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Title: 

ORA Lab Manual Vol. IV Section 15 - Drug and Device Microbiology Page 1 of 77

For the most current and official copy, check QMiS.

Sections in This Document

Purpose ................................................................................................................................. 2

Scope .................................................................................................................................... 3

Responsibility ........................................................................................................................ 3

Background ........................................................................................................................... 3

References ............................................................................................................................ 4

Basics.................................................................................................................................... 4

6.1. Microbiology Techniques............................................................................................. 4

6.2. Media, Reagent, Supply Preparation and Applicable Quality Release Testing ............ 5

6.3. Safety and Hazardous/Infectious Waste...................................................................... 6

6.4. Quality Assurance and Data Integrity .......................................................................... 8

6.5. Working within an ISO 5 Controlled Environment ...................................................... 10

6.5.1. Clean Rooms............................................................................................... 10

6.5.2. Isolator Technology ..................................................................................... 11

6.5.3. Biological Safety Cabinets and Laminar Flow Hoods................................... 13

6.6. Methodology.............................................................................................................. 14

6.6.1. Direct Staining and Visual Screening Method.............................................. 14

6.6.2. Microbiological Examination of Non-Sterile Products................................... 15

Special Assignments/Non-typical......................................................................................... 16

7.1. Non-tuberculosis Mycobacteria (NTM) ...................................................................... 16

7.2. Burkholderia cepacia complex (BCC)........................................................................ 17

7.3. Sterility Testing of Medical Products.......................................................................... 18

7.4. Investigating USP Sterility Testing Failures ............................................................... 20

7.5. Bacterial Endotoxin Testing....................................................................................... 21

7.6. Testing Environmental Monitoring Samples............................................................... 23

7.7. Particulate Matter ...................................................................................................... 24

7.8. Antibiotic Potency Testing ......................................................................................... 25

7.9. Antimicrobial Effectiveness Testing ........................................................................... 27

7.10. Biochemical Identification .......................................................................................... 28

7.11. Polymerase Chain Reaction (PCR) ........................................................................... 30

7.12. Sanger DNA Sequencing .......................................................................................... 31

7.13. Whole Genome Sequencing (WGS).......................................................................... 32

7.14. Developing/Establishing a Rapid Method for Implementation into Regulatory Testing34
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Inspectional Guidance ......................................................................................................... 36

8.1. Drug Inspections ....................................................................................................... 37

8.2. Device Inspections .................................................................................................... 38

8.3. Compounding Pharmacies ........................................................................................ 39

8.4. EM Sample Collections ............................................................................................. 40

Glossary/Definitions............................................................................................................. 41

Supporting Documents ........................................................................................................ 41

Document History ................................................................................................................ 42

Change History.................................................................................................................... 42

Attachments......................................................................................................................... 42

Attachment A - Answer Key ................................................................................................. 43

Purpose

FDA regulatory microbiologists examine products under the purview of the 

Federal Food, Drug, and Cosmetic Act (FD&C Act) for adulteration, conduct 

method development research, respond to outbreaks and other medical 

product emergencies, and participate in team establishment inspections.

Analyses range from the relatively simple to the most complex. In recent years, 

microbiologists have transitioned from sole use of conventional methods to a 

coupling of state-of-the-art rapid methods with traditional methods.

The overall purpose of the training program is to: 

A. Train the analyst to think as a regulatory microbiologist.

B. Introduce typical analytical procedures a regulatory microbiologist 

should know and understand.

C. Show where and how the work performed fits into the regulatory 

framework. 

D. This regulatory framework includes: 

1. The reasons for sample collection.

2. The procedures of inspection and sample collection.

3. The sample analysis procedures.

4. Regulatory follow up actions and relationship of items to the 

FD&C Act.
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Scope

The FDA microbiologist is well termed a regulatory microbiologist because 

everything he or she does is related to the regulation of products and 

manufacturers under the jurisdiction of the Federal Food, Drug, and Cosmetic 

Act (FD&C Act) and Related Acts. Thus, results of his or her work impact 

directly on those products regulated, and therefore on the consumer. The FDA 

microbiologist can also be called a public health microbiologist as removing 

contaminated medical products from the market directly impacts the health and 

welfare of consumers.

Responsibility

A. Trainer

1. Coordinates training with Supervisor(s) and Trainee.

2. Works with trainees to ensure completion of all training needed to 

meet their regulatory responsibilities.

B. Trainee

1. Completes required training within specified timeframes.

2. Reports training received and submits documentation for training to 

supervisor.

3. Reads and complies with standards, regulations, policies, 

procedures, and work instructions.

C. Supervisor

1. Implements and reviews training records.

D. Quality Management

1. Maintains employee competencies matrix records for staff.

Background

This training program is divided into different modules. If all modules are 

completed, the microbiologist will be competent in many procedures in FDA 

microbiology. 

During the first year, there will be several basic training courses offered by 

Office of Training, Education and Development (OTED). Training will also be 
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supplemented by computer-based modules provided online by ORA U. 

However, most instructions will come from the laboratory.

Microbiology methodology is an ever-improving science. Training will be a 

continuing process throughout the microbiologist’s career. Future training will 

reinforce and amplify what the trainee has learned.

References

A. Pharmaceutical Microbiology Manual. (Current ed.) Retrieve through 

QMiS (ORA.007).

B. U.S. Pharmacopeia/ National Formulary (current ed.). 

C. Difco manual of dehydrated culture media and reagents for 

microbiology (current ed.). Detroit, MI: Difco Laboratories, Inc. 

D. FDA Staff Manual Guide, Vol III- General Administration Safety and 

Occupational Health Programs Hazardous Biological Agents and Toxins

E. 21 CFR 211 Current Good Manufacturing Practice for Finished 

Pharmaceuticals

F. ICH Q7 Good Manufacturing Practice Guide for Active Pharmaceutical 

Ingredients

G. Drug Quality Assurance CPG 7356.002

H. Sterile Drug Process Inspections. Retrieve from Compliance Program 

Manual. 

I. Active Pharmaceutical Inspections. Retrieve from Compliance Program 

Manual. 

J. Sterile Drug Products Produced by Aseptic Processing. 

K. Analytical Procedures and Method Validation for Drugs and Biologics. 

L. CPG 7382.845: Inspection of medical device manufacturers

M. CPG 7383.001: Medical Device PMA Preapproval and PMA Postmarket Inspections

N. Guide to inspection of Quality Systems (QSIT)

Basics

6.1. Microbiology Techniques

A. Objective
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Introductory exercises demonstrating the trainee is proficient in basic 

microbiology techniques

B. Assignment

The trainee will discuss and demonstrate basic microbiology skills such as the 

following:

a. Gram staining

b. Aseptic techniques

c. Serial dilutions

d. Quadrant streak

e. Personal protective equipment (PPE)

6.2. Media, Reagent, Supply Preparation and Applicable Quality Release Testing

A. Objectives

1. To introduce the trainee to applicable procedures to be followed for 

preparation of media, reagents and supplies that will be used in medical 

product testing. These procedures will include aseptic technique necessary 

when handling sterilized media.

2. To introduce the trainee to applicable quality assurance procedures used 

for verifying that the media and reagents meet their required specifications.

3. The trainee will learn growth promotion testing in accordance with USP and 

applicable procedures for various media and reagents used in medical 

product testing. This will include the differences between liquid growth 

media and the testing of plated media and specific requirements associated 

with inhibitory testing and indicative testing.

4. To familiarize the trainee with safety concerns, such as use of the 

autoclave(s), hotplate(s) and weighing of powders.

B. Assignment 

1. The trainer will discuss and demonstrate the equipment used in media 

preparation such as balances, hotplates, dispensers, pH meters, pipettors 

and autoclave(s).

2. The trainer will discuss the function of the components that comprise 

commonly used media such as soybean casein digest broth, fluid 

thioglycolate broth, Sabouraud dextrose agar and frequently prepared 

selective agars.

3. The trainer will discuss and demonstrate the growth promotion activities, as 

well as the process for release testing of media.
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4. The trainee will prepare several kinds of media (enrichment broths, 

nonselective plated media and selective plates) and buffer solutions. The 

trainee will perform growth promotion testing on the prepared media and 

buffers. 

5. The trainee will prepare and sterilize supplies including empty bottles, 

tubes and utensils as dictated by individual lab use.

6. The trainer will discuss storage and shelf life of prepared media.

C. Questions

1. Which media should not be steam sterilized and why?

2. Where is agar derived from and what are the special properties of agar that 

make it well suited as a solidifying agent in culture media? 

3. Once agar is melted, what temperature should agar be kept at to prevent it 

from solidifying?

4. What is the purpose of conducting growth promotion testing on media used 

in microbiological testing?

5. In which media and buffer preparations is volume particularly critical?

6. Why is pH important in media and buffer preparation?

7. What are some safety concerns when autoclaving?

6.3. Safety and Hazardous/Infectious Waste

A. Objectives

1. To introduce the trainee to laboratory safety practices.

2. To introduce the trainee to the hazards involved in working with pathogens 

and/or their toxins such as Bacillus cereus toxin.

3. To develop the trainee’s awareness of procedures for the proper 

segregation and disposal of laboratory waste products.

4. To identify resources which can assist the employee with the risk 

assessment process.

B. Assignment

1. Read the following:

a. ORA Lab Manual, Volume III, Section 2 for safety issues

i. Laboratory Chemical Hygiene Plan

ii. Laboratory Hazardous Waste Plan
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iii. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 

current edition

iv. Laboratory Biosafety Plan and applicable SOPs for local site

v. Safety Data Sheets for each chemical used in the analytical 

procedure

C. Questions

1. What are the items of personal protective equipment (PPE), minimally 

needed, in a Biological Safety Level (BSL)-2 biological laboratory?

2. What work practices are to be in place when working in a Biological Safety 

Level (BSL)-2 laboratory? 

3. Describe and give examples of opportunistic microorganisms that cause 

infections, disease, or other health hazards and identify their biosafety 

levels.

4. Describe proper procedures for disposal of used media. Describe how to 

use and operate an autoclave safely. 

5. What is a SDS (Safety Data Sheet)? Where are the SDSs located? What 

information can be found on a chemical reagent hazard label?

6. Does the state regulate medical waste? What laboratory wastes are 

permitted to enter the sewer? What laboratory wastes are incinerated?

7. What are the guidelines for handling microorganisms (mostly bacteria or 

their toxins) in the BMBL? 

8. What are engineering controls? Describe the proper use of these 

engineering controls in a medical product microbiology laboratory. 

9. What are administrative controls? Describe administrative controls 

designed to minimize the risks of hazardous agent exposure to those 

personnel who are not directly involved with their manipulation. List those 

administrative controls that assist in the maintenance of quality control.

10.What safety equipment is normally found within a microbiological 

laboratory? 

11.What types of laboratory procedures have the potential to generate 

aerosols? How can these procedures be contained? How can the 

generation of aerosols be minimized?

12.What decontamination procedures are in place and when are they 

performed?

13.Describe how spills are handled. Are the cleanups following a spill 

documented and the cleanup verified?
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14.What kind of signage is to be in place in a microbiological BSL-2 

laboratory?

15.What are the potential routes of exposure when working with infectious 

organisms?

16.Why is it necessary for a minimum of two people to be working in the 

laboratory at any given time?

17.Has enough training been received to perform assigned tasks and has this 

training been documented?

18.Is the facility designed to prevent infectious organisms from being 

accidentally released to other areas in the building?

19.Is there a sharps safety program in place to reduce hazards when handling 

syringes or pipettes or other sharps? 

D. Exercises

1. Discuss with trainer or supervisor how etiologic isolates are shipped.

2. Discuss with trainer, supervisor, and/or industrial hygienist how 

hazardous waste is managed at the local site.

6.4. Quality Assurance and Data Integrity

A. Objectives

1. To present concepts of a Quality Management System to include:

a. QMiS

b. Accreditation

c. Proficiency Testing

d. Internal Audits

2. To present quality control concepts such as:

a. Method Modifications

b. Contemporaneous documentation

c. Appropriate testing controls

d. Qualified Suppliers, etc. 

3. To present concepts of data integrity to include:

a. Accountability

b. Media/equipment

c. Photography
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d. Labeling

e. FACTS 

f. Analytical worksheets

g. Electronic records 

B. Assignment

1. Read the following:

a. Laboratory Manual of Quality Policies for ORA Regulatory 

Laboratories (Vol. 1 of the ORA Lab Manual) and ISO/IEC 

17025:2017 Sections 6.3, 6.4, 6.5 and 7.1 – 7.8

b. ORA Lab Manual Volume II, Section 2, 

c. ORA Lab Manual Volume III, Section 3, Recording of Results –

Analyst’s Worksheet

d. SOP-000288 Microbiological Controls for Medical Product Sample 

Analysis 

2. Read local Standard Operating Procedures (SOPs) regarding corrective 

actions, preventative actions, nonconforming work, method verification and 

validation and record and data management.

3. Trainer will discuss with trainee the various activities to support quality 

assurance/control such as aseptic technique, quality controls, 

environmental and personnel monitoring, media growth promotion testing, 

suitability testing, proficiency testing, analyst competency, correct media 

volume, pH and formulation, equipment controls, etc. 

4. Trainer will introduce the trainee to the location and content of harmonized 

worksheets. 

5. Trainer will discuss with the trainee the principles of data integrity such as 

accurate calculations, review of controls, real-time recording of 

observations, where to find appropriate acceptance criteria, how to review 

analytical worksheets, how to complete relevant Quality Assurance 

checklists and how to report, evaluate and respond to data 

deviations/nonconformances. 

C. Questions 

1. What are culture controls and why are they required in analyses? 

2. With whom does Data Integrity begin?

3. Why are Quality Assurance and Data Integrity so important?
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4. What is the purpose of environmental and personnel monitoring? 

5. Why are temperatures recorded for incubators, water baths, etc.?

6. Why are worksheets reviewed and signed by an analyst/Supervisor/Team 

Lead who has not worked on the sample?

7. Why are validated methods used?

8. What is QMiS?

6.5. Working within an ISO 5 Controlled Environment

6.5.1. Clean Rooms

A. Objectives

1. Introduce the characteristics of a cleanroom to include the types of 

cleanrooms, the current ISO (International Organization for Standards) 5 

specification, the importance of maintaining appropriate relative humidity 

control and temperature control and pressure differentials.

2. Introduce clean room disinfection procedures.

3. Introduce safety practices, Do’s and Don’ts while working in the clean 

room.

4. Introduce proper sample preparation needed before taking sample into the 

cleanroom.

5. Introduce ways to control contamination in a cleanroom:

a. Control air flow

b. Operator gowning

c. Operator follows strict aseptic procedures

d. Decontamination of all media and equipment used within the cleanroom

e. Follow environmental and personal monitoring procedures

f. Follow effective cleanroom cleaning and testing procedures

B. Assignment

1. The trainer will provide appropriate training materials about cleanroom, 

sterility analysis and hands on training:

a. Disinfection Procedures

b. Media, equipment, and sample prep

c. Gowning
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d. Aseptic Technique

e. Components of quality control

f. Personal monitoring procedures

2. The trainee will work with the trainer to become proficient working in a 

cleanroom environment.

3. The trainee will need to pass gowning qualification and perform satisfactory 

proficiency testing.

C. Questions

1. What environmental characteristics need to be controlled in a cleanroom?

2. Should a cleanroom be under positive or negative filtered air pressure and 

why?

3. Why should a relative humidity in a cleanroom be controlled?

4. What ISO Equivalent Class is equivalent to Class 100 room?

5. Name 3 acceptable agents used to disinfect the cleanroom?

6. Describe the 2-bucket cleanroom disinfection method?

7. Why should you work “wet” while disinfecting the cleanroom?

8. Describe all gowning material (PPE) used for working in the cleanroom?

9. What are the advantages of working as a team “buddy system” within the 

clean room?

10.What are some of the safety practices that should be followed while 

working in the cleanroom?

6.5.2. Isolator Technology

A. Objectives

1. Introduce the key components of what constitutes an isolator.

2. To introduce the principles of vaporized hydrogen peroxide and discuss 

how the sterilant is used within isolators.

3. To introduce the necessary components of quality control for isolators such 

as aseptic technique, environmental monitoring requirements, personal 

protective equipment, glove integrity checks and decontamination cycle 

development.

B. Assignment
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1. The trainer will provide relevant training materials regarding the necessary 

components of an isolator and provide hands on training for the operation 

of the sterility testing isolator in laboratories that are equipped with 

isolators. These activities will be performed by referencing local procedures 

for the operation and maintenance of the sterility testing isolator. The 

trainer will provide one or more training samples for the analyst to process 

into the isolator, conduct sterility testing on and then remove from the 

isolator.

2. Using a validated cycle, the analyst will assist with loading a medical 

product sterility sample into the isolator, executing a decontamination cycle 

and performing sterility testing on the sample within the isolator 

environment with a qualified trainer. If the laboratory does not have access 

to an isolator this assignment can be performed via a discussion with the 

qualified trainer. During the discussion the trainer should discuss the 

importance of proper loading of an isolator and provide details on the types 

of supplies, reagents and test equipment that are needed for a sterility test 

within an isolator.

