or square centimeter)

iii.Standard Plate Count Methods. This technique can be used in those areas in which there
could be either high or low numbers of microorganisms. After sampling the test surface,
aseptically transfer the swab into a sterile test tube containing 10 milliliters of enrichment
broth. Enrichment broth may include neutralizer(s) for sanitizer/disinfectant residues.
Vortex the test tube to release microorganisms from the swab into the broth. If sampling

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 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT SECTION 2

with calcium alginate swabs, sodium citrate or sodium hexametaphosphate may be used
to dissolve the swab and aid in releasing recovered microorganisms from the swab. Re‑
move aliquot(s) of the broth and plate onto a general microbial growth agar medium
and/or onto selective / differential agar media. Agar media may include a neutralizer(s)
for sanitizer/disinfectant residues that may have been picked up by the swab in sampling
test surfaces. If high numbers of microorganisms are expected, further dilute the original
swab dilution sample and plate these aliquots.
To record results, count the colonies on countable plates, multiply by the dilution factor
and record as number of microorganisms per swab, or per unit area (e.g., per square inch
or square centimeter).
2. Contact Sampling: Contact sampling may be performed by using either modified Petri dishes
(i.e., RODAC plates), paddles, or flexible films, which contain a solid agar culture medium
whose convex surface extends above the carrier. Selective and non-selective agar media may be
used. The sterile agar surface is applied to the test surface so that the agar makes total contact
with the area being sampled. An appropriate cleaner such as 70% alcohol is used after sampling
to remove any remaining agar residue. The sampling devices are incubated, after which the
degree of microbial contamination per unit area can be determined. Factors to be considered
when choosing one of these methods are:

2
• Suitability for flat surfaces only

PLANT ENVIRONMENT
EVALUATION OF THE
• Usefulness in remote areas under field conditions
• Commercial availability of disposable units
• Suitability for qualitative/quantitative analysis of environmental cleaning and sanitization
procedures
• Limited shelf life
• Cost
• Problem of confluence, with certain microorganisms, especially if agar surface is wet

3. Rinse Water Method This technique is generally used to sample either equipment interior
surfaces (e.g., kettles, tanks etc.) that cannot be reached using a swab technique or other hard
to access surfaces that come into direct contact with finished product. The rinse water meth‑
od consists of flushing the selected surface with a suitable volume of sterile rinse water, next
collecting a sample of the rinse water, and then quantitatively determining the number of mi‑
croorganisms in the sample. Membrane filtration, pour plates, spread plates or Most Probable
Number (MPN) procedures can be used to quantify the microbial recovery. Advantages of
rinse water method include:

• Surfaces can be selectively tested using this technique

• Chance of introducing testing contamination is minimal
• Allows testing of otherwise inaccessible areas
• Can be used to monitor the efficacy of equipment-sanitizing procedures
• Rinse water monitoring may not detect biofilm bacteria that may adhere to the interior
equipment and transfer line surfaces

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

D. General Applications
Physical surfaces coming into direct contact with the product should be examined for the pres‑
ence of bacteria and fungi that are known to cause product spoilage, or harm to the consumer.
Indirect surfaces such as walls and floors should also be monitored to determine background
levels of microorganisms that are intrinsic to the manufacturing environment.
In general, all equipment (processing and filling), valves, traps and working surfaces should be
monitored on a defined and periodic basis. Transfer lines should be taken apart and tested. Viable
microbial counts should be performed to determine the levels of microorganisms that are present
in these areas.6

AIR SAMPLING
The selection of sites for air sampling is based primarily on the potential for adverse microbiological
effects on the finished product. Routine air monitoring may include selected environmental sites
within the facility as well as sources of compressed air used in manufacturing. Factors to take into
consideration when developing an environmental air monitoring program include room design,
airflow pattern, proximity to vents, and potential for product exposure.19-22
PLANT ENVIRONMENT
EVALUATION OF THE

A. Monitoring Frequency
The air-monitoring program establishes the frequency of routine sampling at each location based
on in-house needs, with the areas of greater microbiological concern being monitored more fre‑
quently. The schedule for air monitoring in each designated area is based upon previously de‑
termined microbial baseline levels. Monitoring frequency is determined in part by the type of
activities in each area, such as machine operation, personnel, physical cleaning, construction, etc.
2

Seasonal changes and climate are also important considerations when establishing an air-sam‑
pling program. Areas of greater microbiological concern, such as exposed product and raw mate‑
rials are usually monitored more frequently.
Supervisory personnel, the plant microbiologist, or other or suitably trained individual should
review and analyze the microbial test data generated during air sampling. These data can be used
for trend analysis and provide a history of the plant environment, which can be used to evaluate
sampling frequency or investigate shifts in microbiological quality.

B. Methods
A variety of methods may be employed for environmental and compressed air sampling. Each is
designed to meet specific needs.23-25 Some sampling methods measure all particulates, including
viable and non-viable microorganisms. Others only measure viable organisms. Consider the fol‑
lowing factors when choosing an air-sampling method:

• Ability to determine change of air contamination over time

• Anticipated bioburden (quantity, viability, type)

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• Collection medium
• Quantitative vs. qualitative measurement
• Ability to determine the number of colony-forming units per unit of time or volume sampled

The monitoring method(s) chosen will be influenced by the requirements of the individual facil‑
ity. The requirement or need to measure only viable microorganisms versus all other particulate
matter should be determined.
1. Viable Methods The most commonly used methods for measuring viable organisms, many of
which are available commercially, are listed below. Also see Table 2-2.
a. Settling Plate A Petri dish containing Trypticase Soy Agar or other suitable general microbial
growth agar is directly exposed in the sampling area (i.e., placed upright in the area with
the lid off). Particles in the air settle out on the agar surface. After a specified exposure time,
the Petri dish is collected, covered with the lid, and incubated. The number of microbial
colonies is counted directly from the plate.
b. Centrifugal Air Sample Air is collected via centrifugation through impeller blades and micro‑
organisms are deposited onto the surface of a nutrient agar medium in a strip. The growth
agar strip is incubated and the number of organisms per volume of air is calculated.

2
c. Sieve Impaction Sampler Air is drawn into the unit through a sieve and over the surface of an

PLANT ENVIRONMENT
EVALUATION OF THE
agar plate. After incubation of the plate, the number of viable microorganisms per unit of
time or volume of air may be determined.
d. Slit-To-Agar Sampler Microorganisms are impinged directly onto a microbial growth agar
surface in a Petri dish that rotates beneath a slit opening. Air is drawn through this slit by
the use of a vacuum. The speed of the Petri dish rotation and the volume of air sampled can
be adjusted. After incubation of the plate, the number of viable microorganisms per unit of
time or volume of air can be calculated.
e. Liquid Impinger Air is drawn through a sampler tube, and particles are collected in liquid
medium. The air rises and is removed from the system. Serial dilutions of the liquid medium
are made, and duplicate aliquots are plated into empty Petri dishes to which a sterile melted
microbial growth agar is then added. The Petri dishes are allowed to solidify and are incu‑
bated. After incubation, the number of microbial colonies is counted per Petri dish, and an
average is calculated for each duplicate serial dilution.
f. Multi- Stage Particle Sizing Sampler The sampler contains up to six plates, arranged vertically.
A measured volume of air is drawn through successively smaller holes in the sieve plates,
resulting in acceleration of the particles at each stage. Viable particle size distribution is then
calculated from the plate counts at each stage.
g. Membrane Filter Air to be sampled is impinged on a gelatin membrane filter, which is then
removed from the filter holder and placed in a dish containing a general microbial growth
medium. After an appropriate incubation period, the number of microbial colonies on the
membrane surface is counted.

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

2. Non-Viable Methods Monitoring of non-viable airborne particulates is outside the scope of

the guideline. Standards for air based on particulate matter counts are addressed elsewhere.25,26
A method that makes use of optical particle counts is commonly employed.
3. Compressed Air Sampling Compressed air that comes into direct contact with the product
process or that could adversely affect the manufacturing environment may be monitored.
The Slit-to-Agar method has been modified for sampling compressed air. This instrumentation,
adapted for sampling compressed gas up to pressures of 125 psi, is based on the impingement
principle of particle capture. The circular sweeping of an agar plate surface is controlled at a
critical distance beneath a laser cut air intake slit and creates a radial undulation over the area of
impingement. The speed of the plate rotation and sampling are precisely controlled so that, after
a period of incubation, the growth on the agar surface can be used to quantitatively measure mi‑
crobial contamination.

EVALUATION OF RESULTS
There are no set criteria for microbiological monitoring of the environment in plants manufactur‑
ing personal care products. Trend analysis performed on the data from microbiological monitoring
PLANT ENVIRONMENT

of surfaces and air in the plant offers a useful evaluation tool. Shifts from established data patterns
EVALUATION OF THE

may indicate changes in the environment or work practices that may have the potential to affect the
microbial quality of the finished product.
In the evaluation of environmental test results, alert and action levels should be established for each
manufacturing area. Levels set will depend on the areas monitored, the historical trend data from
the area, the type of monitoring, and potential effects on finished product quality.
2

A. Factors to Be Considered in Setting Alert and Action Levels

Many factors influence the microbial alert and action levels established for each production area.
These include, but may not be limited to:
1. Objective of the Sampling whether, for example, to measure seasonal changes in air quality, to
determine effectiveness of a sanitization procedure, or to monitor changes in work practices.
2. Area Sampled Microbial criteria for air in a warehouse differ from the criteria for air over a fill‑
ing machine. Criteria for the surface of cleaned and sanitized compounding equipment differ
from those in areas that do not directly contact product.
3. Type of product The type of finished product being manufactured, i.e., hostile vs. microbi‑
ologically susceptible, is an important aspect. Susceptible products that are likely to be more
sensitive to microbial contamination by environmental influences are expected to require more
stringent controls in processing and packaging areas.

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B. Documentation
Documentation is important in establishing the historical trend data in the plant. The trend anal‑
ysis of microbial recovery levels in the environmental may be useful when

• Investigating potential sources of microbial contamination in a product

• Identifying sources of microbial contamination in the plant
• Identifying potential seasonal trends
• Setting and adjusting cleaning and housekeeping schedules
• Choosing sanitizers

Once a program is in place, results generated should be documented by keeping an organized record
system. Records of environmental monitoring should be maintained for appropriate length of time.
These data can be used to establish a normal range of results. Supervisory personnel, a microbi‑
ologist, or other qualified individual should routinely review all results. Based on these results, a
feedback mechanism can be established whereby all departments involved are informed when the
environmental quality is outside of the normal range. The responsibility for documentation and
communication should be addressed by internal standard operating procedures.

2
Refer to “Microbiological Documentation and Validation” (Section 9) and to “Production and

PLANT ENVIRONMENT
EVALUATION OF THE
Control Documentation.”27

C. Interpretation
Once all the data have been collected, consideration of the following information will help in
their interpretation:

• Total number of microorganisms recovered (quantitative analysis)

• Percent of positive results as compared to the total number of areas tested (qualitative analysis);
for example, this might be useful when using qualitative swabs
• Presence or absence of objectionable organisms.

D. Alert / Action Level Response

Regardless of the assessment method used, once a normal range of microbial recovery has been
established, the manufacturer should set alert and action levels for all areas that are routinely
monitored. If microbiological test results reach an alert level, some actions may include the fol‑
lowing: collecting additional samples, observing the manufacturing area, evaluating the process,
increased testing of finished goods, reviewing practices, etc.
1. Investigation If microbiological test results reach an action level, a complete investigation is
indicated, followed by corrective actions. At a minimum, these include:

• Confirm out of specification results

• Microbiology laboratory interview/investigation

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

• Review of product logs/records

• Review of cleaning/sanitization procedures
• Review of product and area history
• Interview personnel (production and laboratory areas)

2. Corrective Actions Depending on the outcome of an investigation, corrective actions may

include:

• Retesting of the affected areas (Depending on the extent of the problem, manufacturing or
filling may be delayed until all environmental testing is complete.)
• Recleaning and resanitization of equipment, floors, walls, etc.
• Additional testing of finished product to ensure its quality
• Retraining/reinforcement of cleaning and sanitization procedures
• Modification of engineering controls
• Modification of practices or processes
• Documentation of corrective action taken
PLANT ENVIRONMENT
EVALUATION OF THE

CONCLUSION
A microbiological monitoring program for the plant environment is a tool to help assure the mi‑
crobiological quality of products manufactured for the Personal Care industry. An environmental
monitoring program includes the microbiological monitoring of surfaces, air, raw materials, and
product. The value of the program depends on adequately trained testing personnel, documentation
of test data, assessment of results, and appropriate response. Once a microbiological profile of the
plant has been established, trend analysis can be used to develop alert and action criteria. Changes
2

in the trend may indicate the need for an investigation, after which a correction action plan may
be implemented. Each step of the process should be accompanied by appropriate documentation.

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Table 2-1

SURFACE-SAMPLING METHODS
Method Description Advantages Disadvantages

Swabs An environmental swab is a • Inexpensive • Leachables from cotton

sampling device comprised • Convenient may inhibit fastidious
of a synthetic (e.g., dacron, • Suitable for irregular microorganisms
calcium alginate) or cotton tip surfaces • Microorganisms may
affixed to a wood or plastic • Calcium alginate tips can be become entrapped in the
stick. It is used to sample dissolved in media to release swab head
discreet areas in difficult to all microorganisms collected
reach locations or irregular • Can be used in highly
surfaces. contaminated areas
• Can be utilized in remote
areas under field conditions
• Can be qualitative or
quantitative

Contact A contact plate may be • Can be used in remote areas • Short shelf life under field
Plating modified Petri dishes, paddles • Samples the same size area conditions
or flexible films containing each time as defined by the • Not suitable for irregular
a solid microbial growth size of the device surfaces
agar whose convex surface • Can be qualitative or • Microbial overgrowth may

2
extends above the carrier. quantitative be a problem
The sampling device may • May leave a residue or
contain any of a number of microbial growth agar on

PLANT ENVIRONMENT
EVALUATION OF THE
various types of microbial the surface after being
growth agar with or without sampled; the agar media
a disinfectant/sanitizer residue needs to be
neutralizing agent. removed.

Rinse Water The rinse water technique • Can be used to test • May not detect the presence
consists of flushing the otherwise inaccessible of biofilm bacteria.
surface to be tested with a areas such as the interior • Not suitable for many
sterile rinse solution such as equipment surfaces of applications.
water. manufacturing equipment. • Extensive manipulation may
• Larger surface areas may be be required.
sampled. • Sample processing may
• Quantitative affect test results

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 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

Table 2-2

AIR-SAMPLING METHODS28
Method Description Advantages Disadvantages

Sedimentation An open Petri dish • Easy • Cannot determine the amount

(i.e., Settling filled with a microbial • Least expensive of air sampled
Plates) growth agar that is • Constant surveillance not • Microbial count cannot be
exposed for a select necessary correlated with air volume
time period (i.e., 15 to • Large number of areas can be • Disposition of colonies is
60 minutes) that is left monitored in a short amount affected by size of particles,
exposed to the air. of time temperature, and flow /volume
• Any type of microbial growth of air passing across surface
Provides a rough agar media can be used • Affected by air movement in
estimate of airborne • No sampling device required area.
contaminants. • If left exposed for a long period
of time, plates can desiccate

Centrifugal Lightweight, portable • High recovery efficiency • Requires special agar strips that
Impactor unit that measures a • Portable are expensive and available only
quantifiable volume • Speed and ease of operation from the manufacturer
of air (1 to 1,000 liters). • Excellent for areas that are • Strips have a limited shelf life
The sampling media difficult to access • Overgrowth of strips in heavily
are agar strips. • Measures concentration of contaminated areas
viable particles as function of
time and unit volume of air
PLANT ENVIRONMENT
EVALUATION OF THE

• Selective/differential agar
strips are available.

Sieve Air is drawn through a • Colony overlapping minimal • May be cumbersome if used
Impaction uniformly perforated • Large air volumes possible with a vacuum
Sampler surface and is • Portable
distributed over an • Air flow can be calibrated
agar surface. • May be used to sample
compresses air when used
with a vacuum
• Choice of agar dish size and
2

media is flexible

Slit to Agar Air is pulled through • Measures concentration of • Vacuum source required
Sampler a slit over a revolving viable particles as function of • Not easily portable
plate. volume of air • Large numbers of sampling
• No serial dilution or plating areas are needed, very time
required consuming.
• Wide application in • Electrical connection required.
surveillance of ambient air • Best suited for clean rooms
contamination • Some systems require 150 mm
• Volume and speed adjustable agar plates
• Constant surveillance not
necessary
• Remote sampling probe can
be used

Liquid Air is drawn through • Samples with high viable • Low sampling rate
Impingement a sampler tube and counts can be diluted for • Vacuum source required
particles are collected enumeration • Time consuming
in liquid medium. • Quantitation is good for • Requires dilution and plating
Microbial counts are spores and vegetative cells • Breaks up bacterial particles
determined in the • Inexpensive • Device may consist of breakable
liquid. glass components

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 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT SECTION 2

Table 2-2

Method Description Advantages Disadvantages

Sieve A specific volume of • Determines size of particles • Limited sampling duration does
Multistage air is drawn through a • Portable not provide entire picture
Particle Sizing series of sieve plates, • Measures concentration of • Requires many plates
Sampler resulting in particle viable particles as a function • Vacuum required
size separation. This of time • Not well adapted for heavily
allows plate counts at • No serial dilution or plating contaminated areas.
each stage. required • Agar desiccation
• Comparable to the impingers

Membrane Air is drawn through • Large volume of air • Equipment cumbersome

Filter a gel filter disc, • 3μm pore size retains Coli- • Additional manipulation of
which is then placed phages membranes
on an agar surface • Gelatin overcomes
for enumeration of desiccation
microorganisms.

2
PLANT ENVIRONMENT
EVALUATION OF THE

PCPC MICROBIOLOGY GUIDELINES | 27

 SECTION 2 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT

REFERENCES
1. United States Pharmacopeia and the National Formulary (USP39-NF34) 2016 (U.S.
Pharmacopeia, 1260 Twinbrook Parkway, Rockville, MD 20852).
2. Parenteral Drug Association. 2014. “Technical Report No. 13 (Revised) Fundamentals of
Environmental Monitoring Program,” PDA. Parenteral Drug Association, Bethesda, MD.
www.pda.org
3. U.S. Food & Drug Administration. 2015. FDA 21 CFR, Part 210 Current Good
Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs; General.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=210
4. U.S. Food & Drug Administration. 2015. FDA 21 CFR, Part 211 Current Good
Manufacturing Practice for Finished Pharmaceuticals. https://www.accessdata.fda.gov/scripts/
cdrh/cfdocs/cfCFR/CFRSearch.cfm?CFRPart=211
5. U.S. Food & Drug Administration. 2001. Guidance for Industry, Q7A Good Manufacturing
Practice Guidance for Active Pharmaceutical Ingredients. http://www.fda.gov/ICECI/
ComplianceManuals/CompliancePolicyGuidanceManual/ucm200364.htm
6. Mortimore, S., and Wallace, C. (eds.) 2013. HAACP: A Practical Approach. Third Edition,
PLANT ENVIRONMENT

Springer, NYC, NY.

EVALUATION OF THE

7. Nikitakis, J.M., ed. 2014. PCPC Quality Assurance Guidelines, The Personal Care Products
Council, Washington, DC 20036
8. Nikitakis, J.M., ed. 2014. Annex 3 – Equipment – Part 1: Packaging, In PCPC Quality
Assurance Guidelines, The Personal Care Products Council, Washington, DC 20036, pp 17-20.
9. Nikitakis, J.M., ed. 2014. “Annex 3 - Equipment: Part 2 – Processing., In PCPC Quality
Assurance Guidelines, The Personal Care Products Council, Washington, DC 20036, pp. 21-30.
2

10. Nikitakis, J.M., ed. 2014. Annex 1 Personnel and Training. In PCPC Quality Assurance
Guidelines, The Personal Care Products Council, Washington, DC 20036, pp1-6
11. Nikitakis, J.M., ed. 2014. Annex 2- Premises(Facility) , In PCPC Quality Assurance Guidelines,
The Personal Care Products Council, Washington, DC 20036, pp. 7-16.
12. Parenteral Drug Association. 2014. Fundamentals of Environmental Monitoring Program
Technical Report No. 13 (Revised). Parenteral Drug Association, Bethesda, MD 20814.
www.pda.org
13. Ibid., p. 8-9
13. Reich, R.R., Miller, M.J. 2003. Developing a Viable Environmental Monitoring Program for
Nonsterile Pharmaceutical Operations. Pharmaceutical Technology 27: 92-100.
14. American Society for Testing Materials. 2015. ASTM Standards on Materials and
Environmental Microbiology. ASTM, West Conshohocken, PA 19428-2959

28 | PCPC MICROBIOLOGY GUIDELINES

 MICROBIOLOGIC AL EVALUATION OF THE PLANT ENVIRONMENT SECTION 2

15. Block, S.S., ed. 2000. Disinfection, Sterilization, and Preservation, 5th Edition, Lippincott
Williams & Wilkins, Philadelphia, PA.
16. Dyer, R.L., Frank, J.F., Johnson. B., Hickey, P., and Fitts, J. 2004. Microbiological Tests for
Equipment, Containers, Water, and Air, In: Standard Methods, For the Examination of Dairy
Products, 17th Edition, Wehr HM and Frank, JF (Eds.), American Public Health Association,
Washington, DC.
17. Lemmen, S.W., Hafner, H., Zolldan, D. Amedick, G., Lutticken, R. 2001. Comparison of
two sampling methods for the detection of Gram-positive and Gram-negative bacteria in the
environment: moistened swabs versus Rodac plates, Int. Journal of Hygiene and Environmental
Health 203: 245-248.
18. U.S. Food and Drug Administration, 2014. Guide to Inspections - Validation of Cleaning
Processes (7/93) www.fda.gov/ICECI/inspections/inspectionguides/ucm074922 p. 5.
19. Parenteral Drug Association. 2014. Fundamentals of Environmental Monitoring Program
Technical Report No. 13 (Revised)., sec. 4.4.3.2.3, p. 20. Parenteral Drug Association, Bethesda,
MD 20814, p. 20.
20. Tille, P. (ed). 2013. Bailey and Scott’s Diagnostic Microbiology, Thirteenth Edition, Mosby/

2
Elsevier, Philadelphia, PA.

PLANT ENVIRONMENT
EVALUATION OF THE
21. Hindiyeh, M., Acevedo, V. and Carroll, K.C. 2001. Comparison of Three Transport Systems
(Starplex StarSwab II, the New Copan Vi-Pak Amies Agar Gel Collection and Transport Swabs,
and BBL Port-A-Cul) for Maintenance of Anaerobic and Fastidious Aerobic Organisms. J. Clin
Microbiol. 39:277-380.
22. Powitz, R. W. 2002. ASampling of Airborne Biological Contaminants: A Rational Approach.
http://www.cemag.us/articles/2002/01/sampling-airborne-biological-contaminants-rational-
approach
23. Mehta, S., Bell-Robinson, D., Groves, T., Stetzenbach L., Pierson, D. 2000. “Evaluation of
Portable Air Samplers for Monitoring Airborne Culturable Bacteria. AIHA Journal 61:850-4.
24. Shelby, S., Grinshpun, S., Willeke, K., Terzieva S., Ulevicius V., Donnelly, J. 1995. Effect
of Impact Stress on Microbial Recovery on an Agar Surface@ Applied and Environmental
Microbiology. 61:1232-1239.
25. ISO 2015. ISO 14644-2 Cleanrooms and associated controlled environments — Part 1:
Classification of air. cleanlinesswww.iso.org.
26. ISO 2015. ISO 14644-2 Cleanrooms and associated controlled environments — Part 2:
Specifications for testing and monitoring to prove continued compliance with ISO 14644-1, ISO,
Geneva, Switzerland (2000). http://www.iso.org.
27. Nikitakis, J.M., ed. 2014. Annnex 5- Production Control In: PCPC Quality Assurance
Guidelines, The Personal Care Products Councilt, Washington, DC 20036, pp. 35-44 (December
1992).
28. Hess, K. 1996. Environmental Sampling for Unknowns, CRC Press, Boca Raton, FL.

PCPC MICROBIOLOGY GUIDELINES | 29

30 | PCPC MICROBIOLOGY GUIDELINES
 SECTION 3

Cleaning and Sanitization

INTRODUCTION
Effective cleaning and sanitization programs are essential to ensure microbial quality in the manu‑
facture of personal care products. Cleaning procedures and sanitization procedures should be val‑
idated in order to consistently meet hygienic manufacturing requirements. The design of cleaning
and sanitization procedures should take into account the product formulation, engineering design
of equipment, and all aspects of manufacturing.

GENERAL CONSIDERATIONS
Specific internal programs for cleaning and sanitization should be established. These programs are
essential to:

• Prevent ingredient cross-contamination

• Assure the microbiological quality of the product
• Meet legal regulations where required

3
• Minimize the microbial load contributed by processing, filling, and storage equipment
• Avoid the time and resources associated with microbial failures

SANITIZATION
CLEANING AND
• Maintain integrity and condition of the equipment

If cosmetics and drug products are manufactured with the same equipment, refer to FDA guide‑
lines for the manufacture of Over-the counter (OTC) drugs.1-3 Factors such as active ingredient and
cleaner residues are important areas of cleaning and sanitization but are beyond the intended scope
of this document. For the purpose of this document, the following definitions apply:

• Cleaning - the process of removing product residue and contaminants such as dirt, dust,
and grease from surfaces. Cleaning is an essential step that needs to be performed before the
performance of a sanitization procedure. It is important that personnel involved in cleaning
have a working understanding of the nature of different types of soils and the chemicals
required for their removal.
• Sanitization - the process utilized to reduce viable microbial contaminants to an acceptable
level such as the microbial count specification of the finished product. All surfaces must be
clean for the sanitization procedure to be effective.
PCPC MICROBIOLOGY GUIDELINES | 31
 SECTION 3 CLEANING AND SANITIZATION

• Validation - the process of substantiating that the process does what it purports to do.
• Documentation - the process of organizing all relevant information in an orderly and easily
understood format. This documentation is required to support the validation of a process
and to maintain a historical record of the process and equipment usage.

Guidance for the development of operating procedures is addressed in each section below, as ap‑
propriate. Written protocols are required prior to attempting to validate any process. For more
information, see the “Microbial Validation and Documentation” (Section 9) and “Microbiologi‑
cal Evaluation of the Plant Environment”(Section 2). Additional information on cosmetics Good
Manufacturing Practices can be found in the Personal Care Product Council’s “Quality Assurance
Guidelines.”4 In addition, an understanding of the importance of biofilms5-7 and application of haz‑
ard analysis of critical control points (HACCP)7-9 should also be considered.

Biofilms
A biofilm5-7 is a community of mixed microorganisms encased in an extracellular polymer matrix,
which they produce and secrete. Bacteria living in a biofilm may behave as a unit or multicellular
organism. Biofilms have the potential to develop in most aqueous systems including process water
systems and on those areas of manufacturing equipment that is difficult to clean and sanitize. Bio‑
films can be also be present on equipment surfaces that appear to be visually clean.

Biofilm Characteristics
• Once formed, biofilms become continuous sources of microbial contamination as microor‑
ganism clumps or aggregates will break from the biofilm and surfaces are colonized down‑
stream and potentially contaminate finished product.
• Although thermal methods (e.g., 65 to 85°C hot water or steam) kill microorganisms within
a biofilm, they are not effective in removing an established biofilm. Killed but intact biofilm
can become a nutrient source for rapid biofilm regrowth.
• Routine microbial sampling of water systems or equipment surfaces may not be able to de‑
tect the presence of biofilms because they are not dispersed homogeneously.

Microorganisms living in a biofilm on equipment are more difficult to kill and subsequently remove
CLEANING AND
SANITIZATION

than free floating organisms because they are protected by the polysaccharide matrix structure of the
biofilm. For chemical sanitization to be effective against biofilms, it must be performed frequently
in order to minimize the amount of biofilm development.

• The less developed and thinner the biofilm, the more effective the biocidal action.
• Compounds such as hydrogen peroxide, peracetic acid or peroxyacetic acid, and ozone oxi‑
3

dize microorganisms and kill biofilm organisms by the formation of reactive peroxides and
hydroxyl free radicals.
• Microorganisms growing in a biofilm can exhibit reduced susceptibility to antimicrobial
agents including disinfectants and sanitizers.
• Microorganisms in a well-developed biofilm can be extremely difficult to kill, even by ag‑
gressive oxidizing biocides due to protection by the polysaccharide layer of the biofilm.

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 CLEANING AND SANITIZATION SECTION 3

Prevention of biofilm formation

Because of the difficulty in treating and removing mature biofilm, the emphasis must be placed on
prevention of biofilm formation. Appropriate equipment design, including adequate drainage and
drying (See sanitary equipment design), validated cleaning and sanitization procedures, and ade‑
quate frequency will help to minimize biofilm development.

HACCP
Hazard analysis critical control point (HACCP8-10) is a systematic preventive approach that address‑
es physical, chemical and biological hazards. HACCP is a risk assessment process that may be used
to identify the critical control points (CCP) of the system. CCP may include areas that are most
difficult to reach for cleaning and sanitization and that are keys to monitoring contamination in
order to maintain microbial control.
HACCP typically consists of 7 steps:

• Identify hazards and control measures

• Determine critical control points
• Define critical limits
• Establish a monitoring system
• Establish corrective actions
• Verify that the control measures operate as defined
• Document procedures, monitoring and events

TRAINING
Personnel should be properly trained in the cleaning and sanitization of the facility and equipment.

3
A training program should be appropriate to the roles and responsibilities of the employee and

SANITIZATION
CLEANING AND
impart an understanding of the elements of cleaning and sanitization and their effect on product
quality. Training should include:

• Basic equipment operation and design

• Concepts of microbial contamination including common sources of contamination
• Proper and safe use and appropriate disposal of cleaning and sanitization agents

For further information on training, see “Microbiology Staff Training” (Section 4) and “Microbial
Validation and Documentation” (Section 9).

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 SECTION 3 CLEANING AND SANITIZATION

VALIDATION
All procedures used for cleaning and sanitization should be validated. For protocol and additional
information, see “Microbial Validation and Documentation (Section 9).

RECORD KEEPING
Ongoing documentation includes routine logs that are necessary to maintain a history of the equip‑
ment usage and cleaning and sanitization practices. This information can also serve as part of a
validation information package. It can be used for trend analysis and evaluating cycle reduction. A
cleaning and sanitizing record should be prepared, maintained, and made readily available for each
piece of equipment.

Equipment Records
Equipment records should include the following information:

• Identification of the equipment

• The product and batch to be made
• Date, start and end times of the cleaning
• Date, start and end times of the sanitization, including expiration time
• Operating procedure, SOP, or procedure number for the cleaning and sanitization being
carried out
• Any variation from the established operating procedure
• Sign off by operator
• Review, approval and sign off by verifier/reviewer
• Time, date and identity of next batch start up
• Date and description of any maintenance, repairs or equipment down time
CLEANING AND
SANITIZATION

Equipment Status
The current status of the equipment should be clearly displayed. Examples of status designation
labels may include:

• In use: Contents and Batch or Lot Number

3

• Needs Cleaning
• Clean Needs Sanitizing
• Sanitized*
• Out of Service

*Sanitization date and expiration time should be included on the label. If expiration time is exceed‑
ed, the equipment should be re-sanitized before being put back into service.

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 CLEANING AND SANITIZATION SECTION 3

MANUFACTURING FACILITY
The environment of the manufacturing facility strongly influences the microbial quality of the fin‑
ished product. Appropriate building design and maintenance are critical. Standard procedures for
facility cleaning should be written and a record of their implementation should be maintained.11,12
For additional information, see “Microbiological Evaluation of the Plant Environment.” (Section 2)

Manufacturing / Production Areas

The frequency of cleaning is determined by the types of activities conducted in any given area of the
facility. Cleaning schedules can be adjusted, and remedial action can be taken as required. Precau‑
tions should be taken to minimize airborne dust from manufacturing equipment and areas during
all cleaning and sanitization. Any spills of raw materials, product, or packaging components should
be cleaned up promptly. Based on product risk, some areas may need to be under greater microbial
control and therefore, may require a higher degree of plant hygiene.

Walls, ceilings, pipes, fixtures, and HVAC systems

Clean on a scheduled basis.

Floors
Clean floors on a scheduled basis and include the following:

• Vacuum and/or sweep frequently

• Wet-mop or machine scrub on a predetermined schedule
• Sanitize as appropriate
• Keep floors dry as much as possible

Cleaning equipment and supply storage

Store cleaning equipment and supplies properly in a clean area. Supplies and equipment used for

3
lavatory cleaning should be stored separately from cleaning supplies and equipment used in manu‑
facturing areas.

SANITIZATION
CLEANING AND
Warehouse Areas
General guidance for the warehouse area includes the following:

• Aisles should be kept neat and clean by sweeping, damp mopping or machine scrubbing. An
appropriate, freshly prepared cleaner should be used.
• An established, monitored, and documented insect and rodent control program should be
in place. For additional information, see “Quality Assurance Guidelines.”4
• Stored materials and containers should be kept clean, orderly, protected and correctly iden‑
tified.

PCPC MICROBIOLOGY GUIDELINES | 35

 SECTION 3 CLEANING AND SANITIZATION

MANUFACTURING AND FILLING EQUIPMENT

Manufacturing and filling equipment13,14 has direct contact with product. For cleaning and saniti‑
zation procedures to be effective, appropriate consideration to equipment design should be made.
It is essential that manufacturing and filling equipment have good drainage and be designed for
ease of conducting proper cleaning and sanitization. In addition, the equipment should be durable
enough to withstand sanitizing chemicals and/or physical agents that are used for cleaning and
sanitization.

Equipment design
The following guidance on overall equipment design is intended to minimize conditions that may
lead to microbial growth in the equipment. It also offers suggestions to reduce the potential degra‑
dation of the equipment by the effects of the cleaners and sanitizers used. It is essential to work with
your engineering department in choosing the appropriate equipment design and specifications for
your facility.

• Design manufacturing and filling equipment to minimize retention of residual product and/
or wash water. Where equipment is not self-draining, the installation of sanitary drain valves
or valves that can be completely disassembled may be of value. Residual water in equipment
will dilute product and/or sanitizer which can lead to microbial growth and the develop‑
ment of adaptable microorganisms.
• Minimize condensation in equipment. Condensation can dilute product and create an en‑
vironment for microbial growth.
• Design equipment so that during cleaning, all internal surfaces are in contact with the clean‑
ing solution. All internal surfaces should be as free as possible of crevices that can harbor
product or microorganisms.
• External surfaces should be easily cleanable.
• Choose materials of construction that are not easily degraded, etched, or reacted when in
contact with the product, cleaners or sanitizers such as 316L stainless steel.
• Choose equipment with a surface finish that is easily cleanable, durable, capable of being
derouged and passivated; e.g., 316L stainless steel with a surface finish of ≤25 Roughness
CLEANING AND
SANITIZATION

Average (RA) or a minimum of 140 grit or better finish.

• Threaded fittings are difficult to clean and can hold finished bulk product residue that can
lead to contamination. Choose equipment without threaded fittings that have a gasket and
clamp connection or use bevel seat connections (outside threads) which are acceptable if
installed and maintained properly to avoid leakage into the outside threads.
3

• Routinely inspect and replace gaskets when necessary, as the gasket interface is readily con‑
taminated when gasket integrity is not maintained.
• Use of sanitary welding techniques such as orbital welding or gas tungsten orbital arc weld‑
ing is recommended to avoid creating crevices or rough surfaces that are difficult to clean.
Chemically welded joints of plastic piping should be checked and be smooth to facilitate
cleaning.

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 CLEANING AND SANITIZATION SECTION 3

Common sanitary design practices for specific types of equipment are listed in the following sec‑
tions.

Tanks/Vessels
• Minimize sharp corners because they are difficult to clean.
• Avoid narrow recesses that could trap product and water.
• Design tanks with a domed head to minimize condensation.
• Choose tanks and vessels with conical or dish shaped bases, with a center drain, as they allow
for complete draining.
• Design vessel openings and surfaces to be easily cleaned.
• Design and maintain covers to fit well and close easily.
• Design vents to minimize debris.
• Eliminate pipes with dead legs.
• Design tanks with spray ball devices that cover the entire surface area and all shadows creat‑
ed by internal components.
• Tanks with mixing capability should be equipped with welded or singe piece mixer blades
and shaft.

Transfer Pipes
• Minimize the length of pipe runs to make cleaning easier and slope the pipe runs to be
self-draining (>1%) to reduce the risk of biofilm formation.
• Choose a pipe diameter that is appropriate to maintain the required flow rate for the clean‑
ing solution.
• In-line filters should be designed for easy cleaning, sanitization and inspection.
• Design piping systems to have a minimal number of T’s.
• Use sanitary welding techniques to avoid the creation of difficult-to-clean crevices and rough

3
surfaces.
• Use sanitary fittings for all connections.

SANITIZATION
CLEANING AND
• Avoid flange and screw-threaded piping that comes in contact with the product.

Valves
Valves should be easily cleanable with no dead spaces to collect product residue or water. Examples
of sanitary valves are diaphragm valves and butterfly valves. The use of valves that can collect prod‑
uct residue or water, such as ball valves, is not recommended due to dead spaces resulting from their
design.

Pumps
Sanitary pumps are recommended. Sanitary pumps are oriented with a vertical inlet and outlet and
passes and pressure relief valves are designed to prevent water and/or product retention. Examples of
sanitary pumps are diaphragm pumps and peristaltic or lobe pumps. Examples of non-sanitary pumps
for moving finished product are centrifugal pumps, gear pumps, or mono pumps. It should be noted

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 SECTION 3 CLEANING AND SANITIZATION

that centrifugal pumps are commonly used in process water systems without having microbial con‑
tamination issues. Pumps should be easily accessible for inspection, cleaning, and sanitization.

Filling Equipment
Fillers should be designed to be easily cleaned and sanitized. Avoid drip pans and water-lubricated
belts. If compressed air is used in filling equipment, the lines should be equipped with microbial
retentive filters and air-line dryers which should be monitored to prevent air-line condensate from
contaminating finished product.

Gaskets
Gasket interfaces are potential sites for contamination. Gasket materials should be compatible with
the product as well as the cleaning and sanitizing solutions. Non-porous, chemically inert materials
such as ethylene propylene diene monomer (EPDM), silicone, and polytetrafluoroethylene (PTFE)
are recommended. Care should be taken to assure that gaskets are properly installed, inspected,
and replaced at a preventative maintenance frequency that is performed prior to wear and damage
causing loss of integrity.

Hoses
Transfer hoses should be of a material that is compatible with product, cleaners and sanitizers to be
used. Common hose materials are:

• Reinforced food grade rubber or neoprene

• TYGON tubing
• Polyethylene
• Polypropylene
• Nylon

They should have flush mounted sanitary fittings composed of 316L stainless steel with rounded
edges and a minimum grit of 180 to prevent migration of product between the fitting and the hose
material. Cleaned and sanitized hoses should be hung in such a manner that they completely drain
CLEANING AND

to dry and visually inspected. Hoses should be capped after drying or stored in a protected area. If
SANITIZATION

hoses are capped, a non-woven microbial barrier is preferred.

Cleaning and Sanitization Schedule

Frequency
3

The frequency of equipment cleaning and sanitization should be determined during validation and
is typically based on several factors including:

• Vulnerability of product to contamination

• Type of equipment used
• Whether continuous process batching is being performed

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 CLEANING AND SANITIZATION SECTION 3

Expiration limit
An expiration limit for cleaning procedures and sanitization procedures should be set for each piece
of manufacturing equipment. This expiration limit reflects the allowable time a piece of equipment
can stand before requiring recleaning and/or resanitization. This will depend on the equipment, the
environment in which the equipment is stored, and methods used for cleaning and sanitization.
Once the cleaning and sanitization procedures have been validated, periodic monitoring of equip‑
ment is essential. For additional information, see “Microbiological Evaluation of the Plant Environ‑
ment (Section 2).

General Procedures
General Housekeeping Practices
• Clean spills immediately and remove debris from the manufacturing areas.
• Use disposable towels and discard immediately after single use. Non-disposable cleaning
cloths should not be used.
• Container exteriors should be cleaned before transferring material into manufacturing areas.

Water
• The water used to make up cleaners and sanitizers should have a low microbial bioburden to
avoid contaminating the cleaner and to avoid consuming the sanitizer.
• Water used to rinse cleansers from cleaned equipment should be fresh, potable water that
has a microbiological quality that meets EPA or equivalent potable water quality standards.15
• Water used to rinse chemical sanitizers from sanitized equipment must have no higher mi‑
crobial bioburden then the microbial specifications of the product to be made in that equip‑
ment.16
• Water hardness should be considered when diluting sanitizers and cleaners because hardness

3
may affect the efficacy of the chemicals used. If an alkaline cleaning agent is used, hardness
ions in the water may precipitate out as calcium carbonate and may cause a white residue on

SANITIZATION
CLEANING AND
the surface. For sanitizer preparation, utilize the water hardness and instructions provided
on the EPA approved sanitizer label.
• The pH of the water may affect the cleaning ability of some cleaners and the antimicrobial
activity of sanitizers/disinfectants.

Equipment cleaning and sanitization

Outer surfaces of equipment should be maintained in a clean state. Clean and sanitize all lines; pro‑
cessing, storage and filling equipment; pumps; pipe connections; flexible hoses and utensils in the
immediate processing and filling areas as follows:

• Remove product residue from all contact surfaces by thoroughly rinsing with water or a
water/detergent solution. Rinse water for sanitized equipment should not contain higher
microbial content than the limits that have been established for the finished product. Tem‑
perature of the rinse solution is dependent on product type, equipment compatibility, and

PCPC MICROBIOLOGY GUIDELINES | 39

 SECTION 3 CLEANING AND SANITIZATION

detergent. NOTE: Nonaqueoustype product residues should be removed by appropriate

predetermined methods.
• Pipeline pigs are devices made of non-porous materials used for recovery of product, prod‑
uct separation, and cleaning of manufacturing pipelines. If pigs are used, assure thorough
cleaning and sanitization of pigging equipment and of the pig itself. When not in use, pigs
must be handled and stored under sanitary, dry conditions. The pig launcher and receiving
station must be sanitary in design as this equipment can easily harbor microbial contami‑
nants.
• Circulate a cleaning solution at an appropriate flow rate for a period of time and at a tem‑
perature capable of effectively removing soil residue in the circuit and/or equipment. All
surfaces not accessible by this cleaning procedure should be cleaned manually and/or by
using special equipment or methods.
• Rinse the cleaning solution thoroughly from the system with microbiologically acceptable
water, as determined by inhouse standards. When water is used to rinse equipment, the
equipment should be drained and used within a validated expiration time.
• All equipment should be sanitized following cleaning or before use according to the written
procedure for the piece of equipment involved and used within the validated expiration
time. See “Special Equipment and Procedures” below.
• If chemical sanitizers are used, rinse water for sanitized equipment should not contain high‑
er microbial content than the limits established for the formulated products.

Equipment should be cleaned as soon after use as possible in order to facilitate product removal.
Product that has dried and hardened onto equipment surfaces can be difficult to remove thoroughly.
Ideally, clean equipment should be sanitized as close to the next use as possible. Cleaned/sanitized
equipment should be properly stored before use to prevent recontamination. In general, equipment
should be drained dry with open ends covered to prevent recontamination. Validated clean hold
times and sanitized hold times should be established for all equipment to be stored prior to re-use.

Special Equipment and Procedures

Special cleaning and sanitizing equipment and methods may be employed for processing and filling
apparatus. The equipment and methods are generally designed to fit the individual needs of each
manufacturing facility. There are several methods for cleaning and/or sanitizing.
CLEANING AND
SANITIZATION

• Manual
Manual methods involve the preparation of cleaning solution and the scrubbing of equip‑
ment or parts using a brush, singleuse cloth or pad. Proper training and appropriate proce‑
dures are critical to obtaining reproducible results.
• Soak
3

This method involves the immersing of utensils or equipment parts in containers of deter‑
gent or sanitizing solution for extended periods of time. Parts should be completely im‑
mersed in the solution with no air bubbles.
• Spray
Low or highpressure sprays are used to remove soil. In most cases, the cleaning action of the
pressure spray is enhanced by the use of detergents. Highpressure spray devices such as spray
balls or injectors may be installed in mixing or storage tanks. Piping that delivers solutions

40 | PCPC MICROBIOLOGY GUIDELINES

 CLEANING AND SANITIZATION SECTION 3

to the spray ball should be sloped to allow adequate drainage. Spray balls or injectors may
become clogged with product residue or debris and should be removed, if possible, and
cleaned periodically. If spray balls or injectors are removed after cleaning and sanitization,
they should be stored in a clean area in a self-draining position.
Highpressure spray wand equipment is also widely used. This type of equipment may be
movable. It is used for general surface cleaning. Spray pressures developed should range
from 200 to 1000 p.s.i.. These devices should not be used in an area or in a manner that
creates an overspray contaminating nearby clean equipment.
• Fog
Fogging is a method of generating a mist for the application of sanitizers. Large areas of
equipment surfaces can be treated by fogging in a very short time using small amounts of
sanitizers. This method of application should only be used when the EPA product label
clearly provides instructions for fogging of hard surfaces. The labeling may only allow fog‑
ging as an adjunct to traditional surface sanitization. Fogging should only be used in closed
systems by properly trained personnel using the appropriate personal protective equipment.
• Clean In Place (CIP)
CIP is a semi or fully automated, self-contained system for the cleaning and sanitizing of
equipment. Cleaning and sanitizing solutions are circulated for a specific time at specified
temperatures. Little or no disassembly of equipment is necessary. Unless properly designed,
installed and maintained, CIP systems can become contaminated. Each system is unique
and to work well it should be properly designed, evaluated and controlled. Factors to con‑
sider when using CIP are: detergent/sanitizer type; detergent/sanitizer concentration; tem‑
perature; and design of equipment. Some equipment design factors include type, number,
positioning of spray devices, pressure to spray device, velocity rates in flow paths, type of
pump, and shadows in tanks created by internal components such as baffles, etc. Portable
and fixed CIP skids should be of sanitary design and have the same validation requirements
as the equipment being cleaned.
• Steam in Place (SIP)
Steam in Place is a semi or fully automated system for the disinfection of equipment. Steam

3
is flushed into the equipment for a specified time at a specified temperature. It is a suitable
system for large volume equipment such as storage tanks, manufacturing vessels, transfer

SANITIZATION
CLEANING AND
pipes, etc. because the elevated temperature can treat complex internal geometries which
may not be reached by sanitizer solutions. Disinfection cycles should be determined for each
piece of equipment in order to take into account their complexity and drainability.

Acceptance Criteria
Prior to validation of the cleaning and sanitization processes for equipment, the acceptance criteria
for each process should be determined. Criteria should take into account the types of finished prod‑
ucts that are being processed by the equipment. Criteria for cleaning include no product residue
and no standing water. Criteria for sanitization typically include no standing water and microbial
bioburden that meet specific requirements or microbial release limits of the finished product. If
chemical sanitizers are being used, analytical specification for detecting the presence of an allowable
limit of sanitizer residue may also be included as part of the acceptance criteria. In general, these are
the minimal criteria that should be considered.

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 SECTION 3 CLEANING AND SANITIZATION

Alert and action levels for microorganisms should be established by quality assurance based on fin‑
ished product microbial content specifications.

CLEANERS
A cleaner can be defined as a chemical or blend of chemicals formulated to remove soils from a con‑
tact surface. These chemicals may be solvents, acids, bases, oxidizers, detergents, and/or water-based
chemical blends.
Aqueous cleaners are defined as blends of water-soluble chemicals designed to remove soils into a
waterbased solution with a water continuous phase during cleaning. These consist of surface active
ingredients and other cleaning chemicals that use detergents to lift soils from surfaces.17

Characteristics of an Efficient Cleaner

Aqueous cleaners are typically formulated to contain several ingredients to allow for maximum
cleaning effectiveness. The ingredient requirements depend on the intended use of the cleaner.
Efficient aqueous cleaners utilize surfactants (anionic, nonionic, cationic and/or amphoteric), dis‑
persants, emulsifiers, wetting agents, builders, chelating agents, sequestering agents, corrosion-in‑
hibiting agents and stabilizers. The surfactants are used for emulsification, wetting and penetration;
builders are used for neutralizing hard water interferences, chelating inorganic soils, and saponifica‑
tion of natural oils; and additives for corrosion inhibition, antiredisposition and good rinseability.
General characteristics to consider in choosing a cleaner:

• Compatibility with equipment, i.e., noncorrosive

• Solubility
• Wetting action
• Penetration properties
• Emulsification and soil-dispersion properties
• Rinsing properties
CLEANING AND
SANITIZATION

• Cost and availability

• Compliance with existing environmental and occupational safety regulations

Table 3-1 gives examples of various types of cleaners. For additional information, see References 13,
14, and 17.
3

Selection of a Cleaner or Cleaners for a Specific Process

Although the characteristics of an efficient cleaner may be more general, the selection of a particular
cleaner for a particular cleaning task requires specific information. The most important consider‑
ations include knowledge of the type of substrate to be cleaned and the type of soil to be removed.
The cleaner type should be matched to the surface to be cleaned (metal, glass, plastic, etc.), the soil

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 CLEANING AND SANITIZATION SECTION 3

type (organic, inorganic, oils, heavy soils, light soils, etc) and the desired cleaning method (manual,
soaking, CIP, power spray wand, etc.). Information on the level of cleanliness required (acceptance
criteria) should also be known. For difficult to clean materials, use of more than one cleaner in a
specific order or regimen may be considered. Several questions can be asked prior to the selection
of a cleaning system:

• Does the cleaner have good detergency on the type of soil to be removed?
• Is the cleaner recommended for the cleaning process to be used?
• Is the cleaner easily rinsed without leaving residuals?
• Does the cleaner have to be treated before being flushed to drain?

Variables Affecting Efficiency

Besides the selection of an efficient cleaner, several other factors are extremely relevant to the success
of a cleaning process. Beyond the cleaner itself, cleaning efficiency is influenced by cleaner con‑
centration, agitation, temperature, cleaning/contact time, rinse method and drying method. These
process variables must be considered, specified, and controlled to ensure a consistent and optimized
cleaning process.

Cleaner Concentration
The concentration of the cleaner and process optimization should be selected through consultation
with the supplier of the material followed by inhouse validation.

Temperature
Temperature should be optimized for the soil being removed as well as the equipment and cleaner
being used. The process should be validated using an appropriate method. Safety considerations
should be addressed if risk of personnel exposure exists. Cleaners efficient at lower temperatures are
now available and may be considered to reduce energy consumption.

Time

3
Cleaning time is dependent on several factors of the process. These factors include mechanical ac‑
tion, temperature, cleaner effectiveness, type of equipment being cleaned, and degree and nature of

SANITIZATION
CLEANING AND
the soil to be removed. For example, the mechanical action of highpressure sprays may require from
seconds to minutes while soaking may require a substantially longer time. Cleaning time should be
determined during the validation of the entire cleaning process/system.

Rinsing
It is important that the rinse procedure removes any residue left during cleaning. The specified
volume of rinse water should be optimized and validated for each particular rinse program. Ensure
there is no cleaner residue remaining.

Drying
To reduce the potential for corrosion, inhibit microbial growth and biofilm formation, and prevent
dilution of chemical sanitizers, it is essential that the equipment be completely drained and dried
after rinsing. Evaporation is the simplest and least-expensive drying method. It is most appropriate
when used after hot water rinses on equipment that can be easily drained such as tanks. Drying by

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 SECTION 3 CLEANING AND SANITIZATION

evaporation is not appropriate for equipment that cannot be completely drained such as filling lines.
Drying by evaporation after ambient water rinses can require longer dry times and may lead to high‑
er risk of microbial contamination. Other methods include circulated hot air, vacuum-drying, and
forced-air blow drying. For these methods, high quality air must be used for drying. The air source
may be filtered (particulate, hydrocarbon, and microbial retentive) to provide high-quality air for
drying. This type of mechanical drying is especially useful for equipment that is used for anhydrous
products where it is essential that no moisture remain in the equipment. Use of alcohol that is free
from spore forming organisms as a finishing step can aid in the evaporation of water. Alcohol can be
used as a dryer/sanitizer although it is not as effective as mechanical drying and is most appropriate
on small pieces of equipment. Caution should always be used when using alcohol on equipment as
it could present a fire and explosion hazard.

Validation of the Cleaning Process

The development of a testing and measurement system is important for optimizing and validating
the effectiveness of a specific cleaning process.17,18 The method selected for measuring the effective‑
ness of the cleaning process should provide information needed to determine that key criteria are
met. Testing of the cleaning process initially requires the development of a baseline level of cleanli‑
ness and an effective method to measure removal of soils and cleaner residues. In many cases, visual
assessments of equipment or simple gravimetric analysis will suffice. Supplemental tools for evalua‑
tion may include video scopes, chemical tracer measurements (fluorescent whiteners, total organic
carbon (TOC) in residual water, or conductivity). The simplest method that provides appropriately
sensitive results should be used.
After the cleaning system has been selected, it should be validated against the targeted product
and on the equipment where the production will occur. Either a quantitative or qualitative meth‑
od may be used to judge the cleaning process, and then acceptance criteria should be established.
Experimentation may occur initially on a smaller bench or pilot-plant scale; however, the cleaning
system should be validated on the actual equipment due to concerns with scaleup. Each variable of
the cleaning process (cleaner concentration, time, temperature, mechanical action, etc.) should be
considered to determine the optimal conditions.

SANITIZERS
CLEANING AND
SANITIZATION

Definition
A sanitizer is either a chemical or physical agent that is effective in reducing microbial contami‑
nation on hard, nonporous contact surfaces. A sanitizer may be considered effective if it reduces
microorganisms to levels established by company standards, with no detectable objectionable mi‑
3

croorganisms, as determined by the cleaning and sanitization protocol.11,17,18

Surfaces should be cleaned and free of residue prior to sanitization since residues can interfere with
activity of both chemical and thermal sanitization.
Factors to consider in choosing a sanitizer:

• Effective against a broad range of microorganisms.

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 CLEANING AND SANITIZATION SECTION 3

• Provides adequate microbial reduction against organisms of concern.

• Effective in a relatively short contact time.
• Stable and efficacious over time, both in concentrate form and at use levels.
• Economical to use.
• Compatible with products and equipment.
• Meets regulatory requirements.
• Environmental impact

Chemical Sanitizers
Combined cleaner/sanitizer agents are available. These “one-step” products are registered by EPA to
be effective in the presence of light to moderate soil; however, heavy soil must be removed prior to
use. When using a “two-step” process where a cleaning agent is used prior to application of a sani‑
tizer, surfaces should be free of residue prior to sanitization since residues can interfere with activity
of chemical sanitization.
Some useful chemical sanitizing agents are chlorine, hydrogen peroxide, peracetic acid, alcohols,
phenolic compounds, and quaternary ammonium compounds. See Table 3-2 for information on
frequently used chemical sanitizers for processing and filling equipment. Chemical sanitizers should
be used according to the manufacturer’s directions and must be shown to be effective for the intend‑
ed use.
For sanitization of process water and process water systems, see “Microbiological Quality for Process
Water” (Section 7). See references #19 and 20 for additional information on chemical sanitizers.

