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Sections in This Document

1. Purpose ................................................................................................................................. 1

2. Introduction............................................................................................................................ 2

3. Current Editorial Board .......................................................................................................... 3

4. Original Authors..................................................................................................................... 3

5. Chapter 1: Antimicrobial Effectiveness Testing ..................................................................... 4

6. Chapter 2: Microbial Examination of Non-Sterile Products..................................................... 8

7. Chapter 3: Sterility Testing ................................................................................................. 14

8. Chapter 4: Investigating USP Sterility Testing Failure......................................................... 28

9. Chapter 5: Bacterial Endotoxin Testing................................................................................ 30

10. Chapter 6: Particulate Matter.............................................................................................. 40

11. Chapter 7: Antibiotic Potency Testing .................................................................................. 44

12. Chapter 8: Bioburden Estimation for Medical Devices......................................................... 59

13. Chapter 9: Environmental Monitoring.................................................................................. 61

14. Chapter 10: Inspectional Guidance..................................................................................... 70

15. Glossary/Definitions............................................................................................................. 90

16. Records ............................................................................................................................... 91

17. Supporting Documents ........................................................................................................ 91

18. Document History ................................................................................................................ 91

19. Change History.................................................................................................................... 92

20. Attachments......................................................................................................................... 92

1. Purpose

The purpose of this Pharmaceutical Microbiology Manual (PMM) is to 

collectively clarify, standardize, and communicate useful analytical procedures 

that are not specifically addressed in the microbiology methods chapters in the 

United States Pharmacopeia. In addition, some sections of this manual can 

serve as a technical reference when conducting microbiological inspections of 

drug, biotechnology and medical device manufacturers. The contents of this 

PMM were collaboration between ORS and CDER in order to maximize the 

efficiency of our analytical results to support CDER’s goal to assure the safety 

and reliability of commercially distributed medical products.
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2. Introduction

The Pharmaceutical Microbiology Manual (PMM) evolved from the Sterility 

Analytical Manual and is a supplement to the United States Pharmacopeia 

(USP) for pharmaceutical microbiology testing, including antimicrobial 

effectiveness testing, microbial examination of non-sterile products, sterility 

testing, bacterial endotoxin testing, particulate matter, device bioburden and 

environmental monitoring testing. The goal of this manual is to provide an 

ORS/CDER harmonized framework on the knowledge, methods and tools 

needed, and to apply the appropriate scientific standards required to assess 

the safety and efficacy of medical products within ORS testing laboratories. 

The PMM has expanded to include some rapid screening techniques along 

with a new section that covers inspectional guidance for microbiologists that 

conduct team inspections.

This manual was developed by members of the Pharmaceutical Microbiology 

Editorial Board and includes individuals with specialized experience and 

training.

The instructions in this document are guidelines for ORS analysts. When 

available, analysts should use procedures and worksheets that are 

standardized and harmonized across all ORS labs, along with the PMM, when 

performing analyses related to product testing of pharmaceuticals and medical 

devices. When changes or deviations are necessary, documentation should be 

completed per the laboratory’s Quality Management System. Generally, these 

changes should originate from situations such as new products, unusual 

products, or unique situations.

This manual was written to reduce compendia method ambiguity and increase 

standardization between ORS laboratories. By providing clearer instructions to 

ORS labs, greater transparency can be provided to both industry and the 

public.

However, it should be emphasized that this manual is a supplement and does 

not replace any information in USP or applicable FDA official guidance 

references. The PMM does not relieve any person or laboratory from the 

responsibility of ensuring that the methods being employed from the manual 

are fit for use, and that all testing is validated and/or verified by the user.

The PMM will continually be revised as newer products, platforms and 

technologies emerge or any significant scientific gaps are identified with 

product testing.

Reference to any commercial materials, equipment, or process in the PMM 

does not in any way constitute approval, endorsement, or recommendation by 

the U.S. Food and Drug Administration.
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U.S. FDA, Office of Regulatory Affairs

Office of Regulatory Science

Office of Medical Products and Specialty Laboratory Operations

3. Current Editorial Board

Editor-In-Chief: Angele Smith, ORS Headquarters 

Rick Friedman, Center for Drug Evaluation and Research

Jennifer Gogley, Pacific Southwest Medical Products Laboratory

Kristy Ford, Detroit Medical Products Laboratory

Marilyn Khanna, ORS Headquarters

Mivoyel Jeanpaul, ORS Headquarters

Sammie La, Pacific Southwest Medical Products Laboratory

Matthew Silverman, Winchester Engineering and Analytical Center

Marla Stevens-Riley, Center for Drug Evaluation and Research

Allison Rodriguez, Winchester Engineering and Analytical Center

Radhakrishna Tirumalai, United States Pharmacopeia 

Theresa To, Winchester Engineering and Analytical Center

4. Original Authors

Rhonda Alexander, Southeast Food and Feed Laboratory

Jasna Braut-Taormina, Northeast Medical Products Laboratory

Jennifer Brzezinski, Forensic Chemistry Center

Marie B. Buen-Bigornia, Denver Food and Feed Laboratory

Rick Friedman, Center for Drug Evaluation and Research

Jennifer Gogley, Pacific Southwest Medical Products Laboratory

Andrew Gonzales, Denver Food and Feed Laboratory

Dennis Guilfoyle (ret), Northeast Medical Products Laboratory

Lawrence James (ret), Northeast Food and Feed Laboratory

Marilyn Khanna, ORS Headquarters

David Lau, San Francisco Food and Feed Laboratory

Eileen Liu, San Francisco Food and Feed Laboratory
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Mercedes Loftis, Denver Food and Feed Laboratory 

Philip McLaughlin (ret), Winchester Engineering and Analytical Center

Zachary Miller, Denver Food and Feed Laboratory

Gary Pecic, Denver Food and Feed Laboratory

Diane Raccasi (ret), Office of Enforcement and Import Operations

George Salem (ret), ORS Headquarters

Angele Smith, ORS Headquarters

Tammara Stephens, Southeast Food and Feed Laboratory

Selen Stromgren, ORS Headquarters

Vilasini Suktankar, Southeast Food and Feed Laboratory

Radhakrishna Tirumalai, United States Pharmacopeia 

Evelyn Wong, Pacific Southwest Medical Products Laboratory 

Jonathon Yenovkian, San Francisco Food and Feed Laboratory

5. Chapter 1: Antimicrobial Effectiveness Testing

Antimicrobial Effectiveness testing is described in USP <51>. Previously this 

chapter was known as “Preservative Effectiveness Testing”. Detailed 

procedure for the performance of the test can be found in USP <51>.

A. Media

For the cultivation of the test organisms, select agar medium that is favorable 

to the rigorous growth of the respective stock culture. The recommended 

media are Soybean Casein Digest Agar/Broth and Sabouraud’s Dextrose 

Agar/Broth. Add a suitable inactivator (neutralizer) for the specific 

antimicrobial properties in the product to the broth and/or agar media used for 

the test procedure whenever needed.

B. Growth Promotion of the Media

Media used for testing needs to be tested for growth promotion by inoculating

the medium with appropriate microorganisms. It is preferable that test 

microorganisms be chosen for growth promotion testing (Section D).

Solid media tested for growth promotion is to be set up using the method that 

will be used to analyze the product (pour plate or spread plate) to determine a

microbial plate count (CFU) which must be ≥ 50% of the microorganism

inoculum’s calculated value.

C. Suitability of the Counting Method in the Presence of Product
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For all product types, follow current USP methodology in chapter <51>, with

the following additional instructions.

Prior to the Antimicrobial Effectiveness testing, determine if any antimicrobial 

properties exist by performing a Suitability testing utilizing microorganisms

used for product testing (section D). Should the Suitability Test fail the results

of Suitability test are invalid and will need to be repeated with proper method

modification to neutralize the inhibiting property.

If multiple samples of the same product from the same manufacturer (same 

amount and form) are collected, one sample may be used for method

suitability for all the samples collected.

D. Test Organisms

All cultures must be no more than 5 passages removed from the original stock 

culture.

1. Candida albicans (ATCC No. 10231)

2. Aspergillus brasiliensis (ATCC No. 16404) 

3. Escherichia coli (ATCC No. 8739)

4. Pseudomonas aeruginosa (ATCC No. 9027)

5. Staphylococcus aureus (ATCC No. 6538)

E. Preparation of Inoculum

Preparatory to the test, inoculate the surface of the appropriate agar medium 

from a recently grown stock culture of each of the above test microorganisms.

Use Soybean-Casein Digest agar for Escherichia coli ATCC 8739,

Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 

6538 and incubate at 32.5 ± 2.5° C for 3 – 5 days. Use Sabouraud Dextrose

agar for Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC

16404 and incubate at 22.5 ± 2.5° C for 3 – 5 days for Candida albicans and 3

- 7 days for Aspergillus brasiliensis.

Harvest the cultures by washing the growth with sterile saline to obtain a

microbial count of about 1x108 CFU/mL (see Microbial Enumeration Tests 

<61> and Tests for Specified Microorganisms <62>). For the A. brasiliensis

ATCC 16404 culture, use sterile saline containing 0.05% polysorbate 80.

Alternatively, cultures may be grown in a liquid medium, i.e. Soybean Casein

Digest Broth or Sabouraud’s Dextrose Broth, (except for the A. brasiliensis 

ATCC 16404 culture) and harvested by centrifugation, washing and suspending 

in sterile saline to obtain a count of about 1 X 108 colony forming units (CFU) 

per mL.
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The estimate of inoculum concentration may be obtained by turbidimetric 

procedures for the challenge microorganisms and later confirmed by plate

count.

Refrigerate the suspension if not used within 2 hours at 2-8° C.

Determine the number of CFU/mL in each suspension using the appropriate

media and recovery incubation times to confirm the CFU/mL estimate.

Use bacterial and yeast suspensions within 24 hr. of harvest. The mold 

preparation may be stored under refrigeration (2-8° C) for up to 7 days.

Note: Alternative commercially available standardized cultures may be used in

lieu of in-house prepared cultures.

F. Procedure

The procedure requires that the test be conducted with a suitable volume of 

product. It is advisable to begin with at least 20 mL of product. Use the

original product containers whenever possible or five sterile, capped

bacteriological containers of suitable size into which a suitable volume of

product has been transferred. If the diluted product exhibits antimicrobial 

properties, specific neutralizers may need to be incorporated into the diluents

or the recovery media. For purposes of testing, products have been divided

into four categories:

Category 1 – Injections, other parenteral including emulsions, otic products, 

sterile nasal products, and ophthalmic products made with aqueous bases or

vehicles.

Category 2 – Topically used products made with aqueous bases or vehicles,

non-sterile nasal products, and emulsions, including those applied to mucous

membranes.

Category 3 – Oral products other than antacids, made with aqueous bases or

vehicles.

Category 4 – Antacids made with aqueous bases or vehicles.

Inoculate each container with one of the prepared and standardized inoculums

and mix. The volume of the suspension inoculums used is 0.5% to 1.0% of the

volume of the product. The concentration of the test organisms added to the

product for Categories 1, 2 and 3 is such that concentration of the test 

preparation immediately after inoculation is between 1x105 and 1x106 colony

forming organisms (CFU) per mL of product. If no suitable neutralizing agent 

or method is found and method suitability requires significant dilution, a higher 

level of inoculum (e.g., 107-108

) may be used so that a 3-log unit reduction can 
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be measured. For category 4 products (antacids) the final concentration of the 

test organisms is between 1x103 and 1x104 CFU/mL of product. 

Immediately determine the concentration of viable organisms in each inoculum 

suspension and calculate the initial concentration of CFU/mL by the plate

count method (see Microbial Enumeration Tests <61>).

Incubate the inoculated containers between 22.5 ±2.5°C in a controlled 

environment (incubator) and sample the container at specified intervals. The 

container sampling intervals include: Category 1 products are sampled at 7,

14, and 28 days and Category 2 – 4 products are sampled at 14 and 28 days. 

Refer to table 3 within USP <51>. Record any changes in appearance of the

product at these intervals. Determine the number of viable microorganisms

per mL present at each of these sampling intervals by the plate count method

utilizing media with suitable inactivator (neutralizer). Calculate the change in

log10 values of the concentration per mL based on the calculated

concentration in CFU/mL present at the start of the test for each 

microorganism at the applicable test intervals and express the changes in

terms of log reductions.

NOTE: The USP does not require a specific volume of product to be added to 

each of the five sterile tubes. It is recommended that 20 mL/tube be used to 

standardize testing for all ORS laboratories.

NOTE: All plate counts should be performed in duplicate (2 plates per 

dilution), and in a dilution series to detect growth inhibited by the preservative

system at the lower dilutions. Carrying the test to the 10-3 dilution would be

sufficient in most cases to overcome preservative inhibition.

G. Interpretation

The criteria for microbial effectiveness are met if the specified criteria are met,

see table below. No increase is defined as not more than 0.5 log10 unit higher 

than the previous value measured.
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6. Chapter 2: Microbial Examination of Non-Sterile Products

This section contains supplemental information for the quantitative

enumeration of viable microorganisms and the determination of the absence of

specified microorganisms in finished pharmaceutical products and raw

materials, previously referred to as Microbial Limits Testing (MLT). The

detailed procedures for these tests are not addressed in this PMM chapter 

because they are found in USP <60> MICROBIOLOGICAL EXAMINATION OF 

NONSTERILE PRODUCTS: TESTS FOR BURKHOLDERIA CEPACIA

COMPLEX, USP <61> MICROBIOLOGICAL EXAMINATION OF

NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS and <62>

MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTS 

FOR SPECIFIED MICROORGANISMS. 

USP Chapter <60> is a test to detect the presence of Burkholderia cepacia

species in a substance or preparation. Products for inhalation use or aqueous 

preparations for oral, oromucosal, cutaneous, or nasal USP <61> describes the 

methods for enumeration of microorganisms from pharmaceuticals and 

includes membrane filtration, conventional plate count (including pour-plate

method, surface- spread method), and the Most-Probable-Number (MPN). 

USP Chapter <62> describes specific enrichment procedures depending on

Category 1 Products

Bacteria: Not less than 1.0 log reduction from the initial calculated 

count at 7 days, not less than 3.0 log reduction from the

initial count at 14 days, and no increase from the 14-day

count at 28 days.

Yeast and Molds: No increase from the initial calculated count at 7, 14, and

28 days.

Category 2 Products

Bacteria: Not less than a 2.0 log reduction from the initial count at 14 

days, and no increase from the 14-day count at 28 days.

Yeast and Molds: No increase from the initial calculated count at 14 and 28 

days.

Category 3 Products

Bacteria: Not less than a 1.0 log reduction from the initial count at 14 

days, and no increase from the 14-day count at 28 days.

Yeast and Molds: No increase from the initial calculated count at 14 and 28 

days.

Category 4 Products

Bacteria, Yeast and

Molds:

No increase from the initial calculated count at 14 and 28 

days.
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the target specified microorganism that must be absent, as required by a

product monograph. Products which are insoluble or immiscible in water must 

be appropriately treated to obtain a suspension suitable for the test 

procedures. USP <1111> is used to identify organism recommendations for 

various drug dosage forms. use—are of concern.

It is important to note that even though the USP delineates methods for the

recovery and identification of specified microorganisms based on monograph

requirements, it is normally necessary to determine if any other microorganisms 

may also be present in the product(s) that may be objectionable and report 

these microorganisms on worksheets. In many cases, these may be

opportunistic or emerging pathogens not targeted for recovery by USP <62>. 

Identification methods, such as VITEK, should be used to identify any 

microbes recovered during USP <62> testing. Alternative methods, advanced 

molecular methods (i.e. PCR, sequencing, etc.) or the use of additional 

general enrichment agar plates or broth without selective properties, may

better suit the screening of test samples. The application of these additional 

agars or methods may need to be considered based on the target population

of the drug or product under analysis and may require a dialogue with the

laboratory supervisor for additional instructions. 

A. Product Storage and Handling

Samples are to be held under the same storage conditions required by the

package label or insert.

1. Prior to product testing, the exterior of the unit container should be

disinfected before transfer to the work station or HEPA filtered

laminar flow hood. If the product container is not hermetically sealed

do not soak the product container in a disinfection solution which

may allow the ingress of bactericidal solution into the product.

2. The work area for opening the unit container should be either a

HEPA filtered laminar flow hood or an alternate controlled

environment to safeguard the exposure of open media and product 

to either environmental or personnel contamination.

3. If the sample is an aqueous based product, the unit(s) should be

shaken prior to transfer to work area to maximize microbial 

dispersement.

4. All subsequent manipulation of test tubes with product or subculturing can be conducted on the laboratory work bench or within a

Biological Safety Cabinet (BSC) if filamentous fungi are suspected.

B. Gowning Requirements
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When conducting the testing, the analyst should wear a clean lab coat, sterile 

sleeves and sterile gloves. Gloves should be frequently disinfected especially 

between opening and handling sample (product) units.

Depending on the type of Laminar Flow Hood or equipment barriers in a particular 

laboratory, it might be beneficial to also wear a surgical mask and hair net.

C. Growth Promotion, Indicative and Inhibitory Properties of the Media

Test each batch of ready-prepared medium and each batch of medium 

prepared either from dehydrated medium or from ingredients for growth 

promotion and where appropriate, for indicative and inhibitory properties, 

following USP <60>, <61> and <62>. Each chapter provides guidance on test

strains to be used for each type of media, refer to Table 1 of USP <60>, <61> 

and <62>. Ensure that seed-lot cultures used are not more than five passages

removed from the original master seed-lot. Test strains suspensions should be

used within 2 hours, or within 24 hours if refrigerated between 2oC and 8oC. 

Spore suspensions (A. brasiliensis, B. subtilis, and C. sporogenes) refrigerated

between 2oC and 8oC may be kept for a validated period. If commercially 

available ready-to-use bacteria or fungal suspension are used, then the 

manufacturer’s instructions should be followed with respect to preparation and 

storage requirements. Additionally, all bacterial and spore suspensions should

be prepared to yield ≤100 CFU. Growth promotion (and suitability test) plates

and tubes should not be incubated in the same incubators used for product 

testing. If this cannot be avoided because of limited space, it is preferable to 

store the “spiked samples” in the lower half of the incubator below the sample

inoculated plates and tubes.

D. Suitability of the Test Method

Suitability demonstrates that the products tested do not exhibit inhibitory

effects on the growth of microorganisms under the conditions of the tests. 

Although the intent is to perform the suitability test before performing the

analysis of the product, it is acceptable to run the product test and the

suitability test concurrently. However, it should be noted that if the suitability

test is run concurrently with the product test and the suitability test should fail,

the results of the product test are invalid and the suitability test as well as the 

product test will need to be repeated with proper method modification to

neutralize the inhibiting property.