3. Using a training cycle or a validated cycle the analyst will independently 

properly clean, load and run a decontamination cycle for the sterility testing 

isolator in preparation for a sample analysis. The trainer will provide the 

analyst with suitable supplies to be used during this training for loading of 

the isolator. If the laboratory does not have access to an isolator this 

assignment can be performed via a discussion with the qualified trainer. 

During the discussion the trainee should describe the proper preparation 

procedures for readying and the isolator for a decontamination cycle and 

provide details on the types of supplies, reagents and test equipment that

are needed for a sterility test within an isolator.

4. Analyze a medical product sample within the isolator environment. The 

trainer will spike one or two units of a sample with aerobic and/or anaerobic 

microorganisms. If the laboratory does not have access to an isolator this 

assignment can be performed via a discussion with a qualified trainer. The 

trainee should provide details on the actions that would be taken to analyze 

a sample within an isolator. 

C. Questions

1. What are two key components of an isolator?

2. What does VHP stand for and what is its purpose in an isolator?

3. Why is aseptic technique still necessary within a sterility testing isolator?

4. What types of environmental monitoring can be performed within a sterility 

testing isolator?
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5. What is the importance of checking the integrity of isolator gloves?

6. Why is a validated loading profile important?

6.5.3. Biological Safety Cabinets and Laminar Flow Hoods

A. Objectives

1. Introduce the key components of what constitutes a biological safety 

cabinet and laminar flow hood.

2. Introduce the principles of the purpose and function of a biological safety 

cabinet and laminar flow hood. 

3. Introduce the necessary quality control related to the operation of biological 

safety cabinets and laminar flow hoods, such as aseptic technique, 

environmental monitoring, personal protective equipment, and 

decontamination. 

B. Assignment

1. The trainer will provide relevant training materials associated with the 

maintenance and disinfection of biological safety cabinets and laminar flow 

hoods. The trainer will also provide the supplies and equipment necessary 

to perform required maintenance and disinfection. Utilizing local 

procedures, the analyst will perform maintenance and disinfection on both 

a biological safety cabinet and laminar flow hood. 

2. The trainer will provide relevant training materials associated with operation 

of biological safety cabinets and a laminar flow hoods and provide hands 

on training related to the operation of the hoods. These activities will be 

performed by referencing local procedures for the operation of biological 

safety cabinets and laminar flow hoods. During the training instruction the 

trainers should discuss the importance of proper placement of items within 

each hood type and cautions related to the disruption of airflow within the 

hood. The trainer will provide training samples for the analyst to process in 

each hood type. 

3. The trainer will provide relevant training materials associated with 

environmental sampling of biological safety cabinets and laminar flow 

hoods. The trainer will supply the necessary supplies needed to perform 

environmental sampling of both types of hoods. Utilizing local procedures, 

environmental sampling activities will be conducted utilizing the applicable 

personal protective equipment to simulate environmental sampling that 

would be required during an aseptic testing event. 

C. Questions
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1. What are the two main differences between a biological safety cabinet and 

a laminar flow hood?

2. Why is personal protective equipment required when working in either a 

biological safety cabinet or laminar flow hood? 

3. Name three ways to prevent an interruption in air flow in a biological safety 

cabinet and laminar flow hood. 

4. How many classes of biological safety cabinets are there?

5. What is an aseptic technique related caution associated with working in an 

open an open-faced laminar flow hood? 

6. Describe the placement of environmental monitoring plates in a biological 

safety cabinet and in a laminar flow hood. 

7. How often do biological safety cabinets and laminar flow hoods require 

certification by a qualified vendor? 

8. Are UV lights in a biological safety cabinet or laminar flow hood an effective 

means of decontamination? 

9. Why is the sample and not the user protected when utilizing a laminar flow 

hood? 

10.What type of filters are used in a biological safety cabinet and laminar flow 

hood? 

6.6. Methodology

6.6.1. Direct Staining and Visual Screening Method

A. Objectives

1. To introduce a direct staining method to include photography for rapid 

determination of the presence of microorganisms in a sterile product.

2. To introduce methods and practices for the visual examination of product 

container/closure defects for sterile medical products. Defects may include 

cracks, punctures or damage that results in a non-intact container/closure 

system.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to direct staining. This may be accomplished through a series 

of designated training samples. Once the trainer is confident that the trainee 

can successfully and independently perform the analysis, the trainee will be 

issued a series of training samples.

1. Read the following:
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a. Pharmaceutical Microbiology Manual (PMM), Chapter 10.

b. Screening Protocol for Direct Staining on Products with Appearance of 

Visible Contamination (ORA-LAB.015). 

2. To analyze two different samples for visual contamination of the 

container/content.

3. To perform Gram staining of aliquots on visually contaminated samples. 

C. Questions

1. What would be some visual clues of microbial contamination?

2. Describe various types of cell morphology.

3. What are fungal hyphae?

4. What are spores?

5. Describe the difference between gram-negative and gram-positive 

organisms.

6.6.2. Microbiological Examination of Non-Sterile Products

A. Objective

1. Introduction to the determination of the quantitative enumeration under the 

conditions described in USP <61>.

2. Introduction to testing for the absence of specified microorganisms under 

the conditions described in the USP <62>. 

3. Introduction of the concept of method suitability testing as described in the 

USP <61> and <62>. 

4. Introduction to USP <61> and <62> concept of overcoming inhibition using 

neutralization and inactivation agents.

B. Assignment

It is the trainer’s responsibility to transfer knowledge both practical and in 

theory related to sample analysis as described in USP <61>, <62>, and the 

PMM. This may be accomplished through a series of designated training 

samples. Once the trainer is confident that the trainee is can independently 

perform the analysis, the trainee will be issued training samples to 

demonstrate proficiency.

1. Read the following:

a. USP <61> (current edition). Microbiological Examination of Nonsterile 

Products: Microbial Enumeration Tests
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b. USP <62> (current edition). Microbiological Examination of Nonsterile 

Products: Tests for Specified Microorganisms.

c. USP <1111> (current edition). Microbiological Examination of 

Nonsterile Products: Acceptance Criteria for Pharmaceutical 

Preparations and Substances for Pharmaceutical Use. 

d. Pharmaceutical Microbiology Manual, Chapter 2.

e. SOP Microbiological Controls for Medical Product Sample Analysis 

(SOP-000288).

2. Perform method suitability testing for two or more samples, such as a solid, 

fluid, water-immiscible fluid (waxes, ointment, creams), or fluid specimen in 

aerosol form for Total Aerobic Microbial Count and Total Yeast and Mold 

Count.

3. Analyze two or more samples such as a solid, fluid, water-immiscible fluid 

(waxes, ointment, creams), or fluid specimen in aerosol form for Total 

Aerobic Microbial Count and Total Yeast and Mold Count.

C. Questions

1. How does the analyst determine which controls to utilize for determination 

of the quantitative enumeration and the absence of specified 

microorganisms’ analysis?

2. What are some agents that can be used for neutralization of interfering 

substances?

3. Why is the Growth Promotion Test performed? 

4. What three tests are performed as part of the Growth Promotion Test? 

5. Why is Method Suitability Testing required prior to performing analysis for 

determination of the quantitative enumeration and the absence of specified 

microorganisms?

6. According to USP <61>, what three methods can be used to enumerate? 

7. What is the purpose of USP <1111>? 

Special Assignments/Non-typical 

7.1. Non-tuberculosis Mycobacteria (NTM)

A. Objectives 

1. To present techniques for the cultivation and identification of NTMs.

2. To present techniques for the recovery of NTMs from non-sterile medical 

products.
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B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a series of training samples. 

1. Read the following:

a. SOP-000059: Procedure for the Isolation and Identification of 

Nontuberculous Mycobacteria from Non-Sterile Medical Products

b. USP <61> Microbiological Examination of Nonsterile Products: 

Microbial Enumeration Tests

c. USP <62> Microbiological Examination of Nonsterile Products: Tests 

for Specified Microorganisms.

d. ORA.007 Pharmaceutical Microbiology Manual, Chapter 2

e. ORA-LAB.018 Biosystems MicroSEQ Microbial ID System

LIB #4569 - Isolation and Identification of Nontuberculous Mycobacteria 

Associated with Tattoo-related Outbreaks

2. Culture and identify the control organisms as described in the method.

3. Analyze a non-sterile medical product for the presence of NTMs as 

described in the method.

C. Questions

1. What are Rapid Growing Mycobacteria and Slow Growing Mycobacteria? 

Give some examples.

2. What media are used for the cultivation of NTMs?

3. What are acid-fast bacteria? 

4. What DNA targets are used in the identification of mycobacteria? 

7.2. Burkholderia cepacia complex (BCC)

A. Objectives

1. Introduce how special assignments may be received by the laboratory from 

CDER in response to surveillance data or adverse events.

2. Introduce the method for Burkholderia cepacia complex testing as it relates 

to non-sterile pharmaceutical products.

B. Assignment
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It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a training sample.

1. Read USP <60> Microbiological Examination of Nonsterile Products- Tests 

for Burkholderia cepacia complex 

2. The trainee should have previously completed or be concurrently 

undergoing training for nonsterile pharmaceutical testing (USP <61> and 

USP <62>) in accordance with this Laboratory Manual Chapter.

3. Working with a trainer observe and assist on testing of non-sterile drug 

products for Burkholderia cepacia complex. Additional training may be 

provided depending on the trainee’s level of understanding of the method.

4. Working independently the trainee will analyze a sample provided by the 

trainer which will consist of multiple units that have been spiked with 

Burkholderia cepacia.

C. Questions

1. What does the phrase Burkholderia cepacia complex mean?

2. What is the primary selective agar used in the method to detect organisms 

within the Burkholderia cepacia complex?

3. What is the typical morphology of Burkholderia cepacia colonies on BCSA? 

4. Why isn’t Cetrimide agar used as the sole agar for the detection of 

Burkholderia cepacia complex?

5. The Burkholderia cepacia complex is comprised of numerous Burkholderia

species. What characteristics do these species have in common?

6. Describe at least two situations in which the detection of Burkholderia 

cepacia complex may be added to a non-sterile analysis.

7.3. Sterility Testing of Medical Products

A. Objectives

1. To analyze medical products that are labeled “sterile” for sterility using 

analytical techniques, such as membrane filtration and direct inoculation.

2. To introduce the following concepts: aseptic technique, method suitability 

testing, and visual examination of particulates.

3. To introduce sample preparation methods for sterility analysis.
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4. To present gowning procedures necessary for work in clean rooms and/or 

isolators.

5. To read and discuss USP sections and supplemental information found in 

the Pharmaceutical Microbiology Manual. 

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a series of training samples. 

1. Read the following:

a. USP <71> Sterility Tests (current edition).

b. Pharmaceutical Microbiology Manual, Chapter 3.

c. Local Standard Operating Procedures

2. If applicable, analyze two different drug samples for sterility using USP 

methodology; one tested by direct inoculation and one tested by membrane 

filtration. Trainer should spike one or two units with aerobic and anaerobic 

organisms and discuss the differences between large volume parenteral 

(LVP) and a small volume parenteral (SVP).

3. If applicable, analyze two different medical device samples for sterility. 

Trainer should spike a few units with aerobic and anaerobic organisms.

C. Questions

1. What is the purpose of method suitability test? 

2. What is the inoculation level in CFUs to be used to inoculate product in 

media?

3. Describe what is done to ensure the work area is acceptable for sterility 

testing?

4. What should an analyst do if the growth in air sample plates exceeds the 

laboratory’s alert/action levels? 

5. When would the incubation time be extended beyond typical 14-day 

incubation period? 

6. What determines if a product is a SVP or LVP?

7. What is the purpose of using two different media?

8. How does Fluid Thioglycollate maintain anaerobic conditions?
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9. What is the function of the red indicator in Fluid Thioglycollate?

10.How is a sterility test by membrane filtration different from a sterility test by 

direct transfer? 

11.What is the difference between Fluid A, D, and K.? When would a 

microbiologist use one over the other?

12.What is the incubation temperature for Fluid Thioglycollate and Soybean 

Casein Digest Medium?

13.What is the purpose of the Pharmaceutical Microbiology Manual (PMM), 

Chapter 3?

14.During the disinfection procedure prior to entering the cleanroom which 

type of products may be soaked in a disinfectant?

15.What are the six strains of microorganisms used for Growth Promotion Test 

and Method Suitability Test?

16.What can you do to eliminate product inhibition in Suitability Testing?

7.4. Investigating USP Sterility Testing Failures 

A. Objectives

1. To identify equipment, materials, media, etc. used during testing and 

evaluate for malfunctions.

2. To identify any deviations from approved methodology.

3. To evaluate analyst qualifications.

4. To identify and evaluate environmental conditions during testing.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sterility failure investigations. This may be accomplished 

through completion of a sterility failure checklist. Once the trainer is confident 

that the trainee can successfully and independently perform the investigation, 

the trainee will be issued a sterility failure analytical package for investigation.

1. Read the following:

a. ORA Laboratory Manual, Control of Nonconforming Work (ORALAB.4.9)

b. Local Nonconformance procedures

c. Pharmaceutical Microbiology Manual Chapter 4.

d. Sterility Failure Checklist (FORM-000115).
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e. Complete sterility failure checklist.

C. Questions

1. What laboratory classification of samples would require a sterility failure 

investigation?

2. What actions should be taken if a negative control is contaminated?

3. What records should be reviewed to determine analyst qualifications? 

7.5. Bacterial Endotoxin Testing 

A. Objectives

1. Introduce principles related to endotoxin testing to include what endotoxin 

is, the purpose of endotoxin testing and the types of products that undergo 

the testing. 

2. Introduce the methodology for USP <85> and USP <161>, as applicable.

3. Introduce the three techniques for the Bacterial Endotoxins Test (BET).

a. Gel Cot Technique

b. Turbidimetric technique

i. Endpoint-Turbidimetric Assay

ii. Kinetic-Turbidimetric Assay

c. Chromogenic Technique

i. Endpoint-Chromogenic Assay

ii. Kinetic-Chromogenic Assay

4. Introduce the following concepts: Endotoxin Limit, Maximum Valid Dilution 

(MVD), Test for Interfering Factors, creation of test dilutions, Control 

Standard Endotoxin (CSE), Reference Standard Endotoxin (RSE), LAL 

Reagent and lysate sensitivity.

5. Introduce and work with the trainee to complete calculations for endotoxin 

limit, Maximum Valid Dilution and potential test dilution series.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a training sample.

1. The trainee is to read the following methods and guidance documents:

a. USP <85> Bacterial Endotoxins Test
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b. USP <161> Medical Devices-Bacterial Endotoxin and Pyrogen Tests

c. Pharmaceutical Microbiology Manual Chapter 5: Bacterial Endotoxin 

Testing

2. The trainee will work with a trainer to complete the following assignments 

related to the Gel Clot Assay:

a. The trainee will observe and assist on at least two gel clot assays.

b. The trainee will work independently to complete the analyst qualification 

for the gel clot assay as required by your laboratory.

c. The trainee will work independently to analyze a sample, using the gel 

clot assay, provided by the trainer which will consist of multiple units 

with varying concentrations of endotoxin.

3. The trainee will work with a trainer to complete the following assignments 

related to the Kinetic Assay:

a. The trainee will observe and assist on at least two kinetic endotoxin 

tests.

b. The trainee will work independently complete the analyst qualification 

for the kinetic assay as required by your laboratory.

c. The trainee will work independently to analyze a sample, using the 

kinetic method, provided by the trainer which will consist of multiple 

units with varying concentrations of endotoxin.

C. Questions

1. What is endotoxin?

2. What is the difference between endotoxin and pyrogens? 

3. Is the USP <85> capable of accurately detecting pyrogens that may be in a 

product? Explain why or why not.

4. What is the assay temperature for Endotoxin Testing?

5. What are some factors that may cause interference in endotoxin testing? 

6. What are some ways to overcome interference in the endotoxin assay?

7. If a product’s endotoxin limit is 0.5 EU/mg, the product’s concentration is 

200 mg/ml and the lysate’s sensitivity is 0.05 EU/ml. What is the maximum 

valid dilution for this product?

8. When calculating the endotoxin limit for a product with the formula, K/M 

what value is used for K for an intrathecal route of administration? What 

value is used for K for routes other than intrathecal?
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9. What is the Gel-Clot technique/method based on?

10.What is the duration of the Endotoxin Testing via Gel-Clot?

11.What tests are performed to ensure the precision and validity of the Gel 

Clot assay?

12.What are acceptable results for system control, negative water control, 

positive product control when running endotoxin testing via Gel-Clot?

13.What are the two types of kinetic methods found in USP <85>. Briefly 

describe each method.

14.According to USP<85>, to perform the confirmation of the lysate sensitivity, 

using the kinetic assay, what is the minimum number of endotoxin 

concentrations that are required for a valid standard curve?

15.For a kinetic assay to be valid per the USP what are the required criteria?

7.6. Testing Environmental Monitoring Samples

A. Objectives

1. Introduction to the qualitative and quantitative assessment of environmental 

monitoring samples collected during inspections of drug 

manufacturers/compounding pharmacies.