Physical Sanitizers
The most common physical sanitizers are steam or hot water. A major advantage of heat is its ability
to penetrate into small cracks and crevices. Heat is also non-corrosive, cost-effective, measurable

3
with recording devices or thermal strips, efficient, effective against a broad range of microorganisms,

SANITIZATION
CLEANING AND
and leaves no residue.
Surfaces should be cleaned and free of residue prior to sanitization since residues can interfere with
activity of thermal sanitization.
See Table 3-3 for information on frequently used physical sanitization methods for processing and
filling equipment.

Factors Affecting Efficacy

Cleaning must always precede sanitization. Inhouse validation is needed to assure efficacy of the san‑
itization process. Roughness of surface, bad welds or other defects can make the equipment difficult
to sanitize. Care should always be taken to follow label directions and manufacturer instructions
and recommendations. Water incorporated into sanitizers should be of appropriate chemical and
microbial quality. The presence of dissolved gasses and solids within water should not be at a level

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 SECTION 3 CLEANING AND SANITIZATION

that inactivates or reduces the efficacy of the sanitizing agent. Operators should be properly trained.
Improper use may give ineffective results, release toxic fumes, or corrode equipment.
The following process variables should be considered, specified, and controlled to ensure consistent
sanitizer performance:

• Condition of equipment surfaces

• Materials of construction
• Concentration of sanitizer
• Contact time
• Temperature
• Optimal pH range
• Mechanical energy (pressure and flow rate)

Rotation of Sanitizers
While rotation of the active ingredients used in sanitizers has been suggested to reduce the potential
for development of bacterial resistance, the published literature has not yet substantiated this recom‑
mendation.21,22 It is critical to assure that the sanitizers are used at the labeled strength through prop‑
er dilution and preparation. Rotation of sanitizers is not a common practice in the manufacture of
personal care products. Where rotation is desired, review the active ingredients listed on the chemi‑
cal sanitizer label to assure that a rotation of active ingredients is achieved when changing products.

SUMMARY
The selection and effective use of a cleaning or sanitizing agent and/or method is dependent on the
manufacturing facility, the type of product processed, and the design and layout of the equipment.
All cleaning and sanitizing procedures should be properly designed and their use documented and
validated. Personnel should receive adequate instruction and training in these areas. With attention
to these details, a cleaning and sanitizing program will ensure a sanitary manufacturing facility.
CLEANING AND
SANITIZATION
3

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 CLEANING AND SANITIZATION SECTION 3

Table 3-1

COMMONLY USED CLEANERS FOR PROCESSING

AND FILLING EQUIPMENT
Cleaner Type pH Range Soils Removed Examples
Water NA Water soluble Potable water
Mineral-Acid 0.2 - 5.5 Heavy scales to inorganic Strong acids:
and Mild Acid salts • Hydrochloric acid
Cleaners Soluble metal complexes • Sulfuric acid
e.g., metal oxides • Phosphoric acid
Weak acids (dilute solutions of organic acids):
• Acetic acid
• Citric acid
Neutral Cleaners 5.5 - 8.5 Light oils Mild, surfactant solutions (may include co-
Small particulates solvents such as alcohols or glycol ethers to
prevent phase separation of the surfactant
solution) without added water softening
agents. Mild surfactants rely on dissolution and
emulsification.
Alkaline 8.5 - 12.5 Oils Ammonium hydroxide
Fats Sodium carbonate
Grease Sodium phosphate
Particulates Borax solutions
Films
Corrosive Alkaline 12.5 - 14 Heavy grease and oils Sodium hydroxide
Pseudomonad biofilm Potassium hydroxide
(alginic acid) Sodium silicates

3
SANITIZATION
CLEANING AND

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 SECTION 3 CLEANING AND SANITIZATION

Table 3-2

COMMONLY USED CHEMICAL SANITIZERS FOR PROCESSING

AND FILLING EQUIPMENT
General types and uses are listed below. Refer to manufacturer’s use directions and material safety data sheets
(MSDS). Appropriate personal protective equipment is required. Comply with existing regulations for use and
disposal. Unless otherwise noted, sanitizers should be rinsed prior to use of equipment. Chemicals should be used
in accordance with the manufacturer’s directions and must be shown to be effective for the intended use.

Type Description Comments

Chlorine-based Sodium hypochlorite, Calcium Better activity at slightly acidic pH (~6.5) and
hypochlorite, Chloramines warmer temperatures23,24.
Reactive with metal surfaces -corrosive if
misused; must carefully regulate exposure time
Too acidic pH will generate toxic chlorine gas
Hydrogen peroxide Purchased as a stabilized Less stable in the presence of light
solution (35% active) Explosive at high levels – may require monitoring
Peroxy-hydrogen peroxide Peroxyacetic acid Generally non-corrosive to stainless steel and
Peracetic acid aluminum. Corrosive to soft metals (iron, copper,
zinc, brass, galvanized steel, etc.)
Breaks down to acetic acid and water
Concentrate is flammable and an explosion
hazard
Alcohols Isopropanol No rinsing required due to evaporation
Ethanol May be used to dry small pieces of equipment or
for anhydrous production
Flammability risk
Phenolic compounds Phenyl and/or chlorinated Working solution may be unstable (use within 2-3
phenols hours)
Quaternary ammonium Quaternary ammonium Has detergent properties
compounds compounds Noncorrosive
May be less effective versus pseudomonads25,26
Inactivated by anionic and non-ionic surfactants
Most effective at neutral or slightly alkaline pH
Requires analysis to confirm effective removal/
rinsing.
CLEANING AND
SANITIZATION
3

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 CLEANING AND SANITIZATION SECTION 3

Table 3-3

COMMONLY USED PHYSICAL SANITIZATION METHODS

FOR PROCESSING AND FILLING EQUIPMENT
Type Description Suggested contact times / comments
Steam Heat27,28 Water at 100°Ca,b,c 20 minutes after temperature has been reached in furthest point of system;
temperature must be validated throughout the system
Clean steam should be used to prevent contamination from boiler
treatment chemicals
Broad spectrum efficacy
Rinsing not required
Minimal risk of microbial resistance
Equipment should be dried after treatment
High energy consumption
Hot Water Water at 80°C 20 minutes after temperature has been reached in furthest point of system;
temperature must be validated throughout the system
Clean steam should be used to prevent contamination from boiler
treatment chemicals
Broad spectrum efficacy
Rinsing not required
Minimal risk of microbial resistance
Equipment should be dried after treatment
High energy consumption

a
Heat may cause equipment damage by expansion of close-fitting and/or moving parts.
b
Heat must be used with thermally stable materials.
c
Steam and scalding water pose a potential hazard.

3
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CLEANING AND

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 SECTION 3 CLEANING AND SANITIZATION

REFERENCES
1. U.S. Food and Drug Administration. 2015. “Current Good Manufacturing Practice in
Manufacturing, Processing, Packing, or Holding of Drugs; General,” FDA 21 CFR, Part 210.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=210
2. U.S. Food and Drug Administration. 2015. “Current Good Manufacturing Practice for
Finished Pharmaceuticals” FDA 21 CFR, Part 211. https://www.accessdata.fda.gov/scripts/cdrh/
cfdocs/cfCFR/CFRSearch.cfm?CFRPart=211
3. U.S. Food and Drug Administration. 2001. Guidance for Industry – Q7A Good
Manufacturing Practice Guide for Active Pharmaceutical Ingredients. http://www.fda.gov/ICECI/
ComplianceManuals/CompliancePolicyGuidanceManual/ucm200364.htm
http://www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM129098.pdf
4. Nikitakis, J.M. (Ed.) 2014. Personal Care Products Council Quality Assurance Guidelines,
Personal Care Products Council, Washington, D.C.
5. Ghannoum, M., and O’Toole, G.A. (Ed.) 2004. Microbial Biofilms. ASM Press, Washington,
DC.
6. Murthy, P.S. and R. Venkatesan, R. 2009. Industrial Biofilms and Their Control. In: Marine
and Industrial Biofouling (H.C. Flemming, P.S. Murthy R. Venkatesan, K. Cooksey eds.),
Springer, NY, pp. 65-102.
7. Clontz, L. and C.M. Wagner (Eds.). 2012. Biofilm Control in Drug Manufacturing, Parenteral
Drug Association, Bethesda, MD.
8. U.S. Food & Drug Administration. 1997. HACCP Principles and Application Guidelines
http://www.fda.gov/Food/GuidanceRegulation/HACCP/ucm2006801.htm, (Updated 2014).
9. Van Scothorst, M. 2004. A Simple Guide to Understanding and Applying Hazard the Hazard
Analysis and Critical Contol Point. ILSI Press, Washington, DC.
10. World Health Organization. 2003. WHO Technical Report Series, No. 908, Annex
7 “Application of Hazard Analysis and Critical Control Point (HACCP) methodology to
pharmaceuticals.”
CLEANING AND
SANITIZATION

11. http://apps.who.int/medicinedocs/documents/s19973en/s19973en.pdf Bloomfield, S. F.,

and R. M. Baird (Eds.) 1996, Microbial Quality Assurance in Cosmetics, Toiletries and NonSterile
Pharmaceuticals, Taylor & Francis, Bristol, PA.,
12. Nikitakis, J.M. (Ed.) 201407 “Annex 2 – Premises (Facility).” In Personal Care Product Council
Quality Assurance Guidelines. The Personal Care Products Council Washington, DC.
3

13. Block, S.S., 2000. Disinfection, Sterilization, and Preservation, 5th Edition, Lippincott
Williams & Wilkins, Philadelphia, PA.
14. Fraise, A., Maillard, J.Y., and Sattar, S. 2013. In Principles and Practices of Disinfection,
Preservation and Sterilization, Wiley-Blackwell, Hoboken, NJ.
15. U.S. Environmental Protection Agency. 2015. Surface Water Treatment Rules
https://www.epa.gov/dwreginfo/surface-water-treatment-rules

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16. Geis, P.A. (ed.) 2006. Cosmetic Microbiology: A Practical Handbook, Taylor & Francis, New
York, NY.
17. McLaughlin, M.C., and Zisman, A.S. 1998. The Aqueous Cleaning Handbook: A Guide to
Critical Cleaning Procedures, Techniques and Validation, Morris-Lee Publishing Group, Rosemont,
NJ.
18. AS TM International 2014. “E1153143 (2010) Standard Test Method for Efficacy of
Sanitizers Recommended for Inanimate Non-Food Contact Surfaces” ASTM International West
Conshohocken, PA. www.astm.org
19. Centers for Disease Control. 2009. Biosafety in Microbiological and Biomedical Laboratories
(BMBL) 5th Edition. Appendix B. (Decontamination and Disinfection). http://www.cdc.gov/
biosafety/publications/bmbl5/
20. United States Pharmacopeia. 2016.USP 39- NF 34 USP <1072> Disinfectants and
Antiseptics. United States Pharmacopeia and National Formulary. Rockville, MD. Pp. 517-520.
21. Martinez, Jose E. 2009. The Rotation of Disinfectants Principle: True or False? Pharmaceutical
Technology, Vol 33, No. 2, p 58-71.
22. Sutton, Scott. 2005. Disinfectant Rotation: A Microbiologist’s View. http://www.microbiol.org/
wp-content/uploads/2010/07/sutton.Controlled.Environ.2005.8.7.9.pdf. Contolled
Environments July 2005: 9-14.
23. Sirtes, G., Waltimo, T., Scaetzle, M., Zehnder, M. 2005. The effects of temperature on sodium
hypochlorite short-term stability, pulp dissolution capacity and antimicrobial efficacy. J Endod.
31:669-71.
24. Dychdala, G. 2001. Chlorine and Chlorine Compunds – Chapter 7. In. Block, S.S
Disinfection, Sterilization, and Preservation – 5th Ed. Lippincott, Williams, & Wilkins,
Philadelphia, PA, pp. 135-158.
25. Sundheim, G., Langsrud, S., Heir, E., Holck, A. L. 1998. Bacterial resistance to disinfectants

3
containing quaternary ammonium compounds. International Biodeterioration & Biodegradation,
41: 235-239.

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CLEANING AND
26. Rorvik, L.M., Aase, B., Langsrud, S., and Sundheim, G. 2000. Occurrence of and a possible
mechanism for resistance to a quaternary ammonium compound in Listeria monocytogenes.
International Journal of Food Microbiology, 62: 57-63.
27. Parenteral Drug Association. 2007. PDA Technical Report 1(TR 1) Validation of Moist
Heat Sterilization Processes Cycle Design, Development, Qualification and Ongoing Control.
Parenteral Drug Association, Bethesda, MD
28. Parenteral Drug Association. 2013. Technical Report. No 61. Steam in Place Parenteral Drug
Association, Bethesda, MD.

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 SECTION 4

Microbiology
Staff Training

INTRODUCTION
The staff of the microbiology department has an essential role in maintaining product quality that
meets development specifications, marketing design, and customer expectations. The knowledge
and skills of this group are crucial. Microbial test results must be accurate and reliable so that deci‑
sions based on the test data can be made with confidence.
Training of the microbiology laboratory staff should cover the following general areas:

• Following documented procedures

• Qualifying staff to perform the analysis
• Adhering to aseptic technique
• Checking equipment function
• Performing routine equipment maintenance
• Laboratory controls and documentation

This training provides confidence that test results are accurate and can be relied upon during the
decision-making process.
Many different types of microbiological tests may be performed in a personal care microbiology
laboratory. These can include content testing of microbiologically susceptible raw ingredients and
finished products, preservative challenge testing of product formulations, and the analysis of envi‑
ronmental test samples such as cleaning and sanitization swabs, air, or water samples from a cosmet‑
ic manufacturing facility. If OTC drugs such as sunscreens are being tested, refer to FDA guidelines
for the manufacture of OTC drugs1,2 and to relevant chapters in the USP.3
There are two goals in having a training program for the employees in a personal care microbi‑
ology laboratory: First, to provide an in-depth, well-rounded program in how and why a certain
microbiological test is to be conducted on a particular test sample; and second, to insure that the
microbiological testing for a particular type of sample will be performed exactly the same way by
4

each employee every time a sample is received for testing. The purpose of this guideline is to provide
information regarding requirements for a microbiology staff training program.
STAFF TRAINING
MICROBIOLOGY

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 SECTION 4 MICROBIOLOGY STAFF TRAINING

ESTABLISHING A PROGRAM
The establishment of a training program should include, but not be limited to, the understanding
of microbiological concepts, review of Standard Operating Procedures (SOPs), and review of test
methods or procedures. It is important that the individual have full understanding of the principles
of aseptic technique. Internal or external training classes can be provided as part of the training
program for an employee. It is recommended that hands-on training be included to demonstrate
proficiency in using laboratory equipment and conducting microbiological test methods. It is rec‑
ommended that a knowledgeable, qualified individual possessing appropriate academic and work
experience should train new employees to the laboratory.

A. Training Frequency
All new laboratory employees should receive training prior to beginning work in the laboratory.
In addition, it is recommended that all current staff employees receive periodic re-training at
intervals most appropriate for keeping them current and proficient in performing the various
procedures for which they are responsible. It is the responsibility of the management to ensure
that each staff member is updated or trained according to the company’s policy or Standard Op‑
erating Procedures.

B. Documentation
For each employee in a personal care microbiology laboratory, a training record or log should be
established. The documentation should include, but not be limited to, training and dates when
proficiency has been demonstrated for each particular test method, technique, policy, or proce‑
dure used by that individual during a workday. It is important that no laboratory staff member
be allowed to perform any laboratory task until documentation is established indicating sufficient
training was received and proficiency was demonstrated.
The trainer should either initial or sign and date the training record or log to verify that the train‑
ing was received and completed for that task. Each training record or log should be periodically
reviewed and initialed or signed and dated by the supervisor of the testing laboratory. Records
should be kept for an appropriate length of time.
It is also important that proper documentation exists that the trainer has the necessary experience
and knowledge for conducting a particular microbial test method, or use of a particular piece of
laboratory equipment.

C. Topics
The training topics will often depend upon the laboratory equipment utilized, testing methods
performed, laboratory function, and individual job responsibilities. The tables in the sections
that follow suggest topics and elements that should be included in a microbiology staff training
program.
STAFF TRAINING
MICROBIOLOGY

54 | PCPC MICROBIOLOGY GUIDELINES

4
 MICROBIOLOGY STAFF TRAINING SECTION 4

LABORATORY ORIENTATION
A general orientation should be given to any new individual as an introduction for entering the mi‑
crobiology laboratory. The topics covered during orientation should remain general in scope, give an
overview of SOPs, and cover guidelines within the laboratory as an introduction. The topics listed in
Table I may be included in a general orientation. Other topics may be added at the discretion of the
person developing the training. More specific topics are discussed in detail in sections that follow.

MICROBIOLOGY LABORATORY
A. Equipment
Equipment availability and usage will vary depending on the testing performed in each labora‑
tory. Most laboratories will contain many of the instruments listed below. Employees should be
trained in the safe and effective use of each piece of equipment needed to fulfill their job function.
The list below is not exhaustive; however, it does contain many of the basic pieces of equipment
that may require calibration. Each laboratory will need a customized list depending on their par‑
ticular testing requirements. Common microbiology laboratory equipment includes:

• Balances
• Sterilizers/Autoclaves
• pH Meter
• Water Baths
• Incubators
• Refrigerators
• Low Temperature Freezers
• Automatic Pipetting/Dispensing Devices (e.g., pipettors, micropipettors,
dispensing pumps, etc.)
• Laminar Flow Hoods/Biological Safety Cabinets
• Microscopes
• Stereoscopes
• Laboratory Water System
• Bunsen Burners
• Colony Counters
• Sample Mixing Devices (e.g., vortexes, Waring® Blenders, etc.)
• Laboratory Shakers
• Centrifuges
4

• Laboratory Ovens
• Air Samplers
STAFF TRAINING
MICROBIOLOGY

• Stopwatches
• Spectrophotometers

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 SECTION 4 MICROBIOLOGY STAFF TRAINING

• Lyophilizers
• Automated Microbial Identification Systems
• Automated Microbial Counting Devices
• Water Activity Instrument
• Dishwasher
• Automated Data Collection System
• Stomacher
• Spril Plater

B. Calibration of Laboratory Equipment

Every testing laboratory has pieces of equipment that will require periodic calibration in order
to verify that they are being maintained and operated in accordance with the manufacturer’s
specifications. Training in verification of the operational status of the equipment, including its
calibration, is important. Some equipment may require external calibration and certification.
Besides learning how to use a piece of laboratory equipment, an employee should be trained in
how to recognize when an instrument is not operating correctly.
The list below is not exhaustive; however, it contains the basic equipment that will need periodic
calibration and is found in most microbiology laboratories. Additional information on calibra‑
tion of microbiological equipment is given in Table 8-1 in “Microbiology Lab Audit” (Section 8).

• Balances (e.g., weight checks)

• pH Meters (e.g., daily)
• Micropipettors
• Thermometers (e.g., test and standard)
• Temperature Recorders
• Water Activity Instruments
• Sterilizers/Autoclave
• Timer
• Temperature Recorders
• Chamber Pressure Gauges
• Heat Distribution and Penetration of Chamber and Chamber Loads
• Stopwatches
• Spectrophotometers
• Laminar/Biological Safety Cabinets
• Air Samplers
• Automated Microbial Identification Systems
• Automated Microbial Counting Devices
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 MICROBIOLOGY STAFF TRAINING SECTION 4

LABORATORY TECHNIQUES
A. Common Techniques
Table 4-2 contains common key elements that should be included in a training program for an
individual responsible for conducting tests in a microbiology laboratory. The list contains key
microbiological techniques that may be employed in the laboratory; however, it is not inclusive
of all the different types of techniques that might be used in every laboratory. Additional tech‑
niques performed in your laboratory should be added to your training program. Specific tests are
discussed in detail in sections that follow.

B. Microbial Content Testing

Microbial content testing is performed on raw ingredients, packaging components, and finished
goods that are susceptible to microbial contamination. It is important that an individual per‑
forming these types of tests be trained and have demonstrated proficiency in using the techniques
listed in Table 4-3.

C. Preservative Effectiveness Testing

With the exceptions of the preparation of microbial challenge inocula and inoculated test sam‑
ples, many techniques used for conducting preservative effectiveness tests are common to the
routine analysis of test samples for microbial content. In addition to these laboratory manipu‑
lations, training should include the calculation of percentage or logarithmic reduction and the
interpretation of acceptance criteria.

ENVIRONMENTAL MONITORING
A. General
To effectively monitor the quality of the personal care product manufacturing and pilot plant
environment, laboratory employees with the responsibility for conducting environmental mon‑
itoring should be trained in all methods currently in use. Environmental testing comprises three
major categories: surface sampling, air sampling, and water analysis. Refer to “Microbiological
Evaluation of the Plant Environment” (Section 2) in these guidelines for information on con‑
ducting environmental monitoring in a manufacturing plant.
Training should be based on written procedures which include:

• Methods and materials

4

• Suggested sites to monitor

• Frequency of testing
STAFF TRAINING
MICROBIOLOGY

• Interpretation of results to include specification levels, where applicable

• Determination of alert and action levels, documentation

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 SECTION 4 MICROBIOLOGY STAFF TRAINING

• Communication of results
• Corrective action procedures

B. Environmental Monitoring Test Methods

1. Surfaces
For monitoring the microbial content of surfaces in a manufacturing plant, a laboratory em‑
ployee should be trained in how to use one or more of the following surface sampling test
methods:

• Swab
• Contact Plate (e.g., Rodac Plates)
• Flexible Films or Contact Slides
• Final Rinse Water

2. Air
For monitoring the microbial content of air in different locations of a manufacturing plant, a
laboratory employee should be trained in how to use one or more of the following air sampling
methods:

• Settling Plate (Sedimentation Plate)

• Centrifugal Air Sampler
• Sieve Impaction Sampler
• Slit-to-Agar Sampler
• Liquid Impinger
• Multi-Stage Particle Sizing Sampler
• Membrane Filter
• Compressed Air

3. Water
For determining the microbial content of water samples in a manufacturing plant, a laboratory
employee should be trained on how to perform the activities listed in one or more of the areas
in Table 4-4.

IDENTIFICATION OF MICROBIAL ISOLATES

Microorganisms may be isolated from environmental sources, raw materials, finished products and
other test samples. At times, there may be a need to identify microbial isolate to either the genus or
species level. Results may determine whether a sample passes or fails and may provide information
on the source of the contamination. It is expected that the individual conducting the testing has the
STAFF TRAINING
MICROBIOLOGY

necessary educational background and proper training to correctly identify a microbial isolate to the

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 MICROBIOLOGY STAFF TRAINING SECTION 4

genus or species. It is strongly recommended that this individual demonstrate proficiency in per‑
forming microbial identifications. The following table contains key microbiological tests that may
be employed to identify microbial isolates. The list may not be all inclusive. Additional microbial
identification tests performed on isolates in different laboratories should be added to the laboratory
training program.

CLEANING AND SANITIZATION

Proper cleaning and sanitization of the manufacturing equipment and the facility are vital to ensure
microbial quality in the manufacture of cosmetic and personal care products. Refer to “Cleaning
and Sanitization” (Section 3) for detailed information for a cosmetic manufacturing facility.
Depending on the structure of the company, the role of the microbiologist and laboratory staff may
include the following:

• Advising on hygienic equipment design

• Cleaning and sanitization procedures and validation
• Performing equipment monitoring to analyze for microbial bioburden
• Auditing cleaning and sanitization procedures
• Interpreting test results
• Advising on action steps

The microbiology department is, by function, an integral part of the cleaning and sanitization pro‑
gram. It is recommended that training include the following:

• Aseptic Sampling
• Testing Methods such as:
– Swabbing
– Direct contact
– Final rinse water
• Validation Protocol
• On-Going Environmental Monitoring Procedures
• Documentation
– Documentation of validation and qualification of cleaning and sanitization procedures
– Logs for equipment cleaning and sanitization history
• Basic Understanding of:
– Cleaning
4

> Chemicals
> Physical methods
STAFF TRAINING
MICROBIOLOGY

– Sanitizers
> Physical methods
> Chemical (including pH range, soil effects, concentration, and contact time)
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 SECTION 4 MICROBIOLOGY STAFF TRAINING

• Basic Understanding of Sanitary Equipment Design and Equipment Function

– Process water system
– Processing equipment
> Mixers/kettles
> Transfer pumps
> Transfer pipes and hoses
> Valves and gaskets
> Storage tanks and vessels
> Ancillary and associated equipment (including scoops, pitchers, funnels)
> Packaging equipment

CONCLUSION
It must be realized that the topics listed above and the suggested elements for a training program for
a microbiology laboratory cannot be all-inclusive. These elements are only for guidance on the com‑
ponents of a microbiology staff training program. If a microbial technique, procedure, or a piece of
laboratory equipment is not listed here and is being performed or used in a microbiology laboratory,
then it should be included in the training record or log for each employee whose job duties include
using the equipment or performing the procedure.
Proficiency testing, as a means of demonstrating competence, is an integral part of a training pro‑
gram.
STAFF TRAINING
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Table 4-1

GENERAL ORIENTATION TO THE MICROBIOLOGY LABORATORY

Topics Areas to be Covered
Organizational Structure • Vice-president
• Director
• Lab manager
• Microbiologists
• Technicians
• Contract temporary staff
• Customers
Introduction to Laboratory • Director
Personnel and Customers • Laboratory manager
• Microbiologists
• Technicians
• Contract temporary staff
• Relevant customer staff
Types of Microbiological • Environmental monitoring
Testing Conducted • Microbial content testing
• Preservative challenge testing
• Selective media
• Culture identification
• Other tests
Laboratory Rules and Safety4 • Laboratory safety manual
• Occupational safety training5
Introduction to Current Cosmetic Good • Documentation of methods and test results
Manufacturing Practices (GMPs)6 • Record keeping rules
• Out-of-Specification investigations and documentation
• Labeling
• Dating
• Signatures
• Expiration dates
• Lot numbers
• Other items as appropriate
Good Laboratory Housekeeping • General organization and cleanliness
• Cleaning schedule
• Cleaning checklist
• Waste handling and disposal
• Others where applicable

4
STAFF TRAINING
MICROBIOLOGY

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 SECTION 4 MICROBIOLOGY STAFF TRAINING

Table 4-2

KEY MICROBIOLOGICAL TECHNIQUES

Technique Detailed List
General • Aseptic technique
• Use of controls (e.g., positive and negative)
• Check expiration dates of media, reagents, etc.
Media and Broth Preparation • Medium/broth selection for application
Diluent Preparation • Ingredient/component weighing
• Water selection
• Equipment selection
• Sterilization
• Sterility/growth promotion controls
• Shelf life
• Documentation
Organism Identification • Isolation
• Gram stain
• Spore stain
• Lacto phenol cotton blue – mold
• Automated methods (as applicable)
• Catalase, oxidase, coagulase testing
Maintenance of Microbial • Lyophilized or frozen culture reconstitution
Culture Stocks • Rotation and generation criteria, including identification criteria
• Preparation of slants
• Documentation for traceability of stocks
Sample Preparation and Testing • Inocula preparation and enumeration
• Sample weighing
• Pour plates
• Streaking
• Broth enrichment
• Incubation temperatures and times
• Colony counting
Waste Disposal • Autoclaving/sterilization
• Labeling
• Chemical and biological hazardous waste handling
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Table 4-3

MICROBIAL CONTENT TESTING

Technique Detailed List
How to Sample • Liquid or granular/solid raw ingredients
• Packaging components (e.g., applicators, sponges, etc.)
• Bulk finished products
• Finished products
Sample Preparation • Water-miscible/dispersible raw ingredients and finished products
• Water immiscible raw ingredients and finished products
• Packaging components
Enumeration • Bacterial and yeast/mold plate count procedures
– Membrane filtration method
– Pour plate method
– Automated methods
Detection • Enrichment testing
Microbiological • Release test specifications/reject procedures
Acceptance Criteria – Raw ingredients
– Packaging components
– Finished goods
Verification of Test • Demonstrate that enumeration and detection methods are capable of recovering
Methods microorganisms from test samples

Table 4-4

WATER MONITORING ACTIVITIES

Area of Training Detailed List
Sample Collection • Key sites in applicable water system:
– Non-circulating hot/cold (with or without chlorine)
– Circulating hot or cold
– Other
• Importance of timing – holding of sample
Use of Chlorine Inactivators • Where Required
Test Methods: • Pour plate method
Total Count • Membrane filtration method
• Paddle Testers (e.g., Membrane Dip Samplers, Agar Dip Slides)
Coliform Screening • Membrane filtration
• Most Probable Number (MPN)
• Presence/Absence Enrichment
• Differential/Selective Agar
Pseudomonas detection • Pour Plate Method
• Membrane Filtration Method
4
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 SECTION 4 MICROBIOLOGY STAFF TRAINING

Table 4-5

MICROBIAL IDENTIFICATION TESTS

Identification of Bacteria Test
Isolation and Staining • Streak plate to isolate organisms
• Gram stain (e.g., Gram-negative and Gram-positive)
• Potassium Hydroxide (KOH) Test - for use on inconclusive Gram stain
results for a bacterial isolate to separate them into Gram-negative and
Gram-positive bacteria groups.
• Morphology (e.g., bacillus or cocci)
• Spore Stain
Biochemical Tests for • Cytochrome Oxidase Test -to separate Gram-negative bacilli into
Gram-negative Bacilli fermentor and non-fermentor groups.
• Oxidation/Fermentation Test -Glucose for Gram-negative bacilli
Biochemical Tests for • Catalase Test - to separate Gram-positive cocci into Catalase Positive and
Gram-positive Cocci Negative groups.
• Coagulase Test - to separate Catalase Positive Gram-positive cocci into
Coagulase Positive and Negative groups.
• Hemolytic Reactions - to identify the various members of Catalase-
negative Gram-positive cocci species.
Specific Biochemical • Assimilation/Utilization of Specific Chemicals
Reactions • Fatty Acid Cell Wall Analysis
Genotypic Methods • DNA Analysis System
Identification of Yeast Test
Staining • Morphology
Use of Specific Biochemical • Assimilation of Specific Chemicals
Reactions • DNA Analysis Systems
Identification of Mold Test
Examination • Morphology/Slide Preparation
• DNA Analysis Systems
STAFF TRAINING
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REFERENCES
1. U.S. Food and Drug Administration. 2015. “Current Good Manufacturing Practice in
Manufacturing, Processing, Packing, or Holding of Drugs; General,” FDA 21 CFR, Part 210.
www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=210
2. U.S. Food and Drug Administration. 2001. Guidance for Industry – Q7A Good
Manufacturing Practice Guide for Active Pharmaceutical Ingredients.
www.fda.gov/ICECI/ComplianceManuals/CompliancePolicyGuidanceManual/ucm200364.htm
3. United States Pharmacopeia and the National Formulary. 2016. (USP39-NF34), U.S.
Pharmacopeia, 1260 Twinbrook Parkway, Rockville, MD
4. Centers for Disease control and Prevention. 2009. Biosafety in Microbiological and Biomedical
Laboratories (BMBL) 5th Edition. http://www.cdc.gov/biosafety/publications/bmbl5/
5. Occupational Safety & Health Administration. 2016. Draft Safety and Health Program
Management Guidelines Document. https://www.osha.gov/shpmguidelines/
6. Nikitakis, J.M. (Ed.) 2014. Personal Care Product Council Quality Assurance Guidelines, Personal
Care Product Council, Washington, D.C.

ADDITIONAL RESOURCES
Akers, M. 1993. cGMP Education and Instruction: A Corporate Approach to Employee Training
Worldwide. Pharmaceutical Technology, 17: 51-60.
Beauchemin, K., Gallup, D., and Gillis, M. 2001. Read and Understand vs. A Competency-Based
Approach to Designing, Evaluation, and Validating SOP Training. PDA Journal of Pharmaceutical
Science and Technology, 55 (1): 10-15.
Deluca, P.P. 1983. Microcontamination Control: A Summary of an Approach to Training. PDA
Journal of Pharmaceutical Science and Technology, 37(6): 218-224.
Gallup, D., Beauchemin, K., and Gillis, M. 1999. A Comprehensive Approach to Compliance
Training in a Pharmaceutical Manufacturing Facility. PDA Journal of Pharmaceutical Science and
Technology, 53(4): 163-167.
Gallup, D., Beauchemin, K., and Gillis, M. 1999. Competency-Based Training Program Design.
PDA Journal of Pharmaceutical Science and Technology, 53(5): 240-246.
Levechck, J.W. 1991. Training for GMPs. Journal of Parenteral Science and Technology, 45(6):
270-275.
Parenteral Drug Association, Inc. 2001.“Technical Report No. 35, A Proposed Training Model
4

for the Microbiological Function in the Pharmaceutical Industry.” 2001. PDA Journal of
Pharmaceutical Science and Technology, 55(6).
STAFF TRAINING
MICROBIOLOGY

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 5
AND ANALYSIS
HANDLING, STORAGE
SECTION 5

Handling, Storage and

Analysis of Raw Materials

INTRODUCTION
Raw materials used by the personal care products industry are not expected to be sterile as received.
Some commodities, especially those of natural origin, may contain large microbial populations. The
incorporation of such raw materials into product formulations is undesirable because the organisms
introduced could:

• Contaminate equipment and environment;

• Present a health hazard;
• Produce undesirable changes in products; and
• Reduce preservative effectiveness.

CATEGORIES
A program to control organisms in raw materials should consider the physical and chemical nature
of the raw materials as well as the subsequent processing involved in the manufacture of quality
products. In general, raw materials may be categorized as:

• Hostile - A raw material that will not support and may inhibit the growth of microorgan‑
isms.
• Inert - A raw material that may act as a carrier of microorganisms but ordinarily will not
promote microbial proliferation.
• Supportive - A raw material that serves as a nutritional substrate and supports microbial
growth.
• Preserved - A raw material to which antimicrobial preservative have been added to control
microbial growth.

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HANDLING, STORAGE

SECTION 5 HANDLING, STORAGE AND ANALYSIS OF RAW MATERIALS

AND ANALYSIS

STABILITY
The stability of raw materials may be affected by the presence of microorganisms. To monitor chang‑
es in microbial content, raw materials should be examined upon receipt and on a regular basis
throughout their shelf life by acceptable microbiological procedures.
5

EXPIRATION
Expiration dates should be established for raw material by product category, history, in‑house ex‑
perience, and manufacturer’s recommendations. An appreciable change from the normal microbial
profile indicates a possible problem, and should be investigated.

RECEIPT
Raw materials received should be properly labeled, placed on quarantine status, and held until re‑
leased by Quality Assurance. For further guidance, refer to “Sampling: Part I: General Provisions
Sampling Plan” in the PCPC Quality Assurance Guidelines.1

STORAGE
Raw materials should be stored under conditions that minimize the possibility for microbial con‑
tamination. Factors to be considered are:

• Control of environmental factors such as temperature, humidity, ventilation and light;

• Proper housekeeping practices;
• Rodent, small animal and insect-control programs;
• Quarantine systems; and
• Special storage conditions where indicated.

Procedures for the control of raw materials should be documented and assigned to a responsible
department. Storage conditions should be periodically monitored by appropriate personnel. Once
established, the procedures should be reviewed on a periodic basis.

TRANSFER
Transfer systems for raw materials include sterilized containers, transfer lines, pumps, and related
equipment. These systems should be evaluated on an individual basis depending on the specific raw
material involved. The raw material categories listed above will aid in this evaluation. For example,
a supportive raw material will require greater consideration (i.e., stringent, sanitary handling) and
more monitoring than a hostile one.

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AND ANALYSIS
HANDLING, STORAGE
HANDLING, STORAGE AND ANALYSIS OF RAW MATERIALS SECTION 5

SAMPLING
Appropriate control procedures are required for sampling raw materials.2

• Personnel - Personnel responsible for sampling raw materials should be trained in aseptic
sampling techniques, preferably by a qualified microbiologist. Individuals with contagious
illnesses or open lesions should not touch or otherwise contact materials being sampled.3
• Containers - All sampling containers should be sterile and of suitable size.
• Utensils - All sampling utensils should be sterile and suited to the particular raw material.
Long‑handled dippers, syringes, sampling tubes, “thieves,” spatulas, spoons, and pipettes are
all examples of sampling utensils. Some of these are commercially available as pre-sterilized
items.
• Techniques - To ensure that samples are representative of the lot or batch, a logical sampling
plan should be developed.1

When samples are obtained for microbiological analysis the following procedures should be ob‑
served.

• Sanitize sample sites externally.

• Obtain subsurface samples of dry raw materials.
• Mix liquids for homogeneity.
• Where possible, take representative samples from the top, middle and bottom of bulk tanks.
• Properly seal containers.

TESTING
Microbiological testing of raw materials can be accomplished according to “Determination of the
Microbial Content of Personal Care Products” (Section 17) or other appropriate method. The na‑
ture of the raw material will determine the method used.

REFERENCES
1. Nikitakis, J.M., ed. 2014. Annex 17, Part 1 – General Provisions Sampling Plan. In: PCPC
Quality Assurance Guidelines, The Personal Care Products Council, Washington, DC 20036, pp
103-106.
2. Nikitakis, J.M., ed. 2014. Annex 17, Part 2 – Sampling and Control Techniques. In: PCPC
Quality Assurance Guidelines, The Personal Care Products Council, Washington, DC 20036, pp
107-110.
3. Nikitakis, J.M., ed. 2014. Annex 1, Part 1 – Personnel and Training. In: PCPC Quality
Assurance Guidelines, The Personal Care Products Council, Washington, DC 20036, pp 1-6.

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 SECTION 6

Microbiological Sampling

6
SAMPLING
MICROBIOLOGICAL
INTRODUCTION
Appropriate microbiological techniques are needed for sampling raw materials, bulk in‑process,
packaging components, and finished goods to ensure personal care product quality. Although each
area has its own specific needs, there are basic similarities that are vital to all. From the time raw
materials arrive until the finished product emerges, product history and proper identification are
essential. In general, aseptic techniques should be followed for valid evaluations of samples. The
frequency, sampling and screening methods may vary, but the need for monitoring by qualified
personnel is of utmost importance.

CATEGORIES OF SUSCEPTIBILITY
All raw materials, packaging components, bulk in‑process, and finished goods differ in susceptibility
to microbial growth. In order to assess the risk of growth occurring in a material, it is helpful to es‑
tablish categories of susceptibility. These categories of susceptibility influence the extent of sampling
and testing required for each material.
Category 1 (High Susceptibility)
High-susceptibility materials include:

• Eye products (aqueous and semi‑aqueous).

• Baby products
• Products for at-risk populations (elderly sick, etc.)
• Emulsions.
• Cream lip preparations (water‑based emulsions).
• Water‑based products.
• Raw materials of natural origin.

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 SECTION 6 MICROBIOLOGIC AL SAMPLING

Category 2 (Medium Susceptibility)

Medium-susceptibility materials include:

• Pressed powders (compact powders, blushing powders).

• Stick preparations, make‑up sticks.
• Loose powders (face).
• Bath powders (dusting talc).
• Some aerosol products.
MICROBIOLOGICAL

• Eye powders, pressed and loose, and stick preparations.

Category 3 (Low Susceptibility)

SAMPLING

Low-susceptibility materials include:

• Alcohol-based preparations (more than 20%).

• Deodorants and antiperspirants.
6

• Bath salts.
• Many aerosol products.
• Raw materials with antimicrobial activity.

Category 4: (Nonsusceptible)
A nonsusceptible material is one that by nature of its components, exclusive of preservatives, will not
support the survival of vegetative organisms.
NOTE: The susceptibility of packaging components and other raw materials should be evaluated
based on their composition and the nature of the product with which they are used.
The above categories are based on the following:

• History - Necessity and frequency of testing a material are based on past microbiological
profiles. Determining the microbial content of a designated number of batches over a period
of time helps to establish the susceptibility category.
• Susceptibility Tests - Materials may be challenged with microorganisms and tested for sus‑
ceptibility.

SAMPLING DEVICES
The following devices for sampling, available from scientific supply houses, may be used:

• Sterile Thief - Can be used for liquid and/or powder.

• Sterile Scoops - Used for powders.
• Sterile Cups - Can be used for liquid and/or powder. Avoid contact of hands with products.

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 MICROBIOLOGIC AL SAMPLING SECTION 6

PERSONNEL
The individual(s) responsible for sampling should be trained in aseptic sampling technique by a mi‑
crobiologist or other qualified person and should be familiar with visual characteristics of containers
and/or materials to be sampled.

6
RAW MATERIALS

SAMPLING
MICROBIOLOGICAL
Designated personnel should be notified of the receipt of each shipment of raw materials. The ship‑
ment should be inspected for physical damage as indicated by leakage of liquids or powders, rusty
or dented containers, and broken or torn containers that expose the contents to outside contamina‑
tion. Tank car shipments may be inspected through the top for gross contamination. Any container
damaged in such a manner that the contents could be contaminated should be rejected and the
supplier notified.1 Each container should be properly labeled.2

Sampling Technique
Aseptic technique should be followed at all times by trained personnel. Sampling should be per‑
formed with sterile equipment, which can be of stainless steel, plastic, or any other microbiologi‑
cally acceptable material. Devices for sampling include ladles, cups, spatulas, scoops, and spoons.
In general, glass devices should be avoided because of danger of breakage. Each container should be
sampled with a separate sterile device.

Techniques for Specific Containers

Bags
1. Place bag in a flat position.
2. Clean and sanitize the area to be opened.
3. Aseptically make opening in bag.
4. Aseptically remove the sample, transfer to a sterile container, and cap the container.
5. Close the opening of the bag carefully and seal it.
6. Label, initial and date the sample container.
7. Identify each bag sampled.2

Tank Car and Storage Tanks

Tank cars and storage tanks present unique problems in sampling. As it is imperative to obtain a
representative sample, it is necessary to sample top and bottom. For example, a thief, such as a sterile
plastic bottle (of unreactive material such as polypropylene) held in a modified water sampler hold‑
er, may be used to transfer the sample to a sterile, properly labeled container.

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 SECTION 6 MICROBIOLOGIC AL SAMPLING

Drums Containing Liquids

1. Clean and sanitize the cover.
2. If the entire lid is removable, a ladle‑type device may be employed. When there is only a
small opening, a dipper‑like device is preferred.
3. Transfer the sample to a sterile, properly labeled container, and cap the container.
4. Identify each container sampled (label, initial and date).2

Drums Containing Dry Materials

MICROBIOLOGICAL

1. Clean and sanitize the cover.

2. Sample from the container using a sterile sampling device.
SAMPLING

3. Transfer the sample to a sterile, properly labeled container and cap the container.
4. Identify each container sampled (label, initial and date).2

Sample Properties
6

The intrinsic properties and microbiological history (internal monographs developed from previous
microbiological assessments) of a raw material are of prime importance in ascertaining sampling
frequency. The type and homogeneity of the material will also play a role in this determination. A
microbiologically nonhomogeneous material generally requires a greater number of samples.
Raw material lots, depending on the type of material, amount ordered, and/or the supplier, are re‑
ceived in various forms: boxcars, truckloads, bags, boxes and drums. A determination of the number
of samples per lot to be taken (whether the lot is in the form of a single boxcar or in the form of
many bags) should be made. Typical sampling plans can be found in the Personal Care Products
Council Quality Assurance Guidelines.2
In most cases, 30‑100 grams of sample are aseptically taken from each container or area of the con‑
tainer chosen by one of the above methods. It is also feasible and practical to test composite sam‑
ples of the same lot number of certain raw materials, which are by previous analysis and/or nature
considered microbiologically homogeneous. If composites of a lot are shown to be unacceptable
by in‑house standards, then all previously sampled containers should be reassayed.1 More extensive
sampling and testing may also be necessary.

Stability
Raw materials should be investigated to determine susceptibility to microbial proliferation, includ‑
ing the effect of storage conditions. Retest intervals should be scheduled to determine the continued
adherence to microbial content specifications.

Stored Unopened Containers

“Low susceptibility” raw materials should be sampled as necessary. “High susceptibility” raw mate‑
rials should be sampled on a defined periodic basis or prior to use. If no history is available, the raw
materials should be retested just prior to use in manufacturing.

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 MICROBIOLOGIC AL SAMPLING SECTION 6

Partially Used Containers

Partially used and resealed raw materials, especially of “high susceptibility,” which have been stored
for a defined, preestablished period of time, should be retested just prior to use in manufacturing.

PACKAGING COMPONENTS

6
Components should be inspected before shipment by the supplier. The burden of correcting prob‑
lems and minimizing defects should be the responsibility of the supplier; however, it is still the

SAMPLING
MICROBIOLOGICAL
personal care product manufacturer’s responsibility to have an acceptable component to give the
consumer.

Sampling
Upon receipt of a shipment of components, a trained quality-assurance sampler should check for
proper identification.
The quality-assurance sampler randomly samples the shipment. The shipment is then sent to a
designated area until it has been released. A visual examination should be done for obvious defects,
such as mold, dust, dirt, insects, or other extraneous materials. If there is any evidence of these, a
microbiological examination should be done. As a rule most components are considered microbi‑
ologically safe and are not routinely tested except for product applicators, brushes and puffs and,
in predetermined cases, those that are in direct contact with “highly susceptible” products. Only
surface areas that come in direct contact with the product are tested.

Sampling Techniques

1. Clean and sanitize area of carton or containers to be opened.

2. Aseptically remove a sufficient number of pieces to ensure that a representative sample is
submitted.
3. Place samples in a suitable bag or container and properly seal to prevent contamination.
4. Place identification label on the outside of the sample containing the following information:
• Name of item.
• Supplier.
• Date received.
• Date submitted to microbiology department.
• Lot number.
• All other pertinent information needed for the identification of the sample.
5. Properly identify, initial and date each carton or container sampled.

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 SECTION 6 MICROBIOLOGIC AL SAMPLING

Storage
When the sampling of components meets all inspecting criteria, the shipment is then accepted. The
warehouse is notified so that the balance of the shipment can be stored in the proper place. Com‑
ponents should be kept elevated from the ground in a relatively clean dust‑free area with a good
rotation system.

BULK IN PROCESS
MICROBIOLOGICAL

Bulk products should be sampled and tested to ensure acceptability of the product before filling, as a
secondary check on sanitary manufacturing practices, to build a product profile, and as an economic
SAMPLING

control to save on labor and component cost.

If at any time after manufacture an adjustment is made to the batch, samples should be resubmitted
for microbiological testing. This applies to both hot and cold mixes.
6

Types of Mixes

1. Cold Mix - No heat applied at any time during manufacture. Sample in accordance with the
procedures stated previously.
2. Hot Mix - Sample after cooling.
3. Aerosols - Sample the concentrate in the same manner as for hot and cold mixes.

In the above three types of mixes, approximately the same sample size should be obtained.
NOTE: If composites of a lot of bulk mixed products are shown to be unacceptable by in‑house
standards, then all samples should be retested individually and if necessary all previously sampled
containers should be reassayed. More extensive sampling and testing may also be necessary. Tanks
should be sampled from the top and bottom before mixing or stirring the contents. Special attention
should be given to the interface of the possible moisture layer on the top of the material. “Low-sus‑
ceptibility” materials (Category 3) should be sampled on a defined periodic basis or prior to use.

Sampling of Bulk Product

• Inasmuch as bulk products are usually stored in tanks, drums, carboys and cartons, a repre‑
sentative sample should be taken regardless of the container size and shape.
• A representative sample may be 50‑100 grams of a well‑mixed product collected in a sterile
container.
• These samples should be sufficient in size to perform all necessary tests, including confirma‑
tory tests.
• Drums and all sub‑units of a manufactured batch of high- and medium- susceptibility ma‑
terials may be sampled according to typical plans as outlined in Personal Care Products
Council Quality Assurance Guidelines, Sampling.2

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 MICROBIOLOGIC AL SAMPLING SECTION 6

• Stored bulk material should be resampled and retested on a defined periodic basis or prior
to use.

FINISHED GOODS
Sampling Intervals and Quantities

6
Samples for testing should be taken at the beginning, middle and end of each shift. If more than

SAMPLING
MICROBIOLOGICAL
one shift fills a batch, samples from each shift should be submitted. In determining the number of
samples taken, consideration should be given to multiple filling lines, container size and extended
downtime and product susceptibility. It is suggested that for possible future reference at least two
unopened samples per batch and/or lot be retained. Retention time should be comparable to that
normally required for other quality control purposes.2

Composites
Composite samples of products from each sampling time may be made; i.e., equal portions of sam‑
ples at the beginning, at the middle, and at the end of the run would be combined to provide three
composite samples.

Frequency of Testing
Samples should be tested as soon as possible after manufacture. In general, the frequency of testing
is determined by the nature of the preparation, efficacy of any antimicrobial agent present, manufac‑
turing process, and experience gained as a result of previous microbiological evaluation. In practice,
it is recommended that, with few exceptions, all susceptible finished products be tested with the
same frequency. This will permit the detection of microbiological problems resulting from formula
changes, errors in compounding or failure of good manufacturing practices.

Microbial Limits
The microbial content for products should follow Establishing Microbiological Quality of Personal
Care Products (Section 12), in‑house specifications, or other appropriate criteria.

Product Release
Finished products should not be released for consumer use until all microbiological testing has been
completed and they are approved for release.

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 SECTION 6 MICROBIOLOGIC AL SAMPLING

REFERENCES
1. Nikitakis, J.M. (ed.) 2014. Annex 8 – Treatment of Product that is Out of Specification In
Personal Care Products Council Quality Assurance Guidelines, Personal Care Products Council,
Washington, DC 20036, pp. 55-58.
2. Nikitakis, J.M. (ed.) 2014. Annex 17 – Sampling. In Personal Care Products Council Quality
Assurance Guidelines, Personal Care Products Council, Washington, DC 20036, pp. 103-110.
MICROBIOLOGICAL
SAMPLING
6

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 SECTION 7

Microbiological Quality
for Process Water

INTRODUCTION
This section is an overview of process water system design and maintenance. It is intended for use by
microbiologists and other technical personnel involved with the design, installation, qualification,
and maintenance of a process water system for the manufacture of personal care products.
Process water systems used in the manufacture of personal care product formulations are typically
comprised of multiple stages. Source water for the system is exposed to various treatments intended

7
to remove chemical, physical, and microbiological impurities and contaminants. The end product

PROCESS WATER
QUALITY FOR
of the system is purified process water in compliance with the manufacturing facilities specifications.
The microbial content of process water should be defined by each company and controlled because:

• It is a major ingredient in aqueous product formulations.

• It can be a major source of microbial contamination for the entire manufacturing system.
• Microbial contaminants in process water can cause adverse physical changes to the product
and may pose a health risk to the consumer.
• A high microbial load may reduce preservative activity or cause preservation failure in the
final product.

DESIGN CONSIDERATIONS FOR PROCESS WATER SYSTEMS

The design of a process water system is dependent on the attributes of the source water for the
proposed new water system. It is recommended that the source water for a process water system be
drinking water that is in compliance with the National Primary Drinking Water Regulations (NP‑
DWR) (40 CFR 141) issued by the U.S. Environmental Protection Agency (EPA) or the drinking
water regulations of the European Union, Japan, or the WHO.1-3 If the water source for the process
water system is from a well on the grounds of the manufacturing facility, then it must be periodically
tested to confirm compliance with regulatory requirements for use as drinking water.4
The supplier of the new process water system must conduct chemical analysis of the raw source water in
order to properly design a system that will provide process water of the appropriate quality. For exam‑
ple, if making OTC drug products, refer to the USP Purified Water monograph.4 Depending upon the
quality of the raw source water, the following components may or may not be used in the process water
system: 1,5 and 6
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 SECTION 7 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER

• Deep-Bed Filtration (Multimedia/Sand Filters) is used to remove suspended material such

as particulates, colloidal matter, and naturally occurring organic matter of heavy molecular
weight.
• Activated Carbon Beds are used to absorb chlorine, chloramines, and organic material from
potable source water.
• Water Softeners are used to remove Calcium, Magnesium, and other cations which cause
water hardness.
• Reverse Osmosis uses a semi-permeable membrane for removing ions from water. Depend‑
ing upon the water quality, a Reverse Osmosis system may be comprised of either a single
pass or a double pass unit. In a double pass Reverse Osmosis unit, two individual systems
are operated in a series where one unit provides feed-water to the second unit.
• Cation/Anion/Mixed Bed Ion-Exchange Columns or Electrodeionization (EDI) is used to
remove ions from water that are not removed by Reverse Osmosis.

During the design process, complete and detailed blueprints, diagrams, and/or flow charts of the
process water system should be prepared and kept on file for reference purposes.7 Any changes to the
system should be similarly documented.

MICROBIOLOGICAL DESIGN CONSIDERATIONS

PROCESS WATER

FOR PROCESS WATER SYSTEMS

QUALITY FOR

The microbiological quality of process water may vary and can be influenced by conditions of man‑
ufacture such as pH, temperature, equipment, and the presence of chemicals. Seasonal variations
in the feed or source water for a process water system may also have an effect on the process water
quality. Although a particular source of water may appear to be suitable for production purposes,
the number and type of microorganisms present may serve as a source of microbiological contam‑
7

ination for the entire manufacturing facility. It is the responsibility of the manufacturer to control
the microbiological and chemical quality of the process water.
In order to help insure the microbial quality of the process water the following must be taken into
consideration during the system’s design: 5,6, and 8

• A continuous, turbulent flow of a minimum of 1 - 2 meters/second must be maintained.

• The entire process water supply and storage system should be designed for proper recircula‑
tion. Recirculation of process water minimizes the development of microbial biofilms and
reduce the tendency of biofilms to shed bacteria into the water.  Furthermore, design and
operational considerations for water storage tanks are required to prevent or minimize the
possibility for the development of microbial biofilms and corrosion. These considerations
for water storage tanks include the use of closed tanks with smooth interior walls, the ability
to spray the tank headspace with recirculated water by using spray balls on the loop return
and the use of heated, jacketed/insulated tanks to heat the water for sanitization.
• Elbows, tees, and bends should be kept to a minimum.
• All piping should be designed to allow for proper drainage.