Neutralizing agents may be used to neutralize the activity of antimicrobial

agents in products, see USP <61> Table 2 for a list of potential neutralizing

agents/methods. The appropriate neutralizing agent should be added

preferably before sterilization of the media. Include a blank control with

neutralizer and without product to demonstrate efficacy and absence of toxicity

for microorganisms.
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USP <60>, <61> and <62> describe the suitability tests necessary for each

analysis. The correct inoculum of not more than 100 CFU is required in 

addition to specific incubation temperatures and durations. Ensure that seedlot cultures used are not more than five passages removed from the original

master seed-lot. For in-house prepared test strain suspensions of vegetative 

bacteria and yeast should be used within 2 hours, or within 24 hours if 

refrigerated between 2oC and 8oC. Spore suspensions (A. brasiliensis, B.

subtilis, and C. sporogenes) can be prepared and maintained between 2oC

and 8oC for up to seven days. Additionally, all bacterial and spore suspensions

should be prepared to yield ≤100 CFU. If commercially available ready-to-use 

bacteria or fungal suspension are used, then the manufacturer’s instructions 

should be followed with respect to preparation and storage requirements. USP 

<60>, <61> and <62> require a control which is without test material to be

included in the suitability test. The following viable ATCC derived cultures may

be used. Please be aware that under the Microbiological Examination of 

Nonsterile Products chapters <60>, <61> and <62> users are allowed

alternative sources of the below listed strains. The organisms below are

recommended for ORS use in order to have a consistent and standard

worksheet format:

USP <60>:

Burkholderia cepacian (ATCC 25416)

Burkholderia cenocepacia (ATCC BAA-245)

Burkholderia multivorans (ATCC BAA-247)

USP <61>:

Pseudomonas aeruginosa (ATCC 9027)

Staphylococcus aureus (ATCC 6538)

Bacillus subtilis (ATCC 6633)

Candida albicans (ATCC 10231)

Aspergillus brasiliensis (ATCC 16404)

USP <62>:

Pseudomonas aeruginosa (ATCC 9027)

Staphylococcus aureus (ATCC 6538)

Escherichia coli (ATCC 8739) 
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Salmonella enterica (ATCC 14028)

Candida albicans (ATCC 10231)

Clostridium sporogenes (ATCC 11437)

USP <60>, <61> and <62> each contain the acceptance criteria for their 

respective suitability test. USP <60> requires that the BCC microorganisms 

must be detected with the indicated reactions. Indicated reactions are 

indicated by the growth of greenish-brown colonies with yellow halos, or white 

colonies surrounded by a pink-red zone on BSCA. For USP <61> the Results

and Interpretation section requires the inoculated product to have a mean

count of any of the test organisms not differing by a factor greater than 2 from 

the control which was without test material. For example, if the control count is 

80 CFU the acceptable counts need to be greater or equal to 40 CFU. USP 

<62> requires the specified microorganisms to be detected with the indicated

reactions. 

E. Test Procedure

Prepare the sample in a manner to achieve a uniform solution or suspension.

This is critical because microbial contamination is not evenly dispersed

throughout a lot or sample of product. Use conventional mechanical and

shaking methods to the extent that original numbers and types of 

microorganisms are not altered in the product.

Use the following general procedures to prepare and handle samples.

1. Analyze samples as soon as possible after receipt. Inspect each

unit visually for integrity of primary container and note any

irregularities. Do not use the product container if it has been

compromised or damaged without supervisor approval. Testing of a

compromised or damaged container should be evaluated on a case

by case basis. Discuss with supervisor if compromised unit

containers need to be tested for forensic purposes (i.e. product 

tampering).

2. Identify units to be tested with Analyst's initials, date, subsample

number, and sample number.

3. Cleanse outer surfaces of sample containers with sterile wipes using

a validated effective antimicrobial agent. Place on a disinfected tray

or surface in a properly disinfected laminar flow hood or biosafety

cabinet. Allow containers to dry.

4. Aseptically open containers and perform weighing procedures in a

laminar flow hood or biological safety cabinet if possible.
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5. Appropriate environmental controls such as air exposure plates

should be used in accordance with local quality procedures.

6. Appropriate negative controls should be run concurrently with the

sample.

F. Interpretation of the Results

USP <60>

The possible presence of Bcc is indicated by the growth of greenish-brown 

colonies with yellow halos, or white colonies surrounded by pink-red zone on 

BCSA. Any growth on BCSA is confirmed by identification tests. Isolates 

should be identified to genus and, if possible, species using rapid identification 

kits or DNA sequencing. The product complies with test if no growth is 

observed or if the confirmatory identification tests are negative.

USP <61>

Regarding USP <61> the acceptance criterion for microbiological quality as it

pertains to quantitative analyses has an allowable variability of the final colony

forming units (CFUs). There is a two-fold tolerance in the final results. For 

example, if the monograph requires a 100 cfu/ml limit, the acceptable upper 

limit for these results would be 200 cfu/ml. Additional information is included in

the “Interpretation of the Results” section of USP <61> that should be read and

understood when reviewing quantitative test results.

USP <62>

Bile-Tolerant Gram-Negative 

The product complies with the test if there is no growth. Any isolates 

should be identified to genus and, if possible, species using rapid 

identification kits or DNA sequencing. 

Escherichia coli 

Growth of colonies indicates the possible presence of E. coli. This is 

confirmed by identification test. Isolates should be identified to genus 

and, if possible, species using rapid identification kits or DNA 

sequencing. The product complies with the test if no colonies are 

present or if the identification tests are negative. 

Salmonella

The possible presence of Salmonella is indicated by the growth of 

well-developed, red colonies, with or without black centers. This is 

confirmed by identification tests. Isolates should be identified to genus 

and, if possible, species using rapid identification kits or DNA 
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sequencing. The product complies with the test if no growth is 

observed or if the confirmatory identification tests are negative. 

Pseudomonas aeruginosa 

Growth of colonies indicates the possible presence of P. aeruginosa. 

This is confirmed by identification test. Isolates should be identified to 

genus and, if possible, species using rapid identification kits or DNA 

sequencing. The product complies with the test if no colonies are 

present or if the identification tests are negative.

Staphylococcus aureus 

The possible presence of S. aureus is indicated by the growth of 

yellow or white colonies surrounded by a yellow zone. This is 

confirmed by identification tests. Isolates should be identified to genus 

and, if possible, species using rapid identification kits or DNA 

sequencing. The product complies with the test if no growth is 

observed or if the confirmatory identification tests are negative. 

Clostridia 

The occurrence of anaerobic growth of rods with or without 

endospores, giving a negative catalase reaction indicates the 

presence of Clostridia. This is confirmed by identification tests. 

Isolates should be identified to genus and, if possible, species using 

rapid identification kits or DNA sequencing. The product complies with 

the test if no growth is observed or if the confirmatory identification 

tests are negative.

Candida albicans

Growth of white colonies may indicate the presence of C. albicans. 

This is confirmed by identification test. Isolates should be identified to 

genus and, if possible, species using rapid identification kits or DNA 

sequencing. The product complies with the test if no colonies are 

present or if the identification tests are negative.

*Note: Regarding USP <60> and <62>, each isolate should be identified to 

genus and, if possible, species using rapid identification kits or DNA 

sequencing.

7. Chapter 3: Sterility Testing

A. Method Suitability Test

For all product types, follow current USP methodology in <71>, with the 

following additional instructions.
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In all cases, even if the product does not include a preservative, the product 

itself may have growth inhibiting properties. All products should undergo a 

prescribed Method Suitability test.

Units selected for suitability testing should be subjected to the same 

disinfection procedure utilized in the sample analysis.

When developing the testing protocol for method suitability the volume of 

product as well as the concentration of the product should be evaluated such 

that the highest volume of product and the highest concentration of product 

should be used for the method suitability testing.

If multiple samples of the same product from the same manufacturer (same 

dosage and form) are collected, one sample may be used for method 

suitability for all the samples collected.

1. When to run Method Suitability:

a. Run the method suitability test prior to conducting the sterility test

in accordance with USP requirements under the following

conditions:

i. If insufficient information about the product exists to judge its

probable growth inhibiting activity.

ii. In all cases, when there is sufficient analytical time available,

i.e., survey type samples.

b. Run the method suitability test concurrently with product sterility

tests when time is critical, and problems associated with 1. above

have been resolved. However, it should be noted that if the

Method Suitability Test is run concurrently with the product

sterility test and the Method Suitability Test should fail, the

results of the product test are invalid and the Method Suitability

Test as well as the product test will need to be repeated with

proper method modification to neutralize the inhibiting property.

c. If an insufficient amount of product is collected and the analysis

is critical, the suitability test can be conducted at the end of the

14-day incubation period. Be sure to use best judgment and

maximum neutralization approach when initially conducting the

product sterility test. If the suitability results indicate inhibition

then the results, if negative, are invalid. However, if the product 

test results indicate microbial presence and the suitability test 

shows inhibition, the results are still valid.

2. Method Suitability Test Procedures
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Method Suitability and positive culture control tests which require the use of 

viable microorganisms, should be performed outside the clean room or 

isolator, in a biosafety cabinet or equivalent.

a. Membrane filtration

i. Pass product fluid through filter membrane. Rinse the

membrane with three 100 ml portions (or more if applicable)

of specified rinse fluid. Do not exceed a washing cycle of five

times 100mL per filter. This step hopefully will neutralize and

remove any antimicrobial residue on the filter membrane.

ii. Add indicated test organisms in specified numbers (less than

100 CFU) into the last 100 ml rinse fluid used.

iii. Filter the rinse fluid and aseptically cut the filter membrane

into two equal parts, transfer one half into each of two 

suitable media. If conducting the sterility test using a closed

canister system, rinse each canister with the inoculated rinse

fluid.

iv. If the available number of test vessels is insufficient for a

complete challenge test for each individual microorganism,

then the test organisms may be composited as necessary.

However, confirmation of growth for the composited

microorganisms will need to be performed.

v. Confirm composited microorganisms by Gram stain, 

microscopic examination, and identification after the

completion of incubation.

vi. See step c. below for additional considerations.

b. Direct inoculation:

For direct inoculation, add the test microorganisms to separate test vessels of

product and culture media if sufficient product is available. See step c. below

for additional considerations.

c. The following test procedures apply to Direct Inoculation and

Membrane Filtration:

i. Inoculate the same microorganism using the same medium 

without the product as a positive control.

ii. For bacteria and fungi, incubate test vessels according to

USP requirements. Ensure that seed-lot cultures used are not 

more than five passages removed from the original master 

seed-lot. For in-house prepared test strain suspensions of 

vegetative bacteria and yeast should be used within 2 hours,
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or within 24 hours if refrigerated between 2oC and 8oC. Spore

suspensions (A. brasiliensis, B. subtilis, and C. sporogenes) 

can be prepared and maintained between 2oC and 8oC for up 

to seven days. Additionally, all bacterial and spore 

suspensions should be prepared to yield ≤100 CFU.

iii. If growth comparable to that of the positive control vessel 

without product is obtained, then you may proceed with the

sterility test. If comparable visible growth is not obtained, the

antimicrobial activity of the product has not been eliminated

under the conditions of the test. Modify the test conditions

and repeat the Method Suitability test. 

d. If product is found to exhibit growth inhibiting activity when

determined concurrently with product testing, the sterility test

must be repeated using a neutralizing agent (or increase media

volume) to modify the conditions in order to eliminate the

antimicrobial activity. 

e. Cultures used for the method suitability test can be purchased

commercially, ready to use, or can be prepared and maintained

locally. Either procedure requires quantitative verification of actual 

CFU’s inoculated at time of use. 

B. Sample Analysis

1. Sample Containers

a. Open the outer sample packaging on a laboratory bench

disinfected with a sporicidal antimicrobial agent. Refer to 

appropriate literature for choosing suitable antimicrobial agents

for use in your facility.

b. Count the number of units received. Compare this number with

the number of units collected.

c. Inside the clean room preparation area located outside the ISO 5

area (if available) remove all outer packaging from subsample

units that will be tested without compromising the sterile integrity

of the product. Remove sample units and place them on a tray

or cart disinfected with an effective antimicrobial agent.

Note: One or more units can be sacrificed to aid in the 

determination for how to aseptically remove test material if the

number of the units received is sufficient.
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d. Examine all units visually for container closure integrity, for the

presence of any foreign matter in the product and other container 

closure defects. Note findings on analyst's worksheet.

e. If foreign matter is observed within the primary container, discuss

with supervisor the employment of ORS procedure Document 

ORA-LAB.015 entitled “Screening Protocol for Direct Staining on

Products with Appearance of Visible Contamination” (see QMiS

for Procedure).

2. Sample Identification

If sample units are not identified by the collector, the analyst should identify

unit with sample #, initials, date, and sub sample # as appropriate for sample

traceability. Otherwise, date and initial each unit.

3. Unit Container Disinfection

a. Cleanse the exterior of all product primary containers using

antimicrobial/sporicidal agents. 

Depending on the clean room design, immediately move the

sample to the clean room on a disinfected designated stainlesssteel cart or place it inside the clean room pass thru for final

preparation. If conducting the sterility test in an isolator, place the

sample on a designated stainless-steel cart. Allow exposure of

the sample to the disinfectant for appropriate time before further 

handling. All units should be disinfected appropriately. The

suggested disinfection procedures can be performed on

commonly encountered units as follows:

i. Ampoules can be wiped with lint free sterile towel/wipes

saturated with disinfectant. Ampoules may be soaked in

disinfectant/sporicidal following manufacturer’s guidance or 

laboratory SOP. 

ii. Vials should not be soaked due to the possibility of migration

of disinfectant under the closure and into the product.

iii. Laminated Tyvek package composed of polyethylene/plastic

laminate can be disinfected with sterile towel/wipes soaked in

disinfectant. Tyvek portion lightly scrubbed with sterile 

particle free dry wipe and air dry in a HEPA filtered laminar

flow hood before testing.

iv. Paper Packages can be disinfected with UV light if possible. 

Wipe where applicable with sterile particle free dry wipes and

air dry as above.
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b. Number of units and/or amount of product tested: 

Follow the current edition of the USP to determine the correct 

number of units to be tested and the amount of product to be

analyzed from each unit. It is preferable to test the entire

contents of each unit if possible. Follow laboratory policy if it 

requires testing more units than the USP requires.

c. If the number of units collected is less than the USP 

requirements, discuss with the laboratory supervisor before

proceeding. Samples collected in a for-cause situation may be

analyzed with a number of units less than the USP requirements.

C. Preparation for the Analysis

1. Media and Rinsing Fluid Preparation: 

Follow current USP when preparing media used for sample analysis. 

Commercially purchased media may also be used for the analysis.

Both prepared and purchased media must meet the requirements of 

the USP growth promotion test of aerobes, anaerobes and fungi.

Media used are:

a. Fluid Thioglycollate medium (FTM) This medium should be

prepared in a suitable container to provide a surface to depth

ratio so that not more than the upper half of the medium has

undergone a color change indicative of oxygen uptake at the end

of the incubation period. If more than the upper third of the

medium has acquired a pink color, the medium may be restored 

once by heating until the pink color disappears. Care should be

taken to prevent the ingress of non-sterile air during cooling.

b. Soybean Casein Digest medium (SCD medium) This media must 

be incubated under aerobic conditions

c. Alternative Thioglycollate medium This type of media must be

incubated under anaerobic conditions.

d. Media for Penicillin and Cephalosporin containing drugs. Add

sufficient quantity of sterile Beta-lactamase to the media to

inactivate the effect of these antibiotics.

e. Diluting and rinsing fluids. These fluid rinses may be filtered

before sterilization to avoid clogging of the filter membrane during

testing.

2. Media storage

For laboratory prepared media, do not use medium for longer 

storage period than has been validated.
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For commercially purchased media, follow the manufacturer’s

recommended storage requirements and expiration date. 

3. Media qualification: 

Perform the following tests on the prepared media before use: 

a. Sterility: The media batch may be used if the sterilization cycle is

validated and monitored with the use of a biological indicator, 

and the batch passes other quality control testing. Also, if

possible, prior to otherwise concurrently, incubate a portion of the

media at the specified temperature for 14 days.

b. Growth promotion test; follow the current USP using

recommended strains of organisms (Table 1, USP <71>). Do not 

use cultures that are more than five passages removed from the

original master seed lot. Commercially prepared and

standardized stable suspension cultures of the recommended

organisms can also be used. Test strains suspensions of 

vegetative bacteria or yeast should be used within 2 hours, or 

within 24 hours if refrigerated between 2oC and 8oC. Spore

suspensions (A. brasiliensis, B. subtilis, and C. sporogenes) 

refrigerated between 2oC and 8oC may be kept for a validated

period of time. If using commercially prepared organisms, follow 

the manufacturer’s instructions. Additionally, all bacterial and 

spore suspensions should be prepared to yield ≤100CFU. All 

bacterial counts must be verified at time of use.

4. Equipment Preparation

Analytical equipment and tools used in sterility analysis and

suitability should be cleaned and sterilized using a validated

sterilization procedure. Commercially purchased equipment and

tools should be labeled sterile and accompanied by a certificate of

analysis for sterility.

D. Clean Room Activities

1. Gowning

Personnel are critical to the maintenance of asepsis in the controlled 

environment. Thorough training in aseptic techniques is required. 

Personnel must maintain high standards each time they deal with 

sterile product. 

a. Personnel gowning qualification should be performed by any

analyst that enters the aseptic clean room. Personnel gowning

qualification must consist of:

i. Training of gowning techniques by a qualified trainer.
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ii. Observation of trainee by trainer while gowning.

iii. General growth media touch plates utilized to analyze if the

trainee gowned correctly without contaminating the sterile

outer gown, sterile gloves and sterile head cover.

b. Some consideration should be taken before entering the clean

room (see below). Follow applicable specific procedures for the

facility.

i. Proper gowning immediately prior to entry the clean room is 

required of all personnel without exception.

ii. Non-linting clean room scrubs that cover as much skin as 

possible is the ideal inner-suit to wear before gowning up for

an aseptic clean room. Street clothes are not permitted.

iii. Remove jewelry and makeup.

iv. Scrub hands (and arms when possible) before gowning.

v. Non-shedding sterile uniform components should be used all

the time.

vi. Use aseptic gowning procedure to don sterile uniform 

components.

vii. Care should be taken to choose gowning that does not 

expose any skin to the aseptic clean room environment.

viii.An appropriate sporicidal/disinfectant is used to sanitize the

gloves.

ix. If possible, post the gowning procedures in the gowning room 

or area to help individuals follow the correct order of gowning.

x. Should an analyst find it necessary to leave the room, he/she

should discard all gowning components and put on new ones

upon re-entry.

xi. If an individual scheduled to enter the clean room for analysis

feels sick or has compromised skin, he/she should talk to

his/her supervisor to postpone entry into the clean room until 

fully healed.

2. Sample Preparation

Repeat disinfection procedure using appropriate 

disinfectant/sporicidal immediately prior to placing product primary

containers in a working certified laminar flow hood. Allow all 

disinfected containers to completely air dry in the laminar flow hood

prior to opening for analysis. Alternatively, if conducting the testing in

an isolator, place the disinfected items into the isolator and proceed
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with the local procedures for the proper decontamination of the

interior of the isolator. 

3. Room Cleaning After Analysis

a. Remove inoculated tubes of media and all controls from the

analytical area by putting them in the pass-thru or on a stainlesssteel cart used for transporting materials in and out of the clean

room.

b. After analysis, all sample containers, equipment wrap, used

equipment and tools are to be removed from the clean room 

before the analyst exits.

c. Sample containers used in the analysis should be returned to the

original outer containers for storage as part of the reserve

sample.

d. Disinfect working area before exiting the clean room.

4. Clean room disinfection and surface monitoring must be conducted

for both aerobic and anaerobic microorganisms on a routine basis. 

The frequency is to be determined by the local laboratory policy. 

E. Method of Analysis

1. Membrane Filtration

Follow the current edition of the USP for the amount of sample to be

tested. 

2. Direct Inoculation

Follow the current edition of USP for the amount of sample and

media to be used. For example: Use 200 ml of each medium when

analyzing solid form products. If the membrane filter method is

unsuitable, certain liquids may be tested by direct inoculation

method.

3. Devices

All devices with only the pathways labeled as sterile are to be tested 

by the pathway with sterile Fluid D and testing the Fluid D via 

membrane filtration. 

4. Incubation of Sterility Test Media

a. Incubate Fluid Thioglycollate (THIO) at 32.5 ± 2.5oC. Do not

shake or swirl test media during incubation or during examination

to minimize aeration of this broth.
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b. Incubate Soybean-Casein Digest Broth (SCD) at 22.5 ± 2.5oC.

Gentle swirling, on occasion is acceptable to increase aeration of

media.

c. Incubation period for THIO and SCD:

i. Not less than 14 days except for products sterilized using

ionizing radiation. If tubes are not read on day 14 due to

holiday or weekend then record the results, even if positive,

on the first available day to observe the tubes.

ii. Additional incubation time may be warranted if the analyst is

made aware of sterilization processes other than heat or 

filtration (e.g. 30 days (at minimum) for products sterilized

using ionizing radiation). This is to allow repair of DNA of 

microorganisms injured by ionizing radiation, if any, that may 

be present).