2. Overview of the concept of aseptic handling and personal protective 

equipment (PPE) practices associated with environmental monitoring. 

3. Introduction to sample, environment, and personnel monitoring.

B. Assignment

It is the trainer’s responsibility to transfer knowledge both practical and in 

theory related to sample analysis as described in the Pharmaceutical 

Microbiology Manual. This may be accomplished through a series of 

designated training samples. Once the trainer is confident that the trainee can 

independently perform the analysis, the trainee will be issued training samples 

to demonstrate proficiency.

1. Read the following:

a. Pharmaceutical Microbiology Manual, Chapter 9 Environmental 

Monitoring

b. USP <1113> Microbial Characterization, Identification, and Strain 

Typing 

2. SOP-000288 Microbiological Controls for Medical Product Sample Analysis
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3. Demonstrate the proper donning of PPE and the various aspects of the 

analysis associated with aseptic technique while working in a biological 

safety cabinet or laminar flow hood. 

4. Demonstrate proper sample, environment, and personnel monitoring to be 

performed during the environmental sample analysis. 

5. Perform environmental monitoring analysis for two or more samples. 

C. Questions

1. What are the five typical types of environmental samples that could be 

submitted for analysis? 

2. What are some of the advantages and disadvantages of the quantitative 

method of environmental monitoring? 

3. What are some of the advantages and disadvantages of the qualitative 

method of environmental monitoring? 

4. What are some of the key elements that an analyst should check the 

environmental monitoring sample for upon the receipt of the sample? 

5. What types of air monitoring plates could be utilized in the biological safety 

cabinet or laminar flow hood during sample analysis?

6. What organism controls are included as part of the analysis? 

7. What is the incubation timeframe and temperature for the environmental 

monitoring sponge or swab samples? 

8. What is the incubation timeframe and temperature for the environmental 

monitoring RODAC Plates and HycheckTM surface samplers? 

9. What are the designated time frames for the observation for growth during 

the environmental monitoring sample analysis? 

10.When growth is observed during an environmental monitoring test what 

media is used to isolate the growth? 

7.7. Particulate Matter

A. Objectives 

1. To understand the basic principles of particulate matter testing.

2. To review the two methods for determination of particulate matter:

a. Light Obscuration Particle Count Test

b. Microscopic Particle Count Test

3. To understand the principles, applications and operation of the HIAC 9703+ 

Liquid Particle Analyzer
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4. To familiarize the trainee with the sample preparation procedure, proper 

equipment uses and proper PPE use.

5. To familiarize the trainee with operation of the HIAC 9703+ Liquid Particle 

Analyzer.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a training sample.

1. The trainee is to read the following:

a. USP <788> Particulate Matter in Injections

b. USP <789> Particulate Matter in Ophthalmic Solutions

c. USP <1788> Methods for the Determination of Particulate Matter in 

Injections and Ophthalmic Solutions

d. USP <771> Ophthalmic Products – Quality Tests

e. Pharmaceutical Microbiology Manual (PMM) Chapter 6: Particulate 

Matter

f. ORA-LAB.019 HIAC 9703+ Liquid Particle Analyzer

g. Any local SOP’s in reference to specific analyzer requirements

2. Working with a trainer, observe and assist on testing a particulate sample 

using HIAC and/or Microscope. 

3. Working independently, the trainee will analyze a particulate sample using 

HIAC and microscope. The sample will be provided by the trainer.

C. Questions

1. What is a particulate matter?

2. What are the two methods for the determination of particulate matter?

3. What is the HIAC 9703+ Liquid Particle Analyzer flushed with and at what 

volume?

4. What troubleshooting techniques can be implemented if a large particle 

reduces or stops the liquid flow?

7.8. Antibiotic Potency Testing

A. Objectives 
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1. Introduce the purpose of performing potency testing using a microbial 

assay.

2. Introduce the two methods for potency determination: Cylinder Plate 

Method and Turbidimetric Method. 

3. Introduce the use of USP monographs for the preparation of the antibiotics 

involved in USP <81> testing. 

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a training sample.

1. Read the following methods and guidance:

a. USP <81>, Antibiotics- Microbial Assays

b. Pharmaceutical Microbiology Manual, Chapter 7

2. Working with a trainer observe and assist on suitability testing of an 

antibiotic to determine the appropriate inoculum of the specified 

microorganism needed to achieve a zone of inhibition of 14-16mm.

3. Working with a trainer observe and assist on testing an antibiotic using the 

cylinder plate method or the turbidimetric method. Additional training may 

be provided depending on the trainee’s level of understanding of the 

method.

4. Working independently the trainee will analyze an antibiotic for potency. 

The antibiotic will be provided by the trainer.

C. Questions

1. How do you determine the preparation steps necessary to test neomycin 

ointment for potency?

2. What is the purpose of the standard curve and how many standards must 

be included on the standard curve?

3. What is the significance of the Zones of Inhibition?

4. Why is it important to know if the bench you are working on for the 

preparation of plates for the Cylinder Plate method is level?

5. What are some indicators, that can be observed on the agar plates used in 

the Cylinder Plate method, that the bench was not level?
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6. How many times must the analysis be independently conducted for a given 

sample and why?

7.9. Antimicrobial Effectiveness Testing

A. Objectives

1. Introduce principles of the Antimicrobial Effectiveness Test to 

include its purpose, the types of products that undergo the testing 

and the calculations that are performed to report results.

2. Introduce the analytical techniques necessary to complete the 

Antimicrobial Effectiveness test in accordance with USP <51>.

3. Introduce the following concepts: preservatives found in 

pharmaceutical products, inactivating agents and enumeration 

techniques.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a training 

sample.

1. Complete necessary training for USP <61> analysis. 

2. Read the following methods and guidance:

a. USP <51>, Antimicrobial Effectiveness Testing.

b. USP <1227>, Validation of Microbial Recovery from 

Pharmaceutical Articles

c. Pharmaceutical Microbiology Manual Chapter 1

3. Working with a trainer observe and assist in the method suitability 

testing and the product testing. Due to the length of the antimicrobial 

effectiveness testing the timepoints found within USP <51> may be 

shortened to complete the training samples in a timely manner.

4. Working independently analyze a sample beginning with method 

suitability and then product testing. As with the trainer led samples, 

due to the length of the antimicrobial effectiveness testing the 

timepoints found within USP <51> may be shortened to complete 

the training sample in a timely manner.

C. Questions
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1. How many categories of products does the USP <51> list? Briefly 

describe each category.

2. Describe the types of containers the test can be performed in?

3. What is the standardized volume of product FDA laboratories should 

try to test for each microorganism?

4. What microorganisms are required by the USP <51> method and in 

what circumstances might additional microorganisms be added?

5. At what time points are aliquots removed and plated or filtered?

6. With regard to the Criteria for Tested Microorganisms in USP <51>, 

how is the term “No increase” defined?

7. At what point in the method should the analyst refer to USP <1227> 

to determine what additional steps may be needed? 

7.10. Biochemical Identification 

A. Objectives

1. To understand the basic principles and applications of biochemical 

identification utilizing VITEK® 2.

2. To understand the basic principles and applications of biochemical 

identification using supplemental tests identified by VITEK® 2.

3. To familiarize the trainee with the techniques, procedures, and equipment 

used in biochemical identification utilizing VITEK® 2. 

4. To familiarize the trainee with the techniques, procedures, and equipment 

used in biochemical identification using supplemental tests identified by 

VITEK® 2.

5. To identify bacteria recovered from medical products utilizing biochemical 

identification utilizing VITEK® 2.

6. To identify bacteria recovered from medical products utilizing biochemical 

identification using supplemental testing identified by VITEK® 2.

B. Assignment

It is the trainer’s responsibility to transfer knowledge both practical and in 

theory related to sample analysis as described in the Pharmaceutical 

Microbiology Manual. This may be accomplished through a series of 

designated training samples. Once the trainer is confident that the trainee is 

can independently perform the analysis, the trainee will be issued training 

samples to demonstrate proficiency.

1. Review the following:
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a. Any applicable site SOPs

b. USP <1113> Microbial Characterization, Identification, and Strain 

Typing

c. VITEK® 2 Manuals

2. The trainer will demonstrate the preparation of isolates for biochemical 

analysis on the VITEK® 2. The trainee will be required to demonstrate 

proper isolate preparation. 

3. The trainer will demonstrate how to perform a Gram stain by a manual 

method and/or automated method. The trainee will be required to 

demonstrate Gram stain proficiency for the various Gram types, such as 

Gram positive, and Gram negative.

4. The trainer will provide an overview of the VITEK® card selection for 

biochemical analysis. The trainee will be required to demonstrate the 

understanding of how to select which VITEK® card is required for 

biochemical analysis via the VITEK® 2.

5. Trainer will demonstrate the proper use of the DensiCHEKTM. The trainee 

will be required to demonstrate the ability to achieve the appropriate optical 

density of a microorganism suspension utilizing the DensiCHEKTM. 

6. The trainee will be required to perform biochemical analysis via VITEK® 2 

on the several different isolates provided by the trainer.

7. Trainer will demonstrate how to determine when supplemental testing is 

required based on the identification report generated by the VITEK® 2. The 

trainer will also instruct the trainee on how to perform the supplemental 

identification tests. 

8. The trainer will demonstrate how to interpret the results generated by the 

VITEK® with and without supplemental tests. The trainee wee be required 

to properly interpret the results of their isolate identifications.

C. Questions

1. Why is it important to have a quality Gram stain prior to starting the 

identification process? 

2. How is the acceptable optical density range of the microorganism 

suspension determined?

3. How does the analyst know if a supplemental test is required to complete 

the biochemical identification?

4. When preparing the microorganism suspension to load into the VITEK® 2 

cassette, what is the maximum hold time? 
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5. When preparing to use the VITEK® 2 cassette what are some of the factors 

that must be considered? 

7.11. Polymerase Chain Reaction (PCR)

A. Objectives

1. To understand the basic principles and applications of the polymerase 

chain reaction. 

2. To familiarize the trainee with the techniques, procedures, and equipment 

used in PCR analysis.

3. To amplify DNA regions commonly used for microbial identification. The 

amplified regions will serve a template for DNA sequencing. 

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a series of training samples.

1. Review the following:

a. ORA-LAB.017 Fungal Identification by DNA Sequencing

b. ORA-LAB.018 Applied Biosystems (AB) MicroSEQ® Microbial 

Identification System

2. Amplify a specific region of a microbial gene using PCR. 

3. Confirm the presence of the amplicon (e.g. gel electrophoresis, DNA 

sequencing, etc.)

4. Practice using a micropipette to dispense small liquid quantities.

5. Practice using proper molecular biology techniques while opening and 

closing small reaction tubes and dispensing reagents.

6. Learn how to calculate correct reagent quantities needed for master mix 

preparation. 

7. Trainer will demonstrate how to use a thermal cycler.

8. Trainer will discuss which organisms can be analyzed using PCR.

C. Questions

1. What are the three major steps (processes) involved in the PCR cycle?

2. What components are needed in the PCR reaction mixture and what 

function does each element serve?
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3. Why is selection of the primers so important to the success of the reaction?

4. Why are there forward and reverse primers?

5. Why does a microbiologist need an excess quantity of primers in the 

reaction mixture?

6. Why always run a reagent control?

7. List three ways to prevent contamination when performing PCR.

8. In what ways can PCR be inhibited?

7.12. Sanger DNA Sequencing

A. Objectives

1. To understand the basic principles and applications of DNA sequencing

2. To familiarize the trainee with the techniques, procedures, and equipment 

used in DNA sequencing

3. To identify bacteria and fungi recovered from medical products utilizing 

DNA sequencing

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. This may be accomplished through a 

series of designated training samples. Once the trainer is confident that the 

trainee can successfully and independently perform the analysis, the trainee 

will be issued a series of training samples.

1. Review the following:

a. ORA-LAB.017 Fungal Identification by DNA Sequencing

b. ORA-LAB.018 Applied Biosystems (AB) MicroSEQ® Microbial 

Identification System

2. Practice using a micropipette to dispense small liquid quantities.

3. Learn how to calculate correct reagent quantities needed for master mix 

preparation. 

4. Trainer will demonstrate the proper use of equipment necessary for DNA 

sequencing methods such as: Centrifuge, thermal cycler, a sequencer, etc.

5. Trainer will demonstrate how to analyze sequencing data using 

MicroSeqID® and Geneious ®.

6. Trainer will demonstrate how to search sequences on the NCBI and 

alternative databases.
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7. Identify a bacterial and a fungal isolate using DNA sequencing

C. Questions

1. What regions are targeted for bacterial and fungal identification by DNA 

sequencing?

2. What are the major steps (processes) involved in DNA sequencing? 

3. Why is the amplicon PCR cleaned up using prior to cycle sequencing?

4. How is cycle sequencing different from conventional PCR? 

5. What libraries are used for identification of bacterial and fungal sequences 

generated?

7.13. Whole Genome Sequencing (WGS)

A. Objectives

1. To familiarize the trainee with wet lab & data analysis protocols

2. To familiarize the trainee with GalaxyTrakr tools used for data analysis. 

3. To familiarize the trainee with protocols.io document repository.

4. To identify microbial isolates originating from medical products & devices 

using WGS & corresponding data analysis tools. 

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to sample analysis. WGS is an advanced analysis, and the 

trainee should already be competent in fundamental analytical skills such as 

proper use of a micropipette. Once the trainer is confident that the trainee can 

successfully and independently perform the analysis, the trainee will be issued 

a series of training samples, or the trainee can shadow another trained analyst 

during WGS analysis of isolates from regulatory samples with supervisory 

approval.

1. Review the following documents: 

a. Genomic DNA Extraction from Gram-Negative and Gram-Positive

Bacterial Cells using the Qiagen DNeasy Blood and Tissue Kit, 

CFSAN/ORS/DM Document 1231.152.72

b. Genomic DNA Extraction from Gram-Negative and Gram-Positive 

Bacterial Cells using the QIAcube Automated System, CFSAN/ORS/DM 

Document 1231.152.41

c. DNA Quantification using the Qubit Fluorometer, Protocols.io
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d. Illumina DNA Prep (M) Tagmentation Library Preparation for use on an 

Illumina MiSeq Sequencer V.2, protocols.io

e. Procedure for Operation and Maintenance of the Illumina MiSeq for 

Whole Genome Sequencing, protocols.io

f. SOP-000792, GalaxyTrakr MicroRunQC Bioinformatic Workflow for 

WGS QA/QC

g. SOP-000795, GalaxyTrakr CFSAN SNP Pipeline with Assembly (Lite) 

Bioinformatic Tool for within Scheme Type (ST) Comparison

h. JA-000500, Instructions for Using ARGOS Database

i. JA-000501, WGS Data Analysis Tool, BTyper3

j. JA-000502, Kraken2 Instructions

k. JA-000503, Instructions for MetaPhlAn3

l. JA-000504, Instructions for SendSketch

2. The trainer is responsible for demonstrating the proper use and 

maintenance of equipment that is specific to WGS: QIAcube, Qubit, & 

MiSeq.

3. The trainer is responsible for demonstrating the proper use & storage of 

reagents specific to WGS analysis including (but not limited to) Qiagen 

DNeasy Blood and Tissue Kit, Illumina library kits, etc. 

4. The trainer is responsible for demonstrating the proper way to perform 

calculations needed through the WGS analysis. 

5. The trainer is responsible for demonstrating use of GalaxyTrakr tools as 

well as interpreting data obtained from the tools.

6. The trainer is responsible for demonstrating the proper means of evaluating 

various quality parameters.

7. The trainer is responsible for demonstrating the correct way to assemble a 

WGS worksheet.

C. Questions

1. What are the general steps in WGS analysis using the Illumina DNA Prep 

protocol?

2. How should an idle MiSeq be maintained? “Idle” in this scenario means out 

of use for ≥7 days. 

3. What are acceptable run metrics for WGS performed using a v3 600 cycle 

cartridge? 
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4. Upon completion of a WGS run, run metrics are visible on the MiSeq 

Control Software. Where else can run metrics be viewed? 

5. What are acceptable quality metrics values generated using MicroRunQC? 

6. What GalaxyTrakr tool should be used to further assess isolates that have 

the same ST value as a control or as an isolate from a previously analyzed 

sample? 

7.14. Developing/Establishing a Rapid Method for Implementation into Regulatory 

Testing 

A. Objectives

1. To familiarize the trainee with the advantages of using rapid methods.

2. To familiarize the trainee with platforms designed to rapidly identify any 

growth.

3. To educate the trainee on the increased sensitivity of rapid method for 

sterility testing. 

B. Assignment

1. Read the following:

a. USP <1225> Validation of Compendial Procedures

b. USP <1223> Validation of Alternative Microbiological Methods 

c. USP <71> Sterility Testing

d. USP <1071>

e. PDA Tech Report No. 33

f. Online vendor-specific rapid screening methodology reference materials 

g. ORA-LAB.016 Medical Products and Tobacco Program Review and 

Clearance

2. The trainer will provide theoretical knowledge related to rapid sterility 

screening methods, through reference materials available through specific 

vendors, online references and/or methods published in the literature 

(PubMed).