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 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER SECTION 7

• U-bends should be avoided unless they are inverted so as not to form a pocket of water that
can stagnate. Pressure sensors should not be mounted directly in line with the piping; a
separator should be used.
• Unused valves or branch lines (dead legs) should be removed from the system as they are a
microbial contamination risk.
• Sanitary type unions and valves should be used to ensure that they may be properly cleaned.
• 316L Stainless steel surface finishes of the water storage tank and distribution loop should
be greater than150 grit or have a roughness average less than 0.8 to be considered sanitary.6
• When 316L stainless steel is used, passivation is recommended to prevent chemical attack
by deionized water. If the system is modified and new stainless steel is installed, the system
needs to be re-passivated.6
• While less expensive than stainless steel, plastic piping is not generally recommended for use
in process water systems. If plastic is used or being considered for use, the following must be
taken into account:
– Where plastic piping is used, 316L stainless steel is still recommended for use in the dis‑
tribution loop.
– With the exception of polyvinylidene fluoride (PVDF) or polytetrafluoroethylene (PT‑

7
FE),plastic piping is known to be intolerant to the use of high heat and ozone as a sani‑
tizer.8

PROCESS WATER
QUALITY FOR
– In the case where PVDF and/or PTFE piping is planned for use to distribute heated wa‑
ter:
> The piping will need more structural support than stainless steel.
> Expansion sections will be needed (PVDF and PTFE expand when heated)
> Insulation will be needed to maintain temperature.
– Polyvinyl chloride (PVC) piping is incompatible with most chemical sanitizers.8
– Plasticizers (phthalates) can be leached from PVC piping and are able to serve as micro‑
bial food source by microbial biofilms.8
• The PVC piping joints cannot be heat welded together. They must be joined with cou‑
plings that use a solvent to partially dissolve the joints. This process forms rough welds that
are known to be favorable sites for the development of microbial biofilms.8 Stainless steel,
PVDF, and PTFE are not known to have this issue. 316L stainless steel is the ideal material
for use in process water system piping as it is compatible with most sanitizing systems and
chemistries, is tolerant over a wide range of temperature, and may be easily cleaned. When
properly designed and maintained, 316L stainless steel is highly effective for minimizing
microbial growth.

MICROBIAL CONTROL METHODS FOR PROCESS WATER SYSTEMS

Process water used for the manufacture of personal care products must meet the microbiological and
chemical test specifications set by the manufacturer. The following microbial control methods may
be used for routine process water treatment and sanitization:

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 SECTION 7 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER

• Chlorination, at 0.5 to 2 parts per million (ppm):5,6 ,and 8

– The presence of residual chlorine in process water must be generated using sodium hypo‑
chlorite solution as the chlorine source.7
– The concentration of hypochlorous acid (HOCl) and hypochlorite ion (OCl-) is pH
dependent.9 When chlorination is used, the pH of the process water must be between 5
and 7 to maximize hypochlorite ion content.
– Hypochlorite solution stability is temperature dependent.9
– Chlorine is inactivated by trace organic residuals and at pH levels above 8.5.9
– Deionization resins can become oxidized in the presence of free chlorine; chlorinated wa‑
ter should not be re-circulated back to the generation portion of a process water system.6
– Medium wavelength UV light (e.g., 185-254 nanometers)will cause the destruction of
chlorine.1 and 5
• Ozone, at 0.1 to 1.0 mg/ml or 0.1 to 1.0 parts per million (ppm):10
– Ultraviolet radiation at a wavelength of 254 nanometers may be used to remove ozone,
however ultraviolet radiation intensity of 100,000 µW-sec/cm2 is required to ensure com‑
plete destruction.10 and 11
– Ozone should only be used in purified water storage tanks and distribution loops which
are composed of 316L stainless steel due to its excellent compatibility with ozone.10
– High water temperatures will greatly reduce the typical half-life of ozone to oxygen from
approximately 20-minutes to a matter of a few minutes or seconds. To prevent this rapid
PROCESS WATER
QUALITY FOR

half-life degradation of ozone into oxygen due to high water temperatures, it is recom‑
mended to control the temperature of the ozonated water to be <25°C.6 and 10
– Ozone must be inactivated by ultraviolet light at either the point of use or after a storage
tank in which the ozone is injected. Ozonated water should not be re-circulated back to
the generation portion of a process water system.
7

– Use of ozone is not suitable in water that contains more than 0.5 parts per million (ppm)
of Manganese (Mn) as: ozone converts soluble Mn to an insoluble form.12
• Other Oxidizing Biocides
– Besides chlorination and ozone, other oxidizers like bromine, iodine, chlorine dioxide,
hydrogen peroxide, hypobromus acid, or sodium bromide may be used.
– It is recommended that oxidizing biocides only be used with piping and storage tanks
comprised of 316L stainless steel since oxidizing biocides have been known to cause cor‑
rosion of some other materials.
– Effective addition levels must be found and validated before use.
– Materials used in formulations produced using water which was treated with these bio‑
cides must be compatible.
• Ultraviolet Light (UV), at 254-265 nanometers wavelength
– Disinfection ultraviolet radiation intensity is typically between 30,000 to 35,000µW-sec/
cm2 5 and 8
– Energy output of the UV lamp(s) must be monitored for the system to maintain antibac‑
terial efficacy. When the bulb drops below 50%, output it must be replaced.

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 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER SECTION 7

– The UV cell housing must be periodically cleaned to ensure that UV light is able to pen‑
etrate into the flowing water.
– The absorbed UV dose is dependent upon the depth and turbidity of the water, flow rate,
lamp intensity, and temperature.
– It is recommended that micro-filtration be used in conjunction with UV light since UV
light by itself is only 90% effective under ideal conditions at killing microorganisms that
are present in water.
• Heat, at ≥ 80°C 6 and 8
– Process water may be heated to a temperature ≥ 80°C in the storage tank or by passing it
through a heat exchanger.2
– Best practice is to continuously re-circulate hot water in a distribution loop.
• Microbial Filtration, with 0.45 micron or smaller pore size
– If used in conjunction with ozone, the microbial filtration cartridge should be composed
of PTFE and PVDF to prevent degradation.6

The individual microbial control methods listed above are recommended to be used in combination
with other microbiological control methods for broad spectrum activity.

7
PROCESS WATER
QUALITY FOR
PROCESS WATER SYSTEM MAINTENANCE
A program of regular maintenance should be devised in order to ensure proper performance of the
process water system. In-line microbial filters, ultraviolet lamps, chlorinating equipment or any
other equipment requiring special attention should be serviced and/or replaced according to the
supplier’s specifications and recommended intervals.8 In addition, regular cleaning of filters, storage
tanks, valves, pumps, piping, meters, and points of use should be implemented. Routine preventa‑
tive maintenance checks should be performed to minimize leaks, ensure pumps are functional, flow
rates are adequate for recirculation, meters are calibrated, and points of use are kept clean and well
maintained.
Maintenance frequency is established during validation of the process water system. A log or elec‑
tronic record should be kept as documentation. Alert and action levels established using historical
data and process water system specifications should be included as part of the plan.
Treatment components of the process water system intended to remove impurities may become a
source of microbial contamination if not properly maintained. Regular microbial maintenance of
these systems should be included in the overall plan in order to guarantee process water quality.

• Deep-Bed Filtration (Multimedia/Sand Filters) units have a high surface area in which
microbial nutrients and microorganisms will be either desorbed or absorbed. Microbial
biofilms may form in these units and shedding can occur. As a microbial control measure,
a periodic back purge of the units is recommended to reduce microbial nutrients and to
prevent the formation of microbial biofilms.5
• Activated Carbon Beds have a high degree of bacteria adherence and shedding due to the
presence of a high surface area. When present, chlorine is removed in the upper portion

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 SECTION 7 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER

of the Carbon Beds which may allow bacterial growth in these units. Periodic water back
flushes, a 90°C hot water flush, or a steam flush can be used for microbial control.5
• Regenerating brine (NaCl) solutions for water softeners should not be relied on for an‑
timicrobial control; it is recommended that these units be periodically back flushed to
remove microorganisms.1

ALERT AND ACTION LEVELS

Alert and action levels should be established based on desired microbiological quality and data col‑
lected during validations. Alert and action levels serve as guidance indicators which help determine
when further investigation and or maintenance is needed based on parameters measured during reg‑
ular system checks.2 For example, when an alert level is exceeded at a process water sampling point,
this indicates that the process water system may have started to drift from its normal operating con‑
ditions. This alert level constitutes a warning to the manufacturing facility and does not necessarily
require the performance of corrective action. However, if the alert level is continually exceeded at
the sampling point, additional testing may be warranted and corrective action or preventive main‑
tenance to the process water system should be considered. When an action level has been exceeded
at a process water sample point, immediate action is required. Action typically includes a root cause
analysis and an evaluation of product impacted. A corrective action plan should be developed based
on a root cause analysis and preventive maintenance to the process water system may be required.
PROCESS WATER
QUALITY FOR

For microbial maintenance of a process water system alert and action levels are particularly import‑
ant. Each manufacturer must establish their own alert and action levels for the process water system
sample points based upon historical microbiological test data. Specifications including number and
type of organism appropriate to the product being manufactured should be generated. It is recom‑
mended that the microbial quality of process water contain a limit that is no higher than the micro‑
bial limits that a manufacturer has established for finished products.
7

Microbial alert levels are microbiological count recoveries for process water that are within the test
specifications but which are higher than normal recoveries from a specific sample point. Microbi‑
al action levels are microbiological count recoveries for process water that are not within the test
specifications from a specific sample point. For example: Historical microbiological test data might
indicate the establishment of an alert level of 25 Colony Forming Units (CFU) per milliliter sample
and an action level of 75 CFU per 1 milliliter sample. Sanitization of the process water system must
be considered when microbial count recoveries have approached an alert or action level. Sanitization
of a process water system should follow the instructions of internal SOPs.

WATER SAMPLING
Best practice for building historical data on the microbiological environment in a process water
system or for pinpointing sources of microbial contamination is to obtain microbiological test data
from various locations in the system.1 The following areas of the generation and supply distribution
system are important and should be considered when taking microbiological samples:1

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 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER SECTION 7

• The incoming raw water.

– Note: The incoming raw water should be treated and be in compliance with local micro‑
biological and chemical drinking (potable) water standards or requirements.
• After passing through carbon or charcoal beds.
• After passing through the Ion exchange resin beds.
• Before and after water storage tanks.
• After each ultraviolet light.
• After each microbial micron filter.
• Each use point in the process water distribution loop.
• In the case of a reverse osmosis system, after the reverse osmosis membrane.
• Any other point in the system where information on the microbiological status is deemed
necessary.

Each process water sampling point must be aseptically sampled and tested for microbial content
in a manner consistent with actual use in manufacturing. For example, if a hose is used with the
process water in production, a sample should be taken from the hose. Also, if the process water use
point is sanitized and flushed before use in production, the same sanitize and flush process should

7
be completed before a sample is taken.

PROCESS WATER
QUALITY FOR
Microbiological test results of the process water system should be routinely monitored for trends
which will indicate if there are increasing microbial counts at each use point. If chemical treatment
is being used as a microbial control method, then daily tests for checking chemical levels are strongly
recommended. Microbiological water test results are typically determined after the process water has
been used for production purposes; therefore it is critical that the process water system be validated
to ensure that the microbial quality of process water that is used in production is satisfactory.

WATER TESTING
There is no single method capable of detecting all the potential microbial contaminants in a water
system.1 The methods used for microbial monitoring should be capable of isolating the number and
type of organisms that have been deemed significant for in process system control and finished prod‑
uct release. When selecting a method to monitor the microbial content of a process water system,
method detection sensitivity, range of detectable microorganisms and types and species or organisms
should be considered.1
Identification of recovered microbial isolates from a process water use point may be important in in‑
stances where specific waterborne microorganisms could potentially be detrimental to the products
or processes in which the water is used.1 This information may also be useful when identifying the
source of microbial contamination in a product or process. However, it is up to the manufacturer to
decide whether or not a particular type of microorganism that is recovered in a process water system
sample is objectionable or not.

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 SECTION 7 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER

Corrective action in accordance with the manufacturing facilities’ maintenance plan must be taken
when objectionable organism(s) or unacceptable microbiological counts are found at alert and/or
action levels at a use point.

PROCESS WATER SYSTEM VALIDATION

A manufacturing facilities’ process water system should be validated to ensure its functionality; it
is the responsibility of the manufacturer to develop and establish a validation protocol.1,5, and 7 Any
changes to the system after validation should be evaluated to determine if revalidation is required. If
a process water system is modified, the affected drawings, diagrams, manuals, and standard operat‑
ing procedures should be revised accordingly.
A validation protocol for a new or modified process water system should include the following:
Installation Qualification (IQ), Operational Qualification (OQ), and Performance Qualification
(PQ).1,5, and 7 Acceptance criteria for each qualification should be defined within the validation pro‑
tocol.
Installation Qualification is the first validation document that should be prepared for a process water
system. Installation Qualification (IQ) verifies that all unit components are installed as per system
specifications and according to the design drawings. The following components are part of the IQ:
PROCESS WATER

• Verification of utilities.
QUALITY FOR

• System description
• Verification that each piece of equipment ordered and received is identical to those specified
in the system drawings.
• Verification of sanitary design and materials of construction.
• Examination and documentation of welds.
7

• Inspections for correct pipe slopes.

• Verification of cleaning and stainless steel passivation.
• Verification of absence of leaks by conducting vacuum testing, or by the use of pressurized
air or water or other acceptable methods.
• Calibration of instrumentation.

Operational Qualification (OQ) verifies the capability of the equipment to perform within opera‑
tional requirements. The following components are part of the OQ:

• Description of the system operation.

• Verification of alarm conditions for utilities such as low steam pressure or instrument air,
diverter conditions resulting from low condensate resistivity, differential pressure levels, etc.
• Formalization and documentation of procedures regarding maintenance, adjustment, cali‑
bration, monitoring, and control of system equipment.
• Verification of system functionality based on operational criteria.

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Performance Qualification (PQ) is a process of rigorous testing which demonstrates the effectiveness
and reproducibility of the process water system. The PQ provides documentation that a system is
consistently performing as designed when it is operated under normal conditions. Performance
Qualification for a process water system consists of three validation phases: prospective, concurrent,
and retrospective.
Phase one (prospective validation): The purpose of this phase is to develop a data set for establishing
standard operating procedures (SOPs) and confirming that these SOPs are adequate. Prospective
validation includes:

• Daily sampling of the water and chemical and microbiological analysis for each component
at each point of use.
• Analysis of the data set to determine system performance against acceptance criteria.
• Determination of the sampling and testing schedule for concurrent phase (Phase Two).
• Development of SOPs for the water system.

Phase two (concurrent validation): The purpose of this phase is to establish the short-term consis‑
tency of the water system when it is operated according to the SOP’s developed during the prospec‑
tive phase. During this phase, water from the process water system may be used for production or

7
cleaning/sanitization. Concurrent validation includes:

PROCESS WATER
QUALITY FOR
• Sampling done according to the schedule/SOP decided upon in the prospective phase.
• Testing and documentation of conformance to microbial limits (Action/Alert levels).
• Testing and documentation of conformance to analytical specifications.

Phase three (retrospective validation): The purpose of this phase is to establish the long-term consis‑
tency of the water system. Long term testing will determine the variability of the feed water supply,
water system operation, and the effect of deterioration on system components. Retrospective vali‑
dation includes:

• Sampling done according to SOP schedule.

• Review of data collected over a year period. Any outlying data must be addressed and a root
cause analysis should be performed.
• Adjustment of alert and action levels based on the long term data.
• Adjustment of analytical specifications based on the long term data.
• Adjustment of the sampling and testing schedule, SOPs, maintenance/calibration activities
and schedule.

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 SECTION 7 MICROBIOLOGIC AL QUALIT Y FOR PROCESS WATER

REFERENCES:
1. United States Pharmacopeia. 2015. <1231> Water for Pharmaceutical Purposes. USP 38-NF-
33. Rockville, Md.
2. U.S. Food & Drug Administration. 2014 “ Guide to Inspection of High Purity Water Systems.”
http://www.fda.gov/ICECI/Inspections/InspectionGuides/ucm074905.htm
3. U.S. Environmental Protection Agency. 1998 “National Primary Drinking Water Regulations:
Disinfection and Disinfection Byproducts.” 40 CFR Parts, 9, 141, and 142.
http://www.epa.gov/safewater/mdbp/dbpfr.pdf. http://www.fda.gov/ICECI/Inspections/
InspectionGuides/ucm074905.htm
4. United States Pharmacopeia. 2016.<1231> Water for Pharmaceutical Purposes. USP 39-NF 34.
Rockville, Md.
5. Collentro, W.V. 1999. Pharmaceutical Water: System Design, Operation and Validation.
Interpharm/CPC, New York, New York.
6. Meltzer, T.H. 1997. Pharmaceutical Water Systems. Tall Oaks Publishing, Inc. Littleton,
Colorado.
7. International Society for Pharmaceutical Engineering (ISPE). 2014. Approaches to
Commissioning and Qualification of Pharmaceutical Water and Steam Systems. ISPE, Tampa,
Florida.
PROCESS WATER
QUALITY FOR

8. Soli, T.C. “Design and sanitization of Water Systems to Prevent Contamination.” In Madsen
R.E, and Moldenhauer, J. Editors. Designing and Controlling Water Systems. 2014. Parenteral Drug
Association, Bethesda, Md.
9. Dychdala, G.R. “Chlorine and Chlorine Compounds.” In: Block, S.S. 2001. Disinfection,
Sterilization and Preservation. Lippincott, Williams & Wilkens. Philadephia, Pa.
7

10. International Society of Pharmaceutical Engineering [ISPE] 2012. Good Practice Guide:
Ozone Sanitization of Pharmaceutical Water Systems. ISPE, Tampa, Florida.
11. Nebel, C. and Nenel, T. 1984. “Ozone: The Process water Sterilant.” Pharmaceutical
Manufacturing 1 (2), pp 16-22.
12. Rice, R.G., Robson, G., Miller, W.M. and Hill, A.G. 1981 Uses of Ozone in Drinking Water
Treatment. American Water Works Association 73 (1), pp 49-57.

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ADDITIONAL READING
Cruver, J.E. “Water Disinfection-A Comparison of Chlorine, Ozone, UV Lights, and Membrane
Filtration” Eight Annual Membrane Technology/Planning Conference & First High-Tech
Separations Symposium, Newton, Ma. (October 15-17, 1990).
Huttquist, A. 2007. Practical Guidelines for Qualifying and Purified Water System.
Pharmaceutical Technology Europe, Vol. 19, Issue 12.
International Society for Pharmaceutical Engineering (ISPE). 2011. ISPE Baseline Guide: Water
and Steam Systems, 2nd. Edition, ISPE, Tampa, Florida.
Maltias, J.B. et al. 1990. An Evaluation of Various Biocides for Disinfection of Reverse Osmosis
Membranes and Water Distribution Systems. Ultrapure Water 7(3): 37-40.
Soli, T.C. 2008. “Pharmaceutical Water, Microbiology” In Pharmaceutical Manufacturing, Vol. 1,
2nd Edition, Prince, E.R. (ed.), DHI Publishing, LLC., Bethesda, Md.
Soli, T.C. 2012. Sanitization Approaches for Biofilm Control.  In Biofilm Control in Drug
Manufacturing. Clontz, L. and Wagner, C.M. (eds.). Parenteral Drug Association. Bethesda. Md.
U.S. Food and Drug Administration 2015. , Reverse Osmosis, http://www.fda.gov/ICECI/

7
Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072913.htm

PROCESS WATER
QUALITY FOR
U.S. Food and Drug Administration. 2015. “Water for Pharmaceutical Use. http://www.fda.gov/
ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072925.htm
U.S. Environmental Protection Agency. 1989. Drinking Water; National Primary Drinking Water
Regulation; Total Coliform (Including Fecal Coliform and E. coli, Federal Register, Vol. 54, No.
124, 40 CFR Parts 141 and 142.

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 SECTION 8

Microbiology
Laboratory Audit

INTRODUCTION
Within the personal care industry, quality assurance, development, and contract microbiology te‑
sting laboratories provide supportive microbiological data related to product safety and quality.
Microbiological aspects of formula development, from product conception to finished goods, need
to be addressed to determine if microbiological standards are met. Examples include the develop‑
ment and evaluation of preservative systems and the examination of the microbial content in raw
materials, intermediates, finished goods, and the manufacturing environment.
Laboratory practices should be evaluated at regular intervals to ensure that the generation of data
is reproducible, accurate, and reliable. An audit serves to review existing practices, systems, and
equipment to ensure that they perform as expected. Microbiology lab audits should be conducted
by individuals familiar with the functions and processes that typically occur in a microbiology la‑
boratory. This document provides guidance for conducting an audit of both in-house and contract
personal care microbiology testing laboratories. If personal care and over-the-counter (OTC) drugs
are tested in the same laboratory, refer to FDA guidelines for the manufacture of OTC drugs and to
relevant chapters in the US Pharacopeia.1-3
Based on the information presented in this document, an example of a personal care Microbiology

8
Laboratory Audit Checklist can be found in Table 8-1.

LABORATORY AUDIT
MICROBIOLOGY
PERSONNEL
Supervisory personnel have the responsibility to ensure that operating systems are consistent with
existing personal care regulations and Cosmetic Good Manufacturing Practices (GMPs).4 Labora‑
tory supervisors are to be familiar with applicable regulations and current industry practices. Initial
and continuing training programs are valuable means to ensure that employees are qualified for
their roles and responsibilities and are informed about all laboratory procedures. The following do‑
cumentation may be considered in recording an employee’s qualifications and is most useful when
maintained on file for reference:

• Job descriptions for all laboratory positions, which may describe the qualifications, primary
and secondary responsibilities, and job functions

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 SECTION 8 MICROBIOLOGY LABORATORY AUDIT

• Organizational chart showing each staff position and each incumbent identified by position,
name and reporting relationship
• Skills checklist or applicable training documentation for each employee detailing the mi‑
crobiological techniques and procedures that an individual has been trained and certified to
perform on a routine basis (See, for example, Table 8-2)
• Records of initial and continuing training for each employee

LABORATORY FACILITIES
Adequate laboratory facilities should be provided to minimize errors in test results due to contami‑
nation, inaccuracy in data interpretation, equipment failure, or sampling mistakes. It is recommen‑
ded that rooms used for microbiological testing be of suitable size, construction, and location to
facilitate proper operation. In general, the attributes of an adequate microbiology laboratory facility
include, but are not limited to the following:

• The laboratory contains separate areas for microbiological analysis, support functions (e.g.,
media prep, sample prep, sample login, clerical work), and storage of personal belongings.
• The area for microbiological analysis is used exclusively for testing aspects such as sample
handling, performing procedures, transfer of cultures, counting of colonies, etc.
• All surfaces in the laboratory are nonporous, cleanable, and sanitizable.
• The facility design minimizes exposure of laboratory areas to air currents.
• Laboratory access is restricted to minimize foot traffic by non-laboratory personnel.
• Microbiological quality of the laboratory environment is controlled by appropriate me‑
chanical and physical means such as use of positive-pressure room air, germicidal lamps,
high-efficiency particulate air filters, and/or laminar flow stations.
• Workspaces such as laboratory benches and laminar flow hoods are sanitized at the begin‑
ning and end of the workday, as well as between individual procedures.
• Adequate ventilation and lighting is provided, especially over work areas.
LABORATORY AUDIT

• Electrical service is appropriate for the equipment used in the laboratory and associated
areas to avoid power outages and equipment failure.
MICROBIOLOGY

• Adequate sink areas with hot and cold water service are provided.
• Sufficient counter and shelf space are available so that all procedures can be performed while
preventing overcrowding, safety hazards, or any cross contamination.
• Clean uniforms or lab coats are provided.
• Microbiologically contaminated materials are decontaminated prior to disposal.
8

• Floors are kept clean by regular mopping and be sanitized by using a disinfectant detergent.
• Adequate containers that are appropriately labeled are provided for microbiological waste,
non-hazardous waste, and general trash disposal. General trash is removed from the labora‑
tory each working day.

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SAFETY
A written safety policy that addresses laboratory personnel, equipment and processes should be
readily available. This policy should reference a published laboratory safety manual.5,6

LABORATORY EQUIPMENT
It is recommended that standard operating procedures as well as supporting documentation (e.g.,
equipment manuals, calibration data) be readily available to personnel for each piece of equipment.
Equipment is to be maintained in accordance with the manufacturer’s guidelines and routinely
calibrated, with clearly visible calibration stickers or logs containing calibration date, calibrators
name, and next scheduled calibration date. Personnel should be adequately trained on each piece of
equipment necessary to their function prior to their routine use of that equipment. A summary of
calibration recommendations can be found in Table 8-3. The following specific attributes for some
types of common laboratory equipment are recommended as a minimum guideline:

Incubators
Incubators should maintain a uniform and constant temperature within predetermined limits;
±2.0°C is recommended for most laboratory applications. An accurate thermometer with a bulb
continuously immersed in liquid (water, mineral oil or propylene glycol) is maintained at appro‑
priate location(s) (e.g., hot and cold spots detected through temperature mapping) in the incu‑
bator. Daily temperature readings are recorded on laboratory workdays. Daily humidity readings
are recorded for humidity-controlled incubators. Temperature recording devices or maximum and
minimum registering thermometers within the incubator are recommended to record temperature
variations. Provision is made for humidity control (e.g., placing a beaker of water in the incubator).
The temperature setting is appropriate for the types of organisms being incubated.

Autoclaves

8
Laboratory autoclaves are capable of maintaining a uniform and constant temperature of 121° -
123°C and reach target temperature within 30 minutes. In order to allow sufficient heat distribu‑

LABORATORY AUDIT
MICROBIOLOGY
tion and penetration for sterilization, autoclaves are properly sized and loaded to prevent crowding
of items. Autoclaves are equipped with accurate thermometers or temperature recorders, pressure
gauges, and properly adjusted safety valves. If a time-temperature recorder is not available, an alter‑
native temperature monitoring device or bioindicator is used. Autoclave tape is not an indicator of
sterility. A maintenance program is in place, which includes an annual certification of temperature
and pressure gauges, timers, and temperature recording devices. In addition, individual run records
(e.g., time-temperature recording) are maintained. Records are routinely reviewed for deviation. An
autoclave log is maintained detailing run conditions and allowing traceability of autoclave run doc‑
umentation with specific loads. Temperature controllers, temperature recording devices, pressure
gauges, and timers must be certified for accuracy to insure proper operation.

Colony Counters
A standard colony counter or comparable instrument may be used. Automated colony counters may
be used where accuracy and reliability have been validated. Automated colony counter accuracy is
checked each day of use and a log-in book is maintained.

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pH Meter
pH meters are capable of accurately measuring the hydrogen ion concentration to 0.1 pH units. The
pH meter should be calibrated each day of use by using at least two certified pH buffer solutions
that bracket the pH range of the sample. Buffers should not be used past their expiration dates.
Probes for taking the pH of a sample should be appropriate for the material being analyzed.

Balances
Balances used for routine weighing (e.g., media, samples, etc.) are accurate for the utilized task.
Balances and weights are calibrated according to a regular preventative maintenance schedule with
weight (mass) standards traceable to NIST Reference standards.7 Weight checks should be per‑
formed with calibrated weights.

Labware
Disposable or reusable labware is inert to the materials with which it may be used. For glassware
items, borosilicate glass is recommended because of its ability to withstand high temperatures. The
following are recommendations for selected examples of commonly used labware:

• Pipettes - sterile glass or disposable plastic pipettes may be used. The accuracy of the pi‑
pettes will depend on the volume (i.e., 50-1,000ul pipette accuracy ±0.6%). If used, micro‑
pipettors should be calibrated annually.
• Dilution bottles and tubes - bottles and tubes are single use or are made of autoclav‑
able material. Screw caps are equipped with inert liners. All reusable items are thoroughly
cleaned and rinsed using a protocol that assures no detectable detergent residue.
• Media preparation utensils - clean borosilicate glass, stainless steel, or other suitable inert
labware are recommended.
• Petri dishes - sterile borosilicate glass or disposable plastic Petri dishes are recommended.

Microscopes
A monocular or binocular microscope suitable for the intended purpose is recommended. The
microscope should be capable of a minimum total magnification (combined ocular and objective
LABORATORY AUDIT

lenses) of 1000x. A dissecting scope may be used for lower magnification. An annual maintenance
check is recommended by which lenses, alignment and mechanism aspects are checked.
MICROBIOLOGY

Refrigerator/Freezers
Commercially available refrigerators are suitable unless there is a need for an explosion-proof model.
Refrigerator temperature should be maintained at 2-8°C and routinely checked. No food should be
stored in laboratory refrigerators or freezers. Standard freezers have autodefrost. Ultra-freezers (e.g.,
-80°C) are maintained within 5°C of the desired temperature.
8

Water Baths
Water baths have thermostatic controls to deliver temperatures from ambient to 100°C ±2.0°C. An
accurate, calibrated thermometer should be placed in the water bath to monitor the temperature.
Temperature readings are recorded daily when in use and also after equilibrium has been reached
following any adjustment of the temperature controls. Biocide may be added to the water to pre‑
vent/control microbial growth.

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 MICROBIOLOGY LABORATORY AUDIT SECTION 8

Laminar Flow Hood

Laminar flow hood operation is certified at least annually to verify adequacy of airflow, condition of
pre-filters, HEPA filter integrity, and particle size exclusion limit. An appropriate sanitizer is recom‑
mended for use to disinfect interior hood surfaces before and after use. UV lights (if installed) are
maintained and operated according to manufacturer’s recommendations and are replaced or verified
annually.

Biological Safety Cabinet

A biological safety cabinet is used when a material is suspected of being highly contaminated, when
manipulating high concentrations of microorganisms that are considered to be a biohazard, or when
there is a potential for aerosolization of microorganisms.5 Operation is certified at least annually, or
after 5000 hours of use, to verify the cabinet performance for airflow, condition of prefilters, HEPA
filter efficiency, and particle size exclusion limit. UV lights (if installed) should be maintained and
operated according to manufacturer’s recommendations, and should be replaced or verified annu‑
ally.

Water Treatment
The laboratory has an appropriate system for producing the desired grade of water (e.g., deionized,
distilled) for use in microbial growth media and other applications. The water treatment unit is
maintained and operated consistent with manufacturer’s recommendations. Water is periodically
monitored to ensure it meets chemical and microbiological quality. If a UV light is part of the water
system, it should be maintained and operated according to the manufacturer’s directions.
Note: If personal care and over-the-counter (OTC) drugs are tested in the same laboratory, refer to
FDA reference materials and the USP Guidelines for Water.1,2,3

Freeze Dryers (Lyophilizers)

Vacuum and temperature records are maintained for each lyophilization operation. Temperature,
vacuum, and timing systems should be calibrated every six months.

8
Thermometers

LABORATORY AUDIT
MICROBIOLOGY
Thermometers are of appropriate range for the application. Measurement accuracy is initially estab‑
lished and rechecked at least annually by using a NIST certified thermometer.8

Spectrophotometers
Calibration is performed against standard solutions every six months or as recommended by the
manufacturer.

Centrifuge
Centrifuges are calibrated for temperature and rotor speed annually.

Water Activity Devices

Devices for the measurement of water activity should be calibrated each day of use against known
standards.

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 SECTION 8 MICROBIOLOGY LABORATORY AUDIT

Media Dispensers
The dispensing volume should be verified on a regularly scheduled basis.

Chemical Storage
Appropriate storage should be used for chemicals used in laboratory (i.e., flammable cabinets for
flammable material).

For more guidance on calibration of microbiological equipment; see PCPC Quality Assurance Guide-
lines.9

DOCUMENTATION
The purpose of documentation is to provide the written record of a laboratory operation. A history
of operation is maintained through the retention of documents (e.g., logbooks, worksheets, cali‑
bration records, etc.). Establishment of a document retention program is highly recommended; it
defines the policy of the company, retention time, form place of storage, etc.
Any documentation should describe the function of the laboratory operation with properly prepa‑
red and regularly updated procedures. Scheduled review of laboratory procedures is made to ensure
that procedures are as described. Discrepancies are corrected and procedures amended when appro‑
priate. The investigation and procedure change are documented. See “Microbiological Validation
and Documentation” (Section 9).
Documentation of the following quality control checks is recommended as part of laboratory pro‑
cedures:

• Methods Validation
All microbiological methods are validated and documented to ensure the accuracy of the
test response.
• Media
Media performance is documented via positive and negative controls on each prepared
LABORATORY AUDIT

batch of media.
MICROBIOLOGY

• Equipment Checks (see Table 8-3)

Calibration, performance and maintenance logs are maintained for each piece of laboratory
equipment. All entries should be dated and initialed.
• Personnel
Documentation of microbiology laboratory personnel training is maintained as well as the
documentation described in the Personnel Section of this guideline. A current signature list
8

to conform to signatures that are issued in the laboratory is maintained. Retraining docu‑
mentation is maintained.
• Forms
Forms used to document test results are periodically reviewed and updated as necessary to
reflect current procedures.

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 MICROBIOLOGY LABORATORY AUDIT SECTION 8

• Procedures
Written standard operating procedures and test methods reviewed and updated on a regular
basis to document changes. All procedures include an effective-date and supercedes-date to
identify the current version. All changes are communicated to laboratory personnel.
• Investigation and Discrepancies
An out of specification investigation procedure exists that includes the steps outlining the
process, detailing the findings, and reporting the results. Any discrepancies in the microbio‑
logical test results and/or procedures are investigated, documented and evaluated by appro‑
priate personnel.

8
LABORATORY AUDIT
MICROBIOLOGY

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 SECTION 8 MICROBIOLOGY LABORATORY AUDIT

Table 8-1

AUDIT CHECKLIST (EXAMPLE)

Note: This list gives examples of points to consider when performing an audit or when preparing to receive an
audit. It is not intended to be all-inclusive or to infer that all points are applicable to all laboratories.

General
1. Is there an organizational chart present showing each employee and reporting relationship?
2. Is the signature list up-to-date?
3. Are there written job descriptions for each laboratory position?
4. Is there a training and development program for laboratory members?
5. Is there formal training documentation?
6. Are individual qualifications for laboratory members on file and updated regularly?

Laboratory Facilities
1. Is access to the microbiology laboratory controlled?
2. Are laboratory facilities clean and orderly?
3. Does the laboratory have adequate workspace, ventilation, and light?
4. Is the laboratory free of dust, drafts and temperature extremes?
5. Are there adequate facilities for cold storage, microbial media, and storage of samples?
6. Review records for sanitization and cleaning?

Laboratory Safety
1. Are laboratory coats worn only in laboratory areas?
2. Are proper shoes and clothing worn in laboratory areas?
3. Is a safety committee and/or advisor established and functional?
4. Are all laboratory members provided with or have access to the laboratory safety manual?
5. Are the members of the janitorial staff provided appropriate training suited to the microbiology area?
6. Does the laboratory provide for the following:
a. Designated containers for broken glass, sharp objects, etc.?
b. First aid kits accessible and maintained?
c. Operational eyewash and deluge shower stations conveniently located?
d. Are fire extinguishers and blankets maintained up-to-date and readily accessible?
e. Are emergency exits clearly marked?
f. Are emergency telephone numbers widely and conveniently located?
7. Is microbiologically contaminated waste decontaminated before disposal?

Laboratory Equipment
1. Review records for equipment calibration and preventative maintenance.
2. Review records for calibration of standards and equipment daily calibration check logs (evaluate where
corrective actions where taken when calibration failed).
3. Review cleaning log records (e.g., incubators, water baths, etc.).
LABORATORY AUDIT

4. Review sterilization cycle log records for autoclaves.

5. Are autoclave controls present (e.g., biological indicator test results)?
MICROBIOLOGY

6. Is each piece of laboratory equipment properly identified and tagged with its calibration, preventative
maintenance, and operational status?
7. Review temperature monitoring log records for incubators, refrigerators, and controlled areas.
8. Review laminar flow hood HEPA filter certification records.
9. Are up-to-date equipment operating instructions available?
10. Is appropriate laboratory/instrumentation available for use in accordance with required methodology?
11. Are there service contracts for maintaining equipment?
a. For what types of laboratory equipment?
8

Environmental Monitoring (EM)

1. Is there a monitoring program for equipment cleaning (e.g., swab tests)?
2. Review records for environmental monitoring.
3. Review periodic evaluation of trended environmental monitoring data.
4. Are environmental isolates from swabs and air samples characterized (e.g., gram stain and or identified)?

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 MICROBIOLOGY LABORATORY AUDIT SECTION 8

Table 8-1 (contd.)

Microbial Media, Buffers, and Reagents

1. Are expiration dates for microbial media, buffers, and reagents assigned and updated?
2. Review microbial media growth promotion test results.
3. After sterilization, is the pH recorded for each prepared lot/batch of microbial growth media?
4. Are microbial growth media, buffers, and reagents traceable to preparation records?
5. Review quality control and quarantine practices for purchased and laboratory prepared microbial growth media.
6. Review records for receipt, storage, preparation, sterilization, and storage of microbial growth media, buffers,
and reagents.

Microbial Content Testing

1. At the time of testing, are negative controls performed to verify the sterility of media, buffers, and materials used
as well as aseptic technique?
2. Whenever appropriate, are positive test controls (e.g., media) performed?
3. Are inoculum counts verified at the time of test (e.g., positive controls and validations)?
4. Is work performed in a laminar flow hood or biological safety cabinet?
5. Is test data analyzed for aberrant/out of specification (OOS) results?
6. Are test methods followed as written?
7. Are isolated microorganisms characterized as needed?
8. Has microbial growth media used in testing been released from quarantine?
9. Have the microbiological test methods for determining microbial content been validated?
10. Have the microbiological test method data been properly documented?
11. Is microbiological testing performed on susceptible raw ingredients?
12. Is in-process bulk batch testing performed?
13. Are sampling requirements for finished products followed?

Culture Maintenance
1. Are ATCC or standard culture collections microbial cultures verified upon receipt (prior to use)?
2. Is record keeping and traceability of microorganisms used in testing in place?
3. Is the (e.g., not more than five passages from original) source culture reculturing being monitored.

Water
1. Is there a source of distilled or deionized water?
2. Is water monitored routinely for chemical and microbiological quality?
3. Review data generated to include evaluation of corrective action plans when test results are aberrant.
4. Review sanitization and preventative maintenance log records for laboratory water system.
5. Review procedure for taking microbiological test samples of water.
6. Are representative water isolates identified?

8
Sample Handling
1. Are there adequate written procedures for receipt, storage and handling of test samples?

LABORATORY AUDIT
MICROBIOLOGY
2. Are there established sample turnaround times/targets?
3. Are sample submission forms stamped with date/time upon receipt?
4. Are samples given an unambiguous sample number when logged?
5. Does a permanent record exist for sample log-in data?
6. Are appropriate chain-of-custody procedures documented and followed, when required?
7. Are there established operating procedures available for disposal of samples?

Quality (QA/QC) System

1. Is the Quality Assurance Manual readily available to all staff members?
2. Is the Quality Assurance Manual updated regularly?
3. Does the quality assurance officer operate independently of analyses?
4. Does the laboratory have periodic system audits?
5. Does the laboratory evaluate its performance through proficiency testing?
6. Do proficiency testing programs have feedback and corrective action programs, procedures, and protocols in
place?
7. Does the laboratory provide in-house training on quality?
8. Are QA policies, protocols, and procedures documented for the following:
a. Methods?
b. Sample collection and handling?

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 SECTION 8 MICROBIOLOGY LABORATORY AUDIT

Table 8-1 (contd.)

c. Quality Control for each type of test performed?

d. Procurement and inventory control?
e. Operation and calibration of laboratory equipment?
f. Preventative Maintenance?
g. Records Management?
9. Is data retrievable and identifiable for each test result?
10. Are applicable computer software programs documented, and back-up copies secured?
11. Are any analyses subcontracted?
12. Are subcontracted laboratories evaluated for Quality Assurance?
a. Are they audited, when and how often?

Records Management
1. Is a system in place that provides for retrievability and traceability of sample source, methodology of analyses,
results, person performing analysis, and date?
2. Are records and reports adequately secured and retained for the required length of time to ensure their
integrity?
3. Are all laboratory notebooks, when completed, filed in a secure, controlled archive area and from which they can
be easily retrieved?
4. Are all laboratory equipment/instrument maintenance logs uniquely identified and stored for easy retrieval?
5. If the laboratory operates a computerized data/information management system (LIMS), are there backups to
ensure integrity and availability of data/information in the event of a system/power failure?

Test Reports
1. Do the laboratory’s reports accurately and clearly present test results and all other relevant information?
2. Does each test report include the following:
a. Identification of laboratory issuing the report?
b. Identification of client. if applicable?
c. Sample identification and description (e.g., sample name and lot number)?
d. Dates/times of sample collection or receipt. Receipt and types of testing performed?
e. Identification of microbiological test methods used in the analysis?
f. Description of sampling procedure, where relevant?
g. Any deviations, additions, or exclusions from a test method?
h. Disclosure of any subcontractor used?
i. Results and any failures identified?
j. Identity of person accepting responsibility for the testing?
k. Is the laboratory report format understandable?
l. Are corrections or additions made to test reports after being issued?
m. Is there a policy/protocol to handle inquires and complaints about test reports and results?
n. Is there a policy/protocol in place outlining the checking and authorization for data release to clients?
LABORATORY AUDIT
MICROBIOLOGY
8

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 MICROBIOLOGY LABORATORY AUDIT SECTION 8

Table 8-2

MICROBIOLOGY SKILLS CHECKLIST (EXAMPLE)

This Microbiology Skills Checklist provides an example of guidance for training areas for skills and the various basic
duties and responsibilities assigned in the microbiology laboratory. The need for proficiency in specific tasks is
dependent on an employee’s work level and area of responsibility. The skills trainer/assessor should be proficient
in all areas being reviewed. This list may be modified as needed based on equipment and function of the lab.
Microbiological laboratory skills include but are not limited to the following:

SKILL

Techniques and Analyses

• Aseptic technique
• Pipetting B traditional and micropipettor
• Streak for Isolation
• Gram stain/KOH string test
• Catalase test
• Oxidase test
• Microbial identification B traditional & rapid
• Water testing and sample collection
• Individual test method proficiency

Media
• Preparation and storage
• Media quality control
• Media remelt in autoclave/microwave
• Use of differential media

Equipment Use and Operation

• Microscope
• Water bath
• Autoclave
• Colony counter
• Spectrophotometer
• Water activity meter
• Lyophilizer
• Anaerobic chamber and jar

Microorganisms
• Inoculum preparation

8
• Organism maintenance techniques

LABORATORY AUDIT
MICROBIOLOGY

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 SECTION 8 MICROBIOLOGY LABORATORY AUDIT

Table 8-3

MICROBIOLOGY LAB B EQUIPMENT CALIBRATION

AND PERFORMANCE
Daily/Continuously

Temperature controlled devices including incubators, Verify temperature control

waterbaths, refrigerators, freezers, centrifuges

Humidified chambers Verify humidity control

Autoclave Sterility indication - each day of use

pH meters Calibration - each day of use

Water activity device Calibration - each day of use

Weekly

Treated water Chemical and microbial testing

Six Months

Balances Calibration

Spectrophotometers Calibration

Dispensers Calibrate dispensing volume

Freeze Dryer (Lyophilizer) Vacuum, temperature, timing calibration

Annually

Autoclave Temperature calibration

Centrifuge Calibration (temperature, rotor speed)

Laminar flow/biohazard hood Certify airflow and operation

Thermometers Accuracy vs. NIST thermometer

LABORATORY AUDIT

Pipettors Dispensing accuracy & precision calibration

MICROBIOLOGY

Stomachers Efficacy

Sonicators Efficacy

Microscope Inspection - alignment, lenses, mechanical aspects

UV lamps Certification or replacement

8

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REFERENCES
1. U.S. Food and Drug Administration 2015. FDA 21 CFR, Part 210 - Current Good
Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs; General.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=210
2. U.S. Food and Drug Administration 200 Guidance for Industry, Q7A Good Manufacturing
Practice Guidance for Active Pharmaceutical Ingredients. http://www.fda.gov/ICECI/
ComplianceManuals/CompliancePolicyGuidanceManual/ucm200364.htm
3. United States Pharmacopeia and the National Formulary 2016. (USP39-NF34) Pharmacopeia,
1260 Twinbrook Parkway, Rockville, MD 20852).
4. Nikitakis, J.M. (ed.) 2014. ISO 22716. Cosmetics good Manufacturing Processes. In PCPC
Quality Assurance Guidelines, The Personal Care Products Council, Washington, DC 20036
5. Fleming, D.O. and Hunt, D.L., Eds. 2006. Biological Safety: Principles and Practices, Fourth
Edition ASM Press, Washington, D.C.
6. Department of Health and Human Services, Centers for Disease Control and Prevention and
National Institutes of Health, 2009. Biosafety in Microbiological and Biomedical Laboratories
(BMBL) 5th Edition, U, US Government Printing Office Washington: http://www.cdc.gov/
biosafety/publications/bmbl5/bmbl.pdf
7. National Institute of Standard and Technology 2016. Specifications, Tolerances, and Other
Technical Requirements for Weighing and Measuring Devices. http://www.nist.gov/pml/wmd/
pubs/hb44.cfm
8. National Institute of Standard and Technology 1996. Specifications and Tolerances for
Thermometers. www.nist.gov/pml/wmd/upload/105-6.pdf
9. Nikitakis, J.M. (ed.) 2014. Annex 16 Calibration Systems. In PCPC Quality Assurance
Guidelines, The Personal Care Products Council, Washington, DC 20036, pp. 99-102.

8
LABORATORY AUDIT
MICROBIOLOGY

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 SECTION 9

Microbiological Validation
and Documentation

INTRODUCTION
Validation assures that the methods and procedures used by the personal care microbiology labora‑
tory are accurate and reproducible. Documentation provides the record of the validation.
In order for results to be meaningful, methods and procedures must be validated. Otherwise, the
conclusions drawn could be erroneous. For example, failure to properly neutralize the preservative
system during testing can result in false negative results where lack of organism recovery may be due
to inhibition of the organism.
A variety of techniques for validating and documenting methods and procedures is available. In
addition to the test methods and procedures used in the laboratory, the microbiological aspects of
process water systems and of cleaning and sanitizing procedures can also be validated by the micro‑
biologist. The results of a validation should then be documented in an organized record keeping
system.
Once these methods and procedures are validated and documented, there is a high degree of confi‑
dence that they can consistently produce accurate and reliable results. This completes the full circle
of quality assurance and is necessary to assure product quality.
Personnel responsible for any aspect of the validation must be adequately trained by education and/
or experience.

GENERAL CONSIDERATIONS
Validation is a process designed to establish documented evidence that a method or procedure does
what it purports to do. A validation or revalidation should be performed:

• When a new method or procedure is developed.

• If there is a significant change in procedure, supplier, product formulation or equipment.
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• As part of a recommended revalidation schedule.

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A. Validation Format
The validation format should contain the following elements:
1. Scope
The scope identifies the area being validated, describes the purpose of the validation, and tells
what it encompasses. This should include the types of validation that will be performed. Three
types of validation include:
a. Prospective
Prospective validation establishes documented evidence that a process does what it purports
to do based on a preplanned validation protocol. All information and results are gathered
before implementation.
b. Concurrent
Concurrent validation establishes documented evidence that a process does what it purports
to do based on information generated during actual implementation of the process. The test
methods, procedures (such as cleaning and sanitizing), systems (such as deionized water sys‑
tem) and equipment are being used while data are being gathered to support the validation.
c. Retrospective
Retrospective validation consists of presenting documented evidence that, based on a review
and analysis of historical data and information, a process does what it was meant to do and
that, all things being equal, can be expected to continue performing properly.
It should be noted, concurrent and retrospective validation can be performed simultaneously.
2. Description
A detailed step by step description of the procedure or method is provided.
3. Requirements
The requirements and acceptance criteria for the areas to be validated are described. For example:
a. Equipment cleaning and sanitization requirements include specific sites, number of swabs
before and after, and defined performance criteria.
b. Laboratory autoclave requirements include number of heat penetration studies, defined ac‑
ceptable results, and negative controls acceptability criteria.
c. Test methods criteria include the number of replicate platings and the allowable variance.
4. Protocol
A written protocol for each method or procedure to be validated should be included.
5. Documentation
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A validation protocol should include appropriate documentation of all methods, procedures

and results.

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6. Conclusion
Each validation protocol should include a conclusion that indicates the results of the valida‑
tion. If the results are satisfactory, a statement may be made that the validation is satisfactory
and that the method or procedure can produce accurate and reliable results. If the results are
unsatisfactory, the next steps or modification required to complete a satisfactory validation
should be indicated.
A final written report should be prepared once the validation has been completed. Any revali‑
dation information can be added as it is generated.

B. Documentation
Once a procedure or method is validated, all results generated during its use should be document‑
ed by keeping an organized record system. They can be organized by product or type of test and
can include:

• Material identification/description
• Identification/code number
• Vendor/in‑house lot number
• Reference to the validated procedure used
• Acceptance criteria
• Tested by
• Date tested
• Reviewed and approved by.

All results should be routinely reviewed by supervisory personnel. Records should be kept for
an appropriate length of time. Refer to the PCPC Quality Assurance Guidelines, Production and
Control Documentation.1
Documented results can be maintained as part of a product or raw material profile in the devel‑
opment of an historical data base. Validation procedure results should be included as part of this
data base. Results which were compiled in an historical data base prior to validation may be useful
as part of a retrospective validation. The data base can also be used as a guide in interpreting test
results and establishing test guidelines and requirements.

LABORATORY AUTOCLAVE
An autoclave is an instrument that uses moist heat supplied by steam under pressure to sterilize
9

materials. The contents, whether liquid or solid, are exposed to saturated steam at the required tem‑
perature and period of time. Pressure serves as a mechanism for obtaining higher temperatures than
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otherwise could be obtained.

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A. Validation
1. Preliminary Considerations
The accurate measurement of both time and temperature are necessary to confirm the steriliza‑
tion of the autoclave chamber contents. Before an autoclave cycle can be validated, tempera‑
ture controllers, temperature recording devices, pressure gauges, and timers of the autoclave
must be certified for accuracy to insure proper operation.
The autoclave load configuration and contents are important elements. The autoclave cycle will
vary based on volume of containers, number of containers, container type, media type, etc.
2. Heat Measuring Devices
Biological indicators and/or chemical and/or mechanical heat measuring devices can be used
to validate an autoclave cycle. The use of biological indicators (BIs) for certain media cycles
may be problematic if minimal sterilization cycles are required. BIs are available with different
populations. Select the most appropriate population.
a. Mechanical Devices
These include calibrated autoclave thermometers or thermocouples.
b. Biological Indicators
Due to their high resistance to moist heat, Bacillus stearothermophilus spores on a paper strip
or as a spore suspension in a glass ampule are the most commonly used biological indicator
used for monitoring autoclave performance.
At the end of the autoclave cycle, the Bacillus stearothermophilus biological indicators
are removed and incubated per manufacturer’s directions. These are usually incubated at
56.0‑60.0°C for 7 days to account for the possibility of slower growth following exposure to
sublethal heat. Use appropriate media if spore strips are used. If BIs are used, they should be
certified against the label claim.
Negative and positive controls should be incubated along with the autoclaved biological
indicator samples. An unautoclaved biological indicator should be incubated as a positive
control. If spore strips are used, a negative media control should be also included.
c. Chemical/Physical Indicators
Chemical and physical indicators are used as a secondary check to monitor a validated cycle.
They are not intended to be used as primary indicators to validate a cycle. Autoclave tape
indicates it has been exposed to an autoclave cycle when, for example, black stripes or the
word “autoclaved” appear on the tape. Ampules which contain a material that melts and
changes color when exposed to the proper temperature may also be used to monitor an
autoclave cycle.
3. Heat Distribution and Penetration
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Heat penetration and/or distribution studies should be performed on empty (or minimum)
and maximum load configurations of the autoclave chamber by using temperature measuring
devices and biological/chemical indicators. The biological/chemical indicators and/or the heat

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measuring devices should be evenly distributed throughout the autoclave chamber for the
monitoring of representative areas. At minimum, the monitoring devices should be placed at
the four corners and center of the autoclave. The actual number may vary based on the size of
the chamber and the load pattern.
a. Heat Distribution
The purpose of a heat distribution study of an autoclave is to determine the uniformity of
temperature in the load. Heat distribution studies should be performed on load configu‑
rations of the autoclave chamber by using temperature measuring devices. It is suggested
that a minimum of three heat distribution studies be done on the load configuration being
validated.
A mean chamber temperature is taken from all the distributed temperature readings. Cham‑
ber temperature uniformity can be considered acceptable if individual temperature readings
deviate less than 1.0°C from the mean chamber temperature. A mean temperature devia‑
tion greater than 2.5°C versus the set temperature may indicate equipment malfunction.
Chamber uniformity range should be based on the manufacturer’s recommendation for the
autoclave capability.
b. Heat Penetration
The purpose of a heat penetration study is to assure that all the containers within a loading
pattern will consistently be exposed to a sufficient amount of heat for sterilization. A mini‑
mum of three heat penetration studies is suggested.
Heat penetration studies may be performed by placing chemical/biological indicators in
containers of media distributed in a load pattern throughout the autoclave. Biological and/
or chemical indicators should be placed throughout the chamber including areas considered
to be the most difficult to sterilize.
4. Documentation
The following information should be recorded for each validation run:
a. Date of validation
b. Autoclave identification or number
c. Run number
d. Sterilization time
e. Sterilization temperature
f. Load contents (media, broth or agar)
g. Number of containers, configuration
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h. Indicators used and the lot number

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i. Placement of indicators, temperature measuring devices

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j. Results of indicators, measured temperatures

k. Control results.
A cycle is considered to be validated when all the chemical and/or biological indicators show
appropriate reactions or no growth after the process and all heat measuring devices read within
acceptable parameters.2

B. Routine Monitoring
Each autoclave cycle should be monitored for the following:

• Temperature and time - Confirm temperature and length of cycle on recording charts or
cycle printouts.
• Pressure - Observe pressure on gauge and confirm if recorded on cycle printouts.
• Biological indicators - Monitor using BIs at least quarterly where applicable for monitoring
a validated cycle.
• Chemical/Physical indicators - Temperature-sensitive indicators, such as autoclave tape,
should be used with each load.

Preventive maintenance should be performed on a routine basis by the manufacturer or other

qualified personnel. Records of maintenance should be retained.

MEDIA
A. General
Microbiological culture media contain growth-promoting substances such as available sources of
carbon, nitrogen and inorganic salts. The quality of the growth characteristics of microorganisms
in culture media depends upon the care taken in the preparation of the medium. To insure satis‑
factory microbial culture media for use in the microbiology laboratory, a validation and quality
control program should be established for freshly prepared or received media as well as for estab‑
lishing the shelf life of the media.
This applies whether the culture media are prepared from commercially dehydrated products or
purchased from a supplier in prepared form. This program is needed to ensure that the media can
consistently perform as expected over its shelf life. This program should include the identification
and control of those factors which affect the performance of the media. Some of these factors
include:

• Preparation of media
• Temperature
• pH
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• Storage conditions
• Shelf life

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B. Validation and Quality Control Program

Elements of a media validation and quality control program include:
1. Inventory Control and Storage of Dehydrated Media
Write the date received and the date opened on the containers of dehydrated media.
Follow the manufacturer’s expiration dates and storage conditions. Most dehydrated micro‑
bial culture media can be stored in a cool, dry place, preferably at a temperature below 30°C.
Manufacturers recommend storing certain types of dehydrated microbial culture media at a
refrigerated temperature (2‑8°C).
Rotate laboratory stock of culture media so that the oldest container is used first. This practice
will allow a turnover in the dehydrated media containers. It will help keep media stock fresh
and within the manufacturer’s expiration dates. Media with outdated expiration dates should
be discarded.
Protect laboratory culture media that is in dehydrated form from absorbing additional mois‑
ture from the environment during storage. A high moisture content could possibly cause the
degradation of various ingredients of the media.
Supplements and additives, where used, should be stored per manufacturer’s recommenda‑
tions. For example, store under refrigeration if the material is sensitive to heat degradation, or
protect from light until use if the material is light sensitive.
2. Preparation of Dehydrated Media
a. Preparation
Follow manufacturer’s directions for the preparation of dehydrated media. Factors in pre‑
paring dehydrated media which may affect its performance include:
• Water for reconstitution - Use purified water, such as distilled, deionized or reverse osmo‑
sis, to reconstitute dehydrated culture media. At a minimum, the water should meet the
Environmental Protection Agency guidelines for drinking water standards.3 Microbiolog‑
ical and chemical evaluation of purified water is recommended. The quality of purified
water may be checked for specific chemical parameters, for example using current USP.
or in‑house requirements, on a regular basis.4,5
• Agar temperature - Monitor temperature when pouring agar plates for streak plates. The
correct temperature should be employed since an incorrect agar temperature can result
in the alteration of the final water content of the medium by excessive evaporation and
medium shrinkage or excessive condensate.
• Additive temperature - Temperature of additives or supplements which are required to be
added after sterilization are important. They should be added at the correct temperature
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to avoid the chemical degradation or destruction of the additive or supplement.