F. Analysis of Medical Devices (ex. Purified Cotton, Gauze, Sutures and 

Surgical Dressings)

The USP method for analysis of surgical dressing/cotton/gauze (in packages) 

calls for a minimum quantity of 100 mg, to be tested in each medium. It is 

recommended that an entire unit shall be tested in each medium for individually 

packaged single-use articles.

1. Gauze, Purified Cotton, Sutures and Surgical Dressings

a. Using media containers as large as quart jars analyze entire unit

of product.

b. If unit is too large for the container, analyze as much of unit as

can be placed in container and covered by the medium.

2. Compositing of Medical Devices

a. Devices may be tested in composites (2 – 4 units/composite) as 

long as they meet the specifications of Chapter 71 of the current 

USP with regards to minimum quantity of a test unit and 

minimum number of units to be tested. All composited units must 

be the same lot number.

b. Devices may be composited only if they successfully pass the 

Method Suitability test. If composited units do not pass Method 

Suitability test, then the product cannot be composited.

G. Control Systems

The objective of a control system is to ensure the sterility, within designated

limits, of all items, media, rinsing fluids, and equipment used in a sterility test.
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The control systems which will accompany all sterility analyses are outlined

below.

1. System Control

A "system control" is used to demonstrate maintenance of sample integrity

during all analytical manipulations. Any piece of equipment that comes in

contact with the product under analysis, along with any manipulations by the

analysts, must be controlled. Thus, all equipment, fluids, and culture media for 

the "system control" must be handled in a manner which duplicates, as closely

as possible, the manipulations of the actual sample being analyzed. All 

materials used as system controls must be sterilized by the analyzing

laboratory. However, the method of sterilization need not be the same as for 

the product, but they must render the material sterile.

The first choice for the system control is the actual product, if enough test units

are available. When complex medical devices must be sacrificed in order to

design a suitable sterility test, consider using them for a system control after 

cleaning, repacking and sterilizing.

When there are viable alternatives, a product unit should not be sacrificed for

use as a system control if this will reduce the number of units available for

sterility testing below USP requirements or ORS policy requirements, except

as provided in the preceding paragraph. If using a product unit would reduce

the subsamples examined below the number required by USP or ORS policy,

the analyzing laboratory should prepare a control from other material than a

unit of the sample product whenever possible.

a. Membrane Filtration: A filter funnel from the vacuum source 

connection on each manifold used in the test is used for the

system control. Alternatively, if a closed canister system is used

to conduct the sterility test a canister set from the same lot used

during the analysis should be used for the system control.

i. Filterable Materials (liquids, soluble solids, etc.)

Use a material similar to the product under test. The control material must be

of the same volume, and similarly packaged as the test product. Filtersterilized and autoclaved Peptone water (USP Fluid A) may be useful for this 

purpose in many cases.

ii. Devices with sterile Fluid Pathway

Use tubing or other containers similarly fitted with needles, valves, connectors, 

etc., as the product under test. Use USP Fluid D to flush lumens.

b. Materials tested by direct inoculation (devices, insoluble solids,

and other non-filterable materials)
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Use materials similar in size, shape, and texture, and similarly packaged as 

product under test. Replicate as nearly as possible pertinent, unusual features

that may reflect on the credibility of the sterility test.

In designing "system controls" for sterility testing, care must be taken to 

duplicate the sample product for most aspects, as nearly as possible. Be 

novel and innovative to meet this requirement and make the system control 

meaningful.

2. Equipment Controls

All equipment items used in the analysis listed below will be controlled

individually. One item from each autoclave lot of equipment is tested in each

medium used in the test. Therefore, for a sample tested in THIO and SCD, 

one item from each sterilizer load (oven or autoclave) is tested in each medium 

giving a total of two controls for each forceps, syringe, etc., used in the test.

Forceps

Syringes

Scissors

Scalpels

Swabs

Pipettes

Membranes (dry, directly from the package). If membranes are sterilized in

place, this control may be omitted.

Hemostats

Other special items that may be required by a specific test.

3. Media and Rinse Fluid Controls

a. An uninoculated media and rinse fluid control are analyzed to 

ensure sterility at time of use.

Alternatively, controls for these materials are accomplished as part of the

"system control" for each manifold. This will also include membrane cutters,

and other items that contact the product but cannot be individually controlled.

4. Environmental Controls

a. Open Media Controls

Tubes of each medium (THIO and SCD) used in the sterility analysis are 

exposed to the immediate environment of the test (e.g., laminar flow hood) for 
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the duration of the test. Alternatively, a laboratory may use agar settling plates 

as detailed in section b.

b. Agar Settling Plates

Plastic Petri dishes containing an effective non-selective medium (based on

test requirements) are exposed in the hood for a period not to exceed four

hours during the analysis. After four hours, plates should be replaced to 

continue monitoring (as appropriate).

Plates should be incubated for 48 hours at 35o C, and an additional 5 days at 

25o

C in order to detect mold contamination.

c. Controls within an Isolator

When conducting the sterility test within an isolator, if it has been designed to

allow for a connection to an air sampler and particle counter this sampling may

be performed for the duration of the sample analysis in lieu of the

environmental samples described above. If the isolator is unable to 

accommodate an air sampler and/or particle counter or the instruments are 

unavailable the environmental controls described in section a. and b. should 

be used. Isolator gloves should be examined before and after a testing 

session to ensure integrity of the gloves were maintained. This examination 

should be documented. Additionally, prior to each decontamination cycle a 

leak test of the isolator system must be performed with passing results.

5. Personnel Monitoring

Personnel monitoring must be performed after analysts conclude sterility

testing and prior to exiting the aseptic clean room. The analyst shall use

general media touch plates to monitor the sterile condition of their clean room

attire and to ensure aseptic techniques were followed.

For example, a minimum of five touch plates should be used for the following 

personnel gowning sites:

RH glove finger tips.

LH glove finger tips.

Chest

Left Forearm

Right Forearm

General media touch plates will be incubated for 5 days at 30-35oC.

NOTE: The numerical values for personnel monitoring limits and specifications

are established on the basis of a review of actual findings within the facility. All 

isolates are to be identified by local laboratory procedure to ensure that the
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analyst did not contaminate the sample. Analysts should be sanitizing their

gloves throughout the sterility analysis and changing gloves when needed. 

However, changing gloves prior to performing personnel monitoring is

unacceptable. Each laboratory is required to monitor and trend data to ensure 

compliance and detect any abnormalities. 

H. Sub-culturing Primary Media

Daily observations of primary test media (THIO and SCD) containing product

should be performed without unnecessary disturbance. All handling of positive

tubes, streaked plates, or subsequent inoculations of additional media will be

done outside the clean room. These culture transfers are to be performed

within a HEPA filtered biosafety cabinet or equivalent outside the ISO5 area 

which has been cleansed with an effective sporicidal/disinfectant anti-microbial 

agent. The analyst should be gowned with at least sterile gloves, sterile 

sleeves and a mask to minimize any possible cross contamination.

1. Record on Analyst's worksheets the day that the primary isolation

media, Fluid Thioglycollate Broth (THIO), or Soybean-Casein Digest 

Broth (SCD) is turbid and inform supervisor. Streak tubes on the

day they first appear positive and again at 14 days to determine the

presence of other possible slow-growing (i.e., fungi) microorganisms.

2. Within a HEPA filtered biosafety cabinet or equivalent outside the

clean room, streak turbid tubes onto Modified Soybean-Casein

Digest Medium [SCD broth + 1.5% agar] (Modified SCDA) or other 

non-selective agar plate.

3. All streaked plates are incubated for a period at least as long as

required for growth in original isolation media (THIO or SCD) not to 

exceed seven days.

4. Subculturing from Fluid Thioglycollate Broth (THIO)

a. Subculture Thioglycollate broth to general medium agar plates in 

duplicate. Streak two plates; incubate one aerobically, and one

anaerobically, each at 32.5 ± 2.5 oC. NOTE: It is suggested to

transfer an aliquot of media from close to the bottom of the tube

to maximize the recovery of strict anaerobes. 

i. Note if any growth is observed on the anaerobic plate which 

differs from growth on the aerobic plate. Pick a single 

representative colony and perform an aero-tolerance test in 

order to determine if a strict anaerobe has been recovered. 

Proceed with identification of any strict anaerobes recovered 

when isolation is complete. 
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ii. Note if any growth is observed on aerobic plate and compare 

to growth on anaerobic plates. Proceed with identification 

when isolation is complete. 

5. Subculturing from Soybean Casein Digest broth (SCD)

a. Sub culture SCD broth to general growth medium and incubate 

aerobically. Streak one plate; incubate aerobically at 22.5 ± 2.5

oC.

i. Note if any growth is observed on general growth medium 

plate. Proceed with identification when isolation is complete. 

ii. Each organism should be identified to genus and species, if 

possible, using rapid identification kits or DNA sequencing. 

I. Product-Induced Turbidity in Primary Test Media

When product-induced turbidity prevents the confirmation of visual 

observation of growth, the following instructions apply 

1. Record "T" for any subsample which is turbid due to product-medium 

mixture. 

2. On the daily observation page, indicate the meaning of "T" as: "T = 

product induced turbidity”. 

3. At the end of the initial 14 days of incubation, transfer portions of the

medium (not less than 1 ml) to a fresh container of the same

medium and then incubate the original and transferred containers for 

not less than 4 days. Note: Follow the current edition of the USP for 

any changes concerning subculturing and incubation of turbid

samples.

4. Examine original product inoculated media and the subcultured

media for growth daily when possible for not less than 4 days of

incubation and record the results on a new daily observation

continuation sheet.

8. Chapter 4: Investigating USP Sterility Testing Failure

A. Introduction

When microbial growth is detected in a pharmaceutical or medical device 

product, the product is considered non-sterile, pending an investigation. 

Because of the public health importance of a non-sterility finding, preliminary 

results should be reported by your laboratory management, without delay, to 

ORS and the appropriate Center (e.g., Office of Compliance/OCTEC). 

Concurrently, a laboratory review should be conducted to answer the following 

question: Was the result true product contamination or was there a clear 
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laboratory error that caused contamination of the sample during the analysis? 

The Out-of-Specification (OOS) investigation will review and document

whether the test results are based on sound laboratory operation.

B. Investigations

Whenever a sterility positive occurs, lab supervisors are responsible for 

starting the investigation immediately. Four factors should be evaluated in the

basic investigation:

1. Equipment:

Determine whether equipment malfunctioned or was not operated properly. If

a malfunction occurred, determine whether it was likely to cause the

contamination. Determine if monitoring records and any checklists or logs 

indicate that the ISO 5 environment was in good state of control (serious 

environmental quality or equipment repair issues) at the time of the sterility

test. Be aware of the most likely failure modes in the equipment (e.g., laminar 

flow hood, glovebox, or isolator) used.

2. Adherence to Analytical Method:

Determine whether there were any anomalies or deviations from the analytical 

method. Adherence to method should be verified at the time of analysis, and

any major breach of sterility test procedure should also be documented at that

time. If any method breaches occurred, determine whether it was likely to

cause the contamination. Be aware of any possible weaknesses in the test

method (e.g., kit, manifold, etc.) used.

3. Analyst:

Evaluate the analyst’s qualifications, including proficiency, personnel 

monitoring results, training record, and experience. Also note whether the

sterility testing practice of the analyst was observed during this or a recent

analysis.

4. Cleanroom and ISO 5 (Class 100) Environmental Conditions:

Determine if disinfection/decontamination of the ISO 5 environment was 

properly done.

Determine whether there was adverse environmental data. Note that a 

negative control failure, on its own, is not necessarily cause for invalidating a 

result. If a negative control was contaminated, consider whether the microbe 

identified is similar to, or the same as, the sterility test isolate and also consider 

whether there are other adverse environmental trends. 

It is advisable to summarize this review process in a standard report and

maintain a sufficient record to reflect that these areas were investigated. In
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addition to the four considerations listed above, overall cleanroom design is 

also an important consideration. There are differences in the construction, 

configuration, and material flow of laboratory cleanrooms. There may be 

differences in size, number of rooms, shape, air handling system, pass through 

autoclaves, gowning room accommodations (sink, HEPA filtration, adequate 

space, bench, etc.). Proper practices and conditions should be assured by

mitigating contamination hazards potentially presented by layout and other 

design provisions. These include appropriate procedures for room and

material disinfection, proper cleanroom uniforms (disposable or reusable), 

sample preparation area, etc. Cleanroom risks can be mitigated by the design 

of ISO 5 testing environment. Equipment that provides barrier protection can

mitigate risks of the surrounding cleanroom environment.

If an investigation finds that the conduct of the analysis included errors or 

events that caused the test specimens to be contaminated by the lab

environment, the Sterility Test result would be invalid, and the substandard 

laboratory practice should be corrected to prevent this problem from recurring. 

For more information on how to judge investigational findings to make this

evaluation, see Section XI.C1.and 2 of FDA’s guidance on Sterile Drug

Products Produced by Aseptic Processing for principles and expectations for 

investigating a sterility positive.

http://www.fda.gov/downloads/Drugs/.../Guidances/ucm070342.pdf

Also see the current version of USP <71>, which provides some guidance

limited on investigations under Interpretation of Data.

C. Conclusion

This suggested list of areas and conditions to review should not be considered

as comprehensive. Additional areas of review may need to be added based on 

some of the unique features or procedures employed by individual ORS 

laboratories.

9. Chapter 5: Bacterial Endotoxin Testing

Bacterial endotoxin is a lipopolysaccharide found in the cell membrane of 

Gram-negative bacteria which may cause adverse events in patients such as, 

but not limited to fevers, headaches, inflammation, nausea, chills, vomiting, 

hypotension, lung toxicity, Toxic Anterior Segment Syndrome, abortion or 

death. Therefore, all sterile drugs, medical devices and combination products 

must meet bacterial endotoxin specifications. Historically, in vitro Limulus 

Amoebocyte Lysate (LAL) assays have been used to detect and quantify 

bacterial endotoxin in pharmaceutical products.
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NOTE: The Gel-Clot Method is considered the “referee method” per the USP. 

If there is a disagreement between the photometric turbidimetric or 

chromogenic methods, the gel-clot method results are reported.

This chapter of the Sterility Analytical Manual is intended to supplement the

methodology procedures found in the USP <85> BACTERIAL ENDOTOXINS

TEST and USP <161> MEDICAL DEVICES-BACTERIAL ENDOTOXIN AND 

PYROGEN TESTS. The USP <85> test is based on a reaction between 

bacterial endotoxin and the LAL reagent. Requirements for the LAL test 

includes optimal pH, ionic strength, temperature and incubation time. The USP 

<85> methods include the gel-clot LAL method and photometric technique 

methods such as the turbidimetric and chromogenic kinetic LAL methods. The 

USP <161> chapter describes the bacterial endotoxin extraction procedure for 

medical devices, if required. After the medical device extraction is performed, 

the USP <85> chapter is followed to detect and quantify any bacterial 

endotoxin present in the sample.

The Gel-Clot Method is a qualitative assay that detects Gram-negative 

bacterial endotoxin based upon a reaction between lysate and endotoxin which 

results in a firm clot formation. For samples with endotoxin, the endotoxin 

amount present in a test sample is calculated by diluting the sample to 

determine the assay endpoint where a clot does not form. If no clot forms in 

the verified dilution from the inhibition and enhancement testing, the sample 

does not contain detectable endotoxin. 

Photometric techniques include the turbidimetric and chromogenic endpoint 

and kinetic assays. The assays involve a change in turbidity or color 

(depending on the assay reagents used) over time for kinetic assays or at the 

end of the incubation period for endpoint assays. For kinetic assays, the onset

time for turbidity or color change is inversely proportional to the concentration

of endotoxin in the solution, i.e. more rapid change at higher endotoxin

concentrations. An instrument is used to read the changes. The analytical 

test results are calculated based on the linear relationship between the 

endotoxin concentration and the turbidity or color development. The standard 

curve plots the log onset time against the log endotoxin concentration. These 

assays are rapid and sensitive, allowing large numbers of samples to be

assayed quickly. However, the gel-clot method is the reference method per 

USP and should be used if there are any doubts or disputes, unless otherwise

indicated in the monograph for the product tested.

New microbiologists should review the references at the end of this chapter.

A. Gel-Clot Method

1. Reference Standard Endotoxin and Control Standard Endotoxins
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The potency of the control standard endotoxin (CSE) with respect to the

reference standard endotoxin (RSE) is determined by the CSE manufacturer.

This information is found in the associated package insert and need not be

repeated.

NOTE: Follow manufacturers’ recommendations for the storage, reconstitution,

and preparation of CSEs, lysates, and other Limulus Amoebocyte Lysate 

(LAL) reagents. In case of a dispute, final decision is based on results

obtained with the USP Endotoxin RSE.

The field laboratories are encouraged to mix the reconstituted CSE for at least 

5 minutes and for at least 1 minute between dilutions.

2. Negative Controls 

Run appropriate negative controls with each sample tested. This assures that 

the equipment and solutions used in the test contain no extraneous detectable

endotoxin. This assay requires two negative controls: negative water control 

and negative system accessory control. The negative water control (blank) 

consists of the pyrogen-free water (Water for BET or LAL Reagent Water) 

used in the assay and is tested undiluted. Run a system negative (accessory) 

control to test the detectable endotoxin level, if any, of accessories used in the 

assay (i.e. beaker full of pyrogen- free test tubes stored for an extended period, 

pipette tips, pipettes, sample tubes, syringes, spatulas, etc.). Rinse all 

accessories used in the assay with pyrogen-free water and test undiluted prior 

to completing all testing for a particular sample. If an alternative diluent is 

used, run an alternative diluent control prior to completing all testing for a 

particular sample. Record these results on the worksheets.

When using commercially purchased pyrogen-free water for product dilutions,

it is recommended to transfer a working volume from the original stock 

container to an individual pyrogen-free test tube or flask in order to minimize 

back contamination. Run a negative control for the working volume for each

sample run.

NOTE: Pyrogen free pipettes, micropipettor tips, test tubes, and other 

accessories are commercially available.

3. Test for Confirmation of Labeled LAL Reagent Sensitivity

Prior to use in the test, the labeled LAL reagent sensitivity must be confirmed. 

Prepare a control standard endotoxin dilution series having at least four 

concentrations equivalent to 2 λ, λ, 0.5 λ, and 0.25 λ. Inoculate four replicates 

from each control standard endotoxin tube with equal amounts of reconstituted 

lysate per manufacturer’s recommendation. Multiple dilution series are not 

required. The geometric mean of the endpoints must be within the limits of
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labeled claim. The acceptable variation is one half (0.5 λ) to two times (2 λ) the

labeled sensitivity (λ).

4. Inhibition or Enhancement Test/Test for Interfering Factors

The suitability of the test results for bacterial endotoxin require an adequate 

demonstration that specimens of the article or of solutions to which the test is

to be applied do NOT of themselves inhibit or enhance the reaction or 

otherwise interfere with the test.

USP states to perform this test “on aliquots of the specimen... in which there is

no detectable endotoxin”. However, this characteristic of the product cannot 

be ascertained prior to the analysis because the specimens are unknown

samples. Because of this limitation, any positive result below the 0.5 lambda

level may not be an enhancement trait of the product, but instead a positive

reaction due to contamination in the sample. The evidence for this conclusion

should be obvious with the results of the assay tubes containing product only.

A large percentage of small volume parenterals appear to be inhibitory to the

LAL gel- clot method because of low pH, or some excipient / active component

of the product. In order to expedite the neutralization of this interfering trait, 

determine the lowest product dilution overcoming the interference but still

within the Maximum Valid Dilution (MVD). The detailed description of this

protocol is delineated in LIB No. 2433 (July 25, 1980), “A condensed

procedure for diluting product in determining compatibility with the Limulus

Amebocyte Lysate test for endotoxin”. In addition, the use of neutralizers such

as sodium laurel sulfate or PyrosperseTM has also been described (see

references).

LAL manufacturers recommend the test sample to have a pH range of 6.0 to 

8.0 for optimal assay performance. Since the lysate is buffered, sample 

dilutions in pyrogen-free water may be enough to test the sample with the LAL 

assay. Determine the pH of the sample with the added lysate and document 

the results. If pH adjustment is needed, use pyrogen-free acid, base or buffers.