Rapid and alternative sterility test methods have progressed significantly, 

but no single method has emerged as dominant and/or suitable for every 

product class. 

The primary technological platforms for Rapid Micro Methods (RMM) fall 

mainly into four categories:
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a. Growth-based methods: e.g., carbon assimilation, enzymatic 

reactions, carbon dioxide generation or ATP bioluminescence, 

(Instrument BacT/Alert®, BioMerieux)

b. Cellular component methods, e.g., fatty acid analysis using gas 

chromatography, specific portions of the microbial cell, cellular ATP, 

membrane filter incubation as part of cell enrichment, (Instrument 

MilliflexTM Rapid, Millipore)

c. Nucleic acid-based methods: e.g., PCR and automated Southern 

blotting

d. Viability-based methods: e.g., fluorescent labelling methods, such as 

flow fluorescence cytometry; immunofluorescence and fluorescent 

nucleic acid stains used as a viability marker (ScanRDI® Instrument, 

BioMerieux).

Each technique has its advantages and limitations and selection of the 

most appropriate instrument platform/methodology that fits a product type, 

must be considered.

3. Learn how to request a USP-NF account and maneuver through 

specific chapters. 

4. The trainer will inform the trainee on the processes involved to develop 

a new method, prior to its implementation as a regulatory method in the 

lab.

a. Determine the processes/guidelines for development of a new 

method.

b. Determine validation criteria/requirements for method 

qualification.

c. Determine laboratory competency.

5. The trainer will inform the trainee of the process for review and approval 

of research projects to include following local processes, entry of the 

project into CARTS and approval from the Office of Research 

Coordination and Evaluation (ORCE).

6. The trainer will provide the trainee with information on how to navigate 

the CARTS tracking system to enter a research project for approval, 

using the following instructions:

a. Go to the ORA Applications intranet page and select the CARTS link

b. CARTS Intranet Page

c. CARTS ‘help’ functionality
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C. Questions

1. What are some of the advantages of rapid methods?

2. How does the rapid sterility screening method differ from the current 

USP compendial method?

3. As per the guidance outlined in USP chapter <1223> what are the two 

general categories used to evaluate an alternative method? 

4. As per USP chapter 1223 what are the validation requirements used to 

evaluate a qualitative method? 

5. What does ORCE stand for? 

Inspectional Guidance

Microbiologists are sometimes requested to accompany investigators on 

inspection of establishments that produce medical products. The trainee will 

accompany an investigator and an experienced microbiologist, on an initial 

inspection when possible. The investigator has the basic responsibility for an 

establishment inspection and the microbiologist is the subject matter expert 

regarding microbiology, but will participate fully in the inspection, covering 

applicable sections of the compliance program for a given inspection and any 

topics assigned by the lead investigator. The microbiologist will have the 

responsibility of documenting the areas covered for the inspection report and 

drafting FDA Form 483 observations made by the microbiologist and participating 

in the final discussion with management. The investigator oversees the inspection.

The trainee should talk with the investigator prior to the inspection and work 

closely with the senior microbiologist, when possible, to prepare for the inspection 

which will include review of the following, when applicable: previous establishment 

inspection reports (EIR), the inspectional assignment, the compliance program 

and relevant sections of the CFR. It may be necessary to prepare sample 

collection materials or obtain proper protective clothing for the establishment.

A. Objective 

1. To introduce the policies and procedures involved in the inspection of 

medical product manufacturers. 

2. To provide instruction and practice in the preparation of an 

Establishment Inspection Report (EIR) 

3. To provide instruction and guidance to the trainee in drafting FDA Form 

483 observations to include determining the appropriate CFR cite for an 

observation. 

B. Assignment
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1. Analyst must have valid FDA credentials. Request or renew them as 

needed prior to the inspection.

2. If possible, meet with the investigator (Consumer Safety Officer (CSO)) 

and other team members before beginning the inspection. Discuss the 

goals of the inspection and what your specific role will be.

3. Read the following:

a. eNSpect assignment for the upcoming inspection.

b. EIR from the last inspection performed at the firm.

c. Compliance Program(s) pertinent to the inspection.

d. Investigations Operations Manual (IOM), Chapter 1 Administration, 

Subchapter 1.6 – Public Relations, Ethics and Conduct; Chapter 4 

Sampling, and Chapter 5 Establishment Inspections.

e. Pharmaceutical Microbiological Manual, Chapter 11 Inspectional 

Guidance.

f. Guide to Inspections of Quality Systems, August 1999, if applicable.

4. Record what is reviewed and observed during the inspection 

electronically or in a bound notebook.

5. Determine if 483 observations from the previous were adequately 

addressed.

6. Collect exhibits to be used to describe processes you reviewed or 

support your observations.

7. Prepare your observations (Form FDA 483) for presentation and 

discussion with Firm management. 

8. Draft portions of the Establishment Inspection Report as appropriate. 

C. Questions

1. What are some of the different types of inspections? 

2. Why is it useful to review the previous EIR?

3. What are 483 observations?

4. How does FDA classify inspection reports?

8.1. Drug Inspections

A. Objectives 

1. Introduce the different types of pharmaceutical inspections, for example 

good manufacturing practice (GMP) inspections, pre-approval inspections, 
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Active Pharmaceutical Ingredient inspections and For-Cause Assignment 

inspections.

2. Introduce the areas to be covered by a microbiologist during 

pharmaceutical inspections.

3. Introduce 21 CFR 210 and 21 CFR 211 regulations.

B. Assignment

1. The trainee will work with the trainer to draft an outline for various 

pharmaceutical inspection types such as GMP inspections, pre-approval 

inspections and API inspections. The GMP and pre-approval inspection 

outlines will focus on a sterile manufacturer.

2. The trainee will work with a trainer to identify potential FDA Form 483 

observations from a set of mock situations. The trainee will draft their 483 

observations, identify the relevant 21 CFR 210 or 211 citations and provide 

a list of the types of exhibits necessary to establish evidence for their 

observations.

3. The trainee will accompany an experienced microbiologist or investigator 

on inspection that requires expertise in microbiology and contribute to the 

inspectional report.

C. Questions

1. List two to three objectives for a pre-approval inspection.

2. Explain the importance of covering good manufacturing practices during a 

pre-approval inspection.

3. For a GMP inspection of a sterile manufacturer what is the main system 

from the Compliance Program Guidance Manual 7356.002A that the 

microbiologist will be expected to cover?

4. A microbiology laboratory lacks method suitability for sterility testing. What 

is a potential 21 CFR 211 cite that may be used to support these 483 

observations?

5. If you were asked to inspect a microbiology laboratory for a sterile 

injectable manufacturer what three finished product release tests would the 

microbiology laboratory be responsible for?

8.2. Device Inspections

A. Objectives 

1. To introduce the different types of medical device inspections. 
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2. To introduce the techniques used in the inspection of medical device 

manufacturers. 

3. To discuss the areas of the inspection typically covered by a microbiologist 

on a team inspection.

4. To introduce the 21 CFR sections applicable to medical devices.

B. Assignment

1. The trainee will work with the trainer to develop an inspectional plan 

specific to medical device manufacturers.

2. The trainee will identify objectionable conditions from a set of mock 

situations provided by the trainer. From this set, the trainee will draft FDA 

Form 483 observations, identify the relevant 21 CFR citations and provide 

a list of the types of exhibits necessary to establish evidence for their 

observations.

3. The trainee will accompany an experienced microbiologist or investigator 

on a medical device manufacturer inspection and contribute to the 

inspectional report.

C. Questions

1. How are medical devices classified?

2. What is QSIT?

3. How do different types of devices obtain market approval from FDA? 

8.3. Compounding Pharmacies

A. Objectives 

1. Introduce the concept of compounding.

2. Introduce the concept of insanitary conditions.

3. Introduce the differences between 503A and 503B compounding 

pharmacies.

4. Introduce the concept of assignments for compounding pharmacy 

inspections. 

5. Introduce the areas to be covered by a microbiologist during compounding 

inspections.

B. Assignment

1. Read the following:

a. An example of compounding assignment focusing on micro-related 

activities 
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b. Previous EIR

c. 503A and 503B in the Act

d. Compounding guidance document

2. The trainee will work with the trainer to draft an outline of mock situations.

3. The trainee will work with a trainer to identify potential FDA Form 483 

observations from a set of mock situations. The trainee will draft their 483 

observations, identify the relevant citations and provide a list of the types of 

exhibits necessary to establish evidence for their observations.

4. The trainee will accompany an experienced microbiologist or investigator 

on inspection that requires expertise in microbiology and contribute to the 

inspectional report.

C. Questions

1. What is compounding? 

2. What are some areas to inspect/observe during Compounding Inspection 

of sterile drug products?

3. What can be a risk and cause of improperly compounding drugs? 

4. Provide an example of a serious compounding related outbreak. 

5. Under 503B in Food, Drug, and Cosmetic Act (FDCA) if compounder 

becomes an outsourcing facility what must they comply with?

6. What effect did the Compounding Quality Act of the Drug Quality and 

Security Act have on compounding facilities? 

7. If a compounder is not registered as outsourcing facility what conditions 

must they meet?

8. How does FDA, CDER implement the compounding provisions of the 

FD&C Act and conduct oversight to protect public health?

8.4. EM Sample Collections

A. Objectives 

1. Introduce the rationale for conducting an EM collection. 

2. Introduce the steps taken to prepare for an EM collection.

3. Introduce the process at the firm.

4. Introduce the process of preparing and delivering the samples to the 

servicing laboratory. 

B. Assignment
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1. The trainee will work with the trainer to prepare a mock: EM request, EM 

sampling plan and EM supply request. 

2. The trainee will work with the trainer to review all the steps of the EM 

collection checklist.

3. The trainee will work with the trainer to go over aseptic techniques, gloving 

techniques, gowning procedures.

4. The trainee will work with the trainer to go over sampling methods and 

location priorities.

5. Analyst going on an EM collection must have valid FDA credentials. 

Request or renew them as needed prior to the inspection.

6. The trainee will accompany an experienced microbiologist to perform an 

EM collection.

C. Questions

1. In what location order do you perform an Environmental Monitoring (EM) 

collection? 

2. Why is Day/Engley (D/E) Neutralizing Broth suitable for Environmental 

Monitoring (EM) collections? 

3. What are the advantages of using sponges/swabs (qualitative method) for 

EM monitoring?

4. What are the advantages and disadvantages of using RODACS/

HychecksTM (quantitative method)?

Glossary/Definitions

A. FACTS – Field Accomplishments and Compliance Tracking System

B. FDA – United States Food and Drug Administration

C. ORA – Office of Regulatory Affairs

D. ORA U – Office of Regulatory Affairs University

E. QMiS – Quality Management information System

F. USP – United States Pharmacopeia

Supporting Documents

A. ORA Laboratory Manual. (Current ed.) 

B. ORA-LAB.016 Medical Products and Tobacco Program Review and 

Clearance
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C. Applicable local procedures for media preparation, growth promotion 

testing and the laboratory quality assurance practices.

D. SOP-000288 Microbiological Controls for Medical Product Sample 

Analysis

Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

00 I See QMiS 

InfoCard LMEB LMEB

01 R See QMiS 

InfoCard

Shannon Ruelle, Biologist

Allison Rodriguez, 

Microbiologist

LMEB

* - D: Draft, I: Initial, R: Revision

Change History

Revision 

# Change

00 New document

01

Added the word “Sanger” to DNA Sequencing

Removed all information for PFGE & replaced with WGS information

Correct spelling of “Burkholderia cepacia” throughout document

Document reformatted

Attachments

List of Attachments

Attachment A - Answer Key...................................................................................... 43
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Attachment A - Answer Key

6.2 Media, Reagent, Supply Preparation and Applicable Quality Release Testing 

A. Which media should not be steam sterilized and why? Examples of culture 

media that are not steam sterilized are Xylose Lysine Deoxycholate (XLD) and 

Violet Red Bile Glucose Agar (VRBG). Autoclaving as well as boiling longer than 

needed destroys the selectivity of the medium.

B. Where is agar derived from and what are the special properties of agar that 

make it well suited as a solidifying agent in culture media? Agar is the dried 

mucilaginous substance extracted from various species of algae. The plants are 

found primarily off the coasts of Japan, China, and southern California. Agar is 

insoluble in cold water but slowly soluble in hot water to give a viscous solution. A 

1% solution melts at 100°C and sets at 35°C to 50°C to a firm gel. Since agar is 

used by a nutrient source by relatively few bacteria, it is the most satisfactory 

solidifying agent for the growth and isolation of bacterial and fungal species.

C. Once agar is melted, what temperature should agar be kept at to prevent it 

from solidifying? Agar should be kept at 55°C to prevent it from solidifying. Prior 

to performing analysis, melted agar used for pour-plating should be tempered to 

45°C.

D. What is the purpose of conducting growth promotion testing on media used 

in microbiological testing? The purpose of the growth promotion test is to 

determine the suitability of culture media. The medium is challenged with a small 

number of microorganisms to assure the nutritive properties. The criteria must be 

met for each new batch of medium to be accepted for use in microbial tests such 

as the colony forming units (CFU) on the new batch of medium must be within a 

factor of two of the count on the previously approved medium.

E. In which media and buffer preparations is volume particularly critical? 

Volume of both buffer and media are important to maintain a proper ratio of 

product to media or buffer. Volume is particularly critical in preparing buffer 

solutions intended for use in dilutions for plate counts. Incorrect volume in the

buffer blank can result in erroneous plate counts.

F. Why is pH important in media and buffer preparation? One of the selective 

factors in favoring growth of one organism over another is pH. For example, 

media for detection of Burkholderia Cepacia Selective Agar (BCSA), pH is used to 

differentiate between organisms. Phenol red is used as a pH indicator. 

Burkholderia cepacia complex colonies will be yellow and surrounded by a pink 

yellow zone. Colonies that absorb the crystal violet dye will appear purple to 

purple gray. Acid production from carbohydrate oxidation will create a yellow 

zone in the medium while peptone utilization will result in pink zone.
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G. What are some safety concerns when autoclaving?

1. Autoclave vessels with vented closures only. Do not use crimped seals.

2. Use only Pyrex® glass vessels and other types of autoclavable materials.

3. For safety, always use the appropriate cycle for sterilization: “Gravity” for 

equipment and “Liquid” for liquids. 

4. Follow manufacturer’s instructions for proper opening of the door and end of 

cycle.

5. Do not allow hot bottles to be jolted. This can cause hot bottle explosion. Do 

not move bottles if any boiling or bubbling is present.

6. Analysts are required to wear appropriate personal protective equipment 

(PPE) such as full-length face shields, water impervious aprons and rubber 

gloves whenever removing materials from the autoclave.

6.3 Safety and Hazardous/Infectious Waste

A. What are the items of personal protective equipment (PPE), minimally 

required, in a Biological Safety Level (BSL)-2 biological laboratory? Each 

person working in a biological laboratory is minimally required to be using safety 

glasses with shields (or goggles, face shield, other splatter guards), a protective 

lab coat, disposable gloves, and protective footwear (closed-toe shoes). 

B. What work practices must be in place when working in a Biological Safety 

Level (BSL)-2 laboratory? 

1. Laboratory access is limited or restricted. Personnel at increased risk for 

acquiring infection, or for whom infection would have serious consequences, 

are not allowed in the laboratory or animal rooms.

2. Laboratory doors are kept closed when experiments are in process.

3. Personnel wash their hands after handling microorganisms, when removing 

gloves, and before leaving the laboratory. Soap and disposable towels are 

readily found at sinks in the microbiology laboratories.

4. No eating, drinking, chewing gum, smoking, handling medications, handling 

contact lenses, or applying cosmetics is permitted in the laboratory. All 

foodstuffs are stored outside of the laboratory. 

5. Mouth pipetting is prohibited.

6. There is a program for handling and disposing of sharps using special sharps’ 

containers.

7. Work surfaces are decontaminated on completion of work and at the end of 

the day. This usually involves a system of various decontamination sprays, 
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such as bleach, iodine, quaternary ammonium materials, amphyll, etc. Any 

splashes or spills of viable material are immediately cleaned up with 

disinfectants that are effective against the organism of concern. Contaminated 

equipment is to be decontaminated before it is sent for repair, sent for 

maintenance, placed in surplus, or shipped out of the lab. When placing the 

equipment in surplus, the equipment is also labeled in accordance with the 

DHHS requirements for property management to show that the equipment is 

clean.

8. All cultures, stocks, and other regulated wastes are autoclaved, or otherwise 

decontaminated, to destroy any viable organisms. All materials for autoclaving 

are placed in red (or otherwise equivalent) bags that are closed for transport 

out of the laboratory. These bags are set in leak-proof totes. Any materials to 

be decontaminated off-site are packaged in accordance with applicable local, 

state, and federal regulations beforehand. 

9. When working with organisms with more hazardous risk, consideration is 

given to chemically decontaminating the wastes within the laboratory.

10.An insect and rodent control program is in place.

11.All procedures are conducted in a manner to minimize aerosols. 