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• pH - The pH of the microbial culture media is critical. There should be a laboratory pro‑
cedure that lists the acceptable pH ranges for a liquid or agar medium. In general, the pH
reading should be taken after autoclaving and subsequent cooling to room temperature
and should meet established requirements.

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b. Media Records
Establish a batch record for each lot of culture medium that is prepared in the laboratory.
The following information should be included on the batch sheet:

• Medium name
• Date of preparation
• Manufacturer’s lot or batch number
• Quantity prepared
• Signature of preparer
• Method of preparation
• pH after autoclaving
• pH adjustment, if required
• Sterilization time and temperature
• Volume dispensed
• Number of units dispensed
• General comments (e.g., appearance of the medium during preparation)
• Performance test results and disposition (acceptable/unacceptable).

Where available and/or appropriate obtain a certificate of analysis the supplier of prepared
media.
3. Labeling and Storage of Prepared Media
a. Labeling
Label all microbial culture media, whether commercially prepared or prepared in the labo‑
ratory. Include the following information:

• Date of preparation or date received of commercially prepared media

• Type of media
• Shelf life or an expiration date, either on the label or on a separate list.

b. Storage
Establish a shelf life, an expiration date for the culture media. In general, the shelf life of a
particular medium is dependent upon how well that medium will continue to support the
growth of test microorganisms. The shelf life can be determined by growth promotion stud‑
ies over time and storage conditions. This can be accomplished by comparing the growth
promotion abilities of the stored versus freshly prepared microbial culture media. Factors
which affect the shelf life of prepared media include:
• Form - The formulation and packaging of the medium will decide its basic susceptibility
to deterioration during storage. In most cases, the shelf life of an agar medium in a Petri
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dish will be shorter than the same dehydrated agar in a sealed container or bottle.

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• Storage temperature - The optimum storage temperature for the majority of prepared mi‑
crobial culture media is about 2‑8°C. Most liquid media will keep for many months at
2‑8°C, but have a tendency to form deposits. This is especially true for culture media at
double strength. If stored at room temperature, shelf life may be shortened.
• Light exposure - Dye‑containing media may fade if exposed to light.
• Evaporation - A volume check on liquid media should be made on older stocks which may
change due to evaporation.
• Dehydration and contamination - Prepared solid media can be stored for many months
in an airtight container. The storage of agar Petri dishes presents two main problems:
dehydration and contamination. The length of storage time that agar Petri dishes can be
kept for use will depend upon the ability to avoid microbial contamination and the loss
of moisture. To prevent moisture loss, agar Petri dishes can be wrapped in plastic bags for
storage at 2‑8°C.
4. Performance Testing of Microbial Culture Media
Performance testing is conducted to confirm that a given media yields expected results when
inoculated with applicable microorganisms.
Usually on the same batch preparation sheet or on a separate coordinated sheet, the following
performance testing details are listed:

• Medium
• Batch/preparation date
• Date tested
• Sterility evaluation
• Specific microorganisms
• Growth promoting, differential, and inhibitory ability
• Pass/Fail
• Operator’s signature and date.

A sterility check should be performed for each lot of prepared microbial culture medium. In‑
cubate a sufficient number of units at the temperature and time for which the medium is going
to be used in the laboratory testing. The sterility test for each lot of prepared medium will
document that it was sterilized properly. This test will ensure that any microbial contamination
detected during a test procedure using this lot of prepared medium was not caused by a lot of
improperly sterilized media.
Each lot of dehydrated microbial culture medium or commercial or laboratory-prepared lot should
be tested, as appropriate, for its ability to support, differentiate, or inhibit the growth of microor‑
9

ganisms. For general selection media, the choice of which microorganisms should be used to vali‑
date the ability of that medium to support growth is up to the individual laboratory. It is preferable
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to use representative strains of different types of organisms. However, selective or differential media
should utilize microorganisms that will demonstrate the characteristics of that medium. A negative
control organism may be included to determine that there are no false positive reactions.

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If all control parameters of the preparation process are validated and monitored, testing may
be done less frequently, but on a regularly scheduled basis.
Two methods used by industry to validate the growth promotion ability of a microbial culture
medium are described below.

• One method involves inoculating a culture medium by streaking solid media or pipetting
into liquid media with a dilution yielding 10‑100 colony forming units (CFU) prepared
from a 24-hour culture of a known microbiological strain.
• The second method consists of inoculating by streaking or pipetting and spreading a solid
culture medium surface with a 10‑3 dilution of a 24-hour culture of a known microor‑
ganism(s).

The choice of method is up to the individual laboratory. There are many factors to consider
including the type of test and the sensitivity and/or detection limits of the test procedures for
which the media will be used. The known microbiological strains used may be American Type
Culture Collection (ATCC) strains of a particular microorganism. In either case, the inoculat‑
ed media should be incubated at the same temperature and time for which it will be used in
laboratory testing. After incubation, the inoculated liquid media is examined for the presence
of turbidity and general solid media for the presence of growth. Selective and enrichment agars
are examined for typical color and colony morphology of the strain used to inoculate them.
If the lot is within the correct pH range, passes the sterility test, and passes the growth promo‑
tion test, it can be used for testing in the microbiology laboratory. If the medium’s pH is out
of range, if the medium does not pass the sterility test, or if it fails to support the growth of the
test microorganism(s), the medium fails the criteria for use in the laboratory.
There may be times when performance testing is conducted concurrently with the use of the
media for laboratory testing. If a lot of media fails the performance test and was used in the
laboratory, all laboratory testing should be repeated with acceptable media.
Results of performance testing should be documented and reviewed for accuracy.

TEST METHODS
A. General Considerations
Validation of a microbiological method is the process by which it is established through labora‑
tory studies that the performance characteristics of the method meet the requirements for the in‑
tended application. Protocols should be designed to generate reliable, accurate and reproducible
results. Microbiological methods are designed to detect and/or identify microorganisms when
present. These methods include conventional plate count/streak plate procedures, as well as rapid
or automated methods. These methods are intended to perform within a wide range of variables,
some of which are:
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• Different types of product

• Different types of media

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• Incubation conditions
• Organisms
• Detection limits

B. Elements of a Validation Protocol

A protocol should be designed to incorporate all variables inherent to the method. Each step of
the protocol or procedure should be documented and include (when applicable):

• Reference to protocol or test procedure number

• Incubation conditions (time or temperatures)
• Neutralizing agents for preservatives or other growth-inhibiting agents
• Confirmation of preservative neutralization
• Identity of personnel performing each step
• Type of media
• Media lot numbers
• Test organisms ‑ known profile (biochemical, morphological, etc.)
• Test organism inoculum level
• Dilutions tested
• Acceptance criteria ‑ including test and control sample. For example, one acceptance criteri‑
on is recovery of artificially introduced microorganisms into a test and control sample. Data
is analyzed comparing test and control sample results.
• Test personnel’s signature upon test completion
• Reviewer’s signature
• Test organisms used should be maintained, as recommended, by the supplier of the organ‑
ism.
• Subculturing should exhibit unique, demonstrable characteristics of the organisms to ensure
a pure and known culture.
• Organism storage, transfers and subcultures should be documented.

Modifications to the protocol should be documented.

The accuracy of a test method can be validated by performing repeated tests on the same sample.
Its reproducibility is usually confirmed by designing a collaborative test whereby a number of
different laboratories perform the same procedure on material from the same batch.
A file should be maintained for all data generated by the test procedures.
In addition to the above elements and general considerations, the following items apply to spe‑
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cific test procedures.

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C. Microbial Content Testing

• Microbial content testing may be performed on raw materials, components, bulk product,
in-process material and finished goods.
• Microbial limit guidelines, sample size and frequency of testing should be established.
• The established testing guidelines should have appropriate documentation.

Refer to “M-1 Determination of the Microbial Content of Personal Care Products”(Section 18),
“Raw Material Microbial Content” (Section 11), and “Establishing Microbiological Quality of
Personal Care Products” (Section 12)

D. Preservative Efficacy Testing

1. Method
The method of preservative challenge testing which is adopted for use should predict preserva‑
tive efficacy under routine good manufacturing practice and normal consumer use conditions.
The preservative system is not intended to compensate for poor GMPs.
2. Test Procedure
The type of test procedure used should be determined. For example:

• Semi-quantitative ‑ Swab or Streak plate Method

• Quantitative ‑ Total Plate Count.
3. Protocol
The elements of the protocol should include:

• Length of the test

• Test organism(s)
• Media
• Sampling frequency
• Confirmation of the neutralization of the preservative
• Test parameters (incubation time, temperature, etc.)
• Sample size
• Acceptance criteria.

Refer to “M‑3 A Method for the Preservation Efficacy Testing of Water‑Miscible Personal Care
Products” (Section 20) and “M‑4 Method for the Preservation Testing of Eye Area Personal Cares”
(Section 21).
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E. Environmental
Ambient air, compressed air, and equipment surfaces in the manufacturing plant are monitored
for the presence of microorganisms using apparatus such as settling plates, air samplers, swabs,
and contact plates. Equipment should be evaluated by reviewing manufacturer’s technical infor‑
mation.
Surface monitoring procedures can be validated in the laboratory by applying known types and
levels of organisms to sample surfaces simulating production surface materials. Recovery using
the various surface monitoring techniques can then be compared to initial types and levels of
organisms to determine the acceptability of the procedure.

• Equipment should be maintained per manufacturer’s specifications and or recommenda‑

tions.
• Validate that the media used supports organism growth.
• Media should neutralize any sanitizer present.
• Establish action and alert levels based on historical data.

Refer to AMicrobiological Evaluation of the Plant Environment.”

ORGANISM IDENTIFICATION
A. Identification systems
Microorganisms may be identified using classical methods. Identification via commercial identi‑
fication kits and/or automated systems is common practice in personal care microbiology labo‑
ratories. Systems used for microbial identification should be validated to ensure they consistently
and accurately identify microorganisms tested.
A validated system should show equivalence with a known method. Introduction of an alternate
identification system requires a validation vs. the current system to ensure performance is compa‑
rable or better than the current system(s). Some of the factors to include:

• Side-by-side comparison testing of the standard routine cultures used in the laboratory and/
or ATCC cultures.
• Side-by-side comparison testing of unknown organisms such as those isolated from the
plant product.

Standard cultures of microorganisms, such as ATCC cultures, should be used. A performance

check should be done on each new lot of kits received. It is important to follow the directions
supplied with the systems. The manufacturer of the identification systems may recommend a
9

series of QA microorganisms to test reliability and accuracy. QA test organisms should be chosen
to give a positive response to each of the tests. Organisms used for the QC test should include
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the following:

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• Positive Control
Inoculate with organisms the manufacturer claims are identifiable by the kit.
• Negative Control/Identification System Control
If applicable, inoculate with sterile saline or appropriate diluent. Do not inoculate with any
organisms. Follow manufacturer’s recommendations.

A system can be considered valid if the known organisms are properly and consistently identified
and the negative controls/identification system controls, as applicable, do not demonstrate any
reactions.
Records should be kept specifying the following:

• System tested
• Manufacturer
• Lot number
• Date tested
• Organisms tested
• Results
• Acceptance criteria ‑ action taken if system or performance check not valid
• Initials or signature of test performer
• Initials or signature of reviewer, as appropriate.

Refer to AM‑2 Examination for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa,
and Candida albicans (Section 19).

MANUFACTURING, EQUIPMENT CLEANING AND SANITIZING

PROCEDURES
A. Introduction
Validation of cleaning and sanitization methods is a procedure of establishing performance char‑
acteristics of a cleaning and sanitizing process and to document that this process will consistently
yield equipment that meets microbiological, physical and analytical chemistry requirements. The
microbiological validation process basically consists of:

• Determining requirements
• Writing a protocol
• Following the written protocol
• Testing and documenting the results.

Results should consistently demonstrate that the process is in control and all steps should be
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carefully documented. The protocol should be approved by the appropriate personnel including
the Microbiology Department. Performing the tests and evaluating the results should be the re‑

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sponsibility of the Microbiology Department. This activity should be coordinated with analytical
chemistry, manufacturing, engineering and key personnel in other departments.

B. Validation Protocol
Components of the validation protocol include:
1. Determine specification or outcome desired.

• What results are required to indicate that the procedure will result in equipment that meets
requirements?

2. Outline cleaning and sanitizing procedure to be validated.

a. Identify specific equipment and testing locations.
b. What pumps, valves, filters, tanks, lines will be looked at and where?
c. List cleaning methods and agents, concentration, contact time, temperature.
d. Equipment must be clean before it can be sanitized.
e. List method(s) of sanitization.
f. Be sure that the sanitizer has itself been evaluated.
g. Map the cleaning and sanitization process; for example:

• Tank #XXX and associated pipes, pumps & lines ‑ identify them.
• Rinse with hot DI (deionized) water for XXX minutes to remove product residue. Disas‑
semble pumps/equipment when necessary to remove product residue.
• Drain rinse water.
• Fill tank with ?% solution XX.
• Recirculate/mix for XXX minutes.
• Drain wash solution.
• Rinse to remove wash solution.
• Sanitize (follow directions for use of sanitizing agent).

h. Establish a time limit for use of prepared sanitizing solution.

i. Establish frequency of sanitization.
j. Establish a time limit for resanitization.
3. Determine what steps should be evaluated, document test procedure (be specific) and
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determine what data is required.

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4. Determine test methods used to provide the data and acceptance criteria required in item
3 (previous page) to confirm the efficacy of the cleaning and sanitizing procedures. Sever-
al types of methods include:

• Visual examination
Visual inspection to detect product residue.
• Examination for odor
Inspect to detect the odor of residual perfume or sanitizer.
• Swabs
Swabs are used to detect microorganisms.
• Rodac plate
Contact plates are used to detect microorganisms.
• Rinse water
Tests on rinse water to detect microorganisms and/or chemical residue.

5. Compare results of tests performed before and after the cleaning and sanitizing proce-
dures to confirm the efficacy of the process.
6. Validation testing should be performed a minimum of three times.
Once the validation for a piece of equipment is completed, it need only be repeated if there is
a change in process, equipment or any parameter that may affect the outcome of the process,
such as product type. To remain aware of any changes, a Process Audit should be routinely
done and communication with key personnel maintained. There should be a routine review,
for example, annually, to insure and document that there have been no changes.
After a process is validated, routine testing, such as equipment monitoring, can be used to
monitor the process.
Refer to “Processing Equipment” and “Packaging Equipment” in the PCPC Quality Assurance
Guidelines.1

PROCESS WATER SYSTEM

A. Validation
The purpose of validating a process water system is to generate documented evidence to provide
a high degree of confidence that the process water system will consistently produce water that
meets established guidelines. This is especially important with a water system since microbiolog‑
ical test results may not be available before the water is used in manufacturing.
The following elements should be included:
1. System description
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Describe the system and give an explanation of the purpose of each element. These should
include:

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a. The type of system

• Deionizing (DI) ‑ carbon beds, deionizing beds (separate, mixed or both)

• Reverse Osmosis
• Distillation Unit.

b. The flow rate of the system.

c. Description of water flow system:

• Type of piping
• Number of outlets
• Points of use
• Recirculation route if applicable.

d. Description of the storage tanks, if applicable.

e. Description and location of any filters present:

• Micron size
• Prefilter
• Final filter.

f. Description and location of any treatment element:

• UV light with rated throughput

• Chlorination with concentration
• Heat with temperature requirements
• Ozonation with concentration.

g. Description and location of sampling and use points.

h. A schematic of the system should also be included.
2. System maintenance
Describe the system maintenance. Include the following elements:
a. Frequency of regeneration, backflush of carbon beds and/or changes of the DI bed and/or
filters
b. Frequency of cleaning and sanitizing the system
c. Method of cleaning and sanitizing, such as heat treatment or use of cleaning or sanitizing
9

solutions
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d. Description of cleaning and/or sanitizing solution(s) used.

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3. The validation protocol

a. Type of validation performed may be:

• Prospective
• Concurrent
• Retrospective
• Combination.

b. The conditions under which a revalidation will be performed should be listed, whether
periodically, after a significant system change, or under other circumstances. Suggested re‑
validation requirements include:

• Perform a complete revalidation if there is a significant change such as in equipment,

procedure, etc.
• Perform a reduced or modified revalidation at a preset interval, for example, annually, to
ensure that the system is still performing as expected.

A periodic revalidation may also be accomplished by a formal review of the data and system
to document that there are no significant changes, and that the system continues to perform
as intended.
c. Time period covered
Samples should be tested more frequently, a minimum of two or three times per week
during this period. Time period covered should include seasonal changes so that all variables
are part of the validation.
d. Test methods
Include a detailed step‑by‑step description of the methods used or a reference to established
test methods. For example, tests commonly performed are:

• Chemical, as per USP4 or internal requirements.

• Microbiological, including total plate count and any other tests needed to support the
criteria as per established internal or compendial methods.

e. Test criteria
Include the criteria used and any action limits that are established. Also specify what actions
are to be taken.
f. Results
The validation protocol should include copies of all test results during the validation test
period.
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4. Discussion of Results
A discussion of the test results should include any investigations or actions taken if results do
not meet expected criteria.
5. Conclusion
A determination based on an analysis of the results of whether or not the system can consis‑
tently produce water which meets requirements if the key parameters are followed.

B. Documentation
Once established as part of the validation, critical system parameters, such as frequency of regen‑
eration, frequency of cleaning/sanitizing, names and concentrations of cleaners/sanitizers used,
and filter changes, should also be checked on a routine basis and documented to ensure the prop‑
er operation of the system. This information is usually recorded as part of the system maintenance
record. This record should include:

• Dates the system was cleaned and sanitized

• Names and concentrations or conditions of the cleaners/sanitizers used
• Maintenance of UV light if applicable
• Dates the system was regenerated, backflushed and/or filters or resin beds were changed
• Dates and explanation of any repairs to the system.

These parameters should be reviewed as part of an investigation if results do not conform to the
guidelines.
After the water system has been validated, samples should be tested on a routine basis to monitor
its performance. These results should be recorded and trends noted. This information should
include:

• Sampling points
• Reference to the validated test procedure/method
• Acceptance guidelines
• Any investigation results and/or actions taken if results do not conform to guidelines
• Tested by and date tested
• Reviewed and approved by.

A record tracing the use of the water (batch numbers, product) should also be maintained. This
is usually maintained as part of the manufacturing record. Refer to Microbiological Quality for
Process Water. (Section 7)
9
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LABORATORY EQUIPMENT AND INSTRUMENTATION

Diverse laboratory equipment and instrumentation is utilized in the microbiology laboratory to
assist in preparing and performing microbiological analyses. All instrumentation should be regularly
monitored to ensure its proper performance and reliability. A master logbook of equipment requir‑
ing monitoring and the observed results should be maintained. In addition, a file should be kept for
each piece of equipment including:

• Name of instrument
• Model number
• Serial number
• Purchase date
• Manufacturer and/or distributor
• Maintenance and operational manuals
• Service representative information.

A list of suggested equipment and instrumentation to be monitored includes but is not limited to:

• Laboratory autoclave (as above)

• Laminar flow hoods/biological safety cabinets
- Air Flow
- Integrity of HEPA filter
- Particle size
• Balances
- Standard weights
• Centrifuges
- Rotor speed (rpm)
- Timer
• Incubators/refrigerators
- Temperature
• Water baths
- Temperature
• Microscopes
- Optical alignment
- Calibration for scale
- Cleaning and maintenance
• Automatic colony counters
- Standard template
• pH meters
DOCUMENTATION
VALIDATION AND

- Standard pH buffers

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• Spectrophotometers
- Standard solution
• Water activity devices
• Standardized salt solution
• Thermometers

Information pertaining to proper control steps for carrying out performance checks and preven‑
tive maintenance should be found in the operator’s manual. To maintain equipment accuracy and
reliability, these tests and maintenance should be performed at regularly scheduled intervals. The
documentation for such testing should include the following:

• Frequency of performance (monitoring schedule)

• Criteria for acceptance/rejection
• Performance against standards
• Results
• Noted deficiencies
• Corrective action if necessary
• Documentation of corrective action and “fit for purpose” performance should be reviewed
and signed
• Initials or signature of test performer and reviewer.

During the validation of standard operation, all results should be recorded accurately including
deviations and corrective action initiated, if necessary. Records should be reviewed periodically by
supervisory personnel.
All equipment should receive regular preventative maintenance. Specialized checks, such as auto‑
clave servicing, centrifuge calibration and laminar flow hood maintenance should be performed by
trained personnel or an authorized representative of the equipment manufacturer.
Refer to Calibration Systems, PCPC Quality Assurance Guidelines.1

REFERENCES
1. Nikitakis, J.M. (ed.) 2014. In PCPC Quality Assurance Guidelines, 2014 The Personal Care
Products Council Washington, DC 20036
2. PharmOut. 2016. Top 10 Considerations when Validating an Autoclave,
http://www.pharmout.net/downloads/white-paper-autoclave-validation.pdf
9

3. United States Environmental Protection Agency 2016. EPA 40 CFR Part 141.
http://www.ecfr.gov/cgi-bin/text-idx?tpl=/ecfrbrowse/Title40/40cfr141_main_02.tpl
DOCUMENTATION
VALIDATION AND

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4. U.S. Pharmacopeia. 2016. Water for Pharmaceutical Purposes, United States Pharmacopeia
and the National Formulary (USP 39 – NF 34) (U.S. Pharmacopeia, 1260 Twinbrook Parkway,
Rockville, MD 20852.
5. U.S. Food and Drug Agency 2014. Guide to Inspections of High Purity Water Systems,
@ July 1993, U.S. Food & Drug Administration. http://www.fda.gov/ICECI/Inspections/
InspectionGuides/ucm074905.htm

Note: Title 40, U.S. Code of Federal Regulations (40 CFR) and other U.S. Government publica‑
tions are available from Superintendent of Documents, U.S. Government Printing Office, Wash‑
ington, D.C. 20402.
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SECTION 10

Maintenance and Preservation

of Test Organisms

INTRODUCTION
Personal care product microbiological laboratories have an ongoing need to maintain and to pre‑
serve cultures for research, bioassays, training, and industrial testing purposes. Cultures may be
used in challenge testing, efficacy and stability testing, growth promotion testing, quality control
evaluations, etc. Microbial cultures that are used for the conductance of compendial microbial
testing should be sourced from either a national culture collection or a qualified secondary supplier
of microbial cultures that is in a form such as frozen, freeze dried, on microbial growth agar slants,
ready-to-use, or that which is most appropriate for use in the laboratory.
The major goals for a maintenance and preservation program of microbial cultures are to ensure:

• Culture Viability
• Culture Purity
• Retention and consistency of genotypic and phenotypic characteristics of a microbial strain

Some important considerations with regards to the maintenance and preservation program for mi‑
crobial cultures include:

• Understanding the advantages and disadvantages of the method(s) chosen and their appli‑
cability
• Matching the laboratory expectations to the method(s) chosen for factors such as ease of use,
laboratory expertise needed, overall value, etc.
• While a number of methods are applicable to a broad array of microorganisms, no method
is ideal for all microbial species. The practitioner is encouraged to consult the scientific liter‑
ature to help in providing understanding of the specific requirements for having a successful
maintenance and preservation program, especially when there is question as to the applica‑
bility of a preservation method for a given microbial strain.

For the purposes of this guideline, methods are divided into long and short term categories. Long
term methods are considered those which routinely ensure culture viability of greater than 3 years.
Short term methods include those methods which routinely ensure a varying degree of culture via‑
bility, from as little as 1 week up to 3 years. Not every method is represented in this chapter but any
validated methods are acceptable.

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SHORT-TERM METHODS
Subculturing
Maintaining microorganisms on or in a microbial growth media with periodic transfer of pure mi‑
crobial colonies is the most common and simplest method of preserving a microbial culture line. It
10

has the advantage of not requiring special equipment. However, it is also the method that has the
greatest possibility in having the accidental introduction of contamination with other microbial
strains as well as causing strain variation. By using a fluorescent amplified fragment length polymor‑
phism analysis, Cross, Russel, and Desai showed that years-old microbial working control cultures
from several accredited microbial food testing laboratories were indeed genetically different from the
original strains that had been obtained from the National Culture Type Collection.1

Storage Conditions
Store cultures for up to four weeks at 2-8°C or at a temperature appropriate for the microorganism.2,3
A few fastidious microbial strains, such as Streptococcus pneumoniae, will require shorter intervals be‑
tween subcultures to maintain viability. In general, length of storage should be the maximum time
that ensures a viable, unaltered culture.
In order to prevent drying, cultures grown on agar plates can be wrapped with sealing film. Alter‑
natively, microbial cultures may be stored on a microbial growth agar slant or as a stab in microbial
growth agar that is present in a screw-top test tube with a rubber lined cap.

Special considerations
The rate of microbial mutation will vary from strain to strain with subculturing. Some strains easily
lose plasmids, which confer properties such as antimicrobial resistance.3 In order to avoid genetic
drift, passages or transfers should be tracked to prevent excessive subculturing for a working mi‑
crobial culture. The United States Pharmacopeia (USP) recommends that the number of passages
from the reference culture be five or less for certain compendial tests such as the Antimicrobial
Effectiveness Test.4
Conditions required for microbial survival will vary from species to species. Sometimes, microbial
survival will also vary from strain to strain. Each microbial testing laboratory will need to determine
the maintenance conditions that are necessary for those microbial strains that are used routinely for
testing.
Frequent check for maintaining the unique characteristics for a microorganism such as pigment
color, colonial morphology, biochemical or other traits should be performed to ensure variations or
contamination with other strains has not occurred.

Advantages
• Expensive equipment is not required.
• No special training is needed.

Disadvantages
• There is a risk of mutation and contamination with every transfer.

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• Most bacterial cultures survive only a few months. Some fungal species may survive longer.
• Requires extensive refrigeration space.
• Need appropriate back-ups and contingencies to ensure refrigerator does not fail.

Overlay Techniques
As an extension of common short-term subculture methods, an overlay of mineral oil can be used.
By overlaying with mineral oil, it reduces the availability of air to those microorganisms that have
been overlayed which causes the slowing of their metabolic rate. After microbial growth for a cul‑
ture on either a microbial growth agar slant or as a stab in microbial growth agar, medicinal-grade
mineral oil is added to a depth of at least 1-2 centimeters (cm) above the highest point of growth.
Overlayed microbial cultures may then be stored at either room temperature (20 to 25°C) or under
refrigeration (2 to 8°C).
A common source for causing microbial contamination of overlayed cultures is by using mineral
oil that is not sterile. To prevent microbial contamination of cultures that have been overlayed with
mineral oil, mineral oil can be heated to 170°C for 1 to 2 hours for sterilization.5
Recovery of an organism that has been overlayed with mineral oil can be accomplished by removing
the mineral oil layer and aseptically removing a small amount of microbial growth by using a sterile
inoculating needle or loop and transferring it to an appropriate microbial growth medium.

Advantages
• This technique has been used extensively for many years because it has been very successful
in maintaining fungal cultures.
• Bacterial cultures can be maintained by this technique for 1 to 2 years, but fungi may sur‑
vive longer.

Disadvantages
• It is a messy procedure.
• This method may not be suitable for some microbial species.

Freezing
In culture preservation by freezing, water is made unavailable to the cell, and the dehydrated cells are
stored at low temperatures. Several factors may affect the viability of a preserved microbial culture
that has been frozen. They include:

• The microbial species

• Final storage temperature
• Freezing rate
• Selection and use of a suitable cryoprotectant agent
• Rewarming rate

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The two primary factors affecting cell viability for freezing techniques are solute concentration and
ice crystal formation. During freezing, extracellular water freezes first. As this happens, solutes ex‑
ternal to the cell begin to concentrate. As a result, water begins to migrate out of the cell to balance
osmotic pressure. However, excessive removal of water from the interior of the cell may result in
harmful concentrations of solutes within the cell. If excessive intracellular water is present in a cell, it
10

can result in the formation of ice crystals that can further damage the interior of the cell. While all of
the above factors play a role, freezing rate and the use of cryoprotectants are of particular importance
in minimizing damage from intracellular solute concentration and ice crystal formation.

Ordinary Freezing (0°C to -20°C)

The freezing temperatures for refrigerators or ordinary freezers typically range between 0°C and
-20°C. Ordinary freezers may vary considerably in their rate, efficiency and consistency of freezing.
In addition, frost-free features employ alternating cooling and warming cycles. In addition, this
temperature range may not readily lend itself to use of cryoprotectants. Taken together, culture
preservation and maintenance by ordinary freezing will generally yield poor results for viability
compared to techniques employing lower temperatures.

Advantages
• Cost
• Minimal expertise

Disadvantages
• Poor culture viability
• Freezer requires power back up, generally little time to respond to power outage before cul‑
tures would thaw
• Temperature cycling may cause ongoing cell damage
• Not amenable for routine culture resuscitation

Deep Freezing (-70°C to -130°C)

The temperature for deep freezing is typically between -70°C and -130°C. A deep freezing method
provides for superior culture viability in comparison to what can be obtained by ordinary freezing,
and is applicable to most bacteria and fungi. This method employs the use of a mechanical freezer in
which a cryoprotectant is used to minimize the occurrence of cellular damage due to the formation
of ice crystals that could occur during the freezing process.

Cryoprotectants
The incorporation of cryoprotectants as part of the freezing process helps maintain viability of the
cells by minimizing:

• Formation of ice crystals can cause physical damage to the cells.

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• High localized intracellular concentration of solutes as water freezes, creating incompatibil‑

ities to cellular systems

The most common cryoprotectants used for bacteria and fungi are dimethylsulfoxide (DMSO) and
glycerol. While a range of DMSO concentrations have been reported depending on the specific
organism, DMSO concentrations (v/v) in the range of 5-10% are most frequently used. Higher
DMSO concentrations can be toxic to microorganisms resulting in a decrease in the viability of a
frozen microbial culture. A wide range of glycerol concentrations (v/v) have also been reported with
the most common concentration being in the range of 10-15%. Glycerol is used most often as a
cryoprotectant for fungi. Other commonly used cryoprotectants include skim milk (used primarily
for bacteria) and blood serum. The practitioner is encouraged to consult the scientific literature to
help understand specific cryoprotectant approaches can be used to have a successful maintenance
and preservation of a given microorganism.6

Basic Method
A cell suspension of the microorganism is prepared in the appropriate media, routinely as an overnight
broth culture. After centrifugation, the pellet is resuspended in fresh sterile broth, supplemented with
an appropriate type and level of cryoprotectants (see above). If microbial growth agar slants are used
as the source of cells, growth is washed from the slant by using a sterile microbial growth broth con‑
taining a cryoprotectant. The selection of cryoprotectant and its concentration may be determined in
advance by examining for any toxic effects on the microorganism. The choice in the type of cryopro‑
tectant being used may vary depending upon the species of the microorganism being preserved.
Aliquots of the resuspended cells (e.g. 0.5 to 5.0 ml) are aseptically placed into sterile vials with suf‑
ficient space for expansion of the liquid during freezing. If using glass beads as part of this method,
refer to guidance below. The vials are then transferred to an ultra-low-temperature freezer, typically
at -70°C to -90°C. Duplicate vials of each microbial strain should be prepared to have one set of
cultures remain frozen at all times as part of a permanent microbial culture collection.

Revival
To recover microorganisms that have been frozen, a vial is removed from the freezer, opened, and a
small amount of frozen material is scraped from the surface with a sterile stick or inoculating nee‑
dle. After scraping, the material is inoculated into an appropriate microbial growth medium that is
incubated at an appropriate temperature and aerobic or anaerobic condition. The unused portion
of the frozen suspension should not be allowed to thaw or re-warm. Thawing can be minimized
through the use of glass beads to optimize culture resuscitation, or by keeping the vial in a bed of
dry ice when working with it.

Advantages
• Reliable, preserves genotypic and phenotypic characteristics
• Viability typically 1-3 years
• Amenable to maintaining and preserving large culture collections
• Does not require specialized skillset
• Not labor intensive
• Easy culture resuscitation when coupled with glass beads (see next section)

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Disadvantages
• Initial cost of freezer
• Need appropriate back-ups and contingencies to ensure freezer does not fail
• May be less reliable for long term viability than ultra-freezing and lyophilization
10

Storage on Beads
This method involves low temperature storage of microbial suspensions in cryovials containing a
cryoprotectant and porous glass or plastic beads. By using this method, most microorganisms will
typically remain viable up to 1 year.

Basic Method
The microorganism to be stored should be grown on the surface of an appropriate non-selective
microbial growth agar Petri dish or slant by incubating for 18 to 24 hours. An isolation streak
should be performed as a purity check for possible microbial contaminants. A dense suspension is
prepared by adding a heavy loopful of microorganism to the cryoprotectant which is added to the
(sterile) cryovial containing the beads. Alternatively, a 1 ml aliquot of an appropriate sterile micro‑
bial growth broth containing a cryoprotectant may be added to a microbial growth agar Petri dish
and mixed to form a thick suspension. After forming this suspension, it is aseptically transferred
to sterile cryovials that contain beads. The cryovial is shaken to distribute the suspension among
the beads. After allowing appropriate time for the organism to bind to the bead (~2 – 5 min), any
additional cryoprotectant/microorganism suspension is aseptically removed using a pipette. The vial
is then ready to be placed in low temperature freezer (-20°C to -70°C).7,8

Revival
To recover a microorganism that is frozen to a bead, it is aseptically transferred from the storage vial
and placed onto microbial growth agar and is rubbed over the surface. Alternatively, the transferred
bead can be placed into a sterile container containing an appropriate microbial growth broth. The
inoculated microbial growth agar or broth is incubated at an appropriate temperature and aerobic
or anaerobic conditions.7,8

Advantages
This method has the same advantages as the traditional frozen suspension techniques. A few addi‑
tional advantages are:

• This method is convenient for frequently used microorganisms.

• Storage on beads eliminates the problem of repeated freezing and warming of frozen suspen‑
sions when subcultures are made; each bead may be removed individually without warming
the remaining beads.
• Many ready-to-use cryovial bead storage systems are commercially available.

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Disadvantages
• Initial cost of freezer
• Need appropriate back-ups and contingencies to ensure freezer does not fail
• May be less reliable for long term viability than ultra-freezing and lyophilization

LONG-TERM METHODS
Ultra-freezing (Below -130°C)
Cryopreservation or ultra-freezing by using liquid nitrogen is a method that offers long-term stor‑
age of microorganisms to a laboratory. Ultra-freezing can preserve the original characteristics of a
microorganism and is simple to perform. Furthermore, storage in liquid nitrogen provides a stable
culture collection of critical sub-units that are easily accessible when needed or be preserved many
years, provided storage temperature is maintained below - 130°C.9,10

Preparation
Cells are grown to the late logarithmic stage by using a nutritive, nonselective microbial growth me‑
dia. After reaching logarithmic stage of the cell cycle, cells are suspended in a fresh microbial culture
medium that contains a cryoprotectant in order to be aseptically distributed into either sterile glass
or plastic ampoules. Mold cultures are harvested after they have sporulated. DMSO or 5 to 10%
glycerol is commonly used as a cryoprotectant for preserving bacteria and fungi because they lessen
the effects of internal ice crystal formation.9

Basic Method
A slow cooling rate of 1° to 10°C per minute until a temperature of -30°C is reached will help main‑
tain culture viability.11 This can be achieved by putting the vials in a controlled-rate freezing cham‑
ber or, more inexpensively, in a commercially available, insulated chamber placed in a mechanical
freezer at -70°C or colder for at least 24 hours.9
Microorganisms can be stored in nitrogen in either the liquid phase (-196°C) or in the vapor phase,
which is above the liquid. Storage of cultures in the vapor phase of liquid nitrogen has the advantage
in reducing the risk of having microbial contamination when compared to the use of submerged
tubes in liquid nitrogen. For liquid nitrogen vapor storage, it is important to note that the tempera‑
ture of the vials will rise further away from the surface of the liquid nitrogen.9

Revival
To revive a frozen culture after withdrawal from ultra-cold storage, place the ampule in a 37°C water
bath.9,10 When the frozen culture is thawed, the contents of the vial or ampoule should be imme‑
diately transferred aseptically to an appropriate microbial growth medium in order to prevent any
harmful effects from the cryoprotectant.

Special considerations
The use of liquid nitrogen requires safety precautions. Ampules that are stored in the liquid phase
can present the risk of explosion if liquid nitrogen penetrates an imperfect seal and expands rapidly

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when warmed. Polypropylene screw-cap ampules reduce the hazard that glass ampules pose, as well
as eliminating several steps of preparation. A plastic face shield and protective gloves needs to be
worn while depositing or withdrawing microbial cultures from a liquid nitrogen containing storage
vessel. Failure to maintain a minimal level of nitrogen can result in the loss of an entire collection. To
prevent this, liquid nitrogen levels within the storage vessel will need to be monitored on a regular
10

basis and an alarm should be installed to detect low levels.9

Advantages
• Microorganisms are viable for up to 30 years provided the storage temperature is maintained
below -130°C.10
• Passages are kept to a minimum which helps prevent phenotypic drift.

Disadvantages
• Expertise and time are required.
• Freezer and supplies are expensive.
• Cost to run freezer is high.
• Frozen cultures can be harmed or ruined by thawing due to power outages. Frequent mon‑
itoring necessary.
• Safety equipment such as protective gloves and a face shield is required when placing or
removing cultures.

Freeze-drying (Lyophilization)
Freeze-drying has been widely used to preserve a broad range of bacterial and fungal species. It
involves the removal of water from a suspension of cells by a process called sublimation. During
sublimation, frozen liquid is converted directly into gas: there is no liquid phase. By removing water
from vegetative cells and spores, it halts cell metabolism which reduces the risk in having damage
due to prolonged exposure ice crystallization. This process can result in having a stable preparation
for a microbial culture that can be easily resuscitated.9

Preparation
To prepare for freeze-drying or lyophilization, vegetative microbial cells are grown on a nutritive and
non-selective microbial growth medium to the late logarithmic stage or early stationary stage of the
cell cycle.9 Cells are harvested and resuspended in a protective medium containing components such
as skim milk or sucrose.11 This medium will protect microbial cells from injury that could occur
during the freezing and drying. Recovery is best when the protective medium is inoculated heavily
so that it contains 106 to 1010 cells per ml.12 The medium is dispensed into vials for lyophilization.

Basic Method
Freeze-drying involves three steps. In the first step, the cell suspension is frozen slowly (for example,
-1°C per minute), allowing the formation of ice crystals which create channels from which the water
vapor can escape. In the second step that is called primary drying, ice crystals are sublimed from a
solid to a gaseous form by using a high efficiency vacuum pump. The water vapor travels to a con‑

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denser, which is much cooler than the microorganism sample. This leaves only bound water in the
cells, which in the third step, called secondary drying, is removed with additional heat.13

Storage and Revival

Freeze-dried microorganisms are extremely sensitive to moisture and oxygen.9 For this reason, the
material should be stored in a protective vessel impervious to moisture. A desiccant, added to the
vessel, will prevent cells from trapping water. Survival rates may be improved by using sealed glass
ampoules. A microbial suspension in a freeze-dried vial should be stored at 2°-8°C since oxygen
exposure increases with temperature. Survival rates vary with the species, cryoprotectant, lyophiliza‑
tion process, and storage method but given the right conditions, freeze-dried microorganisms may
remain viable for 30 years. Gram-negative organisms tend to outlive Gram-positive organisms.12
Freeze-dried microorganisms should be rehydrated by using a sterile microbial growth broth.9 It is
recommended that nonselective microbial growth medium and favorable growth conditions such as
temperature and air exposure be used for growing rehydrated microbial cultures.

Special considerations
There is not one freeze-drying protocol for all microorganisms. The process for each particular mi‑
croorganism should be empirically determined to provide for the greatest recovery of the original
population. Several factors in the process should be considered in reaching this goal including:

• Growth phase of the culture

• Temperature of growth
• Composition of the growth and suspension medium
• Freezing temperature
• Rate and duration of the freeze-drying process
• Final moisture content
• Rehydration processes

There is the potential for microbial contamination if microorganisms are freeze-dried without
the use of some type of protective barrier because microorganisms in the suspension can escape
from their vials or trays during processing. Some type of bacteriological filter should be used when
freeze-drying more than one strain at a time and equipment should be decontaminated after use.13
Freeze-drying could cause the loss of plasmids in some bacterial species that could result in changing
the characteristics of a microorganism. Microbial species that are sensitive to the freeze-drying tech‑
nique may lose some viability. Quality control testing is essential. New batches of strains should be
checked for purity, viability, and phenotypic, proteotypic, and/or genetic characteristics.
Safety glasses should be worn when opening flame-sealed ampoules containing the freeze- dried
material.5

Advantages
• After a culture has been freeze-dried or lyophilized, it can be stable for up to 30 years.
• Special storage conditions are not necessary.

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TEST ORGANISMS

• Freeze-dried microorganisms can be easily rehydrated by using a sterile microbial growth

medium.
• Microorganisms may be shipped at room temperature.
• Power outages do not have an adverse effect on freeze-dried microorganisms in comparison
to those microorganisms that have been frozen.
10

Disadvantages
• A major disadvantage is the high initial capital cost for equipment.
• Expertise and time are required.
• There is not one method for all microorganisms because different species have different re‑
quirements.

IN HOUSE ISOLATES
Personal care product manufacturers strive to maintain clean and hygienic facilities so that they
may produce microbiologically safe products. CFR Title21 Part 211 Current Good Manufacturing
Practices for Finished Pharmaceuticals is a specific manufacturing guideline for personal care and
cosmetic manufacturers which are regarded as best practice.14 Although clean, personal care facilities
are not completely sterile, microorganisms are often still present in process water, raw materials, and
even finished goods. Difficult to sanitize areas in the manufacturing environment like pipes, tanks,
fill lines, air handling systems, and warehouses may present ideal sources for these microorganisms
to grow and adapt. Many times, in house isolates are detected and isolated during regular product
screening or hygiene audits.
In house isolates are useful in the conductance of preservative efficacy testing. The inclusion of in
house isolates may more adequately establish product robustness against the types of microorgan‑
isms that may be introduced during production and consumer usage. In comparison to reference
strains, environmental, raw material and product formulation isolates of a microbial species may
have developed an increased resistance to certain types of material such as preservatives15 or product
conditions such as high pH. Consequently, it is important for the microbiologist to use in house
isolates whenever appropriate during routine product testing and new product development.
In the case of these in house isolates, conventional methods of storage and maintenance may not be
adequate for them to retain their original genotype for exhibiting their phenotypic characteristics.
Spontaneous genetic reversion could occur if these microbial isolates are removed from either their
original environment for propagation on artificial microbial growth medium or if they are subjected
to physical stress. Occasionally, a recovered microbial isolate may not even survive minimal storage
on artificial microbial growth medium. Special attention should be given to the way that these re‑
covered microbial isolates are stored so that their original attributes are not lost and culture viability
is maintained. For those recovered microbial isolates obtained from raw materials or product for‑
mulations, a common strategy for maintaining these isolates is either a periodic subculturing into
the material from which they had been originally isolated or devising an artificial growth medium
to ensure retention of the desired culture trait. For example, Burkholderia cepacia, a common in
house isolate, may grow poorly or not at all on artificial growth medium.16,17 The isolate’s viability

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TEST ORGANISMS
PRESERVATION OF
MAINTENANCE AND PRESERVATION OF TEST ORGANISMS SECTION 10

may be better maintained by storage in the product it was isolated from or by utilizing a specialized
enrichment agar. Key traits which are unique to a recovered microbial isolate should be thoroughly
characterized and frequently monitored to provide an added measure of assurance of strain stability.
Deviation from the expected biological profile may indicate that the isolated organism no longer
retains its resistant characteristics.

CONCLUDING REMARKS
This guideline provides an overview of some commonly used techniques currently acknowledged
as satisfactory for the maintenance and storage of microbial cultures. There are many other less
common techniques, including those developed by companies for internal use, which may also be
acceptable. The objective for any maintenance and storage program is to maintain culture viability
and purity while ensuring the retention of desired culture characteristics. Microbial strains sourced
from culture collections should be maintained in a way that is consistent with the recommendations
of the supplier. It is the responsibility of the microbiologist to select and properly execute appro‑
priate methods for the organism(s) in question as well as maintain any applicable documentation
relating to the culture collection.

REFERENCES
1. Cross, L.J., Russell, J.E., and Desai, M. 2011. Examining the genetic variation of reference
microbial cultures used within food and environmental laboratories using fluorescent amplified
fragment length polymorphism analysis. FEMS Microbiology Letters 321:100–106.
2. ISO 11133. 2014. Microbiology of food, animal feed and water – Preparation, production,
storage and performance testing of culture media. www.iso.org
3. CLSI. 2015. M07- A10, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Grow Aerobically; Approved Standard—Tenth Edition. Clinical Laboratory Standards
Institute, Wayne, PA.
4. United States Pharmacopeia and National Formulary. 2016. USP 39 NF 34 (2016) Chapter
<51> Antimicrobial Effectiveness Test. United States Pharmacopeia and National Formulary,
Rockville, MD, pp. 67-69.
5. Petti, C.A. and Carroll, K.C. 2011. “Procedures for the Storage of Microorganisms” In: Manual
of Clinical Microbiology 10th Edition (pp. 124-131). Versalovic, J., Carroll, K.C., Funke, G.,
Jorgensen, J. H., Landry, M.L., Warnock, D.W. (Eds.) ASM Press, Washington, DC.
6. Hubálek, Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology
46(3):205-229
7. Pro-Lab Diagnostics. 2016. Microbank™: Product instructions. Retrieved from
http://www.pro-lab.com/inserts/PL170.pdf

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TEST ORGANISMS

8. Saraiva Giampaglia, Carmen Maria, Artemir Oelho De Brito, Maria Conceicao Martins, Suely
Yoko Mizuka Ueki, Fabio OliveirLatrilh, Rosangela Siquira De Oliveira, Jonas Umeoka Yamauchi,
and Maria Alice De Silva Telles. 2009. “Maintenance of Mycobacterium tuberculosis on Glass
Beads.” Annals of Clinical & Laboratory Science 39.1: 51-54.
10

9. American Type Culture Collection® (2015) ATCC® Bacterial Culture Guide: Tips and
techniques for culturing bacteria and bacteriophages. Manassas, VA. https://www.atcc.org/~/
media/PDFs/Culture%20Guides/Previews/ATCC_Bacterial_Culture_Guide_Preview.ashx
10. De Paoli, P. 2005. Biobanking in microbiology: From sample collection to epidemiology,
diagnosis, and research. FEMS Microbiology Reviews 29: 897-910.
11. Malik, K.A. and Claus, D. 1987. Bacterial Culture Collections: Their Importance to
Biotechnology and Microbiology. Biotechnology and Genetic Engineering Reviews, 5:1,137-198.
12. Miyamoto-Shinohara, Y., Imaizumi, T., Sukenobe, J., Murakami, Y., Kawamura, S. and
Komatsu, Y. 2000. Survival Rate of Microbes after Freeze-Drying and Long-Term Storage,
Cryobiology 41, 251-255.
13. LABCONCO. 2010. A Guide to Freeze Drying for the Laboratory. http://www.labconco.
com/literature/brochures/freeze-dry-systems
14. U.S. Food & Drug Administration. 2015. CFR Code of Federal Regulations. Title 21,
Chapter 1, Part 211, Current Good Manufacturing Practices for Finished Pharmaceuticals.
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=211
15. Geis, P.A., Krowka, J.F. 2013. The microbiology of personal care and cosmetic products.
SIMB NEWS, November/December 2013, V.63 N.6, pp.197-200. www.simbhq.org
16. Torbeck, L., Accasi, D., Guilfoyle, D.E., Friedman R.L., Hussong D. 2011. Burkholderia
cepacia: This Decision Is Overdue. PDA Journal of Pharmaceutical Science and Technology
September–October 2011, Vol. 65, No. 5, pp.535-543.
17. Barlasov, J., Sutton, S. Jakober, R. 2014. Recovery of Stressed (Acclimated) Burkholderia
cepacia Complex Organisms. American Pharmaceutical Review, April 2014, Vol. 17, No 3.
www.americanpharmaceuticalreview.com

ADDITIONAL INFORMATION
U.S. Food & Drug Administration (2016) Guidance for Industry and FDA Staff: Current Good
Manufacturing Practice Requirements for Combination Products. Draft Guidance.
http://www.fda.gov/RegulatoryInformation/Guidances/ucm126198.htm
U.S. Food & Drug Administration (2008)Good Manufacturing Practice (GMP) Guidelines/
Inspection Checklist.
http://www.fda.gov/Cosmetics/GuidanceRegulation/GuidanceDocuments/ucm2005190.htm

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TEST ORGANISMS
PRESERVATION OF
MAINTENANCE AND PRESERVATION OF TEST ORGANISMS SECTION 10

Gherna, R.L. 1994. Culture preservation. In Methods for General and Molecular Bacteriology (pp.
278-292). Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R. (Eds.), American Society
for Microbiology, Washington, D.C.
Kumar, S., Kashyap, P.L., Singh, R. and Srivastava, A.K. 2013. Preservation and Maintenance of
Microbial Cultures. In Analyzing Microbes: Manual of Molecular Biology Techniques (pp. 135-152).
Arora D.K., Das S., and Sukumar, M. (Eds.) Springer, Berlin, Heidelberg.
Miyamoto-Shinohara, Y., Sukenobe, J., Imaizumi, T. and Nakahara, T. 2008. Survival of freeze-
dried bacteria, J. Gen. Appl. Microbiol. 54: 9-24.
Morgan, C.A., Herman, N., White, P.A. and Vesey, G. 2006. Preservation of micro-organisms by
drying; A review, Journal of Microbiological Methods, 66, 183-193.
Palková, Z. 2004. Multicellular microorganisms: laboratory versus nature. EMBO Rep. 2004
May 5(5): 470–476.
van Griethuysen A., van Loo, I., van Belkum, A., Vandenbroucke-Grauls, C., Wannet, W., van
Keulen, P. and Kluytmans, J. 2005. Loss of the mecA Gene during Storage of Methicillin-Resistant
Staphylococcus aureus Strains. American Society for Microbiology, J. Clin. Microbiol. 43.3:1361-
1365.

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 SECTION 11

Raw Material
Microbial Content

11
MICROBIAL CONTENT
RAW MATERIAL
INTRODUCTION
In order to minimize the chance of contaminated finished product, it is necessary to control the
microbial content of personal care raw materials along with other physical and chemical attributes.
Personal care manufacturers should evaluate the microbiological quality of their raw materials and
establish appropriate specifications based on the best available scientific information.
Water and water supplies are addressed in the Personal Care Product Council’s “Microbiological
Quality for Process Water” guideline (Section 7). Water systems should be properly validated and
controlled. Quality specifications for water should be set, including alert and action levels.

GENERAL CONSIDERATIONS
When establishing acceptable levels for raw material microbial content, the following criteria should
be considered:

• Chemical composition.
• Physical nature.
• Origin and availability.
• Lot uniformity.
• Intended use of the product.
• Concentration of raw material used in the product.
• Manufacturing process.
• Raw material history.
• Storage conditions.
• Water activity.

Many synthetic raw materials currently used by the industry contain low microbial counts, due to
extremes in pH, low water content, or inherent antimicrobial properties. Others may be supplied as
aqueous dispersions or solutions, and may be susceptible to microbial proliferation. Therefore, it is
important to evaluate susceptible synthetic materials upon receipt to ensure that they have not been
contaminated during the manufacturing process, packaging, transportation and storage. When a

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 SECTION 11 RAW MATERIAL MICROBIAL CONTENT

bioburden reducing treatment is implied with a raw material, the effectiveness of the process should
be evaluated and verified.
Naturally occurring raw materials are likely to contain a high level of microorganisms that may
pose a contamination risk to the finished product if not reduced or eliminated during processing.
The microbial content may vary depending upon the type and source of the raw material. It may be
necessary to treat such materials to reduce microbial levels before use or to purchase already treated
materials.
The criteria set by the manufacturer for the microbial content of a raw material should take into
MICROBIAL CONTENT

consideration the release criteria established for each finished product. For example, the absence of
Salmonella is significant if a raw material is used in an oral product. A raw material microbial con‑
RAW MATERIAL

tent specification is usually not greater than that for the finished product, especially when it is used
at greater than 1% in the formulation. A raw material with a microbial count greater than that set
for the finished product may be acceptable if its use does not compromise the safety and stability of
the formulation and its concentration in the finished product is low. When a bioburden is employed
with raw material, the effectiveness of the process should be evaluated and verified.
11

SPECIFIC CRITERIA
This guideline recognizes the importance of using raw materials of the highest quality in the man‑
ufacture of personal care products. Special conditions may allow or necessitate acceptance criteria
that vary from those recommended below. It is recommended that the minimum test portion be 1
g or 1 mL of sample.
The following are recommended guidelines:

• All synthetic and natural raw materials - not more than 102 CFU per g or mL

NOTE: Interpretation of results

The inherent variability of a plate count should be taken into account, thus the interpretation may
be as follows:

• 102 - may be interpreted as 2 x 102

In addition to these recommended numerical guidelines, no raw material should have a microbial
content recognized as either harmful to the user or able to compromise integrity of the finished
product as recovered by standard plate count, specific pathogen test, or an equivalent automated
procedure.

GENERAL RECOMMENDATIONS
As personal care products and toiletries need not be manufactured from sterile raw materials, it is
important that raw materials are obtained from qualified suppliers and are handled, stored, and

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 RAW MATERIAL MICROBIAL CONTENT SECTION 11

used under conditions designed to deter microbial proliferation or subsequent contamination. The
Personal Care Products Council’s Quality Assurance Guidelines1 are a useful guide for the storage
and handling of raw materials as well as microbiological sampling techniques.2 Sampling techniques
are also described in Section 6 (Microbiological Sampling).
Validated microbiological analytical methods should permit the detection of microorganisms and
ensure the inactivation of the preservative. The presence of objectionable organisms can be deter‑
mined by identification of isolates using procedures such as described in “M-2 Examination for

11
and Identification of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida
albicans” (Section 19).

