Method Validation of U.S. Environmental Protection Agency 

Microbiological Methods of Analysis 

Prepared for: 

The EPA Forum on Environmental Measurements (FEM) 

Prepared by: 

The FEM Microbiology Action Team 

FEM Document Number 2009-01 

October 7, 2009 

Contributors: 

Sandhya Parshionikar, PhD Richard A. Haugland, PhD Orin Shanks, PhD 

Technical Support Center National Exposure Research Laboratory National Risk Management 

Office of Groundwater and Drinking Office of Research and Development Research Laboratory 

Water Cincinnati, OH Cincinnati, OH 

Cincinnati, Ohio 

Margo E. Hunt, PhD Michele Cottrill Ann Grimm, PhD 

Quality Staff OPPTS/OPP/BEAD/Microbiology National Exposure Research 

Office of Environmental Information Laboratory Branch Laboratory 

Washington, DC Ft. Meade, MD Cincinnati, OH 

Fred Genthner, PhD Stephanie Harris, D.V.M. Robin K. Oshiro, PhD 

National Health & Environmental US EPA Region 10 Laboratory Office of Science and 

Effects Research Laboratory Manchester, WA Technology 

Gulf Breeze, FL Washington, DC 

Andrew Lincoff Stacy Pfaller, PhD Mano Sivaganesan, PhD 

US EPA Region 9 Laboratory National Exposure Research Laboratory National Risk Management 

Richmond, CA Cincinnati, OH Research Laboratory 

Cincinnati, OH 
Disclaimer 

This document contains direction developed solely to provide internal guidance to U.S. 

Environmental Protection Agency (EPA) personnel. EPA retains the discretion to adopt 

approaches that differ from this guidance on a case-by-case basis. The guidance set forth in this 

document does not create any rights, substantive or procedural, enforceable at law by a party to 

litigation with EPA or the United States. The use of mandatory language such as “must” and 

“require” in this guidance document reflects sound scientific practice and should not be 

construed to create any legal rights or requirements. The use of non-mandatory language such as 

“may,” “can,” or “should” in this guidance does not connote a requirement but does indicate 

EPA’s strongly preferred approach for validating and peer reviewing EPA methods prior to 

publication for general use. 

References within this document to any specific commercial product, process, or service 

by trade name, trademark, manufacturer, or otherwise does not necessarily imply its endorsement 

or recommendation by EPA. Neither EPA nor any of its employees, makes any warranty, 

expressed or implied, nor assumes any legal liability of responsibility for any third party’s use, or 

the results of such use, of any information, apparatus, product, or process disclosed in this 

manual, nor represents that its use by such third party would not infringe on privately owned 

rights. 

i 
Foreword 

The U.S. EPA Science Policy Council (SPC) established the Forum on Environmental 

Measurements (FEM) in April 2003. The FEM is a standing committee of senior EPA managers 

who provide EPA and the public with a focus for addressing measurement and methods issues 

with multiprogram impact. Action teams are commissioned by the FEM to address specific 

issues. The Method Validation Team was formed in October 2003 and tasked with developing 

Agency-wide, internal guidance for validating and peer reviewing EPA methods prior to 

publication for general use. This document contains guidance for microbiological methods of 

analysis. 

ii 
Acknowledgements 

The authors would like to thank the following reviewers for their contributions to this 

document. 

Reviewers 

Mark Borchardt, PhD 

Marshfield Clinic Research Foundation, Wisconsin 

George Di Giovanni, PhD 

Texas A&M Agricultural Research and Extension Center, Texas 

Yildiz Chambers 

Computer Science Corporation 

Ronald Landy, PhD 

Region 3, Environmental Sciences Center, USEPA 

Nicholas Ashbolt, PhD 

Office of Research and Development, USEPA 

Carrie Miller
 

Office of Groundwater and Drinking Water, USEPA 

James Sinclair, PhD 

Office of Groundwater and Drinking Water, USEPA 

Nichole Brinkman 

Office of Research and Development, USEPA 

Contract Support 

The authors would also like to thank Versar, Inc. and Eastern Research Group, Inc. 

(ERG) for contract support on this document. 

Versar Headquarters 

6850 Versar Center 

Springfield, VA 22151 

(703) 750-3000 

Eastern Research Group, Inc. 

Corporate Headquarters 

110 Hartwell Avenue 

Lexington, MA 02421-3136 

(781) 674-7200 

iii 
1.0 

Page No. 

INTRODUCTION ........................................................................................................... 1-1 

1.1 Microbiology at EPA ........................................................................................... 1-1 

1.2 Purpose................................................................................................................. 1-2 

1.3 Intended Audience ............................................................................................... 1-2 

1.4 Scope of the Guidance ......................................................................................... 1-2 

2.0 GENERAL GUIDELINES FOR METHOD VALIDATION ......................................... 2-1 

2.1 Introduction.......................................................................................................... 2-1
 

2.2 Method Selection ................................................................................................. 2-1 

2.3 Method Optimization ........................................................................................... 2-2 

2.4 Development of Operational Limits and Within-Laboratory Method Performance 

Attributes.............................................................................................................. 2-3 

2.4.1 Specificity and Sensitivity ....................................................................... 2-4 

2.4.2 Precision................................................................................................... 2-5 

2.4.3 Accuracy and Bias ................................................................................... 2-6 

2.4.4 Limit of Detection (LOD) ........................................................................ 2-7 

2.4.5 Linearity................................................................................................... 2-7 

2.4.6 Calibration and Other Performance Attributes of Absolute Quantitative 

Test Methods........................................................................................................ 2-8 

2.4.7 Special Considerations for Culture-based Microbiological Test Methods 2­

9 

2.4.8 Other Operational Limits and Quality Control Measures ........................ 2-9 

2.5 Writing the Method............................................................................................ 2-10 

2.6 Multi-laboratory Validation Studies .................................................................. 2-11 

2.6.1 Tier 1 Validation.................................................................................... 2-11 

2.6.2 Tier 2 Validation.................................................................................... 2-11 

2.6.3 Tier 3 Validation.................................................................................... 2-11 

2.6.4 Types of Multi-laboratory Validation Studies ....................................... 2-12 

2.6.5 Participating Laboratories ...................................................................... 2-13 

TABLE OF CONTENTS
 

iv 
2.6.6 Test Materials ......................................................................................... 2-15 

2.6.7 Replication of Test Materials................................................................. 2-16 

2.7 Development of a Study Plan and Study Implementation ................................. 2-17 

2.8 Reporting Results ............................................................................................... 2-17 

3.0 REFERENCES ................................................................................................................ 3-1 

v 
Appendix A – EMMC Format for Writing a Method 

1.0 Scope and Application 

2.0 Summary of Method 

3.0 Definitions of Method 

4.0 Interferences 

5.0 Safety 

6.0 Equipment and Supplies 

7.0 Reagents and Standards 

8.0 Sample Collection, Preservation and Storage 

9.0 Quality Control 

10.0 Calibration and Standardization 

11.0 Procedures 

12.0 Data Analysis and Calculations 

13.0 Method Performance 

14.0 Pollution Prevention 

15.0 Waste Management
 

16.0 References 

17.0 Tables, Diagrams, Flowcharts, and Validation Data 

Appendix B – Interlaboratory Collaborative Study 

Validation Protocol for the Quantitative Three Step Method 

Appendix C – Guidelines for the Development and Validation of Nucleic Acid Amplification 

(PCR) based Microbiological Method 

Introduction 

1.0 The PCR Method: General Background 

2.0 Method Selection 

3.0 Method Development and Optimization 

3.1 Design of Primers and Probes 

3.2 Optimization of Reaction Conditions
 

3.3 Sample Preparation 

3.4 PCR Inhibition Control 

3.5 Sample Processing Control 

3.6 Preparation and Use of Standards for Quantitative Real-time PCR 

Applications 

3.7 Real-time Quantitative PCR Data Analysis 

4.0 Quality Assurance and Quality Control (QA/QC) 

5.0 PCR Method Performance Criteria 

5.1 Specificity and Sensitivity 

5.2 Precision 

5.3 Accuracy and Bias 

5.4 Limit of Detection (LOD) and Limit of Quantification (LOQ) 

5.5 Sample Limit of Detection (SLOD) 

5.6 Linearity, Range of Quantification (ROQ) and Amplification Efficiency 

5.7 Ruggedness 

vi 
6.0 Multilaboratory Validation Studies 

6.1 Influence of PCR Platform and Reagent System
 

7.0 Potential Differences between PCR Chemistries 

8.0 References 

Appendix D – Guidelines for the Validation of Efficacy Test Methods 

Introduction 

1.0 Selection of Test Method to Generate Product Efficacy Data 

2.0 Validation of Efficacy Test Methods and Study Plan Preparation 

3.0 Pre-Validation Considerations: Factors Affecting Efficacy of Antimicrobial 

Products 

4.0 Desirable Attributes of a Validated Test Method 

5.0 Method Performance Characteristics 

6.0 References 

Appendix E – The Method Approval Process 

 Method Approval through the Rule Making Process 

1.0 Alternate Method Approval Processes 

1.1 Alternate Test Procedure (ATP) Program 

2.0 Expedited Methods Approval 

3.0 Reference Methods and Other Approved Methods 

4.0 References 

vii 
Figure 1-1: Overall Method Validation and Approval Process ................................................... 1-4 

Figure 2-1: Margin of Error for a Method with 80 % Sensitivity Rate and Confidence Interval of 

90% ............................................................................................................................................ 2-14 

List of Figures 

viii 
1.0 INTRODUCTION 

1.1 Microbiology at EPA 

Microorganisms are ubiquitous in the environment. As such, the study of these organisms 

and their impact on human health through environmental exposures is an important part of 

EPA’s mission. Consequently, the Agency is extensively involved in the study and monitoring of 

microorganisms in a variety of matrices, including air, water, soil, sludge and surfaces (fomites). 

In addition, to better manage pathogenic organisms in the environment, EPA is heavily invested 

in determining the impact of various treatment and decontamination procedures on 

microorganisms. The data generated by these efforts help inform EPA in the development of 

regulations and improved pathogen management approaches. 

With specific regard to drinking and source waters, the Agency develops regulations 

designed to address the issue of microbial contamination. The agency develops methods that are 

then validated and used to support regulations. In addition, the agency also develops consensus 

methods, which are not used by industry for compliance but to provide advisory 

information/data. Consensus methods are often developed through collaboration between 

industry and EPA to assist the industry in monitoring un-regulated parameters. A good example 

of the Consensus Method process is the Microscopic Particulate Analysis (MPA) (EPA 910/9­

92-029, 1992), a microscopic technique developed to assist water utilities and State regulatory 

agencies in determining if groundwater sources are under the direct influence of surface water. 

For both implementation of regulations and advisory purposes, EPA publishes microbiological 

methods used by public water utilities, academia, industries and other government agencies. 

Regardless of the purpose, reliable, and accurate methods are needed to ensure the validity of the 

data collected. Methods used for these purposes therefore must be validated before they are 

published as EPA methods. 

Method validation is defined as a process that demonstrates the suitability of an analytic 

method for its intended purpose (Green 1996). This document is intended to provide general 

guidance for the validation of microbiological methods likely to be used in future EPA methods. 

This document is designed to be applicable to all methods pertaining to assaying environmental 

microorganisms as well as to efficacy testing of antimicrobial agents. In exploring requirements 

to validate a specific method, the EPA Forum on Environmental Measurements (FEM) 

Microbiology Action Team concluded that the provision of specific guidance on how to conduct 

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validation studies for every method of analysis was beyond the scope of the document. 

Therefore, detailed validation protocols applicable to specific methods are not covered. Instead, 

guidelines for general validation are provided with some emphasis on certain technologies. 

Wherever appropriate, validation protocols developed by organizations and used by EPA are 

referenced; e.g. International Standards Organization (ISO), American Society for Testing and 

Materials (ASTM) International, and Association of Analytical Chemists (AOAC) International. 

As new microbiological methods of analysis are developed, this document may be 

revised. This text, therefore, should be treated as a living document. 

1.2 Purpose 

The purpose of this document is to provide agency-wide guidance on validating microbial 

methods, thus promoting consistency in the area of measurement methodology and policy across 

EPA program offices and regions. 

1.3 Intended Audience 

This guidance is intended for internal use by EPA personnel who are responsible for the 

development, implementation and review of microbiological methods of analysis used in 

environmental testing. These methods may be published as serially numbered EPA methods, or 

incorporated by reference in regulations or could be used purely for research efforts by EPA’s 

Office of Research and Development and the EPA Regions. It is recognized that this document 

may also be used by clients, contractors or other interested parties who, upon reviewing an EPA 

method for potential use, are interested in the Agency’s process for validation, approval, and 

acceptance of EPA methods. 

1.4 Scope of the Guidance 

This guidance is intended for new EPA microbiological methods that have not yet 

undergone validation prior to being published as EPA methods or adapted as Agency-accepted 

regulatory standards. This guidance includes validation principles for a range of analytes such as 

whole microbes or nucleic acids. A typical method involves sample collection, sample 

processing, extraction or isolation (microbes or nucleic acid) procedures, and analytical detection 

1-2 
of analytes. Validation issues with extraction procedures and detection are addressed in this 

guidance. A separate guidance document is being prepared for microbiological sampling. 

The primary focus of this document is validation guidelines for microbiological methods. 

Emphasis is on issues associated with PCR methods and efficacy test methods for antimicrobials 

in Appendices C and D, respectively. The guidance also provides readers with an understanding 

of EPA’s method approval process (Appendix E) after the methods are validated. The general 

flow depicting overall method validation and approval process is shown in Figure 1. 