Consideration is given to the degree of hazard when loops are used in flames, 

centrifugation, opening screw-capped bottles and wet petri dish covers, use of 

a syringe, needle, and septum, streaking plates, pipetting, slide agglutination, 

etc. Good microbiological techniques, substitution of disposable equipment, 

and use of primary containment devices such as biosafety cabinets reduce 

the risk. 

12.Laboratory management establishes policies and procedures whereby 

personnel in the laboratory are informed of the potential hazards and meet 

special entry requirements, such as immunization.

13.A biohazard sign is posted at the entrance to any laboratory where etiologic 

agents are used. This label identifies what etiologic agents are used, the 

biosafety level, any required immunizations, the responsible person and their 

phone number, what PPE is worn in the laboratory, and any procedures for 

exiting the laboratory.

14.Immunizations or tests are offered to personnel for the agents handled or 

potentially present in the lab.

15.Baseline blood serum samples may be collected and stored, depending on 

what agents are handled.

16.Biosafety procedures are incorporated into individual SOPs or a Biosafety 

Manual is adopted or prepared for the local laboratory. Personnel are advised 
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of special hazards and are instructed to read and follow instructions on 

practices and procedures.

17.Laboratory and support personnel receive training on potential hazards 

associated with the work performed, the precautions to avoid exposure, and 

exposure evaluation procedures. Training is updated annually or when the 

procedure or policy changes.

18.Substitution of glassware for plasticware is recommended.

19.A high degree of caution is taken with any procedure using needles and 

syringes, or any other sharps. Sharps should be restricted whenever possible.

20.Only needle-locking syringes or disposable syringes should be used for 

injections, etc. Needles should not be manipulated in any manner before 

disposal; they should be carefully placed into a conveniently located punctureresistant container used for sharps disposal. Non-disposable sharps are to be 

placed in a hard-walled container for transport to a processing area for 

decontamination, preferably by autoclaving.

21.Syringes that re-sheathe the needle, needleless systems, and other safety 

devices are used when possible.

22.Never handle broken glassware directly by hand. Use a mechanical means, 

such as brush and dustpan or tongs to pick up broken glass pieces. Dispose 

of broken glass in special containers.

23.Containers of broken glass, sharps, and contaminated needles are 

decontaminated prior to disposal, or are shipped off-site in accordance with 

local, state, or federal regulations.

24.Any cultures, tissues, specimens of body fluids, or potentially infectious 

wastes are placed in a container with a cover that prevents leakage during 

collection, handling, processing, storage, transport, or shipping.

25.All spills and accidents that result in overt exposures to infectious materials 

are immediately reported to the Designated Laboratory Official. Medical 

evaluation, surveillance, and treatment are provided as needed and written 

records are maintained in accordance with 29 CFR 1910.1020.

26.Only animals involved with the work being performed are permitted in the 

laboratory.

27.Correct personal protective equipment is worn in the laboratory.

28.Disposable gloves are not reused, washed, or used when touching “clean” 

surfaces, such as the telephone, door handles, keyboards, etc.

29.Properly maintained safety equipment, including biological safety cabinets 

(Class II) or other containment devices are used whenever procedures with a 
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potential for creating infectious aerosols or splashes are conducted, or high 

concentrations or large volumes of infectious agents are used.

30.High concentrations or large volumes of infectious material may be 

centrifuged in the open lab if sealed rotor heads or centrifuge safety cups are 

used and if these rotors or cups are only opened inside of the biosafety 

cabinet.

31.All surfaces inside the laboratory are readily accessible for cleaning and can 

easily be decontaminated. No carpets or rugs are used in the laboratory.

32.The biological safety cabinet is located where fluctuations of the room supply, 

and exhaust air do not cause the cabinet to operate outside its parameters for 

containment. The cabinet is located away from doors, from windows that can 

be opened, from heavily traveled laboratory areas, and from other potentially 

disruptive equipment to maintain the biological safety cabinets’ air flow 

parameters for containment. 

33.Eyewashes are readily found throughout the laboratory and are flushed 

weekly.

34.Outside windows are fitted with screens.

35.Illumination is provided without glare or reflections.

C. What are some opportunistic pathogens that you might encounter when 

analyzing samples? Identify their biosafety levels. A complete list of all 

regulated organisms appears in 42 CFR 72.3.

Most recalls of medical products are due to contamination by Gram negative 

bacteria. Some of the most commonly isolated organisms within this group are 

Burkholderia cepacia, Pseudomonas spp. and Ralstonia picketti. Yeast and mold 

are also commonly isolated. Some of the most commonly isolated genera include 

Candida, Aspergillus, Penicillium, Exserohilum, Fusarium, Bipolaris, and 

Rhizopus. Gram positive bacteria, such as Staphylococcus spp., Enterococcus 

spp. and Bacillus spp. are isolated less frequently. 

Environmental monitoring samples collected from cleanroom environments 

typically contain organisms associated with human skin such as Gram-positive 

cocci, like Micrococcus and Staphylococcus. Microorganisms associated with the 

environment such as Gram-positive rods, like Bacillus spp from soil and Gramnegative rods, like Pseudomonas from water are also detected, although in lower 

numbers. Fungi such as Penicillium, Aspergillus, Rhizopus, Thrycophyton, 

Cladosporium and Curvularia are also commonly found. All the organisms listed 

above are Biosafety level (BSL) 1 and 2.

1. Clostridium botulinum – Biosafety Level 2 practices, containment equipment, 

and facilities are recommended for all activities with materials known to contain 
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or potentially to contain the toxin. Additional primary containment and 

personnel precautions, such as those recommended for Biosafety Level 3, are 

indicated for activities with a high potential for aerosol or droplet production, 

and those involving production quantities of toxin. Animal Biosafety Level 2 

practices, containment equipment, and facilities are recommended for 

diagnostic studies and titration of toxin.

2. Escherichia coli (Cytotoxin-producing (VTEC/SLT) organisms) – Biosafety 

Level 2 practices, containment equipment, and facilities are recommended for 

all activities utilizing known or potentially infectious clinical materials or 

cultures. Animal Biosafety Level 2 facilities and practices are recommended for 

activities with experimentally or naturally infected animals.

3. Salmonella – all serotypes except typhi – Biosafety Level 2 practices, 

containment equipment, and facilities are recommended for activities with 

clinical materials and cultures known to contain or potentially containing the 

agents. Animal Biosafety Level 2 practices, containment equipment, and 

facilities are recommended for activities with experimentally or naturally 

infected animals.

4. Salmonella typhi – Biosafety Level 2 practices, containment equipment, and 

facilities are recommended for all activities utilizing known or potentially 

infectious clinical materials and cultures. Biosafety Level 3 practices and 

procedures are recommended for activities likely to generate aerosols or for 

activities involving production quantities of organisms.

D. Describe proper procedures for disposal of used media. Describe how to 

use and operate an autoclave safely. All BSL-2 cultures, stocks, and other 

regulated wastes are autoclaved or otherwise decontaminated, to destroy any 

viable organisms. All materials for autoclaving are placed in red bags that are 

closed for transport out of the laboratory. These bags are set in leak-proof totes. 

Any materials to be decontaminated off-site are packaged in accordance with 

applicable local, state, and federal regulations beforehand. When working with 

organisms with more hazardous risk, consideration is given to chemically 

decontaminating the wastes within the laboratory. All wastes are autoclaved. All 

bags autoclaved are to allow the heat and steam to effectively penetrate the 

wastes. Metal totes can be used to further circulate the heat for effectiveness. 

The autoclave is professionally serviced as scheduled by the laboratory and 

records are maintained of this service. 

Proper PPE used with this process includes a rubber apron, heat and steam 

resistant gloves, and a face shield. Records are maintained of the destruction of 

any select toxins. 
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Consideration should be given to means of decontaminating equipment. If waste 

is transported out of the laboratory, it should be properly sealed and not 

transported in public corridors.

The manufacturer’s instructions for safe operation of the autoclave are readily 

found or posted near the autoclave

E. What is an SDS (Safety Data Sheet)? Where are the SDSs located? What 

information can be found on a chemical reagent hazard label? Safety Data 

Sheets are required documents in OSHA’s Hazard Communication standard 

prepared by manufacturers or distributors of chemicals received by a facility to 

relay information about the hazards of those chemicals, etc.

SDSs are located where identified for the laboratory and on-line at the web sites 

of the various manufactures or distributors. Chemical labels must relate the 

manufacturer, his address, and phone number in addition to the major hazards of 

the chemical. The health hazards disclosed on the label include, whether the 

material is a carcinogen, a reproductive toxin, a toxic material, a corrosive, 

damaging to organs, a central nervous system depressant, a corrosive, an irritant 

or sensitizer. Any conditions that adversely affect the chemical are also relayed.

F. Does the local state regulate medical waste? What laboratory wastes are 

permitted to enter the sewer? What laboratory wastes are to be 

incinerated? EPA no longer regulates medical waste and its destruction, but 

many states have adapted previous EPA requirements and these requirements 

are still valid. 

G. What are the guidelines for handling microorganisms (mostly bacteria or 

their toxins) in the BMBL? Organisms are mostly BSL-2 and are handled as 

any other BSL-2 organism described above.

H. What are engineering controls? Controls include use of biological safety 

cabinets, isolated work areas, use of equipment and procedures that reduce 

manipulation, and employment of ventilation and air filtering systems. Describe 

the proper use of engineering controls in a medical products microbiology 

laboratory. Properly maintained safety equipment, including biological safety 

cabinets (Class II) or other containment devices are used whenever procedures 

with a potential for creating infectious aerosols or splashes are conducted, or 

high concentrations or large volumes of infectious agents are used. Materials that 

are potentially not contained are managed in the biosafety cabinet. For example, 

infectious material may be centrifuged in the open lab if sealed rotor heads or 

centrifuge safety cups are used and if these rotors or cups are only opened 

inside of the biosafety cabinet. Any operation that potentially generates an 

aerosol is handled in the biosafety cabinet.
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Plastic backed absorbent paper may be used in the cabinet to contain some of 

the aerosol and reduce an extensive cleanup. The cabinet is always 

decontaminated before and after use with an effective disinfectant. The sash is 

preset to 8-10 inches to minimize any air turbulence in the room that might 

adversely affect the cabinet’s performance. Arm movements in and out of the 

cabinet are slow and minimal to not disrupt the performance. All supplies are 

placed so they are readily found during use of the cabinet. No air grills are 

blocked during use. 

All contaminated materials are laid in a flat container with disinfectant for later 

decontamination. No flames are used in the cabinet; disposable loops or 

electronic heaters are used for loops. Personnel are dissuaded from walking in 

front of the cabinet during use. A contractor certifies the cabinets annually.

I. What are administrative controls? Describe administrative controls 

designed to minimize the risk of hazardous agent exposure to those 

personnel that are not directly involved with their manipulation. List those 

administrative controls that assist in the maintenance of quality control. 

Cleaning crews should not be allowed in the lab except when scheduled. Only 

authorized personnel should be allowed in the labs and even tighter controls 

used for animal suites. All infectious materials that are transported should be 

contained. Use of plasticware is recommended to reduce the potential for 

breakage. All wastes are autoclaved to ensure nothing infectious enters the 

sewer system in the building. All materials shipped offsite are specially packaged 

to meet federal requirements. All equipment is disinfected before service or 

surplus.

J. What safety equipment is found within a microbiological laboratory?

Eyewashes, sinks, telephones, paper towels, and liquid soap should be readily 

found in every laboratory. Cabinets used for spill supplies, first aid boxes, safety 

showers, fire pull stations, and fire extinguishers should be found in various 

locations throughout the microbiology area.

K. What types of laboratory procedures have the potential to generate 

aerosols? How can these procedures be contained? How can the 

generation of aerosols be minimized? Blending, opening containers of live 

organisms, using needles and syringes, splattering or spilling infectious liquids, 

injecting needle through septum, opening food containers under pressure, 

splattering droplets of infectious materials in a flame, etc. Analysts should use 

good technique and when needed use containment equipment such as biosafety 

cabinets.

L. What decontamination procedures are in place and when are they 

performed? Work surfaces are decontaminated on completion of work and at 

the end of the day. This usually involves a system of various decontamination 
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sprays, such as bleach, iodine, quaternary ammonium materials, amphyll, etc. 

Contaminated equipment is decontaminated before it is sent for repair, sent for 

maintenance, placed in surplus, or shipped out of the lab. When placing the 

equipment in surplus, the equipment must be also labeled in accordance with the 

DHHS requirements for property management to show that the equipment is 

clean.

M. Describe how spills are handled? Are the cleanups following a spill 

documented and the cleanup verified? Spills are decontaminated with an 

effective disinfectant and then cleaned up with absorbent pads. Any splashes or 

spills of viable material are immediately cleaned up with disinfectants that are 

effective against the organism of concern. The material is collected as chemical 

waste if chemicals are also present. If radioactive materials are present, the spill 

is then handled as radioactive waste. The area is monitored to verify no 

hazardous agents are present afterwards by plating swipes of the area. The 

decontaminated microbial spill is handled as a non-hazardous spill if no 

organisms are present. All areas are routinely decontaminated at the end of a 

project and at the end of the day in the microbiological lab. Routine settling plates 

and air monitoring is done to evaluate if there are any infectious issues in the lab. 

All spills and accidents that result in overt exposure to infectious materials should 

be immediately reported to laboratory management. Medical evaluation, 

surveillance, and treatment are provided as needed and written records are 

maintained in accordance with 29 CFR 1910.1020.

N. What kind of signage is to be in place in a microbiological BSL-2 

laboratory? A biohazard sign should be posted at the entrance to any 

laboratory where etiologic agents are used. The hoods and biosafety cabinets 

should have dated certification stickers. It is recommended that refrigerator and 

freezers have signs that the units are not to be used for food, flammables, or 

other unsafe storage. Any restricted entrance rooms should be posted. The 

animal area has special requirements for posting which should be followed. 

O. What are the potential routes of exposure when working with infectious 

organisms? The most common potential routes of exposure are inhalation, 

ingestion, punctures, and through the skin absorption and exposure to the 

mucous membrane of the eye. Any cuts in the skin are problematic when working 

with organisms. 

P. Why is it necessary for a minimum of two people to be working in the 

laboratory at any given time? This is a standard policy for laboratories.

Q. Has training to perform the assigned tasks been completed and has this 

training been documented? All laboratory personnel receive regularly 

scheduled training on a revolving annual basis on safety issues and waste 

management. This is also part of the training given to all new hires. FDA has 
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started the new ORA U on-line training program with web-based training; 

completion of these modules is documented.

R. Is the facility designed to prevent infectious organisms from being 

accidentally released to other areas in the building? Laboratories have 

different features. Answers should be reflective of the local lab.

S. Is there a sharps safety program in place to reduce hazards when handling 

syringes or pipettes or other sharps? The answer for all labs should be yes 

and defined for that lab.

6.4 Quality Assurance and Data Integrity

A. What are culture controls and why are they required in analyses? Culture 

controls are specific organism strains used to verify system test environment 

(quality control). Testing typically employs positive and negative cultural controls. 

The positive cultural control assures that conditions at the time of analysis were 

satisfactory for the recovery of the target organism and that the media and 

biochemical tests give correct results. The negative cultural control demonstrates 

the atypical results for biochemical tests and growth characteristics and assures 

that differential media can give atypical reactions.

B. With whom does Data Integrity begin? Data Integrity begins with you, the 

analyst. 

C. Why are Quality Assurance and Data Integrity so important? It ensures each 

test provides a complete, accurate, legible account of sample handling and 

analysis: Sample accountability, chain of custody, quality, methods, 

media/reagents/equipment, analytical data, attachments, etc. Quality assurance 

is needed to ensure our work products meet acceptability requirements. QA 

covers all the processes we run. Data integrity is needed to maintain and ensure 

accuracy and consistency of our data over its lifecycle.

D. What is the purpose of environmental and personnel monitoring? The 

purpose of environmental monitoring and personnel monitoring is to ensure 

laboratory conditions and personnel do not adversely impact the sample analysis 

or the environment. 

E. Why are temperatures recorded for incubators, water baths etc.? Checks of 

the temperature of instruments such as incubators and water baths are needed 

to maintain confidence in the calibration status of the instrument. For instruments 

that maintain certain temperatures to encourage microbial growth, these checks 

are to be performed frequently. The ALACC recommends incubators be checked 

twice daily (AM and PM) and water baths be checked daily.

Microorganisms only grow at certain temperature ranges, and each has an 

optimum temperature. If an incubator or waterbath fails to provide that 
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temperature, the target pathogen may not be recovered. It is critical to never 

exceed the temperature range of the organism or it may die off.

F. Why are worksheets reviewed and signed by an analyst/Supervisor/Lead 

who has not worked on the sample? Worksheets are our “work product” and 

are used in regulatory actions by the agency. They are written by the lead analyst 

or electronically generated by the web application and subsequently reviewed by 

a check analyst, supervisor, director, compliance officer, and in some cases by 

our legal staff, judges, jury, etc. Not all the readers are scientists; therefore, the 

worksheet should be presented clearly enough for all to understand. 

A poorly presented worksheet will not hold up in court, despite the elegant 

analytical science that may have gone into it. 