NOTE: Contact LAL manufacturers for recommendation of commercially

available neutralizing buffer to be used with their LAL kits.

5. Test Procedure

The storage and mixing of samples prior to analysis may affect recovery of

endotoxin contamination. Sample (product) bottles should be vigorously

shaken prior to analysis, preferably on a vortex (see reference for supporting

evidence for this step). A minimum of 30 seconds to 1 min on the vortex is

recommended for each product unit.

6. Endotoxin Calculation
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Calculate endotoxin concentration per the USP Bacterial Endotoxins test 

chapters <85> and <161>. For additional information refer to USP <1085>.

NOTE: Adjust the final endotoxin value taking into account the volume of the

rinse solution used in the extraction procedure.

7. Compositing Samples

The Bacterial Endotoxin test <85> does not directly address the issue of 

combining product units (compositing/pooling). The risk of unit composites is

that one unit (vial, ampoule, etc.) may have bacterial endotoxin contamination

at a higher level but the dilution of this one unit with endotoxin-free units of

product may reduce the detectable level of endotoxin below the sensitivity of

the lysate or dilute the level of endotoxin below the acceptable monograph

level. Therefore, when using a composite format for screening drug

products for endotoxin it is important to adjust the MVD calculation to

account for this reduced lysate sensitivity. Secondly, when compositing

is performed for product screening, if a positive result is detected a

repeat test is acceptable under the conditions stated by the Interpretation

section of the USP chapter.

It would be advisable when performing the repeat test from a composite

mixture that, if remaining product is available and had been opened aseptically

under controlled conditions, the repeat test be performed on the original 

individual units. It is strongly advised that the individual units be adequately

shaken to assure that the endotoxin is re-suspended back into solution before

taking the sample test aliquot. If any of the original individual units fail the

USP test at this point, the compendium does not allow any additional

repeat testing unless the test can be proven not to be suitable as defined

by the USP chapter.

8. Relevant excerpts from Guidance for Industry Pyrogen and

Endotoxin testing: Questions and Answers)

Question 1: Can FINISHED product units (vials, ampoules, pre-filled syringes,

etc.) be "Pooled" into a composite and screened for bacterial endotoxin?

Response 1:

Yes. With some exceptions (see below), finished drug product units may be

pooled into a composite sample and assayed for bacterial endotoxins. The

composite sample may be represented by the entire unit or partial aliquots

(equal volumes) of finished product containers from one manufactured lot of

aqueous-based pharmaceuticals. Pooling would generally be accepted for 

small-volume parenterals (those with volumes of 100 mL or less) as long as

the MVD is adjusted to a proportional, lower value because of the potential for

diluting a unit containing harmful levels of endotoxins with other units
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containing lower, less harmful, levels of endotoxins. This “adjusted MVD” is

obtained by dividing the MVD computed for an individual sample by the total 

number of samples to be pooled. FDA suggests pooling no more than three

units per composite in keeping with the concept of testing representative

beginning, middle, and end finished product containers. If this reduction in

MVD results in an inability to overcome product-related assay interference

because of an insufficient dilution, then the samples should be tested 

individually.

Finished medical devices may also be pooled into a composite sample and

assayed for bacterial endotoxins. Testing for medical devices should be

conducted using rinsing/eluting and sampling techniques as described in ISO

10993-1 and ISO 10993-12, as also used for inhibition/enhancement. 

Sampling can be adjusted for special situations. After a suitable eluate/extract

pool is obtained from a finished production lot, this pooled extract should be

kept under conditions appropriate for stability until it is tested in duplicate.

FDA recommends that pooled samples be a composite of aseptically removed 

aliquots (after at least 30 seconds of vigorous mixing) from each of the product 

containers. In this way, the original, individual containers will be available for 

possible retesting in the event the pooled sample displays an OOS result.

Some product types should not be pooled. Two examples are drug products

that have an initial low MVD (see discussion above of “adjusted MVD”) and

products that are manufactured as a suspension, because sample aliquot 

homogeneity may present significant interference issues.

Question 2: Can INTERMEDIATE (IN-PROCESS) sample aliquots be "pooled"

into a composite and screened for bacterial endotoxin?

Response 2:

FDA does not recommend pooling in-process samples from different inprocess stages of the manufacturing process because it may be difficult to

ensure the homogeneity of these materials.

Question 3: Retesting when test failure occurs:

Response 3:

When conflicting results occur within a test run, the analyst should consult USP 

Chapter <85>, Gel-Clot Limits Test, Interpretation, for guidance on repeat 

testing. As specified in Chapter <85>, if the test failure occurred at less than

the maximum valid dilution (MVD), the test should be repeated using a greater 

dilution not exceeding the MVD. A record of this failure should be included in

the laboratory results. If a test is performed at the MVD and an out-ofspecification (OOS) test result occurs that cannot be attributed to testing error, 
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continue product dilution until the actual endotoxin concentration can be

calculated. These results should be recorded on your worksheets.

B. Photometric Quantitative Techniques

Endpoint and kinetic assays are photometric quantitative assays used for the

detection of bacterial endotoxins. Endpoint and kinetic assays may utilize

turbidimetric or chromogenic formats. Endpoint assays measure endotoxin the 

increase in turbidity (turbidimetric) or color (chromogenic) at the end of the 

incubation time.

This section provides procedural information that can be applied to kinetic 

assays. The Kinetic Chromogenic and Turbidimetric reagents are 

commercially available. Assay may be purchased as a kit. A certificate of

analysis should be maintained for the control standard endotoxin, Limulus

Amebocyte Lysate (LAL), and pyrogen free water used in the assay. Other 

materials such as pyrogen free pipettes, micropipettor tips, test tubes, and 96-

well microplates may be purchased from various vendors. Certificates of 

analysis indicating these materials are pyrogen-free should also be maintained 

by the laboratory.

1. Kinetic Assays: The kinetic bacterial endotoxin detection software is

designed to run the following assays.

a. Initial qualification of a lysate/Initial qualification of the testing

analyst

b. RSE/CSE assesses potency of control standard endotoxin (CSE)

in terms of reference standard endotoxin (RSE) 

c. Test for Interfering Factors

d. Sample Test

e. Instrument Calibration Tests

The initial qualification assay verifies the proficiency of the analyst operating 

the Kinetic software and equipment. The initial qualification assay may also be

used to qualify each new lot of kinetic lysate and control standard endotoxin. 

Requirements of USP <85> must be followed with respect to the number of 

endotoxin standards and the number of replicates required for a valid assay.

The RSE/CSE assay may be used to compare the potency of the CSE with the

concentration of the RSE. Normally, the RSE/CSE assay does not need to be

performed, unless there is reason to believe the values in the manufacturer’s

certificate of analysis (COA) are not correct.

The test for interfering factors must be run for each sample.
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The routine assay is designed to test unknown samples for bacterial 

endotoxins. Samples collected for LAL analysis should be run using the 

routine assay, after taking the other three assays into consideration.

NOTE: Contact LAL manufacturer to qualify the kinetic readers (IQ/OQ/PQ) 

prior to use. Furthermore, laboratories should follow scheduled preventative 

maintenance (PM) and/or calibration to ensure the readers perform properly 

for regulatory sample testing.

2. Procedure

Perform the assay according to the instructions that are included with the LAL

test kit or LAL lysate, in accordance with USP <85>. Additional instructions

may be found in reference 8 listed below.

C. Medical Devices

This section applies to medical devices or assemblies or fluid pathways of 

medical devices or assemblies (i.e. solid medical devices such as disposable 

syringes, cartridges, transfusion and infusion assemblies, implants, 

intravenous catheters, dialysis tubing; liquid medical devices such as saline, 

heparin and dialysate; and gel medical devices such as demineralized bone 

matrices and hyaluronic acid devices) that are labeled sterile and 

nonpyrogenic that are in contact directly or indirectly with the cardiovascular 

system, lymphatic system or cerebrospinal fluid. Follow USP <161> for testing 

requirements

If only the device pathways are labeled as sterile and nonpyrogenic, the device 

pathways must come in contact with the extraction fluid for the entire course of 

the extraction per USP<161>. 

Liquid medical devices do not require extractions.

The standard extracting, rinsing and soaking fluid for medical devices is 

pyrogen-free water. The analytical approach for testing medical devices is

covered in USP. If the extraction fluid shows endotoxin interfering results, 

modification of testing may be necessary depending on the product 

configuration. Alternate diluents must be verified to not interfere with the 

endotoxin assay prior to use.

NOTE: The rinse or extract volume may be adjusted for the size and 

configuration of the device. The device or components may be cut into smaller 

pieces using sterile, nonpyrogenic equipment such as forceps, scissors, wire 

cutters, etc. prior to the extraction. 

A collaborated method prepared by an ORS laboratory is available for

extraction of endotoxin from devices and may be used if necessary. Analytical 

verification of the final version should be conducted by the responsible
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laboratory. The ORS laboratory protocol is summarized below for 

convenience.

Perform medical device extractions and final endotoxin concentration 

calculations per USP <161> and USP <1085> Guidelines on the Endotoxins 

Test.

Alternatively, if deemed necessary, perform the ORS laboratory modified 

extraction and analysis of Endotoxin from Medical Devices:

a. Preparation of 1% SLS solution

Prepare a 1% stock solution by placing one (1) gram of sodium lauryl sulfate 

(SLS) into a depyrogenated glass flask and add 99 ml of pyrogen-free water. 

Allow the SLS to fully dissolve. This should be followed by filtration through a

10,000 MW depyrogenation membrane filter into a pyrogen-free glass or

plastic container.

b. Equipment needed: Ultrasonic bath with a range of 150 to 480 

watts. 

c. Extraction procedure

i. Dilute 2mL of 1% SLS stock solution to 20mL (0.1%) using

LAL reagent water in a 20 x 150 mm screw-cap tube.

ii. Dilute 1.5mL of the 0.1% SLS solution to 15 mL (0.01%) using

LAL reagent water in a 20 x 150 mm screw-cap tube.

iii. Prepare the appropriate number of tubes (one tube for each

device) and one as a negative / system control. Preheat in a

water bath to 37°C ±1°C.

iv. Aseptically remove the device from its packaging and cut it 

diagonally into pieces less than 5mm in length. Small metal 

and plastic pieces such as needles and luer-locks should be

tested whole. Pyrogen-free fluid pathways should be flushed 

with extract solution.

Note: Extract volume may need to be adjusted depending on 

medical device size. 

v. Place all pieces into the 20 x 150 mm tube containing 15 mL

of preheated (37°C ±1°C) 0.01% SLS rinse solution.

vi. Vortex the tubes for 30 – 60 seconds or until all pieces of the

device are immersed in the rinse solution.

vii. Sonicate the test containers for 60 minutes (wattage range

150 – 480 watts) at 37°C ±1°C. Do not sonicate more tubes

than can be vortexed within 15 minutes of completion of the

sonication. Make sure the water in the sonicator covers the
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rinse solution in the 20 x 150 mm tubes. Do not allow the

water in the sonicator to exceed 38°C.

viii.Vortex the tubes for 2 minutes. Remove a portion of the

eluate (5 – 10 mL) for LAL testing. If the eluates are not 

tested immediately for endotoxin, they should be refrigerated. 

All eluates must be tested within 24 hours of extraction. Prior 

to analysis vortex at least one minute. 

2. Alternative Diluent Examples (Verify prior to use):

Diluent/Buffer Interfering Factor

PyrosperseTM Endotoxin Binding or Masking

Β-G-Blocker® Enhancement due to β-glucans

Tris Buffer 50mM solution Strongly Acidic (pH < 6) or Basic 

(pH > 8)

Magnesium chloride (MgCL2) 

10mM solution Chelating

Note: Always use LRW as the diluent for the CSE standard serial dilutions.

3. Test the sample eluate from the medical device extraction, using the 

bacterial endotoxins test (BET) assay parameters, procedures, 

standards, and controls for the gel-clot, kinetic chromogenic or 

turbidimetric, or endpoint assays, as directed in USP<85> and 

USP<161>.

D. Endotoxin References

1. United States Pharmacopeia (USP) Chapter <85> Bacteria 

Endotoxins Test. Official, Current Version.

2. USP <161> Medical Devices – Bacterial Endotoxin and Pyrogen 

Tests, Current 

Version.

3. USP <1085> Guidelines on the Endotoxins Test, Current Version.

4. The U.S. Food and Drug Administration, Guidance for Industry, 

Pyrogen and Endotoxins Testing: Questions and Answers, June 

2012

5. Guilfoyle, D.E., Yager, J.F. and S.L. Carito. 1989. The effect of 

refrigeration and mixing on detection of endotoxin in parenteral 

drugs using the Limulus Amebocyte Lysate (LAL) test. J. Parenteral 

Science & Technology. Vol 43, No 4, p. 183-187
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6. Novitsky, T.J., Schmidt-Gengebach, J. and J.F. Remillard. 1986. 

Factors affecting recovery of endotoxin adsorbed to container 

surfaces. J. Parenteral Science & Technology. Vol 40, No 6, P. 

284-286

7. Twohy, C.W., Duran, A.P. and J.T. Peeler. 1986. Extraction of

bacterial endotoxin from medical devices. J. Parenteral Science &

Technology. Vol 40, No 6, p. 287-291

8. Guilfoyle, D.E. and T.E. Munson. 1982. Procedures for improving

detection of endotoxin in products found incompatible for direct

analysis with Limulus amebocyte lysate, in Endotoxins and Their

Detection with the Limulus Amebocyte Lysate Test., Watson, S.W., 

Levin, J., and Novitsky, T.J. (eds), Alan R. Liss Inc., NY, p. 79-90

9. Guilfoyle, D.E., Munson, T. and J.P. Schrade. 1981. Use of

PyrosphereTM for Reducing Product Interference in the Limulus

Amebocyte Lysate Test, Laboratory Information Bulletin, U.S. Food

and Drug Administration, Washington D.C., No. 2503 (115), p. 1-8

10.Tepedino, A., Guilfoyle, D.E. and Munson, T. 1980. A Condensed

Procedure for Diluting Product in Determining Compatibility

(Inhibition / Enhancement test) with the Limulus Amebocyte Lysate

Test for Endotoxin, Laboratory Information Bulletin, U.S. Food and

Drug Administration, Washington D.C., No. 2433, p. 1-17

10. Chapter 6: Particulate Matter

This chapter is intended to supplement the methodology procedures found in

the USP <788> PARTICULATE MATTER IN INJECTIONS and USP <789> 

PARTICULATE MATTER IN OPHTHALMIC SOLUTIONS. In addition, USP 

<1788> DETERMINATION OF PARTICULATE MATTER provides useful 

guidance for these assays. Where appropriate the laboratory wide procedure, 

ORA-LAB.019 “HIAC 9703+ Liquid Particle Analyzer” is referenced to address 

specific requirements for the use of the HIAC instrument.

Particulate matter consists of mobile, randomly-sourced extraneous

substances, other than gas bubbles, that cannot be quantitated by chemical 

analysis due to the small amount of material that it represents and

heterogeneous composition. Injectable solutions, including solutions

constituted from sterile solids intended for parenteral use, are essentially free

from particulate matter observable on visual inspection. The tests described

herein are physical tests performed for the purpose of enumerating sub-visible 

extraneous particles within specific size ranges.
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All large-volume injections for single-dose infusion and those small-volume

injections for which the monographs or product specifications specify such

requirements are subject to the particulate matter limits set forth for the test 

being applied, unless otherwise specified in the individual monograph or 

product specification.

Not all injection formulations can be examined for particles using the light

obscuration method. Any product that is not a pure solution having clarity and

viscosity approximating those of water may provide erroneous data when

analyzed by the light obscuration counting method. Refer to specific

monographs when a question of test applicability occurs. The microscope

counting method may be used to analyze such materials. In some instances, 

the viscosity of a material to be tested may be sufficiently high so as to

preclude its analysis by either method. In this event, a quantitative dilution with

an appropriate diluent may be made to decrease viscosity, as necessary, to

allow the analysis to be performed.

A. Light Obscuration Particle Count Test

The test applies to large-volume injections labeled as containing more than

100 mL and single-dose or multiple-dose small-volume injections labeled as

containing 100 mL or less that are either in solution or in solution constituted

from sterile solids, where a test for particulate matter is specified in the

individual monograph or drug product specification. It counts suspended

particles that are solid or liquid. 

1. Test Apparatus

The apparatus is an electronic, liquid-borne particle counting system that uses

a light- obscuration sensor with a suitable sample feeding device. Critical 

operational criteria consist of the following:

a. Sensor Concentration Limits

Use an instrument that has a concentration limit (the maximum number of 

particles per ml) identified by the manufacturer that is greater than the 

concentration of particles in the test specimen to be counted. 

b. Sensor Dynamic Range

The dynamic range of the instrument used (range of sizes of particles that can

be accurately sized and counted) must include the smallest particle size to be

enumerated in the test articles.

2. Instrument Calibration

The instrument must be calibrated periodically according to the manufacturer’s 

recommendation. The following are parameters that should be evaluated as 
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part of the periodic instrument calibration in addition to other tests 

recommended or routinely performed by the instrument manufacturer.

a. Sample Volume Accuracy

The accuracy of the sample volume must be assessed and found to be within 

the manufacturer’s recommended range. 

b. Sample Flow Rate

Verify that the flow rate is within the manufacturer's specifications for the

sensor used.

c. Calibration and Resolution of the sensor

Verify that the sensor resolution is within the manufacturer’s specifications for 

spherical particles of known size between 10μm and 25μm.

3. Test Environment

a. Glassware, closures and other equipment must be suitably 

cleaned so that the level of particles added has a negligible effect 

on the outcome of the test. 

Note: Glassware can be purchased in particle free packages 

which are a suitable particulate free vehicle for compositing 

liquid samples.

b. Perform the test in an environment that does not contribute any 

significant amount of particulate matter. Preparation of the test 

specimen, glassware, closers and other equipment should be 

performed in an environment protected by HEPA filters. 

c. The analyst should don non-shedding garments and powder-free

gloves throughout the preparation of samples.

4. Instrument Start-up and Environment Blank

a. Following instrument start-up perform an instrument flush using 

Milli-Q water or equivalent in accordance with ORA-LAB-.019. 

b. Prior to beginning a test procedure an environment blank must 

be prepared and tested according to ORA-LAB.019. After 

passing results have been obtained the instrument can now be 

used to perform the test procedure.

5. Test Procedure

a. For containers having volumes of less than 25 mL, test a solution 

pool of 10 or more units to obtain a volume of no less than 25 
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mL. Single units of small-volume injections may be tested 

individually if the individual unit volume is 25 mL or greater.

b. Prepare the test specimens as described in USP <788> and 

<789>. Following the preparation of the test specimen the 

sample can be assayed in accordance with USP <788> and 

<789> in addition to the procedures found in ORA-LAB.019.

c. Containers with removable stoppers may be sampled directly by 

removing the closure. For test specimens that require the 

contents of the container to be removed for testing withdraw the 

contents of the container in the normal or customary manner of 

use, or as instructed in the package labeling. When test 

specimens are to be pooled remove the closure and empty the 

contents into a suitably cleaned container (preferably from 

particle free glassware vendor).

d. Following the completion of the test the instrument will generate 

a report. The report will include the raw data counts, calculated 

values and state if the test specimen met the USP limits for the 

particular test performed.

Note: If the average number of particles exceeds the USP limits 

the prepared specimen must be tested by the Microscopic 

Particle Count Test.

B. Microscopic Particle Count Test

The microscope particulate matter test may be applied to large-volume and 

small volume parenteral injections and to ophthalmic solution products.

The test Apparatus is described in USP <788> with additional information 

found in USP <1788>.

1. Test Environment and Environmental Blank:

Refer to Section A.3 for the requirements of the test environment and 

preparation of the specimen, glassware and equipment used in the assay.

Prior to initiating the test sequence with a specimen, a blank determination is 

required and must be carried out according to USP <788>. The environmental 

blank must meet the requirements set forth in USP <788> in order to initiate 

testing of the specimen.