1-3 
Method Selection

Quantitative Qualitative

Culture Methods

Molecular Methods

Efficacy Test Methods 

 

 

Method Selection 

Quantitative Qualitative 

Culture Methods 

Molecular Methods 

Efficacy Test Methods 

Method Optimization

Sample Collection

Sample Processing

Quality Assurance and Quality Control

Data Analysis

Method Optimization 

Sample Collection
 

Sample Processing
 

Quality Assurance and Quality Control
 

Data Analysis
 

Method Validation

Tier 1 Tier 2 Tier 3

(single lab) (three labs) (nine labs)

Description of Method Performance Attributes

Writing of Final Method

 

 

Method Validation 

Tier 1 Tier 2 Tier 3 

(single lab) (three labs) (nine labs) 

Description of Method Performance Attributes 

Writing of Final Method 

Final Method Approval

Rulemaking Process

Alternate Method Approval

Alternative Test Procedures

Expedited Method Approval

Final Method Approval 


 

Alternate Method Approval
 

Alternative Test Procedures
 

Expedited Method Approval
 

Figure 1. Overall Method Validation and Approval Process 

1-4 
2.0 GENERAL GUIDELINES FOR METHOD VALIDATION 

2.1 Introduction 

Method validation is the process of determining if a method is suitable for its intended 

purpose. Validation can be classified as primary validation and secondary validation according to 

its purpose. Primary validation is an exploratory process for establishing the operational limits 

and performance characteristics of a new, modified or otherwise inadequately characterized 

method. Secondary validation on the other hand, is the process of gathering evidence that a 

laboratory can meet the specifications established in primary validation (ISO 2000). 

This section outlines a series of steps and procedures that are recognized as being integral 

to the selection and validation of an analytical method for an intended use. These steps are 

normally performed sequentially and procedural details may vary for different types of methods. 

It should be noted that successful validation of any analytical method is not possible 

without thorough and systematic planning and preparation. A written study plan should be 

prepared for each step of the validation process and subjected to appropriate review prior to 

implementation. In addition, it is expected that all laboratories involved in each step of a 

validation process will have a Quality System or quality assurance (QA) program in place to 

ensure standardization of laboratory operations, as well as adequate quality control (QC) 

activities. 

2.2 Method Selection 

Some methods may not require formal validation. The decision on whether to proceed 

with validation will often be predicated on an expectation that a method will be widely 

implemented and/or be needed to support a regulatory requirement. It will also require a 

judgment about the expected capability of the method(s) under consideration to meet the 

requirements of its intended use. Information may already be available for the method(s) that can 

be used in the decision process. Other criteria to consider before pursuing validation include 

whether a method: 

Χ Is based on sound underlying scientific principles 

Χ Is applicable for routine analysis of samples 

2-1 
Χ	 Can detect analytes in the concentration range of interest 

Χ	 Has sufficient specificity and sensitivity for intended use 

Χ	 Can meet specific method performance criteria 

Χ	 Has adequate QA/QC controls 

Χ	 Can be performed with readily-available equipment 

Χ	 Can be conducted for a reasonable cost 

Χ	 Addresses the level of expertise required (e.g., are there technique-sensitive areas? Is 

specialized training required?) 

Χ	 Contains necessary aspects of quality assurance (e.g., calibrated equipment, media 

quality, incubation conditions) 

Χ	 Addresses biosafety concerns (e.g., are there specific biosafety practices necessary to 

handle the test pathogens?) (Fleming and Hunt, 2000; Centers for Disease Control, 

2007). 

2.3 Method Optimization 

New or, in many instances, even established methods identified in the selection process 

may require optimization prior to their validation. Optimization involves the identification of 

specific factors employed in the method such as procedures, environmental conditions, and 

reagents most likely to introduce variability in results and the subsequent iterative testing of 

modifications to the method that minimize this variability. Terms commonly used to characterize 

the stability of a method’s results in the presence of variable factors are ruggedness and 

robustness. Ruggedness and robustness are defined as the ability of a method to provide 

acceptable results in the presence of variable factors that are unavoidable and avoidable, 

respectively. Examples of generally unavoidable sources of variability can include different 

analysts, lots or preparations of reagents, and calibration of instruments. Examples of potentially 

avoidable sources of variability could include the use of reagents and/or instruments from 

different sources. In some cases, information about these differences may be desirable, since it 

can allow for more flexibility in the use of the methods. 

2-2 
For effective optimization, it is important to consider the whole method, both the sample 

collection and processing component, as well as the detection assay. A highly sensitive assay 

such as PCR is meaningless if the overall recovery of an organism is poor and/or assay inhibitors 

are not removed. The impact of the sample collection procedure on a microorganism’s viability 

is also important to consider, depending on whether the goal of the method is to specifically 

detect viable organisms or not. Furthermore, while it is possible to optimize individual 

components of methods and thereby estimate a given method parameter such as sensitivity, it is 

critical to empirically determine this value using the complete method. Finally, methods should 

be optimized to minimize the variability in results caused by interferences from different sample 

matrices. When matrix effects are a cause of significant variability and/or when quantification of 

the analyte is sought, use of an internal control should be considered part of the method. 

While a substantial amount of effort can be invested in the optimization process, it is 

beneficial, in minimizing problems that may be encountered in the subsequent steps of method 

validation, particularly in the final interlaboratory testing stage. Standardized experimental and 

statistical designs for testing the ruggedness and robustness of analytical methods are also 

available to facilitate these efforts (Youden and Steiner, 1975; Dols and Ambrecht, 1976). 

2.4	 Development of Operational Limits and Within-Laboratory Method Performance 

Attributes 

Once an analytical method has been developed and optimized, the first step in validating 

it is to determine its operational limits and within-laboratory performance attributes within these 

limits that are relevant to the intended use. As with optimization, this process is often carried out 

in the laboratory where the method was developed, but it may also be conducted by the 

organization intending to implement the method. This process, often referred to as primary 

validation, should provide preliminary baseline specifications (numerical and descriptive) of the 

method’s performance within the laboratory performing the tests. The performance attributes 

requiring determination may differ depending on the nature and applications of the method (e.g., 

culture- vs. microscopy-based, molecular- or chemistry-based, and qualitative vs. quantitative) as 

are the experimental designs that are best suited to make these determinations. A guideline for 

determining performance attributes of several different broad categories of method types are 

available (ISO 1994b, ISO 1994c, ISO 2000, US FDA 2001). To obtain examples of relevant 

2-3 
 

experimental designs that might be employed, new method developers should also consult the 

scientific literature for descriptions of similar previously validated methods. In many instances, 

different terms and definitions have been applied to the same performance attributes in the 

context of different types and applications of methods. With these caveats, some performance 

attributes and operational limits that require determination for primary validation of most 

analytical methods are provided below. 

2.4.1 Specificity and Sensitivity 

Sensitivity and specificity are terms that can have different definitions for different types 

or categories of analytical methods. In a general sense, we define these terms by the extent to 

which a method responds uniquely to the specified target organism or group of organisms. 

Specificity is the ability to discriminate between the target organism and other organisms. It is 

mathematically expressed as: 

Specificity = TNC / (TNC + TPI) Equation (1) 

Where: 

TNC = Number of samples tested negative correctly 

TPI = Number of samples tested positive incorrectly 

Specificity for microbiology methods and media is traditionally demonstrated through the 

use of pure positive and negative control cultures. For example, appropriate American Type 

Culture Collection (ATCC) strains for several groups of enteric control culture bacteria are given 

in Section 5.1.6.4 of EPA’s Manual for the Certification of Laboratories Analyzing Drinking 

Water, 5th Edition (U.S. EPA 2005). Positive cultures listed for Enterococci include 

Enterococcus faecalis ATCC 11700 and Enterococcus faecium ATCC 6057. Appropriate 

negative controls include Staphylococcus aureus ATCC 6538, Esherichia coli ATCC 8739 or 

25922, and Serratia marsecens ATCC 14756. The definition of appropriate target and non-target 

control cultures or other standards for use in both validation and routine QC would be expected 

in the development of any new microbial method. In a robust method, a single target organism 

should be discernable in complex matrices containing potentially millions of non-target 

organisms. 

2-4 
 

 

Sensitivity is the proportion of target organisms that can be detected. It is mathematically 

expressed as: 

Sensitivity = TPC / (TPC + TNI) Equation (2)

 Where: 

TPC = Number of samples tested positive correctly 

TNI = Number of samples tested negative incorrectly (Deep 2006) 

Data to calculate sensitivity are typically generated by repeated testing of serial dilutions 

of a “known” spike standard. 

2.4.2 Precision 

Precision can be defined as the closeness in agreement between independent test results 

obtained under stipulated conditions (ISO 1994a). This term is usually expressed as the variance, 

standard deviation, or coefficient of variation of a series of test results. Precision is often 

expressed as the percent coefficient of variation (%CV), where: 

%CV = (standard deviation of measurements / mean) x 100 Equation (3) 

The expected precision of culture-based microbial methods is typically derived 

mathematically based on the assumption that bacteria are distributed randomly in a well-mixed 

sample and follow a Poisson distribution. For methods involving direct counts, such as 

membrane filtration or plate counts, the 95% confidence limits around any result are given in 

Standard Methods (20th Ed.), 9222B.6.c. For both methods, quantitation is obtained by Most 

Probable Number (MPN) analysis of presence/absence tests of samples divided into multiple 

tubes or wells. Ninety-five (95) % confidence limits for results obtained by MPN tests are given 

in SM 9221C.2. Any new proposed culture-based method would report expected levels of 

precision. 

Precision may be considered at four levels: within-lab repeatability, within-lab 

reproducibility, between-lab repeatability, and between-lab reproducibility. The first two levels 

2-5 
should be addressed in the primary validation of a method over the entire concentration range of 

the analyte that is expected to be relevant to its intended use. 

Repeatability. Repeatability can be defined as the closeness in agreement between results 

of successive measurements of the same analyte carried out under the same conditions of 

measurement over a short interval of time. Repeatability is also termed intra-assay precision. 

Suppose the within laboratory precision is Sr and the between laboratory precision is SL. 

Then the precision SR (including within and between) among laboratories is expressed as 

SR = S 2 

r + S 2 L Equation (4)

Reproducibility. Reproducibility can be defined as the closeness of the agreement 

between the results of measurements of the same analyte carried out under variable conditions of 

measurement. For determination of within-lab reproducibility, some of the variable conditions 

that should be considered include different time intervals between analyses, analysts, lots or 

preparations of reagents, instruments, and different water matrices. 

2.4.3 Accuracy and Bias 

Accuracy can be defined as the closeness of the agreement between a test result and the 

accepted reference value, while bias can be defined as the difference between the expectation of 

the test results and a known or accepted reference value. The term “accuracy,” when applied to a 

set of test results, has been more comprehensively defined as a combination of random 

components (related to random error) and a common bias component (related to total systematic 

error) associated with the method (ISO 1994a). As with precision, this bias component should be 

first characterized at the within-laboratory level as a primary attribute of most methods. By the 

above definition, the determination of bias in the analysis of a material by a new method requires 

the knowledge of either the true value for the analyte in the sample or the assignment of an 

accepted reference value. In some cases these values may be known or assigned through the use 

of fortified or spiked samples, certified reference materials, analysis by another presumably 

unbiased method, or internal controls. When analyzing fortified or spiked samples, bias is often 

2-6 
expressed by the terms recovery or percent recovery, i.e., test result, divided by the expected 

(assigned) value for the added spike material, multiplied by 100. 

In other instances, there may be no direct means of determining the bias of a new 

method’s test results. Under these circumstances the recovery of the analyte by the new method 

is sometimes assessed in relation to the results of an accepted reference method and expressed as 

relative recovery (ISO 2000). If no such reference method is available, relative recovery may be 

defined by the new method itself. An example of this can be found where the test results from 

two simultaneously processed and analyzed samples with unknown quantities of analyte are 

compared. In this case the two test results can be expressed as the ratio of the analyte recovered 

in the two samples. By designating one of these unknown samples as a reference, the relative 

recovery of the analyte from any number of additional simultaneously processed and analyzed 

unknown samples can be determined and compared to each other based on their respective 

recovery ratios with this reference sample. For methods with high variability, this technique is 

still useful since the variation can be quantified through measurement of standard deviation. In 

addition, if the reference sample can be considered to be the same in different test runs of the 

method, i.e., a consistent material containing the same (albeit unknown) quantity of analyte, the 

respective recovery ratios with this sample can also be used for comparing the relative recoveries 

from other unknown samples in these different sets of analyses. 

2.4.4 Limit of Detection (LOD) 

Limit of detection can be defined as the minimum amount of analyte that can be reliably 

detected (i.e., distinguishable from known and characterized background with a given level of 

confidence). The LOD measurements establish a baseline detection value under optimal 

conditions. Culture based methods are typically expected to be capable of detecting single target 

organisms in large samples. Where no organisms are detected, the result is reported as <1 

organism per sample volume or mass. 

2.4.5 Linearity 

Linearity is defined as the ability of the analytical procedure to obtain test results within a 

given range, which are directly proportional to the concentration of analytes (microorganisms or 

nucleic acid) in the sample. 