It is critical that the worksheet be flawless before it leaves the laboratory. The 

lead analyst or additional analysts may not have detected all the mistakes on the 

worksheet. Therefore, the worksheet is thoroughly reviewed by a peer analyst 

who did not work on the sample, is more objective, and thus is less likely to 

overlook an error. If a peer analyst cannot decipher a worksheet, then a nonscientist has no chance. Worksheets are checked for method suitability, accuracy 

of calculations, accuracy of transcribed data, and completeness.

G. Why are validated methods used? 21CFR2.19 states that it is the policy of 

FDA to use official methods published in the AOAC, whenever possible. These 

methods have been validated to provide documented evidence which provides a 

high degree of assurance that a procedure does what it purports to do (the 

requirements for an intended use are fulfilled) and is reproducible by other 

laboratories. Use of validated methods ensures consistency within the various 

laboratories in the agency. Furthermore, use of validated methods ensures that 

our analytical work will hold up in court in defense of our regulatory actions. 

The techniques of validation include using reference standards, comparison of 

results with other methods, interlaboratory comparisons, assessment of factors 

that influence the results, and assessment of uncertainty of the results. Methods 

are to be characterized for detection limit, selectivity, linearity, limit of 

repeatability and/or reproducibility, robustness, and interference from the matrix. 

Microbiological methods are to achieve a low number of false positives, a low 

number of false negatives, high sensitivity, high specificity, and a sensitive limit of 

detection. 

H. What is QmiS? Quality Management Info System (QmiS) is a repository for 

quality system documents. It also tracks quality activities such as document 

changes, corrective and preventive actions, etc.
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6.5 Working within an ISO 5 Controlled Environment

6.5.1 Clean Rooms

A. What environmental characteristics need to be controlled in a 

cleanroom? Cleanrooms are enclosed areas where there is control over 

particulates in the air with temperature, humidity and pressure control.

B. Should a cleanroom be under positive or negative filtered air pressure 

and why? Cleanroom should be under positive filtered air pressure. The 

positive filtered air pressure should be greatest in the testing area, that is the 

cleanest area, lower in the non-analytical area and lower still in the gowning 

area. Positive pressure protects the product.

C. Why should a relative humidity in a cleanroom be controlled? 

Electrostatic charges at low relative humidity can develop with particulate 

matter and microorganisms being attracted to walls and other surfaces. High 

relative humidity can cause rusting of metal equipment. Microorganisms 

(bacteria, yeast and mold) growth can be enhanced at the higher relative 

humidity.

D. What ISO Equivalent Class is equivalent to Class 100 room? Class 100 

room is equivalent to an ISO 5 room.

E. Name 3 acceptable agents used to disinfect the cleanroom. 

 These are some of the agents that can be used to disinfect the cleanroom:

1. 70% Ethanol/Isopropyl Alcohol (ETOH/IPA), 3-5% Hydrogen Peroxide 

(H2O2) (for direct contact surface disinfection)

2. Sodium Hypochlorite (1:10 – 1:100 dilution NOTE! Corrosive to stainless 

steel and other metals)

3. Iodophors

4. Quaternary Ammonium Compound

5. UV irradiation at 256 nanometers wavelength

6. Formaldehyde (May be toxic/carcinogenic)

7. Glutaraldehyde (May be irritating to the eyes, nasal passages upper 

respiratory tract and skin. Also leaves a residue.) 

8. Phenol and phenol derivatives (Toxic and may cause skin sensitization.)

F. Describe the 2-bucket cleanroom disinfection method? Dedicate a 

separate set of buckets and mops for cleanroom disinfection of floors and 

walls. Buckets #1 and #2 contains the clean disinfectant solution. Saturate 

the mop with the cleaning solution from bucket#1. Mop the designated 
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area. Wring the waste solution into (waste) bucket #2. Repeat until all areas 

are clean. When bucket #1 is dirty, replace it with another bucket with fresh 

disinfectant solution. Make the original bucket #1 into bucket #2 (waste 

bucket). Repeat process until all cleanroom surfaces are sanitized. In 

addition to the 2-bucket cleanroom disinfection method there is also a 3-

bucket method that can be also implemented. The third bucket is a rinse 

bucket where the mop is rinsed after the dirty waste solution is wrung out in 

the second (#2) bucket and before adding solution from #1 bucket.

G. Why should you work “wet” while disinfecting the cleanroom? First 

clean a small portion of the floor. Step into the wet area that you just 

disinfected and start cleaning in the recommended pattern while always 

stepping into the wet area just cleaned. This will minimize the stirring up of 

particulate matter and microorganisms. Any microorganisms that then settle 

down on the wet surface will be inactivated.

H. Describe all gowning material (PPE) used for working in the 

cleanroom? The following gowning materials are needed for working in a 

cleanroom environment: shoe covers, sterile bouffant, beard cover if 

applicable, mask, hood, gloves, boot covers, coveralls, cuff tape, googles.

I. What are the advantages of working as a team “buddy system” within 

the clean room? Advantages of working as a team “buddy system” is for 

safety concerns, greater efficiency, assist as a third hand, each member of 

the team can monitor his/her partner so that they can be focused on working 

aseptically within the hood.

J. What are some of the safety practices that should be followed while 

working in the cleanroom? Some of the safety practices that should be 

followed while working within the cleanroom: 

1. Cleanrooms are restricted areas, and access to them must be limited to 

authorized individuals only.

2. Protective clothing is always to be used by individuals entering and 

working in the cleanroom.

3. Smoking, chewing gum, and eating are forbidden in the cleanroom.

4. Avoid wearing jewelry.

5. Avoid mannerisms such as scratching, touching the head, face or body 

areas.

6. If possible, use the “buddy system”.

7. Always utilize aseptic technique.

8. Turn off the UV light upon entering the room.
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9. Watch out for spills on the floor.

10.No running or fast movements. Don’t walk around unnecessarily. 

Movement of cleanroom personnel should be restricted as much as 

possible to prevent stirring up of settled particulate matter on the 

cleanroom surfaces.

11.Be aware of vapors such as from alcohol, bleach or other chemicals 

used in the area.

12.Be careful when handling sharps, syringes, heavy equipment.

6.5.2 Isolator Technology

A. What are two key components of an isolator?

1. Closed working environment that is accessed through glove ports.

2. Interior surfaces are sterilized through a validated decontamination cycle. 

Commonly VHP is used.

3. Working environmental air is supplied by HEPA filters.

B. What does VHP stand for and what is its purpose in an isolator?

1. Vaporized Hydrogen Peroxide

2. VHP is a common surface sterilant that is used to decontaminate the 

surfaces of items placed inside an isolator as well as sterilize the interior 

surfaces of the isolator.

C. Why is aseptic technique still necessary within a sterility testing 

isolator? Although the surfaces of the interior of the isolator have 

undergone a decontamination cycle there may be present a low number of 

microbes, in addition one or more sub samples may have microorganisms 

present and aseptic technique when used can prevent the contamination of 

sub samples that may have not otherwise contain microorganisms.

D. What types of environmental monitoring can be performed within a 

sterility testing isolator? Surface monitoring, non-viable particulate 

monitoring of the air and viable monitoring of the air.

E. What is the importance of checking the integrity of isolator gloves? All 

manipulations of the sample supplies and equipment within the isolator are 

performed by gloves. Over time these gloves may lose integrity. If a glove 

develops a tear or puncture it allows the uncontrolled environment outside the 

isolator to enter the controlled environment which may lead to product 

contamination and a false positive result. Consistent evaluation of the integrity 

of the isolator gloves after each testing session ensures that the controlled 

environment of the isolator is maintained. 
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F. Why is a validated loading profile important? Vaporized hydrogen 

peroxide is surface sterilant. If items are placed within the isolator chamber 

such that they occlude the surface of a nearby object it is possible that 

microbes may survive the decontamination cycle. If too many items that are 

known to absorb VHP are placed into the isolator chamber a decontamination 

cycle may not be as effective since the items may absorb the VHP and 

therefore there would not be enough the VHP present to sterilize all surfaces.

6.5.3 Biological Safety Cabinets and Laminar Flow Hoods

A. What are the two main differences between a biological safety cabinet 

and a laminar flow hood? The direction of the air flow and the protection 

provided to the user and the product. 

B. Why is personal protective equipment required when working in either 

a biological safety cabinet or laminar flow hood? Personnel protective 

equipment is worn to prevent unwanted exposure and to prevent the 

introduction of contamination. 

C. Name three ways to prevent an interruption in air flow in a biological 

safety cabinet and laminar flow hood. Some appropriate responses would 

be, do not rest arms or place equipment on or in front of the grill, proper 

closure of the sash, slow deliberate movements while working in the hood, 

make sure the hood is level, and don’t overcrowd the hood.

D. How many classes of biological safety cabinets are there? Class I, Class 

II, and Class III. 

E. What is an aseptic technique related caution associated with working in 

an open an open-faced laminar flow hood? Leaning head or body into the 

laminar flow hood or talking, coughing or sneezing while directed towards the 

face of the hood.

F. Describe the placement of environmental monitoring plates in a 

biological safety cabinet and in a laminar flow hood. The response would 

be based on the local procedure regarding placement of environmental 

monitoring plates in each type of hood.

G. How often do biological safety cabinets and laminar flow hoods require 

certification by a qualified vendor? Biological safety cabinets and laminar 

flow hoods require annual certification by a qualified vendor. 

H. Are U.V. lights in a biological safety cabinet or laminar flow hood an 

effective means of decontamination? No, U.V. lights are not effective 

means of decontamination. U.V. lights can accumulate dust and dirt that will 

block the effectiveness of the light. Over time U.V. bulbs lose the necessary 

wattage for disinfection purposes.
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I. Why is the sample and not the user protected when utilizing a laminar 

flow hood? Because of the directional air flow and the construction of a 

laminar flow hood. 

J. What type of filters are used in a biological safety cabinet and laminar 

flow hood? High Efficiency Particulate Air (HEPA) filters are used in 

biological safety cabinets and laminar flow hoods. 

6.6.1 Direct Staining and Visual Screening Method

A. What would be some visual clues of microbial contamination? Unusual 

color of solution, turbidity, stringy/thread-like fibers, granular or clumps either 

floating or settling to bottom, etc. 

B. Describe various types of cell morphology. Cell morphology is an 

essential component in the characterization of bacteria. Once visible, a 

colony of cells is observed using microscopy and described based on size, 

structure, shape and form of the cells. Bacterial shapes include bacillus (rodlike), coccus (circular), or spiral (twisted into helices) and are identified 

through Gram staining practices. 

C. What are fungal hyphae? Fungal hyphae are the vegetative growth of a 

fungus represented by long, branching filamentous strands and referred to 

as mycelium.

D. What are spores? Spores are cells produced by either bacteria or fungi that 

can give rise to new adult organisms without sexual fusion. Bacterial spores 

are formed in response to adverse environmental conditions and help in cell 

survival. They have no metabolic activity and are highly resistant to heat and 

disinfectants Fungal spores are microscopic biological particles that allow 

fungi to reproduce, serving a similar purpose to that of seeds in the plant 

world.

E. Describe the difference between Gram-negative and Gram-positive 

organisms. Organisms are differentiated by the structure of their cell walls. 

Gram-positive organisms contain high levels of peptidoglycan which allows 

for the retention of crystal violet stain. Gram-negative organisms lack a cell 

wall which prevents retention of crystal violet but allows for retention of the 

safranin counter stain. 

6.6.2 Microbiological Examination of Non-Sterile Products

A. How does the analyst determine which controls to utilize for 

determination of the quantitative enumeration and the absence of 

specified microorganisms’ analysis? The controls used for USP <61> and 

<62> analyses are outlined in the respective USP chapters.
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B. What are some agents that can be used for neutralization of interfering 

substances? Glycine, Lecithin, Polysorbate, Thioglycolate, Thiosulfate, and 

Mg or Ca ions, among others.

C. Why is the Growth Promotion Test performed? The purpose of the 

Growth Promotion test is to demonstrate the suitability of the media for the 

intended use. 

D. What three tests are performed as part of the Growth Promotion Test?

Test for Growth Promoting Properties, Test for Inhibitory Properties, and Test 

for Indicative Properties.

E. Why is Method Suitability Testing required prior to performing analysis 

for determination of the quantitative enumeration and the absence of 

specified microorganisms? Suitability testing aides in demonstrating that 

the proposed test method adequately detects and enables recovery of 

microorganisms in the presence of a given product under a set of given 

conditions and meets the criteria of the USP. 

F. According to USP <61>, what three methods can be used to 

enumerate? Membrane filtration, plate count, and most probable number. 

G. What is the purpose of USP <1111>? To provide guidance regarding 

acceptance criteria for pharmaceutical preparations and substances for 

pharmaceutical use.

7 Special Assignments/Non-typical 

7.1 Non-tuberculosis Mycobacteria (NTM)

A. What are Rapid Growing Mycobacteria and Slow Growing 

Mycobacteria? Give some examples. NTMs are divided into two groups 

based on their growth rate. Rapid growers develop visible colonies within 

seven days. Some examples of rapid growers are Mycobacterium 

abscesses, M. chelonae and M. fortuitum. Slow growers take more than 

seven days to develop visible colonies. Some examples of slow growers are 

Mycobacterium avium, M. chimera and M. intracellulare.

B. What media are used for the cultivation of NTMs? Middlebrook 7H9 

broth is used for sample enrichment. This is followed by isolation on 

Middlebrook 7H10 agar (for rapid growers) and Middlebrook 7H11 agar (for 

slow growers). M7H11 agar has added antibiotics to select against other 

bacteria and yeast during the prolonged incubation. 

C. What are acid-fast bacteria? Acid fast bacteria have cell walls rich in lipid 

complexes, particularly in mycolic acids, which give them the ‘acid fast’-

specific character when colored by carbol fuchsin during the acid-fast 
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staining procedure. Acid fast bacteria will be red, while nonacid fast bacteria 

will stain blue/green with the counterstain. All mycobacteria are acid fast.

D. What DNA targets are used in the identification of mycobacteria? Both 

16S rRNA and rpoB gene are used. For some species 16S rRNA may 

provide enough species differentiation. For other species, sequencing of the 

rpoB gene may be necessary. 

7.2 Burkholderia cepacia complex

A. What does the phrase Burkholderia cepacia complex mean? The phrase 

Burkholderia cepacia complex (BCC) refers to a group of over twenty 

species of the Genus Burkholderia. Within the complex, Burkholderia 

cepacia is the most common human pathogen.

B. What is the primary selective agar used in the method to detect 

organisms within the Burkholderia cepacia complex? FDA laboratories 

use Burkholderia Cepacia Selective Agar (BCSA), a selective agar for the 

detection of Burkholderia cepacia complex microorganisms. BCSA contains 

crystal violet, polymyxin B, gentamicin and vancomycin as selective agents, 

which inhibit organisms other than BCC.

C. What is the typical morphology of Burkholderia cepacia colonies on 

BCSA? The typical colony morphology of Burkholderia cepacia on BCSA is 

growth with or without a color change of the agar from red-orange to yellow. 

BCC organisms oxidize the lactose and sucrose in the media. Producing 

acid. The phenol red in the media indicates the pH change.

D. Why isn’t Cetrimide agar used as the sole agar for the detection of 

Burkholderia cepacia complex? Cetrimide agar is designed to detect 

Pseudomonas aeruginosa. Burkholderia cepacia complex strains do not 

grow well on the agar and do not produce a typical morphology on the agar.

E. The Burkholderia cepacia complex is comprised of numerous 

Burkholderia species. What characteristics do these species have in 

common? Strains within the Burkholderia cepacia complex are Gram 

negative, catalase positive and do no ferment lactose.

F. Describe at least two situations in which the detection of Burkholderia 

cepacia complex may be added to a non-sterile analysis. Testing for 

Burkholderia cepacia complex may be requested or added to the testing of 

nonsterile medical products due to the following: an adverse event which has 

identified or implicated Burkholderia cepacia complex as the cause, the 

intended patient population may be immunocompromised or consist of 

children, historical trends observed with a product may require the analysis 

to be performed, inspectional findings may indicate the presence of 
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Burkholderia cepacia complex in the manufacturing environment or product 

component or for surveillance purposes. 

7.3 Sterility Testing of Medical Products

A. What is the purpose of the Method Suitability Test? The USP Method Suitability 

Test is a validation step for the USP Sterility test. It demonstrates that the product 

being tested does not exhibit inhibitory effects on the growth of microorganisms under 

the conditions of the test, i.e. the suitability of the test method to recover the test 

microorganisms in the media tested and at the level tested.

B. What is the inoculation level in CFUs to be used to inoculate product in media? 

The inoculum level is not more than 100 CFUs. The importance of inoculating a lowlevel inoculum is to show that the sterility test can detect low level microbial 

contamination.

C. Describe what is done to ensure the work area is acceptable for sterility testing

Clean all operational surfaces within the clean room using an appropriate disinfectant 

and/or sporicidal solution, if needed. The area is uncluttered and exposed to the 

HEPA filtered air. Opened media controls and/or exposure plates are prepared in 

advance and used at the time of analysis.

D. What should an analyst do if the growth in air sample plates exceeds the 

laboratory’s alert/action levels? Inform the supervisor immediately of this finding 

and follow local procedures and record findings. 