MICROBIAL CONTENT
RAW MATERIAL
If raw materials are found to have a microbial content greater than specified, an investigation to
identify and eliminate the source of the contamination can assist in implementing preventative
measures.2
It is recommended that a qualified microbiologist or independent microbiology laboratory be en‑
gaged to:

• Design procedures for the examination of specific raw materials,

• Examine the manufacturer’s raw materials for microbial content on a continuing basis,
• Interpret assay data on a routine basis, and
• Periodically review and update procedures, when applicable.

REFERENCES
1. Nikitakis, J.M. (ed.) 2014. Annex 4 – Raw Materials and Packaging Materials. In Personal Care
Products Council Quality Assurance Guidelines, Personal Care Products Council, Washington, DC
20036, pp. 31-34.
2. Nikitakis, J.M. (ed.) 2014. Annex 17 – Sampling. In Personal Care Products Council Quality
Assurance Guidelines, Personal Care Products Council, Washington, DC 20036, pp. 103-110.

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 SECTION 12

Establishing Microbiological
Quality of Personal Care
Products
INTRODUCTION
Control over the microbial content of personal care products is consistent with the control of other
aspects of quality, and is necessary in order to ensure consumer safety and product integrity and
stability.
Because personal care products may be applied to bacteria-populated skin, microorganisms need

12
to be controlled, but not necessarily eliminated from these products. Therefore, it is appropriate
to assign rational limits to microbial content based on the best available information. Criteria such

QUALITY
MICROBIOLOGICAL
as the products intended use, route of administration, and target population should be taken into
account when establishing microbiological guidelines for safety and quality. The microbiological
quality guidelines presented here are intended to assist manufacturers in establishing best practices
including appropriate microbiological limits for their products that are susceptible to microbial
contamination. Guidance on microbiological quality for personal care products is available from the
Personal Care Products Council1 and Cosmetics Europe.2
It should be recognized that the application of microbial limits alone is not sufficient to guaran‑
tee the microbiological quality of the product. In addition, a microbiological quality management
process must also be implemented to ensure that the products produced by a manufacturer are in
compliance with their microbiological test specifications. This quality management process includes
appropriate product development to ensure consumer safety, supplier quality management, and
adherence to current Good Manufacturing Practices (GMP) in order to minimize the occurrence
of microbial contamination of finished products during manufacturing. Essential to the implemen‑
tation of effective microbiological quality management is the requirement for a microbiological
awareness education and training program for all levels of employees.

GENERAL CONSIDERATIONS
A. Product Development
Personal care product formulations that can either support the proliferation of microorganisms or
allow their survival in finished products should contain a preservative or preservative system that
will prevent the proliferation and/or survival of contaminating microorganisms that are introduced
during consumer use. The manufacturer has the responsibility for producing an effectively preserved

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product. For product formulations that are susceptible to microbial growth and survival as a result
of contamination, preservation efficacy testing should be conducted during the development phase
of a product to ensure that they are adequately preserved.
The function of preservation is to protect consumers and to prevent product spoilage during normal
and reasonably foreseeable product use. Preservatives should not be used in lieu of current Good
Manufacturing Practices. Understanding the root causes of microbial contamination issues during
manufacturing is important. Instituting appropriate corrective measures will help to prevent their
recurrence and eliminating them during production will lead to control and prevention of microbial
contamination.
It must be emphasized that preservation systems cannot be chosen satisfactorily on theoretical
grounds and they require in situ determination of their efficacy by microbiological challenge tests or
other appropriate test systems during product development.
The formulator should develop product formulations that are adequately preserved to control
growth of microorganisms that are introduced into a product during normal consumer usage. If a
preservative is shown to be necessary, it should be selected at an early stage in the development of a
product and be considered an integral part of the formulation.
Water is essential for microbial growth. A preservative system should have solubility and partition
characteristics such that it is available at effective concentration in the aqueous phase of a multiphase
system.
MICROBIOLOGICAL

A preservative system should be effective against a broad spectrum of microorganisms and safe at the
concentration used. Combinations of preservatives can sometimes be more effective than individual
QUALITY

components in providing antimicrobial protection to a product. If a combination of preservatives

is used as the preservative system in a product, the concentration of the individual components
should be at levels that are necessary to ensure effective preservation. Storage temperature, light ex‑
posure, and prolonged storage stability are important. The concentration of the preservative system
12

in a product formulation should be at levels necessary to allow for sufficient antimicrobial activity
to ensure adequate preservation. The preservative system should be compatible with other product
constituents and should be effective at the pH of the formulation. These determinations of com‑
patibility can be obtained by conducting microbial challenge testing during product development.
Lastly, it is important to ensure that critical processing parameters such as pH adjustments, heat
tolerance, and the proper order of addition for preservatives into a product formulation should be
properly documented in the directions for manufacturing. Following these directions is important
to prevent the accidental decomposition of the preservative system and to ensure that the preserva‑
tive system provides effective antimicrobial activity.

B. Raw Materials
Personal care products for use by the general public are not required to be manufactured using ster‑
ile raw materials or under aseptic processing conditions. Therefore, microorganisms found in the
general environment, in raw materials and in formulations components may be introduced into the
product during manufacture. It is important that raw materials and components be handled and
stored under conditions that are designed to deter the introduction of microbial contamination and
limit microbial proliferation.

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Raw materials can contribute a significant level of microbial contamination to the finished product.
It is important to monitor and control them. The monitoring of raw materials should be appropriate
to their susceptibility to microbial contamination, as determined by a risk assessment. If practical,
suppliers of raw materials yielding the lowest population of microbial contamination should be
used. When a necessary raw material fails to have an acceptable level of microbial contamination,
the manufacturer might negotiate with the raw material supplier seeking a reduction of microbial
contamination and develop a specification to maintain the lower level.
Water is one of the major raw materials that are commonly used in most personal care product
formulations. Water can contain large numbers and many different types of microorganisms. These
microorganisms may present a distinct hazard to the microbiological quality and stability of the fin‑
ished products. Therefore, steps must be taken by the manufacturer to ensure that water used either
as a raw ingredient in a formulation or for processing such as cleaning and sanitization is regularly
monitored for microbial content. Appropriate steps should to be taken by the manufacturer when
either high microbial counts or the presence of undesirable microorganisms are found to be present
in water samples.

C. Good Manufacturing Practices (GMP)

12
Current good manufacturing practices (GMP) are necessary to avoid accidental human or envi‑
ronmental microbial contamination during product manufacture. The manufacturing equipment

QUALITY
MICROBIOLOGICAL
should be designed for ease of cleaning and sanitizing, as well as for processing capability. It is
recommended that, wherever possible, the physical plant of the manufacturing facility be designed
and constructed to facilitate the manufacturing operations (compounding or processing, packaging,
storage, and quality assurance) in accordance with GMPs.
In order to comply with GMP, manufacturers of personal care products must define and follow spe‑
cific cleaning, sanitization, and control procedures. This process should include the establishment
of standard operating procedures to control microorganisms in susceptible raw materials, bulk and
finished products, as well as on personnel, equipment, and premises.
Adequate records should be maintained for all aspects of microbiological testing during the development
and manufacture of each product and for all control procedures used at the manufacturing facility. In
particular, documentation during the manufacture of products is an essential component of GMP.

D. Control and Assessment of Bulk and Finished Products

Susceptible formulations should be tested for microbial content on a routine basis, with product
data periodically reviewed for trends. The testing program should take into account microbiologi‑
cally susceptible raw materials, processing steps, and storage conditions.
The manufacturer should take into account the specific nature of the product when establishing
microbiological criteria for safety. It is the responsibility of the manufacturer to ensure that:

• Any microorganisms present are unable to proliferate in the product;

• The species and quantity of microbes do not present a hazard to the consumer when using
the product as directed;

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• Any microorganisms present do not compromise either the aesthetic quality or long-term
stability of the product
• The packaging (including cap or closure) does not facilitate introduction of microorganisms
into the product during its anticipated life.

A sampling plan developed for formulations that are susceptible to microbial contamination during
the manufacturing process should be appropriate to the formulation and take into consideration the
manufacturing process history.
It is further recommended that a qualified microbiologist or independent microbiology laboratory
be engaged to develop validated microbiological procedures to analyze specific products and to pe‑
riodically examine manufacturing procedures and interpret assay data.

SPECIFIC CRITERIA
Acceptance criteria for the various classes of personal care products should be established when
necessary. For microbiologically susceptible products, conformance to the criteria is determined by
using a suitable technique, such as a plate count procedure (See References 3 & 4 and Section 18 –
Determination of the Microbial Content of Personal Care Products). A risk assessment of products
that are not considered microbiologically susceptible may either support a decision not to test some
MICROBIOLOGICAL

products or indicate reduced testing levels for others. An international standard such as ISO 29621
(Cosmetics – Microbiology – Guidelines for the risk assessment and identification of microbiologi‑
cally low-risk products)4 provides useful inputs to such a risk assessment.
QUALITY

A microbial content enumeration method for a particular product should be qualified for the type
of product being tested. The method must permit the detection at a minimum, or the growth of
any relevant microorganisms present. Furthermore, the test method used to quantify the microbial
12

content should adequately inactivate microbial growth inhibitors present in the product and permit
the detection of any relevant viable microorganisms that may be present in the product. It is recom‑
mended that the minimum test portion be 1 g or 1 ml of sample.
No product should have a microbial content that could be recognized as either harmful to the user
or able to compromise product aesthetics such as changes in viscosity, development of malodors,
and presence of visible microbial colonies. Specified organisms can be determined and identified
from colonies recovered on suitable microbial growth media. Alternative procedures can also be
used to detect the presence of specified organisms.
The conditions under which personal care products are manufactured, marketed, and used by the
consumer vary throughout the world. Alternative microbial limits, such as those set by compendia,
governmental bodies, or other recognized authorities, may be applicable in some cases. The appli‑
cation of specific criteria under this guideline should be decided by each national agency, whether
government or association. Where applicable, additional specific microbial content criteria for a
specific country, region, or other area may be necessary in order to comply with local regulatory
requirements.

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Some suggested criteria for microbial content are listed below:

Baby products - not more than 102 CFU per g or ml.

Eye area products - not more than 102 CFU per g or ml
All other products - not more than 103 CFU per g or ml.

NOTE: Interpretation of results:

The inherent variability of a plate count should be taken into account, thus the criteria recommend‑
ed should be interpreted as follows:

102 - maximum limit of acceptance is 2 x 102.

103 - maximum limit of acceptance is 2 x 103.

Any microbial content exceeding acceptance criteria should be investigated to identify and eliminate
the source of the contamination. Preventive measures should then be implemented.
The references included with this document provide different methods and procedures that may be
used in order to make sure that personal care products are in conformance with the recommended

12
limits.

QUALITY
MICROBIOLOGICAL
REFERENCES
1. Nikitakis, J.M. ed. 2014. Personal Care Products Council Technical Guidelines – Quality Assurance
Guidelines, Personal Care Products Council, Washington, DC. www.personalcarecouncil.org
2. COLIPA. 1997. Guidelines on Microbial Quality Management (MQM). The European
Cosmetic Toiletry and Perfumery Association. www.cosmeticseurope.eu
3. ISO 2009. ISO 17516. Cosmetics – Microbiology – cosmetics Microbiology – Microbiological
Limits. www.iso.org.
4. ISO 2010. ISO 29621. Cosmetics – Microbiology – Guidelines for the risk assessment and
identification of microbiologically low-risk products. www.iso.org.
5. Japan Cosmetic Industry Association (JCIA). 2010. Technical Document 119: Methods of
Microbial Limits Test. www.jcia.org/n/en/jcia/d/

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 SECTION 13

Determination of Preservation
Efficacy in Water-Miscible
Personal Care Products
INTRODUCTION
Many personal care products provide conditions that are conducive to microbial growth such as
water, nutrients, and favorable pH. Preservation efficacy testing (PET) is performed to assure that
each personal care product that is susceptible to microbial growth is not affected by the introduction
of microorganisms during normal or reasonably anticipated use by the consumer. However, some
personal care products, due to characteristics that create an environment hostile to microbial growth
and/or survival, are considered low microbiological risk1 (see also Section 14 - Microbiological Risk
Factor Assessment of Atypical Cosmetic Products and Reference #1). These products are not rou‑
tinely subject to preservative efficacy testing.
The design of preservation tests and subsequent interpretation of results is a complex process. The
technical personnel responsible for preservation testing should therefore, be professionally educated
and experienced in conducting challenge test procedures and evaluating the data generated (see Sec‑
tions 4 and 9 – Microbiology Staff Training and Microbial Validation and Documentation).
It is important to remember that microorganisms are ubiquitous and capable of adaptation. No
method can guarantee adequate microbiological control under all conditions. In addition, the im‑
portance of adhering to good manufacturing practices in the production of personal care products

13
cannot be overstated. Preservatives should not be used as a substitute for good manufacturing prac‑
tices.2

PRESERVATION EFFICACY
DETERMINATION OF
Personal care products constitute a wide and expanding variety of formulations and package config‑
urations that differ significantly from each other in their composition, intended use, and physical
characteristics. This guideline reflects current considerations and practices used within the personal
care industry in conducting preservation efficacy testing of water-miscible products. Other stan‑
dardized methods as well as those developed by the user may also be employed to assess preservation
efficacy of water-miscible personal care products.3,4 For guidance on testing atypical products, see
Section 16 - Microbiological Risk Factor Assessment of Atypical Cosmetic Products.

SCOPE
The purpose of this guideline is to provide guidance to those developing and performing PET of
water-miscible personal care products such as are described in Method M-3, A Method for Preserva‑

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 SECTION 13 DETERMINATION OF PRESERVATION EFFICACY IN WATER-MISCIBLE PRODUCTS

tion Testing of Water-Miscible Personal Care Products (Section18) and Method M-7, A Screening
Method for Preservation Testing of Water-Miscible Personal Care Products (Section 21).

GENERAL CONSIDERATIONS
The preservation efficacy test (PET) is an important tool in evaluating the robustness of a formula‑
tion to withstand microbial contamination during consumer use. Various factors can have an effect
on the performance of a preservative system in a formulation and on the interpretation of results
generated from these studies.1,5-7
The following factors may be considered when performing a preservation efficacy test and interpret‑
ing results:

• The nature and compatibility of the raw materials in the formulation.6

• Information on preservation of similar formulations.
• Types of packaging used to contain and deliver the product.1,5,7
• Information on the product’s intended use, including area of application, frequency of use,
shelf-life, intended users, etc.

PRODUCT TESTING
Microbial Content Test
It is recommended that the bioburden of the test sample(s) be determined by a microbial content
test, such as Method M-1 - Determination of the Microbial Content of Cosmetic Products (Section
18), prior to performing the preservation efficacy test. The purpose is to verify that the level and
type of microorganisms in the test sample will not interfere with the recovery of the test organisms
PRESERVATION EFFICACY

or interfere with the interpretation of the challenge test data. Furthermore, an initially high micro‑
bial bioburden in the test sample before inoculation with microorganisms could compromise the
DETERMINATION OF

preservation system.

Neutralization
Antimicrobial or preservative neutralizers are added to microbial plate count diluents and recovery
agars to inhibit or inactivate the antimicrobial properties or activity of the formulas being tested.8
Carryover of antimicrobial activity from the product formulation into the microbial plate count di‑
13

luent and recovery growth agar may partially or completely inhibit the growth of surviving challenge
test microorganisms.9
Antimicrobial or preservative neutralization may normally be accomplished by use of chemical
neutralizing agents, physical dilution, or a combination of both. Prior to the start of the neutral‑
ization step of the test, verification of the lack of neutralizer toxicity to each of the challenge test
microorganisms should be confirmed.10 Once this has been confirmed, this step does not need to

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be repeated during routine neutralization studies of formulations using the same microbial count
diluent or recovery agar. If a new preservative neutralizing agent is added to the microbial count
diluent or recovery agar, verification of the lack of neutralizer toxicity to each of the challenge test
microorganisms should be re-confirmed.
Examples of chemicals and agents that may be used to neutralize the antimicrobial activity of pre‑
servatives are listed in Table 13-1.11 Verification of the antimicrobial or preservative neutralization
is generally performed by inoculating the product dilution and a microbial count diluent without
product (control) with a low level of challenge microorganisms to yield a final count of approx‑
imately 30 to 300 Colony-Forming Units (CFU)/milliliter (ml) for each challenge test microor‑
ganism in a product test dilution and diluent control. Enumeration of the microorganisms from
these dilutions is performed. Neutralization of the antimicrobial or preservative activity is verified if
microbial recoveries of both test and control dilutions are within 50% of each other. If one or more
challenge test microorganisms cannot be recovered, the use of a higher dilution and/or the investi‑
gation of additional chemical neutralizers may be considered.
Table 13-2 provides examples of the test counts, controls, and interpretation of results that may be
encountered when performing neutralization studies during preservative efficacy testing.

TEST PROCEDURE
Organisms
The organisms listed in Table 20-1 of Method M-3 (Section 20) and Table 24-1of Method M-7
(Section 24) are representative types of the microbial species that a formulation may encounter
during manufacture and use: Gram-positive cocci, Gram-negative fermentative bacilli, Gram-neg‑
ative non-fermentative bacilli, yeast and mold. It is recommended that at least one microorganism
from each group be included in the challenge test. Examples of other organism types, relevant to the
formulation, may include microbial isolates that have been recovered from raw materials, formula‑

13
tions returned from consumers or other sources (e.g., product in or after use studies). Either pure
or mixed microbial culture suspensions may be used to conduct challenge testing of formulations.
Decisions to use pure or mixed cultures may be influenced by the factors discussed below.

PRESERVATION EFFICACY
DETERMINATION OF
Inocula consisting of only pure microbial cultures will yield specific data on each test microorgan‑
ism employed in the challenge study. Mixed culture inocula may serve to simulate real world condi‑
tions during use. When conducting mixed culture challenge studies, it is recommended that closely
related types of microorganisms such as Gram-positive bacteria (e.g., cocci), Gram-negative fermen‑
tative bacilli, Gram-negative non-fermentative bacilli, and yeasts and molds be pooled into separate
distinct groups. Antagonism between different types of organisms may occur due to differences in
growth factors and nutritional requirements.12 A rapidly growing organism may impede the detec‑
tion of a more slowly growing organism. For example, Escherichia coli has a shorter generation time
and may obscure detection or growth of microorganisms such as Pseudomonas aeruginosa that have a
longer generation time. Competition for growth factors or production of inhibitory byproducts and
other factors may result in antagonism between different types of microorganisms.12,13

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Inoculation and Enumeration Procedures

A typical challenge test consists of the following steps: inoculation of the test formulation, followed
by enumeration of the inoculated formulation at various time points, and interpretation of data to
determine adequacy of preservation. Details of these procedures may be found in the challenge test
methods of M-3 (Section 20) and M-7 (Section 24). It is recommended that the volume of inoc‑
ulum added to the sample of the formulation does not exceed 1% by volume. Larger volumes of
inocula (e.g., >1.0%) may result in undesirable dilution of the test formulation.
A preservative challenge test usually employs a single inoculation of each microorganism or pool of
microorganisms. A rechallenge consisting of a second inoculation may be considered if more infor‑
mation is desired, e.g., to determine if a formulation is marginally preserved.

OTHER CONSIDERATIONS
Modified Formulations
When changes are made to the composition of a formulation that has already been challenge test‑
ed, a rapid screening test such as described in Method M-7 (Section 24) may be used to determine
whether the change has an adverse effect on the preservation adequacy of the formulation. The
decision to perform additional challenge testing for these types of formulations is dependent upon,
but not limited to the type of finished package used, pH changes to the formulation, the addition
of new or deletion of raw ingredients from the previously tested formulation, and the challenge test
data of the original tested formulation.

Scale-Up/Pilot Batches
Changes in processing conditions during scale-up from laboratory to production size batches may
alter the performance of the preservation system. Processing conditions (e.g., order of raw ingredi‑
PRESERVATION EFFICACY

ent addition, pH of a batch during processing, and temperature of a batch during processing) may
alter the antimicrobial activity or the physical stability of the preservative system in a formulation.
DETERMINATION OF

Therefore, preservation tests may be performed on scale-up batches to confirm the effectiveness of
the preservation system.

Product Stability
During product development, the stability of the preservation system in a formulation should be
considered. Challenge tests may be performed on either bulk material or product from a filled con‑
13

tainer that has been aged by using accelerated aging conditions such as holding at specific tempera‑
ture and/or humidity conditions or real time aging at ambient conditions. Accelerated aging may
cause a formulation to undergo chemical and physical changes more rapidly than would otherwise
occur during real time aging. A decrease in preservative effectiveness over a period of time may occur
due to a variety of factors. These factors include preservative degradation, partitioning, interaction
with other formula ingredients, and chemical reaction with or absorption into the packaging mate‑
rial. The PET may be used to assess the degree of preservative effectiveness after accelerated or real

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time aging of a formulation has occurred. The preservative challenge test criteria for accelerated or
real time aged product may or may not differ from the challenge test criteria for fresh product.

RECOMMENDATIONS
Since many personal care products are used on a regular basis, an effective preservation system
should ensure the reduction of bacteria to a low and steadily decreasing level and fungi should re‑
main static or decrease over time, even after repeated use by the consumer. The following challenge
test criteria are the minimal recommendations for evaluating preservation system performance in
water- based product formulations:

• Vegetative Bacteria
There should be greater than or equal to 3 log (≥99.9%) reduction of vegetative bacteria
by aerobic plate count or quantitative spread plate methods within 7 days following each
challenge and no increase to the end of the test period.
• Yeast and Molds
There should be greater than or equal to 1 log (≥90%) reduction of yeasts and molds by
aerobic plate count or quantitative spread plate methods within 7 days following each chal‑
lenge and no increase for the duration of the test period.
• Spore‑Forming Bacteria
If spore-forming bacteria are included in the test, there should be bacteriostatic activity
against these microorganisms throughout the entire test period.

The above minimal challenge test criteria are suggested to aid manufacturers in evaluating the ad‑
equacy of preservation in personal care products. If a product does not meet these criteria, it is the
responsibility of the manufacturer to select the appropriate challenge test criteria that will ensure
product integrity. For example, single use packaging or use of pressurized delivery systems may be
factors that could be considered in selecting appropriate criteria. More stringent challenge test cri‑

13
teria may be considered where deemed appropriate.

PRESERVATION EFFICACY
DETERMINATION OF

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Table 13-1

EXAMPLES OF ANTIMICROBIAL PRESERVATIVES AND

RECOMMENDED NEUTRALIZING AGENTS
Antimicrobial Preservatives Recommended Neutralizing Agents:
Alcohol Dilution, Nonionic Surfactants (e.g., Polysorbates)
Bronopol (2-Bromo-2-Nitropropane-1,3,-Diol) Sulfhydryl Compounds (e.g., Cysteine, Thioglycollate,
Thiosulfate, and Metabisulfite)
Chlorhexidine Nonionic Surfactants (e.g., Polysorbates) and Lecithin,
Anionic Surfactants
Formaldehyde donors Dilution, Protein, Gelatin, Sodium bisulfite, Histamine,
Histidine, Nonionic Surfactants (e.g., Polysorbates),
Lecithin
Glutaraldehyde Dilution, Sodium bisulfite, Sodium sulfite, Sodium
thioglycollate, Glycine
Iodopropynyl Butylcarbamate (IPBC) Sodium thiosulfate
Isothiazolinones Dilution, Amines, Sulfites, Sodium bisulfite,
Sodium thioglycollate, Mercaptans
Organic Acid Preservatives (e.g., benzoic and sorbic Nonionic Surfactants (e.g., Polysorbates), increasing pH
acids)
Mineral Acids (e.g., sulfuric and hydrochloric acids) Increasing pH, peptones
Parabens Lecithin, Nonionic Surfactants (e.g., Polysorbates),
Phenolic Compounds (e.g., Phenylphenol, Nonionic Surfactants (e.g., Polysorbates) and Lecithin
chloroxylenol, cresols, chlorocresols)
Quaternary Ammonium Compounds Lecithin, Nonionic Surfactants (e.g., Polysorbates),
Protein, Anionic Surfactants
PRESERVATION EFFICACY
DETERMINATION OF
13

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Table 13-2

INTERPRETATION OF DATA FOR NEUTRALIZATION VERIFICATION

Actual counts >50 % criteria

Test Dilution
Control Count Test Dilution Count % Recovery Pass /Fail

100 70 70% Pass

100 50 50% Pass

100 30 30% Fail

100 25 25% Fail

13
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REFERENCES:
1. ISO 2010. ISO 29621, Cosmetics – Microbiology – Guidelines for the risk assessment and
identification of microbiologically low-risk products. www.iso.org.
2. Nikitakis, J.M. 2014. Personal Care Products Council Quality Assurance Guidelines. The
Personal Care Products Council, Washington, DC.
3. United States Pharmacopeia. 2016. <51> Antimicrobial effectiveness testing. USP 39-NF31,
United States Pharmacopeia and the National Formulary. Rockville, MD, pp.67-69.
4. ISO 2012. ISO 11930, Cosmetics – Microbiology – Evaluation of the antimicrobial protection
of a cosmetic product. www.iso.org.
5. McCarty, T.J. 1984. Formulated factors affecting the activity of preservatives. In: Kabara, JJ,
Orth, DS, ed. Cosmetic and Drug Preservation, Principles and Practice. Marcel Dekker Inc., New
York, pp.359-402.
6. English, D.J. 2006. Factors in selecting and testing preservatives in product formulations. In:
Orth, D.S., Kabara, J.J., Denyer, S.P., and Tan, S.K., eds. Cosmetic and Drug Microbiology.
Informa Healthcare, New York, pp. 57-108.
7. Brannan, D.K. and Dille, J.C. 1990. Type of closure prevents microbial contamination of
cosmetics during consumer use. Appl. Environ. Microbiol. 56: 1476-1479.
8. Singer, S. 1987. The use of preservative neutralizers in diluents and plating media. Cosmetics
and Toiletries. 102(12): 55-60.
9. Sutton, S.W. 1996. Neutralizer evaluations as control experiments for antimicrobial efficacy
Test. In: J.M. Ascenzi ed. Handbook of Disinfectants and Antiseptics. Marcel Dekker, Inc., New
York. pp 43-61.
10. ASTM International. 2013. ASTM E1054-08 – Standard test methods for evaluation of
inactivators of antimicrobial agents. http://www.astm.org.
PRESERVATION EFFICACY

11. Anon. 2003. Disinfectants and Antiseptics. Pharmacopeial Forum Vol. 29 (3) May-June, 726-
DETERMINATION OF

735.
12. Fredrickson, A.G. and Stephanopoulos, G. 1981. Microbial Competition. Science: 213: 971-
979.
13. Neilands, JB. Microbial Iron Compounds. Ann. Rev. Biochem. 50: 715-731.
13

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 SECTION 14

Preservation Efficacy
Testing of Eye-Area
Personal Care Products
INTRODUCTION
It is recognized that personal care products may be environments in which microorganisms can
adapt and then proliferate unless proper precautions are taken during formulation and manufacture.
The intended use of eye area personal care products makes it imperative that these products be pre‑
pared with preservative systems that remain effective.1 The alleged incidence of corneal ulceration
due to the periocular use of bacteria‑laden personal care2 has led the Food and Drug Administration
(FDA) to specifically address the adequate preservation of these eye area personal care products3
and the Cosmetic, Toiletries and Fragrance Association ( now called the Personal Care Products
Council) to recommend the same microbial limits as those indicated for baby products.4 Eye area
products are normally free of microbial contamination when purchased. However, some products
may contain organisms that are representative of human skin flora after use by the consumer.5 Mi‑
croorganisms may be introduced into the product from the environment or by the consumer, who
may, for example, add tap water to a product to make it less viscous.
In evaluating the adequacy of preservation of eye area personal care products, it is important to
point out that there is no substitute for judgment by knowledgeable microbiologists. It must also
be recognized that the addition of preservatives to personal care products is an adjunct to, but not a
substitute for, good manufacturing practices.

GENERAL CONSIDERATIONS
A. Developmental Formulations
Formulations that differ in at least one ingredient, e.g., binder or surfactant, should be tested
during the developmental stage using appropriate test microorganisms. If several preservative
systems are to be evaluated, each test formulation should be prepared concurrently from the same
microbiologically acceptable raw materials.
14

B. Pilot Batches
It may be desirable to perform a preservation test on individual pilot batches of product to ver‑
CARE PRODUCTS
TESTING OF EYE-AREA

ify the effectiveness of the preservative system. If feasible, these tests should be accompanied by
analytical determinations of preservative presence and concentration. Tests may be performed on
bulk material or on the filled samples.

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C. Stability
Product should be evaluated for preservative stability in commercial packaging by testing after
storage that simulates warehouse, shipping and shelf‑life conditions.

RECOMMENDATIONS
Since eye area personal care products are usually applied daily, an effective preservation system will
help ensure a low level of microorganisms even after severe microbial insult acquired during product
use or misuse. There are many references recommending preservative efficacy in sterile ophthal‑
mics5,7,8,9,10,11 and several in aqueous eye personal care products.6,9 Given the daily use of eye area per‑
sonal care products, it is recommended that multiple challenges be made to fully ensure adequacy
of preservation.11 The following are recommended as minimal criteria for preservative performance.
A. Aqueous Liquid and Semi‑Liquid Eye personal care products
1. Vegetative Bacteria
There should be greater than 99.9% (3-log) reduction of vegetative bacteria by aerobic plate
count or quantitative spread plate methods within 7 days following each challenge and contin‑
ued reduction to a less‑than‑detectable level by the end of the test period.
2. Yeast and Molds
There should be greater than 90% (one log) reduction of yeasts and molds by aerobic plate
count or quantitative spread plate methods within 7 days following each challenge and contin‑
ued reduction for the duration of the test period.
3. Spore‑Forming Bacteria
There should be bacteriostatic activity against spore‑forming bacteria throughout the entire
test period.
B. Non‑Aqueous Eye Products
1. Vegetative Bacteria
There should be a 99.9% (3-log) or greater reduction of vegetative bacteria by aerobic plate
count or quantitative spread plate methods within 7 days following each challenge and contin‑
ued reduction to a less‑than‑detectable level by the end of the test period.
2. Yeasts and Molds
There should be at least a 90% (1-log) reduction of yeasts and molds by aerobic plate count or
quantitative spread plate methods within 7 days following each challenge and the level should
remain at or below that level for the duration of the test.
TESTING OF EYE-AREA

3. Spore‑Forming Bacteria
CARE PRODUCTS

There should be bacteriostatic activity against spore‑forming bacteria throughout the entire
test period.

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 PRESERVATION EFFICACY TESTING OF EYE-AREA PERSONAL CARE PRODUCTS SECTION 14

These minimal criteria are suggested to aid manufacturers in evaluating the adequacy of preservation
of eye area personal care. Ultimately, it is the responsibility of the manufacturer to select appropriate
criteria that will ensure product integrity.

REFERENCES
1. Wilson, L.A., Kuehne, J.W., Hall, S.W. and Ahearn, D.G. 1971. “Microbial Contamination in
Ocular Personal Cares,” Am. J. Ophthal. 71(6):1298‑1302.
2. Wilson, L.A., and Ahearn, D.G. 1977. “Pseudomonas‑Induced Corneal Ulcers Associated with
Contaminated Eye Mascaras,” Am. J. Ophthal. 84:112‑119 (1977).
3. Madden, J.M., and Jackson, G.J. 1981.“Personal Care Preservation and Microbes: Viewpoint of
the Food and Drug Administration,” Personal Cares & Toiletries 96:75‑77.
4. “Microbiological Limits for Personal Cares and Toiletries” 2001. CTFA Microbiology Guidelines,
Personal Care, Toiletry and Fragrance Association, Washington, DC 20036 (November 2001).
5. Wilson, L.A., Julian, A.I., and Ahearn, D.G. 1975. “The Survival and Growth of
Microorganisms in Mascara During Use,” Am. J. Ophthal. 79(4):596‑601.
6. Tenenbaum, S. 1967. “Pseudomonads in Cosmetics,” J. of Soc. Cosmet. Chem. 18:797‑807.
7. Bean, H.S. 1972. “Preservatives for Pharmaceuticals,” J. of Soc. Cosmet. Chem. 23:703‑720.
8. British Pharm. 1980. Appendix XVIC A192, Vol. II.
9. CTPA Recommended Microbiological Limits and Guidelines to Microbiological Quality Control,
Appendix III (July 1983), Cosmetics, Toiletry & Perfumers Association - London W1M 9HD,
United Kingdom.
10. United States Pharmacopeia and the National Formulary. 2016. (USP39‑NF34), U.S.
Pharmacopeia, 1260 Twinbrook Parkway, Rockville, MD 20852.
11. Preservation Subcommittee of CTFA Microbiology Committee. 1981. “A Study of the Use of
Re‑challenge in Preservation Testing of Cosmetics,” CTFA Cosmet. J. 13:19‑22.

14
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TESTING OF EYE-AREA

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QUALITY AFTER USE
ASSESSMENT OF PRODUCT
SECTION 15

Microbiological
Assessment of Product
Quality After Use
INTRODUCTION
Every personal care product manufacturer has a responsibility in establishing the microbiological
safety of its finished products. The establishment of microbiological safety for a personal care prod‑
uct is a 2-step process. The first step is to provide assurance to the consumer that each personal care
product is free from the numbers and types of objectionable microorganisms that could affect prod‑
uct quality and/or the health of the consumer. This first step in the microbiological safety process
is accomplished by following current Good Manufacturing Practices (GMPs) during the manufac‑
turing of each personal care product. In addition, each manufacturer should have the appropriate
microbial quality control checks on those raw materials and finished products that are susceptible
to microbial contamination. Additional information on GMPs for cosmetics can be found in the
Personal Care Product Council’s Quality Assurance Guidelines.1
The second step is to ensure that each personal care product not be affected by the introduction
of microorganisms during normal or reasonably anticipated use by the consumer. To prevent the
growth of microorganisms in or on a personal care product that are introduced during consumer
use, preservatives may be added to the formulation. For most typical personal care products (e.g.,
water miscible), microbial challenge testing (e.g., “M-3 Method for Preservation Efficacy Testing of
Water-Miscible Personal Care Products” (Section 20), “M-4, Method for Preservation Efficacy Test‑
ing of Eye-Area Personal Care Products” (Section 21), USP Antimicrobial Effectiveness Test,2 ISO
119303 or an in-house challenge test method) is performed to verify that the preservative system of
a formulation can prevent the growth of microorganisms.
In addition, the analysis of used test samples for microbial content from either a clinical or sensory
study may provide additional assurance in the adequacy of the preservative system. Furthermore,
there are atypical personal care products (e.g., anhydrous gels, waxed based sticks, loose or pressed
powders, etc.) for which the traditional preservative challenge test may not yield the appropriate
information regarding either the microbial integrity or susceptibility to contamination of the prod‑
uct by the consumer. For these type products, analysis of samples after an in-use study may be more
appropriate than a traditional challenge test.

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SECTION 15 MICROBIOLOGIC AL ASSESSMENT OF PRODUCT QUALIT Y AFTER USE

ESTABLISHING A PROGRAM
In the development of an adequately preserved product, the nature of the product, directions for ap‑
plication, the microbial quality of the raw materials and the manufacturing process should be taken
into consideration. The microbiologist can exercise control over these factors. However, a personal
15

care product manufacturer does not have control of how a consumer will use a finished product.
In order to establish a program for evaluating the microbiological integrity or susceptibility to con‑
tamination of a personal care product during consumer use, it is necessary to generate information
relating to the way the product may be or should be used. This information can be obtained from
the product manager or through questionnaires, consumer letters, or by consumer market tests.
To determine how to structure an in-use study for a personal care product, the following informa‑
tion should be obtained: application, handling, length of use, and storage. This information will aid
the investigator in selecting the appropriate test panel members, defining usage instructions, and
defining a timetable. Study design should reflect actual product usage as closely as possible. If test
samples of a personal care formulation are going to be used in a clinical study for the establishment
of proposed product claims or to determine actual product safety, the study design should incorpo‑
rate all aspects of Good Clinical Practice.4 Special care should be exercised if this type of in-use study
involves finished products that are intended for use in either sensitive body areas, such as the eyes or
eye area, or by sensitive populations such as children or the elderly.

PANEL SELECTION
Selection of panel test members should be based on consumer habits and practices. Panel structure
must reflect this consumer usage information and should consider such factors as typical consumer
age, sex, geographical distribution, product usage patterns, etc. Panel size should be a function of
the degree of statistical sensitivity desired, with larger panels yielding increased sensitivity. An exact
size-versus-sensitivity determination may be made by consulting an appropriate sampling table.
After the panel structure has been determined, a request for participation should be forwarded to
potential panelists. It is recommended that this request be made to at least 20% more individuals
than are required to participate as a test panel member in order to allow for attrition and the initial
inability of some people to participate. The request for participation should include

• The test panel starting and termination dates;

• A concise, clear, comprehensive outline of what the test panelists would be expected to do
during the study; and
• A form for the test panelist’s signature showing agreement to participate in the study.

No individual should be included on a test panel until this signed form is returned.

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PRODUCT EVALUATION
Before anything is submitted for evaluation by a test panel, it should be confirmed that the formu‑
lation has been reviewed and approved for application and that microbial content testing of unused
test samples has been conducted to ensure that the product is microbiologically safe under the
prescribed conditions of use. In addition, the formulation being tested should have been evaluated
for safety and cleared for usage by the appropriate product safety department. Product identifica‑
tion should be recorded, including any pertinent history, product age, and lot or batch number. To
determine actual usage by a test panel member, it is recommended that each test sample should be
weighed prior to distribution and after it is returned.
When the test samples are distributed, a comprehensive set of use instructions should be prepared
and given to each test panel member. The instructions for using a test sample should include the
following:

• Approximate quantity of material to apply

• Method of application
• Frequency of use
• Handling the product between uses
• When to return the product (intermittent evaluation or after final use)
• Any other instructions pertinent to the product under review
• Name of a person to contact if any questions should arise

EVALUATION OF USED PRODUCT

When used test samples are returned to the test site, each test panelist should complete a question‑
naire or hand in a diary. In designing a questionnaire, the following points should be considered:

• How often was the product used?

• When was the product used?
• Where was the product stored between uses?
• Were there any problems associated with its use?

The questionnaire should be designed to generate information that might be helpful in pinpoint‑
ing the reasons for an aberrant test result. It may provide the investigator or microbiologist with
information regarding the product’s ability to withstand either inappropriate or normal consumer
usage. A sample questionnaire is presented in Appendix I. If a test panelist uses a diary, the following
information should be recorded: time at which product was applied each day; amount of product
used at each application; and where the product was stored between applications.
When evaluating returned test samples, microbiological content testing should be conducted before
any other testing is performed. This is to ensure that recovered microbial contaminants from a test
sample were introduced during consumer usage and not from subsequent handling. The microbi‑
ological evaluation of the used product should be conducted within a reasonable time after the last

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use of the product by test panelists (i.e., 7 days or less). Depending upon the number of individuals
that participate in a test panel, the microbiologist will have to decide whether to test all or a portion
of the returned samples for microbiological content. If a test panel consists of a low number of indi‑
viduals, it is recommended that each returned sample be analyzed for microbiological content. If the
test panel consists of a large number of individuals, all or a portion of the returned samples may be
15

analyzed. If a portion of the returned used test samples are analyzed, the number chosen should be
based upon some type of statistical sampling plan (e.g., the square root plus one). If aberrant micro‑
biological test results are obtained, the microbiologist has the option to analyze additional returned
test samples for confirmation of the result.

MICROBIAL CONTENT OF PRODUCT

The method used for determining the microbial content of used product samples will depend on
the nature of the product. Following are several methods that may be used. Those methods other
than aerobic plate count are considered semi-quantitative. Where possible, quantitative recovery is
preferable to semi-quantitative recovery. Semi-quantitative results should be reported as an estimate
of the microbial content of that unit.
Where feasible, an aliquot of the used test sample may be aseptically removed from each container
and analyzed for microbial content using an aerobic plate count method (e.g., “M-1 Determina‑
tion of the Microbial Content of Personal Care Products” (Section 18) and “M-2 Examination for
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans”(Section 19)
or in-house method). For water immiscible products (e.g., oils or emulsions), a suitable solubilizing
agent may be incorporated into the test diluent or broth to make the sample aliquot miscible with
water in order to recover microorganisms present in the test sample.
For products where microorganisms would only be recovered from the product surface (e.g., sticks,
pressed powders, hot pour products in compacts) only the surface of the product sample should be
tested. For these “atypical products,” the following methods of recovery may be considered.
A sterile moistened applicator may be used to sample the product surface, and then streaked onto a
Petri dish containing solid culture medium.
The product may be sampled by a direct contact method using a contact plate (a modified Petri
dish containing a solid culture medium whose convex surface extends above the carrier), paddles or
flexible film containing solid culture media.
Alternative test methods to those described above may be used. Appropriate preservative neutralizers
should be incorporated into product diluents, liquid or solid media. Whatever method is chosen, it
should be verified for the recovery of microorganisms. The same method should be applied to the
control sample.

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MICROBIAL CONTENT OF APPLICATORS /

UNIQUE PACKAGING ELEMENTS
Product applicators or unique packaging elements (natural or synthetic) that come into direct con‑
tact with the product may be evaluated for estimated microbial content, as these items are the vec‑
tors of microbial contamination into the product. The following semi-quantitative methods may be
used to determine estimated microbial content.

• Aseptically transfer the applicator to a container of sterile diluent or liquid culture medium.
After vigorously shaking, stomaching, or vortexing for a set period of time, perform an aer‑
obic plate count on an aliquot of the diluent or liquid culture medium.
• The applicator may be sampled using a direct contact method (see Microbial Content of
Product section on p. 166).

Alternative test methods to those described above may be used. Appropriate preservative neutral‑
izers, as required, should be incorporated into diluents, liquid or solid media. Whatever method is
chosen, it should be verified for recovery of microorganisms.

IDENTIFICATION OF ISOLATES
It is recommended that recovered microorganisms from test samples be identified. If multiple types
of microbial colonies are obtained, representative microbial colonies may be selected for identifica‑
tion.

INTERPRETATION OF RESULTS
For convenience, it is recommended that all test results be summarized. The interpretation of mi‑
crobiological in-use test results is largely a matter of in-house specifications. The extent to which
microorganism recoveries can be considered significant must be viewed in light of what the ultimate
effect would be on the consumer, the type of product (e.g., typical or atypical product formulation),
and how the product would typically be used by the consumer (e.g., eye versus lip).
Recovery of low levels of microorganisms may be of some significance, especially in water-based
products where the potential for proliferation may exist. Thus, the acceptance criteria for samples of
water-based products returned from in-use studies normally reflect the specification for end product
release. For these products, further investigation into recovery of low levels of microorganisms may
be warranted.
However, for products with low water activity, the potential for proliferation does not exist. For
these types of products, recovery of normal skin flora may be expected. Therefore, the acceptance
criteria for atypical products may be significantly different from those of water based products.
Higher microbial counts may be acceptable in atypical products used in areas of the body that con‑
tain higher microbial populations, and where there is less risk to the consumer. For example, 103 to
104 CFU/gram may be recovered in products applied to the lip area.

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ASSESSMENT OF PRODUCT
QUALITY AFTER USE
SECTION 15 MICROBIOLOGIC AL ASSESSMENT OF PRODUCT QUALIT Y AFTER USE

To ensure that the number of organisms recovered do not increase over time, either retest of initial
samples or of additional samples may be used to confirm stasis or reduction in recoverable levels of
microorganisms. If levels of microorganisms recovered do increase, then formulation and/or pack‑
age design should be reviewed. Any reported result that is aberrant may warrant further investiga‑
tion including performance of non-microbiological testing.
15

In-use studies of test samples cannot give a complete picture of how well a product will withstand
consumer handling and use. However, an in-use study for a proposed product may provide a margin
of added assurance to the manufacturer, as well as alerting them to potential problems that could
occur in the field. Regardless of the nature of the test data generated, consumer in-use studies can
provide meaningful information in how a product may behave during repeated microbiological
insult during consumer usage.

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 15
QUALITY AFTER USE
ASSESSMENT OF PRODUCT
MICROBIOLOGIC AL ASSESSMENT OF PRODUCT QUALIT Y AFTER USE SECTION 15

Appendix I

SAMPLE CONSUMER EVALUATION QUESTIONNAIRE

Product: _________________________________________________________________________

Panelist: _________________________________________________________________________

1. How often did you apply the product?

2. When did you apply the product (e.g., after bath/shower, after housework, before
retiring, etc.)?

3. Did you use the product on areas other than the hands? If so, where?

4. Where was the product kept when not in use?

5. Did you experience any adverse reactions (e.g., a rash)? If so, please explain.

6. List any comments you may have.

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ASSESSMENT OF PRODUCT
QUALITY AFTER USE
SECTION 15 MICROBIOLOGIC AL ASSESSMENT OF PRODUCT QUALIT Y AFTER USE

REFERENCES
1. Nikitakis, J. M. (Ed.) 2014. Personal Care Product Council Quality Assurance Guidelines,
Personal Care Product Council, Washington, D.C.
2. United States Pharmacopeia and the National Formulary. 2016 (USP39-NF34), <51>
15

Antimicrobial Effectiveness Testing, U.S. Pharmacopeia, 1260 Twinbrook Parkway, Rockville,

MD, pp/ 67-69.
3. ISO 2012. ISO11930 Cosmetics — Microbiology — Evaluation of the antimicrobial
protection of a cosmetic product, www.iso.org.
4. U.S. Food and Drug Administration. 1996. Guidance for Industry E6 Good Clinical Practice:
Consolidated Guidance. http://www.fda.gov/downloads/Drugs/.../Guidances/ucm073122.pdf

ADDITIONAL INFORMATION
Brannan, D.K., Dille, J.C., Kaufman, D.J., 1987 “Correlation of In-Vitro Challenge Testing with
Consumer Use Testing for Personal Care Products,” Applied and Environmental Microbiology,
58:1827-1832
Brannan, D.K., Dille, J.C., 1990 “Type of Closure Prevents Microbial Contamination of Personal
Cares During Consumer Use,” Applied and Environmental Microbiology, 56 :1476-1479
Farrington, J.K., Martz, E.L., Wells, S.J., Ennis, C.C., Holder, J., Levchcuk, J.W., Avis, K.E.,
Hoffman, P.S., Hitchins, A.D., Madden, J.M., 1994.“Ability of Laboratory Methods to Predict
In-Use Efficacy of Antimicrobial Preservatives in an Experimental Personal Care,” Applied and
Environmental Microbiology, 60 :4553-4558
Larson, E.L., Gomez-Duarte, D., Lee, L.V., Della-Latta, P., Kain, D.J., Keswick, B.H., 2003.
“Microbial flora of hands of homemakers,” Am.J. Infect. Control, 31 :72-79.
Lindstrom, S.M., Hawthorne, J.D., 1986. “Validating the Microbiological Integrity of Personal
Care Products through Consumer-Use Testing,” J. Soc. Cosmet. Chem. 37:481-428.
Passaro, D.J., Waring, L., Armstrong, R., Bolding, F., Borvier, B., Rosenberg, J., Reingold, A.W.,
McQuitty, M., Philpott, S.M., Jarvis, W.R., Werner, S.B., Tompkins, L.S., Vugia, D.J. 1997.
“Postoperative Serratia marcescens Wound Infections Traced to an Out-of-Hospital Source,”
J. Infect. Diseases, 175:992-995.
Trick, W.E., Vernon, M.O., Hayes, R.A., Nathan, C., Rice, T.W., Peterson, B.J., Segreti,
J., Welbel, S.F., Solomon, S.L., Weinstein, R.A., 2002. Impact of Ring Wearing on Hand
Contamination and Comparison of Hand Hygiene Agents in a Hospital, Clinical and Infectious
Diseases, 36:1383-1390.
Wilson, L.A., Julian, A.I., and Ahearn, D.G. 1975. The Survival and Growth of Microorganisms
in Mascara During Use, Am. J. Ophthal. 79:596-601

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 SECTION 16

Microbiological Risk Factor

Assessment of Atypical
Personal-Care Products

16
ATYPICAL PRODUCTS
ASSESSMENT OF
INTRODUCTION
Every personal care product manufacturer has a dual responsibility relative to the microbiological
quality of its products. The first is to ensure that the product, as purchased, is free from the numbers
and types of microorganisms that could affect product quality and consumer health. The second is
to ensure that microorganisms introduced during normal product use will not adversely affect the
quality or safety of the product.
During product development, the microbiologist may use several tools to evaluate the ability of a
product to prevent the growth of microorganisms introduced during product use. The challenge
test, which involves introducing a known quantity of microorganisms into a formula and monitor‑
ing the rate of kill over time, is frequently used.* A second method of evaluating product quality
during consumer use is by evaluating the product after a use test that simulates “real life” situa‑
tions.** Finally, the microbiologist may perform a microbiological risk assessment of the product.
The risk assessment process is based on a number of factors generally accepted as important in
evaluating the spoilage potential of a product. It is intended to guide the microbiologist and for‑
mulator in determining what level of testing is necessary to assure the quality of the product during
manufacturing and consumer use.
This guideline serves as an aid to the personal care microbiologist in assessing the microbiological
quality of formulations for which the normally recommended method of challenge testing, de‑
veloped for water based formulations, may not yield appropriate information. These include an‑
hydrous formulations, formulations with low water content, or those products where water is the
internal phase.
This guideline also serves as a tool to aid the microbiologist in recommending ways of reducing
product susceptibility to microbial growth. Certain personal care products, depending on their
composition and presentation (packaging), may have negligible potential for microbial prolifera‑
tion during use. Microbial contamination of a personal care product during use is a function of
the physico-chemical characteristics of the product and the way in which it is packaged (i.e., its
presentation). The guideline “ISO 29621 Cosmetics — Microbiology — Guidelines for the risk

* For examples, see “M-3 Method for Preservation Efficacy Testing of Water-Miscible Personal Care Products” (Section 20)
and “M-4 Method for the Preservation Efficacy Testing of Eye Area Personal Care Products” (Section 21). For guidance
on the use of these methods, see “Determination of Preservation Efficacy in Water-Miscible Personal Care Products”
(Section 13) and “Preservation Efficacy Testing of Eye-Area Personal Care Products” (Section 14).
** For guidance, see “Microbiological Assessment of Product Quality after Use” (Section 15).

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 SECTION 16 MICROBIOLOGICAL RISK FACTOR ASSESSMENT OF ATYPICAL PERSONAL-CARE PRODUCTS

assessment and identification of microbiologically low-risk products” describes a variety of factors

than can influence the ability of microorganisms to replicate in a personal care product.1

PRODUCT TYPES
Common examples of atypical products are listed below. In each example, water is not readily avail‑
able to provide an environment that supports growth of microorganisms. Water in the product may
ATYPICAL PRODUCTS

be surrounded by oil or silicone as the external phase, with the water being present as small droplets
and influenced by other water-soluble formula ingredients. Also, the water activity may be too low
ASSESSMENT OF

to support growth, and in some cases, the product might be totally anhydrous.2

• Wax based products

• Products with high oil/low water content
• Siloxane and siloxane derivative based products
16

• Lip balms
• Pomades
• Ointments
• Powders
• Cream to powder make-up

In addition to products with low water activity, products with the physico-chemical characteristics
below may not allow the proliferation of harmful microorganisms:
Products with an alcohol content equal to or greater than 20% (vol/vol)2
Products with a pH of less than 3 or greater than 10.3-6

PRODUCT SUSCEPTIBILITY
Atypical products may contain raw ingredients that do not support the growth of microbial contam‑
inants and therefore may prevent microorganisms from proliferating when the product is subjected
to normal consumer use. In these types of products, organisms may survive, but cannot reproduce.
This may be due to low water activity or low water activity in combination with pH and/or an‑
tagonistic formula ingredients that are water soluble. Water droplet size may also be critical in the
water phase. If water activity reading is low in a product formulation or if the formula is anhydrous,
studies have shown that microorganisms will not proliferate. In fact, this is the basis for the use of
water activity as an assessment tool in determining the risk for microbiological proliferation for food
products, like cereals.7,8
In some atypical products, microbial survival may occur on the outside of the product without ever
permeating and spreading through the product. This observation is also due to the low “free water”
content.

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 MICROBIOLOGICAL RISK FACTOR ASSESSMENT OF ATYPICAL PERSONAL-CARE PRODUCTS SECTION 16

Because most atypical products will not support microbial proliferation, the method of product
delivery may be the vector for transferring microbial contamination from the product back to the
consumer.

RISK FACTOR ASSESSMENT

16
A number of factors need to be evaluated when performing a microbial risk assessment to determine
what type of testing or preservative system may be needed for a particular formulation. Listed below

ATYPICAL PRODUCTS
ASSESSMENT OF
are several factors that need to be considered in determining a products potential risk of microbial
contamination during consumer usage.

Water Activity
The metabolism and reproduction of microorganisms demands the presence of water in an available
form. The most useful measurement of water availability in a product formulation is water activity
(aw). Water activity is defined as the ratio of the water vapor pressure of the product to that of pure
water at the same temperature: 7
aw = p/Po = (n2/(n1 + n2))
where,

p is the vapor pressure of the solution,

Po is the vapor pressure of pure water,
n1 is the number of moles of solute, and
n2 is the number of moles of water.

When a solution becomes more concentrated, vapor pressure decreases, and the water activity falls
from a maximum of 1.00 (aw) for pure water. As the water activity level falls below the optimum
value for each microorganism, the length of the lag phase in the microbial growth cycle will increase
toward infinity unless rehydration occurs.
Listed below are examples of the minimum water activity levels required for growth of selected mi‑
croorganisms.8,9,11

Approximate Minimum Water Activity (aw) Required for Growth of Selected

Microorganisms 1,12

Most bacteria 0.94 – 1.00

Enterbacteraciae >0.93
Pseudomonas species >0.96
Staphylococcus aureus >0.86
Most spoilage yeast >0.70
Most spoilage mold >0.60

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 SECTION 16 MICROBIOLOGICAL RISK FACTOR ASSESSMENT OF ATYPICAL PERSONAL-CARE PRODUCTS

The above water activity values should be considered as reference points since microbial growth may
occur at lower values depending on differences in temperature or nutrient content of the product
formulation. Even though water activity values are important in assisting in the risk analysis for
microbial contamination, water activity should never be used as the sole indicator in determining
whether product testing is necessary for a particular product formulation.