2-7 
2.4.6	 Calibration and Other Performance Attributes of Absolute Quantitative Test 

Methods 

The performance attributes discussed in Sections 2.4.1 thorugh 2.4.5 are generally 

relevant to both qualitative and quantitative analytical methods. They can be characterized either 

directly or with minimal mathematical data transformations on the basis of the response values of 

the detector or instrument utilized in the method, or in the case of culture based methods, the 

bacterial/viral counts obtained. For methods specifically designed to obtain absolute quantitative 

measurements of an analyte, several additional operational limits and performance attributes may 

require determination and in many instances the relationships between detector or instrument 

response and analyte concentrations must also be established through the development of a 

calibration curve using reference standards of known concentration. The concentration of analyte 

in a reference standard can be established by several alternative approaches including: 

assignment or certification based on experimental work by some national or international 

organization; consensus or certification based on collaborative experimental work under the 

auspices of a scientific or engineering group; or in some cases, assignment based on analysis 

results of a reputable commercial or other non-commercial establishment. As mentioned in 

Section 1.4, analytical detection is only a part of the complete method and wherever appropriate, 

calibration to establish complete method recovery may be necessary. 

For standard curve development, the simplest model (e.g., linearity) that adequately 

describes the concentration-response relationship should be used. The calibration curve is also 

used to establish the limit of quantification of the method (i.e., the lowest amount of analyte that 

can be quantitatively determined with suitable precision and accuracy) and the quantitative range 

of the method (i.e., the interval between and including the upper concentration and limit of 

detection of the analyte over which the concentration-response model provides suitable precision 

and accuracy). During this process, the statistical dispersion of the standard measurements by the 

instrument or detector (instrument precision) is combined with the model assumptions to 

generate a calibration equation. A statistic such as R-squared combined with a confidence 

interval can be used to provide a measurement of reliability or uncertainty for an estimate from 

an “unknown” sample based on the calibration curve model. 

2-8 
2.4.7 Special Considerations for Culture-based Microbiological Test Methods 

Historically, most microbial work has involved cultivation (viability testing) of 

microorganisms in a selective medium for enumeration or for the determination of presence or 

absence of the target microorganism(s). EPA or other regulatory requirements determine whether 

quantitative or qualitative results are required and may specify which methods are allowed. 

While not strictly considered as absolute quantification methods, due to the near impossibility of 

knowing the true amount of viable microorganisms in a sample, quantitative culture-based 

microbiological methods have performance attributes and operational limits that are the same as, 

or are analogous to, most of the terms defined above, including specificity, sensitivity, precision, 

recovery, limit of detection, linearity and limit, and range of quantification (ISO 2000). Unlike 

chemical analytical methods, however, many of the performance attributes of culture methods 

are based upon particle distribution (i.e., distribution of the microorganisms) as defined by the 

Poisson distribution or where over-dispersion occurs (often in environmental waters due to 

clumping or simply the natural variability of target microbes), using a negative binomial 

distribution model (ISO 2000). Due to the often skewed distribution of target microbes in waters, 

when using methods that are based on normality of count data, it is generally necessary to 

convert the data to log-base 10 (or in other ways transform) to meet basic statistical assumptions 

when describing metrics such as the mean and standard deviation (Youden and Steiner 1975; 

ISO 2000; APHA 2005). 

Over-dispersion in a culture method can also be caused by a number of technical factors 

including variability in collection and/or preparation of the starting sample, and its dilution. 

Other factors such as physico-chemical properties and overall microbial composition of the 

sample, incubation stress, quality, and origin of detection media components and incubation 

conditions can also contribute to variability and uncertainty in measurements by these methods. 

2.4.8 Other Operational Limits and Quality Control Measures 

Analyte stability may have an important effect on the test results of many analytical 

methods. Consequently, the determination of acceptable holding times and storage conditions of 

samples and (if applicable) reference standards may be an important operational limit that should 

be determined during the primary validation process. Parameters that should be investigated 

using experimental designs are similar to those employed for determining the precision and bias 

2-9 
or relative recovery of the method (see above). The objective is to identify the limits of 

conditions that produce no significant differences in test results compared with those obtained 

from analyses of fresh samples at a specified confidence level. Different sample matrices may 

also affect test results. Thus, a number of sample matrices that are representative of the intended 

use of the method need to be tested to determine the range and/or proportion of these samples 

compared to those obtained from a specified reference matrix (i.e., no significant difference at a 

specified confidence level). 

Many analytical methods employ the use of matrix spike or internal control materials as 

surrogates of the true analyte for the routine determination of the efficiency of extraction from 

sample matrices and interferences in the analyses. If these quality control materials are utilized in 

a method, studies should be performed to determine whether their recoveries and responsiveness 

to interferences is representative of the true analyte. Another important consideration is to ensure 

that control material does not interfere with the detection of the true analyte at low concentration. 

2.5 Writing the Method 

The next step in the validation process is to prepare a complete written description of the 

method. Historically, EPA methods have been written in a format that includes the following 

components: scope and application; method summary; definitions; interferences; health and 

safety; equipment and supplies; reagents and standards; sample collection, preservation and 

storage; quality control; calibration and standardization; procedural steps; calculations and data 

analysis; method performance; pollution prevention; and waste management. Each component of 

this format is described in greater detail in Appendix A of this report. Note that this is a 

recommended format, and not a requirement. In this guidance, we further recommend inclusion 

of the numerical and descriptive specifications of the method’s operational limits and 

performance attributes determined from the within-laboratory testing results during primary 

validation. This is justified since for some methods the primary validation process should be 

sufficient for meeting the intended use of the method. In addition, if subsequent validation of the 

method in a multi-laboratory collaborative study is needed, the inclusion of the primary 

validation study specifications in the written method will serve to inform the design and 

interpretation of results of the collaborative studies and may also be useful for establishing 

analytical quality control systems. 

2-10 
2.6 Multi-laboratory Validation Studies 

EPA has historically recommended the use of interlaboratory collaborative studies for the 

validation of methods that are expected to see widespread use or to support regulatory activity. A 

tiered approach developed under the streamlining initiative (U.S. EPA 1996) has typically been 

used by EPA for the validation of microbiological methods. This approach takes into 

consideration the level of intended use for a method and as such will minimize the validation 

requirements of limited-use methods (single-laboratory and single-industry use) and instead 

focuses resources on validation of methods that are intended for nationwide use. Because QC 

acceptance criteria are developed from validation studies and validation requirements vary with 

each tier, the statistical procedures used to develop the criteria will vary by tier as well. 

2.6.1 Tier 1 Validation 

Tier 1 refers to new methods or method modifications that will be used by a single 

laboratory for one or more matrix types (i.e., air, water, soil). Validation requirements for Tier 1 

have reflected this limited use and typically require single-laboratory testing in the matrix types 

for which the method will be used. Under Tier 1, single laboratories are able to use modified 

methods without the burden of conducting an interlaboratory method validation study. 

2.6.2 Tier 2 Validation 

Tier 2 refers to new methods or method modifications that will be used by multiple 

laboratories analyzing samples of one matrix type. Validation for Tier 2 typically requires a 

three-laboratory interlaboratory study using the matrix in which the method will be used. 

2.6.3 Tier 3 Validation 

Tier 3 refers to new methods or method modifications that will be used by laboratories on 

a nationwide basis for all matrix types. Validation at Tier 3 requires a nine-laboratory 

interlaboratory study on nine matrix types. Validation must be performed on a minimum of nine 

matrix types in each sample for which the method will be applied. 

This tiered approach guidance continues in this document with the recognition, however, 

that decisions concerning the numbers of participating laboratories in different collaborative 

2-11 
studies may be influenced by a number of additional factors. Such factors, as detailed further 

below, can include the specific objectives of a study including statement of a testable hypothesis, 

the availability of laboratories that have instrumentation for and experience with the method, the 

complexity of the method, the underlying statistical theory and subjective judgments. Study 

design must be peer-reviewed by qualified members of the scientific community including 

statistician(s) before the study is implemented. 

2.6.4 Types of Multi-laboratory Validation Studies 

While the ultimate goal of interlaboratory validation is to demonstrate the suitability of 

an analytical method for its intended purpose under conditions of widespread use, several 

different types of collaborative studies may be required before making this final assessment. 

Some methods may require a collaborative study to simply establish an acceptable reference 

standard or material as one of the steps in this process (Horwitz 1995, Holmes et al., 2001). 

Primary Validation or Laboratory Performance Study. Collaborative studies have been 

developed into a widely used tool for testing performance characteristics of chemical methods, 

but this process is normally associated with the widespread availability of laboratories that 

already have extensive experience with these methods. This may not be the case for many newer 

microbiological methods (ISO 2000). For methods of this type, the more prudent next step in the 

validation process may be to conduct a collaborative laboratory performance study. Studies of 

this type are usually conducted with the objective of evaluating or improving laboratory 

performance of the method. Reported results by different individual analysts or laboratories are 

compared with those from other laboratories or with the known or assigned reference values of a 

limited number of test samples with a simple, well-defined matrix. These studies may also allow 

for assessments of method robustness, the clarity of the test procedures provided in the written 

method, and more generally, whether the method appears ready for widespread implementation. 

More than one study of this type may be required if correctable deficiencies related to any of the 

above aspects of the method are discovered. This type of study may also be called primary 

validation. These studies should not be confused with laboratory proficiency testing, which is 

normally associated with the evaluation or accreditation of laboratories in performing a 

previously validated and accepted method. 

2-12 
Secondary Validation or Method Performance Study. The type of collaborative study 

that is normally conducted in the validation process is a method performance study. The 

objective of these studies is normally to provide specifications for the methods performance 

attributes across a representative sample population of laboratories that would be expected to 

employ the method for its intended purpose. A second or alternative objective may be to 

demonstrate equivalency of a new method with an established reference method. This type of 

study is also sometimes referred to as secondary validation and guidelines on the experimental 

and statistical design of; preparation for; and implementation and analyses of results of studies of 

this nature have been published by a number of organizations (ISO, AOAC, ASTM, IUPAC, 

FDA, NCCLS, and others). A synopsis of recommendations that generally are consistent in these 

different guidance documents and other recommendations that may be specifically associated 

with the validation of microbiological test methods are provided in the following sections. 

2.6.5 Participating Laboratories 

The decision on how many laboratories to include in a method performance study often 

takes into account the effect of sample size on the estimation of confidence intervals for the 

performance parameters being tested. These intervals can be predicted based on statistical theory 

for different numbers of test results from different laboratories (Youden and Steiner 1975, 

AOAC 1989, ISO1994a). 

A relationship exists between the number of participating laboratories, the number of test 

samples, the acceptable margin of error (precision), and the desired level of confidence when 

considering the number of independent laboratories required for the validation of a method. 

There are a number of documents (McClure, 1990) that discuss these relationships for 

quantitative method validation, however, there is very little guidance regarding qualitative 

methods. In general, binomial data sets (e.g., presence/absence) generated from qualitative 

methods require more independent laboratories and test samples to achieve equivalent levels of 

margin of error and confidence levels when compared to quantitative data. Following is a 

mathematical model to estimate the margin of error (p2) (precision) given a certain number of 

laboratories (L) and test samples (m) for a method that is required to correctly classify at least 

2-13 
 

(80%) (p1 ) of the positive test portions (sensitivity rate), with a confidence limit of 90%. The 

equation is as follows: 

p2 = 100 Zα S

ˆ / [L1/2 m (p1/100)] Equation (5) a 

Where: 

L = 2, 3....., 25 

m = 3, 5, 10 

α = .05 (corresponds to Zα = 1.645 or 90% confidence limit) 

S

ˆ = estimated standard deviation (simulation method) of the number of positive a 

responses per laboratory and 80% sensitivity rate to estimate the margin of error 

(see Figure 2). 

Figure 2. Margin of Error for a Method with 80 % Sensitivity Rate and Confidence 

Interval of 90 % 

Because of the diminishing return in decreasing uncertainty that is associated with 

increasing numbers of independent test results from different laboratories, a common 

compromise is 8-15 laboratories to participate. The expectation is that a small number of these 

data sets may be statistical outliers or may be obtained from incorrect performance of the method 

and thus will be excluded. However, there are limitations to applying this purely statistically 

based approach to microbiological methods. Ultimately decisions on sample size (i.e., number of 

2-14 
laboratories) in a study must be based to some degree on a subjective judgment as to whether the 

results of the study will be effective in demonstrating method suitability (ISO 2000). Method 

complexity and the resource availability may also be important factors in making this decision. 

It is generally recommended that the selection of laboratories for a method performance 

study not be restricted to only laboratories with extensive experience with the specific method 

under consideration. Conversely, it is highly recommended that all participating laboratories 

have extensive experience with the techniques and/or instrumentation utilized in the method. 

Criteria for selection should include precision and accuracy of the laboratory when performing 

the technique on blind samples (rsd). For newer methods, it has been suggested that the 

performance attribute specifications obtained in these studies be reported as preliminary or initial 

estimates because they could further improve as laboratories gain experience with the method 

(ISO 1994a). 

2.6.6 Test Materials 

Test materials in a validation study have been previously defined as specific 

analyte/matrix/concentration combinations to which the method performance parameters apply 

(Horwitz 1995). Similar to within-lab method validation studies, the test materials used for 

collaborative method performance studies should be representative of the range of matrices 

intended for analysis by the method. In most cases natural samples of the materials containing 

naturally occurring analytes are preferred. Several different analyte concentrations should be 

tested and the range of analyte concentrations should be within the operational range of the 

method as specified from primary validation study results and should also be representative of 

the expected range of concentrations associated with the intended use of the method. If a specific 

concentration of analyte is known or predicted to be associated with a regulatory requirement, 

the concentrations tested should include and bracket this value. Larger numbers of test materials 

are generally required for method equivalency studies (ISO 2004). 