2. Test Procedure and Interpretation of Data

a. For large volume parenterals, single units are tested. For small 

volume parenterals less than 25 ml in volume, the contents of 10 

or more units are combined in a cleaned container; the test 
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solution may be prepared by mixing the contents of a suitable 

number of vials and diluting 25 ml with particle-free water or with 

an appropriate particle-free solvent when particle-free water is 

not suitable. Small volume parenterals having a volume of 25 ml 

or more may be tested individually.

b. Proceed with the test as delineated in USP <788> recording the 

number of particles that are equal to or greater than 10μm and 

the number of particles that are equal to or greater than 25μm. 

As an alternative a partial membrane filter count and 

determination of the total filter count by calculation is allowed. 

Once a particle count is determined the mean number of 

particles for the examined specimen is calculated. Note: For test 

specimens which are covered by USP <789> the number of 

particles that are equal to or greater than 50μm must also be 

counted and a calculated average reported. 

c. The calculated data is evaluated according to USP <788> or 

USP <789> considering the solution type and the container 

volume.

11. Chapter 7: Antibiotic Potency Testing

A. General Information

USP <81> Antibiotics- Microbial Assays, is the primary reference for bioassay 

of human antibiotic potency testing. Prior to testing, the current USP <81> and 

respective drug monograph should be reviewed for testing requirements, 

acceptance criteria and applicability. USP <81> lists specific human antibiotics 

to be tested for microbial potency; this list can change at any time. All other 

human antibiotics not listed in USP <81> are typically tested using CFR 21 

Parts 300-499 or USP HPLC methods. Lastly, animal antibiotics are typically 

tested using chemistry methods as published in JOAC, AOAC, Laboratory 

Information Bulletins (LIBs) and/or manufacturer’s methodology. 

Improved manufacturing technology (e.g. purification methods) has evolved 

potency testing from a simple biological assay to different chemical assays. 

Most chemical assays are based in the segregation and quantification of 

antibiotic components through the use of high-performance liquid 

chromatography (HPLC). However, chemical assays do not demonstrate 

biological activity and antimicrobial efficacy of a test antibiotic, particularly an 

antibiotic which may contain numerous active components, each exhibiting 

different antimicrobial activities. Chemical potency assay does not require the 

use of a live test microorganism. For the remainder of this chapter, antibiotic 

potency refers to USP <81> testing only. 
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Antibiotic potency testing is a biological assay whereby varying concentrations 

of antibiotic are tested against a live microorganism. The resulting biological 

response is measured and evaluated against a median reference standard 

[S3] and standard curve [S1], [S2], [S4] and [S5]. The biological response is 

referred to as antibiotic activity or potency. Antibiotic potency is dependent 

upon antibiotic-microorganism specificity and is physically expressed by the 

inability of a microorganism to grow under optimal conditions in the presence 

of a specific test antibiotic. 

Antibiotic potency testing is a multi-variable test dependent on a variety of 

factors. Factors may include: 1) Test microorganism growth requirements and 

inoculum levels 2) Test antibiotic dose and 3) Technical competency in 

preparation and/or use of equipment, growth media, reagent, test organism 

and antibiotic standards. Potency testing requires a basic knowledge of 

laboratory safety, analytical chemistry, microbiology and aseptic techniques. 

Potency testing is a manual, multi-step and multi-day process performed with 

common laboratory equipment. At minimum, two employees are required for 

preparation and sample setup. Due to the multiple stages of preparation and 

testing, only qualified (i.e. initial and routine trainings and evaluations) 

laboratory personnel should be authorized to perform USP <81> testing. 

Antibiotic potency testing is performed either by the plate (cylinder-plate or 

diffusion) or tube (turbidimetric) method. Both plate and tube methods 

demonstrate measurable levels of growth inhibition. For example, zones of 

inhibition (ZOIs) are observed and measured during cylinder-plate testing. 

And, turbidity is observed and measured during tube testing. Growth inhibition 

measurements are tabulated and integrated into a linear regression curve, 

resulting in extrapolated antibiotic potency values. 

Potency is denoted in units (U) or μg of activity and may or may not be exact in 

equivalence to the μg (weight) of the active compound. The following three 

reasons may explain this weight-activity discrepancy: 1) activity may be 

caused by the antibiotic’s free base or salt form and activity is denoted in either 

form 2) the antibiotic may contain similar chemical components but differ in 

activity or 3) the antibiotic activity is represented by a heterogeneous family of 

antibiotics and not a single analog. 

An internal quality control is built into each plate and tube tested. Since 

antibiotics have different listed dosages per label, the test antibiotic/unknown 

sample is diluted to a known sample concentration ([U3]). The unknown 

sample [U3] has an equivalent concentration of the median reference standard 

([S3]). The median reference standard ([S3]) is the median concentration (i.e. 

mid-point) of the five-point standard curve ([S1], [S2], [S4] and [S5]). The five-
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point standard functions as the testable detection range and is prepared from a 

traceable and within expiry USP Reference Standard (RS). 

The USP <81> states the following criteria for both the Plate and Tube 

Methods: 

1. The calculated potency of the test antibiotic/unknown sample ([U3]) 

must be 80% to 125% of the median reference standard ([S3]);

2. Relative standard deviation for all measured (i.e. millimeters or 

absorbance) and calculated data (e.g. averages) is NMT 10%; and, 

3. Testing is performed in triplicate over a period of three independent 

test runs 

The USP <81> Plate Method further states: 

1. The percentage coefficient of determinations (%R2) for each standard 

curve will be NLT 95% (i.e. correlation coefficient of NLT 0.9750); and, 

2. ZOIs for all media reference standard ([S3] will measure between 14-16 

mm). 

The USP <81> Tube Method further states: 

1. The percentage coefficient of determinations (%R2) for each standard 

curve will be NLT 90% (i.e. correlation coefficient of NLT 0.950); and, 

2. Absorbance values of the media reference standard ([S3]) are 

predetermined per antibiotic. Refer USP <81> for testing parameters and 

acceptable data requirements. 

B. Equipment

Antibiotic potency testing is a quantitative test dependent upon the preparation 

and use of growth media, reagents, test organism and antibiotic standards; 

therefore, Class A volumetric glassware is used in the preparation of the test 

antibiotic/unknown sample, reference standard and dilutions of the reference 

standard. Class A glassware is physically labeled either “to deliver” (TD) or “to

contain” (TC). An understanding of Class A glassware prior to laboratory use is 

required. For example, a high viscous liquid (e.g. antibiotic ointment) cannot be 

delivered from a “to deliver” Class A pipette. “To deliver” Class A pipettes 

solely rely on gravity to assist in evacuation. In the case of the antibiotic 

ointment, the ointment would remain in the Class A pipette at time of 

evacuation. However, USP <81> states sterile and disposable glassware can 

also be used for any measurement of test antibiotic/unknown sample,

reference standard and dilutions of the reference standard.

Standard laboratory glassware such as beakers, funnels, flasks, roux and 1-2

liter bottles are also required. Sterile and disposable equipment such as test 
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tubes, petri-plates and serological pipettes may be used so long as the use of 

this equipment does not affect the quantitative aspect of potency testing. 

Additional equipment includes a stainless steel penicylinders, penicylinder 

dropper, cuvettes, pipettor and micropipette, pH meter, hot plate, adjustabletemperature water bath, incubator and a manual/automatic plate reader or UVVIS spectrophotometer. 

Equipment in direct contact with the test microorganism should be clean (i.e. 

residue free) and sterile. Residues (e.g. antibiotic or detergent) may interfere 

with antibiotic potency testing. Methods of equipment cleaning and dry and 

heat sterilization should have appropriate validation and verification checks to 

ensure glassware is clean and sterile. See USP <81> for penicylinder cleaning 

instructions and USP <1051> Cleaning Glass Apparatus, for glassware 

cleaning instructions. 

Equipment used to provide a unit of measurement should have the appropriate 

validation and frequent verification checks to ensure reliable and reproducible 

results; examples of such equipment include a weight scale, pH meter, 

autoclave, micrometer, manual/automatic plate reader and UV-VIS 

spectrophotometer. For more information on manual and automatic plate 

readers, see section titled Antibiotic Potency Testing: Plate Method. 

Testing can be performed on a laboratory benchtop and does not require setup 

in a clean room or a laminar flow hood. However, it is important to exercise 

aseptic technique when working with general growth media and test organisms 

to prevent cross contamination. 

When performing the plate method, it is important to use a laboratory bench 

top that has been checked with a level. The bench top will be used to prepare 

single and bi-layer agar plates. If the bench top is not level, liquid agar could 

potentially pool unevenly within a petri plate. Unevenly distributed agar causes 

uneven antibiotic diffusion. Zones of inhibition formed after diffusion should be 

uniform in shape (i.e. circular). Irregular shaped ZOIs do not have a diameter 

that can be measured accurately. Irregular shapes include ovoid, elliptical 

and/or any shape without a uniform and defined perimeter. 

C. Test Organism, Inoculum Preparation and Standardization

Antibiotic potency is dependent upon antibiotic-microorganism specificity. USP 

<81> identifies specific microorganisms and correlating antibiotics for testing. 

Prior to use in test, the test microorganism must be characterized as pure and 

robust. Primary and working cultures must be aseptically prepared and 

dedicated to preventing contamination of the primary test microorganism. If the 

primary and/or working culture becomes contaminated, perform a visual 

inspection and basic microscopy for typical growth characteristics and 

morphology. Additionally, AOAC approved rapid identity testing methods such 
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as API or VITEK should be performed. Contaminated primary and/or working 

cultures cannot be used for antibiotic testing; likewise, all antibiotic potency 

data generated using contaminated cultures will be considered as invalid. 

A test inoculum is prepared from a working culture. Inoculum preparation is a 

multi-step and multi-day process. Preparation of the inoculum requires a basic 

knowledge of microbiology, aseptic technique and laboratory safety. Refer to 

USP <81> Antibiotics- Microbial Assays, for test organism and inoculum 

preparation. The test inoculum is a solution containing the live test 

microorganism diluted with a method specific diluent. To be viable, the 

microorganisms used in the test inoculum must be within 5 passages of the 

primary test microorganism. 

Verification of the test inoculum is performed prior to sample testing. 

Verification is a preliminary test which evaluates the potency, purity and 

robustness of the test inoculum when challenged against a known median 

reference standard ([S3]) and standard curve ([S1], [S2], [S4] and [S5]). Refer to 

USP <81> for inoculum verification, testing parameters and acceptable data 

requirements and Section D, Antibiotic Standard and Sample Solution 

Preparation. As previously stated, acceptance testing requirements and 

acceptance criteria are as follows:

The USP <81> states the following criteria for both the Plate and Tube 

Methods: 

1. The calculated potency of the test antibiotic/unknown samples ([U3]) 

must be 80% to 125% of the median reference standard ([S3]);

2. Relative standard deviation for all measured (i.e. millimeters or 

absorbance) and calculated data (e.g. averages) is NMT 10%; and, 

3. Testing is performed in triplicate over a period of three independent 

test runs 

The USP <81> Plate Method further states: 

1. The percentage coefficient of determinations (%R2) for each standard 

curve will be NLT 95% (i.e. correlation coefficient of NLT 0.9750); and, 

2. ZOIs for all media reference standard ([S3] will measure between 14-16 

mm). 

The USP <81> Tube Method further states: 

1. The percentage coefficient of determinations (%R2) for each standard 

curve will be NLT 90% (i.e. correlation coefficient of NLT 0.950); and, 
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2. Absorbance values of the media reference standard ([S3]) are 

predetermined per antibiotic. Refer USP <81> for testing parameters and 

acceptable data requirements. 

Primary and working cultures should be identified with the microorganism 

specie name, ATCC (American Type Culture Collection) number and 

preparation and expiration date. Recommended storage requirements for 

primary and working inoculum stock are specified in USP <81>. To reduce 

assay variability, a primary stock culture of ≤ 14 days should be used to 

prepare a working stock culture; also, a working stock culture of ≤ 7 days 

should be used to prepare a test inoculum. The test microorganism bioactivity 

is known to decrease over time and adjustments to the test inoculum volume 

may be required to meet testing acceptance criteria (e.g. ZOI diameters 

between 14-16 mm as exhibited by the median reference standard ([S3]). 

Therefore, to reduce assay variability, it is recommended to use fresh (e.g. ≤ 2 

day old) primary and working cultures when possible.

D. Antibiotic Reference Standards ([S1] – [S5]) and Unknown Sample (U3])

Preparation

Antibiotic potency testing requires the use of a standard curve to test an 

unknown sample. The reference standard (RS) must be from a verified source 

such as U.S. Pharmacopeia (USP). Procedures for reference standard 

preparation can be found in USP <81>. A single RS will be used to prepare a 

five-point ([S1] – [S5]) standard curve. Typically, the five standard solutions 

increase in concentration by a ratio of 1:1.25. For example, [S1] – [S5] 

standards can be represented in test as the following standard concentrations: 

6.40 μg/mL, 8.00 μg/mL, 10.0 μg/mL, 12.5 μg/mL and 15.6 μg/mL. See 

Diagram 1 for further details. Labeling accompanying the RS will contain 

preparation, storage and expiration information; this information contains 

specific handling instructions and should be followed in order to achieve 

reproducible and reliable potency test data. A new standard curve must be 

prepared each day of inoculum verification (i.e. test plates) and/or each of the 

three independent test runs. 

Antibiotic potency samples will vary in physical form, dose and administration; 

examples of differing sample types include tablets, powders, solutions or semisolids. Regardless of these physical and chemical attributes, the test sample 

and reference standard must be diluted prior to testing. Each dilution for the 

test sample and reference standard must be considered. The dilution factor 

(for each dilution) and the total dilution (for multiple dilutions) are data required 

to calculate potency. 
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Diagram 1. Example of the Plate Method and placement of the standard curve 

and unknown sample onto triplicate agar plates. 6/19/17 MBB.

Prior to testing, the unknown sample [U3] must be diluted to a known 

concentration. See USP <81> for the list of recommended concentrations of 

median reference standards used in test. The target concentration is typically 

equivalent to the median reference standard [S3] of the standard curve. 

Diluting the unknown sample [U3] in this manner ensures a detection limit 

within the linear portion of the standard curve. For example, if the median 

reference standard [S3] has a concentration of 10.0 ug/mL, the unknown 

sample [U3] will also be diluted to a concentration of 10.0 ug/mL. See Diagram 

1 for an example of one independent test run for the Plate Method and 

placement of the standard curve and unknown sample onto triplicate agar 

plates. 

Quality control (QC) plates may include a test of respective diluents used for 

the reference, standard curve ([S1]-[S5]) and/or test/unknown sample [U3]. 

One of three QC plates should be dedicated for the evaluation of the inoculum 

used in test; specifically, this plate will only contain the test inoculum, will be 

free of penicylinders and should exhibit uniform growth (i.e. lawn) on top and 

within the agar. Microbial growth other than the lawn indicates the inoculum 

used in test may be contaminated and/or the technique used in preparing the 

single and agar plates may have been compromised the agar plates. The 

remaining two of three QC plates should be dedicated to each specific diluent 

used in test; for example, Water for Injection (WFI) and Buffer No. 4 are used 

in Vancomycin testing and therefore two QC plates, each containing WFI or 
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Buffer No. 4 are prepared. Diluents are specific to each antibiotic and are listed 

in USP <81>.

E. Growth Media and Additional Test Solutions

In order to culture a pure and robust test organism, proper use and preparation 

of growth media (agar or broth), buffers and diluents is required. Components 

or final preparations of growth media, reagents or diluents may be prepared 

on-site and/or purchased from outside sources. Regardless of origin, any final 

product made from growth media components and/or preparations must be 

verified for identification, expiry, sterility and growth promotion, prior to use in 

test. Refer to USP <81> for preparation, use and storage of all growth media, 

buffers and diluents.

F. Antibiotic Potency Testing: Plate Method 

The Plate Method is the most commonly used USP <81> test. The Plate 

Method uses a solid medium (agar) to demonstrate antibiotic activity (i.e. 

zones of inhibitions (ZOIs)). This method requires the use of a five-point 

standard curve [S1] – [S5], median reference standard [S3], test antibiotic/

unknown sample [U3], verified inoculum, stainless steel penicylinders and 

petri-plates containing growth agar. See Diagram 1 for an example of one 

independent test run for the Plate Method and placement of the standard curve 

and unknown sample onto triplicate agar plates. 

Depending upon the antibiotic, either a single layer or bi-layer agar plates are 

used in test. See USP <81> for preparation for single and bi-layer agar plates. 

1. Single Layer Plates: Prior to solidification, the growth agar is 

inoculated with a known volume of the verified test microorganism. 

The inoculated agar is thoroughly mixed, poured into a petri-plate 

and allowed to solidify. 

2. Bi-layer Plates: Prior to solidification, a portion of the growth agar is 

poured into the base of a petri-plate and allowed to solidify; this is 

the base layer of the test plate. The remaining portion of growth agar 

is further cooled and inoculated with a known volume of verified test 

organism. This inoculated agar is thoroughly mixed, poured onto the 

cooled base layer and allowed to solidify. 

Once the agar solidifies (either single layer or bi-layer agar plate), stainless 

steel penicylinders are applied to the agar surface using a penicylinder 

dispenser. Penicylinders are applied in an equidistant and upright fashion. 

Each penicylinder should be immediately dosed with two concentrations of 

antibiotic, the diluted unknown sample [U3] or the median reference standard 

(S3). Further, both antibiotics are dosed in equal volume into alternating 

penicylinders. See Diagram 2 for further details. 
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Diagram 2. Example of plated and dosed penicylinders containing the diluted unknown 

sample and median reference standard. 6/19/17 MBB.

After dosing, all plates prepared in one independent test run are incubated at 

the same time. As the plates incubate, the antibiotics diffuse through the agar, 

creating a zone of clearing below and around penicylinder perimeter; this 

clearing is referred to as the zone of inhibition (ZOI). The ZOI demonstrates 

the antibiotic activity of the diluted unknown sample [U3] and the median 

reference standard [S3]. See Diagram 3 for further details. 

Diagram 3. Example of plated and dosed penicylinders on incubated plates 

showing zones of inhibition. 6/19/17 MBB.
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After the specified incubation time, the penicylinders are removed, 

decontaminated (i.e. autoclaved) and washed with soap and water. The 

penicylinders are then heat sterilized prior to re-use in test. The diameter of 

each ZOI is measured with a manual/automatic plate reader. ZOIs should only 

be measured by qualified laboratory personnel. ZOI measurements are taken 

with the use of a manual or automatic plate reader. Examples of a manual 

reader include a Fisher-Lily zone reader or manual/electronic calipers; manual 

reads require data entry (e.g. handwritten and/or electronic) onto hardcopy 

and/or into electronic logbooks and/or spreadsheets. Examples of automated 

plate readers include OMNICON or Trinity V3. Unlike manual reads, 

automated plate readers allow a computerized system to measure the plates

and do not require data entry of raw data.

Diagram 4. Example of a plated diluted unknown sample and median 

reference standard exhibiting equivalent ZOI diameters. 6/19/17 MBB.

The zones of inhibition exhibited by the unknown sample [U3] and the median 

reference standard [S3] should be approximately equivalent. See Diagram 4 

for further details. The ZOI comparison between the diluted unknown [U3] and 

the median reference standard [S3] can be described mathematically as the 

percentage of reference concentration. The calculated potency of the unknown 

sample [U3] must have a percentage of reference concentration of 80% -

125%. If the concentration of the diluted unknown sample [U3] falls outside this 

range of 80%-125%, the same unknown sample must be retested using an 

estimated dilution to obtain an equivalent concentration of the median 

reference standard [S3]. Diluting the unknown sample [U3] in this manner 
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ensures a final detection limit within the linear portion of the standard curve. 

Test sample preparation, storage and expiration require the dose and 

administration information (as per label claim) and procedures prescribed in 

USP <81> and the appropriate USP antibiotic monograph. 

