Microbial Limit and Bioburden Tests : Validation Approaches and Global Requirements,Second Edition book cover
2nd Edition

Microbial Limit and Bioburden Tests
Validation Approaches and Global Requirements,Second Edition

ISBN 9781420053487
Published October 14, 2008 by CRC Press
280 Pages 97 B/W Illustrations

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Book Description

In recent years, the field of pharmaceutical microbiology has experienced numerous technological advances, accompanied by the publication of new and harmonized compendial methods. It is therefore imperative for those who are responsible for monitoring the microbial quality of pharmaceutical/biopharmaceutical products to keep abreast of the latest changes. Microbial Limit and Bioburden Tests: Validation Approaches and Global Requirements guides readers through the various microbiological methods listed in the compendia with easy-to-follow diagrams and approaches to validations of such test methodologies.


Includes New and Updated Material

Now in its second edition, this work is the culmination of research and discussions with technical experts, as well as USP and FDA representatives on various topics of interest to the pharmaceutical microbiologist and those responsible for the microbial quality of products, materials, equipment, and manufacturing facilities. New in this edition is an entire chapter dedicated to the topic of biofilms and their impact on pharmaceutical and biopharmaceutical operations. The subject of rapid methods in microbiology has been expanded and includes a discussion on the validation of alternative microbiological methods and a case study on microbial identification in support of a product contamination investigation.

Substantially updated and revised, this book assists readers in understanding the fundamental issues associated with pharmaceutical microbiology and provides them with tools to create effective microbial contamination control and microbial testing programs for the areas under their responsibility.

Table of Contents

I. Microbial Life and Ecology

An Overview of Microbial Life

Microbial Phylogeny

Microbial Taxonomy

Microbial Growth and Survival

Growth Curve


Energy Sources



Cell Shape


Bacterial Growth and Reproduction

Cell Structures

The Phyla Gram-Positive Bacteria and Proteobacteria

Gram-Positive Bacteria


The Gram-Staining Method

KOH Test

Catalase Test


Cell Structures

Fungal Growth and Reproduction



Microorganisms of Interest

Genus Staphylococcus

Staphylococcus aureus

Genus Pseudomonas

Pseudomonas aeruginosa

Genus Burkholderia

Burkholderia cepacia

Genus Ralstonia

Ralstonia pickettii

Genus Comamonas and Genus Stenotrophomonas

Family Enterobacteriaceae

Genus Escherichia

Escherichia coli

Genus Salmonella

Genus Shigella

Genus Serratia

Genus Klebsiella

Genus Bacillus

Bacillus subtilis

Bacillus cereus

Genus Clostridium

Clostridium perfringens

Clostridium sporogenes

Candida albicans

Zygosaccharomyces rouxii

Genus Aspergillus

Aspergillus niger

Genus Penicillium


II. Microbial Contamination and Control

Microbiological Contamination

Product Recalls

Nonsterile Products

Microbial Limit Standards

The Preservation of Pharmaceutical Products

Antimicrobial Activity and Efficacy

Types of Preservatives


Benzalkonium Chloride

Benzoic Acid and Salts

Boric Acid and Salts



Dowicil 200




Sorbic Acid Salts

Microbiological Control

Risk Assessment

Objectionable Organisms

Sanitization and Disinfection Practices

Definitions and Types of Chemical Products

Factors in Choice and Use of Disinfectants

Rotation of Disinfectants

Qualification of Disinfectants

In Situ Testing

In Vitro Testing

Expiration Date for Disinfectant Solutions

Sanitizers Used for Equipment Cleaning

Neutralization and Microbial Recovery Studies

Requalification and Change Control



III. The USP Microbial Limit Tests

History of the Revision and Harmonization Process

USP Chapter <61>: Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

Sample Preparation

Total Aerobic Microbial Count

Total Combined Yeasts and Molds Count

Bioburden Tests

Two-Media Bioburden Test

One-Medium, Dual-Temperature Incubation Bioburden Test

TAMC and TYMC Tests via Plate-Count Methods

Pour-Plate Method

Spread-Plate Method

Incubation and Results Calculation

Test Controls

TAMC and TYMC Tests via Membrane Filtration Method

Test Controls

TAMC Test by the Multiple Tube Method


Interpretation of the TAMC and TYMC Test Results

USP Chapter <62>: Microbiological Examination of NonSterile Products:

Tests for Specified Microorganisms

Sample Preparation for Direct Inoculation Tests

Test for Absence of Escherichia coli

Test for Absence of Salmonella spp

Test for Absence of Bile-Tolerant Gram-Negative Bacteria

Test for Absence of Pseudomonas aeruginosa

Test for Absence of Staphylococcus aureus

Test for Absence of Candida albicans

Test for Absence of Clostridia

Quantitative Test for Bile-Tolerant Gram-Negative Bacteria



IV. Pharmaceutical Waters

Types of Water for Pharmaceutical Purposes

Microbial Quality Attributes

Testing of Pharmaceutical Waters

Sampling Program

Sample Collection and Preservation

Bioburden Testing

Recovery Media

Coliform Testing

Identification of Waterborne Microorganisms

Establishing Alert and Action Levels

Validation of Water Systems

Microbial Control and Sanitization


V. Environmental Monitoring

Cleanroom Classification

Occupancy State

Routine EM Program

Testing Frequency and Sampling Sites

Setting Alert and Action Levels

Test Methods and Equipment

Surface Sampling for Viable Particles

Active Air Sampling for Viable Particles

Active Air Sampling for Nonviable Particles

Microbial Identification Program

Data Analysis

EM During Facility Validation Activities

Room Occupancy

EM of Isolators

Microbial Control in Cleanrooms


VI. Bioburden Considerations In Equipment-Cleaning Validation

Biocontamination Control

Disposable and Single-Use Equipment

Equipment-Cleaning Methods

Validation of Cleaning Methods

Sampling Recovery Methods

Swabbing of Equipment

Rinsing of Equipment

Qualification of Sampling Methods

Recovery Study Using the Wet Method

Recovery Studies Using the Dry Method

Effects of Product and/or Cleaning Agent Residue on the Recovery of Microorganisms