A highly important prerequisite of these studies is that the test samples that are analyzed 

by the different laboratories should originate from materials in which the analyte is 

homogeneously distributed. This is often challenging with microbes. This property can be 

established by the use of certified reference materials or determined from analyses of replicate 

samples with materials in the laboratory that procures them or prepared by a reference 

2-15 
laboratory. In the latter instances, precision estimates from these analyses should be compared 

with the method’s primary validation study precision estimates under repeatability conditions. It 

is important to remember that in the case of most microbiological methods, this property will be 

based upon particle distribution (i.e., distribution of the microorganisms) unlike in chemical 

methods. If this property can not be established with reasonable certainty in natural materials or 

if natural materials can not be readily found that contain the analyte, then another commonly 

employed alternative is the utilization of spiked materials. For laboratory prepared spikes, 

documentation of how precise the spikes are will be useful. Homogeneity of the analyte in the 

spiked materials should also be confirmed as above. If analyte recovery is a performance 

attribute that is being evaluated, spike levels should be sufficiently high to be readily 

differentiable from the levels of any naturally occurring analytes in the materials based on the 

method’s primary validation study precision estimates under reproducibility conditions. 

2.6.7 Replication of Test Materials 

Precision and bias (or recovery) are the performance attributes that are most commonly 

characterized in collaborative studies of quantitative methods. Accuracy as defined by ISO (ISO 

1994a) is influenced by both of these parameters and precision in turn is influenced by both 

between-lab repeatability and reproducibility components. As shown above, the number of 

replicate test results per test material will influence the uncertainty of estimates for repeatability 

and reproducibility standard deviations. The latter uncertainty value is also shown to be a 

function of the ratio of these two standard deviations. It is commonly observed with many 

chemical analytical methods that this ratio is relatively high, i.e., the variability of test results 

obtained under repeatability conditions is considerably smaller than that obtained under 

reproducibility conditions (AOAC 1989). When this situation occurs, analyses of more than two 

replicate samples of the different study materials will provide relatively little additional 

information. Microbiological analytical methods may differ in this regard, however, and analysis 

of additional replicate samples is warranted. Repeatability and reproducibility precision 

specifications generated from the primary validation study or results from collaborative 

laboratory performance studies can be used to determine the most appropriate number of 

replicate test sample analyses to perform. While the use of known replicate samples is a common 

2-16 
practice, the use of blind replicate samples is generally recommended to eliminate the potential 

for any selectivity by the laboratories in their procedures and reporting of test results. 

2.7 Development of a Study Plan and Study Implementation 

Once the study design has been determined it should be documented in a study plan that 

provides a detailed description of the study’s objectives and the clear statement of a testable 

hypothesis(es). These objectives should include management and implementation of the study; 

establishment of requirements for data reporting; documentation and review by laboratories; 

means for assessment of adherence to instructed validation methodologies, accuracy, and 

completeness; and specific instructions for data analysis including statistical tests and anticipated 

metrics for the results. Any study-specific procedural instructions that are not fully explained in 

the written method (e.g., sample storage conditions, when to perform analyses, and how to 

record and report the data) should be written in detail and be provided with the method to the 

laboratories well in advance of the study. New study organizers can also consult study plans for 

previously validated EPA methods or from the general scientific literature, if applicable. An 

example of study plan for a previously conducted validation study by EPA is given in 

Appendix B. Additional guidance that may be useful in the study planning and implementation 

process can be found in documents from several other organizations (AOAC 1989, ISO 2000, 

ASTM 2001). 

2.8 Reporting Results 

Results from the method performance study should be disseminated and made available 

to the scientific community for peer review before presentation at a scientific meeting, peerreviewed publication, and/or publication of a technical manual. Studies completed for the 

purposes of method approval (via publication in a final ruling) in the Federal Register may fulfill 

this requirement by inclusion in the docket for the proposal of the method. 

The report should identify the participant laboratories and describe all test materials 

together with how they were collected and/or prepared. It should also describe all reagents, 

spiking materials and reference standards used in the study, including their commercial or 

organizational sources, if applicable. If any of these materials were custom-prepared for the 

study, the report should include a description of how they were prepared and documentation of 

2-17 
their homogeneity. In many instances (e.g., when submitting for peer-reviewed publication), the 

report will also provide a summary of the study design, procedures and statistical methods used 

for analyses of the results. 

A critical component of the report is a presentation and discussion of the numerical and 

descriptive specifications for the performance attributes of the methods that were determined 

from the collaborative study. These specifications summarize the performance of the method 

under the specified conditions of the study and are the primary source of information for 

determining the suitability of the method for its intended purpose in the context of widespread 

use. The report may also include the development of quantitative QC criteria for initial and 

ongoing method/laboratory performance assessments. Lastly, if possible, a statistical assessment 

of the method’s comparability with any available reference method should be provided. Criteria 

and guidelines for the establishment of equivalence between microbiological test methods have 

been developed (ISO 2004). 

2-18 
3.0 REFERENCES 

AOAC. 1989. Guidelines for collaborative study of procedure to validate characteristics of a 

method of analysis. J. Assoc. Off. Anal. Chem. 72, 694–704. 

AOAC. 2006. AOAC Task Force Recommends Best Practices for Validation of Microbiological 

Methods; Final Report Submitted. Inside Laboratory Management. November/December 

p 10. 

American Public Health Association (APHA) 1998. Standard Methods for the Examination of 

Water and Wastewater (20th Ed,) 922B.6.c. 

APHA, 2005. Part 9020. Quality Assurance/Quality Control. In Standard Methods for the 

Examination of Water and Waste Water, 

http://standardmethods.org/store/BrowseSM.cfm. 

ASTM, Designation E691-99, “Standard Practice for Conducting an Interlaboratory Study to 

Determine the Precision of a Test Method,” Annual Book of ASTM Standards, Vol. 14.02, 

ASTM, West Conshohocken, PA, 2001, Section 17.1. 

Centers for Disease Control and Prevention and National Institutes of Health, 2007. Biosafety in 

Microbiological and Biomedical Laboratories, 5th edition. U.S. Department of Health and 

Human Services. U.S. Government Printing Office, Washington, D.C. 

Deep, R. 2006. Probability and Statistics. Academic Press, New York. 

Dols, T and B. Ambrecht, 1976. Simplex optimization as a step in method development. J. 

Assoc. Off. Anal. Chem. 59: 1204-1207. 

Fleming, D.O. and Hunt, D.L. eds. 2000. Biological Safety: Principles and Practices. ASM Press, 

Washington, D.C. 

Green, J.M. 1996. A Practical Guide to Analytical Method Validation. Analytical Chemistry 

68:305A-309A. 

Holmes H, C. Davis, A. Heath, I. Hewlett, N. Lelie. 2001. An international collaborative study to 

establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based 

techniques. J. Virol. Methods 92:141-150. 

ICH. 1996. Validation of analytical procedures: Methodology ICH-Q2B. International 

Conference on Harmonization of Technical Requirements for the Registration of 

Pharmaceuticals for Human Use. 

International Organization for Standardization. 1994a. ISO 5725-1:1994. Accuracy (trueness and 

precision) of measurement methods and results - Part 1: General principles and 

definitions. Geneva, Switzerland. 

3-1 
International Organization for Standardization. 1994b. ISO 5725-2:1994. Accuracy (trueness and 

precision) of measurement methods and results - Part 2: Basic method for the 

determination of repeatability and reproducibility of a standard measurement method. 

Geneva, Switzerland. 

International Organization for Standardization. 1994c. ISO 5725-3:1994. Accuracy (trueness and 

precision) of measurement methods and results - Part 3: Intermediate measures of the 

precision of a standard measurement method. Geneva, Switzerland. 

International Organization for Standardization. 2000. ISO/TR 13843:2000(E). Water quality – 

Guidance on validation of microbiological methods. Geneva, Switzerland. 

International Organization for Standardization. 2004. ISO 17994:2004. Water quality - Criteria 

for establishing equivalence between microbiological methods. Geneva, Switzerland. 

McClure F.D. 1990. Design and analysis of Qualitative Collaborative Studies: Minimum 

Collaborative Program, J. Assoc. Off. Anal. Chem. Vol. 73, No. 6. 

Niemela, S. I. 2003. Uncertainty of quantitative determinations derived by cultivation of 

microorganisms. Centre for Metrology and Accreditation, Helsinki, Finland. 

USEPA. 1992. Consensus Method for Determining Groundwaters Under the Direct Influence of 

Surface Water Using Microscopic Particulate analysis (MPA). EPA 910/9-92-029. 

USEPA. 1996. Guide to Method Flexibility and Approval of EPA Water Methods. 

EPA 821D96004. 

USEPA. 2004. Quality Assurance/Quality Control Guidance for Laboratories Performing PCR 

Analyses on Environmental Samples EPA 815-B-04-001. United States Environmental 

Protection Agency. 

USEPA. 2005. Manual for the Certification of laboratories Analyzing Drinking Water. EPA 815­

R-05-004. 

US FDA, Center for Drug Evaluation and Research (CDER). 2001. Guidance for industry. 

Bioanalytical method validation. http://www.fda.gov/cder/guidance/index.htm 

Youden and Steiner, 1975. Statistical Manual of the Association of the AOAC, Association of 

Official Analytical Chemists. Arlington, VA. 

3-2 
Appendix A – Environmental Monitoring Management Council (EMMC) 

Format for Writing a Method 
The components of the Environmental Monitoring Management Council (EMMC) format 

for writing a method are detailed below: 

1.0 Scope and Application 

Include the matrices to which the method applies, a generic description of method 

sensitivity and the data quality objectives which the method is designed to meet. Much of this 

material may be presented in a tabular format. 

2.0 Summary of Method 

Summarize the method in a few paragraphs. The purpose of the summary is to provide a 

succinct overview of the technique to aid the reviewer or data user in evaluating the method and 

the data. List sample volume, concentration, other preparation steps employed, the analytical 

instrumentation, and the techniques used for quantitative determinations. 

3.0 Definitions of Method 

Include the definitions of all method-specific terms here. For extensive lists of 

definitions, this section may simply refer to a glossary attached at the end of the method 

document. 

4.0 Interferences 

This section should discuss any known interferences, especially those that are specific to 

the performance-based method. If known interferences in the reference method are not 

interferences in the performance-based method, this should be clearly stated. 

5.0 Safety 

Χ Above and beyond good laboratory practices 

Χ Disclaimer statement (look at ASTM disclaimer) 

Χ Special precautions 

Χ Specific toxicity of reagents 

Χ Not appropriate for general safety statements 

A-1 
This section should discuss only those safety issues specific to the method and beyond 

the scope of routine laboratory practices. Reagents that pose specific toxicity or safety issues 

should be addressed in this section. 

6.0 Equipment and Supplies 

Use generic language wherever possible. However, for specific equipment do not assume 

equivalency of equipment that was not specifically evaluated, and clearly state what equipment 

and supplies were tested (i.e. incubators, water baths). 

7.0 Reagents and Standards 

Provide sufficient details on the concentration and preparation of reagents and standards 

to allow the work to be duplicated, but avoid lengthy discussions of common procedures. 

8.0 Sample Collection, Preservation and Storage 

Provide information on sample collection, preservation, shipment, and storage conditions. 

9.0 Quality Control 

Describe specific quality control steps, including such procedures as laboratory control 

samples, QC check samples, instrument checks, etc., defining all terms in Section 2.0. 

10.0 Calibration and Standardization 

Discuss initial calibration procedures here. Indicate frequency of such calibrations, refer 

to performance specifications, and indicate corrective actions that must be taken when 

performance specifications are not met. This Section may also include procedures for calibration 

verification or continuing calibration. 

11.0 Analytical Procedure 

Provide a general description of the sample processing and instrumental analysis steps. 

Discuss those steps essential to the process, and avoid unnecessarily restrictive instructions. 

A-2 
12.0 Calculations and Data Analysis 

Describe qualitative and quantitative aspects of the method. List identification criteria 

used. Provide equations used to derive final sample results from typical instrument data. Provide 

discussion of estimating detection limits, if appropriate. 

13.0 Method Performance 

A precision/bias statement should be incorporated in the Section, including: 

Χ Detection limits 

Χ Source/limitations of data 

Provide detailed description of method performance, including data on precision, bias, 

detection limits (including the method by which they were determined and matrices to which 

they apply), statistical procedures used to develop performance specification, etc. Where 

performance is tested relative to the reference method, provide a side-by-side comparison of 

performance versus reference method specifications. 

14.0 Pollution Prevention 

Describe aspects of this method that minimize reagents or prevent pollution that may be 

attributable to the reference method. 

15.0 Waste Management 

Cite how waste and samples are minimized and properly disposed. 