Formula Review
ATYPICAL PRODUCTS

Every formula contains raw materials that have an impact on the susceptibility of the formula to
microbiological contamination. Raw materials can be classified as susceptible, hostile, or neutral to
ASSESSMENT OF

microbial growth, and their concentration will affect the susceptibility of the formulation to micro‑
bial contamination.***
It is recommended that a microbiologist review formulas to determine their potential susceptibility
to microbial contamination. If a formulation tends to absorb moisture, samples of these types of
atypical products should be microbiologically evaluated (including aw) after being exposed to high
16

humidity conditions (i.e., 50-75% for about 3 weeks or until equilibrium is demonstrated).
Anhydrous products may contain binders or other hygroscopic materials that are able to absorb
moisture. In addition, consideration of water on the surface of products may occur under high hu‑
midity conditions. Consumers may introduce water during normal use or during misuse.
The physical product form will affect whether microbial contaminants will be introduced at the
product surface or be mixed throughout the product during consumer use.
Factors to be taken into consideration are:

• Raw material susceptibility

• Raw material microbial load
• Percent concentration of raw material
• Presence of preservative inactivators
• Presence of preservative potentiators
• Presence of fragrance and other ingredients that may act as preservatives
• Binder level in powders (The higher the binder percentage, the more hydrophobic the product
will be.)
• Product physical form (solid or liquid; melting point)

Site of Application
The risk assessment needs vary, depending upon the body area for product application. The site of a
product’s application is an important risk factor in determining the level and type of microbiological
testing that would be required for a personal care product. For example, an eye area product presents
a much greater potential risk of microbiological contamination to the consumer than a product that
is applied to other areas of the body. Lip products, under normal conditions, generally come into
contact with higher numbers of microorganisms which are part of the normal microflora present in
the consumer’s lip area, but do not pose a health risk.
*** For examples, see “Raw Material Microbial Content” (Section 11)

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Some points to consider when determining the risk in relation to site of application for a product
formulation include:

• Is the site on the body to which the formulation is to be applied to an area where microbial
levels are high (lip) or low (eye)?
• Is the product applied under moist (higher risk) or dry (lower risk) conditions?
• What is the mode of application (brush, sponge, or finger)?

16
• How frequently is the product used?

ATYPICAL PRODUCTS
ASSESSMENT OF
Applicators and the Mode of Application
The mode of application plays a large role in determining the risk factor of a product. Even though
the product may not support growth, the method of delivery may be a vector of microbial bacterial
contamination.
Applicators could provide an environment that might be suitable for microbial proliferation. For
example, porous sponge applicators may be a concern due to their ability to absorb moisture and re‑
tain sebum and dirt from the skin. With the presence of water, sebum, and dirt from the skin, there
may be enough water and nutrients present in a used applicator to allow for microbial proliferation.
In those applicators that are to be used in conjunction with a wet/dry product, the incorporation
of an antimicrobial agent may be considered to prevent microbial proliferation. The preservatives
system of a product must not be expected to inhibit microbial growth in or on a product applicator
Some typical questions that need to be asked before microbiological testing is conducted on appli‑
cators are as follows:

• Are applicators such as puffs, brushes, or pads used with the products?
• Could these applicators act as a breeding ground for microorganisms or a vector for micro‑
bial contamination of the product and/or consumer?
• Does the product and component come in direct contact with the consumer (lips, eyes, fingers)?
• Do these applicators contain an anti-microbial agent?
• Is the applicator stored in direct contact with the product?
• Are there directions given on how to store or clean applicators between uses?

When evaluating and determining the risk factor for microbiological contamination in product
applicators, the following additional factors are to be considered:

• Type of applicator
• Type of material used
• Treated or not treated with an antimicrobial agent.
• The efficacy of a treated applicator should be tested via a zone of inhibition test or other
appropriate method.10,11
• Wet/dry application of product

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 SECTION 16 MICROBIOLOGICAL RISK FACTOR ASSESSMENT OF ATYPICAL PERSONAL-CARE PRODUCTS

Packaging
The type of packaging used for a finished product is a critical risk factor in determining the overall
potential for microbial contamination during consumer usage. The use of packaging can provide
additional protection by restricting direct access to the product.
The following factors are among those taken into consideration when assessing product risk with
regards to packaging:

• Single or multiple use packaging?

ATYPICAL PRODUCTS

• What is the size of the package?

ASSESSMENT OF

• What is the mode of dispensing?

• What is the predicted use-up rate?
• Does the package type allow for direct consumer contact?
• Is the package pressurized?
16

Confirmation of the microbial integrity of the applicator and finished product can be determined
from conducting an in-use study.

Manufacturing Process
Certain aspects of the manufacturing process (e.g., high temperature) may affect the microbiological
contamination susceptibility for a personal care product. It is useful for both the microbiologist and
personal care chemist to review the manufacturing process to determine the potential risk of micro‑
bial contamination to the formulation.
Factors to be considered:

• Are there processing factors that could affect the efficacy of the preservative system?
• What is the temperature of the manufacturing process?
• What is the microbial content of the raw materials?
• Are hostile raw materials used to make the product?
• What is the order of addition of the raw materials?

PRODUCT TESTING
General
After evaluating the above factors, the microbiologist can determine what level and type of microbi‑
ological testing is necessary. If it is determined that microbiological testing is necessary, the follow‑
ing information should be taken into account to select the most appropriate test method.

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Challenge Tests
General Considerations
The recommended preservative challenge test methods that are used for determining the preserva‑
tive adequacy of aqueous based products may not be suitable for evaluating certain atypical product
formulations. When testing and assessing preservative challenge test data for atypical products, the
following factors are important points to consider:

16
• A test in which an aqueous based inoculum is introduced into an anhydrous sample may
change the physical dynamics of the product and, therefore, may not predict its microbial

ATYPICAL PRODUCTS
ASSESSMENT OF
stability.
• Most preservatives are water-soluble. In emulsions, preservatives are used in the water phase
because contaminating microorganisms require water to proliferate.
• For an emulsion in which the external phase is water immiscible and an aqueous challenge
inoculum is used, the water-soluble preservatives will be unable to penetrate the water im‑
miscible phase. In these cases, the preservatives will be unavailable to either inhibit prolifer‑
ation or have cidal activity against each of the challenge microorganisms.

Possible Test Method Modifications

If preservative challenge testing is performed on an atypical product formulation, aqueous based
challenge protocols may need to be modified to take into account a number of factors. For specific
test methods see M-4, M-5, and M-6 (Sections 21, 22, and 23, respectively). A number of possible
modifications to these methods are discussed below.

• Reduction in Inoculum Concentration

For anhydrous products, a reduction in the challenge inoculum size to 103 to 104 Colo‑
ny-Forming Units (CFU) per gram or milliliter may be used instead of the inoculum con‑
centration of 105 to 106 CFU per gram or milliliter that is recommended in the aqueous
based challenge test methods (See M-3, Section 20). By reducing the inoculum size, it is
easier to measure stasis or quantify an increase in the microbial count.
• Reduction in Inoculum Volume
Reduction of the ratio of microbial inoculum suspension to the volume of product may also
be considered. The recommended ratio of inoculum suspension for aqueous based products
is no more than 1.0% for a challenge sample. For atypical product formulations, the ratio
of inoculum suspension to product may need to be reduced to 0.1% in order to minimize
changes in the physical dynamics of the product.
• Surface Inoculation and Sampling
For solid atypical products, such as anhydrous sticks and powders, inoculation and sampling
of the product surface instead of the whole product more closely simulates potential con‑
sumer contamination. This modification also maintains the physical product integrity. In
these types of products, the microorganisms are not able to penetrate into the interior and
will always be found on the outer-most layer of the product after consumer usage.
Note: If performing challenge testing on a solid anhydrous stick or powder product, inocu‑
late a sufficient number of samples to obtain a unique sample for each sampling time-point.

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• Inoculum Delivery Systems

For liquid, anhydrous, atypical product formulations, one may consider using an oil soluble
carrier system, such as light mineral oil or other suitable oil carrier, to deliver and disperse
the microbial challenge inoculum into liquid atypical product formulations to form a ho‑
mogeneous mixture.
If using this technique, the absence of inhibitory or toxic properties of the oil soluble carrier
system should be verified for each of the challenge organisms.13
• Sampling Diluents
ATYPICAL PRODUCTS

The recovery procedure for determining the microbial counts from inoculated challenge
samples of an atypical product may need to be modified from those that are commonly used
ASSESSMENT OF

in aqueous based challenge test methods. For example, water-in-oil emulsions and anhy‑
drous products are not readily miscible with water. It has been demonstrated that 1.0-gram
aliquots of an anhydrous product solubilizer in a 1.0-gram aliquot of sorbitan monostearate
(Tween 80) and this mixture were dispersed further by increasing the volume with an aque‑
ous diluent to make a 1:10 dilution.14
16

Challenge Test Acceptance Criteria

It is the responsibility of the manufacturer to set the challenge test criteria for the product type and
form. In the performance of challenge testing of atypical products, the pass/fail criteria may need
to be modified in comparison to the preservative challenge test criterion that is commonly used
for aqueous based products. For example, the challenge acceptance criteria for anhydrous atypical
products may be stasis for certain types of challenge microorganisms, because these organisms do
not need a source of water to survive.
If criteria other than the aqueous based challenge criteria are used to show adequate preservation for
an atypical product formulation, a risk assessment needs to be conducted by the microbiologist to
justify the use of these alternate preservative challenge test criteria.

In Use Studies
General
In addition to or in place of a product challenge test, an in use study may provide sufficient data to
conduct a risk assessment on some products. An in-use study may be used to evaluate the microbi‑
ological integrity of a product during consumer use. The study design should reflect actual product
use as closely as possible. For further information, refer to “Microbiological Assessment of Product
Quality after Use.” (Section 15)

Testing
When samples are returned from an in-use study, an aerobic plate count must be conducted before
any other tests are performed in order to ensure that any microbial contaminants recovered were
introduced by the panelists and did not arise from subsequent handling in the laboratory. Useful
microbial content information may be obtained by a similar evaluation of the components (such as
applicators) that come into direct contact with the product during use.

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 MICROBIOLOGICAL RISK FACTOR ASSESSMENT OF ATYPICAL PERSONAL-CARE PRODUCTS SECTION 16

Microbiological analysis of these samples can be conducted by using either standard in-house meth‑
ods or the PCPC, “M-1 Determination of the Microbial Content of Personal Care Products” (Sec‑
tion 18) and “M-2 Examination for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa,
and Candida albicans” (Section 19). It is recommended that microbiological evaluation take place
within 7 days after the last consumer use.

Criteria

16
The pass/fail criteria for in-use return samples normally reflect the microbiological test specifications
that are used for quality control end product release. The pass/fail criteria for atypical products or

ATYPICAL PRODUCTS
ASSESSMENT OF
for water-based products may vary significantly depending on the product type and area of use. For
example, it may be acceptable that products that have been used in the lip area may contain a higher
microbial level than products used in the eye when evaluated after use.
To ensure that the number of microorganisms recovered after usage does not increase over time,
these types of atypical products should be tested at a prescribed time interval. If microbial counts
do increase over time, the formulation and/or package design should be reviewed to determine what
steps could be taken to prevent them from increasing.

REFERENCES
1. ISO 2010. ISO 29621 Cosmetics — Microbiology — Guidelines for the risk assessment and
identification of microbiologically low-risk products. Geneva, Switzeland. www.iso.org
2. Yablonski, J.I. and Mancuso, S.E. 2002. “Preservation of Atypical Cosmetic Product Systems,”
Cosmetics and Toiletries, 117 :31-40.
3. Ali, Y. et al. 2001. “Alcohol” in S.S. Block, Disinfection, Sterilization, and Preservation, Fifth
Edition, pp. 229-253.
4. Brannan, D.K. 1997. Cosmetic Microbiology, CRC Press, New York, pp. 47-50.
5. Kabara, J.J. (ed.). 1984. Food grade chemicals in a systems approach to cosmetic preservation,”
In: Cosmetic and Drug Preservation: Principles and Practice, Marcel Dekker, New York, p. 391.
6. Kabara, J.J., and Orth, D.S. 1996. Preservative-Free and Self-Preserving Cosmetics and Drugs,
Marcel Dekker, New York, p. 245-246.
7. Silliker, J.H., et al., Eds. 1980. International Commission on Microbiological Specifications for
Food. Microbial Ecology of Foods, Vol. 1, Academic Press, Orlando, FL, pp. 76-91.
8. Food Microbiology Fundamentals and Frontiers (ed Michael P. Doyle, Larry R. Beuchat, and
Thomas J. Montville); ASM Press, 1997, ISBN 1-55581-117-5.
9. Jay, J.M. (ed.) 2000. Modern Food Microbiology, Sixth Edition, Aspen Publishers, Gaithersburg,
MD, pp. 38-44, especially p. 42.
10. Brannen, D.K. (ed.) 1997. Cosmetic Microbiology, CRC Press, Boca Raton, FL, pp. 47-50.

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 SECTION 16 MICROBIOLOGICAL RISK FACTOR ASSESSMENT OF ATYPICAL PERSONAL-CARE PRODUCTS

11. Hartman, P.A. 1968. Miniaturized Microbiological Methods, Academic Press.

12. Curry, J. 1985. “ Water Activity and Preservation,” Cosmetic and Toiletries, 100; pp. 53-54.
13. ASTM International 2013. E1054-08 Standard Test Methods for Inactivators of Antimicrobial
Agents.
14. McConville, J.F., Anger, C.H., D.W. Anderson 1974, “Method for Performing Aerobic Plate
Counts of Anhydrous Cosmetics Utilizing Tween 60 and Arlacel 80 as Dispersing Agents,” Applied
Microbiology, 27, No.1, pp 5-7.
ATYPICAL PRODUCTS
ASSESSMENT OF

OTHER SOURCES OF INFORMATION

Kabara, J. J., and Orth, D. S. 1996. Preservative-Free and Self-Preserving Cosmetics and Drugs,
Marcel Dekker, New York, pp. 45-73.
Yablonski, J.I. 2002. “Preservation of Atypical Skin Care and Related Cosmetic Product Systems,”
16

Cosmetics and Toiletries, p. 117.

Brannan, D.K., Dille, J.C., Kaufman, D.J. 1987. “Correlation of In-Vitro Challenge Testing with
Consumer Use Testing for Cosmetic Products,” Applied and Environmental Microbiology, 58 (3):
1827-1832
Farrington, J.K., Martz, E.L., Wells, S.J., Ennis, C.C., Holder, J., Levchcuk, J.W., Avis, K.E.,
Hoffman, P.S., Hitchins, A.D., Madden, J.M. 1994. “Ability of Laboratory Methods to Predict
In-Use Efficacy of Antimicrobial Preservatives in an Experimental Cosmetic,” Applied and
Environmental Microbiology, 60 (12): 4553-4558
Lindstrom, S.M. 1986. “Consumer Use Testing: Assurance of Microbial Product Safety,” Cosmetics
and Toiletries, 101: 73-74
Lindstrom, S.M., Hawthorne, J.D. 1986. “Validating the Microbiological Integrity of Cosmetic
Products through Consumer-Use Testing,” J. Soc. Cosmet. Chem. 37: 481-428
Orth, D.S., Barlow, R.F., Gregory, C.A. 1992. “The Required D-Value: Evaluating Product
Preservation in Relation to Packaging and Consumer Use/Abuse,” Cosmetics and Toiletries, 107
(12): 39-43
Orth, D.S. 1993. Handbook of Cosmetic Microbiology, Marcel Dekker, p. 151.
Orth, D.S. and Milstein, S.R. 1989. “Rational development of preservative systems for cosmetic
products,” Cosmetics and Toiletries, 104(10): 91-103.
Wilson, L.A., Julian, A.I., and Ahearn, D.G. 1975. “The Survival and Growth of Microorganisms
in Mascara During Use,” Am. J. Ophthal. 79(4): 596-601

180 | PCPC MICROBIOLOGY GUIDELINES

 SECTION 17

Determination of Preservation
Efficacy in Nonwoven Substrate
Personal Care Products
INTRODUCTION
Nonwoven substrate personal care products, commonly called wipes, constitute a wide and expand‑
ing variety of items that differ significantly from other types of personal care products in their com‑
position, intended use, and physical characteristics.1 The nonwoven matrix or substrate is composed
of fibers or filaments that are bonded together mechanically, thermally, or chemically and is used for
the delivery of cosmetics or other product systems.

17
In view of the differences between wipes and other types of personal care products, the standard

IN NONWOVEN SUBSTRATE
PRESERVATION EFFICACY
preservation efficacy tests for aqueous-based (See Section 20: M-3 A Method for Preservation Effica‑
cy Testing of Water-Miscible Personal Care Products) or atypical (See Section 23: M-6 Method for
Preservation Testing of Atypical Personal Care Products) personal care products are not suitable for
testing these product forms. The two major test method differences have to do with the procedure
for inoculum introduction and the procedure for the recovery of introduced microorganisms. It is
recommended that, when developing preservation efficacy methods and testing protocols, the cos‑
metic microbiologist be aware of the factors listed below under “General Considerations” and how
they may affect the reliability of the test method under development.
This document is intended to be used in conjunction with “M-5 Methods for Preservation Testing
of Nonwoven Substrate Personal Care Products” (Section 22).

GENERAL CONSIDERATIONS
A. Components
A nonwoven personal care product is composed of the following components:

• Substrate - nonwoven carrier including coatings or finishes applied to that carrier

• Add-ons - personal care formulation applied to a substrate; liquids and lotions are the most
common
• Packaging - final container for delivering the finished nonwoven substrate product

Any change to the composition or nature of any of these components may affect the overall pres‑
ervation efficacy of the final product and may require retesting.

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 SECTION 17 PRESERVATION EFFICACY IN NONWOVEN SUBSTRATE PERSONAL CARE PRODUCTS

1. Substrate
The nature and composition of substrates can have a decided effect upon preservative-substrate
interaction as well as subsequent preservative system performance. A substrate2,3 is a nonwoven
web of long and short fibers held together by some means of bonding other than weaving.
Fibers can be natural or synthetic. The substrate functions as the carrier for a variety of prod‑
uct-specific add-ons. Generally, substrates are composed of any one or a combination of vari‑
ous fiber types including natural materials such as cellulose or wood pulp or synthetic materials
such as rayon or viscose, polyester and polypropylene polymer extrusions or bicomponent ma‑
terials. Bonding technologies include mechanical entanglement, chemical or adhesive binding,
thermal melting and hydrogen bonding.
Binders can significantly affect the preservative system.4,5 For example, anionic binders, com‑
monly used in some substrates, have the potential to inactivate or seriously disrupt most cat‑
ionic preservative systems. Alternatively, binders may contain preservatives which may result
in a more robust product.
Other substrate issues that can affect preservation efficacy may include the fiber type and
composition, the web forming process, the web bonding process, the proportion of pulp to
binder, the composition and ionic nature of the chemical binding agent, and the presence and
IN NONWOVEN SUBSTRATE
PRESERVATION EFFICACY

nature of substrate finishes or coatings. Depending on the nature and degree of reactivity of
fiber surfaces, preservatives may become chemically or physically bound and their antimicro‑
bial activity may be reduced. This may also be the case with certain finishes, coatings and other
substrate surface treatments that can react with preservatives.
2. Add-Ons
An add-on is the formulation applied to a nonwoven substrate. The add-on can be of varied
composition and may be in the form of a liquid, lotion, emulsion, powder, cream, ointment,
oil or other material. Although preservation efficacy demonstrated for the add-on may provide
17

useful information, it may not be predictive of the preservation efficacy of the final nonwoven
substrate product. Substrate and packaging may also influence preservation efficacy. The ratio
of the weight of the liquor to the weight of the substrate is a critical factor in the performance
of the preservative. Ratios can vary from 5:1 to 1:1 depending on product. The lower the ratio,
the more preservative required, especially against mold. If the liquor ratio is changed, then a
new test is required.
3. Packaging
The packaging size and type, e.g., tubs, canisters, soft packages, single pack, etc., should be tak‑
en into consideration in developing the most appropriate protocol for the preservation efficacy
testing of the final product. How the product changes over time may be dependent on the type
of package chosen, e.g., evaporation of the add-on through the package or adsorption of the
add-on to the package can occur, potentially affecting preservative stability.

B. Intended Use and Delivery of Product

Intended use and delivery of the product may influence the test procedure. The type of packag‑
ing, e.g., open tub or pack, and the number of wipes in the package may influence the number

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 PRESERVATION EFFICACY IN NONWOVEN SUBSTRATE PERSONAL CARE PRODUCTS SECTION 17

of inoculations in the test method. The length of the test should be representative of packaging
design. The selection of challenge organisms ideally reflects the final use of the product. For exam‑
ple, challenge organisms for baby wipes (coliforms) may differ from those for an eye area product
(Pseudomonads), (See Section 16 – Microbiological Risk Factor Assessment of Atypical Personal
Care Products).
End use and delivery of the product may determine acceptance criteria. For example, there may
be different acceptance criteria for single pack versus multi pack products.

C. Preservative Stability
It is recommended that preservative stability be evaluated in the finished product packaging be‑
cause of possible interactions of preservative, add-on, substrate, and package. The stability of the
preservative system in the add-on does not necessarily reflect its stability in the finished product.
It is recommended that accelerated aging studies on finished wipe products be confirmed with
real time studies. See the Personal Care Products Council Stability Testing Guidelines6 for more
detail.

17
PRODUCT TESTING

IN NONWOVEN SUBSTRATE
PRESERVATION EFFICACY
A. Preliminary Considerations
Due to the unique nature of non-woven products, molds are organisms of particular concern due
to their ability to degrade cellulose fibers and the exposure of the large surface area of the wipe
to the environment during manufacture and use. A high microbial load may reduce preservative
activity or cause preservative failure in the final product.
A sedimentation study7 to determine fluid migration through a vertical stack of wipes may be
useful information for some test protocols, for instance if the inoculum is delivered by filter car‑
rier. This data is useful in determining the distribution, or degree of sedimentation, of the add-on
within packages of saturated wipe products packaged in stacks.
A period of time for equilibration of the add-on and the substrate before testing is recommended.
This allows time for total saturation of the add-on and distribution of the preservative. An ana‑
lytical approach to the determination of equilibration may be considered.  For example, if one is
conducting the sedimentation study for fluid distribution, an analytical evaluation of preservative
distribution (either by direct measurement of the preservative or by measuring a surrogate analyte
which would mimic preservative migration) can be done in parallel. 
It is recommended that all aspects of product testing, such as organism recovery, neutralization,
inoculation, etc, be verified (See Section 9: Microbial Validation and Documentation) for meth‑
od suitability.

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 SECTION 17 PRESERVATION EFFICACY IN NONWOVEN SUBSTRATE PERSONAL CARE PRODUCTS

B. Organisms
Organism strains recognized by United States Pharmacopeia (USP)8 and/or used in the industry
for preservation efficacy testing are recommended. (See Section 10: Maintenance and Preserva‑
tion of Test Organisms and Section 22: M-5 Methods for Preservation Testing of Nonwoven
Substrate Personal Care Products – Table 22.1- Suggested Challenge Organisms) Additional ref‑
erence strains and/or organisms appropriate to the product, including spores, may be used where
deemed necessary.
Pure or mixed culture inocula may be used. However, if different microorganisms are pooled,
antagonism among microbes may occur or it may be difficult to differentiate between types of
survivors.(See Section 20 M-3- A Method for Preservation Efficacy Testing of Water-Miscible
Personal Care Products - 5.2.1 )
Some products may not require a full preservation efficacy test. For example, dry wipes, as defined
by water activity measurement, may require limited or alternative testing.

C. Inoculation Procedures
IN NONWOVEN SUBSTRATE

The choice of inoculation site, inoculum volume, and distribution of the inoculum onto the
PRESERVATION EFFICACY

product should be determined with the anticipated consumer use and packaging of the product
in mind. The procedure used should be representative of product use.
There are several aspects to consider when choosing an inoculation procedure:
1. Packaging
Although testing in the final product packaging is preferred, there are situations where it is
impractical or impossible to do so. In these cases, testing outside of the final package is an ac‑
ceptable alternative. If the same product is delivered in different packaging, i.e., tubs, canisters,
17

soft packages, etc., it is recommended that each package type be tested.

2. Inoculum Volume
It is recommended that a consistent inoculum volume be chosen to achieve a set organism level
at time zero. This volume is dependent on the method of inoculation (See Section 22: M-5
Methods for Preservation Efficacy Testing of Nonwoven Substrate Personal Care Products).
Keeping the inoculum volume to a minimum will avoid dilution of the add-on. Care should
be taken to ensure that the volume provides sufficient inoculum across the wipe.
3. Inoculation Site / Distribution
The nonwoven substrate can be inoculated using a variety of methods. It is important to verify
that the inoculum site, distribution, and inoculum recovery is appropriate to the final packag‑
ing and use.
4. Reinoculation
Reinoculation of the nonwovens during the preservation efficacy test may be influenced by
how the product is used by the consumer. If a reinoculation is performed, ensure that there are
adequate numbers of wipes to complete assays for the required test period.

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 PRESERVATION EFFICACY IN NONWOVEN SUBSTRATE PERSONAL CARE PRODUCTS SECTION 17

D. R ecovery Procedures
1. Neutralization Verification
It is recommended that neutralization studies be conducted with all challenge organisms used
in the test. See ASTM 1054-08 for details.9
2. Recovery Verification
The nonwoven substrate material is likely to entrap some microorganisms resulting in a less
than complete recovery of the inoculum. The level of recovery may change from product to
product, depending on the combination of substrate and add-on. In most cases, mechanical or
other action, is necessary to release microorganisms from the substrate (See Section 22: M-5
Methods for Preservation Efficacy Testing of Nonwoven substrate Personal Care Products).
Addition of a surfactant and / or multiple extractions from the same sample may be necessary
to optimize recovery. It is recommended that the interpretation of results take into account the
established recovery efficiency which is based on a time zero count.

17
RECOMMENDATIONS
It is recommended that a risk assessment be used to establish acceptance criteria for a specific prod‑

IN NONWOVEN SUBSTRATE
PRESERVATION EFFICACY
uct (See Section 16 – Microbiological Risk Factor Assessment of Atypical Cosmetic Products). The
risk assessment should reflect the final product and its intended use and may take into consideration
many factors including, but not limited to, the following:

• Test method and microorganisms

• Nature of the add-on
• Nature of the substrate
• Degree of saturation
• Degree of microbial recovery from the substrate
• Design and size of the packaging
• Intended use and target consumer
• Performance and history of similar products

Whatever the product, an effective preservative system for a personal care wipe should prevent pro‑
liferation of introduced microorganisms. The following criteria may be used as guidelines, but may
be modified based upon the method used and the risk assessment.

Vegetative Bacteria
There should be greater than 99.9% (3 log) reduction of vegetative bacteria by aerobic plate count,
quantitative spread plate or spiral plate methods within 14 days following each challenge and no
increase for the duration of the test period.

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 SECTION 17 PRESERVATION EFFICACY IN NONWOVEN SUBSTRATE PERSONAL CARE PRODUCTS

Yeast and Molds

There should be greater than 90% (1 log) reduction of yeasts and molds by aerobic plate count,
quantitative spread plate or spiral plate methods within 14 days following each challenge and no
increase for the duration of the test period.

Spore-Forming Bacteria
If spore-forming bacteria are used in challenge testing, there should be bacteriostatic activity against
spore-forming bacteria throughout the entire test period.

REFERENCES
1. Lochhead, Robert Y. 2006. Emerging Technologies for Cosmetic and Personal Care Wipes.
Cosmetics and Toiletries 121: 47-52.
2. See the INDA (International Nonwovens & Disposables Association) website at www.inda.org
IN NONWOVEN SUBSTRATE

for information on the nonwovens industry.

PRESERVATION EFFICACY

3. See the EDANA (European Disposables and Nonwovens Association) website at

www.edana.org for information on nonwovens and the European nonwovens industry.
4. Sutton, S. 1996. Neutralizer Evaluations as Control Experiments for Antimicrobial Efficacy
Tests. In Handbook of Disinfectants and Antiseptics (Ascenzi, J. M., ed.), Marcel Dekker, Inc., pp.
43-62.
5. McCarthy, Terrence J. 1984. Formulated Factors Affecting Activity of Preservatives. In Cosmetic
and Drug Preservation Principles and Practice. (Jon J. Kabara, ed.), Marcel Dekker, Inc., pp. 359-
17

388.
6. Nikitakis, J.M. (ed.) 2014. Guideline for Industry: The Stability Testing of Cosmetic Products,
Personal Care Products Council, Washington, DC, 2010.
7. Cremieux, A., S. Cupferman, and C. Lens. 2005. Method for the Evaluation of the Efficacy on
Antimicrobial Preservatives in Cosmetic Wet Wipes, International Journal of Cosmetic Science, 27:
223-236.
8. United States Pharmacopeia and the National Formulary (USP 39 – NF34). 2016.
Antimicrobial Effectiveness Testing,” U.S. Pharmacopeia, 1260 Twinbrook Parkway, Rockville,
MD, 2499-2500.
9. ASTM 1054-08, 2016. “Standard Test Methods for Evaluation of Antimicrobial Agents,”
Annual Book of ASTM Standards, Volume 11.05, ASTM.

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 SECTION 18

M-1
Determination of the
Microbial Content Of
Personal Care Products
1 Scope
1.1 This section is an acceptable plate count procedure for determining the microbial content of
personal care products. Microbial content testing may be performed on raw materials, bulk
products, in-process materials, and finished goods.

2. Applicable Documents
2.1 “M-2 Examination for and Indentification of Staphylococcus aureus, Escherichia coli and Pseu-
domonas aeruginosa).” (Section 19)
2.2 Establishing Microbiological Quality of Personal Care Products (Section 12)
2.3 Raw Materials Microbial Content (Section 11)
2.4 Microbial Validation and Documentation (Section 9)

18
3. Suggested Materials

THE MICROBIAL CONTENT

M-1 DETERMINATION OF
3.1 Media for the enumeration of bacteria or fungi
3.1.1 Media for the enumeration of bacteria or fungi*
Letheen Agar
Microbial Content Agar
Nutrient Agar
Standards Methods Agar with Lecithin and Polysorbate 80
Trypticase Soy Agar (Soybean-Casein Digest Agar)

* It must be demonstrated that the test method adequately inactivates antimicrobial substances present in the product. It is
recommended that a neutralizer be present in the diluent or agar or both.1

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 SECTION 18 M-1 DETERMINATION OF THE MICROBIAL CONTENT OF PERSONAL CARE PRODUCTS

Trypticase Soy Agar with Lecithin and Polysorbate 80

Tryptic Soy Agar (Soybean-Casein Digest Agar)
Tryptone Soya Agar or equivalent
Other media as needed
3.1.2 Media for the enumeration of fungi*
Mycophil Agar with low pH
Potato Dextrose Agar
Sabouraud Dextrose Agar or equivalent
Other media as needed
3.1.3 Media used as diluents.*
Acto Tryptone (1%)
Letheen Broth
Nutrient Broth
Trypticase Azolectin Tween Broth Base
D/E Neutralizing Media or equivalent
Other media or diluents as needed
3.1.3.1 Prepare dilution bottles containing 80 mL of diluent for water-immisci‑
ble products and 90 mL for water-miscible products.
3.1.3.2 Sterile wide-mouth dilution bottles containing 10 mL of Polysorbate 80.
3.2 Equipment
THE MICROBIAL CONTENT
M-1 DETERMINATION OF

3.2.1 Autoclave
3.2.2 Sterile Petri dishes, 15 × 100 mm
3.2.3 20 mL, 10 mL, and 1.0 mL sterile syringes and/or pipettes, spatulas and other
sampling devices
3.2.4 Water bath capable of maintaining a temperature range of 45°-50°C
3.2.5 Microbiological incubators at 20°-25°C and 30°-34°C
18

3.2.6 Colony counter

3.2.7 Compound light microscope with 1000X oil immersion lens
3.2.8 Stereo microscope

* It must be demonstrated that the test method adequately inactivates antimicrobial substances pres‑
ent in the product. It is recommended that a neutralizer be present in the diluent or agar or both.1

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 M-1 DETERMINATION OF THE MICROBIAL CONTENT OF PERSONAL CARE PRODUCTS SECTION 18

4. Procedure for Aerobic Plate Count

4.1 Use sterile materials, equipment and aseptic techniques.
4.2 For water-miscible products, transfer by means of a syringe, pipette or spatula 10mL or g
of the well-mixed product to a dilution bottle containing 90 mL of diluent (this is a 1:10
dilution). Mix or vortex product and diluent until homogeneous.
4.3 For water-immiscible products, transfer by means of a syringe, pipette or spatula 10 mL or g
of the well-mixed product to a dilution bottle containing 10 mL of Polysorbate 80. Disperse
the product within the Polysorbate 80 with a spatula. Volume to 100 mL with diluent (this
is a 1:10 dilution). Mix or vortex product and diluent until homogeneous.
4.4 The precise volume or weight of sample and diluent may be varied. A dilution ratio of 1:10
with a minimum sample size of 10 mL or g is recommended.
4.5 When the same agar is used for bacterial and fungal assays, dispense 1 mL of the dilution
into each of three Petri dishes and 0.1 mL into three additional Petri dishes (to give triplicate
plates of 1:10 and 1:100 dilutions). Add 15 to 20 mL melted agar medium kept at 44°-48°C
and rotate plates sufficiently to disperse the product. Allow the agar to solidify and invert
plates. Incubate one plate of each dilution as follows:
a) At 30°-35°C for a minimum of 2 days for the bacterial assay.
b) At 20°-25°C for a minimum of 5 days for the fungal assay.
c) In a refrigerator to prevent growth. Or: Dispense 1 mL of the dilution into two Petri
dishes and 0.1 mL into two additional Petri dishes (to give duplicate plates of 1:10 and
1:100 dilutions). Add melted agar medium (as above) and incubate one plate of each
dilution as follows:
(1) At 30°-35°C for a minimum of 48 hours followed by a minimum of 48 hours at 20°-
25°C.

18
(2) In a refrigerator to prevent growth.
A diagram of this dilution and plating scheme is shown in Figure 18-1

THE MICROBIAL CONTENT

M-1 DETERMINATION OF
4.6 When separate agars are used for bacterial and fungal assays, dispense 1 mL of the dilution
into each of four Petri dishes and 0.1 mL into four additional Petri dishes (to give quadru‑
plicate plates of 1:10 and 1:100 dilutions). Add 15-20 mL of agar medium for bacterial assay
kept at 44°-48°C to two plates of each dilution. Add 15-20 mL of agar medium for fungal
assay kept at 44°-48°C to two plates of each dilution. Rotate all plates sufficiently to disperse
the product. Allow the agar to solidify and invert the plates. Incubate one plate of each dilu‑
tion as follows:
a) Bacterial assay medium at 30°-35°C for a minimum of 2 days
b) Fungal assay medium at 20°-25°C for a minimum of 5 days
c) Remaining bacterial and fungal medium plates in a refrigerator to prevent growth
A diagram of this dilution and plating scheme is shown in Figure 18-2

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 SECTION 18 M-1 DETERMINATION OF THE MICROBIAL CONTENT OF PERSONAL CARE PRODUCTS

4.7 Testing in duplicate may be performed for increased accuracy. Wide disparity between du‑
plicate tests may invalidate results.
4.8 To ensure comparable results between different types of microorganisms (bacteria, yeasts,
and molds), plates should be incubated and read at times consistent between separate tests.
4.9 Include a laboratory control using apparatus, dilution blank (without product), media and
appropriate incubation. Concurrent contamination on test and control plates invalidates the
test. Find and eliminate the source of contamination. Repeat both control and product tests.
4.10 Count the colonies. If there is difficulty in distinguishing colonies from material, compare to
the refrigerated plates, or examine the colonies under a stereo microscope. (With experience,
the refrigerated plates can be eliminated from the procedure.)
4.10.1 General Counting Rules: if the number of colonies appears to exceed 250 CFU,
the plate may be sectioned for an estimated count. For plates where colonies are
too numerous and unable to be clearly distinguished, the plate may be marked as
“Too Numerous to Count” (TNTC).
4.11 The number of colony forming units (CFU) per mL or g is the colony count multiplied by
the appropriate dilution factor (10 or 100).
4.12 Neutralization of antimicrobial activity should be evaluated for each product tested.Car‑
ryover of antimicrobial activity from the product formulation into the plate count diluent
and recovery growth agar may occur. This may inhibit the growth of surviving challenge test
microorganisms, resulting in a false negative microbial count. To avoid a false negative result,
neutralization of the antimicrobial properties of the formulation must take place in the plate
count diluent and/or the recovery growth agar. Antimicrobial neutralization may normally
be accomplished by use of chemical neutralizing agents, physical dilution, or a combination
of both. Verification of neutralization is generally performed by inoculating the product di‑
lution with a low level of challenge microorganisms and performing the enumeration meth‑
od. Side-by-side dilutions with and without a product formulation are made. Enumeration
THE MICROBIAL CONTENT

of the microorganisms from these dilutions is performed. Neutralization is verified if micro‑

M-1 DETERMINATION OF

bial recoveries are similar. If one or more challenge microorganisms cannot be recovered, the
use of a higher dilution and/or the investigation of additional chemical neutralizers may be
considered.1-3
4.13 Morphologically suspect colonies can be further identified by the methods described in
“M-2 Examination for and Identification of Staphylococcus aureus, Escherichia coli, Pseudo-
monas aeruginosa, and Candida albicans (Section 19).”
18

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 M-1 DETERMINATION OF THE MICROBIAL CONTENT OF PERSONAL CARE PRODUCTS SECTION 18

REFERENCES
1. ASTM International 2013. ASTM E1054-08 Standard Practices for Evaluating Inactivators of
Antimicrobial Agents. ASTM International, West Conshohocken, PA.
2. U.S. Pharmacopeia & National Formulary. 2016. USP 39- NF 34. <61> “Microbial Limit
Tests.” U.S. Pharmacopeia, Rockville, MD. pp. 83-88
3. U.S. Pharmacopeia & National Formulary. 2016. USP 39- NF 34. <1227> Validation of
Microbial Recovery from Pharmacopeal Articles.” U.S. Pharmacopeia, Rockville, MD. pp. 684-
686.

18
THE MICROBIAL CONTENT
M-1 DETERMINATION OF

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 SECTION 18 M-1 DETERMINATION OF THE MICROBIAL CONTENT OF PERSONAL CARE PRODUCTS
Figure  18-­‐1.    Aerobic  Plate  Count  Using  the  Same  Agar  

  FIGURE 18-1. AEROBIC PLATE COUNT USING THE SAME AGAR

 

  Step  1:    1:10  Dilution  of  the  Product  

 

Step  2:    Plate  Dilutions  (1.0  mL  and  0.1mL)  

1.0  mL  

1.0  mL   1.0  mL   1.0  mL  

0.1  mL  

0.1  mL   0.1  mL   0.1  mL  

Step  3:    Incubate  Plates  

THE MICROBIAL CONTENT
M-1 DETERMINATION OF

1:10  Dilution   1:100  Dilution  

Bacteria   30  -­‐35°C  >  2  days   Bacteria  

18

Fungal   20  -­‐25°C  >  5  days   Fungal  

Control   Refrigerated  Plates   Control  

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 M-1 DETERMINATION OF THE MICROBIAL CONTENT OF PERSONAL CARE PRODUCTS SECTION 18

FIGURE
Figure  18-­‐2  18-2. AEROBIC
 Aerobic   Plate  Count  PLATE COUNT
Using  Separate   USING SEPARATE AGARS
Agars  

 
 
Step  1:    1:10  Dilution  of  the  Product  
 

Step  2:    Plate  Dilutions  (1.0  mL  and  0.1mL)  

1.0  mL  

1.0  mL   1.0  mL   1.0  mL   1.0  mL  

0.1  mL  

0.1  mL   0.1  mL   0.1  mL   0.1  mL  

Step  3:    Incubate  Plates  

1:10  Dilution   1:100  Dilution  

18
Bacteria   30  -­‐35°C  >  2  days   Bacteria  

THE MICROBIAL CONTENT

M-1 DETERMINATION OF
Fungal   20  -­‐25°C  >  5  days   Fungal  

Bacteria   Bacteria  
Control   Refrigerated  Plates   Control  

Fungal   Fungal  
Control   Refrigerated  Plates    
Control  

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 SECTION 19

M‑2
Examination for and
Identification of Staphylococcus
aureus, Escherichia coli,
Pseudomonas aeruginosa, and
Candida albicans

INTRODUCTION
For cosmetics and other personal care products, the identification of Staphylococcus aureus, Esche-
richia coli, Pseudomonas aeruginosa, and Candida albicans may be relevant because they may affect
product integrity and consumer safety. The methods described here represent current industry prac‑
tices for identification of these microorganisms. The identification of other microorganisms, such as
Burkholderia and Enterobacter species, may also be of interest.

1. Scope
1.1 The following document gives general guidelines for identifying Staphylococcus aureus, Esch-
erichia coli, Pseudomonas aeruginosa, and Candida albicans that may have been isolated from
raw materials and personal care products using M-1 (See Guidelines - M-1) or other enu‑
meration or detection methods. Alternative methods (e.g., ISO Cosmetic Microbiology
Standards (see Additional Information), biochemical and/or genotypic procedures) may be
substituted for these methods.

2. Suggested Materials:
2.1 General, Selective, and Differential Microbial Growth Media
19

Table 19-1 summarizes suitable media that may be used for presumptive identification of
these specified microorganisms. Procedures for use of these media in the identification of in‑
dividual types of microorganisms will be described in the following sections. Confirmation
MICROORGANISMS
M‑2 IDENTIFICATION OF

of the result of each of these tests by biochemical or other methods is recommended.

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 SECTION 19 M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS

2.2 Reagents
2.2.1 Cytochrome Oxidase Test. Use filter paper impregnated with 1% tetraethyl
phenylenediamine dihydrochloride (Kovacs’ oxidase reagent)
2.2.2 Materials for Coagulase Test. Mammalian plasma, preferably rabbit or horse, with
or without suitable additives.1
2.2.3 Materials for Latex Agglutination Test. Kits are available from numerous commer‑
cial sources. The exact composition of materials and reagents may vary by manu‑
facturer.
2.2.4 Gram Stain Kit containing:
2.2.4.1 Gram Crystal Violet Solution
2.2.4.2 Gram Iodine Solution
2.2.4.3 Gram Decolorizer Solution
2.2.4.4 Gram Safranin Solution
Kits are available from numerous commercial sources. The exact composition of
reagents may vary by manufacturer. Follow the manufacturer’s procedure for stain‑
ing.
2.3 Equipment
2.3.1 Water baths at 37°C, 42°C and 45.5°C
2.3.2 Ultraviolet light (488 nanometers)
2.3.3 Compound light microscope with 1000X magnification, oil immersion lens
2.3.4 Microbiological incubator(s) at temperature(s) appropriate for specific test(s)

3. Procedure
3.1 The following procedures should be performed only with isolated colonies. If microbial
growth is observed on Petri dishes from enumeration or detection procedures, representative
colonies should be streaked for isolation onto non-selective agar. Incubate plates at tempera‑
ture used in enumeration or detection procedure. Before use in presumptive identification
testing, it is recommended that batches of selective/differential agars should be tested for
their microbial growth promotion ability and appearance of the medium or colonies using
the microbial strains that have been recommended by the manufacturer. It is recommend‑
ed that positive and negative control test microorganisms be used in conjunction with the
unknown microbial isolate to verify the accuracy of biochemical tests. Schematic diagrams
M‑2 IDENTIFICATION OF

for the identification of Staphylococcus aureus, Escherichia coli, Pseudomonas aeurginosa and
Candida albicans are shown in Figures 19-1 through 19-5.
MICROORGANISMS

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 M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS SECTION 19

3.2 Gram Stain

3.2.1 Gram staining should be performed on colonies appearing after 18-24 hours incu‑
bation according to the Gram Stain supplier’s instructions. It is recommended that
a known Gram-positive cocci and a Gram-negative bacilli be used as a positive and
negative control. Furthermore, commercially prepared microscope slides contain‑
ing fixed gram positive cocci and Gram-negative bacilli can be used as controls if
18-24 hour cultures are not available.
3.2.1.1 Gram-positive bacteria have a bluish to purple color Gram stain reac‑
tion.
3.2.1.2 Gram-negative bacteria have a pink to red color Gram stain reaction.
3.3 Test for Staphylococcus aureus2-4
The main elements for presumptive identification are Gram positive cocci which produce
catalase, are coagulase positive and exhibit characteristic growth on selective and differential
media. On general purpose media (e.g., Soybean-Casein Digest Agar Medium or Nutrient
Agar), S. aureus growth appears as smooth colonies generally pigmented yellow.
Gram‑positive cocci should be tested for the presence of catalase. Catalase-positive organ‑
isms should then be tested using the latex agglutination test. If this test is not available, refer
to the following sections for determining coagulase activity.
3.3.1 Catalase
3.3.1.1 With a sterile plastic inoculating loop or wooden stick, transfer a small
amount of an 18-24 hour pure culture from a general purpose media to
the surface of a clean, dry glass microscope slide.
3.3.1.2 Add 1 or 2 drops of the 3% hydrogen peroxide solution onto a portion
of a Gram-positive cocci microbial colony on the glass microscope slide.
3.3.1.3 Immediate bubbling of gas is indicative of a positive catalase test result
for the presence of a Staphylococcus species.
The non-appearance of bubbles immediately is indicative of a catalase
negative test result (Gram-positive cocci species that are Catalase nega‑
tive are non Staphylococcus aureus).
Note: Some bacteria may possess enzymes other than catalase that can
decompose hydrogen peroxide. Therefore, the appearance of a few bub‑
bles forming after 20 or 30 seconds is not considered to be a positive test
result.
3.3.1.4 For a positive and negative catalase test control, known ATCC cultures
19

of Staphylococcus and Streptococcus species should be used when an un‑

known Gram-positive coccus is tested.
MICROORGANISMS
M‑2 IDENTIFICATION OF

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3.3.2 Coagulase
3.3.2.1 After 24 hours, transfer characteristic colonies (see Table 19-1) from the
surface of the medium to individual tubes each containing 0.5 mL of
coagulase test plasma. Simultaneously assay coagulase positive and nega‑
tive cultures. Incubate at 35.0 ± 2.0°C, examining the tubes for clots at 3
hours and at subsequent intervals up to 24 hours. If commercial kits are
used, manufacturers’ instructions should be followed. While most S. au-
reus are coagulase positive, the latex agglutination test should be used for
confirmation of Gram-positive, catalase-positive cocci.4,5 If the reactions
of the controls are not correct, the assay results are invalid.
3.3.2.2 For a positive and negative Coagulase test control, a known ATCC cul‑
ture of Staphylococcus aureus and Staphylococcus epidermidis should be
used when an unknown Gram-positive coccus is tested.
3.3.3 Latex Agglutination Test
3.3.3.1 A 18-24 hour Gram positive cocci isolate that is catalase positive on a
general purpose media should be used to perform the test.
3.3.3.2 Because of the availability of numerous commercial latex agglutination
test kits, see manufacturer directions in how to perform the test.
3.3.3.3 For positive and negative latex agglutination test controls, known ATCC
culture of Staphylococcus aureus and Staphylococcus epidermidis should be
used when an unknown Gram-positive, catalase positive coccus is tested
3.3.4 Selective/Differential Media
As an alternative to the above biochemical tests, Catalase-positive, Gram-positive
cocci isolates may be streaked onto one of the following selective/differential agars
(see Table 1) to gain further information for the presumptive identification of an
isolate as Staphylococcus aureus:
3.3.4.1 Vogel-Johnson Agar (VJA) – the presence of black microbial colonies
surrounded by a yellow zone.2
3.3.4.2 Mannitol Salt Agar (MSA) – the presence of yellow microbial colonies
with yellow zones.2
3.3.4.3 Baird Parker Agar (BPA) – the presence of black, shiny microbial colo‑
nies surrounded by clear zones.2
3.3.5 If desired, confirmatory identification of an isolate as Staphylococcus aureus may be
accomplished using a commercially available identification kit for Gram-positive
cocci.
M‑2 IDENTIFICATION OF
MICROORGANISMS

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3.4 Test for Escherichia coli

The main elements for presumptive identification are Gram negative rods which are cyto‑
chrome oxidase negative, ferment lactose, can grow in the presence of bile salts and exhibit
characteristic growth on selective or differential media. On general purpose media growth
usually appears as smooth, convex, moist grey colonies.
3.4.1 Cytochrome Oxidase Test
If a microbial isolate is determined to be a Gram-negative rod or bacillus, the
Cytochrome Oxidase Test is performed to differentiate Gram-negative bacilli into
the following 2 groups: non-fermentors and fermentors. Escherichia coli is a cyto‑
chrome oxidase-negative fermentative species.3
Add a few drops of Kovacs’ oxidase reagent to a strip of filter paper. Smear a loopful
of an 18 – 24 hour culture grown on non-selective media onto the reagent soaked
filter paper using a platinum or plastic loop. (Do not use a nichrome wire loop, as
it may cause a false positive.) A dark purple-black color that develops within 15
seconds is a positive reaction.2-3
Known ATCC cultures of Escherichia coli and Pseudomonas aeruginosa should be
used as positive and negative control microorganisms for the Cytochrome Oxidase
Test when an unknown Gram-negative bacillus is tested.
3.4.2 Selective/Differential Media
Streak isolates onto Eosin Methylene Blue (EMB) or MacConkey Agar Plates
(Mac) and incubate at 35.0 ± 2°C for 24 hours. Escherichia coli will appear as
pink to red colonies with bile precipitation on MacConkey agar or as blue-black
colonies with dark centers and a green metallic sheen under a transmitted light on
EMB Agar.2
3.4.3 If desired, confirmatory identification of an isolate as Escherichia coli may be ac‑
complished using a commercially-available identification kit for Gram-negative
bacilli.
3.5 Test for Pseudomonas aeruginosa.
The main elements for presumptive identification are Gram-negative rods which are cy‑
tochrome oxidase-positive, produce diffusible fluorescent blue-green pigments and exhibit
characteristic growth on selective or differential media.
3.5.1 Cytochrome Oxidase Test
If a microbial isolate is determined to be a Gram-negative rod or bacillus, the Cy‑
tochrome Oxidase Test is performed to differentiate Gram-negative bacilli into the
19

following 2 groups: non-fermentors and fermentors. Pseudomonas aeruginosa is a

cytochrome oxidase-positive non- fermentative species.3
MICROORGANISMS
M‑2 IDENTIFICATION OF

Add a few drops of Kovacs’ oxidase reagent to a strip of filter paper. Smear a loopful
of an 18 – 24 hour culture grown on non-selective media onto the reagent soaked
filter paper using a platinum or plastic loop. (Do not use a nichrome wire loop, as

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 SECTION 19 M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS

it may cause a false positive.) A dark purple-black color that develops within 15
seconds is a positive reaction.2,3
Known ATCC cultures of Escherichia coli and Pseudomonas aeruginosa should be
used as positive and negative control microorganisms for the Cytochrome Oxidase
Test when a unknown Gram-negative bacillus is tested.
3.5.2 Selective/Differential Media
Streak isolates onto Cetrimide or Pseudomonas Isolation Agar (PIA) plates and
incubate at 35.0 ± 2.0°C for 24 hours.
3.5.2.1 Note: After incubation, Pseudomonas aeruginosa colonies will appear as
yellow-green to blue microbial colonies on Cetrimide Agar and green to
blue-green microbial colonies on Pseudomonas Isolation Agar.2
3.5.3 If desired, confirmatory identification of an isolate as Pseudomonas aeruginosa may
be accomplished using a commercially available biochemical identification kit for
Gram-negative bacilli.
3.6 Test for Candida. albicans 6,7
The main elements for presumptive identification are yeasts which produce chlamydospores
on Corn Meal Agar and exhibit characteristic growth on selective and differential media. On
general purpose media, growth appears as white to beige creamy convex colonies.
3.6.1 Gram-positive, short ovoid or elongated cells, sometimes with buds should be
tested to determine the presence of Candida albicans. (Figure 19-4).
3.6.2.1 Chlamydospore production on Corn Meal Agar with 1% Polysorbate
806
3.6.2.1.1 Remove a small portion of the yeast colony with an inoculat‑
ing wire and streak-inoculate the surface of the medium across
the center of the plate. Place a sterile coverglass over the inoc‑
ulum streak. Incubate at 25.0 ± 2.5°C for up to 3 days.
3.6.2.1.2 Inspect plates daily by removing the dish lid and examine the
growth through the coverglass under the microscope with
magnification of 100 to 400X.
3.6.2.1.3 Candida albicans produces large, highly refractile, thick-walled
chlamydospore which may be seen terminally or on short lat‑
eral branches.2
3.6.2.2 Chromogenic Agar
3.6.2.2.1 Candida albicans Chromogenic agar is a specialized microbial
M‑2 IDENTIFICATION OF

growth agar which contains 1 or more chromogenic substrates

MICROORGANISMS

that can be converted to a pigment by a targeted enzyme(s)

that can be used differentiate Candida albicans from other
Candida species.

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3.6.2.2.1.1 There are several types of Candida albicans Chro‑

mogenic Agars that are commercially available.
3.6.2.2.2 Streak yeast isolate onto chromogenic agar and incubate aero‑
bically at 35.0 +2.0°C for 36 to 48 hours in an inverted posi‑
tion.
3.6.2.2.3 After incubation, examine the presence of microbial growth for
pigmentation that is characteristic for the growth of Candida
albicans from other yeast species.
3.6.2.3 BiGGY Agar
3.6.2.3.2 Streak yeast isolate onto BIGGY Agar and incubate at 30.0 +/-
2.0°C for 18-72 hours.
3.6.2.3.2 After incubation, examine growth for the presence of brown to
black colonies with no sheen and no diffusion of the pigment
into the medium.2
3.6.2.3.3 If desired, confirmatory identification of a yeast isolate as
Candida albicans may be accomplished by using a commercially
available biochemical identification kit for yeasts.

19
MICROORGANISMS
M‑2 IDENTIFICATION OF

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Table 19-1

SELECTIVE/DIFFERENTIAL MEDIA FOR STAPHYLOCOCCUS AUREUS,

ESCHERICHIA COLI, PSEUDOMONAS AERUGINOSA, AND CANDIDA
ALBICANS(2)
Selective/Differential Selective/Differential Selective/Differential Selective/Differential
Media for E. coli Media for S. aureus Media for P. aeruginosa Media for C. albicans
Eosin-Methylene Blue Vogel-Johnson Agar (VJA) Pseudomonas Isolation BiGGY Agar
(EMB) Agar plates plates Agar (PIA)
MacConkey Agar (Mac) Mannitol Salt Agar (MSA) Cetrimide Agar Chromogenic Agar

Baird-Parker Agar (BPA) Corn Meal Agar with 1%

Polysorbate 80

Other media may be substituted providing that their equivalence has been demonstrated.
M‑2 IDENTIFICATION OF
MICROORGANISMS

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REFERENCES
1. United States Pharmacopeia. 2016. <62> Microbial Examination of Nonsterile Products: Tests
for Specified Microorganisms. United States Pharmacopeia and the National Formulary. USP394-
NF34. Rockville, MD., 75-80.
2. Difco & BBL Manual. 2009. 2nd Edition. Divison of Becton Dickinson and Company, Sparks,
Maryland.
3. Tille, P.M. 2013. Bailey & Scott’s Diagnostic Microbiology, 13th Edition. Mosby, New York, New
York.
4. Essers, L., and Radebold, K., 1980. Rapid and Reliable Identification of Staphylococcus aureus
by a Latex Agglutination Test. J. Clin. Microbiol. 12: pp 641-643.
5. Roberts, J.I.S., and Gaston, M.A. 1987. Protein A and Coagulase Expression in Epidemic and
Non-epidemic Staphylococcus aureus. J. Clin. Pathol. 40: pp 837-840.
6. Kelly, J.P. and Fungiello, F. 1959. Candida albicans: A Study of Media Designed to Promote
Chlamydospore Production. J. Lab. & Clin.. Med., 53, pp 807-809.
7. Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L., Pfaller, M.A. 2015. Manual of Clinical
Microbiology 11th Edition, Volume 1; ASM Press.