E. When would the incubation time be extended beyond typical 14-day incubation 

period? The incubation time would be extended beyond 14-day incubation period in 

the following instances:

1. If the product was sterilized by irradiation usually medical devices such as 

catheters or bulk powders.

2. If the product material being tested renders the medium turbid so that the 

presence or absence of microbial growth cannot be readily determined by 

visual examination. 

3. If the product contains sub lethally damaged cells may require additional 

growth factors because of damaged enzymes or transport systems.

4. If the organism of concern is a slow-grower requiring additional incubation 

time.

F. What determines if a product is an SVP or LVP? According to the USP a Small 

Volume Parenteral (SVP) is a product volume of 100 ml or less, A Large Volume 

Parenteral is a product volume greater than 100 ml.

G. What is the purpose of using two different media? The Soybean Casein Digest 

(SCD) broth is used to culture aerobic bacteria, yeast and mold. The Fluid 
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Thioglycollate Medium (FTM) is used to culture anaerobic or microaerophilic 

bacteria.

H. How does Fluid Thioglycollate maintain anaerobic conditions? Sodium 

Thioglycollate lowers the oxidation-reduction potential of the media and the low 

percentage of agar reduces the diffusion of oxygen into the liquid media.

I. What is the function of the red indicator in Fluid Thioglycollate? The red 

indicator (resazurin) indicates the status of the oxidation or aerobiosis of the broth. If 

red indicator is more than 1/3 of total media, the FTM may be reheated once per 

USP.

J. How is a sterility test by membrane filtration different from a sterility test by 

direct inoculation? Sterility test by membrane filtration allows the liquid drug 

product to pass through a membrane filter to collect the microorganisms from the 

product and be subsequently cultured in an enrichment medium. In Sterility testing 

by direct inoculation the product is added directly to the enrichment medium. 

K. What is the difference between Fluid A, D, and K? When would one use one 

over the other? Fluid A is 1 gram of peptic digest of animal tissue in water to make 

1 liter. Fluid D is the same as Fluid A except it contains 1 ml of polysorbate 80 

(Tween) added to the 1 liter. Fluid K is 5 grams of peptic digest of animal tissue, 3 

grams of beef extract and 10 grams of polysorbate 80 added to 1 liter of water. For 

the membrane filtration method, Fluid A is used to rinse aqueous solutions and 

soluble solids. Fluid D is used to rinse oils and oily solutions of low viscosity or 

devices with pathways labeled sterile. Fluid K is for rinsing viscous oils, ointments 

and creams.

L. What are the incubation temperatures for Fluid Thioglycollate Medium and 

Soybean Casein Digest Medium? Fluid Thioglycollate Medium is incubated at 

32.5°C+/-2.5°C and Soybean Casein Digest Medium is incubated at 22.5°C+/-2.5°C.

M. What is the purpose of the Pharmaceutical Microbiology Manual (PMM), 

Chapter 3? The purpose of the 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 PMM supplements the USP 

for FDA analysts and provides detailed procedures for performing the sterility test.

N. If the product being tested renders the medium turbid, when should the

medium be subcultured to fresh medium, according to the USP? If the product 

being tested renders the medium turbid so that the presence or absence of microbial 

growth cannot be readily determined by visual examination, 14 days after the 

beginning of incubation transfer portion (each not less than 1 mL) of the medium to 
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fresh vessels of the same medium, and then incubate the original and the transfer 

vessel for not less than 4 days.

O. In the event of a dispute between an alternative method and the USP Sterility 

method, which method would prevail? The USP allows the use of alternative 

methods if the alternative method or technology for the specific test is suitable and 

validated and meets the criteria of traditional USP by being at least equivalent or 

better than the official compendial method. In the event of a dispute, only the result 

obtained using the method or procedure published in the USP is conclusive. The 

alternative method will not serve as a legal replacement for the official USP method 

which serves as the referee test.

P. During the disinfection procedure prior to entering the cleanroom which type 

of products may be soaked in a disinfectant? Ampoules

Q. What are the six strains of microorganisms used for growth promotion test 

and Method Suitability Test? Aerobic bacteria-Staphylococcus aureus; Bacillus 

subtilis, and Pseudomonas aeruginosa; Anaerobic bacterium – Clostridium 

sporogenes and Fungi – Candida albicans and Aspergillus brasiliensis.

R. What can you do to eliminate product inhibition in Suitability Testing? To 

eliminate product inhibition in Suitability Testing you can neutralize the activity of 

antimicrobial agents by dilution and or by using neutralizing agents such as Lecithin, 

Polysorbate, Glycine, Sodium bisulfite, Thioglycollate, Thiosulfate, Mg or Ca ions.

7.4 Investigating USP Sterility Testing Failures 

A. What laboratory classification of samples would require a sterility failure 

investigation? Laboratory Class 3

B. What actions should be taken if a negative control is contaminated? 

Determine whether the organism recovered from the negative control is consistent 

with organisms recovered from the product or the positive control to ensure no 

cross-contamination occurred. 

C. What records should be reviewed to determine analyst qualifications? 

Proficiency tests or training records.

7.5 Bacterial Endotoxin Testing

A. What is endotoxin? Endotoxin is the lipopolysaccharide component found in the 

outer membrane of Gram-negative bacteria. 

B. What is the difference between endotoxin and pyrogens? Pyrogens are 

substances that cause fever when introduced into the blood stream or cerebrospinal 

fluid. Bacterial endotoxins are one subset of the larger category of pyrogens.

C. Is the USP <85> capable of accurately detecting pyrogens that may be in a 

product? Explain why or why not. No, the USP <85> is not intended to detect all 
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pyrogens. USP <85> is a method to detect bacterial endotoxin which is only one 

category of pyrogens.

D. What is the assay temperature for Endotoxin Testing? 37 +/- 1°C

E. What are some factors that may cause interference in endotoxin testing? 

Interfering factors may be caused by product related physical inhibitors (i.e., ionic, 

viscosity, adsorption or sequestration of endotoxin) or chemical inhibitors (i.e., 

chelation, protein denaturation, pH perturbation) or environmental inhibitors (i.e., 

temperature, vibration) during assay in heating blocks, plastic containers (i.e., 

polypropylene) that adsorb endotoxin out of the product, micelle/agglutination and 

hydrophobic traits of lipopolysaccharides causing them to adhere to container 

surfaces (i.e., glass vials) or container closure system (i.e., rubber stopper).

F. What are some ways to overcome interference in the endotoxin assay? 

Interfering factors may be overcome via product dilution within the calculated MVD 

or the use of endotoxin dispersing agents, detergents, dialysis, ultrafiltration.

G. If a product’s endotoxin limit is 0.5 EU/mg, the product’s concentration is 200 

mg/ml and the lysate’s sensitivity is 0.05 EU/ml. What is the maximum valid 

dilution for this product?

MVD= Endotoxin limit x Product Concentration

Lysate sensitivity

MVD= 2000

H. When calculating the endotoxin limit for a product with the formula, K/M what 

value is used for K for an intrathecal route of administration? What value is 

used for K for routes other than intrathecal? For an intrathecal route of 

administration K=0.2 USP-EU/Kg of body weight. For any other route of 

administration K= 5 USP-EU/kg of body weight.

I. What is the Gel-Clot technique/method based on? The test is based on clotting 

of the limulus blood in the presence of endotoxins. The presence of a clot indicates 

a positive test.

J. What is the duration of the Endotoxin Testing via Gel-Clot? 60 minutes +/- 2 

minutes

K. What tests are performed to ensure the precision and validity of the Gel Clot 

assay? 1) Bacterial Endotoxins Test Confirmation of Labelled Reagent (Lysate 

Sensitivity) 2) Bacterial Endotoxins Test Product Compatibility Test for Interfering 

Factors, Inhibition/Enhancement Test for Gel-Clot Techniques.

L. What are acceptable results for system control, negative water control, 

positive product control when running endotoxin testing via Gel-Clot? When 

running Endotoxin Testing via Gel-Clot a negative result, indicated by the absence 
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of a clot, is expected for both the System Control and the Negative Water Control. 

Duplicate sample dilution aliquots are spiked with CSE as Positive Product Controls 

and the spike amount is 2λ. A firm clot is formed.

M. What are the two types of kinetic methods found in USP <85>. Briefly 

describe each method. The two kinetic methods are the kinetic turbidimetric 

method and the kinetic chromogenic method. The kinetic turbidimetric method is an 

assay that measures the increases in turbidity of a product in the presence of 

endotoxin lysate. The kinetic chromogenic method measures the amount of 

chromophore that is released from a suitable chromogenic peptide by the reaction 

of endotoxin in the product and the endotoxin lysate.

N. According to USP<85>, to perform the confirmation of the lysate sensitivity 

what is the minimum number of endotoxin concentrations that are required 

for a valid standard curve? USP <85> requires a minimum of three endotoxin 

standard concentrations to generate a valid standard curve.

O. For the kinetic assay to be valid per the USP what are the required criteria?

For a valid assay the following criteria must be met:

1. The absolute value of the correlation coefficient of the standard curve must be 

greater than or equal to 0.980.

2. The result for the negative water control must be less than the endotoxin 

detection limit of the lysate used.

3. The maximum percent for the Coefficient of Variation for Standards (%CV) must 

be less than 10%.

7.6 Testing Environmental Monitoring Samples

A. What are the five typical types of environmental samples that could be 

submitted for analysis? The five typical types of environmental samples that 

could be submitted are sponges, sponge applicators, swabs for qualitative 

analysis and RODAC and HycheckTM surface samplers for quantitative analysis. 

B. What are some of the advantages and disadvantages of the quantitative 

method of environmental monitoring? An advantage of using the quantitative 

method of environmental monitoring analysis is the ability to determine the relative 

numbers of microorganisms within a finite area of a surface. Some disadvantages 

of the quantitative method of environmental monitoring analysis are that the plates 

or paddles cannot be used in cervices, cracks or other difficult to reach locations, 

colony overgrowth can make enumeration difficult, and the quantitative method 

only recovers a small percentage of the contact surface flora. 

C. What are some of the advantages and disadvantages of the qualitative 

method of environmental monitoring? Some advantages of the qualitative 

method of environmental monitoring analysis are that a sponge or sponge 
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applicators enable the sampling of a larger surface area, cotton tip swabs allow for 

sampling of cracks, crevices, and other tight areas, and sponges, sponge 

applicators and swabs provide the ability to apply mechanical contact pressure, 

which increases recovery of microorganisms. A disadvantage of the qualitative 

method of environmental monitoring analysis is that microbes can become 

trapped in the pores of the sponge or the fibers of the cotton tipped swabs.

D. What are some of the key elements that an analyst should check the 

environmental monitoring sample for upon the receipt of the sample? Three 

key elements that the analyst needs to check for upon sample receipt is that the 

sample was received in the proper packaging, at the proper temperature, and 

within 48 hours of sample collection. Additionally, the analyst should check the 

sample for signs of tampering, leakage, or the potential for cross contamination.

E. What types of air monitoring plates could be used in the biological safety 

cabinet or laminar flow hood during sample analysis? The types of air 

monitoring plates are Modified Letheen Agar, Sabouraud Dextrose Agar or TSA 

w/5% Sheep Blood Agar.

F. What organism controls are included as part of the analysis? The five 

organism controls are included in the environmental monitoring analysis are 

Aspergillus brasiliensis ATCC 16404, Candida albicans ATCC 10231, Bacillus 

cereus ATCC 14579, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus 

aureus ATCC 6538. 

G. What is the incubation timeframe and temperature for the environmental 

monitoring sponges or swab samples? All environmental sub samples are 

incubated for 14 days at 25-30°C. 

H. What is the incubation timeframe and temperature for the environmental 

monitoring RODAC Plates and Hycheck surface samplers? RODAC Plates 

and HycheckTM surface samplers are incubated at 30-35°C for two days, then 

incubated at 20-25°C for five days or longer when contaminants are suspected to 

be slow growing. 

I. What are the designated time frames for the observation for growth during 

the environmental monitoring sample analysis? The sub samples must be 

observed every business day and returned to the incubator post observation. If 

turbidity is observed in the sub sample bag, the sub sample will be processed for 

isolation of the growth, and then the sub sample will be returned to incubation. On 

day 5, 6, or 7 and on day 14 all the sub samples must be streaked regardless of 

turbidity or lack of turbidity

J. When growth is observed during an environmental monitoring test what 

media is used to isolate the growth? Subculture all EM samples onto a 

combination of non-selective media (i.e. MLA, etc.) and selective/differential 
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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.

7.7 Particulate Matter

A. What is a particulate matter? Particulate matter consists of mobile, randomlysources, extraneous substances, other than gas bubbles, that cannot be 

quantitated by chemical analysis due to the small amount of material that it 

represents and to its heterogeneous composition.

B. What are the two methods for the determination of particulate matter? 

1. Light Obscuration Particle Count Test

2. Microscopic Particle Count Test

C. What is the HIAC 9703+ Liquid Particle Analyzer flushed with and at what 

volume? HIAC 9703+ Liquid Particle Analyzer is flushed with ultra-pure water 

such as Milli-Q water, GenPure System, Nano-Pure System, double filter water 

or equivalent and with at least 10X the sample volume.

D. What troubleshooting techniques can be implemented if a large particle 

reduces or stops the liquid flow? A flush in the reverse direction can push the 

particle out through the sample probe by performing a backflush of the system.

7.8 Antibiotic Potency Testing

A. How do you determine the preparation steps necessary to test Neomycin 

Ointment for potency? The USP monograph for Neomycin Ointment should be 

referenced to determine the appropriate preparation of the ointment for potency 

testing. 

B. What is the purpose of the standard curve and how many standards must 

be included on the standard curve? There are five standards on the standard 

curve. The standard curve is used to extrapolate the potency of the test sample. 

C. What is the significance of the Zones of Inhibition? The Zones of Inhibition 

are the clearings observed on an agar for the Cylinder Plate Method. The 

diameter of the Zones of Inhibition is used to determine the potency of the test 

product. 

D. Why is it important to know if the bench you are working on for the 

preparation of plates for the Cylinder Plate method is level? The Cylinder 

Plate method requires that single layer or bi-layer plates be used. The layers of 

agar are considered relatively thin and a level bench is necessary to ensure even 

agar layers and therefore consistent, round zones of inhibition.

E. What are some indicators, that can be observed on the agar plates used in 

the Cylinder Plate method, that the bench was not level? Some indicators 
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that a bench which was used to pour agar plates for the Cylinder Plate method 

was not level include: oval zones of inhibition, pooling of the agar against one 

side of the petri dish and unevenness of an agar layer. 

F. How many times must the analysis be independently conducted for a given 

sample and why? Each analysis must consist of three independent test runs 

with each test run consisting of triplicate plates or tubes. The analysis entails 

multiple variables and is dependent on multiple factors, such as inoculum levels. 

Therefore, three independent test runs are required to ensure accurate results.

7.9 Antimicrobial Effectiveness Testing

A. How many categories of products does the USP <51> list? Briefly describe 

each category. The USP <51> has four categories of products. The categories 

are: Category 1- Injections; other parenterals including emulsions, otic products, 

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

vehicles. Category 2- Topically applied products made with aqueous bases or 

vehicle; nonsterile 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 an aqueous base.

B. Describe the types of containers the test can be performed in? When 

possible, the test should be carried out in the original container. If the original 

container cannot be used the test should be carried out in a container that is 

inert to the antimicrobial agent(s) present in the product. A suitable container 

may be sterile, large screw-cap glass test tubes.

C. What is the standardized volume of product FDA laboratories should try to 

test for each microorganism? The standardized volume FDA laboratories use 

is 20mls.

D. What microorganisms are required by the USP <51> method and in what 

circumstances might additional microorganisms be added? The following 

are the five microorganisms required by the method: Candida albicans, 

Aspergillus brasiliensis, Escherichia coli, Pseudomonas aeruginosa and 

Staphylococcus aureus. Additional microorganisms may be added to the assay 

by the request of the Center. Additional microorganisms might be included due 

to claims the labeling contains or due to an ongoing adverse event occurrence.

E. At what time points are aliquots removed and plated or filtered? At the 

following time points an aliquot should be removed for assay: time 0, 7 days, 14 

day and 28 days. There may be additional time points added or the USP <51> 

time points adjusted based on recommendations from the Center.

F. How does the USP <51> define “No increase”? “No increase” is defined as 

not more than 0.5 log10 unit more than the value to which it is compared.
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G. At what point in the method should the analyst refer to USP <1227> to 

determine what additional steps may be needed? USP <1227> provides

useful information and guidance that would be helpful during the design of the 

test particularly during the suitability portion of the test method. USP <1227> 

provides guidance on how to demonstrate that any neutralizers used during the 

assay are shown to not inhibit the growth of the microorganisms. 

7.10 Biochemical Identification

A. Why is it important to have a quality Gram stain prior to starting the 

identification process? A Gram stain is performed to determine which VITEK®

card to select for biochemical identification. If the Gram stain is not prepared 

correctly the wrong VITEK® card could be chosen and an incorrect identification 

obtained. 