16.0 References 

17.0 Tables, Diagrams, Flowcharts, and Validation Data 

A-3 
Appendix B – Interlaboratory Collaborative Study 
Interlaboratory Collaborative Study 

Validation Protocol for the Quantitative Three Step Method 

Prepared by: 

Stephen F. Tomasino, Ph.D., Study Director 

Rebecca M. Fiumara, Co-Study Director 

U.S. Environmental Protection Agency 

Office of Pesticide Programs Microbiology Laboratory 

Environmental Science Center 

701 Mapes Road 

Fort Meade, Maryland 20755-5350 

Study Director Phone: 410-305-2976 

FAX: 410-305-3094 

Email: tomasino.stephen@epa.gov 

CONTROL COPY #: 7.20.06 (final) 

1 
SECTION PAGE

1. INTRODUCTION 3 

2. COLLABORATORS 5 

3. STUDY DESIGN 7 

4. TEST SAMPLE PREPARATION 11 

5. QUALITY ASSURANCE 12 

6. REFERENCE METHOD – Revised AOAC Method 966.04 14 

7. QUANTITATIVE THREE STEP METHOD 23 

8. REPORTING RAW DATA 36 

9. STATISTICAL ANALYSIS 36 

10. REFERENCES 39 

11. LIST OF APPENDICES 40 

A. EPA Quality Assurance Project Plan 

B. AOAC Method Data Sheets 

C. TSM Data Sheets 

D. Media and Reagent Preparation Sheets 

E. Parameters for Testing Chemicals 

F. TSM Log Reduction Plan 

G. Material Safety Data Sheets (MSDS)

 H. Safety Checklist 

TABLE OF CONTENTS 

2 
Section 1. INTRODUCTION 

In response to the intentional release of spores of Bacillus anthracis in 2001, and the 

associated need for determining the performance of sporicidal and sterilant chemicals for use in 

the decontamination of buildings, the U.S. Environmental Protection Agency (EPA) Office of 

Pesticide Programs (OPP) initiated a research program to evaluate and improve laboratory 

efficacy test methods for sporicides and sterilants. Due to limitations and deficiencies associated 

with the Agency's reference method for efficacy testing, AOAC Method 966.04, a key priority 

related to EPA's Homeland Security efforts has been the development of a quantitative 

methodology to replace or augment Method 966.04, with emphasis on the regulatory aspects of 

verifying the performance of chemicals used in building decontamination. The most significant 

concerns associated with Method 966.04 are the qualitative nature of the method, the use of raw 

garden soil extract as a source of minerals for spore production, the carrier materials (unglazed 

porcelain and silk suture loops), the lack of a standardized procedure for enumeration of spores, 

spore wash-off, and the long incubation time (21 days). Several of the deficiencies were recently 

addressed by EPA in a collaborative study to modify the AOAC method. 

In a pre-collaborative study, two quantitative carrier-based test methods for determining 

the efficacy of liquid sporicides and sterilants on a hard surface, the Standard Quantitative 

Carrier Test Method – American Society for Testing and Materials (ASTM) E 2111-00 and an 

adaptation of a quantitative micro-method (the Three Step Method) as reported by Sagripanti and 

Bonifacino, were compared (1). By conducting the study in a comparative, standardized manner 

across multiple laboratories, valuable statistical information on the performance of two 

quantitative methods was generated. Based on the statistical parameters measured and the 

additional test method attributes related to logistics and human resources, the Three Step Method 

(TSM) was determined to be the more suitable method, and will be advanced to the AOAC 

validation process. The flexibility in the TSM for incorporating coupon materials other than 

glass was considered as an advantage, and that the practice time and training issues (identified by 

analysts who performed the method) could be resolved through the re-organization of the 

protocol and the use of a flowchart and process checklist. As the development and use of 

quantitative test methods for sporicides and sterilants proceeds, additional studies will be 

necessary to develop meaningful and relevant performance standards (i.e., pass/fail criteria with 

a minimum LR). 

Although the details of the method have been published, the TSM is considered a new 

method (i.e., for the purpose of this initiative) with a limited amount of historical use in the 

regulatory arena. The TSM is a quantitative procedure for hard surfaces – considered as a 

significant advantage over qualitative procedures and suspension-based tests. The TSM uses 5 × 

5 × 1 mm glass coupons to deliver spores into the sporicidal agent (400 μL) contained in 1.5 mL 

microcentrifuge tubes, 3 coupons per chemical treatment. Following exposure to the test 

chemical and neutralization, spores are removed from the carriers in three fractions by sonication 

and vortex-mixing. Liquid from each fraction is plated on recovery medium for viable spore 

enumeration. Control counts (water control) are compared to the treated counts and the level of 

efficacy is determined by calculating the Log10 reduction (LR) of spores; LR = log10 (mean 

3 
spores/control carrier) - log10 (mean spores/treated carrier). The original procedures for the TSM 

were reported by Sagripanti and Bonifacino (2). However, during the course of the precollaborative studies, EPA refined and altered the method slightly, and finalized the protocol for 

the purpose of this validation study. It should be noted that following the completion of the precollaborative study, a similar version of the TSM was accepted and published by ASTM 

International (3). 

The purpose of this Interlaboratory Collaborative Study (CS) is to evaluate the TSM 

according to AOACI Official Methods of Analysis (OMA) procedures for official method 

validation. The applicability will be limited to one spore-forming microorganism (Bacillus 

subtilis), one hard surface (glass), liquid formulations of sporicides and sterilants, and without 

organic burden added to the spore inoculum. The test chemicals used in the CS represent three 

chemical classes of sporicides: sodium hypochlorite, a combination of peracetic acid and 

hydrogen peroxide, and glutaradehyde. The suitability of the TSM for porous materials and 

gaseous formulations will require additional collaborative studies. If the TSM is validated for B. 

subtilis, the Study Director will propose language in the validation report to allow the testing of 

other Bacillus species, such as B. anthracis, with the TSM. 

The overall objective of the CS is to evaluate the performance of the TSM by generating 

and comparing control counts and efficacy data and assessing the degree and source(s) of 

variability associated with the data, both within and between laboratories, when the method is 

used in actual practice. AOAC Method 966.04, Sporicidal Activity of Disinfectants, is 

recognized as the reference method. Method 966.04 provides a qualitative measure of product 

efficacy against spores of B. subtilis and Clostridium sporogenes dried on two types of carriers, 

porcelain penicylinders and silk suture loops. Sixty-carrier tests on three lots of product are 

required for an EPA registration – all carriers must show no growth to support a sporicidal claim. 

For the purpose of this CS, only the Bacillus and hard surface (porcelain penicylinders) 

components of Method 966.04 will be evaluated. The comparative testing with the reference 

method is problematic due to its qualitative nature. In this study, chemical treatments from one 

of three replications per product will be tested using both the reference method and TSM. The 

cost of conducting Method 966.04 is a limiting factor and it would not be feasible to generate 

enough inoculated porcelain carriers to do each treatment and replication. 

Prior to initiation of this collaborative effort, the AOAC INTERNATIONAL will 

assemble a review panel, the AOAC Sporicidal Method Expert Review Panel (ERP), to evaluate 

the CS protocol. The ERP, along with members of the AOAC INTERNATIONAL Methods 

Committee on Microbiology, will be engaged early in the development of the study design to 

ensure the protocol format and test design are acceptable and meet the goals established for the 

AOACI Official Methods process. 

4 
Section 2. COLLABORATORS 

A total of eight to eleven laboratories will participate in this study. The laboratories 

selected will have existing microbiology programs, appropriately trained personnel, and have the 

capability of conducting the validation protocol within the timeframe established by the Study 

Director. The OPP Microbiology Laboratory is the lead laboratory. Two Food and Drug 

Administration (FDA) laboratories are currently collaborating with EPA per an Interagency 

Agreement (IAG) and will also participate in the study. A third FDA laboratory is expected to 

participate under the IAG. At least five laboratories who have not been involved with the 

development or evaluation of the TSM will be included. The name and associated contact 

person for each participating laboratory are provided in Table 1. 

Table 1. List of collaborators 

Lab Number Laboratory Name Contact Information 

1 U.S. EPA

 OPP Microbiology Laboratory 

Stephen Tomasino, Ph.D., Study Director 

Rebecca Fiumara, Co-Study Director 

U.S. EPA 

Environmental Science Center 

701 Mapes Road, Ft. Meade, MD 20755-5350 

Phone for S. Tomasino: 410-305-2976 

Phone for R. Fiumara: 410-305-2635 

FAX: 410-305-3094 

Email: tomasino.stephen@epa.gov 

2 U.S. FDA 

Denver District Laboratory 

Elizabeth Gonzales 

U.S. FDA

 Denver Federal Center 

6th Avenue and Kipling Street, Building 20 

Denver, CO 80225-0087 

Phone: 303-236-9608 

Fax: 303-236-3100 

Email: egonzale@ora.fda.gov 

3 

U.S. FDA 

Winchester Engineering and 

Analytical Center (WEAC) 

Allison Rodriguez 

U.S. FDA 

Winchester Engineering and Analytical Center 

109 Holton Street, Winchester, MA 01890 

Phone: 781-729-5700 

Fax: 781-729-3593 

Email: arodrigu@ora.fda.gov 

4 

U.S. FDA 

Office of Science and 

Engineering Laboratory (OSEL) 

- White Oak Campus 

Victoria Hitchins, Ph.D. 

U.S. FDA 

Office of Science and Engineering Laboratories 

Center for Devices and Radiological Health 

10903 New Hampshire Avenue 

Silver Spring, MD 20993 

Phone: 301-796-0258 

Email: victoria.hitchins@fda.hhs.gov 

5 
5 Advanced Sterilization 

Microbiology Laboratory 

Harriet Chan-Myers 

Advanced Sterilization Microbiology Laboratory 

33 Technology Drive, Irvine, CA 92618 

Phone: 949-453-6330 

6 STERIS Corporation 

Dan Klein 

STERIS Corporation 

PO Box 147, Saint Louis, MO 63166-0647 

Phone: 314-290-4777 

Fax: 314-725-5687 

Email: Daniel_Klein@steris.com 

7 MicroBioTest 

Donna B. Suchmann 

MicroBioTest Inc., Unit B 

105 Carpenter Dr, Sterling, VA 20164 

Work Phone: 703-925-0100 

Fax: 703-925-9366 

8 ATS Labs 

Dave Rottjakob/Doug Anderson 

ATS Laboratories 

1285 Corporate Center Drive, Suite 110 

Saint Paul, MN 55121 

Tel. +1651.379.5510 

Email: dave.rottjakob@ats-labs.com 

9 Bioscience Labs 

Daryl Paulson 

BioScience Laboratories Inc. 

300 N. Wilson Ave, Suite 1, Bozeman, MT 59715 

Phone (406) 587-5735 

Fax (406) 586-7930 

Email: dpaulson@biosciencelabs.com 

10 Ohio Department of Agriculture 

Jim Agin 

Ohio Department of Agriculture 

Bldg 3, Consumer Analytical Lab 

8995 E Main St, Reynoldsburg, OH 43068 

Work Phone: 614-728-0198 

Fax: (614) 728-6322 

Email: agin@odant.agri.state.oh.us 

6 
Section 3. STUDY DESIGN 

A.	 The Study Director, Dr. Stephen Tomasino, is responsible for organizing the CS and 

assessing the preparedness of each collaborating laboratory prior to initiation of research. 

Toward that effort, the Study Director and the OPP Microbiology Laboratory Quality 

Assurance Unit will conduct a readiness review of each participating laboratory to ensure 

compliance with EPA's Quality Assurance Project Plan 2003-01 (Appendix A). The CoStudy Director, Rebecca Fiumara, will serve as the technical lead for the TSM. 

B.	 The method protocols, standardized test forms and data sheets (Appendices B and C), 

media preparation sheets (Appendix D), selected media and reagents, test chemicals, and 

inoculated porcelain penicylinders will be provided by the Study Director. Test 

parameters for each chemical treatment describing the conditions for testing (e.g., 

dilution, neutralizer, contact time, temperature, etc.) will be provided to each laboratory 

by the Study Director (Appendix E). 

C.	 The Study Director recognizes that it is desirable to distribute the chemical treatments to 

the testing laboratories in containers that are marked only with a treatment code. 

However, due to the instability of test chemicals such as diluted sodium hypochlorite, it 

will be necessary for each lab to prepare the actual test formulations (i.e., perform 

dilutions) on-site. The test chemicals will be provided by the Study Director. The test 

scheme will be randomized to account for potential subjectivity by the analysts. 

D.	 In order to have a balanced design that will be conducive to a straight-forward statistical 

analysis, the labs will be asked to perform the same number of tests per day. The study 

design calls for testing three chemicals, three levels each (high, medium and low), one 

chemical per day. Three replications are required. Assuming no repeat testing, 9 test 

days will be required to complete the CS. Water controls (control carrier counts) will be 

included each test day for the TSM. Each treatment will be evaluated by the TSM; 

however, Method 966.04 will be conducted concurrently on the first replication only for a 

total of nine 30-carrier AOAC tests per laboratory. 

E.	 Testing must be initiated within 4 weeks after the readiness review, practice/training and 

receiving supplies, and must be completed within 12-14 weeks after initiation of the first 

test. 

F.	 Two or three technicians will be required per test day for approximately 6 hours. Each 

laboratory will be encouraged to establish a technician team which will conduct all tests, 

i.e., it is important to design the study so that the differences among technician teams do 

not affect the outcome. If the same technician team always conducted the testing, the 

technician effect will not confound the results. Practice runs will be encouraged in 

advance of testing to ensure analyst proficiency in performing each method. 

7 
G.	 It is important to randomize any steps in which subjective decisions or unknown factors 

could affect the conclusions. The experimental results could be criticized because the 

order of experimentation was systematic; any systematic trend in environmental 

conditions would affect the results. It is preferable to have a numbering system or 

positioning arrangement that gives each item a unique identification. In this study, the 

Study Director will provide the randomized order of testing of chemicals for each lab 

using an acceptable method of randomization. The randomization will be done before the 

experimentation is initiated. 

H.	 Test chemicals used in the study are: 

1.	 Sodium hypochlorite (reagent grade, Sigma-Aldrich sodium hypochlorite 

solution, 12% available chlorine) 

2.	 A combination of peracetic acid and hydrogen peroxide (Spor-Klenz Ready to 

Use, an EPA-registered commercial sporicide) 

3.	 2.6% glutaraldehyde (Metricide 14-Day, a commercially available sterilant). 

For the purpose of this CS, the test chemicals are experimental components only and are 

not being tested to support or verify product label claims. Each chemical will be tested at 

three levels: high (efficacious), medium (moderate efficacy) and low (non-efficacious), to 

provide a range of efficacy. The test conditions used to generate the range are shown in 

Table 2. Efficacy data generated by EPA to support the test conditions are presented in 

Appendix F. 