ADDITIONAL INFORMATION
European Pharmacopeia 8th Edition, 2014. <2.6.13> Microbiological Examination of Non-sterile
products (Test for Specified Micro-organisms, Council of Europe, Strasbourg, France.
US Food and Drug Administration. 2016 Bacteriological Analytical Manual, http://www.fda.gov/
Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.
ISO 18415, Cosmetics - Microbiology — Detection of Specified and Non-specified
Microorganisms
ISO 18416 Cosmetics- Microbiology – Detection of Candida albicans
ISO 21148 Cosmetics – Microbiology General Instructions for Microbiological Examination
ISO 21150 Cosmetics – Microbiology - Detection of Escherichia coli
ISO 22717 Cosmetics – Microbiology - Detection of Pseudomonas aeruginosa
ISO 22718 Cosmetics – Microbiology - Detection of Staphylococcus aureus
19
MICROORGANISMS
M‑2 IDENTIFICATION OF

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 M‑2 IDENTIFICATION OF
19 MICROORGANISMS

Recovered
Microbial Isolate
SECTION 19

Streak onto a Non-Selective Microbial Growth Agar for obtaining 18-24

CANDIDA ALBICANS

hour Pure Microbial Colonies

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Gram Stain

Gram-positive bacilli
isolate Gram-positive cocci Gram-negative bacilli Yeast isolate
isolate isolate

No further additional See Figure 2 Cytochrome Oxidase See Figure 5

testing is required Test

Negative Positive
AUREUS, ESCHERICHIA COLI, PSEUDOMONAS AERUGINOSA AND

See Figure 3 See Figure 4

FIGURE 19-1 - IDENTIFICATION SCHEMATIC FOR STAPHYLOCOCCUS
M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS


AUREUS

Gram-positive cocci
isolate

Biochemical
Identification Path Presumptive Identification
Path

Streak onto one of the following

Catalase Test Staphylococcus aureus
Selective/Differential Microbial
Growth Agars
Positive Negative

Vogel-Johnson Mannitol Baird Parker

Coagulase Test/ Latex Non-Staphylococcus Agar (VJA) Salt Agar Agar (BPA)
Agglutination Test aureus isolate
S. aureus colonies S, aureus S. aureus
will appear as colonies will colonies will
Positive Negative black colonies appear as appear as
surrounded by a yellow black, shiny
yellow zone colonies
colonies
with yellow
M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS

Staphylococcus Non–Staphylococcus surrounded by

zones
aureus isolate aureus isolate clear zones
FIGURE 19-2 – IDENTIFICATION SCHEMATIC FOR STAPHYLOCOCCUS
SECTION 19

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MICROORGANISMS 19
M‑2 IDENTIFICATION OF
 M‑2 IDENTIFICATION OF
19 MICROORGANISMS

Cytochrome Oxidase
Negative , Gram-
SECTION 19

negative bacilli isolate

Biochemical Presumptive
Identification Path Identification Path

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Use a commercial Streak growth onto to one of the
Gram-negative bacilli following Escherichia coli
biochemical Selective/Differential Growth Agars
identification kit to
confirm identification

Eosin-Methylene MacConkey Agar

Blue Agar (EMB) (Mac)

E coli colonies will appear E. coli colonies will

as blue-black colonies with appear as pink to red
dark centers and a green colonies with bile
metallic sheen under precipitation
transmitted light.
FIGURE 19-3 – IDENTIFICATION SCHEMATIC FOR ESCHERICHIA COLI
M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS


AERUGINOSA

Cytochrome Oxidase Positive,

Gram-negative bacilli isolate

Biochemical Presumptive
Identification Path Identification Path

Use a commercial Streak growth onto to one of the

Gram-negative bacilli following Pseudomonas aeruginosa
biochemical Selective/Differential Growth Agars
identification kit to
confirm identification

Cetrimide Agar Pseudomonas Isolation

Agar (PIA)

Pseudomonas aeruginosa colonies

M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS

will appear as yellow, blue-green or

blue in color
FIGURE 19-4 – IDENTIFICATION SCHEMATIC FOR PSEUDOMONAS
SECTION 19

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MICROORGANISMS 19
M‑2 IDENTIFICATION OF
 M‑2 IDENTIFICATION OF
19 MICROORGANISMS

Gram-Stain
SECTION 19

Gram-positive, short-ovoid or elongated cells

Presumptive Identification Path

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Biochemical Identification Path

Streak onto one of the following

Selective/Differential Growth Agars

Use a commercial yeast biochemical

identification kit to confirm
identification Corn Meal Agar with Chromogenic BiGGY Agar
1% Polysorbate 80 Agar

Microscopic examination Candida albicans Candida albicans

for the presence of colonies will will appear as
chlamydospores is appear with a brown to black
indicative for pigment that can colonies with no
Candida albicans be used to sheen and no
distinguish from diffusion of
other yeasts. pigment into agar
FIGURE 19-5 – IDENTIFICATION SCHEMATIC FOR CANDIDA ALBICANS
M‑2 EXAMINATION FOR AND IDENTIFIC ATION OF MICROORGANISMS
 20
WATER MISCIBLE PRODUCTS
M-3 EFFICACY TESTING OF
SECTION 20

M-3
A Method for Preservation
Efficacy Testing of Water
Miscible Personal Care Products
1. Scope
1.1 This is an acceptable procedure for determining the preservative efficacy of water-miscible
personal care product formulations.1-5
1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents
2.1 “Determination of Preservation Efficacy in Water-Miscible Personal Care Products” (Section 13).

3. Materials
3.1 Selection of Challenge Microorganisms
The microbial strains listed in Table 20-1 may be considered for use in developing preserva‑
tion efficacy data for Personal Care products.
Either pure or mixed microbial culture suspensions may be used to challenge test formula‑
tions. Inocula consisting of only pure microbial cultures will yield specific data on each test
microorganism employed in the challenge study. When conducting mixed culture challenge
studies, it is recommended that closely related types of microorganisms such as Gram-posi‑
tive bacteria, Gram-negative bacteria, and yeasts and molds be pooled separately.
3.2 Maintenance of Challenge Microorganisms
Refer to Section 10 (Maintenance and Preservation of Test Organisms), ATCC culture
maintenance recommendations, available on their website (www.atcc.org), and to other
sources.6-8
Storage of other organisms relevant to the product in the original product or incorporation
of product into maintenance medium is often the only way to retain its unique characteris‑
tics. This method is especially appropriate where the isolate is subsequently inoculated into
a similar material.

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SECTION 20 M-3 EFFICACY TESTING OF WATER MISCIBLE PERSONAL CARE PRODUCTS

3.3 Test Media

3.3.1 Inocula Suspending Fluids
Suspending fluids are used to prepare the bacterial and fungal suspensions for in‑
oculating the test product. The following may be used:
20

• Phosphate Buffer (pH 7.0)

• 0.85% Sodium Chloride Solution (Normal Saline)
• Sodium Chloride Peptone Solution (1% peptone in normal saline)
Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate 80
or other surfactant to the suspending fluid is recommended to aid in dispersion of
mold spores.
3.3.2 Microbial Plate Count Diluents
Plating diluents serve to disperse the sample and dilute it to levels that permit re‑
covery of surviving microorganisms from an inoculated product formulation. The
choice of diluent depends on its ability to meet the requirements of preservative
neutralization (Section 4.1). The following are examples of diluents that may be
used:
• Buffered Sodium Chloride Peptone Solution
• Dey/Engley (D-E) Neutralizing Broth
• Eugon Broth
• Letheen Broth
• Modified Letheen Broth
• Phosphate Buffer, pH 7
• Soybean-Casein Digest Medium (Tryptic Soy Broth)
• Trypticase Azolectin™ Tween™ (TAT) Broth
• Saline-Tween-Lecithin Diluent
Other suitable diluents may also be used.
Addition of neutralizers may be necessary to demonstrate adequate preservative
neutralization. If neutralizing systems other than those listed above are used, ab‑
sence of neutralizer toxicity should be verified.
3.3.3 Recovery Agars
Many factors affect organism viability. Therefore, it is important for the agar to
provide optimum nutritional support for the recovery of the challenge organisms.
The following have been found suitable for preservation studies:

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M-3 EFFICACY TESTING OF
M-3 EFFICACY TESTING OF WATER MISCIBLE PERSONAL CARE PRODUCTS SECTION 20

• Eugon Agar
• Letheen Agar
• Microbial Content Agar
• Modified Letheen Agar
• Plate Count Agar
• Soybean Casein Digest Agar Medium (Tryptic Soy Agar)
• Microbial Content Agar with Tween
Addition of neutralizers may be necessary to demonstrate adequate preservative
neutralization. Other suitable agars and neutralizing agents may be used.
If the above agars do not support the growth of fungi, one of the following agars
may be considered:
• Malt Agar
• Malt Extract Agar
• Mycological Agar
• Potato Dextrose Agar
• Sabouraud Dextrose Agar
Other suitable agars and neutralizing agents may be used.

4. Preliminary Tests
4.1 Preservative Neutralization
Carryover of antimicrobial activity from the product formulation into the plate count dilu‑
ent and recovery growth agar may occur. This may inhibit the growth of surviving challenge
test microorganisms, resulting in a false negative microbial count. To avoid a false negative
result, neutralization of the antimicrobial properties of the formulation must take place in
the plate count diluent and/or the recovery growth agar.
Antimicrobial neutralization may normally be accomplished by use of chemical neutralizing
agents, physical dilution, or a combination of both.
Verification of neutralization is generally performed by inoculating the product dilution
with a low level of challenge microorganisms and performing the enumeration method Side-
by-side dilutions with and without a product formulation are made. Enumeration of the
microorganisms from these dilutions is performed. Neutralization is verified if microbial
recoveries are similar. If one or more challenge microorganisms cannot be recovered, the
use of a higher dilution and/or the investigation of additional chemical neutralizers may be
considered.9,10

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SECTION 20 M-3 EFFICACY TESTING OF WATER MISCIBLE PERSONAL CARE PRODUCTS

4.2 Microbial Content Test

It is recommended that that a microbial content test be performed on the test sample pri‑
or to performing the preservative efficacy test (See Section 18 and References 11 and 12).
Verification of neutralization of the antimicrobial properties of the test sample should be
20

demonstrated (See Section 4.1 on page 211 and References 9 and 10).

5. Inoculation Procedures
5.1 Preparation of Inocula
Freshly prepared cultures should be used for inoculating test samples.
In general, culture conditions in Table 20-2 should be considered when preparing the inoc‑
ula. Refer to the ATCC website (www.atcc.org) for optimal growth media and conditions
for specific microorganisms. Inclusion of cellulose degrading molds may necessitate longer
incubation periods and require a paper source for growth.
5.1.1 Preparation of Initial Bacteria and Yeast Suspensions
Broth cultures or cultures grown on solid agar media are acceptable for use. For
reference strains such as the ATCC strains, no more than five transfers from the
stock culture are recommended.12 Broth cultures should be centrifuged and then
re-suspended in the chosen suspending fluid. (See 3.3.1) Microbial growth on a
solid medium is transferred to the chosen suspending fluid.
5.1.2 Preparation of Initial Mold Suspensions
The mold inoculum is prepared by washing the sporulating agar culture with the
chosen suspending fluid (See 3.3.1) and filtering the spore suspension through
sterile gauze or glass wool. Sterile glass beads can be used as an aid in the dispersion
of spores in the suspending fluid.
5.1.3 Preparation of Bacterial Spore Suspensions
If spore-forming bacteria are to be included in the test, the inocula may be pre‑
pared as indicated in the AOAC Sporicidal Test.3 Some strains are commercially
available as prepared spore suspensions.
5.1.4 Preparation of Challenge Inocula
5.1.4.1 Inoculum levels
The recommended inoculation levels for challenge testing are:
• 1x106 Colony-Forming Units (CFU) of bacteria pergram of product
• 1x105 CFU of yeast per gram of product
• 1x105 CFU of mold spores per gram of product
The inoculum level for the challenge microorganisms should be verified
by standard microbiological techniques such as pour plate methods.

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5.2 Product Challenge

5.2.1 Pure and mixed-culture challenge
Either pure or mixed cultures may be used to challenge test formulations. Pure
culture challenge, although more time-consuming, will yield specific data on each
microorganism employed in the study. Mixed-culture challenge, on the other
hand, can be used to obtain rapid pass-or-fail decisions on preservative adequacy
and reduce the workload. However, antagonism among organisms may occur. It is
recommended that broadly related types of microorganisms such as Gram-positive
bacteria, Gram-negative bacteria, or molds be pooled separately when conducting
mixed-culture challenge.
All products should be thoroughly mixed manually or mechanically after inocula‑
tion to distribute the challenge microorganisms uniformly. It is recommended that
the volume of the inoculum be < 1% of the sample weight and should not alter the
character of the product being challenged. Challenged formulations should then
be stored at ambient temperature for the duration of the test.
5.3 Sampling the Challenged Product
5.3.1 Sampling interval
Challenged formulations should be sampled for viable microorganisms at selected
time intervals after inoculations. The frequency of sampling should follow a set
pattern to facilitate future comparison of test results between different product
formulations or samples, for example, weekly up to 28 days after inoculation.
5.3.2 Sampling and plating methods
The inoculated product should be thoroughly mixed just prior to sampling to ensure
that the sample is representative. In some cases, the inoculum can thrive in “pockets”
of growth in the formulation while other areas are relatively free of microorganisms.
Many aerobic microorganisms grow especially well at the formulation-air interface.
Often it is very difficult to break up the “pockets” of growth, and special procedures
are needed. The following mixing methods have been used to overcome this problem:
• Vigorous mixing with a stirring rod
• Capping and shaking vigorously by hand
• Mixing in a vortex mixer
• Mixing with a magnetic stirrer
• Mixing with a propeller stirrer
• Mixing with a non-aerating stirrer
• Mixing in a micro blender
• Mixing in a stomacher
• Gentle mixing in a tissue grinder

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Sample size will in part determine the minimum detectable level. A sample sizeo‑
fatleastonegramoronemilliliterofproductforthequantitativepourplatemethodis rec‑
ommended. Aseptic techniques must be employed.
5.3.2.1 Quantitative pour plate method
20

Serial dilutions are prepared from the aliquot recovered from the chal‑
lenged sample unit. Each serial dilution is thoroughly mixed, and an ali‑
quot is transferred to a Petri dish. Melted agar maintained at 44-48°C is
added to the Petri dish, and the dish is rotated to uniformly disperse the
product dilution. The agar plates are allowed to solidify, then inverted
and incubated under conditions appropriate for the test microorganisms
(see Table 20-2).
After incubation, the number of microbial colonies is counted, and the
resulting figure is multiplied by the appropriate dilution factor to obtain
the number of microorganisms per sample unit.
5.3.2.2 Quantitative spread plate method
The quantitative spread plate method is performed in a manner similar
to the pour plate method; however, an aliquot of each dilution is trans‑
ferred directly onto the surface of solidified microbial growth agar. The
sample aliquot is then evenly spread over the agar surface. The agar plates
are allowed to dry, then inverted and incubated under conditions appro‑
priate for the test microorganisms (see Table 20-2).
After incubation, the number of microbial colonies is counted, and the
resulting figure is multiplied by the appropriate dilution factor to obtain
the number of microorganisms per sample unit.
5.2.3.3 Quantitative Spiral Plate Method.
The quantitative spiral plate method is performed in a similar manner
to spread plate method; however, an aliquot of the test solution is trans‑
ferred to a spiral plate machine and it is diluted onto an agar plate in
a spiral pattern. The agar plates are allowed to dry, then inverted and
incubated under conditions appropriate for the test organisms.

6. Other Considerations
6.1 Length of Test Procedure
It is recommended that preservation tests be carried out for a minimum of 28 days. In some
cases, numbers of challenge microorganisms may be reduced below detectable levels during
the early stages of the test only to adapt to the preservative system and later proliferate. A
final judgment of preservative adequacy should not be made until all the data are obtained.

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WATER MISCIBLE PRODUCTS
M-3 EFFICACY TESTING OF
M-3 EFFICACY TESTING OF WATER MISCIBLE PERSONAL CARE PRODUCTS SECTION 20

6.2 Rechallenge
Consideration may be given to rechallenge. A rechallenge is useful for determining if a
formulation is marginally preserved and identifying which types of microorganisms may be
potential problems for that particular formulation.

Table 20-1

TABLE 20-1: SUGGESTED CHALLENGE MICROORGANISMS

Type Microorganism (ATCC5 Number) Recommendation
Gram-Positive Cocci Staphylococcus aureus (6538)* Select at least one
Staphylococcus epidermidis (12228)
Fermentative Klebsiella pneumoniae (10031) Select at least one
Gram-Negative Bacilli Enterobacter cloacae (13047))
Escherichia coli (8739)*
Enterobacter gergoviae (33028)
Non-Fermentative Pseudomonas aeruginosa (9027)* Select at least one
Gram-Negative Bacilli Burkholderia cepacia (25416)
Pseudomonas fluorescens (13525)
Pseudomonas putida (31483)
Yeasts Candida albicans (10231)* Recommended
Molds Aspergillus brasiliensis (16404)* Select at least one
Penicillium species
Spore-Forming Bacilli Bacillus subtilis (6051) Optional
Other Other organisms relevant to product Optional
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans
(10231) and Aspergillus brasiliensis (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial
Effectiveness Testing method.4

Table 20-2

TABLE 20-2: CULTURE CONDITIONS FOR PREPARATION OF INOCULA

Cultures Media Temperature Time
Bacteria Soybean Casein Digest (Tryptic Soy) Broth/Agar Medium 30-37°C 18-48 hours
Nutrient Broth/Agar
Eugon Agar/Broth
Other Suitable media
Yeasts Sabouraud Dextrose Agar 25-35°C 24-48 hours
Soybean Casein Digest (Tryptic Soy) Broth/Agar Medium
Mycophil (Mycological) Broth/Agar
Other Suitable media
Molds Sabouraud Dextrose Agar 20-30°C 7-28 days
Potato Dextrose Agar
Mycophil (Mycological) Agar
Malt Extract Agar
Other Suitable media

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SECTION 20 M-3 EFFICACY TESTING OF WATER MISCIBLE PERSONAL CARE PRODUCTS

REFERENCES
1. AOAC International 2007. AOAC 998.10-2009, Efficacy of preservation of non-eye area water-
miscible cosmetic and toiletry formulations. AOAC International, Rockville, MD
2. ASTM International 2012. ASTM E640-06 (2012) Standard Test Method for Preservatives in
20

Water-Containing Cosmetics. ASTM International, West Consohocken, PA

3. AOAC International. 2013. AOAC Method 966.04 Sporicidal Activity of Disinfectants. AOAC
International, Rockville, MD
4. U.S. Parmacopeia & National Formulary. 2016. USP 39- NF 34. <51> Antimicrobial
Effectiveness Testing, USP-NF, Rockville, MD,pp. 67-69.
5. ISO 2012. Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a
cosmetic product, ISO, Geneval, Switzerland, www.iso.org)
6. Brown, M.R. and Gilbert, P. (Eds.) 1995. Microbiological Quality Assurance: A Guide Towards
Relevance and Reproducibility of Inocula, CRC Press, Boca Raton, FL.
7. Kirsop, B.E. and Doyle, A. (Editors) Maintenance of Microorganisms and Cultured Cells, Second
Edition, Academic Press, New York, NY, 1991.
8. Reichgott, M. 2003. “Reference Strains: How Many Passages Are Too Many?,” ATCC
Connection 23, available at: https://www.atcc.org/~/media/PDFs/Technical%20Bulletins/tb06.
ashx
9. U.S. Parmacopeia & National Formulary. 2016. USP 39- NF 34. <1227> Validation of
Microbial Recovery from Pharmacopeal Articles.” U.S. Pharmacopeia, Rockville, MD.pp. 684-
686
10. ASTM International 2013. ASTM E1054-08 Standard Practices for Evaluating Inactivators of
Antimicrobial Agents. ASTM International, West Conshohocken, PA.
11. U.S. Pharmacopeia & National Formulary. 2016. USP 39- NF 34. <61> “Microbial Limit
Tests.” U.S. Pharmacopeia, Rockville, MD. pp. 83-88
12. ISO 2014. ISO 17516 Cosmetics — Microbiology — Evaluation of the antimicrobial
protection of a cosmetic product. ISO. Geneva, Switzerland. www.iso.org

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 SECTION 21

M-4
Method for Preservation
Efficacy Testing of Eye Area

21
Personal Care Products

OF EYE AREA PRODUCTS

M-4 EFFICACY TESTING
1. Scope
1.1 This is an acceptable procedure for determining the preservative on efficacy of eye area per‑
sonal care products.1,2
1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents
2.1 “Preservation Efficacy Testing of Eye Area Personal Care Products” (Section 14).

3. Materials
3.1 Selection of Challenge Microorganisms
The types of microorganisms shown in Table 21-1 should be given consideration in develop‑
ing preservation data. Additional microorganisms may also be included in the test procedure
if preservation problems may be encountered with such microorganisms.
3.2 Maintenance of Challenge Microorganisms
See also Section 10 (Maintenance and Preservation of Test Organisms) and Reference 3.
Table 21-2 shows conditions recommended for culture maintenance. Alternatively, cultures
may be freeze-dried, frozen or grown on a slant and overlaid with sterile mineral oil. Al‑
though initially more time-consuming, these methods eliminate the necessity of frequent
transfers and help ensure better culture stability. The viability of cultures must be checked
regularly.
Other isolates may present unique maintenance problems. Storage in the original product or
in corporation of product in the maintenance medium may be necessary to retain viability
and continued resistance. This method is especially appropriate where the isolate is subse‑
quently inoculated into a similar product.

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3.3 Test Media

3.3.1 Plating diluents
Plating diluents serve to disperse the sample and dilute it to levels that permit bet‑
ter recovery of the microbial population of a challenged formulation. Ideally, the
diluent should contain both neutralizing agents and a biologically inert surface-ac‑
tive agent.
OF EYE AREA PRODUCTS

The following diluents have been found suitable for preservation studies:
M-4 EFFICACY TESTING

• Letheen Broth
• Thioglycolate Broth
• TAT Broth
• Dey/Engley (D-E) Neutralizing Broth
Other diluents may also be used
21

The addition of lecithin and an appropriate polysorbate to commercially available

dehydrated broth formulations is also acceptable.
3.3.2 Recovery media
It is important that the recovery medium provide adequate nutritional support for
the growth of damaged cells. It is recommended that neutralizing agents be incor‑
porated into the agar to counteract preservative carry-over from the diluent to the
recovery medium. Letheen agar is an example of a commercially available medi‑
um containing neutralizers. It can be used in the recovery of bacteria, yeasts and
molds. D-E Medium (DIFCO) is useful when the preservative system is unknown
or when several different types of preservatives are present. In most cases, the ad‑
dition of lecithin and an appropriate polysorbate to any nutritionally adequate
growth medium for bacteria, yeasts or molds is sufficient to achieve preservative
neutralization.
3.3.3 Evaluating preservative neutralization4
The presence of active preservatives carried over from the challenged formulation
into the plating diluent and recovery medium may inhibit viable organisms and
result in false-negative readings. Neutralizing agents should be incorporated into
the plating diluent and/or recovery medium in order to inactivate preservatives
and permit accurate enumeration of the microbial content. Methods to evaluate
neutralizer effectiveness are as follows:
If growth is not obtained on the dilution plates after incubation, inoculate the
surface of the 10-1 and 10-2 plates with approximately 100 CFU of a mixed culture
of Gram-positive bacteria. Perform the same procedure with a mixed culture of
Gram-negative bacteria, a mixed culture of yeasts and a mixed culture of molds.
If growth is not apparent on any one of the streaks after incubation, neutraliza‑
tion of the preservative system is inadequate and an appropriate neutralizer must

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 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS SECTION 21

be found. Where neutralizers are not available or effective, physical dilution or

membrane filtration to recover surviving microorganisms from the sample may be
necessary.

4. Procedure
4.1 Aseptic technique should be practiced, and all test materials and media should be sterile.

21
4.2 Aqueous Liquid and Semi-liquid Eye Products

OF EYE AREA PRODUCTS

M-4 EFFICACY TESTING
4.2.1 Inoculum preparation
Fresh cultures should be used for challenging preservative systems. Either broth
cultures or cultures grown on solid media are recommended. After two or three
consecutive daily transfers, bacterial and yeast cultures may be used to challenge
the product. If log-phase cells are desired, an incubation period of 18-24 hours is
usually adequate. Broth cultures may be used directly or centrifuged and resus‑
pended in phosphate buffer (pH 7.0) or 0.85% saline. Growth on a solid medium
is transferred to phosphate buffer prior to use.
The mold inoculum is prepared by washing the 7- to 14-day-old agar slants with
phosphate buffer or 0.85% saline and filtering the spore suspension through gauze.
Low concentrations of polysorbates (e.g., 0.05% polysorbate 80) can be added to
the saline to aid in spore dispersal. In addition, harvesting spore suspensions with
glass beads in the saline aids in their dispersal.
In-house isolates should be inoculated into the test formulation directly from con‑
taminated product. If this is not feasible, in-house isolates may be cultured in
broth or on solid media as described above.
Table 21-3 shows conditions that should be considered when preparing the inoc‑
ulum:
If spore-forming bacteria are to be employed, inocula can be prepared as indicated
in the AOAC sporicidal test.5
4.2.2 Sample Preparation
The amount of product required to perform a preservation study should be a
minimum of 20 grams for each test microorganism or pool of microorganisms.
Sufficient product is needed to sample at each evaluation time and to have some
material remaining in the event that additional platings are required. When rechal‑
lenges are used, it will be necessary to increase the amount of product to be tested.
During the early developmental stage, a formulation may be challenged in glass
containers. Subsequent preservation tests should be conducted on product in the
final package to ensure its compatibility with the preservative system. It is recom‑
mended that all formulations be examined for microbial content prior to initiating
preservation studies.

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 SECTION 21 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS

4.2.3 Product challenge

4.2.3.1 Pure and mixed-culture challenge
Either pure or mixed cultures may be used to challenge test formula‑
tions. Pure-culture challenge, although more time consuming, will yield
specific data on each microorganism employed in the study. Mixed-cul‑
ture challenge, on the other hand, can be used to obtain rapid pass-or-
fail decisions on preservative adequacy and reduce the workload. How‑
OF EYE AREA PRODUCTS
M-4 EFFICACY TESTING

ever, antagonism among organisms may occur. It is recommended that

closely related types of microorganisms such as Gram-positive bacteria,
Gram-negative bacteria, or yeasts and molds be pooled separately when
conducting mixed culture challenge.
4.2.3.2 Inoculum levels
A reasonable challenge should be larger than the total challenge expected
during consumer use. On this basis, the following levels are recommend‑
21

ed:
• 1x106 CFU of bacteria per gram of product
• 1x105 CFU of yeast per gram of product
• 1x105 CFU of mold spores per gram of product
Other levels may be used as needed
After inoculation, all products should be thoroughly mixed manually or
mechanically to uniformly distribute the challenge microorganisms. The
volume of the inoculum should not alter the character of the product
being challenged. Challenged formulations should then be incubated at
controlled room temperature under appropriate conditions of humidity
for the duration of the test.
4.2.4 Sampling the challenged product
4.2.4.1 Sampling intervals
Challenged formulations should be sampled for viable micro‑
organisms at selected time intervals after inoculations. The fre‑
quency of sampling should follow a set pattern to facilitate
future comparison of results. Sampling is recommended im‑
mediately after inoculation (0 hour) and at 1-3, 7, 14, 21 and
28 days. When rechallenged, sampling should be once a week
thereafter for at least 3 weeks.
4.2.4.2 Sampling methods
The inoculated product should be thoroughly mixed just pri‑
or to sampling to ensure that the sample is representative. In
some cases, the inoculum can thrive in “pockets” of growth in

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 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS SECTION 21

the formulation while other areas are relatively free of microor‑

ganisms. Manyaerobic microorganisms grow especially well at
the formulation-air interface. Often it is very difficult to break
up the “pockets” of growth, and special procedures are needed.
The following mixing methods have been used to overcome
this problem:
• Vigorous mixing with a stirring rod

21
• Capping and shaking vigorously by hand

OF EYE AREA PRODUCTS

M-4 EFFICACY TESTING
• Mixing in a vortex mixer
• Mixing with a magnetic stirrer
• Mixing with a propeller stirrer
• Mixing with a non-aerating stirrer
• Mixing in a micro blender
• Gentle mixing in a tissue grinder
• Mixing in a stomacher
Sample size will in part determine the minimum detectable
level. A sample size of one-gram or one-milliliter of product
for the quantitative pour plate method is recommended.
4.2.4.3 Plating (enumeration) methods
Quantitative pour plate method
Serial tenfold dilutions are prepared from the one-gram or
one-milliliter aliquot withdrawn from the challenged formu‑
lation. Each dilution is thoroughly mixed, and one milliliter
from each dilution is transferred by pipette to a Petri dish.
Melted agar maintained at 43-46°C is added to the Petri dish,
and the dish is rotated to uniformly disperse the product. So‑
lidified plates for bacteria and yeast are incubated at 30-37°C
for 48-72 hours. Mold plates are incubated at 20-25°C for 5-7
days. After incubation, the colonies are counted, and the re‑
sulting figure is multiplied by the dilution factor to obtain the
number of microorganisms per gram or milliliter of product.
Quantitative spread plate method
The quantitative spread plate method is performed in a manner
similar to the pour plate method; however, 0.1-0.2 ml of each
dilution is pipetted directly onto the surface of solidified agar.
The sample is then evenly spread over the surface of the agar
with a glass “hockey stick.” The plates are allowed to dry and
then incubated, and colonies are counted as described above.

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 SECTION 21 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS

4.3 Non-Aqueous Eye Personal Care Products

4.3.1 Inoculum preparation
Aqueous inoculum - See 4.2.1.
Emulsified aqueous inoculum - This is an aqueous inoculum emulsified with not
more than 1% of dispersing agent such as polysorbate, sorbitan oleate or glycerol.
OF EYE AREA PRODUCTS

Oil inoculum - Challenge cultures may be resuspended in light mineral oil.

M-4 EFFICACY TESTING

4.3.2 Inoculation methods

4.3.2.1 Mixing
See 4.2.4.2 above. A glass rod, tongue depressor, or mechanical mixer
may be necessary to uniformly disperse test microorganisms. Inoculated
product that has collected on the mixing device or on the container’s
inner surfaces or edges must be worked back into the sample to prevent
21

excessive loss of product.

4.3.2.2 Surface inoculation
Swabbing - A swab is dipped into an inoculum of known concentration
and swabbed across the entire product surface. Spreading - A known
volume of inoculum is pipetted onto the surface of the product and uni‑
formly spread using a glass rod or other instrument.
Dipping - The product in its container is dipped into an inoculum of
known concentration for a predetermined length of time.
Spraying - The product is sprayed with a suspension of inoculum using
an atomizer. Appropriate safety precautions should be taken.
4.3.3 Product challenge
4.3.3.1 Oils and water-in-oil emulsions
Procedure
Prepare enough of the formulation to permit adequate sampling at each
test interval. At least 20 mL or 20 grams of the product should be chal‑
lenged with each test microorganism or mixture of test microorganisms.
Use containers that can be sealed to prevent excessive evaporation and
are large enough to allow for adequate mixing. The containers should
not react with the product.
Inoculum
The inoculum volume should be 0.1% to 1.0% of the sample volume
to keep the sample as water-free as possible. The inoculum may be an
aqueous or oil suspension added as a liquid or spray.

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 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS SECTION 21

4.3.3.2 Loose powders

Procedure
At least 20 grams of product should be challenged with each test organ‑
ism or mixture of test microorganisms. This sample size is usually large
enough to permit numerous samplings. Standardized containers that can
be loosely capped and are large enough to allow for adequate mixing

21
should be used. The containers should not react with the product.
Inoculum

OF EYE AREA PRODUCTS

M-4 EFFICACY TESTING
A fine spray or a liquid inoculum (volume 1% to 5% of the test sample)
should be added to the product and thoroughly mixed.
4.3.3.3 Pressed powders
Pressed powders treated as loose powders
Procedure
Pressed powders may be removed from containers, ground
(e.g., mortar and pestle) into fine particles, and processed as
described above. A minimum sample size of 20 grams should
be prepared for each challenge microorganism or pool of mi‑
croorganisms.
Inoculum
See 4.3.3.2.
Pressed powders in pans or cakes
Procedure
For pressed powders inoculated on the surface, a suitable num‑
ber of pans or cakes should be prepared for adequate sampling
for each sampling interval. It is suggested that each pan or cake
contain one gram of sample.
Inoculum
These samples are surface inoculated using any of the methods
under “Surface Inoculation” above.
4.3.3.4 Wax-based products
Bulk samples
Procedure
For bulk samples, a minimum size of 20 grams should be pre‑
pared for each challenge microorganism or pool of microor‑
ganisms. Briefly warming the bulk product to no more than
45°C may aid in the dispersion of the challenge inoculum.

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 SECTION 21 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS

Inoculum
An oil or emulsified aqueous inoculum is suggested. It may be applied as
a liquid or a spray and mixed as in “Mixing” above.
Pan, cake or stick samples
Procedure
OF EYE AREA PRODUCTS

For pans, cakes or sticks that are surface inoculated, see 4.3.3.3.
M-4 EFFICACY TESTING

Pressed powders in pans or cakes

Inoculum
See 4.3.2.2.
4.3.4 Sampling the challenged product
See 4.2.4.
21

Table 21-1

SUGGESTED CHALLENGE MICROORGANISMS

Type Microorganism (ATCC Number) Recommendation
In-house Isolates As appropriate one or more
Gram-Positive Cocci Staphylococcus aureus (6538) at least one
Staphylococcus epidermidis (12228)
Fermentative Klebsiella pneumonia (10031) at least two
Gram-Negative Rod Enterobacter cloacae (13047)
Escherichia coli (8739)
Enterobacter gergoviae (33028)
Non-Fermentative Pseudomonas aeruginosa (9027) at least one in addition to
Gram-Negative Rod Burkholderia cepacia (25416) P. aeruginosa
Pseudomonas fluorescens (13525)
Pseudomonas putida (12633)
Flavobacterium species
Acinetobacter species
Yeasts Candida albicans (10231) at least one
Candida parapsilosis (22019)
Molds Aspergillus brasiliensis (16404) at least one
Penicillium species
Spore-Forming Bacteria Bacillus subtilis (6051) optional

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 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS SECTION 21

Table 21-2

SUGGESTED CULTURE CONDITIONS

Cultures Maintenance Media Storage Conditions Transfer Frequency
Bacteria Nutrient Agar Refrigeration 4-8°C Weekly, Biweekly, or Monthly
Trypticase Soy Agar

21
Yeasts Trypticase Soy Agar Refrigeration 4-8°C Biweekly or Monthly
Potato Dextrose Agar
Mycophil (Mycological) Agar

OF EYE AREA PRODUCTS

M-4 EFFICACY TESTING
Molds Sabouraud Dextrose Agar Refrigeration 4-8°C Biweekly or Monthly
Potato Dextrose Agar
Mycophil (Mycological) Agar

Table 21-3

SUGGESTED INOCULUM CONDITIONS

Cultures Media Temperature Time
Bacteria Trypticase Soy Broth/Agar Medium* 30-37°C 18-48 hours
Nutrient Broth/Agar
Yeasts Trypticase Soy Broth/Agar Medium* 30-37°C 24-48 hours
Mycophil (Mycological) Broth/Agar
Molds Sabouraud Dextrose Agar 20-25°C 7-14 days
Potato Dextrose Agar
Mycophil (Mycological) Agar
* Soybean Casein Digest Medium

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 SECTION 21 M-4 PRESERVATION EFFICACY TESTING OF EYE AREA PERSONAL CARE PRODUCTS

ADDITIONAL INFORMATION
Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” Journal of the Society of Cosmetic
Chemistry 23:703-720. 1972.
Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” Journal of the Society of Cosmetic
Chemistry 18:797-807.
Wilson, L.A., Kuehne, J.W., Hall, S.W. and Ahearn, D.G. 1971. “Microbial Contamination in
OF EYE AREA PRODUCTS
M-4 EFFICACY TESTING

Ocular Cosmetics,” American Journal of Ophthalmology, 71(6):1298-1302.

REFERENCES
1. ASTM International. 2012. ASTM E640-06 (2012) Standard Test Method for Preservatives in
Water-Containing Cosmetics. ASTM International, West Consohocken, PA.
2. AOAC International. 2007. AOAC 998.10-2009, Efficacy of preservation of non-eye area
21

water-miscible cosmetic and toiletry formulations. AOAC International, Rockville, MD

3. Brown, M.R., and Gilbert, P. (Eds.) 1995., Microbiological Quality Assurance: A Guide Towards
Relevance and Reproducibility of Inocula, CRC Press, Boca Raton, FL.
4. ASTM International. 2013. ASTM E1054-08 Standard Practices for Evaluating Inactivators of
Antimicrobial Agents. ASTM International, West Conshohocken, PA.
5. AOAC International. 2013. AOAC Method 966.04 Sporicidal Activity of Disinfectants. AOAC
International, Rockville, MD

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 SECTION 22

M-5
Methods for Preservation
Testing of Nonwoven Substrate
Personal Care Products
1. Scope
1.1 These methods cover a variety of procedures currently used within the personal care products
industry to evaluate preservative efficacy of different types of nonwoven substrate, wipe, or
towelette products.

22
These methods apply to nonwoven substrate personal care products that contain an aque‑
ous-based add-on solution. For nonwoven personal care products containing non-aqueous

NONWOVEN SUBSTRATE
M-5 TESTING OF
add-on materials or concentrates, it is important that critical consideration be given to
the typical use of the finished product and risk assessment and testing be completed as
detailed in “Microbiological Risk Factor Assessment of Atypical Personal Care Products”
(Section 16).
It is recommended that the method chosen reflect consideration of the manufacturing pro‑
cess, the type of packaging used, and the end use of the product.
1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents
2.1 “Determination of Preservative Adequacy in Nonwoven Substrate Products” (Section 17).

3. Materials
3.1 Selection of Challenge Microorganisms
The microbial strains listed in Table 22-1 may be considered for use in developing preserva‑
tion data of nonwoven substrate, wipe, or towelette products.
3.2 Maintenance of Challenge Microorganisms
See Section 10 (Maintenance and Preservation of Test Organisms)

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 SECTION 22 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS

3.3 Test Media

3.3.1 Inocula Suspending Fluids
Suspending fluids are used to prepare the bacterial and fungal suspensions for in‑
oculating the test product. The following may be used:
• Phosphate Buffer (pH 7.0)
• 0.85% Sodium Chloride Solution (Normal Saline)
• Sodium Chloride Peptone Solution (1% Peptone in Normal Saline)
Other suitable fluids may be used. To aid in dispersion of mold spores, adding 0.05%
– 0.1% polysorbate 80 or other surfactant to the suspending fluid is recommended.
3.3.2 Microbial Plate Count Diluents
Plating diluents serve to disperse the sample and dilute it to levels that permit re‑
covery of surviving microorganisms from an inoculated product formulation. The
choice of diluent depends on its ability to meet the requirements of preservative
neutralization (Under “Preliminary Tests” later in this section). The following are
examples of diluents that may be used:
NONWOVEN SUBSTRATE

• Buffered Sodium Chloride Peptone Broth

M-5 TESTING OF

• Dey/Engley (D-E) Neutralizing Broth

• Eugon Broth
• Letheen Broth
• Modified Letheen Broth
22

• Phosphate Buffer, pH 7
• Soybean Casein Digest Medium (Tryptic Soy Broth)
• Trypticase Azolectin™ Tween™ (TAT) Broth
• Saline-Tween-Lecithin Diluent
Other suitable diluents may be used.
The addition of neutralizers may be necessary to demonstrate adequate preserva‑
tive neutralization (See 4.2).
3.3.3 Recovery Agars
Many factors affect organism viability. Therefore, it is important for the agar to
provide optimum nutritional support for the recovery of the challenge organisms.
The following have been found suitable for preservation studies:
• Eugon Agar
• Letheen Agar

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 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS SECTION 22

• Microbial Content Agar

• Modified Letheen Agar
• Plate Count Agar
• Soybean Casein Digest Agar Medium (Tryptic Soy Agar)
• Microbial Content Agar with Tween
The following media are specifically recommended for the recovery of yeasts and
molds during preservation studies:
• Malt Agar
• Malt Extract Agar
• Mycological Agar
• Potato Dextrose Agar
• Sabouraud Dextrose Agar

22
Other suitable agars may be used. The addition of neutralizers may be necessary to
demonstrate adequate preservative neutralization.

NONWOVEN SUBSTRATE
M-5 TESTING OF
4. Preliminary Tests
4.1 Initial Count
It is recommended that all formulations be examined for microbial content prior to initia‑
tion of preservation studies.
4.2 Neutralization of Antimicrobial Activity
Carryover of antimicrobial activity from the product formulation into the plate count dilu‑
ent and recovery growth agar may occur. This may inhibit the growth of surviving challenge
test microorganisms resulting in a false negative microbial count. To avoid a false negative
result, neutralization of the antimicrobial properties of the formulation must take place in
the plate count diluent and/or the recovery growth agar.
Antimicrobial neutralization may normally be accomplished by the use of chemical neutral‑
izing agents, physical dilution, or a combination of both.
Verification of neutralization is generally performed by inoculating the product dilution
with a low level of challenge microorganisms and performing the enumeration method (See
Section 6.2). Side-by-side dilutions with and without a product formulation are made. Enu‑
meration of the microorganisms from these dilutions is performed. Neutralization is verified
if microbial recoveries are within 1 log. If one or more challenge microorganisms cannot be
recovered, the use of a higher dilution and/or the investigation of additional chemical neu‑
tralizers may be considered. Refer to ASTM E1054-081 or USP2 for additional details.
In some cases, low recovery of organisms may be due to poor recovery efficacy rather than
failure to neutralize the preservative system.
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 SECTION 22 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS

4.3 Recovery Efficiency

Recovery of microorganisms from the nonwoven substrate is a separate issue from antimi‑
crobial neutralization. The substrate may entrap the microorganisms resulting in incomplete
recovery of the microbial population by the use of conventional dilution and plating tech‑
niques. Therefore, additional techniques may be used to verify the consistent recovery of
microorganisms from the substrate material (Section 6.1.3).

5. Inoculation Procedures
5.1 Preparation of Inocula
Freshly prepared cultures should be used for inoculating test samples. In general, culture
conditions in Table 22-2 should be considered when preparing the inocula. Refer to the
ATCC Web site (www.atcc.org) for optimal growth media and conditions for specific micro‑
organisms. Inclusion of cellulose degrading molds may necessitate longer incubation periods
and require a paper source for growth.
5.1.1 Preparation of Initial Bacteria and Yeast Suspensions
NONWOVEN SUBSTRATE

Either broth cultures or cultures grown on solid agar media are acceptable for use.
For reference strains such as the ATCC strains, no more than five transfers from
the stock culture are recommended.3 Broth cultures should be centrifuged and
M-5 TESTING OF

then re-suspended in the chosen suspending fluid (See 3.3.1). Microbial growth
on a solid medium is transferred to the chosen suspending fluid.
5.1.2 Preparation of Initial Mold Suspensions
The mold inoculum is prepared by washing the sporulating agar culture with the
chosen suspending fluid (See 3.3.1) and filtering the spore suspension through
22

sterile gauze or glass wool. Sterile glass beads can be used as an aid in the dispersion
of spores in the suspending fluid.
5.1.3 Preparation of Bacterial Spore Suspensions
If spore-forming bacteria are to be included in the test, the inocula may be pre‑
pared as indicated in the AOAC Sporicidal Test.4 Some strains are commercially
available as prepared spore suspensions.
5.1.4 Preparation of Challenge Inocula
5.1.4.1 Inoculum Levels
The recommended inoculation levels for challenge testing are:
• 1x106 Colony-Forming Units (CFU) of bacteria per sampling unit
of product
• 1x105 CFU of yeast per sampling unit of product
• 1x105 CFU of mold spores per sampling unit of product

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 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS SECTION 22

The inoculum level for the challenge microorganisms should be verified

by standard microbiological techniques such as pour plate methods.
5.1.4.2 Culture Suspensions
Either pure or mixed microbial culture suspensions may be used to chal‑
lenge test formulations. Inocula consisting of only pure microbial cul‑
tures will yield specific data on each test microorganism employed in
the challenge study. When conducting mixed culture challenge studies,
it is recommended that closely related types of microorganisms such as
Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds
be pooled separately. These suspensions may be used directly for inocu‑
lation or dried onto filter carriers as described below.
5.1.4.3 Dried Inoculum on Filter Carriers
Dried inoculum carriers are prepared by filtering the culture suspensions
onto 13 mm 0.45 micron membrane filters (such as cellulose ester mem‑
branes) to achieve inoculum levels recommended after drying. The filters
are placed in a covered Petri dish and dried at 37°C for 20 to 30 minutes.

22
The number of viable microorganisms on dried carriers must be equivalent
to the recommended levels. If necessary, the volume of filtered culture sus‑

NONWOVEN SUBSTRATE
M-5 TESTING OF
pension may be increased to take into account mortality due to desiccation.
5.2 Sample Preparation
In addition to the quantity required for the test, it is recommended that extra sample units
be prepared in the event they are needed. An unpreserved control should be included if pos‑
sible. If product rechallenge is desired, sufficient sample units must be prepared prior to the
start of the test. The sample reporting unit used depends on the inoculation and sampling
method chosen. If applicable, determine and record the weight and/or the average area of
the nonwoven substrate product sample, e.g., 1 g or 1 cm2.
If possible, preservative challenge testing should be conducted on product in the final pack‑
age to ensure compatibility with the preservative system and to represent the marketed prod‑
uct. Where the product does not lend itself to testing in the container/package, other ap‑
proaches may be employed, as detailed below in Section 5.2.4.
5.2.1 Tubs
Aseptically open packages and inoculate the product as received according to the
inoculation procedure (Section 5.3). Reseal the packages and follow the sampling
procedure under Section 6.1.
5.2.2 Soft Packages
Aseptically open packages, and inoculate the product as received according to
the inoculation procedure (Section 5.3). Some inoculation techniques may allow
for the aseptic introduction of the inocula directly into the package. Reseal the
packages and follow the sampling procedure (Under “Sampling the Challenged
Product” in Section 6.1 below).

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 SECTION 22 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS

5.2.3 Canisters
Aseptically open canister, remove the roll, and inoculate the top sheets of the prod‑
uct according to an appropriate inoculation procedure (Section 5.3). Reinsert the
roll into the canister. Seal the canisters and follow the sampling procedure (Section
6.1).
5.2.4 Transferred Samples
Aseptically open packages and transfer an appropriate number of nonwoven sub‑
strate units to sterile, resealable containers for inoculation. Follow the inoculation
procedure (Section 5.3). Seal the containers and follow the sampling procedure
(Section 6.1).
5.3 Methods for Inoculation
Inoculation of nonwoven substrates can be accomplished in a variety of ways. Methods for
inoculating product are described below. In each case, verification of microorganism recov‑
ery (described below) is an important component of the method verification.
5.3.1 A specific volume of an inoculum suspension is delivered by pipette using a point
delivery over the sample unit in a predetermined pattern. (For example, place 0.1
NONWOVEN SUBSTRATE

ml in five different areas of the substrate such as the four corners and the center.)
After inoculation, the package is sealed. This inoculation method can be used to
M-5 TESTING OF

inoculate one or a series of multiple sample units in one package.

5.3.2 A specific volume of an inoculum suspension is delivered by multi-channel pipette
using a point delivery over the sample unit in a predetermined pattern. After in‑
oculation, the package is sealed. This inoculation method can be used to inoculate
one or a series of multiple sample units in one package.
22

5.3.3 A specific volume of an inoculum suspension is aseptically introduced onto the

substrate in a straight line down the center of the substrate sample. The inoculum
must be applied to the substrate so that uniform cross sections may be cut off of
the substrate(s) for sampling. After inoculation, the package is sealed. This inocu‑
lation method can be used to inoculate one or a series of multiple sample units in
one package.
5.3.4 By means of a syringe, a specific volume of an inoculum suspension is aseptical‑
ly introduced into the package containing a sample unit. After inoculation, the
package is sealed, and the inoculum is well mixed by massaging the package. This
inoculation method is quantitative for single unit soft packages and qualitative for
multiple unit soft packages. This method is not suitable for tubs or canisters.
5.3.5 In a Class 2 (or greater) Biological Safety Cabinet, the inoculum is sprayed evenly
over the entire surface of a pre-determined area of the substrate, (e.g., a 9 cm2 sam‑
ple in a 10 cm2 Petri dish), by using an airbrush or other suitable spraying device.
The substrate sample is sprayed for an appropriate time to deliver the target inocu‑
lum. The quantity of inoculum delivered must be calculated for the specific spray
device. The inoculated sample should be sealed in a plastic bag or other suitable
container to prevent drying.

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 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS SECTION 22

5.3.6 Dried inocula on membrane filter carriers are placed between two substrate layers
in the package. Placement of the filter may be determined by conducting a sedi‑
mentation study. After inoculation, the package is resealed.
5.4 Storage of Inoculated Samples
Challenged formulations can be stored at controlled or ambient temperature undercondi‑
tions of humidity considered appropriate for the final product packaging for the duration of
the test.

6. Recovery Procedures
6.1 Sampling the Challenged Product
6.1.1 Sampling Intervals
Challenged formulations should be sampled for viable microorganisms at selected
time intervals after inoculations. The frequency of sampling should follow a set
pattern to facilitate future comparison of test results between different product
formulations or samples, for example, weekly up to 28 days after inoculation.

22
6.1.2 Sampling Sites

NONWOVEN SUBSTRATE
M-5 TESTING OF
The method of sampling chosen will depend upon several factors including the
method of inoculation. Below are several sampling methods that may be used.
6.1.2.1 For most inoculation methods, the top nonwoven substrate in the stack
or the outer most nonwoven substrate in the roll may be sampled from a
product package.
6.1.2.2 For a product challenge method where multiple nonwoven substrates
in a product package are inoculated, the inoculated substrate units per
package should be sampled at the appropriate time.
6.1.2.3 For a product challenge method where an inoculated nonwoven sub‑
strate is aseptically transferred to a secondary package (See “Transferred
Samples” in Section 5.3), one product package per sampling interval
may be sampled.
6.1.2.4 For a product challenge method where the inoculum is evenly distribut‑
ed across the nonwoven substrate, a uniform cross section (e.g., 1 g) of
the substrate may be sampled at each sampling interval.
6.1.2.5 For a product challenge method using dried inocula, a membrane filter
carrier is sampled at each interval. Additionally, one or two substrates
above and one or two below the filter may be sampled separately at each
sampling interval to evaluate migration of organisms through the sam‑
ple.

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 SECTION 22 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS

6.1.3 Recovery Methods

Aseptically remove the inoculated product or membrane filter carrier from the
container(s) and thoroughly mix with the preservative neutralizing diluent. Care
must be taken to sample the areas of the product that have been inoculated.
Organisms may be recovered from the sample using the following processing tech‑
niques:
• Mixing with diluent and glass beads by means of a mechanical wrist shaker
or reciprocal shaker for a predetermined period.
• Mixing with diluent in a vortex mixer is recommended when sample sizes
are small, e.g., 1 g or less.
• Mixing in a Stomacher with a diluent, e.g., 1 to 2 minutes at medium speed.
Other methods that may be employed include manual shaking or the use of an
orbital mixer or a blender. The addition of glass beads may improve recovery of
microorganisms, although they are not recommended for use in plastic bags or
with a blender.
NONWOVEN SUBSTRATE

6.2 Enumeration Methods

6.2.1 Quantitative Pour Plate Method
M-5 TESTING OF

Serial dilutions are prepared from the aliquot recovered from the challenged sam‑
ple unit. Each serial dilution is thoroughly mixed and an aliquot is transferred to
a Petri dish. Melted agar maintained at 44-48°C is added to the Petri dish, and
the dish is rotated to uniformly disperse the product dilution. The agar plates are
allowed to solidify, then inverted and incubated under conditions appropriate for
the test microorganisms (see Table 22-2).
22

After incubation, the number of microbial colonies is counted and the resulting
figure is multiplied by the appropriate dilution factor to obtain the number of
microorganisms per sample unit.
6.2.2 Quantitative Spread Plate Method
The quantitative spread plate method is performed in a manner similar to the pour
plate method; however, an aliquot of each dilution is transferred directly onto the
surface of solidified microbial growth agar. The sample aliquot is then evenly spread
over the agar surface. The agar plates are allowed to dry, then inverted and incubated
under conditions appropriate for the test microorganisms (see Table 22-2).
After incubation, the number of microbial colonies is counted, and the resulting
figure is multiplied by the appropriate dilution factor to obtain the number of
microorganisms per sample unit.

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 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS SECTION 22

7. Reporting
Calculate and report the percent reduction of inoculum counts per substrate, per gram of prod‑
uct, or per unit area for each organism or organism pool.

Table 22-1

SUGGESTED CHALLENGE MICROORGANISMS

Type Microorganism (ATCC Numbers) Recommendation
Gram-Positive Cocci Staphylococcus aureus (6538)*
Staphylococcus epidermidis (12228)

22
Fermentative Gram- Klebsiella pneumoniae (10031) Select at least one
Negative Bacilli Enterobacter cloacae (13047)

NONWOVEN SUBSTRATE
M-5 TESTING OF
Escherichia coli (8739)*
Enterobacter gergoviae (33028)
Non-Fermentative Pseudomonas aeruginosa (9027)* Select at least one
Gram-Negative Bacilli Burkholderia cepacia (25416)
Pseudomonas fluorescens (13525)
Pseudomonas putida (31483)
Yeasts Candida albicans (10231)* Select at least one
Candida parapsilosis (22019)
Molds Aspergillus brasiliensis (16404)* Select at least one
Chaetomium globosum (6205)**
Trichoderma reesei (13631)**
Cladosporium oxysporum (76499)**
Penicillium species
Spore-Forming Bacilli Bacillus subtilis (6051) Optional
Other In-house isolates Optional
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans
(10231), and Aspergillus brasiliensis (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial
Effectiveness Testing Method.5
**Inclusion of cellulose degrading molds may require longer incubation periods and require a paper source for
growth.