B. How is the acceptable optical density range of the microorganism 

suspension determined? The VITEK® 2 Systems Product Information Manual 

contains the designated ranges for optical density of the microorganism 

suspension based on the VITEK® card being used for analysis

C. How does the analyst know if a supplemental test is required to complete 

the biochemical identification? The VITEK® result report will indicate a low 

discrimination or a slash line result, and list the supplemental tests required to 

separate the closely related microorganisms.

D. When preparing the microorganism suspension to load into the VITEK® 2 

card, what is the maximum hold time? The maximum hold time of the 

microorganism suspension is 30 minutes. 

E. When preparing to use the VITEK® 2 card what are some of the factors 

that must be considered? Allow the VITEK® card to come to room 

temperature prior to taking it out of the package. Do not use the card if the 

package liner is damaged or if there is no desiccant present. Do not touch the 

straw when removing the card from the package. 

7.11 Polymerase Chain Reaction (PCR)

A. What are the three major steps (processes) involved in the PCR cycle? 

Denaturation, annealing (complementary binding of primers), and extension.

B. What components are needed in the PCR reaction mixture and what 

function does each element serve?

1. Template: provides source of target DNA

2. Primers: bind to specific locations on the target DNA

3. Nucleotides: provide the building blocks of DNA structure

4. Polymerase: extends the DNA strands
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C. Why is selection of the primers so important to the success of the 

reaction? The primers will bind on either side of the target DNA. Where they 

bind will determine what portions of the DNA strand are amplified and how 

large the PCR product will be. 

D. Why are there forward and reverse primers? Primers are short DNA 

sequences complementary to the single-stranded DNA templates from which 

the polymerase begins replicating. PCR uses forward and reverse primers that 

bind to opposite strands of DNA, flanking the region that is to be amplified. 

DNA synthesis at one primer is directed toward the other, resulting in the 

replication of the desired sequence. 

E. Why does one need an excess quantity of primers in the reaction 

mixture? To ensure the denatured DNA strands anneal to the primers, not to 

each other. The quantity of primers in the reaction also partly determines how 

many times the DNA can be amplified.

F. Why always run a reagent control? “DNA free” reagent control should 

always be run to show that there was no DNA present in the reagent to 

contaminate the PCR reaction.

G. List three ways to prevent contamination when performing PCR.

1. Prepare PCR solutions in an area different from the amplification area.

2. Use pipettes and supplies that are dedicated for PCR use only.

3. Use gloves. 

4. Aliquot stock solutions and prepared solutions into small quantities and 

small containers to avoid contamination of a large volume of solution.

5. Avoid spilling and splashing of reagents.

6. Use filtered pipette tips.

H. In what ways can PCR be inhibited? PCR inhibitors can interfere with the 

cell lysis needed for extraction of DNA, interfere by nucleic acid degradation or 

capture, and can inhibit polymerase activity of amplification of target DNA. 

Potential PCR inhibitors include: components of body fluids (e.g. hemoglobin, 

urea, and heparin), environmental compounds (phenolic compounds, humic 

acids, and heavy metals), constituents of bacterial or fungal cells, non-target 

DNA, pollen, glove powder, laboratory plastic ware, and cellulose.

7.12 Sanger DNA Sequencing

A. What regions are targeted for bacterial and fungal identification by DNA 

sequencing? For bacterial identification we target the first 500 base pairs of 

the 16S ribosomal RNA (16S rDNA) bacterial gene. For fungi we target the 

internal transcribed spacer (ITS) regions and the D1D2 regions of the 
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eukaryotic rRNA gene complex. These gene regions are commonly used for 

identification as they contain sequence stretches ranging from highly 

conserved to variable.

B. What are the major steps (processes) involved in DNA sequencing? 

Amplicon PCR, amplicon PCR clean up, cycle sequencing, cycle sequencing 

clean-up and capillary electrophoresis

C. Why is the amplicon PCR cleaned up using prior to cycle sequencing? 

To produce amplicon free of extra dNTPs and excess primers that might 

interfere with the sequencing reaction. ExoSAP-ITTM utilizes two hydrolytic 

enzymes, Exonuclease I and Shrimp Alkaline Phosphatase. Exonuclease I 

removes residual single-stranded primers and any extraneous single-stranded 

DNA produced in the PCR. Shrimp Alkaline Phosphatase removes the 

remaining dNTPs from the PCR mixture.

D. How is cycle sequencing different from conventional PCR? Cycle 

sequencing takes advantage of the ability of DNA polymerase to incorporate 

2 ́,3 ́-dideoxynucleotides (ddNTPs). These are deoxynucleotide (dNTP) base 

analogs that lack the 3 ́-hydroxyl group essential in phosphodiester bond 

formation. The cycle sequencing reaction requires DNA template, a 

sequencing primer, DNA polymerase, dNTPs, ddNTPs, and buffer. Each type 

of ddNTP (ddA, ddC, ddG, or ddT) is labelled with a different fluorescent dye. 

As the denaturation, annealing, and extension cycles are performed, DNA 

polymerase adds a dNTP or the corresponding ddNTP at each step of chain 

extension. When a dNTP is added to the 3 ́ end, chain extension can continue. 

However, when a ddNTP is added to the 3 ́ end, chain extension terminates. 

Cycle sequencing results in the formation of extension products of various 

lengths terminated with fluorescently labelled ddNTPs at the 3 ́ end.

E. What libraries are used for identification of bacterial and fungal 

sequences generated? For bacteria we use the MicroSEQ® Microbial 

Identification System proprietary libraries. For fungi we use two publicly 

available databases, NCBI and Mycobank.

7.13 Whole Genome Sequencing (WGS)

A. What are the general steps in WGS analysis using the Illumina DNA Prep 

protocol? The general steps in WGS analysis are as follows: Extraction of 

genomic DNA; DNA quantitation; tagmentation of input DNA; PCR 

amplification; PCR cleanup; library pooling; MiSeq loading; MiSeq run; and 

data analysis. 

B. How should an idle MiSeq be maintained? “Idle” in this scenario means 

out of use for ≥7 days. If the instrument is idle, a standby wash must be 

completed, and then the instrument can be left in standby / idle mode. 
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Additionally, a standby wash should be completed every 30 days the 

instrument is idle.

C. What are acceptable run metrics for WGS performed using a v3 600 cycle 

cartridge? Acceptable run metrics in this scenario would be Q30 (%) ≥70, 

cluster density = 1200-1400 K/mm2, and clusters passing filter ≥80%. Run 

metrics vary based on the type of cartridge used. 

D. Upon completion of a WGS run, run metrics are visible on the MiSeq 

Control Software. Where else can run metrics be viewed? Run metrics are 

available on the MiSeq Control Software once a run has completed, and run 

metrics are also available using Sequence Analysis Viewer (SAV).

E. What are acceptable quality metrics values generated using 

MicroRunQC? Estimated Coverage must be ≥ 30X, unless stated otherwise in 

SOP-000792. MeanQ_R1 and R2 must be ≥30.

F. What GalaxyTrakr tool should be used to further assess isolates that 

have the same ST value as a control or as an isolate from a previously 

analyzed sample? If an isolate has the same ST value as a control, or as an 

isolate from a previously analyzed sample, the CFSAN SNP Pipeline with 

Assembly Lite tool should be used for further assessment.

7.14 Developing/Establishing a Rapid Method for Implementation into Regulatory 

Testing 

A. What are some of the advantages of rapid methods? They provide quicker 

results, are easy to use, may provide increased accuracy and sensitivity and 

are amenable to automation.

B. How does the rapid sterility method differ from the current USP 

compendial method? The rapid method has a fast turnaround time with less 

operator handling and provides automated results making this less prone to 

human error. The compendial method is operator driven, requires daily 

monitoring and takes more than 14 days to complete. 

C. As per the guidance outlined in USP chapter <1223> what are the two 

general categories used to evaluate an alternative method? The two 

general categories described are:

1. Qualitative Methods which include assays to demonstrate the presence or 

absence of microorganisms in a test article.

2. Quantitative methods yield a numerical result related to the microbial 

content in the test article. 
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D. As per USP chapter 1223 what are the validation requirements used to 

evaluate a qualitative method? The validation requirements for a qualitative 

test method includes:

1. Limit of detection

2. Specificity

3. Robustness

4. Repeatability

5. Ruggedness

6. Equivalency to the reference method 

E. What does ORCE stand for? The Office of Research Coordination and 

Evaluation (ORCE) is the research and review arm of the Office of Regulatory 

Science (ORS). It supports collaborative scientific activities that help advance 

regulatory science.

8. Inspectional Guidance

A. What are some of the different types of inspections? Surveillance 

inspections are conducted as routine assignments to evaluate the firm’s 

compliance with quality regulations. They are conducted every 2 years for 

Class II and Class III devices. Compliance inspections are conducted to verify 

the adequate correction of previous violations, to document continuing 

violations, or to support future regulatory action. For-cause inspections are 

carried out in response to specific information such as consumer complaints, 

recalls, or laboratory results, among others. Pre-Approval Inspections are 

conducted after a company submits an application to FDA to market a new 

product. These inspections verify the data included in the application and 

confirm that the facility can manufacture the product.

B. Why is it useful to review the previous EIR? The previous EIR provides an 

overview of the establishment’s operations and products as well as an 

understanding of their compliance history. You should examine the previous 

inspectional findings to determine any follow-up that would need to occur at 

the next inspection. 

C. What are 483 observations? The FDA 483 Inspectional Observations are 

intended to notify the inspected establishment’s top management in writing of 

significant objectionable conditions, relating to products and/or processes, or 

other violations of the FD&C Act and related Acts which were observed during 

the inspection.

D. How does FDA classify inspection reports? Inspections are classified as 

No Action Indicated (NAI). Voluntary action indicated (VAI) some deficiencies 
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identified but not serious. Official Action Indicated (OAI) serious deficiencies 

identified, and FDA must act to assure correction.

8.1 Drug Inspections

A. List two to three objectives for a pre-approval inspection. The following 

are the three objectives for a pre-approval inspection: 1) Readiness for 

commercial manufacturing; 2) Conformance to the submitted drug application; 

3) Data integrity audit.

B. Explain the importance of covering good manufacturing practices 

(GMPs) during a pre-approval inspection. As part of a pre-approval 

inspection the manufacturing practices of the firm should be evaluated to the 

extent possible to determine if the firm is operating in accordance with cGMPs. 

A firm must be in compliance with GMPs as part of the assessment of the 

firm’s ability to manufacture and market the drug product for which they have 

submitted for FDA approval.

C. For a GMP inspection of a sterile manufacturer what is the main system 

from the Compliance Program Guidance Manual 7356.002A that the 

microbiologist will be expected to cover? The microbiologist would be 

expected to provide complete coverage of the Laboratory Control System with 

respect to the microbiology laboratory. 

D. If a microbiology laboratory lacks method suitability for sterility testing 

what is the potential 21 CFR 211 cite that may be used to support an 

observation? Although several citations may be used the following citation, 

21 CFR 211.165I. “The [accuracy] [sensitivity] [specificity] [reproducibility] of 

test methods have not been [established] [documented]. Specifically, ***” 

addresses the specific need for the test method to be suitable and fit for use.

E. If you were asked to inspect a microbiology laboratory for a sterile 

injectable manufacturer what three finished product release tests would 

the microbiology laboratory be responsible for? The three finished 

product release tests what would likely be completed by the microbiology 

laboratory or a contract testing laboratory are: sterility testing, endotoxin 

testing and sub-visible particulate matter testing.

8.2 Device Inspections

A. How are medical devices classified? Medical devices are classified as 

Class I, Class II, or Class III based on the risks associated with their use. 

Class I devices present minimal potential for harm to the user. Examples of 

Class I devices include enema kits and elastic bandages. Class II devices 

generally present a moderate risk of harm to the user. Examples of Class II 

devices include powered wheelchairs and some pregnancy test kits. Class III 

devices are those that sustain or support life, are implanted, or present 
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potential high risk of illness or injury. Examples of Class III devices include 

implantable pacemakers and breast implants.

B. What is QSIT? Medical device routine inspections are generally conducted 

using the Quality System Inspection Technique (QSIT). QSIT is a tool 

designed to streamline medical device inspections. It divides the firm’s quality 

system into four subsystems: management controls, design controls, 

preventive and corrective actions and production and process controls. QSIT 

uses a “top down” approach that looks at procedures and ask questions first, 

then reviews records.

C. How do different types of devices obtain market approval from FDA?

Class I devices can generally be sold without preapproval. Most Class II 

devices must receive prior clearance from the FDA before they can be sold in 

the U.S. The clearance process is known as “premarket notification” and the 

application is referred to as a “510(k) application”. In the 510K, the 

manufacturer must demonstrate that the device to be marketed is at least as 

safe and effective as, or substantially equivalent to, a legally marketed device 

that is not subject to PMA. Most Class III devices must undergo a more 

rigorous process known as “premarket approval” (PMA) before they can be 

sold in the U.S. In the PMA, the manufacturer must demonstrate that the 

device is safe and effective for its intended purpose. 

8.3 Compounding Inspections

A. What is compounding? Compounding is a practice in which a pharmacist or 

doctor combines, mixes, or alters ingredients of a drug to create a medication 

tailored to the needs of an individual patient.

B. What are some areas to inspect/observe during a Compounding 

Inspection of sterile drug products? Some of the areas to cover during a 

Compounding Inspection of sterile drug products are: aseptic technique, 

process and facility design, environmental and personnel monitoring, product 

inspection, quality systems, equipment, containers, and closures, etc.

C. What can be a risk and cause of improperly compounding drugs? Drugs 

that are compounded improperly, or that are compounded under sustained 

conditions, can pose serious health risks (i.e. compounded drugs that are 

produced under insanitary conditions can become contaminated, and errors 

made during compounding can result in sub or super potent products. 

D. Provide an example of a serious compounding related outbreak. An 

outbreak of fungal meningitis in late 2012 was one of the most serious in a 

long history of outbreaks associated with contaminated compounded drugs. It 

was a multistate outbreak where over 750 patients were diagnosed with a 
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fungal infection after receiving injections of the contaminated drugs and 

numerous patients died.

E. Under 503B in Food, Drug, and Cosmetic Act (FDCA) if compounder 

becomes an outsourcing facility what must they comply with? Under 

503B a compounder who becomes an outsourcing facility must comply with 

current good manufacturing practices requirements, must be inspected by FDA 

according to a risk-based schedule, must meet certain conditions, including 

reporting adverse events to FDA associated with their compounded drugs, 

must be registered as an outsourcing facility, is engaged in the compounding 

of sterile drugs, complies with all of the requirements in section 503B, including 

labeling provisions.

F. What effect did the Compounding Quality Act of the Drug Quality and 

Security Act have on compounding facilities? It removed certain 

previsions from section 503A related to solicitation of prescriptions and 

clarified that section 503A is applicable to compounders nationwide. It also 

added new section 503B: “Outsourcing Facilities”.

G. If a compounder is not registered as outsourcing facility what conditions 

must they meet? Compounders who do not register as outsourcing facilities 

must meet the conditions under section 503A or be regulated as conventional 

drug manufacturer.

H. How does FDA, CDER implement the compounding provisions of the 

FD&C Act and conduct oversight to protect public health? Implement the 

compounding provisions of the FD&C Act, as amended by the Drug Quality 

and Security Act (DQSA), conduct oversight through compounding inspection 

enforcement actions where appropriate to protect public safety, such as 

warning letters, letters referring to inspectional findings, recall, voluntary 

temporary or permanent cessations of operations by compounders, civil 

enforcement actions in collaboration with the Department of Justice.

8.4 EM Sample Collections

A. In what location order do you perform an Environmental Monitoring (EM) 

collection? When collecting EM sample, you always start in locations that are 

under the greatest control and then move to sample to lesser controlled area. 

Work from cleanest to dirtiest. For example, start with ISO 5 HEPA filtered 

laminar flow hood then move to to outside the hood, room ISO 7 area.

B. Why is Day/Engley (D/E) Neutralizing Broth suitable for Environmental 

Monitoring (EM) collections? 

1. D/E neutralizes a broad spectrum of antiseptics and disinfectants which 

may be present in the surfaces being sampled such as quaternary 
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ammonium compounds, phenolics, iodine, chlorine preparations, mercurial, 

formaldehyde and glutaraldehyde.

2. The media contains nutrients necessary to support microbial growth.

C. What are the advantages of using sponges/swabs (qualitative method) 

for EM monitoring?

1. Can monitor a larger surface area

2. Can reach tight or remote areas that are excellent conditions for microbial 

contamination or build up that can be difficult to clean

3. The contact pressure increases the total number of organisms recovered. 

The mechanical process of swabbing dislodges the microbial biofilm.

D. What are the advantages and disadvantages of using 

RODACS/HychecksTM (quantitative method)?

D.1 Advantages

Assess the relative number of microorganisms present within a certain 

area (usually four-square inches).

D.2 Disadvantages

1. Cannot reach small areas such as crevices, cracks or other hard to 

reach locations. These areas may be more likely to be areas of 

contamination.

2. Low efficiency or recovery of microorganisms transferred to the 

agar surface plate.

3. Can only be used on flat surfaces.