I.	 Table 2. Test chemicals and conditions for testing 

Test Chemical 

Treatment Level and Test Parameters* 

High (LR ≥ 6) Medium (LR 2-6) Low (LR 0-2) 

1) Sodium hypochlorite ▪6000 ± 300ppm 

▪adjusted pH (7±0.5) 

▪30 ± 1 min 

▪6000 ± 300ppm 

▪unadjusted pH 

▪10 min ± 10 sec 

▪3000 ± 300ppm 

▪unadjusted pH 

▪10 min ± 10 sec 

2) 0.08% peracetic acid and 

1.0% hydrogen peroxide ▪30 ± 1 min ▪10 min ± 10 sec ▪1 min ± 5 sec 

3) 2.6% glutaraldehyde ▪180 ± 3 min ▪60 ± 1 min ▪10 min ± 10 sec 

*Test conditions expected to generate three levels of efficacy when tested with the TSM. 

J.	 Test chemicals and the Material Safety Data Sheets (Appendix G) will be provided to 

each laboratory by the Study Director. For sodium hypochlorite treatments, each 

laboratory will prepare the diluted test chemical from a reagent-grade product per the 

preparation sheets provided by the Study Director – HACH Test Kits will be used to 

verify available chlorine for diluted samples. The Study Director will request the use of a 

single lot for each test chemical. In advance of testing, an analysis of formulation 

chemistry will be performed on each lot (one container) by OPP chemists to confirm the 

percent active ingredient. 

8 
K.	 The test microbe is Bacillus subtilis (ATCC # 19659) obtained directly from a reputable 

supplier (e.g., ATCC). Each lab will initiate a new stock culture. Note: An existing 

stock culture may be used if it meets the quality control standards (e.g., proper 

documentation, confirmation testing) – Study Director approval is required in this case. 

L.	 Presque Isle Cultures, 3804 West Lake Rd, Erie, PA 16505 will provide inoculated 

porcelain carriers (approx. 4,000 total for the entire study) to each laboratory per the 

modified methodology for AOAC Method 966.04. The vendor, per instructions provided 

by the Study Director, will follow the revised method (i.e., the use of nutrient agar 

amended with manganese sulfate) for generation of spore suspensions and inoculation of 

carriers. No organic burden will be added to the spore inoculum. Carrier counts and HCl 

resistance will be determined by the vendor, and must meet AOAC method 

specifications, in advance of shipping the carriers to the collaborative laboratories. A 

minimum of 1.0 × 105

 (log10 density = 5.0) and a maximum of approximately 1.0 × 106 

spores/carrier will be required. Multiple lots of inoculated carriers are anticipated. 

M.	 Spores for use in the TSM will be produced by each collaborating laboratory per the 

method provided. The mean target carrier load for the TSM is 1.0 × 107 spores/carrier or 

7.0 logs per carrier – a level suitable for measuring a log reduction of ≥ 6 logs. No 

organic burden will be added to the spore inoculum. Carriers will be inoculated from one 

spore preparation per laboratory; enough carriers will be inoculated to perform the entire 

study (approx. 150). 

N.	 The basic CS design: 

o 8-11 laboratories 

o One microbe 

o Three chemicals, each with three levels of efficacy 

o Water control for TSM (control carriers) 

o Three replications for TSM, one replication for AOAC method 

o One carrier type for the TSM (glass) 

o One carrier type for the AOAC method (porcelain) 

o TSM uses 3 carriers per treatment 

o Each AOAC test will use 30 carriers 

o Target carrier counts established for each method 

O. Example of a test scheme for one laboratory with test chemicals randomized for three 

replications – see Table 3. 

9 
Table 3. Example of the test scheme 

Replication Treatment and Levels Test Method Performed 

1. Sodium Hypochlorite TSM* AOAC 966.04** 

Rep 1 (Day 1) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

No 

2. Peracetic acid/hydrogen peroxide TSM AOAC 966.04 

Rep 1 (Day 2) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

No 

3. Glutaraldehyde TSM AOAC 966.04 

Rep 1 (Day 3) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

No 

2. Peracetic acid/hydrogen peroxide TSM AOAC 966.04 

Rep 2 (Day 4) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

No 

No 

No 

No 

3. Glutaraldehyde TSM AOAC 966.04 

Rep 2 (Day 5) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

No 

No 

No 

No 

1. Sodium Hypochlorite TSM AOAC 966.04 

Rep 2 (Day 6) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

No 

No 

No 

No 

Rep 3 (Day 7) 3. Glutaraldehyde TSM AOAC 966.04 

10 
 

 

 

Table 3. Example of the test scheme (Continued) 

Replication Treatment and Levels Test Method Performed 

1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

No 

No 

No 

No 

1. Sodium Hypochlorite TSM AOAC 966.04 

Rep 3 (Day 8) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

No 

No 

No 

No 

2. Peracetic acid/hydrogen peroxide TSM AOAC 966.04 

Rep 3 (Day 9) 1. High 

2. Medium 

3. Low 

4. Water Control 

Yes 

Yes 

Yes 

Yes 

No 

No 

No 

No 

*Three carriers per treatment; **30-carriers per treatment 

P. Table 4. Randomized Order of Testing (based on 10 labs) 

Rep* Random Order of Test Chemicals** 

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 

Rep 1 2, 3, 1 1, 3, 2 2, 3, 1 1, 2, 3 2, 3, 1 2, 1, 3 1, 2, 3 2, 3, 1 1, 2, 3 3, 2, 1 

Rep 2 1, 3, 2 3, 1, 2 2, 3, 1 3, 2, 1 1, 2, 3 1, 3, 2 3, 2, 1 1, 2, 3 1, 3, 2 1, 2, 3 

Rep 3 2, 3, 1 2, 3, 1 3, 2, 1 2, 3, 1 1, 3, 2 3, 2, 1 1, 2, 3 3, 2, 1 3, 2, 1 2, 3, 1 

*Three total tests days per replication; one chemical class tested per day 

**1 = sodium hypochlorite, 2 = hydrogen peroxide/peracetic acid, and 3 = glutaraldehyde; order within a 

 test day will be High, Medium, Low, and Water Control 

Section 4. Test Sample Preparation 

Prepare test chemicals as follows: 

1. Sodium hypochlorite (Sigma Aldrich reagent grade solution, approx. 12% available 

chlorine, product no. 425044), diluted to 6000 ± 300ppm with reagent grade water, pH 

adjusted (pH 7.0 ± 0.5) with 5% acetic acid. pH adjusted treatment must be used in test 

within approximately 120 min after preparation; used for the high treatment. 

2. Sodium hypochlorite (Sigma Aldrich reagent grade solution), diluted to 6000 ± 300ppm 

with reagent grade water, unadjusted pH (pH ~10.0). Test within approximately 120 min 

after preparation; used for medium treatment. 

11 
3. Sodium hypochlorite (Sigma Aldrich reagent grade solution), diluted to 3000 ± 300ppm 

with reagent grade water, unadjusted pH (pH ~10.0). Test within approximately 120 min 

after preparation; used for low treatment. 

4. 0.08% peracetic acid/1.0% hydrogen peroxide product; ready to use product, must initiate 

testing within approximately 3 hr after dispensing. 

5. 2.6% glutaraldehyde product, activate according to product directions, 14-day shelf-life 

post activation. The activated product may used for testing during the 14-day period. 

Section 5. Quality Assurance 

The sponsor of this project is the EPA Office of Research and Development. Document 

archiving will be adequate to ensure that all studies are supported by complete, accurate, 

consistent, and chronological records from initial collection of raw data to final analysis 

interpretation and reporting of results. 

The preparedness of each laboratory will be assessed by the Study Director and Quality 

Assurance Unit (QAU) for each laboratory prior to initiation of the study to ensure compliance 

with a project-specific EPA Quality Assurance Project Plan (Appendix A). The expected level 

of quality assurance is consistent with EPA Good Laboratory Practices. Numerous guidance 

documents, standard methods and Standard Operating Procedures (SOPs) will be used to 

maintain data quality. Proper record keeping and archiving will be performed to ensure the 

defensibility and reconstructibility or reanalysis of the study. No specific certification is required 

for this study; however, staff performing the assays must be familiar with standard 

microbiological techniques such as aseptic transfer, serial dilutions, plate counts and microbe 

identification. Scientists and analysts involved in testing shall be familiar with each efficacy 

method and associated procedure (e.g., carrier counts, neutralization confirmation) and will be 

proficient in conducting each designated efficacy test method. 

In-house practice sessions will be required for each laboratory to build proficiency with 

each method prior to official testing. The Lead laboratory (OPP Microbiology Laboratory) will 

conduct a series of conference calls with the participating laboratories to discuss the details of 

each method protocol. Documentation of practice and training for projects will be maintained in 

a training file for each participating scientist or analyst. 

Research documentation shall include project identification, data, and researcher. Preprinted forms will be used. Research documentation shall be in ink and the use of a single line 

for correcting entries with the date and initials of the person making the correction and the reason 

for the change. Project-specific documentation shall be maintained in a project file, or the 

project file must identify where the documents are stored. Where possible, EPA SOPs and 

standard forms shall be used for those operations which have become or will become routine, 

including test methodology, analytical procedures and calibration procedures. SOPs that are 

comparable to EPA’s may be used; however, this will require concurrence by the Study Director 

or the EPA Quality Assurance Unit. The purpose of the SOPs is to facilitate the uniform 

12 
performance of routine procedures. The Standard Operating Procedures for quality control 

activities are located at: http://www.epa.gov/oppbead1/methods/atmpa2z.htm. Selected 

electronic spreadsheets and email will be considered as official documentation and will be 

maintained and archived accordingly. 

All preparations of test chemicals, media and reagents will be tracked using an assigned 

media preparation number. Samples, test chemicals, will be maintained to ensure their integrity. 

Test chemicals will be stored away from standards, media, and reagents to prevent crosscontamination. No official chain of custody documentation will be required for test chemicals 

evaluated in this research; however specific information on source, identification, and volume 

received will be maintained and archived for all test chemicals. All supplies and materials 

considered “critical” to the quality of the research such as media, reagents, carriers, and test 

chemicals shall be inspected prior to use to ensure that the shipment has not been damaged or 

compromised in any way. For pre-sterilized lab supplies, the manufacturer’s statement of 

sterility is acceptable for quality control documentation for sterility; no further testing is 

required. For growth media, performance testing (sterility and suitability to support growth of B. 

subtilis) must be performed a minimum of one time, preferably on the first batch prepared per 

lot. 

Suppliers (vendors) of testing materials and components with specific requirements such 

as sodium hypochlorite and inoculated carriers will be requested to provide verification of the 

desired specifications. The information and data will be maintained and archived in the project 

file. 

Upon completion of each study, a peer review of the data entry/tabulation will be 

performed by laboratory personnel. A draft report of the findings or data summary will be 

compiled and forwarded to each lab’s Quality Assurance personnel for review. The designated 

QAO at each facility will review and comment on the data and supporting information before 

submission to the statistician. Critical findings will be immediately communicated to the Study 

Director. Data may be rejected if microbial contamination occurs at a level unacceptable to the 

Study Director. 

13 
Section 6. The Reference Method 

Note: Laboratories will only conduct the efficacy component (see C. g) 

AOAC Official Method 966.04; Sporicidal Activity of Disinfectants 

Modified Method 

First Action 2006 

Applicable to testing sporicidal activity of liquid disinfectants using modified method 

966.04 against Bacillus subtilis on a hard surface (porcelain carrier). Performance criteria for 

product efficacy are not impacted. This method has been validated for products containing 

sodium hypochlorite, peracetic acid/hydrogen peroxide, and glutaraldehyde. See results of the 

collaborative study supporting the modifications to Method 966.04. 

(1) All manipulations of the test organism are required to be performed in accordance 

with biosafety practices stipulated in the institutional biosafety regulations. Use the equipment 

and facilities indicated for the test organism. For recommendations on safe handling of 

microorganisms refer to the Centers for Disease Control/National Institutes of Health 

(CDC/NIH) Biosafety in Microbiological and Biomedical Laboratories manual. (2) Disinfectants 

may contain a number of different active ingredients, such as heavy metals, aldehydes, 

peroxides, and phenol. Personal protective clothing or devices are recommended during the 

handling of these items for purpose of activation, dilution, or efficacy testing. A chemical fume 

hood or other containment equipment may be employed when appropriate during performing 

tasks with concentrated products. The study analyst may wish to consult the Material Safety 

Data Sheet for the specific product/active ingredient to determine best course of action. (3) 

References to water mean reagent-grade water, except where otherwise specified. (4) 

Commercial dehydrated media made to conform to the specified recipes may be substituted. (5) 

These microbiological methods are very technique sensitive and technique-oriented, thus exact 

adherence to the method, good laboratory practices, and quality control (QC) are required for 

proficiency and validity of the results. (6) Detergents used in washing glassware may leave 

residues which are bacteriostatic. Test for inhibitory residues on glassware periodically. For 

procedure, refer to Standard Methods for the Examination of Water and Wastewater, Section 

9020, Quality Assurance/Quality Control. 