USP <81> states the following criteria for both the Plate and Tube Methods: 1) 

The calculated potency of the test antibiotic [U3] must be 80% to 125% of the 

median reference standard [S3] 2) Relative standard deviation for all 

measured and calculated data is NMT 10% and 3) Testing is performed in 

triplicate over a period of three independent test runs. The Plate Method 

further states: 1) The percentage coefficient of determinations (%R2) for each 

standard curve will be NLT 95% (i.e. correlation coefficient of NLT 0.9750) and 

2) ZOIs for all media reference standard (S3 will measure between 14-16 mm). 

Refer to USP <81> for testing parameters and acceptable data requirements. 

Diagram 5 below summarizes the operations prior, during and after testing 

using the Plate (Diffusion) Method (Hewitt and Vincent 1989).
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Diagram 5. Example of operations conducted prior, during and after testing 

using the Pate (Diffusion) Method (Hewitt and Vencent 1989). 

G. Antibiotic Potency Testing: Tube Method

Of the two USP <81> methods, the Tube Method is less commonly used. The 

tube method uses a liquid medium (growth broth) to demonstrate antibiotic 

activity. This method requires the use of a test antibiotic/unknown [U3], fivepoint (minimum) standard curve ([S1]-[S5]) and verified inoculum. Growth 

inhibition is measured by qualified laboratory personnel with the use of a UVVIS spectrophotometer. Test antibiotic will be analyzed in triplicate over a 

period of three independent test runs. Refer to USP <81> for Tube Method 

procedures.
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Test antibiotic/unknown [U3] tubes will contain the growth broth, inoculum and 

the test antibiotic/unknown [U3] in triplicate. The standard curve ([S1]-[S5]) 

tubes will contain the growth broth, inoculum and each of the respective 

standard curve concentrations [S1], [S2], [S3], [S4], and [S5] in triplicate. The 

QC tubes will contain additional median reference standard [S3], will be used 

to perform preliminary absorbance checks and serve as a quality check for 

diluents and inoculum used in test. See Diagram 6 for an example of one 

independent test run for the Tube Method and placement of the standard curve 

and unknown sample into triplicate tubes.

Diagram 6. An example of one independent test run for the Tube Method and 

placement of the standard curve and unknown sample into triplicate tubes. 

6/22/17 MBB.

When a microorganism is placed into a broth containing the appropriate 

nutrients to support growth, the microorganism flourishes and the broth 

becomes turbid. Turbidity is typically a simple visual indicator of microbial 

growth; this growth can be quantified by a UV-VIS spectrophotometer by 

measuring values absorbance or transmittance exhibited by the broth. 

Test tubes containing growth broth, inoculum, test/unknown sample and 

standard curve ([S1] – [S5]), will be prepared and analyzed the same day. The 

test tubes are then placed into a circulating water bath for NMT 5 hours to 

reach a specified turbidity (i.e. absorbance). It is recommended, one or more 

QC tubes be used to perform an absorbance check at timed interval to assure 

the required absorbance is not exceeded; this activity may require the 

preparation of more than three QC tubes. After the specified absorbance is 
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achieved, formaldehyde or a heat treatment is immediately added/applied to 

each test tube to inhibit additional microbial growth prior to absorbance reading 

and use of the UV-VIS. The absorbance or transmittance is read ad 580 nm or 

530 nm. 

USP <81> states the following criteria for both the Plate and Tube Methods: 1) 

The calculated potency of the test antibiotic [U3] must be 80% to 125% of the 

median reference standard [S3] 2) Relative standard deviation for all 

measured and calculated data is NMT 10% and 3) Testing is performed in 

triplicate over a period of three independent test runs. The Tube Method 

further states: The percentage coefficient of determinations (%R2) for each 

standard curve will be NLT 90% (i.e. correlation coefficient of NLT 0.950) and 

2) Absorbance values of the media reference standard [S3] are predetermined 

per antibiotic. Refer USP <81> for testing parameters and acceptable data 

requirements. 

Diagram 7 below summarizes the operations prior, during and after testing 

using the Tube Method (Hewitt and Vincent 1989).
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Diagram 7. Example of operations conducted prior, during and after testing 

using the Tube (Turbidimetric) Method (Hewitt and Vencent 1989). 

H. Calculations

Potency calculations are listed in USP <81> and are specific to either the Plate 

or Tube Method. Calculations may be performed by traditional hand math or by 

electronic spreadsheet such as Microsoft Excel. In order to achieve reliable 

and reproducible results, electronic spreadsheets should be validated. 

Likewise, automated readers performing calculations should also be validated 

prior to use in testing.

I. Inspectional Objectives

When conducting an inspection of a human antibiotic manufacturer , contract 

manufacturer or test laboratory, inspectional objectives should include 
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coverage of high risk product/s (unless stated otherwise through special 

assignment or investigation) and a comparison of both USP <81> and USP 

monograph requirements and acceptance criteria to three types of in-house 

practice and procedures related to: 1) General laboratory and analytical 

operations 2) Initial training and requalification and 3) Data generation, review 

and archival. Employee practices should be documented in established written 

procedures. And, the original test data should match the final and/or released 

test results. All laboratory practices, procedures and test data should be based 

in sound science and meet the requirements and acceptance criteria of both 

USP <81> and related USP monograph. 

Specifically, a CSO and/or Analyst should conduct observations and make a 

procedural comparison of media, reference standard, standard curve, culture 

control, unknown sample and equipment preparation and use; also, a review 

or practice and procedure of equipment calibration, maintenance (preventative 

and major) and use should be conducted. Practice and procedure 

comparisons should be made for laboratory personnel involved with data entry, 

transcription and/or review should be contemporaneously observed, followed 

by an immediate review of related documents for thoroughness and accuracy; 

this includes data transcription from handwritten and/or electronic data into 

logbooks, batch records and/or certificates of analyses. Additionally, it is 

necessary to understand which document serves as the original record; 

request for the most responsible person employed at the firm to identify the 

original record/s containing the raw data. 

If the firm utilizes contract manufacturing services, is a contract manufacturer 

and/or a contract test laboratory, an active Quality Agreement, as established 

by the firm and customer, may provide insight to the roles and responsibilities 

of each party for testing and data generation, review and archival. 

J. References:

1. Hewitt, W. & Vincent, S. (1989) Theory and Application of 

Microbiological Assay, Academic Press, Inc.

12. Chapter 8: Bioburden Estimation for Medical Devices

According to FDA Compliance program 7382.845, Inspections of Medical

Device Manufacturers, Part IV, - “Bioburden testing is to be performed in

accordance with the guidance provided in ISO 11737-1, Sterilization of medical 

devices – Microbiological methods – Part I: Estimation of population of 

microorganisms on products. The methodology used for estimating the

bioburden is to be validated. Twenty units are to be tested.” 1

The term “bioburden” is commonly used to describe the population of 

microorganisms present on unsterilized material or products. The bioburden
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quantity and types of bioburden organisms present can impact the sterilization

process of the material or product. It is important to develop procedures which

provide accurate, precise, and reproducible measurement of the bioburden 

population associated with the material or product. There are several 

approaches to remove microorganisms from a medical device. Some

examples of these recovery methods include: filtration followed by plating;

ultrasonic/shaking followed by filtration then placing on an agar medium; 

stomaching/rinsing/flushing followed by filtration and plating on an agar 

medium; if all else fails perform a direct swabbing or contact plate.

The bioburden estimation of a medical device generally consists of four distinct 

stages:

1. Collection of microorganisms from the medical device. (See Annex A and

B)

2. Enumeration of the collection sample containing recovered

microorganisms.

3. Bioburden characterization.

4. Application of the correction factor(s) determined during bioburden

recovery studies in order to calculate the bioburden estimate from the raw

presterilization count. The correction factor is derived from the 

determination of the recovery efficiency of a method. The calculation of a 

correction factor is illustrated in Appendix C, Section C.2.

It is not possible to define a single microbial collection technique because of

the wide variety of materials used in health care products. Furthermore, the

selection of conditions for enumeration will be influenced by the types of

microbial contamination which may be anticipated.

The current method “ANSI/AAMI/ISO 11737-1:2018/(R) 2011 sterilization of

health care products – Microbiological methods – Part 1: Determination of the

population of microorganisms on product” has the latest revisions and

provides a great deal of information that will guide an analyst to the method

needed for a particular type of product.

Annex A contains a decision tree “that addresses designing a bioburden

method based on the nature of the product being tested and includes guidance

for choosing such things as agitation techniques or filtration versus direct 

plating.”

2 Annex A also addresses the procedures (repetitive recovery method,

product inoculation method) available for the validation of the method for

determining bioburden.
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Annex B has a comprehensive list of the different removal techniques that can

be employed and alternatives for samples where removal of microorganisms

by elution is not used.

Annex C has a more in-depth explanation of the validation of the repetitive

recovery and product inoculation methods.

A. References:

1. FDA Compliance Program 7382.845 Inspections of Medical Device

Manufacturers, February 2, 2011.

2. ANSI/AAMI/ISO 11737-1:2018/(R) 2011, Sterilization of health care

products – Microbiological methods – Part 1: Determination of the

population of microorganisms on product.

3. PDA Technical Report No. 21, Bioburden Recovery Validation. 1990

13. Chapter 9: Environmental Monitoring

ORS microbiologists may be required to assist CSOs during on-site

inspections of pharmaceutical facilities to perform environmental monitoring

(EM) sampling of those facilities to assess the microbiological bioburden of

critical surfaces. This chapter describes a suggested procedure for conducting

this activity. This should only serve as a guide with some modifications

depending on the specific facility or special instructions from ORS 

Headquarters. Be sure to confirm with ORS Headquarters which ORS 

laboratory was designated to receive the EM samples. These procedures allow

for a qualitative and quantitative assessment of environmental monitoring 

samples for the microbial presence in critical processing or laboratory area(s) 

being monitored. 

A. Materials/Equipment

1. Sampling Materials (Disinfect all materials being brought into the 

ante-room, cleanroom, or area designated for

compounding/processing by the firm.)

a. Sterile polyester or cotton swab with a sterile transport media

solution. 

b. Sterile sponge with detachable handle

c. *Hycheck or equivalent surface samplers

d. *RODAC/contact plates (Replicate Organism Detection And 

Counting)

*Use media containing lecithin and tween neutralizers
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e. Sterile Whirl-pak® bags.

f. Sterile water for irrigation or sterile saline held in screw cap

containers. 

g. Dey/Engley (D/E) neutralizing broth

h. Letheen Broth

i. Sterile Disinfectant in a spray bottle

j. Sterile 70% alcohol spray bottle or wipes

k. Black marker (permanent, fine point). 

l. Digital Camera

2. Testing Equipment and Materials

a. Biological Safety Cabinet (BSC) with HEPA filtration 

b. Laminar Flow Hood (LFH) with HEPA filtration

c. 10% Bleach or appropriate disinfectant/sporicidal

d. Sterile 70% ethanol (ETOH) or Isopropyl Alcohol (IPA) 

e. Sterile Sleeves 

f. Sterile Gloves 

g. Hair Net

h. Lab Coat/Sterile Gown

i. Beard Cover and/or Mask

j. Incubator set at 32.5oC ± 2.5oC. Incubator set at 22.5oC± 2.5oC. 

k. Modified Letheen Broth (MLB) 

l. Modified Letheen Agar (MLA) 

m. Sabouraud Dextrose Broth 

n. Sabouraud Dextrose Agar

o. Malt Extract Agar w/chlorotetracycline 

p. TSA w/5% Sheep Blood Agar

q. MacConkey Agar 

r. RODAC plates

s. Hycheck slides

t. Soybean Casein Digest Agar (TSA) 
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u. Soybean Casein Digest Broth (with neutralizers) 

v. Neutralizers (i.e. lecithin, tween, etc...) 

B. Sampling Preparation

1. Don appropriate personal protective equipment (PPE) as follows:

Note: All ORS full gowning and procedures should be performed prior to 

conducting the collection. The firm’s procedures/guidelines should be adhered

to when entering classified area; however, it is the expectation that the 

microbiologists entering the classified area, in order to perform sampling, will 

don sterile garments. If there is a dispute regarding the PPE to be donned for 

aseptic sampling, ORS Headquarters should be contacted prior to commencing 

gowning.

a. Nonsterile Hair net and beard cover (if needed) 

b. Sterile mask 

c. Shoe covers (non-sterile)

d. Sterile Gloves

e. Sterile Disposable Coverall

f. Sterile Hood

g. Sterile Goggles

h. Sterile Boot Covers

2. Sampling Equipment Controls

a. Aseptic Technique Control: Place one (1) sterile Dacron or cotton

swab into sterile water and place back into its sterile transport 

media solution. Place this negative control into a sterile Whirlpak® bag.

Note: This control may not be needed if the sponges and/or 

swabs are received pre-moistened. There is no surface contact 

for this control.

b. Swab/Sponge Sterility Control: Place an intact unused swab (or 

sponge) unit into a sterile Whirl-pak® bag. 

c. RODAC/Hycheck Sterility Control: Place an unused RODAC 

plate and/or Hycheck plate within a sterile Whirl-pak® bag. 

d. Whirl-pak® bag Sterility Control: Include one unopened Whirlpak® bag as a closed control. 
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e. Glove Sterility Control: If the sampler uses ORS sterile gloves

then have an intact unit containing gloves placed into a sterile

plastic bag and sent as a control. 

f. Sterile Equipment Control: Include any other sterile equipment

used during EM sampling (i.e. sterile specimen cup, sterile

media, etc.). (Coveralls, masks, and boot covers do not need to 

be included). 

C. EM Sampling Procedure

It is recommended that the investigative team bring equipment for both

qualitative and quantitative EM methods. Qualitative methods utilizing

sponges/ swabs are used for hard to reach areas. RODACs or Hychecks are

employed for the quantitative method to enumerate microbes on open flat work

surfaces. Each analyst is to perform the same role throughout the collection. 

(Ex. One analyst collects swabs and second analyst serves as an assistant.) 

See suggested sampling locations listed in section E of this procedure.

1. Disinfect gloved hands with a suitable sanitizing agent (i.e. sporicidal

agent or sterile 70% alcohol). 

a. Repeat this step between each EM sample.

b. Allow gloves to air dry so no disinfectant is dripping from gloves.

c. Some swab/sponge sampling packages include a secondary set

of sterile gloves. In these instances, the secondary glove can be

aseptically used on top of the primary gloves to expedite the

sampling process. The secondary gloves should not be 

removed, but additional gloves should be added and disinfected 

as needed. 

d. When sampling an ISO 5 location.

i. Verify that the LFH/BSC certification is current.

ii. Allow LFH/BSC to run approximately 10 minutes before

initiating sampling

iii. Wipe down all outer sampling containers with a suitable

sanitizing agent before placement in the LFH/BSC

iv. Do not open sampling materials outside the LFH/BSC

2. Qualitative Swabbing

a. Open a sterile swab (or sponge). Dampen with wetting agent 

(sterile water, saline, or D/E neutralizing broth) and squeeze off

excess by pressing against the inside of the container holding the

wetting agent.
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b. Apply swab (or sponge with handle) to surface (or equipment) 

being monitored with firm application pressure.

c. When sampling (monitoring) flat surfaces allow the swab (or 

sponge with handle) to firmly rub an area of approximately 24 to

30 cm2

.

d. Apply the swab (or sponge) within this contact area in both a

horizontal and vertical direction for approximately 10 seconds.

e. Place the swab (or sponge) back into the carrier container (if it

came with one) and place into the additional sterile Whirl-pak®

bag. Be sure to break off the handle portion of the sponge

applicator stick.

3. Quantitative RODAC/Hycheck Sampling

a. Carefully remove the lid of RODAC plate or loosen the cap on 

the Hycheck slide tube. Take care not to touch the agar surface.

Note: Examine agar for contamination and or dehydration

b. Gently but firmly touch the RODAC agar surface against the area

being sampled, exert moderate, even, vertical pressure and then

carefully replace lid. Avoid using rubbing motions of the plate at

the sample site as this may break the agar.

c. When using Hycheck press down on the spike to bend the

paddle at the hinge line gently lowering the slide and press the

agar to the surface with firm and even pressure. Repeat this step

using the 2nd agar surface on an area adjacent to the initial test 

site. Replace slide in container and close tightly.

4. Sampling of Irregular Surfaces

A classified area may have an exposed irregular surface (i.e. particle board, 

wood, broken laminate, etc.) that requires sampling. In these situations, a 

colorless transport media such as letheen broth, saline or sterile water should 

be used to wet the sponge or swab prior to sampling. The microbiologist 

should avoid the use of D/E neutralizing broth which is a dark purple color and 

may discolor the irregular surface. 

At the microbiologist’s discretion, it may be determined that a RODAC plate 

should be used to sample the irregular surface. In this situation, a RODAC 

plate comprised of colorless agar such as tryptic soy agar would be 

appropriate. A colored agar such as D/E agar should be avoided.

5. Place the EM sample into a sterile Whirl-pak® bag and identify the

bag immediately after.
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6. Assign a consecutive number to the sample (i.e.1, 2, 3, etc.), in

addition include the date, location of sample site (be specific) and

your initials. Record in your inspectional record book the swab

number and the location of the swab site.

7. After the sampling, the sampled area should be disinfected with 

sterile 70% alcohol. A sterile lint free wipe may be used to help 

remove sampling residue and expedite drying of the area.

8. As soon as possible, place the double-bagged subs inside an 

insulated cooler, with pre-frozen gel packs to keep the samples cold, 

but not frozen, and transport/ship the sample to the servicing lab for 

analysis, within 24 hours of collection. 

9. Place the sample into a suitable mailing container to prevent 

crushing or physical damage to the swabs. The container should

have some insulation capacity to prevent extreme temperature

(freezing or excessive heat).

10.Contact with the receiving laboratory in advance regarding pending

samples. This will ensure they have appropriate personnel and

materials for sample set up within 48 hours of collection.

D. Recommended Environmental Monitoring Sites

When on an inspection, do not allow the firm to disinfect the work area prior to

sampling. The facility and the equipment should be sampled during an inprocess state as determined by the firm. The presence of disinfectant on the

swab may reduce the microbial bioburden or increase inhibition during broth

incubation. When collecting EM samples start in locations that are under the

greatest control (ISO 5- HEPA filtered LFH/BSC or Isolator) and move to lesser 

controlled areas (areas outside the work station but still within the room).

1. Swab the frequently utilized surfaces within the controlled work 

station such as:

a. Hooks for intravenous bags in the LFH or isolator

b. Center of work surface

c. Fingertips & sleeves of Isolator gloves

d. Peristaltic pumps

e. Storage bins inside work station

f. Shelving inside work station or any other stationary items

g. Equipment control panels including on/off switches of LFH/BSC

h. Flexible plastic curtains used to separate multiple workstations
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2. Swab corner crevices inside the HEPA Filtered work station.

3. Swab the handle, squeeze-trigger and nozzle of any bottle kept in

the clean room or work station used for spraying (i.e., 70% alcohol,

disinfectant solutions, etc.).

4. Swab the underside of the chair in front of the work station. 

Specifically, on the front bottom rim where personnel would hold to

pull up the chair.

5. Swab tables or benches within the controlled room where product 

container(s) or post sterilized product may be held outside of the

HEPA filtered workstation.

6. Swab the air in-take grid on each of the HEPA filtered work stations.

Usually located on top of the unit holding the course filters.

7. Swab the exhaust (return) grid for the room air handling system that 

is connected to the facility air supply where the product

manufacturing or compounding occurs.

8. Swab the light switch and door knob or handles leading into and out 

of the clean room and the phone or intercom base.

9. Swab any cardboard boxes, handles of plastic containers, tools

(crimpers) or scissors, key pads on weighing scales, calculators or 

computers kept in the cleanrooms, keyboard, mouse and touch 

screen monitors.

10.Swab the exterior cuffs of the used lab coats worn by personnel 

during manufacturing or compounding. They may be hanging in the

entry (ante) gowning room.

11.Swab the bottom horizontal window sill within the clean room.

12.Swab any area under open or dislodged ceiling panels.

13.Sample areas of discoloration, stains or water and oil droplets.