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 SECTION 22 M-5 PRESERVATION TESTING OF NONWOVEN SUBSTRATE PRODUCTS

Table 22-2

CULTURE CONDITIONS FOR PREPARATION OF INOCULA

Cultures Media* Temperature Time
Bacteria Soybean Casein Digest (Tryptic Soy) Broth/Agar Medium 30-37°C 18-48 hours
Nutrient Broth/Agar
Yeasts Sabouraud Dextrose Agar 25-35°C 24-48 hours
Soybean Casein Digest (Tryptic Soy) Broth/Agar Medium
Mycophil (Mycological) Broth/Agar
Molds Sabouraud Dextrose Agar 20-30°C 7-28 days
Potato Dextrose Agar
Mycophil (Mycological) Agar
Malt Extract Agar
* Available in dehydrated forms from commercial sources.
NONWOVEN SUBSTRATE

REFERENCES
M-5 TESTING OF

1. ASTM International. 2013. ASTM E1054-08 Standard Practices for Evaluating Inactivators of
Antimicrobial Agents. ASTM International, West Conshohocken, PA.
2. U.S. Parmacopeia & National Formulary. 2016. USP 39- NF 34. <1227> Validation of
Microbial Recovery from Pharmacopeal Articles. USP-NF, Rockville, MD,pp. 684-686.
3. Reichgott, M. 2003. “Reference Strains: How Many Passages Are Too Many?,” ATCC
22

Connection 23, available at: https://www.atcc.org/~/media/PDFs/Technical%20Bulletins/

tb06.ashx
4. AOAC International. 2013. AOAC Method 966.04 Sporicidal Activity of Disinfectants. AOAC
International, Rockville, MD
5. U.S. Parmacopeia & National Formulary. 2016. USP 39 - NF 34. <51> Antimicrobial
Effectiveness Testing. USP-NF, Rockville, MD, pp. 79-81.

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 SECTION 23

M-6
A Method for Preservation
Testing of Atypical Personal
Care Products
1. Scope
1.1 This general method reflects a variety of approaches currently used within the cosmetics
industry and serves as an acceptable procedure for determining the preservative efficacy of
atypical personal care products, such as oils, powders, or other formulations that have low
water content and/or are not miscible with water. The recommended preservative challenge
test methods used for determining the preservative efficacy of aqueous-based products (See
References 1–3 and Section 20) may not be suitable for evaluating certain atypical prod‑
uct formulations. When testing and assessing preservative challenge test data for atypical
products, the following factors are important points to consider (See “Microbiological Risk
Factor Assessment of Atypical Personal Care Products” in Section 16):
• A test in which an aqueous-based inoculum is introduced into an hydrous product may
change the physical dynamics of the product and, therefore may not predict its microbial
stability.
• Most preservatives are water soluble. In emulsions, preservatives are used in the water
phase because contaminating microorganisms require water to proliferate.

23
• For an emulsion in which the external phase is water immiscible (emulsions in which

ATYPICAL PRODUCTS
M-6 TESTING OF
water is not the external or continuous phase) and an aqueous challenge inoculum is
used, the water-soluble preservatives may not be able to migrate into the aqueous phase.
In these cases, the preservatives may not be available to inhibit proliferation or have cidal
activity against each of the challenge microorganisms.
1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents
2.1 “Microbiological Risk Factor Assessment of Atypical Personal Care Products” (Section 16).
2.2 “Determination of Preservative Adequacy in Personal Care Product Formulations” (See
Section 13).

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 SECTION 23 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS

3. Materials
3.1 Selection of Challenge Microorganisms.
The microbial strains listed in Table 23-1 may be considered for use in developing preserva‑
tion data for personal care products.
Either pure or mixed microbial culture suspensions may be used to challenge test formula‑
tions. Inocula consisting of only pure microbial cultures will yield specific data on each test
microorganism employed in the challenge study. When conducting mixed culture challenge
studies, it is recommended that separate pools of closely related types of microorganisms such
as Gram-positive bacteria, Gram-negative bacteria, and yeasts and molds be maintained.
3.2 Maintenance of Challenge Microorganisms
Refer to Section 10 (Maintenance and Preservation of Test Organisms), the ATCC cul‑
ture maintenance recommendations, available on their website (www.atcc.org), and to other
sources.4,5
3.3 Test Media
3.3.1 Inocula Suspending Fluids
Suspending fluids are used to prepare the bacterial and fungal suspensions for in‑
oculating the test product. The following may be used:
• Phosphate Buffer (pH 7.0)
• 0.85% Sodium Chloride Solution (Normal Saline)
• Sodium Chloride Peptone Solution (1% peptone in normal saline)
Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate 80
or other surfactant to the suspending fluid is recommended to aid in dispersion of
mold spores.
3.3.2 Microbial Plate Count Diluents
ATYPICAL PRODUCTS

Plating diluents serve to disperse the sample and dilute it to levels that permit re‑
M-6 TESTING OF

covery of surviving microorganisms from an inoculated product formulation. The

choice of diluent depends on its ability to meet the requirements of preservative
neutralization (See Section 4.1 “Preservative Neutralization”). The following are
examples of diluents that may be used:
• Buffered Sodium Chloride Peptone Solution
23

• Dey/Engley (D-E) Neutralizing Broth

• Eugon Broth
• Letheen Broth
• Modified Letheen Broth
• Phosphate Buffer, pH 7

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 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS SECTION 23

• Soybean-Casein Digest Medium (Tryptic Soy Broth)

• Tryptone-Azolectin-Tween® (TAT) Broth
• Other suitable broth
Addition of neutralizers may be necessary to demonstrate adequate preservative
neutralization. Other suitable diluents may be used.
3.3.3 Recovery Agars
Many factors affect organism viability. Therefore, it is important for the agar to
provide optimum nutritional support for the recovery of the challenge organisms.
The following have been found suitable for preservation studies:
• Eugon Agar
• Letheen Agar
• Microbial Content Agar
• Modified Letheen Agar
• Plate Count Agar
• Soybean-Casein Digest Agar Medium (Tryptic Soy Agar)
The addition of neutralizers may be necessary to demonstrate adequate preser‑
vative neutralization. Other suitable agars may be used.
If the above agars do not support the growth of fungi, one of the following agars
may be considered:
• Malt Agar
• Malt Extract Agar

23
• Mycological Agar

ATYPICAL PRODUCTS
M-6 TESTING OF
• Potato Dextrose Agar
• Sabouraud Dextrose Agar
Other suitable agars may be used.

4. Preliminary Tests
4.1 Preservative Neutralization
Carryover of antimicrobial activity from the product formulation into the plate count dilu‑
ent and recovery growth agar may occur. This may inhibit the growth of surviving challenge
test microorganisms resulting in a false negative microbial count. To avoid a false negative
result, neutralization of the antimicrobial properties of the formulation must take place in
the plate count diluent and/or the recovery growth agar.

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 SECTION 23 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS

Antimicrobial neutralization may normally be accomplished by use of chemical neutralizing

agents, physical dilution, or a combination of both.
Verification of neutralization is generally performed by inoculating the product dilution
with a low level of challenge microorganisms and performing the enumeration method.6
Side-by-side dilutions with and without a product formulation are made. Enumeration
of the microorganisms from these dilutions is performed as described.6 Neutralization is
verified if microbial recoveries are similar. If one or more challenge microorganisms cannot
be recovered, the use of a higher dilution and/or the investigation of additional chemical
neutralizers may be considered.
4.2 Microbial Content Test
It is recommended that that a microbial content test (Section 18) be performed on the test
sample prior to performing the preservative efficacy test. Verification of neutralization of
the antimicrobial properties of the test sample should be demonstrated (See Section 4.1 and
Reference 7) at the same time as the microbial content test.

5. Inocula Preparation
5.1 Preparation of Inocula
Freshly prepared cultures should be used for inoculating test samples.
In general, culture conditions in Table 23-2 should be considered when preparing the inocula.
5.1.1 Preparation of Initial Bacteria and Yeast Suspensions
Either broth cultures or cultures grown on solid agar media are acceptable for use.
For reference strains such as the ATCC strains, no more than five transfers from
the stock culture are recommended.7
5.1.1.1 Aqueous Inoculum
Broth cultures should be centrifuged and then re-suspended in the cho‑
ATYPICAL PRODUCTS

sen suspending fluid. (See Section 20) Microbial growth on a solid me‑
M-6 TESTING OF

dium is transferred to the chosen suspending fluid.

5.1.1.2 Emulsified Aqueous Inoculum
An aqueous emulsified inoculum may be prepared by adding not more
than 1% of dispersing agent such as polysorbate, sorbitan oleate or glyc‑
erol to the aqueous inoculum.
23

5.1.1.3 Oil Inoculum

Challenge cultures may be resuspended in light mineral oil.
Note: If using this technique, the absence of inhibitory or toxic proper‑
ties of the dispersing agent or oil soluble carrier system should be verified
for each of the challenge organisms.

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 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS SECTION 23

5.1.2 Preparation of Initial Mold Suspensions

5.1.2.1 Aqueous Mold Inoculum
The mold inoculum is prepared by washing the sporulating
agar culture with the chosen suspending fluid and filtering the
spore suspension through sterile gauze or glass wool. Sterile
glass beads can be used as an aid in the dispersion of spores in
the suspending fluid.
5.1.2.2 Emulsified Aqueous Mold Inoculum
An aqueous emulsified inoculum may be prepared by adding
not more than 1% of dispersing agent such as polysorbate,
sorbitan oleate or glycerol to the aqueous inoculum.
5.1.2.3 Oil Inoculum
Challenge cultures may be resuspended in light mineral oil.
5.1.3 Preparation of Bacterial Spore Suspensions
If spore-forming bacteria are to be included in the test, the inocula may
be prepared as indicated in the AOAC Sporicidal Test.8 Some strains are
commercially available as prepared spore suspensions.
5.1.4 Inoculum Levels
Inoculum challenge levels ranging from 1 x 104 to 1 x 108 CFU per gram
or ml of product have been reported in the literature for preservative
system evaluation.10-12
For some atypical products, (e.g., anhydrous products), a reduction in
the challenge inoculum size to 103 to 104 Colony-Forming Units (CFU)

23
per gram or milliliter may be used instead of the inoculum concentration
of 105 to 106 CFU per gram or milliliter that is recommended in the
aqueous based challenge test methods.

ATYPICAL PRODUCTS
M-6 TESTING OF
Note: By reducing the inoculum size, it is easier to measure stasis or
quantify an increase in the microbial count.

6. Inoculation Procedures for Test Samples

Depending on the product form, the following carriers for inocula may be considered. The vol‑
ume of the inoculum should not alter the character of the product being tested.
• Aqueous inoculum (See Section 20)
• Emulsified aqueous inoculum
• Oil inoculum

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 SECTION 23 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS

6.1 Inoculum Dispersed into Product

6.1.1 Oils, water-in-oil emulsions, water in silicone and semisolid products (<20% wa‑
ter)
Procedure
Prepare enough of the formulation to permit adequate sampling at each test inter‑
val. At least 20 mL or 20 grams of the product should be challenged with each test
microorganism or mixture of test microorganisms.
Inoculum
The inoculum volume should be 0.1% to 1.0% of the sample volume in order to
keep the sample as water-free as possible. The inoculum may be an aqueous or oil
suspension.
6.1.2 Loose Powders
Procedure
At least 20 grams of product should be challenged with each test organism or
mixture of test microorganisms. This sample size is usually large enough to permit
numerous samplings.
Inoculum
The inoculum (volume 0.1% to 1% of the test sample) should be added to the
product and thoroughly mixed.
6.1.3 Pressed Powders
Procedure
Pressed powders can be inoculated on the surface or removed from containers,
ground (e.g., mortar and pestle) into fine particles, and processed.
ATYPICAL PRODUCTS

A minimum sample size of 20 grams should be prepared for each challenge micro‑
organism or pool of microorganisms.
M-6 TESTING OF

For wet dry pressed powders, up to 5% water may first be added to the product,
prior to inoculation.
6.1.4 Mixing
A glass rod, tongue depressor, or mechanical mixer may be necessary to uniformly
23

disperse test microorganisms. Inoculated product that has collected on the mixing
device or on the container’s inner surfaces or edges must be worked back into the
sample to prevent excessive loss of product (See Section 20).

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 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS SECTION 23

6.2 Surface inoculation

Swabbing
A swab is dipped into an inoculum of known concentration and swabbed across the entire
product surface.
Spreading
A known volume of inoculum is pipetted onto the surface of the product and uniformly
spread using a glass rod or other instrument.
Dipping
The product in its container is dipped into an inoculum of known concentration for a pre‑
determined length of time.
Spraying
The product is sprayed with a suspension of inoculum using an atomizer. Appropriate safety
precautions should be taken.
6.3 Wax based solid products
For solid atypical products, such as anhydrous sticks or pans, inoculation and sampling of
the product surface instead of the whole product more closely simulates potential consumer
contamination. This modification also maintains the physical product integrity. In these
types of products, the microorganisms are not able to penetrate into the interior and will
always be found on the outer-most layer of the product after consumer usage. Although this
type of product is not usually susceptible to microbial contamination, surface inoculation
may be used.
Note: If performing challenge testing on a solid anhydrous stick or powder product, inocu‑
late a sufficient number of samples to obtain a unique sample for each sampling time-point.

23
6.4 Storage of Inoculated Samples
Inoculated samples should be stored under ambient conditions

ATYPICAL PRODUCTS
M-6 TESTING OF
6.5 Sampling the Challenged Product
6.5.1 Sampling interval
Challenged formulations should be sampled for viable microorganisms at selected
time intervals after inoculations. The frequency of sampling should follow a set
pattern to facilitate future comparison of test results between different product
formulations. Sampling intervals should be based on microbial contamination risk
as demonstrated by product water activities and other microbiological related at‑
tributes.
• Water in oil and/or silicone emulsions
Three sampling points, such as 7, 14, 28 days

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 SECTION 23 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS

• Semisolid products (<20% water)

Three sampling points, such as 7, 14, 28 days
• Oil or silicone based products (anhydrous)
Three sampling points, such as 2,7,14,28 days.
• Loose, wet dry and pressed powders
Three sampling points, such as 2,7,14,28 days
• Wax based and other solid products
Three sampling points, such as 2,7,14,28 days.
6.5.2 Sampling and plating methods
The inoculated product should be thoroughly mixed just prior to sampling to en‑
sure that the sample is representative. For water-immiscible products (e.g., oils and
emulsions), a suitable solubilizing agent may be incorporated into the test diluent or
broth to make the sample aliquot miscible with water in order to recover microor‑
ganisms present in the test sample. For solid products, the surface may be sampled by
removing the top layer. For products where microorganisms would only be recovered
from the product surface (e.g., sticks, pressed powders, hot pour products in com‑
pacts), only the surface of the product sample should be tested. For these “atypical
products,” the following methods of recovery may be considered.
• A sterile moistened applicator may be used to sample the product surface, and
then streaked onto a Petri dish containing solid culture medium.
• The product may be sampled by a direct contact method using a contact plate
(a modified Petri dish containing a solid culture medium whose convex surface
extends above the carrier), paddles, or flexible film containing solid culture media.
In some cases, the inoculum can thrive in “pockets” of growth in the formulation
while other areas are relatively free of microorganisms. Many aerobic microorgan‑
isms grow especially well at the formulation-air interface. Often it is very difficult
to break up the “pockets” of growth, and special procedures are needed. The fol‑
ATYPICAL PRODUCTS

lowing mixing methods have been used to overcome this problem:

M-6 TESTING OF

• Hand mixing with a stirring rod

• Capping and shaking vigorously by hand
• Mixing in a vortex mixer
• Mixing with a magnetic stirrer
23

• Mixing with a propeller stirrer

• Mixing with a non-aerating stirrer
• Mixing in a micro blender
• Mixing in a stomacher

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 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS SECTION 23

Sample size will in part determine the minimum detectable level. A sample size of
at least one gram or one milliliter of product for the quantitative pour plate meth‑
od is recommended. Aseptic techniques must be employed.
6.5.2.1 Quantitative pour plate method
Serial dilutions are prepared from the aliquot recovered from the challenged sam‑
ple unit. Each serial dilution is thoroughly mixed, and an aliquot is transferred to
a Petri dish. Melted agar maintained at 45-48°C is added to the Petri dish, and
the dish is rotated to uniformly disperse the product dilution. The agar plates are
allowed to solidify, then inverted and incubated under conditions appropriate for
the test microorganisms (see Table 23-3).
After incubation, the number of microbial colonies is counted, and the resulting
figure is multiplied by the appropriate dilution factor to obtain the number of
CFU per gram or milliliter of sample.
6.5.2.2 Quantitative spread plate method
The quantitative spread plate method is performed in a manner similar to the pour
plate method; however, an aliquot of each dilution is transferred directly onto
the surface of solidified microbial growth agar. The sample aliquot is then evenly
spread over the agar surface. The agar plates are allowed to dry, then inverted and
incubated under conditions appropriate for the test microorganisms (see Table 23-
2).
After incubation, the number of microbial colonies is counted, and the resulting
figure is multiplied by the appropriate dilution factor to obtain the number of
CFU per gram or milliliter of sample.
6.5.2.3 Quantitative Spiral Plate Method.
The quantitative spiral plate method is performed in a similar manner to spread

23
plate method; however, an aliquot of the test solution is transferred to a spiral plate
machine and it is diluted onto an agar plate in a spiral pattern. The agar plates are

ATYPICAL PRODUCTS
M-6 TESTING OF
allowed to dry, then inverted and incubated under conditions appropriate for the
test organisms.

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 SECTION 23 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS

Table 23-1

SUGGESTED CHALLENGE MICROORGANISMS

Type Microorganism (ATCC Number) Recommendation
Gram-Positive Cocci Staphylococcus aureus (6538)* Select at least one
Staphylococcus epidermidis (12228)
Fermentative Gram-Negative Bacilli Klebsiella pneumoniae (10031) Select at least one
Enterobacter cloacae (13047))
Escherichia coli (8739)*
Enterobacter gergoviae (33028)
Non-Fermentative Gram-Negative Pseudomonas aeruginosa (9027)* Select at least one
Bacilli Burkholderia cepacia (25416)
Pseudomonas fluorescens (13525)
Pseudomonas putida (31483)
Yeasts Candida albicans (10231)* Recommended
Molds Aspergillus brasiliensis (16404)* Select at least one
Penicillium species
Spore-Forming Bacilli Bacillus subtilis (6051) Optional
Other organisms relevant to the Optional
product
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans
(10231) and Aspergillus brasiliensis (16404) are specified in the United States Pharmacopeia (USP) Antimicrobial
Effectiveness Testing method.9

Table 23-2

CULTURE CONDITIONS FOR PREPARATION OF INOCULA

Cultures Media Temperature Time
Bacteria Soybean-Casein Digest Medium/Soybean-Casein Digest Agar 30-37°C 18-48 hours
ATYPICAL PRODUCTS

Medium (Tryptic Soy Broth/Agar)

Nutrient Broth/Agar
Other Media
M-6 TESTING OF

Yeasts Sabouraud Dextrose Agar 25-35°C 24-48 hours

Soybean-Casein Digest Medium/Soybean-Casein Digest Agar
Medium (Tryptic Soy Broth/Agar) Medium
Other Media
Molds Sabouraud Dextrose Agar 20-30°C 7-28 days
Potato Dextrose Agar
Malt Extract Agar
23

Other Media

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 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS SECTION 23

Table 23-2

TABLE 23-3: INCUBATION CONDITIONS FOR RECOVERY OF

MICROORGANISMS
Cultures Media Temperature Time
Bacteria For recovery agars, see 30-37°C 24-72 hours
Section 3.3.3
Yeasts 25-35°C 48-72 hours
Molds 20-30°C 3-7 days

ADDITIONAL INFORMATION
Bean, H.S. 1972. “Preservatives for Pharmaceuticals.” J. of Soc. Cosmet. Chem. 23:703-720.
Tenenbaum, S. 1967. “Pseudomonads in Cosmetics.” J. of Soc. Cosmet. Chem. 18:797-807.
Wilson, L.A., Kuehne, J.W., Hall, S.W., and Ahearn, D.G. 1971. “Microbial Contamination in
Ocular Cosmetics,” American Journal of Ophthalmology 71(6):1298-1302.
Yablonski, J.I. and Mancuso, S.E. 2002. “Preservation of Atypical Cosmetic Systems.” Cosmetics &
Toiletries 2: 41.

REFERENCES
1. ASTM International 2012. ASTM E640-06 (2012) Standard Test Method for Preservatives in

23
Water-Containing Cosmetics. ASTM International, West Consohocken, PA.

ATYPICAL PRODUCTS
M-6 TESTING OF
2. AOAC International 2007. AOAC 998.10-2009, Efficacy of preservation of non-eye area water-
miscible cosmetic and toiletry formulations. AOAC International, Rockville, MD
3. ISO 2012. ISO 11930 Cosmetics — Microbiology — Evaluation of the antimicrobial
protection of a cosmetic product. ISO. Geneva, Switzerland. www.iso.org
4. Brown, M.R. and Gilbert, P. (Eds.) 1995. Microbiological Quality Assurance: A Guide Towards
Relevance and Reproducibility of Inocula, CRC Press, Boca Raton, FL.
5. Kirsop, B.E. and Doyle, A. (Editors) 1991. Maintenance of Microorganisms and Cultured Cells,
Second Edition, Academic Press, New York, NY.
6. ASTM International 2013. ASTM E1054-08 Standard Practices for Evaluating Inactivators of
Antimicrobial Agents. ASTM International, West Conshohocken, PA.
7. Reichgott, M. 2003. “Reference Strains: How Many Passages Are Too Many?,” ATCC Connection
23, available at: https://www.atcc.org/~/media/PDFs/Technical%20Bulletins/tb06.ashx

PCPC MICROBIOLOGY GUIDELINES | 247

 SECTION 23 M-6 A METHOD FOR PRESERVATION TESTING OF AT YPIC AL PRODUCTS

8. AOAC International. 2013. AOAC Method 966.04 Sporicidal Activity of Disinfectants. AOAC
International, Rockville, MD
9. U.S. Parmacopeia & National Formulary. 2016. USP 39 - NF 34. <51> Antimicrobial
Effectiveness Testing, USP-NF, Rockville, MD, pp. 67-69.
ATYPICAL PRODUCTS
M-6 TESTING OF
23

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 SECTION 24

M-7
A Screening Method for
Preservation Testing of Water-
Miscible Personal Care Products
Scope
1. Introduction
1.1 This procedure allows a more rapid determination of preservation efficacy in water-miscible
personal care products in comparison to traditional methods. This procedure is intended to
be used as a screening test during product development to quickly differentiate between pre‑
servative systems that may be capable of providing adequate preservation and those which
have insufficient anti-microbial activity to protect the product. It is not intended to provide
the definitive information on the adequacy of preservation of the final formulation. This
information is obtained through standard tests, such as those described in Methods M-3
(Section 20), M-5 (Section 22), and M-6 (Section 23).
This procedure may also be used to more quickly qualify products to which minor formula‑
tion changes have been made. However, to assure that the product is adequately preserved,
more stringent criteria for the elimination of microorganisms over the course of the test than
those used in conventional tests should be adopted.
1.2 Aseptic techniques and sterile materials must be employed.

2. Applicable Documents
2.1 “Determination of Preservative Efficacy in Water-Miscible Personal Care Products” (Section
13).

3. Materials
3.1 Selection of Challenge Microorganisms
24

The microbial strains listed in Table 24-1 may be considered for use in developing preserva‑
tion data for personal care products.
MISCIBLE PRODUCTS
M-7 SCREENING WATER-

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 SECTION 24 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS

3.2 Maintenance of Challenge Microorganisms

See Section 10 and the website of the American Type Culture Collection (www.atcc.org)
Storage of other organisms appropriate to the product under test in the original product
or incorporation of product into maintenance medium is often the only way to retain its
unique characteristics. This method is especially appropriate where the isolate is subsequent‑
ly inoculated into a similar material.
3.3 Test Media
3.3.1 Inocula Suspending Fluids
Suspending fluids are used to prepare the bacterial and fungal suspensions for in‑
oculating the test product. The following may be used:
• Phosphate Buffer (pH 7.0)
• 0.85% Sodium Chloride Solution
• Sodium Chloride Peptone Solution (1% peptone in 0.85% saline)
Other suitable fluids may be used. The addition of 0.05% – 0.1% polysorbate 80
or other surfactant to the suspending fluid is recommended to aid in dispersion of
mold spores.
3.3.2 Microbial Plate Count Diluents
Plating diluents serve to disperse the sample and dilute it to levels that permit re‑
covery of surviving microorganisms from an inoculated product formulation. The
choice of diluent depends on its ability to meet the requirements of preservative
neutralization (Section 4.1). The following are examples of diluents that may be
used:
• Buffered Sodium Chloride Peptone Solution
• Dey/Engley (D-E) Neutralizing Broth
• Eugon Broth
• Letheen Broth
• Modified Letheen Broth
• Phosphate Buffer, pH 7
• Soybean-Casein Digest Medium (Tryptic Soy Broth)
• Tryptone-Azolectin-Tween® (TAT) Broth
M-7 SCREENING WATER-

Other suitable diluents may also be used

MISCIBLE PRODUCTS

Addition of neutralizers may be necessary to demonstrate adequate preservative

neutralization.

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 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS SECTION 24

3.3.3 Recovery Agars

Many factors affect organism viability. Therefore, it is important for the agar to
provide optimum nutritional support for the recovery of the challenge organisms.
The following have been found suitable for preservation studies:
• Eugon Agar
• Letheen Agar
• Microbial Content Test Agar
• Modified Letheen Agar
• Plate Count Agar
• Soybean-Casein Digest Agar Medium (Tryptic Soy Agar)
Addition of neutralizers may be necessary to demonstrate adequate preservative
neutralization.6 Other suitable agars may be used.
If the above agars do not support the growth of fungi, one of the following agars
may be considered:
• Malt Agar
• Malt Extract Agar
• Mycological Agar
• Potato Dextrose Agar
• Sabouraud Dextrose Agar
Other suitable agars may be used.

4. Preliminary Tests
4.1 Preservative Neutralization
Carryover of antimicrobial activity from the product formulation into the plate count dilu‑
ent and recovery growth agar may occur. This may inhibit the growth of surviving challenge
test microorganisms resulting in a false negative microbial count. To avoid a false negative
result, neutralization of the antimicrobial properties of the formulation must take place in
the plate count diluent and/or the recovery growth agar.
Antimicrobial neutralization may normally be accomplished by use of chemical neutralizing
agents, physical dilution, or a combination of both.
24

Verification of neutralization is generally performed by inoculating the product dilution with

MISCIBLE PRODUCTS
M-7 SCREENING WATER-

a low level of challenge microorganisms and performing the enumeration method Side-by-side
dilutions with and without a product formulation are made. Enumeration of the microor‑
ganisms from these dilutions is performed. Neutralization is verified if microbial recoveries

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 SECTION 24 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS

are similar. If one or more challenge microorganisms cannot be recovered, the use of a higher
dilution and/or the investigation of additional chemical neutralizers may be considered.
4.2 Microbial Content Test
It is recommended that that a microbial content test (See Section 18) be performed on the
test sample prior to performing the preservative efficacy test. Verification of neutralization of
the antimicrobial properties of the test sample should be demonstrated (See Section 4.1) at
the same time as the microbial content test.

5. Inoculation Procedures
5.1 Preparation of Inocula
Freshly prepared cultures should be used for inoculating test samples.
In general, culture conditions in Table 24-2 should be considered when preparing the in‑
ocula. Refer to the ATCC website1 for optimal growth media and conditions for specific
microorganisms.
5.1.1 Preparation of Initial Bacteria and Yeast Suspensions
Either broth cultures or cultures grown on solid agar media are acceptable for use.
For reference strains such as the ATCC strains, no more than five transfers from
the stock culture are recommended.7 Broth cultures should be centrifuged and
then re-suspended in the chosen suspending fluid. Microbial growth on a solid
medium is transferred to the chosen suspending fluid.
5.1.2 Preparation of Initial Mold Suspensions
The mold inoculum is prepared by washing the sporulating agar culture with the
chosen suspending fluid (See Section 3.3.1 above) and filtering the spore suspen‑
sion through sterile gauze or glass wool. Sterile glass beads can be used as an aid in
the dispersion of spores in the suspending fluid.
5.1.3 Preparation of Bacterial Spore Suspensions
If spore-forming bacteria are to be included in the test, the inocula may be pre‑
pared as indicated in the AOAC Sporicidal Test.8 Some strains are commercially
available as prepared spore suspensions.
5.1.4 Preparation of Challenge Inocula
5.1.4.1 Inoculum levels
The recommended inoculation levels for challenge testing are:
M-7 SCREENING WATER-

• 1x105 to 1×106 Colony-Forming Units (CFU) of bacteria per gram

MISCIBLE PRODUCTS

of product
• 1-5×105 CFU of yeast per gram of product
• 1-5×105 CFU of mold spores per gram unit of product

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 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS SECTION 24

The inoculum level for the challenge microorganisms should be verified

by standard microbiological techniques such as pour—or streak—plate
methods. It is recommended that the volume of the inoculum be <1%
of the sample weight/volume and should not alter the character of the
product being challenged.
5.2 Product Challenge
Either pure or mixed microbial culture suspensions may be used to challenge test formula‑
tions. Inocula consisting of only pure microbial cultures will yield specific data on each test
microorganism employed in the challenge study. When conducting mixed culture challenge
studies, it is recommended that closely related types of microorganisms such as Gram-posi‑
tive bacteria, Gram-negative bacteria, and yeasts and molds be pooled separately.
All products should be thoroughly mixed manually or mechanically after inoculation to
distribute the challenge microorganisms uniformly. The volume of the inoculum should not
alter the character of the product being challenged. Challenged formulations should then be
stored at ambient temperature for the duration of the test.
5.3 Sampling the Challenged Product
5.3.1 Sampling interval
To obtain rapid results, it is suggested that challenged formulations be sampled for
viable microorganisms at 1, 2 or 3 and 7 days following inoculation.
5.3.2 Sampling and plating methods
The inoculated product should be thoroughly mixed just prior to sampling to
ensure that the sample is representative. In some cases, the inoculum can thrive
in “pockets” of growth in the formulation while other areas are relatively free of
microorganisms. Many aerobic microorganisms grow especially well at the formu‑
lation-air interface. Often it is very difficult to break up the “pockets” of growth,
and special procedures are needed. The following mixing methods have been used
to overcome this problem:
• Vigorous mixing with a stirring rod
• Capping and shaking vigorously by hand
• Mixing in a vortex mixer
• Mixing with a magnetic stirrer
• Mixing with a propeller stirrer
• Mixing with a non-aerating stirrer
24

• Mixing in a micro blender

MISCIBLE PRODUCTS
M-7 SCREENING WATER-

• Mixing in a stomacher
• Gentle mixing in a tissue grinder

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 SECTION 24 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS

Sample size will in part determine the minimum detectable level. A sample size of
at least one gram or one milliliter of product for the quantitative pour plate meth‑
od is recommended.
5.3.2.1 Quantitative pour plate method
Serial dilutions are prepared from the aliquot recovered from the chal‑
lenged sample unit. Each serial dilution is thoroughly mixed, and an ali‑
quot is transferred to a Petri dish. Melted agar maintained at 45-48°C is
added to the Petri dish, and the dish is rotated to uniformly disperse the
product dilution. The agar plates are allowed to solidify, then inverted
and incubated under conditions appropriate for the test microorganisms
(see Table 24-2).
After incubation, the number of microbial colonies is counted, and the
resulting figure is multiplied by the appropriate dilution factor to obtain
the number of microorganisms per gram.
5.3.2.2 Quantitative spread plate method
The quantitative spread plate method is performed in a manner simi‑
lar to the pour plate method. However, an aliquot of each dilution is
transferred directly onto the surface of solidified microbial growth agar.
The sample aliquot is then evenly spread over the agar surface. The agar
plates are allowed to dry, then inverted and incubated under conditions
appropriate for the test microorganisms (see Table 24-2).
After incubation, the number of microbial colonies is counted, and the
resulting figure is multiplied by the appropriate dilution factor to obtain
the number of microorganisms per gram.
5.2.3.3 Quantitative Spiral Plate Method.
The quantitative spiral plate method is performed in a similar manner
to spread plate method; however, an aliquot of the test solution is trans‑
ferred to a spiral plate machine and it is diluted onto an agar plate in
a spiral pattern. The agar plates are allowed to dry, then inverted and
incubated under conditions appropriate for the test organisms.
M-7 SCREENING WATER-
MISCIBLE PRODUCTS

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 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS SECTION 24

Table 24-1

SUGGESTED CHALLENGE MICROORGANISMS

Type Microorganism* (ATCC Number) Recommendation
Gram-Positive Cocci Staphylococcus aureus (6538)* Select at least one
Staphylococcus epidermidis (12228)
Fermentative Gram- Klebsiella pneumoniae (10031) Select at least one
Negative Bacilli Enterobacter cloacae (13047))
Escherichia coli (8739)*
Enterobacter gergoviae (33028)
Non-Fermentative Gram- Pseudomonas aeruginosa (9027)* Select at least one
Negative Bacilli Burkholderia cepacia (25416)
Pseudomonas fluorescens (13525)
Pseudomonas putida (31483)
Yeasts Candida albicans (10231)* Recommended
Molds Aspergillus brasiliensis (16404)* Select at least one
Penicillium species
Spore-Forming Bacilli Bacillus subtilis (6051) Optional
Other organisms relevant Optional
to the product
*Staphylococcus aureus (6538), Escherichia coli (8739), Pseudomonas aeruginosa (9027), Candida albicans
(10231) and Aspergillus brasiliensis (16404) are specified in the United States Pharmacopeia (USP)
Antimicrobial Effectiveness Testing method.2

Table 24-2

CULTURE CONDITIONS FOR PREPARATION OF INOCULA

Cultures Media** Temperature Time
Bacteria Soybean-Casein Digest Medium/Soybean- 30-37°C 18-48 hours
Casein Digest Agar Medium (Tryptic Soy Broth/Agar )
Nutrient Broth/Agar
Eugon Broth/Agar
Yeasts Sabouraud Dextrose Agar 25-35°C 24-48 hours
Soybean-Casein Digest Medium/Soybean-
Casein Digest Agar Medium (Tryptic Soy Broth/Agar )
Mycophil (Mycological) Broth/Agar
Molds Sabouraud Dextrose Agar 20-30°C 7-28 days
Potato Dextrose Agar
Mycophil (Mycological) Agar
Malt Extract Agar
24

** Available in dehydrated forms from commercial sources.

MISCIBLE PRODUCTS
M-7 SCREENING WATER-

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 SECTION 24 M-7 SCREENING OF WATER-MISCIBLE PERSONAL C ARE PRODUCTS

Table 24-3

INCUBATION CONDITIONS FOR RECOVERY OF MICROORGANISMS

Cultures Media Temperature Time
Bacteria For recovery agars, see Section 3.3.3 30-37°C 24-72 hours
Yeasts 25-35°C 48-72 hours
Molds 20-30°C 3-7 days

REFERENCES
1. ASTM International. 2013. ASTM E1054-08 Standard Practices for Evaluating Inactivators of
Antimicrobial Agents. ASTM International, West Conshohocken, PA.
2. ASTM International. 2012. ASTM E640-06 (2012) Standard Test Method for Preservatives in
Water-Containing Cosmetics. ASTM International, West Consohocken, PA.
3. AOAC International. 2007. AOAC 998.10-2009, Efficacy of preservation of non-eye area
water-miscible cosmetic and toiletry formulations. AOAC International, Rockville, MD
4. ISO 2012. ISO 11930 Cosmetics — Microbiology — Evaluation of the antimicrobial
protection of a cosmetic product. ISO. Geneva, Switzerland. www.iso.org
5. Brown, M.R. and Gilbert, P. (Eds.) 1995. Microbiological Quality Assurance: A Guide Towards
Relevance and Reproducibility of Inocula, CRC Press, Boca Raton, FL.
6. Kirsop, B.E. and Doyle, A. (Editors) 1991. Maintenance of Microorganisms and Cultured Cells,
Second Edition, Academic Press, New York, NY.
7. Reichgott, M. 2003. “Reference Strains: How Many Passages Are Too Many?,” ATCC
Connection 23, available at: https://www.atcc.org/~/media/PDFs/Technical%20Bulletins/tb06.
ashx
8. AOAC International. 2013. AOAC Method 966.04 Sporicidal Activity of Disinfectants. AOAC
International, Rockville, MD
9. U.S. Pharmacopeia & National Formulary. 2016. USP 39 - NF 34. <51> Antimicrobial
Effectiveness Testing, USP-NF, Rockville, MD, pp. 67-69.
M-7 SCREENING WATER-
MISCIBLE PRODUCTS

256 |PCPC MICROBIOLOGY GUIDELINES

24
 SECTION 25

Glossary
A Anaerobic
able to grow in the absence of oxygen.
Action Level
level or range that, when exceeded, indicates Anhydrous
that a process has deviated from its normal op‑ without water.
erating condition, and that requires corrective
action to be taken to bring the process back into Antimicrobial
its normal operating condition. a chemical agent, either produced by a micro‑
organism or by synthetic means, that is capable
Adaptation of killing or suppressing the growth of micro‑
a change or changes in an organism or popu‑ organisms.Antimicrobial Preservative Efficacy
lation of organisms, through which the organ‑ Test. See Preservation Test.
ism(s) become more suited to the prevailing en‑
vironmental conditions. Antiseptic
a substance for use on living tissue that either
Add-on destroys or inhibits the growth of microorgan‑
in a non-woven substrate based product, the isms.
formulation added to the substrate, e.g., a lo‑
tion, solution, emulsion, oil, or other material. Aqueous
containing water.
Aerobic
requiring oxygen for growth. Aseptic
free of microorganisms that are capable of caus‑
Agar ing infection or contamination.
a gelatinous colloidal extract from algae consist‑
ing of agarose and agaropectin that is used in Aseptic Technique
microbial growth media to make it a semi‑solid precautionary measures taken in microbiologi‑
at room temperature. cal work to prevent the contamination by extra‑
neous microorganisms.
Air Sampling
a technique by which the quantity of viable mi‑ Atypical Product
croorganisms or particulate matter present in a a product in which water is not readily avail‑
voume of air is determined or isolated. able to provide an environment that supports
growth of microorganisms, a product in which
Alert Level the water activity is too low to support growth,
a level or range that, when exceeded, warns that or a product having other physico‑chemical
24

a process may have deviated from its normal op‑ characteristics that do not allow growth of mi‑
erating condition. croorganisms.
MISCIBLEGLOSSARY
M-7 SCREENING WATER-

Ambient Air
environmental or room air.
PRODUCTS

PCPC MICROBIOLOGY GUIDELINES | 257

 GLOSSARY

B C
Bactericide CFU
a chemical or physical agent that destroys viable See Colony-forming Unit
bacteria.
Calibration
Bacteriostat the set of operations and conditions that estab‑
an agent that inhibits the growth of bacteria. lishes the relationship between values produced
by a measuring instrument or system, or val‑
Bacterium (pl. bacteria) ues obtained from a material measure, and the
a single‑celled, prokaryotic microorganism that corresponding values from a known reference
multiplies by cell division. standard.

Biochemical Characteristics Challenge Organisms

biochemical reactions that are indicative for a microorganisms used in preservative challenge
particular microbial species. tests.

Biocide Challenge Test

a chemical or physical agent that destroys all vi‑ See Preservation Test.
able microorganisms.
Chlorination
Biofilm the use of chlorine or chlorine-donating com‑
a complex structure consisting of diverse micro‑ pounds to effect sanitization or to control mi‑
colonies of various microorganisms embedded crobial levels.
in a matrix of extracellular organic polymers ad‑
hering to moist surfaces. Cleaner
a chemical or blend of chemicals formulated to
Biological Indicator remove undesirable soils from a surface; may
a characterized preparation of a specific micro‑ be a solvent, acid, base, detergent, and/or wa‑
organism resistant to a particular sterilization ter-based mixture.
process.
Cleaning
Biostatic Activity the process of separating and eliminating gen‑
the ability of a chemical agent or a physical con‑ erally visible dirt from a surface, accomplished
dition that inhibits the growth of microorgan‑ using, in variable proportions, chemical action,
isms. mechanical action, temperature and duration of
application.
Bulk in Process
a product in a partial state of completion or fin‑ Cleaning Agent
ished goods before filling. an agent designed to remove visible and non‑vis‑
ible foreign matter from surfaces.
Bulk Product
WATER-

product that has completed the processing steps Coliform Organisms

PRODUCTS

up to final packaging but has not been placed in Gram‑negative, nonspore‑forming bacteria of
MISCIBLEGLOSSARY

the final package. intestinal origin that ferment lactose with gas
M-7 SCREENING

formation.

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 GLOSSARY

Colony Culture Medium

a macroscopically visible growth of microorgan‑ a nutrient‑containing liquid (broth) or solid
isms on a solid culture medium. (agar) that supports the growth of microorgan‑
isms.
Colony‑forming Unit (CFU)
an organism or cluster of organisms that causes Culture, Mixed
a visible colony. the presence of more than one species of micro‑
organism in a culture medium.
Compressed Air
air under pressure greater than that of the at‑ Culture Slant
mosphere. an inclined agar medium in a test tube used for
growth of microorganisms.
Concurrent Validation
the generation of current test data that will be Culture Stability
used to document that a process or procedure maintenance of biochemical or other desirable
does what it is intended to do. characteristics of bacteria or fungi over time and
through repeated subcultures.
Contact Plate
See Rodac Plate.
D
Cosmetic
Dead End
articles intended to be rubbed, poured, sprin‑
See Dead Leg.
kled, or sprayed on, introduced into, or oth‑
erwise applied to the human body...for cleans‑
Dead Leg
ing, beautifying, promoting attractiveness,
any area within a piping system that allows ma‑
or altering the appearance” See [FD&C Act,
terial to accumulate and then stagnate.
sec. 201(g)(1) http://www.fda.gov/Regulatory
Information/Legislation/FederalFoodDrugand
Deionization
CosmeticActFDCAct/default.htm
a water-treatment process that removes ions
from water by passing it through cationic and
Contact Time
anionic resin beds.
the time during which a microorganism or mi‑
crobial growth medium is in the presence of a
Diluent
test surface or chemical agent.Contamination
a medium or vehicle used to reduce a material
the presence of undesirable organisms.
to a less concentrated form.
Culture, Fresh
Disinfection
a population of a single species of a microorgan‑
the destruction of disease‑causing or objection‑
ism that has been recently cultivated either in a
able microorganisms, with the exception of
liquid medium or on an agar medium.
spores, on inanimate surfaces by chemical or
physical means.
24

Culture Maintenance
a process that keeps a microorganism alive, un‑
Distillation
MISCIBLEGLOSSARY
M-7 SCREENING WATER-

contaminated, and without variation or mu‑

a process that consists of driving gas or vapor
tation, so that it is as close as possible to the
from a liquid or solid by heating and then con‑
original isolate.
densing to liquid.
PRODUCTS

PCPC MICROBIOLOGY GUIDELINES | 259

 GLOSSARY

Documentation Fungicide
records containing all relevant information or‑ a chemical or physical agent that kills fungi.
ganized in an orderly and easily understood for‑
mat, usually applied to a process, method, or Fungistat
equipment usage. an agent that inhibits the growth of fungi.

Fungus
E a saprophytic, symbiotic, or parasitic, hetero‑
trophic, eukaryotic microorganism, i.e., a yeast
Enteric Organisms
or mold
microorganisms associated with the intestinal
tract.
G
Eucaryotic
a type of cell that has a well‑defined nuclear Genotype
membrane. genetic material contained in the entire comple‑
ment of alleles (chromosomes).

F Genus (pl. genera)

a specific biological classification of very closely
Fecal Contamination
related species of organisms, ranking between a
adulterated by feces, often inferred from the
family and a species.
presence of coliform bacteria, but may be pres‑
ent whether or not coliforms are present.
Glutaraldehyde
a saturated dialdehyde that is chemically related
Filamentous Fungus
to formaldehyde.
an organism that exhibits mycelial or threadlike
morphology. Gram‑negative
bacteria that retain a red color after the Gram
Filtration
stain procedure.
the removal of particulates from a fluid by an
appropriately sized filter.
Gram‑positive
bacteria that retain a violet color after the Gram
Finished Goods
stain procedure.
any manufactured cosmetic product or compo‑
nent that is suitable for use, whether or not it is Gram Stain
packaged or labeled. differential staining procedure used for bacterial
classification.
Finished Product
a manufactured cosmetic product that has un‑ Growth
dergone all stages of production, including
an increase in the number of microorganisms.
packaging in its final container as placed on the
market and made available to the consumer.
WATER-

Growth Phase
PRODUCTS

See Log Phase.

MISCIBLEGLOSSARY

Formalin
M-7 SCREENING

a clear 37% aqueous solution of formaldehyde.

260 |PCPC MICROBIOLOGY GUIDELINES

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 GLOSSARY

H of the proper installation of utilities for a piece

of equipment.
Halogenated Compounds
chemical compounds containing either chlo‑ Iodophore
rine, bromine, iodine, or fluorine. a chemical complex of iodine and a surface
active agent that functions as a disinfectant
Heat Distribution through slow release of iodine.
measurement of temperature uniformity within
an autoclave chamber with empty and loaded Isolate (verb)
chamber configurations. to separate a mixed population of microorgan‑
isms to obtain pure cultures.
Heat Penetration
measurement of temperature uniformity with‑ Isolate (Noun)
in items for sterilization of a loaded autoclave a microorganism cultured from an ingredient,
chamber configuration. finished product, or the environment
Homogeneous
uniform throughout, in structure or composi‑ L
tion.
Log Phase
Hostile Environment a pattern of microbial growth in which there is an
any material or condition unfavorable for sur‑ exponential increase in the number of viable cells.
vival or growth of microorganisms.
Lyophilized
freeze‑dried.
I
Indigenous M
occurring naturally within a particular environ‑
ment. Maintenance
support and verification operations, either pe‑
Innocuous riodic or unplanned, intended to keep facility
microorganisms commonly considered harm‑ and equipment in proper working condition.
less.
Membrane Filter
Inoculation a pliable filter or filter unit containing pores of
the introduction of microorganisms into a a known size used to separate out microorgan‑
product formulation or onto a substrate. isms from a fluid.

Inoculum Membrane Filtration

material containing living microorganisms used removal of particulates (including microorgan‑
for inoculation. isms, depending on the filter’s rating) using a
24

membrane.
Installation Qualification (IQ)
MISCIBLEGLOSSARY
M-7 SCREENING WATER-

a description of the physical characteristics, Microbes

drawings, specifications, operating manuals, microorganisms, including bacteria, yeasts and
calibration of instrumentation, and verification molds.
PRODUCTS

PCPC MICROBIOLOGY GUIDELINES | 261

 GLOSSARY

Microbial Content O
the number of viable microorganisms present in
a specific volume or quantity of material. Objectionable Organism
an organism that can be harmful to the user
Microbial Proliferation Rate based upon the nature of the product, its in‑
reproduction rate of microorganisms. tended use and its potential hazard, or is able
to compromise the physical integrity or appear‑
Microbiological Limits ance of the product.
maximum microbial content and specific mi‑
crobial type restrictions established for raw ma‑ Operational Qualification (OQ)
terials or finished products. a list of critical components, operating ranges,
as defined by the specification, and actual per‑
Microbiological Specification formance for a piece of equipment.
a statement of microbial content limits.
Ozone
Mold a highly reactive allotropic triatomic form of
filamentous fungus. oxygen used in disinfection and deodorization.

Most Probable Number Ozonation

a counting technique that utilizes a multiple the addition of ozone to a water system to re‑
“dilution” to extinction approach in microbial duce microbial levels.
growth medium and a mathematical formula to
estimate the number of microorganisms present
in a sample. P
Package Compatibility
Motility the absence of a detrimental interaction be‑
the movement of a bacterial cell in any medium. tween a product and its package.

Pathogen
N a disease‑causing organism.
Natural Raw Materials
substances of plant, animal, or mineral origin Peroxygen Compounds
that may be minimally processed before use. chemical compounds that contain the bivalent
group O‑O and are used as sanitizing agents.
Negative Control
a sample in a test series without the test agent Personal Care Product
used as a standard of comparison in judging ex‑ Articles including cosmetics, as defined by the
perimental effects. U.S. food and Drug Administration, as well as
some products such as suncreens that are clas‑
Neutralizing Agent sified as srugs by the agency. See: http://www.
a chemical substance added to a medium to in‑ fda.gov/ForIndustry/FDABasicsforIndustry/
ucm238796.htm
WATER-

activate an antimicrobial agent.

PRODUCTS
MISCIBLEGLOSSARY

Pipeline Pig
M-7 SCREENING

a device made of non-porous materials that is

used to remove and clean product from manu‑

262 |PCPC MICROBIOLOGY GUIDELINES

24
 GLOSSARY

facturing pipelines; it fits the internal radius of Process Water

a pipe, is inserted, launched and moved through treated water used as a raw material in the man‑
the pipe length pushed by air, product or water. ufacture of a product.

Plate Count Process Water System

the number of viable colony‑forming units a manufacturing system used to make process
(CFU) per plate; a method of determining how water.
many CFU per measure (usually per gram or
ml) of the sample being evaluated. Proliferation
microbial growth.
Positive Control
a sample in a test series with a test agent that has Prospective Validation
a known observable effect for use as a standard the establishment of documented evidence that
of comparison. a process does what it purports to do, based on
a preplanned validation protocol.
Potable Water
water that is suitable for drinking. Purified Water
process water obtained by distillation, ion‑ex‑
Pour Plate change treatment, reverse osmosis, or other
a plate count method in which a test material suitable means, the quality of which may be
is introduced and dispersed uniformly after the checked for specific chemical parameters, for
addition of molten agar medium to a Petri dish. example using current USP or in‑house require‑
ments.
Preservation Test
a method in which a material is inoculated with
selected microorganisms to determine the anti‑ Q
microbial effectiveness of a preserved or an un‑
Quaternary Compound
preserved formulation.
a quaternary ammonium compound in which
the ammonium hydrogen atoms have been re‑
Preservative
placed with organic radicals, generally used as a
a chemical agent that kills microorganisms or
surface active agent and/or a biocide.
prevents microbial growth.Preservative Efficacy
Testing. See Preservation Test.

Preservative Failure
R
deterioration in the effectiveness of the preser‑ Raw Material
vative system in a product that allows for micro‑ any ingredient used in the manufacture of a
bial growth or survival. product.

Preservative System Resistance

the agent(s) incorporated into a product to re‑ the ability of microorganisms to maintain via‑
24

duce or prevent microbial growth. bility in adverse environmental conditions.

MISCIBLEGLOSSARY
M-7 SCREENING WATER-

Procaryotic Retrospective Validation

a type of cell without a nuclear membrane. the use of historical test data to document that a
process does what it is intended to do.
PRODUCTS

PCPC MICROBIOLOGY GUIDELINES | 263

 GLOSSARY

Rodac Plate Semi‑quantitative

an agar plate used for determining surface con‑ less than quantitative measurement, often re‑
taminants by direct contact. ferring to a measurement on an arbitrary scale,
e.g., 0 to ++++.

S Shelf Life
a period of time during which a raw material
Sample
or finished product may be stored and remain
one or more representative portions or items se‑
suitable for use.
lected from a set to obtain information about
that set.
Species
one kind of microorganism; a subdivision of a
Sampling
genus.
operations relating to the taking and prepara‑
tion of samples.
Specification
clear and accurate description of the essential
Sampling Devices
technical requirements for items, materials, or
tools and equipment used to aseptically sample
services; in the microbiology laboratory, often
materials, surfaces, or an environment.
refers to a statement of microbial limits.
Sampling Personnel
Spore
individuals trained in proper sampling tech‑
a highly resistant form of a microorganism, e.g.,
niques to prevent extraneous microbial contam‑
mold or bacilli.
ination.
Spread Plate
Sanitary
a plate count method in which a small volume
hygienic.
of test material is dispersed, by means of a sterile
spreader, over the entire surface of a solid agar
Sanitary Manufacturing Practice
medium in a Petri dish.
guidelines to maintain clean and sanitary condi‑
tions within the manufacturing environment.
Stabile Product
a finished product that maintains acceptable
Sanitization
characteristics over a specified time and under
the process utilized to reduce viable microor‑
defined environmental conditions.
ganisms on a surface to an acceptable level;
surfaces must be clean for the sanitization pro‑
Standard Operating Procedures
cedure to be effective.
written procedures detailing how to operate
equipment or execute a process.
Sanitizer
a chemical agent used for sanitization of clean
Sterile
surfaces.
free from viable microorganisms and spores.
WATER-

Selective medium
Sterilization
PRODUCTS

a medium that allows the growth of certain

MISCIBLEGLOSSARY

the use of either physical or chemical agents to

types of microorganisms in preference to others.
M-7 SCREENING

destroy all viable microorganisms and spores

from a material or equipment.

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 GLOSSARY

Streak Plate Transport Swab

a method in which inoculum is spread across a swab designed to maintain, under specified
the surface of a prepared solid agar medium in a conditions of time and temperature, the viabil‑
Petri dish, to produce isolated colonies. ity and numbers of microorganisms recovered
from a sampling procedure.
Subculture
preparation of a fresh culture from an existing Trend Analysis
culture. review and analysis of routine data for patterns
and variations.
Submicron Filtration
a filtration process that uses a filter with a pore
size smaller than 1 micron. U
Ultrafiltration
Suitable Medium
a water-treatment process in which a selective
a medium used for optimal growth of microor‑
permeable membrane filter is used to separate
ganisms, or one that will suppress the growth of
dissolved molecules based on size. Ultraviolet
certain organisms, while allowing the growth of
referring to electromagnetic radiation with
“target” organisms.
wavelengths shorter than visible light and lon‑
ger than X‑rays, generally between 200 and 400
Surface Monitoring
nanometers.
periodic determination of the microbial content
of a surface.
Ultraviolet lamp
a light that produces ultraviolet radiation for
Susceptible
disinfection.
subject to microbial contamination.

Swab
a wad of absorbent material usually wound
V
around or attached to the end of a small stick Validation
or applicator and used for removing material or substantiation and verification that a specific
microorganisms from a surface area. See also process or test method consistently does what it
Transport Swab. is intended to do.

Swabbing Validation Protocol

the process of wiping a surface with a moist, an approved written plan that details the means
sterile applicator, in order to collect viable mi‑ by which validation will be achieved and de‑
croorganisms. fines the acceptance criteria for a process or test
method.

T Vectors
carriers of microorganisms.
24

Taxonomy
the classification of organisms, based on mutual
similarities. Vegetative Bacteria
MISCIBLEGLOSSARY
M-7 SCREENING WATER-

viable bacterial cells not in a resting state.

Total Plate Count
See Plate Count.
PRODUCTS

PCPC MICROBIOLOGY GUIDELINES | 265

 GLOSSARY

Verification Water Activity

evidence that establishes or confirms that the the ratio of the vapor pressure in a product to
specified application of a process or test meth‑ that of pure water; this ratio is used to evaluate
od consistently and accurately does or measures the susceptibility of a water‑based product to
what it is intended to do or measure; evidence microbial contamination.
that establishes or confirms the accuracy of a
procedure or criterion. Wipes

Viable
capable of living. Y
Yeast
W a single‑cell fungus that reproduces primarily by
budding.
Waste
any material or product that its holder intends
for disposal.
WATER-
PRODUCTS
MISCIBLEGLOSSARY
M-7 SCREENING

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PCPC MICROBIOLOGY GUIDELINES | 267
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