Establishing Limits

Execution of Equipment-Cleaning Validation Protocol

Validation of Cleaned Equipment Hold Time

Validation of Dirty Equipment Hold Time

Ongoing Verification of Cleaning

Validation of Holding Time/Shipping Conditions


VII. Method Validation and Media Suitability Testing

Suitability Test Design

Representative Challenge Organisms

Maintenance and Preparation of Test Organisms

Preparation of Working Cultures

Validation of Storage Period for Working Cultures

Recovery of Injured Organisms

Suitability Testing By Direct Inoculation/Plating Methods

Validation of Screening for Specified Microorganisms

Modifications to the Direct Inoculation Method

Validation of the TAMC and TYMC Tests

Approach 1

Approach 2

Modifications to the Plate Method

Suitability Testing for Membrane Filtration Methods

Validation of Screening for Specified Organisms

Validation of TAMC and TYMC

Modifications to the Membrane Filtration Method

Suitability of Microbiological Media

Growth Promotion Testing for Microbial Enumeration Media

Growth Promotion Testing for Selective Media

Validation of Rapid Microbiological Methods

The Validation Package

Validation Criteria

Validation of Quantitative Methods




Limit of Quantitation


Limit of Detection




Validation of Qualitative Methods


Limit of Detection



Accuracy and Precision

Validation of Automated Microbial Identification Methods





Final Thoughts

Points to Consider


VIII. Microbiological Quality of Pharmaceutical and Biopharmaceutical Products and Raw Materials

Microbiological Testing

Raw Materials

Biopharmaceutical Products

Nonsterile Finished Drug Products

USP Chapter <1111>

Testing Frequency

Stability Testing

Water Activity

Measuring Water Activity

The Chilled-Mirror/Dew Point Method

Capacity Sensors

Pharmaceutical Applications for Water Activity

International Harmonization

Looking Ahead


IX. Rapid Testing and Alternative Methods in Microbiology

Rapid Method Technology Platforms

Impedance/Conductance Technology

Gas Consumption or Generation

ATP Bioluminescence

The Celsis ATP Bioluminescence Systems

Automated Biochemical Assays

The VITEK® System

The Biolog Systems

Fatty Acid Analysis Using Gas Chromatography

The MIDI System

Enzyme-Linked Immunosorbent Assay (ELISA)


Analysis of Biomolecules Using Mass Spectrometry

Polymerase Chain Reaction (PCR)

Detection of Microbial Contamination Using PCR Technology


The Riboprinter®

Fluorescent Labeling Assays

Scan® RDI Microbial Detection


Biosensors and Microarrays

Laboratory-on-a-Chip Technology

Barriers to Implementation

Regulatory Climate

Future Trends

Case Study: Genotypically Similar Staphylococci

Contaminant Isolate and Environmental Sampling

Ribosomal Gene Sequencing

Phenotypic Analysis

Genetic Subtyping—PFGE

Results and Reporting


X. Biofilms

Biofilm Definition

Biofilm Structure

The Biology of Biofilms

Biofilm Formation

Quorum Sensing

Cell Adhesion

Smooth versus Rough Surfaces

Hydrophobic versus Hydrophilic Surfaces

Electrostatic Charge Properties

Low-Shear versus High-Shear Environments

Biofilm Dispersion

Biofilm Resistance and Phenotypes

Pharmaceutical Production Equipment and Materials Prone to Biofilm Formation

Water Systems

Production Equipment

Ultrafiltration/Diafiltration (UF/DF) Systems

Chromatography Systems

Miscellaneous Parts and Materials

Biofilm Control and Prevention

Heat Treatment

Chemical Treatment

Prevention of Biofilms

Methods for Detection and Recovery of Biofilm Organisms

Qualification of Chemical Sanitization Using Biofilm Cells

Types of Biofilm Reactors

Choosing a Biofilm Reactor

Testing Sanitizers Using the CDC Biofilm Reactor

Setting up the Biofilm Reactor

Exposure of Biofilm to Disinfectant Solution

Harvesting Biofilm Cells

Sanitizer/Disinfectant Efficacy Evaluation

Method Qualification and Test Controls

Testing Sanitizers Using a Static Biofilm Reactor

Industrial Significance of Biofilms

The Future in Biofilm Research


XI. Handling Aberrant and Out-of-Specification Microbial Data

Historical Overview of Investigating OOS Results

Out-of-Specification (OOS) Result

Laboratory Investigations

Conducting the Investigation

Retesting and Resampling

Testing for Outliers

Repeat Testing

Concluding the Investigation

Product Lot Disposition

OOS Investigations and FDA Citations



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