14.Use your discretion to sample any other high-risk surface locations.

15.Photograph surfaces or equipment that display gross signs of

contamination (i.e., particulate matter, fungi, discoloration, etc.). Try

to include a distant picture of the targeted area along with a focused

close-up. Be sure to sample this location, as well.

E. Analysis Preparation conducted by ORS laboratory

1. All processing of swabs must be aseptically performed within a 

HEPA Filtered II Biological Safety Cabinet (BSC) or HEPA Filtered 
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Laminar Flow Hood (LFH) with an air classification at the same 

rating or better than that from which the swab was collected. 

2. All surfaces within the BSC or LFH must be thoroughly disinfected

with a sporicidal disinfectant followed by filter sterile 70% ethanol or 

IPA prior to placing swabs under the hood and beginning analysis.

3. In order to assure that the BSC/LFH and media are free of microbial 

contamination, standard open and closed controls used for sterility

testing should be performed concurrently with analysis.

4. Don as appropriate the following PPE:

a. Hair net

b. Disposable lab coat

c. Sterile disposable sleeves

d. Sterile gloves

e. Mask/beard covers

f. Lab Safety Glasses

5. Sterile gloves must be decontaminated between the processing of

each individual swab. Sterile gloves and sleeves should be

discarded and replaced as needed.

6. Sample Preparation

a. Examine swab containers for closure integrity to ensure

tampering, leakage, or potential cross contamination has not 

occurred.

b. Carefully disinfect exterior of each swab container and place into

the sanitized BSC/LFH and allow to air dry.

7. Media Selection

a. Neutralizing additives (i.e. Tween/Polysorbate, Lecithin, etc.) are

utilized to neutralize inhibitory disinfectant residues transferred to

the swab during sampling that might inhibit microbial growth. 

b. For a broad-spectrum recovery of microorganisms, utilize a 

nutrient rich general-purpose media containing neutralizers (i.e.

MLB, MLA, etc.).

c. When targeting fungal populations only, it is necessary to use an 

appropriate fungal media such as Sabouraud Dextrose or Malt

Extract media. It is beneficial to use an antibiotic (i.e.

Chlortetracycline) which will help to selectively inhibit bacterial
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growth and restrict the size and height of colonies of more rapidly

growing molds.

d. TSA w/5% Sheep Blood Agar is beneficial for cultivating 

fastidious microorganisms. 

e. MacConkey Agar is used for the isolation and differentiation of 

Gram negative and enteric organisms.

f. RODAC plates and Hycheck slides are used for the detection 

and quantification of microbiological contamination.

F. Analytical Procedure

1. Approximately 100 ml of sterile media, MLB or other suitable media, 

should be aseptically added to each plastic bag containing a square

sponge swab. Mix or swirl thoroughly.

2. Approximately 10 ml of sterile media, MLB or other suitable media, 

should be aseptically added to a sterile container to which the swab 

and its transport media are added. Mix or swirl thoroughly.

3. All swabs are incubated at 25oC- 30oC for at least 14 days to allow

for the resuscitation of potentially stressed microbes.

4. Hycheck slides and RODAC plates should promptly be incubated at 

30-35oC for 2 days and then 20 oC -25 oC for 5 days. Longer 

incubation times may be required when contaminants are suspected 

to be slow growing. Check plates daily for colony formation to 

minimize obscuring visualization of smaller colonies by over growth.

a. Count and record the number of colony forming units for RODAC 

plates. A representative of each colony type should be picked

and re-streaked for purity and subsequently identified.

b. Count the number of colonies on both sides of the paddle for the

Hycheck slide. Report the colony counts for each side of the

paddle and a representative of each all colony type should be

picked and re-streaked for purity and subsequently identified.

5. Check all swabs/sponges daily for turbidity and subculture for 

isolation as turbidity is observed. All subculturing must be performed 

under LFH or BSC. If container does not allow for turbidity 

observation to be made, then subculture all environmental samples 

between Day 5 and 7. All environmental samples will be 

subcultured following day 14 incubation regardless of previous 

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6. Subculture all EM samples onto a combination of non-selective 

media (i.e. MLA, etc.) and selective/differential media (i.e. 

MacConkey agar, MEA, etc.). It is recommended to include TSA 

w/5% Sheep Blood Agar as one of the differential medias for 

subculturing. A minimum of two agars should be utilized, 1 must be 

a nonselective agar.

a. Fungal media should be incubated at 20o to 25o for 5 to 7 days. 

In some cases, extended incubation times may be appropriate, 

but generally not beyond 14 days unless there is a specific 

scientific justification.

b. All other culture media should be incubated at 30o to 35o for 2 to

3 days.

7. Re-incubate all cultured swabs until the full incubation (14 days) 

timeframe are met.

8. Incubate all negative controls, such as system controls, media 

controls, under the same conditions as the sample.

9. Process and incubate submitted collector’s controls under the same 

conditions as the sample. 

10.Perform microbial characterization and identification following USP 

<1113> Microbial characterization, Identification and Strain Typing, 

as guidance. Typically, rapid identification systems (i.e. VITEK) are

employed after primary screening and characterization are 

performed. Other identification platforms such as DNA sequencing

may be beneficial if acceptable identification is not obtained through

biochemical testing.

14. Chapter 10: Inspectional Guidance

A. Microbiological Issues for Inspection of Pharmaceutical Laboratories

The following topics should be reviewed and evaluated during an inspection of 

a pharmaceutical microbiology laboratory.

1. Finished product testing using USP or Non-compendia method

2. Review the original results for the following: sterility, bacterial 

endotoxin, microbiological examination of nonsterile products:

specified microorganisms and enumeration, antimicrobialeffectiveness test, bioburden determination, water quality control 

testing.

3. Method Suitability (sterility), preparatory test (bacterial endotoxin), 

validation of method used for bioburden and water analysis
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4. Reagents and media- proper storage, expiration date, and growth 

promotion

5. Equipment and Instrumentation- (Steritest, manifold,

automated/molecular identification system, Vitek, isolator and biodecontamination system) review calibration, maintenance, validation

(IQ, OQ, PQ)

6. Sterility testing area design, operational procedures, monitoring,

aseptic technique, gowning procedures, proper sample container

disinfection, surface/air monitoring, HEPA filter certification, etc.

7. Method description, modifications, and verification along with

recording of sample results and appropriate review and evaluation

by management

8. Qualifications, training and identification of the personnel conducting

each step of the analysis

9. Qualification and training of management to critically review data

and interpret its significance

10.Microbial specifications set for raw material, finished product, water 

bioburden, and EM for analytical areas. Note: During pre-approval 

inspections questions about the appropriateness of finished product, 

raw material and in-process specifications should be discussed with 

the Center reviewer of the application. 

11.Integrity and accuracy of the laboratory information management 

system (LIMS) for microbiology data entry, review and approval

12.Selection, handling, and storage of Biological Indicators (BIs)

13.Private (contract) testing laboratory quality agreements, data review,

and associated problems; Have there been any changes in contract

labs and why?

14.Proper use and control of In-vitro diagnostic test kits, positive and

negative controls, interpretation and reliability of results

15.Risk assessment of microbiological results for non-sterile products

16.A list of the entire laboratory’s microbiological data deviations (Out of 

Specification (OOS)/ (Out of Limits (OOL) results) and Corrective

Action Preventative Actions (CAPA) since last FDA on-site 

inspection

17.Stability Testing – sample storage conditions, missed sampling

dates, etc.
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B. Microbiological Issues for Inspection of Pharmaceutical Manufacturing

Facilities

The following topics should be reviewed and evaluated by a microbiologist 

although some aspects will also be covered by the Consumer Safety Officer 

during the inspection.

1. Product sterilization or bioburden reduction stage and validationaseptic/filtration, steam, ETO, radiation, and other chemical 

processes

2. Depyrogenation- dry heat ovens for glass containers, wash/rinse for

stoppers, adequacy of validation using spiked endotoxin, recovery

studies before depryrogenation, filtration and column applications

3. Environmental monitoring- Types of equipment, calibration,

operation and maintenance; surface, air, personnel and water; 

critical work areas for aseptically filled products (ISO 5, isolators, 

etc.); surface contact, surface sanitizer neutralizing media (e.g., TSA 

w/ Lecithin & Polysorbate 80), observe sampling technique, sample

must represent dynamic/operational conditions, and trending/CAPA.

4. Process simulation (media fills) studies - growth promotion testing to 

include when is it performed and which microorganisms are 

included, who is responsible for reading turbidity, volume adequacy 

in the product containers and accountability of product containers 

during and after incubation periods. 

5. Disinfection and sanitization- agents used (sporicidal?), preparation

problems (over dilution); applicator (i.e., mop, spray. aerosol), time

of exposure, areas of contact, supervision; residues, UV lights, water 

systems, filling equipment, work surfaces, process columns,

verification and validation

6. Room design and Equipment- accessibility for disinfection and

cleaning; aseptic filling critical area; HEPA filter certification and

maintenance, air flow patterns/smoke studies, change evaluation/recertification (rearranging cleanroom, adding equipment, HVAC, etc.), 

test during dynamic/operational conditions with maximum number of

personnel in place, personnel and equipment flow, room differential 

pressure and temperature; adequacy of primary and secondary

barriers

7. Water purification and delivery system: vulnerability of distillation

process, RO, deionizers, cartridge filters, etc.; UV lights, dead legs,

biofilm; corrosion (heat exchangers); waterborne microorganisms
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(nanobacteria) and endotoxin production; cold system problems, 

disinfection problems

8. Personnel- training procedures for aseptic technique, gowning

procedures, cleaning and maintenance personnel training for ISO 5

room entry; glove and garment monitoring procedures

9. Product sampling: a representative sample is selected based on lot 

size as per USP <71> guidelines; quantity per container & units per 

batch, sample storage (time and temperature), sampling port 

sanitization or sterilization problems; be aware of skip lot testing on

raw material.

10.Maintenance records- determine dates, and location of equipment 

failures or out-of- service equipment that may have an impact on

microbial contamination of product; looks for signs of roof leaks and

water stains on ceiling panels, the degree of dirt and dust 

accumulation on supply and exhaust vents. Ask about new

construction, plumbing or air handling system and the reason for

change.

11.Compressed air systems—sterile process air, microbial particulate

filtration (0.2 μm, hydrophobic), condensate causing blockage and

microbial growth, routine point-of-use sampling, maintenance, filter

integrity test

C. Sample Data Review – When all results are negative

The following should be reviewed when all the firm’s sterility, nonsterile

product and/or environmental monitoring test results indicate no microbial

growth for validity and/or accuracy:

1. Media- growth promotion; adequacy of the documented pH; low

agar or broth volume in container, incubator temperature not set

correctly; improper medium storage after QC (crystals from freezing, 

inadequate mixing prior to dispensing, agar plates dried out during

incubation, etc.)

2. Method suitability testing- validation for sterility; preparatory testing

(BET). Appropriateness of neutralizers, product dilution, filtration;

presence of toxic chemical contaminants in the water used to 

prepare the buffers, Not following method (excess of product added

to broth during test but not during suitability testing, etc.)

3. Improper tube or agar plate examination- check filter surface on

submerged filter membrane (mold budding); surface film, light hazy

growth in Thio broth, microbes settle to bottom of tube, pinpoint
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colonies (microaerophilic); medium not inoculated. Review 

disinfection process to assess the potential of antimicrobial residue

being introduced during sample preparation; Review gas used for 

Isolator sterilization with medium inside chamber to assess the 

potential penetration into liquid broth and/or test product packaging.

4. Bioburden - Review the products bioburden for the presence of 

fastidious microorganisms which may require inclusion of special 

additives in the medium, such as halophilic contaminants in

bicarbonate or high salt products need medium supplements with

essential salts for survival. Where appropriate, the laboratory 

extend incubation time to improve the detection and recovery of 

fastidious microorganisms as part of their environmental monitoring 

program or part of investigation. 

5. Water test method- Evaluate the length of storage and storage 

temperature for collected samples prior to testing. Evaluate the type 

of collection bottles and their compatibility with the testing, for 

example endotoxin testing.

6. Adherence to methods - Review data for adequacy of incubation

time (for example 14 days for USP <71>), temperature (USP 

required temperatures) or appropriate media usage.

7. Worksheets - Review possible falsification or incorrect entry onto

worksheets or Laboratory LIM system.

Compare LIMs database entries to the analyst’s laboratory

notebook; Phrase the question “When you get a positive test 

result....” not “If you get a positive test result...” Inspect the

laboratory refrigerator or freezer for evidence of stored sample 

isolates. If they lyophilize sample isolates, ask to review the spread

sheet data storage directly from the computer screen; hard copies

could be obtained later. Review the Vitek or Micro Id isolate log book

for all microorganisms identified and work backwards to the product

lot number, filling rooms, equipment used, components or raw

material used for that lot. This may allow you to find other lots 

associated with the contaminated lot.

8. Personnel- review training records, personnel qualifications and

experience. Observe analysts during sample collection, preparation,

etc., and look for errors which may inhibit microbial recovery.

9. Laboratory - Visit the microbiology laboratory and look in the

refrigerators, incubators, look at discarded plates from that day’s

work or request speciation log book and determine if microbial 
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recovery has occurred, but not recorded on official worksheets or

entered into LIMs. Determine where plates or other growth media 

are stored pending microbial identification. Review available plates 

or growth media to determine if the findings match those 

documented in the laboratory records.

D. Sample Data Review – When Microbial Growth Is Indicated

When you encounter inspectional evidence that the firm has manufactured a 

microbiologically contaminated product, the few suggestions listed below

should help you evaluate and proceed with this information. It is recommended 

that this evidence be communicated with the lead Consumer Safety Officer on 

the inspection so that appropriate communications with the responsible 

compliance office and the Center can occur. In addition, the Consumer Safety 

Officer can assist with the inspection of the manufacturing operations.

1. Documentation- Review and obtain copies of all records for lots

indicating contamination; determine if there are other lots

manufactured either before or after the “bad” lot(s). Review all 

associated activity and equipment related to the contaminated lot. 

There may be common water sources, mixing tanks, piping, raw

material, sterilizers, filters, etc. that may have been cross

contaminated and transferred microbes to subsequent lots of

products.

2. Validation - Review current established validation studies for

product/component sterilization (disinfection for non-sterile

products); has there been any equipment changed or modified; has

there been a change of personnel or training; any new source

material for equipment (vent filters, gaskets, filter manufacturer, 

etc.); any processing changes or room modifications, construction

elsewhere in the facilities, etc.

3. Environmental Monitoring (EM) - Review of environmental monitoring

(EM) procedures and results for manufacturing and laboratory areawould the product contaminant grow on the EM medium; was the

product contamination found in the manufacturing area; growth

promotion potential of contaminant in other medium (i.e., TSB and

Thio)

4. Speciation- Record and copy the method of identification (i.e. API, 

Vitek, etc.). Determine if there were possible secondary

contaminants that were not identified or recorded (check original

plates, or isolates); verify accuracy of entry into the LIM system.
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5. Source of potential contamination – Determine the potential source 

of contamination, which may include Staphylococcus (skin, insect, 

etc.); Pseudomonas (water, plants, etc.); yeast and mold (spores) 

(environmental)

6. Investigation Report – Review firm’s investigation report of 

microbiological out of specification (OOS) test results; source of 

contamination; where there corrective action(s) taken; if any repeat 

testing performed; batch disposition; does it include related lots and 

ancillary systems? Was the product rejected or released? If released 

ask why? Evaluate justification. 

7. Product Test Failure - When a firm has a final product or in-process

test result that indicates a failure (USP test failure, OOS, etc.) ask–

Were the results due to laboratory error or a true process

contamination?

8. Focus Areas - During the investigation, there are two areas for 

the review to focus: the manufacturing site and the laboratory that 

determined the OOS result. The investigations may run 

concurrently between manufacturing and the laboratory. For ease 

of review the questions listed in #9 (below) deal with laboratory 

data and those in Section E concern the manufacturing review. 

The laboratory section covers those questions that should be 

asked for a critical review of the microbiological data 

accumulated for sterility failures, non-sterile medical product 

failures, etc. Section E covers the manufacturing area and is 

divided into aseptic manufacturing (high risk) and terminally 

sterilized products (low risk). 

9. Laboratory Facility and Analytical Review

a. Review QC records for proper/validated sterilization of all 

equipment and media used during the sterility test method: 

manifold/ Steritest; rinse fluid, culture media, canister kits, etc.

b. Review the EM data acquired during sterility testing (i.e., settling 

plates, RODAC), simulation system controls, etc. What are the 

microbial species and their determined normal habitat (i.e., 

water, plants, people, etc.?)

c. Review training records and qualification of analysts performing 

the test; interview and/or observe analysts

d. Review the qualification of the bio-clean room facilities or isolator 

chamber used during testing. Are there any leaks in the gloves, 

improper sanitization of product container before placement into 
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work station or isolator? Has the isolator been evaluated for 

leaks?

e. Review cleaning and sterilization requirements for reusable 

glassware and equipment. Poorly cleaned glassware will make 

sterilization of equipment more difficult and possibly shelter 

trapped microbes from the killing effect of the sterilant.

f. Review laboratory areas used for sub-culturing the sterility test 

medium onto enrichment plates. Cluttered work space or unsanitized surfaces may cause plate contamination.

g. Check the original plates used for isolation for possible preexisting contamination (i.e. growth in non-streaked locations on 

the agar surface, subsurface growth)

h. Check to see if the medium had been recalled or has had past 

problems with contamination during manufacturing.

i. It may be necessary to perform a genotype identification on the 

two isolates (product source and manufacturing area isolate) if 

they are the same species.

E. Manufacturing Facility Review

1. Aseptically filled pharmaceuticals

a. Check environmental monitoring (EM) data taken from production

areas and the testing environment (i.e., S-T-A, settling plates, 

RODAC, etc.) for microbial contamination that matches the

microbe isolated from the finished product sterility test

If no microorganisms are detected, check the adequacy of the

EM method used during manufacturing for proper sensitivity and

applicability, for example

i. Are they using proper medium (i.e. non-selective medium)?

ii. Have they performed growth promotion?

iii. Did they use appropriate incubation time and temperatures?

iv. Are they sampling in the appropriate room locations, during

dynamic conditions, longest time between

cleanings/sanitation and at frequency to assure reliability of 

the results?

v. If they recovered an anaerobic bacterium from the sterility

test, (Thioglycollate broth) do they perform EM for anaerobic 

bacteria?
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b. Have they performed a filter integrity test on the membrane used

for the product sterilization? Review the products pre-filtration

bioburden levels to assure that the concentration of bacteria in

the bulk has not exceeded the membrane filtration capacity that 

was determined in their validation studies. Have they changed

the source or model for the membrane filter cartridge used in the

process?

c. Has the firm manipulated or excluded some of the data used in

the final QC report? Perhaps raw data was averaged to bring the

bioburden count below the alert or action levels. It can be helpful 

to request electronic Excel sheet version of data, to allow sorting 

(by frequency of organism, location, etc.) and trend analysis;

hard copies can be requested later, if necessary.

d. Review the media simulation studies. Did the microbial species

recovered in past simulation studies match the microbe(s) 

recovered from the current product test failure?

e. Has there been a change or breach in the personnel barrier 

system to protect the product? Were there any interventions by

maintenance or other staff personal during the manufacturing of

the contaminated lots? Review glove/uniform monitoring results.

f. Review the antimicrobial effectiveness challenge studies for the

product if it multiple dose. Where there any changes to the

container/closure component source or requirements?

g. Were there any changes to the disinfection procedure, reagents

use, new personnel, application, equipment (mops, aerosols,

etc.) etc.?

2. Terminally sterilized drug product

a. Check autoclave validation studies for sterilization process- cold

spot, heat penetration (challenged inside dry tubing, 

connectors/caps/stoppers, largest liquid volume, etc.), changes

in chamber load configuration, etc.

b. Check maintenance records for house steam, records for 

autoclave repair, new plumbing

c. Check Biological Indicator (BI) information- improper storage of

BIs; changes in the culture (inoculum level and/or BI organism 

species) and incubation parameters

d. Evaluate the heat resistance characteristics of the microbial 

isolate found in the product during sterility testing and determine
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if it can survive during the process conditions, review product 

container/closure integrity data and possible recent supply

source changes to vials or rubber stoppers; check possible post 

sterilization package integrity problems- mostly medical device

issue.