A. Media and Reagents 

(a) Culture Media.–(1) Nutrient broth.–For use in preparing nutrient agar. Add 5 g beef extract 

(paste or powder), 5 g NaCl, and 10 g peptone (anatone, peptic hydrolysate of pork tissues, 

manufactured by American Laboratories, Inc., 4410 S 102nd St., Omaha, NE 68127) to 

approximately 1 L water. Boil mixture for 20 minutes with constant stirring. Readjust volume 

to 1 L with water and allow cooling to around 50oC. Adjust pH to 6.8 ± 0.2 with 1N HCL or 1N 

NaOH, if necessary. Filter through paper (e.g., Whatman filter paper No. 4). Dispense 10 mL 

portions into 20 × 150 mm culture tubes or 20 mL portions into 25 × 150 mm culture tubes. 

14 
Dehydrated nutrient broth may be substituted – prepare according to the manufacturer's 

instructions. (2) Nutrient agar.–For stock cultures slants. Add 1.5% (w/v) Bacto-agar to 

unsterilized nutrient broth. Boil mixture until agar is dissolved. Adjust pH to 7.2 ± 0.2 if 

necessary. Dispense 5 mL portions into 16 x 100 mm screw cap tubes. Larger tubes may be 

used as well. Autoclave for 20 minutes at 121oC. Remove from autoclave and slant tubes to 

form agar slopes. (3) Nutrient agar with 5μg/mL MnSO4:H20 (amended nutrient agar).–For 

spore production. Suspend 11.5 g nutrient agar in 495 mL water, add 5 mL 500 ppm 

MnSO4:H20. Dissolve by boiling. Adjust pH to 6.8 ± 0.2 if necessary. Autoclave for 15 

minutes at 121oC. Pour agar into plates. (4) Trypticase soy agar.–Suspend 40 g dehydrated 

trypticase soy agar in 1 L water and heat gently while stirring. Boil one minute or until 

completely dissolved. Adjust pH to 7.3 ± 0.2. Autoclave 15 minutes at 121oC. Pour agar into 

plates. (5) Fluid thioglycollate medium (FTM).–Suspend 29.5 g of dehydrated fluid 

thioglycollate medium in 1 L water. Heat to boiling to dissolve completely. Adjust pH to 7.1 ± 

0.2 if necessary. Dispense 10 mL portions into 20 ×150 mm culture tubes and autoclave for 15 

minutes at 121oC. Store at room temperature. Protect from light. Note: If after autoclaving the 

aerated portion of media consumes more than one third of tube, media must be re-boiled by 

placing tubes in beaker of boiling water. Media can only be re-boiled once. (6) Fluid 

thioglycollate medium with 1M NaOH (modified FTM).– For subculturing spores exposed to 2.5 

M HCl. Suspend 29.5 g of fluid thioglycollate medium in 1 L water. Heat boiling to dissolve 

completely. Cool and adjust pH to 7.1 ± 0.2 if necessary. Add 20 mL 1M NaOH, mix well. 

Check final pH and record (pH between 8 and 9 is typical). Dispense 10 mL into 20 × 150 mm 

culture tubes and autoclave for 15 minutes at 121oC. Store at room temperature. Protect from 

light. Note: If after autoclaving the aerated portion of media consumes more than one third of 

tube, media must be re-boiled by placing tubes in beaker of boiling water. Media can only be reboiled once. Note: Media can be stored for up to two months. 

(b) Manganese Sulfate Monohydrate.–500 ppm. Add 0.25 g of manganese sulfate to 500 mL 

water. Filter sterilize for use. 

(c) Dilute hydrochloric acid.–2.5M. Use to determine resistance of dried spores. Standardize and 

adjust to 2.5M as in 936.15. 

(d) Sterile water.–Use reagent-grade water. Reagent-grade water should be free of substances 

that interfere with analytical methods. Any method of preparation of reagent-grade water is 

acceptable provided that the requisite quality can be met. Reverse osmosis, distillation, and 

deionization in various combinations all can produce reagent-grade water when used in the 

proper arrangement. See Standard Methods for the Examination of Water and Wastewater for 

details on reagent-grade water. 

(e) Triton X-100 

(f) Ethanol (40%) 

(g) Test organism.–Bacillus subtilis (ATCC No. 19659) obtained directly from a commercial 

supplier (e.g., ATCC). 

15 
 

B. Apparatus 

(a) Carriers.–Penicylinders, porcelain, 8 ± 1 mm OD, 6 ± 1 mm ID, 10 ± 1 mm length 

(Available from CeramTec Ceramic, Laurens, SC, www.ceramtec.com, Cat. No. LE15819.) 

(b) Glassware.– For disinfectant, 25 × 150 mm or 25 × 100 mm culture tubes (Bellco Glass Inc., 

Vineland, NJ; reusable or disposable 20 × 150 mm (for cultures/subcultures); 16 × 100 mm 

screw cap tubes for stock cultures. Cap with closures before sterilizing. Sterilize all glassware 2 

hr in hot air oven at 180o C or steam sterilize for a minimum of 20 min at 121oC with drying 

cycle. 

(c) Sterile centrifuge tubes.–Polypropylene, 15 mL conical tubes with conical bottoms 

(Corning), from Fisher, or equivalent. 

(d) Water bath/chiller unit.–Constant temperature for test chemical, capable of maintaining 20 ± 

1oC temperature or specified temperature for conducting the test. 

(e) Petri dishes.–Plastic (sterile) 

(f) Filter paper.–Whatman filter paper #2; placed in Petri dishes for storing carriers. 

(g) Test tube racks.–Any convenient style. 

(h) Inoculating loop.–Any convenient inoculation/transfer loop for culture transfer. 

(i) Wire hook.–For carrier transfer. Make 3 mm right angle bend at end of 50 – 75 mm 

nichrome wire No. 18 B&S gage. Have other end in suitable holder. 

(j) Centrifuge.–Non-refrigerated (e.g., Eppendorf 5804 R). 

(k) Sonicator.–Ultrasonic cleaner (e.g., Branson Model 1510). 

(l) Orbital shaker.–speed range from 25 to 500 rpm (e.g., VWR DS 500). 

(m)Vacuum desiccator.–For carrier storage. With adequate gauge for measuring 27” (69 cm) of 

Hg and fresh desiccant. 

(n) Certified biosafety cabinet (Class I or II).–Recommended for use to maintain aseptic work 

environment. 

(o) Certified Timer.–For managing timed activities, any certified timer that can display time in 

seconds. 

16 
 

 

 

C. Operating Technique 

(a) Culture initiation.– Initiate B. subtilis culture (e.g., use nutrient broth to re-hydrate a 

lyophilized culture, and incubate the broth culture for 24 ± 2 hours at 36 ± 1oC prior to streak 

inoculation). Streak inoculate a set (e.g., six) nutrient agar slopes and incubate 24 ± 2 hours at 

36 ± 1oC. Concurrently, perform purity and identification confirmation testing for QC (e.g., 

colony morphology on TSA, Gram stain, or use of other identification systems). Following 

incubation, store at 2-5oC. Maintain stock culture on nutrient agar slants by monthly (30 ± 2 

days) transfers. 

(b) Production of B. subtilis spore suspension.–Using growth from a stock culture tube, inoculate 

10 mL tubes (e.g., 2 tubes, depending on the amount of spore preparation desired) of nutrient 

broth and incubate tubes on an orbital shaker for 24 ± 2 hours at approximately 150 rpm at 36 ± 

1oC. Use this culture to inoculate amended nutrient agar plates. Inoculate each plate with 500 μl 

of broth culture and spread the inoculum with a sterile bent glass rod or suitable spreading 

device. Wrap each plate with parafilm or place in plastic bags. Incubate plates inverted for 12­

14 days at 36 ± 1oC. Following incubation, harvest the spores by adding 10 mL cold sterile 

water to each plate. Using a spreader (e.g. bent glass rod), remove growth from plates and pipet 

suspensions into 15 mL sterile conical tubes (10 plates = 14 tubes, ~10 mL each). Centrifuge 

tubes at 5000 rpm for approximately 10 minutes at room temperature. Remove and discard 

supernatant. Re-suspend pellet in each tube with 10 mL cold sterile water and centrifuge at 5000 

rpm for approximately 10 minutes. Remove and discard supernatant. Repeat twice. Re-suspend 

the pellet in each tube with 10 mL sterile water. Store the spore suspension at 2-5oC. Examine 

spore suspension with a phase contrast microscope or by staining to assess quality of the spores. 

Examine a minimum of five fields and determine ratio of spores to vegetative cells (or 

sporangia). Percentage of spores versus vegetative cells should be at least 95%. Spore 

suspension from multiple plates can be combined and re-aliquoted into tubes for uniformity. 

Prior to inoculation of carriers, determine spore titer of the concentrated spore suspension by 

plating serial dilutions (e.g., 1.0 × 10-6 through 1.0 × 10-8) using pour or spread plating on TSA 

plates. For pour plating, add molten TSA tempered to 45-55oC to each plate, swirl, and allow 

agar to solidify. Incubate plates for 24 ± 2 hours at 36 ± 1oC and determine titer. Note: When 

harvested and processed, ten plates of amended nutrient agar should provide 80 - 100 mL of 

concentrated spore suspension (approx. 109

 CFU/mL). Diluting the suspension prior to carrier 

inoculation will be necessary; a titer of 1.0 × 108

 to 5.0 × 108 CFU/mL should be adequate to 

achieve the target carrier count. 

(c) Preparation of porcelain carriers.–Prior to use, examine porcelain carriers individually and 

discard those with scratches, nicks, spurs, or discolorations. Rinse unused carriers gently in 

water three times to remove loose material and drain. Place rinsed carriers into Petri dishes 

matted with 2 layers of filter paper in groups of 15 carriers per Petri dish. Sterilize 20 minutes at 

121oC. Cool and store at room temperature. Note: Handle porcelain carriers with care when 

placing in Petri dishes. Minimize carrier movement and avoid excessive contact between 

carriers that might result in chips and cracks. Wash carriers with Triton X-100 and rinse with 

water 4 times for reuse. 

17 
(d) Inoculation of Porcelain Carriers.–Dilute the concentrated spore suspension as necessary 

with sterile water to achieve carrier counts between 1.0 × 105

 and approximately 1.0 × 106 

spores/carrier. Dispense 10 mL diluted spore suspension into an appropriate number of 25 × 150 

mm tubes. Add 10 sterile carriers to each tube containing 10 mL spore suspension, slightly 

agitate, and let stand 10-15 minutes. Remove each carrier with sterile hook and place upright in 

sterile Petri dish lined with two sheets of filter paper, no more than 30 carriers per Petri dish. Air 

dry in biological safety cabinet for approximately 30 ± 2 minutes. Place Petri dishes containing 

inoculated carriers in vacuum desiccator containing CaCl2 and draw vacuum of 69 cm (27”) Hg. 

Dry carriers under vacuum for 24 ± 2 hours before use in HCl resistance, efficacy testing or 

carrier counts. Maintain under vacuum for up to three months. Carriers may be used after three 

months if they meet the acceptable HCl resistance and carrier count criteria. Inoculated carriers 

should not be used after one year of storage. Sterilize and reuse if necessary (see C.c). 

(e) Spore Enumeration (Carrier Counts).–Prior to use, determine the carrier counts for each 

preparation of carriers. Assay 5 randomly selected carriers per preparation. Place each 

inoculated carrier into a 50 mL plastic, polypropylene conical centrifuge tube containing 10 mL 

of sterile water. Sonicate carriers for 5 minutes ± 30 seconds. Note: For sonication, place tubes 

into an appropriately sized beaker with tap water to the level of sterile water in the tubes. Place 

beaker in sonicator (ultrasonic cleaner) so that water level in the beaker is even with water level 

fill line on sonicator tank. Fill tank with tap water to water level fill line. Suspend beaker in 

sonicator tank so it does not touch bottom of tank and so all three water levels (inside test tubes, 

inside beaker, and sonicator tank) are the same. Following sonication, vortex tubes for 2 minutes 

± 5 seconds. Dilute spore suspensions by transferring 1 mL aliquots to tubes containing 9 mL 

sterile water. Dilute spore suspensions out to 1.0 ×10-5 and plate dilutions 1.0 × 10-2 through 1.0 

× 10-5. Plate each dilution in duplicate using pour or surface spread plating with TSA. For pour 

plating, add molten TSA tempered to 45-55oC to each plate. Swirl pour plates to distribute 

spores evenly and allow agar to solidify. Invert plates and incubate for 24-48 hours at 36 ± 1oC. 

Count colonies (by hand or with colony counter). Use dilutions yielding between 30 and 300 

CFU per plate (target counts) for enumeration; however, record all counts less than 30. Report 

plates with colony counts over 300 as TNTC (Too Numerous to Count). Average spore counts 

per carrier should be between 1.0 × 105

 and approximately 1.0 × 106 spores/carrier. Do not use 

carriers with counts outside this range. 

(f) HCl resistance.–Equilibrate water bath to 20 ± 1oC. Pipet 10 mL of 2.5M HCl into two 25 × 

100 mm tubes, place into water bath, and allow to equilibrate. Start timer and rapidly transfer 4 

inoculated penicylinders into an acid tube (2.5 M HCl) with flamed hooks or forceps. Do not 

allow carriers or transfer device to contact inside of wall of acid tube. Transfer individual 

carriers after 2, 5, 10, and 20 minutes of HCl exposure to a separate tube of modified FTM. 

Rotate each tube vigorously by hand for approximately 20 seconds and then transfer carrier to a 

second tube of modified FTM. For viability control, place one unexposed inoculated carrier in a 

separate tube of modified FTM. For media sterility, use one tube of modified FTM. Incubate all 

test and control tubes for 21 days at 36 ± 1oC. Record results as growth (+) or no growth (0) at 

each time period. Spores should resist HCl for ≥ 2 minutes to be qualified as resistant test 

spores. Discard carriers if not resistant and repeat preparation of carriers as previously 

described. 