F. Inspectional Elements listed in the six (6) Inspectional Systems covered 

by the CP 7356.002 that cover ONLY Microbiological Issues

Some of the key coverage elements in five of the six Inspectional Systems 

listed in FDA Compliance Program Guidance Manual Program 7356.002 that 

relate to microbiological issues. Some examples are included for 

clarification. (Labeling system not included).

1. Quality System

a. Discrepancy and failure investigations related to manufacturing

and testing: are they documented, evaluated and investigated in

a timely manner; including corrective actions where appropriate.

b. Validation: status of required validation/revalidation (e.g.,

computer, manufacturing process, laboratory methods).

c. Training/qualification of employees in quality control unit

functions.

2. Facilities and Equipment System

a. Facilities

i. Cleaning and maintenance

ii. Facility layout and air handling systems for prevention of

cross-contamination (e.g. penicillin, beta-lactams, steroids, 

hormones, cytotoxics, etc.)

iii. Specifically, designed areas for the manufacturing operations

performed by the firm to prevent contamination or mix-ups

iv. General air handling systems

v. Lighting, potable water, washing and toilet facilities, sewage

and refuse disposal

vi. Sanitation of the building, use of rodenticides, fungicides, 

insecticides, cleaning and sanitizing agents

b. Equipment

i. Adequacy of equipment design, size, and location

ii. Equipment surfaces should not be reactive, additive, or

absorptive
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iii. Appropriate use of equipment operations substances, 

(lubricants, coolants, refrigerants, etc.) contacting

products/containers/etc.

iv. Cleaning procedures and cleaning validation

v. Controls to prevent contamination, particularly with any

pesticides or any other toxic materials, or other drug or nondrug chemicals

vi. Qualification, calibration and maintenance of storage

equipment, such as refrigerators and freezers for ensuring

that standards, raw materials, reagents, etc. are stored at the

proper temperatures

3. Materials System

a. Representative samples collected, tested or examined using

appropriate means

b. Testing or validation of supplier's test results for components, 

containers and closures

c. Rejection of any component, container, closure not meeting

acceptance requirements. Investigate the firm's procedures for

verification of the source of components.

d. Appropriate retesting/reexamination of components, containers, 

closures

e. Water and process gas supply, design, maintenance, validation

and operation

f. Containers and closures should not be additive, reactive, or

absorptive to the drug product

g. Documented investigation into any unexpected discrepancy

4. Production System

a. Training/qualification of personnel

b. Validation and verification of cleaning/sterilization/

depryrogenation of containers and closures

c. Established time limits for completion of phases of production

(i.e. microbial growth potential of product)

d. Implementation and documentation of in-process controls, tests,

and examinations (e.g., bioburden determination pH, adequacy

of mixing)
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e. Justification and consistency of in-process specifications and

drug product final specifications

f. Prevention of objectionable microorganisms in non-sterile drug

products

g. Equipment cleaning and use logs

h. Process validation, including validation and security of

computerized or automated processes (i.e. simulation studies)

i. documented investigation into any unexpected discrepancy

5. Laboratory Control System

a. Training/qualification of personnel

b. Adequacy of staffing for laboratory operations

c. Adequacy of equipment and facility for intended use

d. Calibration and maintenance programs for analytical instruments

and equipment

e. Validation and security of computerized or automated processes

f. Reference standards; source, purity and assay, and tests to

establish equivalency to current official reference standards as

appropriate

g. System suitability checks on chromatographic systems (e.g., GC 

or HPLC)

h. Specifications, standards, and representative sampling plans

i. Adherence to the written methods of analysis

j. Validation/verification of analytical methods

k. Control system for implementing changes in laboratory

operations

l. Required testing is performed on the correct samples

m. Documented investigation into any unexpected discrepancy

n. Complete analytical records from all tests and summaries of 

results

o. Quality and retention of raw data (e.g., chromatograms and

spectra)

p. Correlation of result summaries to raw data; presence of unused

data
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q. Adherence to an adequate Out of Specification (OOS) procedure

which includes timely completion of the investigation

r. Adequate reserve samples; documentation of reserve sample

examination

s. Stability testing program, including demonstration of stability

indicating capability of the test methods (i.e. container/closure, 

AET)

G. Sample Collection During an Establishment Inspection

Samples of defective product constitute persuasive evidence that significant

CGMP problems exist. Physical samples may be an integral part of a CGMP 

inspection where control deficiencies are observed. Physical samples should

be correlated with observed control deficiencies. The investigator should 

consult Center and/or ORS Headquarters for guidance on quantity and type of

samples (in-process or finished) to be collected. Documentary samples may be

submitted when the documentation illustrates the deficiencies better than a

physical sample. Divisions may elect to collect, but not analyze, physical 

samples, or to collect documentary samples to document CGMP deficiencies. 

Physical sample analysis is not necessary to document CGMP deficiencies.

When a large number of products have been produced under deficient 

controls, collect physical and/or documentary samples of products which have

the greatest therapeutic significance, narrow range of toxicity, or low dosage

strength. Include samples of products of minimal therapeutic significance only

when they illustrate highly significant deficiencies.

H. Appendix A: Literature and Resources

A Comprehensive List of Only Microbiological Regulatory and Scientific

Literature Resources

The scope of this reading material will ONLY include microbiological scientific

and regulatory publications or websites for conventional drugs, biologics and

combinatorial products. Some references to medical device regulations will be

included if relevant during an FDA investigation that covers microbiology.

1. Legal requirements and regulations

a. CFR 210 & 211 “cGMPs for finished Pharmaceuticals” 

b. CFR 210 & 211 amended effective Dec 2008 (several changes

that include microbiological requirements of aseptically filled

products)

c. CFR 610 General Biological Product standards” (Not covered

during this review) 
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d. CFR 820 “Quality Systems Regulation (Devices, not covered) 

e. CFR 314.81(b)(3)(ii)- Applications for FDA approval to market a

new drug (revised April 1, 2008) For submission of an alternate

microbiological method with a comparability study 

f. CFR 1271 Human cells, tissues, and cellular and tissue-based

products

2. FDA Compliance Program Guidance Manuals

a. FDA Compliance program Guidance Manual for FDA Staff: Drug 

Manufacturing Inspections program 7356.002

During an inspection this program designated six (6) critical systems for 

review. They include: Quality System (always covered during an FDA 

inspection); Facilities &Equipment; Material; Production; Packaging and

labeling; and Laboratory control systems.

b. FDA Compliance program Guidance Manual for FDA Staff:

Sterile Drug Process Inspections 7356.002A

The sections entitled “Inspectional” and “Analytical” and “Attachment A” are 

pertinent to an FDA microbiologist. 

3. Compliance Policy Guides

a. Sec. 100.550- Status and Responsibilities of Contract Sterilizers

Engaged in the Sterilization of Drugs and Devices (CPG

7150.16) (Oct 2006) 

b. Sec. 490.100 Process Validation Requirements for Drug Products 

and Active Pharmaceutical Ingredients Subject to Pre-Market 

Approval (CPG 7132c.08) (3/2004) 

c. Manual of Policies and Procedures, CDER, MAPP 5040.1

Product Quality Microbiology Information in the Common

Technical Document - Quality (CTD- Q)

d. Compliance Policy Guidance for FDA Staff- Sec. 280.110

Microbiological Control Requirements in Licensed Anti-Human

Globulin and Blood Grouping Reagents

4. US Pharmacopeia (USP) Compendium

Review all relevant product monographs (not all have microbiological 

requirements); the following are regulatory chapters that contain enforceable

microbiology requirements

General Notices and Requirements (page 1-13), Chart 10- Microbiology
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a. <1> Injections

b. <51> Antimicrobial Effectiveness test

c. <55> Biological Indicators-Resistance Performance tests

d. <60> Microbiological Examination of Nonsterile Products: Tests 

for Burkholderia cepacia complex

e. <61> Microbiological Examination of Nonsterile products:

Microbial enumeration tests

f. <62> Microbiological Examination of Nonsterile products: Tests

for Specified Microorganisms

g. <63> Mycoplasma

h. <71> Sterility Tests

i. <81> Antibiotics-Microbial Assays

j. <85> Bacterial Endotoxins Test

k. <151> Pyrogen Test

l. <161> Transfusion and Infusion Assemblies and Similar Medical 

Devices

m. <171> Vitamin B12 Activities Assay

n. <797> Pharmaceutical Compounding-Sterile Preparations

Dietary Supplements General Chapters Information

a. <2021> Microbial Enumeration Test-Nutritional and Dietary

Supplements

b. <2022> Microbiological Procedures for Absence of Specified

Microorganisms- Nutritional and Dietary Supplements

c. <2023> Microbiological Attributes of Non-sterile Nutritional and

Dietary Supplements

USP Informational chapters<1000>through <1999> are informational chapters 

and provide guidance to the industry. These Informational chapters will help

explain or expand on scientific principles established in the regulatory

chapters.

a. <1035> Biological Indicators for Sterilization

b. <1072> Disinfectants and antiseptics
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c. <1111> Microbiological Examination of Nonsterile Products: 

Acceptable Criteria for Pharmaceutical Preparations and

Substances for Pharmaceutical Use

d. <1112> Application of water activity Determination to Non-sterile

pharmaceutical products

e. <1113> Microbial Characterization, Identification, and Strain

Typing

f. <1116> Microbiological evaluation of clean rooms and other

controlled environments

g. <1117> Microbiological Best Laboratory Practices

h. <1207> Sterile Product Packaging—Integrity Evaluation

i. <1208> Sterility Testing –Validation of Isolator Systems

j. <1209> Sterilization—Chemical and Physicochemical Indicators

and Integrators

k. <1211> Sterilization and Sterility Assurance of Compendial 

Articles

l. <1223> Validation of alternative microbiological methods

m. <1227>Validation of Microbial Recovery from Pharmacopeial 

Articles

n. <1237> Virology Test Methods

5. AOAC international Includes chapters on disinfectants evaluation 

(i.e., Phenol coefficient Methods; Hard surface carrier test methods; 

Use-Dilution Method) 

6. Association for the Advancement of Medical Instrumentation (AAMI)/ 

International Organization for Standardization (ISO).

There are over fifty (50+) documents available through AAMI/ISO on the topic

of “Sterilization Processes and Validation”. These are internationally

recognized standards and procedures recognized by FDA and Industry.

AAMI/ISO Guidance documents- are available at the FDA intranet.

7. FDA Inspection Guidance documentsListed below are all the FDA guidance documents that contain only

microbiological information relevant to inspection. In most cases these will be

listed in the general website for CBER or CBER guidelines.
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a. Submission of Documentation in Applications for Parametric

Release of Human and Veterinary Drug Products Terminally

Sterilized by Moist Heat Processes (draft 8/2008) 

b. Validation of Growth-Based Rapid Microbiological Methods for 

Sterility Testing of Cellular and Gene Therapy Products (draft

guidance, 2/2008) 

c. Guidance for Industry- Container and Closure system Integrity

Testing in Lieu of Sterility Testing as a Component of the

Stability Protocol for Sterile Products (2/2008) 

d. Guidance for Industry- Quality Systems Approach to

Pharmaceutical cGMP Regulations (9/2006) 

e. Draft Guidance for Industry and FDA Staff- Nucleic Acid Based In

Vitro Diagnostic Devices for Detection of Microbial Pathogens

(12/2005) Docket number 2005D-0434 

f. Guidance for Industry- Manufacturing Biological Drug

Substances, Intermediates, or Products using Spore-forming

Microorganisms (2/2005) 

g. Sterile Drug Products Produced by Aseptic Processing —

Current Good Manufacturing Practice, 9/2004

h. Comparability Protocols - Chemistry, Manufacturing, and

Controls Information, Required for industry interested in

substituting an automated/Rapid Microbiological method in place

of the USP compendial method cited in their original application

(2/2003)

i. Guidance for Industry- Sterility Requirement for Aqueous- Based

Drug Products for Oral Inhalation—Small Entity Compliance

Guide (11/2001) 

j. Guide to Inspections of Quality Systems-Medical Device (8/1999)

k. Guidance for Industry-Content and Format of Chemistry, 

Manufacturing and Controls Information and Establishment 

Description Information for a Vaccine or Related Product

(1/1999) 

l. Guide to Inspections of Lyophilization of Parenterals (10/18/97) 

m. Guide to Inspections of Cosmetic Product manufacturers

(2/1995) 
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n. Guide to Inspections of Sterile Drug Substance Manufacturers, 

(7/1994) 

o. Guide to Inspections of Topical Drug Products (7/1994) 

p. Guidance for Industry- Submission Documentation for

Sterilization Process Validation in Applications for Human and

Veterinary Drug Products (11/1994) 

q. Guideline for the manufacture of In Vitro Diagnostic Products

(1/1994) 

r. Guide to Inspections of Microbiological Pharmaceutical Quality

Control Laboratories (7/1993) 

s. Guide to Inspections of High Purity Water Systems, (7/1993) 

t. Guide to Inspections of Validation of Cleaning Processes

(7/1993) 

u. FDA Biotechnology Inspection Guide, Reference materials and

training aids (11/1991) 

v. Guidance for Industry, Pyrogen and Endotoxins Testing: 

Questions and Answers (June 2012)

8. Inspectors technical guidance (ITG)-

The following are the ITGs that were written regarding microbiological issues.

a. PYROGENS, STILL A DANGER (1/12/79 Number: 32) 

b. HEAT EXCHANGERS TO AVOID CONTAMINATION (7/31/79

Number: 34) 

c. REVERSE OSMOSIS (10-21-80 Number: 36) 

d. BACTERIAL ENDOTOXINS/PYROGENS (3/20/85 Number: 40) 

e. LYOPHILIZATION OF PARENTERALS (4/18/86 Number: 43) 

f. WATER FOR PHARMACEUTICAL USE (12/31/86 Number: 46) 

g. MICROBIOLOGICAL CONTAMINATION OF EQUIPMENT

GASKETS WITH PRODUCT CONTACT (12/31/86 Number: 48) 

9. Miscellaneous FDA Documents and References

This list of references may not be entirely microbiology but very important none

the less.

a. FDA Inspectional Operational Manual 

b. FDA Warning Letters and Responses 
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c. FDA Bacteriological Analytical Manual (BAM) (1/2001) 

10. Important Government and International organizations:

a. National Institute of Health (www.nih.gov)

b. Center for Disease Control and Prevention (www.cdc.gov)

c. CDC report on environmental monitoring

(http://www.cdc.gov/ncidod/dhqp/gl_environinfection.html)

d. World Health Organization (www.who.org) International 

pharmaceutical regulations along with monitoring of disease

outbreaks around the globe may be important if assigned to work

in a high-risk area.

11. Industry Technical referencesParenteral Drug Association (PDA) Technical Reports- Although the scientific 

recommendations in these technical reports are not enforceable by FDA, they

do contain industry current manufacturing practices and scientifically sound

principles that support regulatory concerns. The following is a selection of PDA 

technical reports that may be useful:

Report No. Title Date

1 Validation of Moist Heat Sterilization Processes: Cycle Design,

Development, Qualification and Ongoing Control

July

2007

3 Validation of Dry Heat Processes Used for Sterilization and

Depyrogenation

1981

4 Design Concepts for the Validation of Water-for-Injection Systems 1983

5 Sterile Pharmaceutical Packaging: Compatibility and Stability 1984

7 Depyrogenation 1985

11 Sterilization of Parenterals by Gamma Radiation 1988
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13 Fundamentals of an Environmental Monitoring Program 1990

(Revised

2001)

15 Industrial Perspective on Validation of Tangential Flow Filtration in

Bio-pharmaceutical Application

1992

20 Report on Survey of Current Industry Gowning Practices 1990

21 Bioburden Recovery Validation 1990

22 Process Simulation Testing for Aseptically Filled Products 2011

23 Industry Survey on Current Sterile Filtration Practices 1996

26 Sterilizing Filtration of Liquids 2008

28 Process Simulation Testing for Sterile Bulk Pharmaceutical

Chemicals

2006

(revised)

29 Points to Consider for Cleaning Validation 2012

30 Parametric Release of Pharmaceuticals Terminally Sterilized by

Moist Heat

1999

33 Evaluation, Validation and Implementation of New Microbiological

Testing Methods

2000

34 Design and Validation of Isolate Systems for the Manufacturing and

Testing of Health Care Products

2001

35 A Proposed Training Model for the Microbiological Function in the

Pharmaceutical Industry

2001

36 Current Practices in the Validation of Aseptic Processing -- 2001 2002

40 Sterilization Filtration of Gases 2005

41 Virus Filtration 2005

45 Filtration of Liquids using Cellulose-based depth filter 2008

57 Analytical Method Validation and Transfer for Biotechnology products 2012
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61 Steam in place 2013

12. Books and Commercial Trade reports:

a. ASM, Manual of Clinical Microbiology;

b. Disinfection, Sterilization, and Preservation, by S Block; Bergey’s

manual systematic Bacteriology

c. Remington’s Pharmaceutical Sciences

d. F-D-C Monthly Reports

Excellent summary of conferences, FDA regulation changes, Key Industry

personnel and often a list of the most recent Product Recalls and regulatory

actions by FDA. Need to sign up for email membership. Instructions for 

membership enrollment are available at FDA website below. 

(http://inside.fda.gov/Library/ElectronicResourcesWebLERN/Alphabeticallist/in

dex.htm)

The “Gold Sheet”- Pharmaceutical & Biotechnology Quality Control The “Pink

Sheet”- Prescription Pharmaceuticals and Biotechnology The “Gray Sheet”-

Medical Devices Diagnostics & Instrumentation The “Silver Sheet”- Medical 

Device Quality Control reports

13. Free Trade publications available on linePharmaceutical Technology (www.pharmtech.com)

Controlled Environments (www.cemag.us)

American Pharmaceutical Review (www.americanpharmaceuticalreview.com)

International BioPharm (www.biopharminternational.com/)

XIV. Professional memberships

These organizations have available searchable references. 

International Society of Pharmaceutical Engineers (www.ispe.org)

American Society for Microbiology (www.asm.org)

Parenteral Drug Association (www.pda.org)

15. Glossary/Definitions

None
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16. Records

None

17. Supporting Documents

None

18. Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

1.1 I 4/25/2014 Angele C. Smith, 

Microbiologist

1.2 R 3/30/2015 Angele C. Smith, 

Microbiologist

1.3 R 6/7/2016 Angele C. Smith, 

Microbiologist

Selen Stromgren, Deputy 

Director MPTSS

1.4 R 1/31/2018 Angele C. Smith, 

Microbiologist

George Salem, Staff Director 

OMPSLO

02 R 7/27/2020 Angele C. Smith, 

Microbiologist

Bryan Gamble, Acting Deputy 

Associate Director OMPSLO

* - D: Draft, I: Initial, R: Revision
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19. Change History

Revision 

# Change

1.1

ii.- updated

Chapter 3. A. 2. a. iv. – revised

Chapter 8. D. 1st paragraph – revised

Chapter 10. H. 5. – author information updated

1.2

Chapter 3, D. 1. a. - revised 

Chapter 7 – Antibiotic Potency Assay (inserted)

1.3

Chapter 9, A. 1. i – revised

Chapter 9, B, B.1.a, C.4, C.7, C.9, C.10, E.1, E.2 – revised

Chapter 10, A.6 – removed

Appendix A, removed links

1.4 Updated highlighted (grey) in document

1.5

Updated to new template

Updated author information

Updated to 508 Compliance

Updated Chapter 2 – revised to match new USP method

Updated Chapter 3 – revised for clarification

Updated Chapter 4 – revised for clarification

Updated Chapter 5 – revised for clarification; added two USP references

Updated Chapter 9 – revised for clarification

Updated Chapter 10 – revised for clarification

20. Attachments

None