18 
 

(g) Efficacy Test.–Aseptically prepare disinfectant samples as directed. Prepare all dilutions with 

sterile standardized volumetric glassware. For diluted products, use 1.0 mL or 1.0 g of sample 

disinfectant to prepare the use-dilution to be tested. Use v/v dilutions for liquid products and w/v 

dilutions for solids. For a 30-carrier test, place 10 mL product at dilution recommended for use 

or under investigation into each of six 25 × 150 mm or 25 × 100 mm test tubes, or use 

appropriate number of tubes assuming 5 test carriers per tube of test chemical. Place tubes in 20 

± 1oC water bath and let equilibrate to temperature. Using a sterile hook (or forceps), transfer 

inoculated carriers sequentially at 2 minute intervals in groups of 5 from Petri dish to test tubes 

containing sporicidal agent. Use a certified timer to monitor time. Flame hook and allow 

cooling after each transfer. When lowering carriers into test tube, neither carriers nor wire hook 

may touch sides of tubes. If interior sides are touched, note tube number. Do not count carrier 

set if any carrier from that group of 5 yields a positive result; testing another set of five carriers is 

recommended. Carriers must be deposited into test tubes within ± 5 seconds of the prescribed 

drop time. Return tubes to water bath immediately after adding carriers. After contact period 

has been achieved, transfer carriers in same sequential timed fashion into primary subculture 

tubes containing appropriate neutralizer (10 mL in 20 × 150 mm test tubes). Remove the carriers 

one at a time from the test tube with sterile hook, tap against interior side of tube to remove 

excess sporicidal agent, and transfer into neutralizer tube (primary tube). All five carriers must 

be transferred during each 2 minute interval. Flame hook between each carrier transfer. Move 

remaining carriers into their corresponding neutralizer tubes at appropriate time. Carriers may 

touch interior sides of neutralizer tube during transfer, but contact should be minimized. After 

each carrier is deposited, recap neutralizer tube and gently shake to facilitate adequate mixing 

and efficient neutralization. Within one hour from when last carrier was deposited into 

primaries, transfer carriers using sterile wire hook to second subculture tube (secondary tube) 

containing 10 mL of appropriate recovery medium, one carrier per tube. Move carriers in order, 

but movements do not have to be timed. Gently shake entire rack of secondary tubes after all 

carriers have been transferred. Incubate primary (neutralizer) and secondary subculture tubes for 

21 days at 36 ± 1oC. Report results as growth (+) or no growth (0). A positive result is one in 

which medium appears turbid. A negative result is one in which medium appears clear. Shake 

each tube prior to recording results to determine presence or absence of growth/turbidity. 

Primary and secondary subculture tubes for each carrier represent a “carrier set”. A positive 

result in either primary or secondary subculture tube is considered a positive result for the carrier 

set. 

Media sterility controls and system controls (check for aseptic technique during carrier 

transfer process) are recommended. For media controls, incubate 1-3 unopened subculture 

medium tubes with the test sample tubes for 21 days at 36 ± 1oC. For system controls, use sterile 

forceps or needle hooks to transfer 3 sterile carriers into a tube of test chemical. Transfer system 

control carriers to neutralizer medium as follows: at start of sample test (prior to first tube), 

transfer 1 sterile carrier to tube of neutralizer medium. After one half of test carriers have been 

transferred to neutralizer tubes, transfer a second sterile carrier to tube of neutralizer medium. 

After all test carriers (last tube) have been transferred to neutralizer tubes, transfer third sterile 

carrier to tube of neutralizer medium. Transfer system control carriers to secondary subculture 

medium as follows: immediately prior to initiating transfer of test carriers into secondary 

subculture medium tubes, transfer first system control sterile carrier from neutralizer medium to 

tube of subculture medium. After one half of test carriers have been transferred to secondary 

19 
subculture medium tubes, transfer second system control sterile carrier to tube of subculture 

medium. After all test carriers have been transferred to secondary subculture medium tubes, 

transfer third system control sterile carrier to tube of subculture medium. For each test, include a 

positive carrier control by placing one inoculated carrier into tube of secondary subculture 

medium. Incubate controls and test sample tubes together for 21 days at 36 ± 1oC. 

Perform identification confirmation on a minimum of three positive carrier sets per test, if 

available, using Gram stain and/or plating on TSA. Additional confirmation may be performed 

using VITEK, API analysis or comparable method. If fewer than three positive carrier sets, 

confirm growth from each positive carrier set. If both tubes are positive in carrier set, select only 

one tube for confirmatory testing. For test with 20 or more positive carrier sets, confirm at least 

20% by Gram stain. If Gram stains are performed from growth taken directly from positive 

tubes, the staining should be performed within 5-7 days of conducting the efficacy test. 

(h) Neutralization Confirmation Procedure.– A neutralization confirmation test must be 

performed in advance or in conjunction with efficacy testing. This assay is designed to simulate 

the conditions (i.e., neutralizer, subculture medium, contact time, diluent, concentration of test 

substance) of the efficacy test and to demonstrate the recovery of a low level of spores (e.g., 5 – 

100). Diluted inoculum (e.g., spores of B. subtilis) is added directly to the various sets of 

subculture media tubes (see Table 1). This assay provides for a quantitative approach to 

assessing the effectiveness of the neutralizer and any bacteriostatic action resulting from the 

neutralizer itself or neutralizer – disinfectant interactions. 

Produce a spore preparation according to the procedure for amended nutrient agar. 

Harvest growth from plates (e.g., five plates) per the method, except re-suspend pellet after final 

centrifugation step in approximately 100 ml aqueous (40%) ethanol. Determine spore count by 

serial dilution and plating on TSA. Desirable target of the initial working suspension is 1.0 × 108 

to 1.0 × 109

 CFU/mL. The suspension may require adjustment to reach target titer. Prepare 

serial ten-fold dilutions of the inoculum in sterile water out to 10-8. Use 10-6, 10-7 and 10-8 

dilutions to inoculate the neutralizer and subculture media tubes – the target number of spores to 

be delivered per tube in this assay is 5–100 per tube. Determine spore titer by plating (spread 

plate or pour plate) each of three dilutions in duplicate on TSA agar. Incubate plates inverted for 

24-48 hours at 36 ± 1oC. Count colonies (by hand or with colony counter). Report plates with 

colony counts over 300 as TNTC (Too Numerous to Count). Note: A standardized spore 

preparation adjusted to deliver 5–100 spores/mL may be substituted for the three dilutions of 

spore inoculum. In addition, spores sheared from inoculated carriers may be used as a working 

suspension. 

Use 5 sterile porcelain carriers (only 3 to be used in the assay). Within 5 seconds, place a 

set of 5 carriers into a test tube (25 × 150 mm or 25 × 100 mm) containing test chemical; transfer 

carriers according to section (g). Allow carriers to remain in test chemical per the specified 

contact time and temperature. After the contact time is complete, aseptically transfer three of the 

five carriers individually into tubes containing the neutralizer per section (g). This set of tubes is 

20 
 

the Neutralizer/Primary Subculture treatment. Following the transfer of the last carrier into 

neutralizer tube, transfer each carrier, in sequence, into tube containing secondary subculture 

medium. This portion of assay is not timed, but should be made as soon as possible. This set is 

the Secondary Subculture treatment. Following carrier transfer, inoculate each tube 

(Neutralizer/Primary and Secondary Subculture treatment tubes) with one mL of each of three 

inoculum dilutions (10-6, 10-7 and 10-8). For controls, use three fresh unexposed tubes of 

neutralizer and three tubes of the secondary subculture medium; also inoculate each control tube 

with one mL of each of three inoculum dilutions. Include one uninoculated tube of neutralizer 

and secondary subculture media to serve as sterility controls. See Table 1 for tube inoculation 

scheme. Incubate all tubes 5-7 days at 36 ± 1oC. Record results as growth (+) or no growth (0). 

Note: The lack of complete neutralization of the disinfectant or bacteriostatic activity of the 

neutralizer itself may be masked when a high level of inoculum (spores) is added to the 

subculture tubes. 

Table 1. Neutralization confirmation procedure – inoculating treatment and control tubes 

with diluted spore suspension* 

Neutralizer-Primary 

Subculture Treatment 

Secondary Subculture 

Treatment (with Carrier) 

Neutralizer-Primary 

Inoculated Control 

Secondary Subculture 

Inoculated Control 

1 mL of 10-6  Tube 1 

1 mL of 10-7  Tube 2 

1 mL of 10-8  Tube 3 

1 mL of 10-6  Tube 1 

1 mL of 10-7  Tube 2 

1 mL of 10-8  Tube 3 

1 mL of 10-6  Tube 1 

1 mL of 10-7  Tube 2 

1 mL of 10-8  Tube 3 

1 mL of 10-6  Tube 1 

1 mL of 10-7  Tube 2 

1 mL of 10-8  Tube3 

*1.0 × 10-6 through 1.0 × 10-8 based on an approx. starting suspension of 108 spores/mL 

Confirm a minimum of one positive per treatment and control (if available) using Gram 

staining and colony morphology on TSA. For each treatment and control group, conduct 

confirmation testing on growth from tube with fewest spores delivered. B. subtilis is a Gram 

positive rod and colonies on TSA are opaque, rough, dull, round, with irregular margins, and low 

convex. Colonial variation may be observed and is typical for this strain. Growth in the 

inoculated controls verifies the presence of the spores, performance of the media, and provides a 

basis for comparison of growth in the neutralizer and subculture treatment tubes. Note: There 

may be cases when the neutralizer is significantly different from the secondary subculture media; 

in these cases, growth may not be comparable. The uninoculated control tubes are used to 

determine sterility, and must show no growth for the test to be valid. 

The occurrence of growth in the Neutralizer/Primary Subculture and Secondary 

Subculture treatment tubes is used to assess the effectiveness of the neutralizer. No growth or 

growth only in tubes which received a high level of inoculum (e.g., the dilution with plate counts 

which are too numerous to count) indicates poor neutralization and/or presence of bacteriostatic 

properties of the neutralizer or neutralizer-disinfectant interactions. For a neutralizer to be 

deemed effective, growth must occur in the Secondary Subculture treatment tubes which 

received lower levels of inoculum (e.g., 5-100 CFU/mL). Growth in the Secondary Subculture 

inoculated Control verifies the presence of the spores, performance of the media, and provides a 

basis for comparison of growth in the neutralizer and subculture treatment tubes. No growth or 

only growth in tubes which received high levels of inoculum (e.g., a dilution with plate counts 

which are too numerous to count) indicates poor media performance. Growth in the Neutralizer21 
Primary inoculated Control should be comparable to the Secondary Subculture inoculated 

Control if the neutralizer is the same as the secondary subculture media. There may be cases 

when the neutralizer is significantly different from the secondary subculture media. In these 

cases, growth may not be comparable to the Secondary Subculture inoculated Control. The 

Neutralizer-Primary and Secondary Subculture uninoculated Control tubes are used to determine 

sterility, and must show no growth for the test to be valid. 

Note: For product registration, the U.S. EPA requires the following to demonstrate 

sporicidal/sterilant-level efficacy: Using AOAC method 966.04, sixty carriers representing each 

of two types of surfaces (porcelain penicylinders and silk suture loops) must be tested separately 

against spores of both Bacillus subtilis (ATCC 19659) and Clostridium sporogenes (ATCC 

3584) on three samples representing three different batches of product, one of which must be at 

least 60 days old (2 carrier types × 2 test microorganisms × 60 carriers/type = 240 carriers per 

batch sample; 3 product batches × 240 carriers/batch = total of 720 carriers). The product must 

kill all of the test spores on all of the 720 carriers without any failures. 

References 

ASTM International Method E 1054 – Standard Test Methods for Evaluation of Inactivators of 

Antimicrobial Agents 

Standard Methods for the Examination of Water and Wastewater. 21st Ed. American Public 

Health Association, 1015 15th Street, NW, Washington, DC 

Biosafety in Microbiological and Biomedical Laboratories. 4th Ed. U.S. Department of Health 

and Human Services, Public Health Service, Centers for Disease Control and Prevention and 

National Institutes of Health 

22 
 

Section 7. Quantitative Three Step Method 

Note: Laboratories will conduct the method in its entirety. 

Determining the Efficacy of Liquid Sporicides and Sterilants Against Spores 

of Bacillus subtilis on a Hard Surface Using the Quantitative Three Step 

Method 

The Quantitative Three Step Method (TSM) is suitable for determining the sporicidal 

activity of liquid sporicidal agents against the genus Bacillus on a hard surface. For the purpose 

of this protocol, the terms sporicide and sterilant are considered synonymous. The TSM is an 

adaptation of a quantitative micro-method as reported by Sagripanti and Bonifacino (1). See 

results of the pre-collaborative study supporting the use of the TSM (2). A similar version of the 

TSM was recently accepted and published by ASTM International (3). 

Note: All manipulations of the test organism are required to be performed in accordance with 

biosafety practices stipulated in the institutional biosafety regulations. Use the equipment and 

facilities indicated for the test organism. For recommendations on safe handling of 

microorganisms refer to the CDC/NIH Biosafety in Microbiological and Biomedical 

Laboratories manual (4). Disinfectants may contain a number of different active ingredients, 

such as heavy metals, aldehydes, peroxides, and phenol. Personal protective clothing or devices 

are recommended during the handling of these items for purpose of activation, dilution, or 

efficacy testing. A chemical fume hood or other containment equipment may be employed when 

appropriate during performing tasks with concentrated products. The study analyst may wish to 

consult the Material Safety Data Sheet for the specific product/active ingredient to determine 

best course of action. References to water mean reagent-grade water, except where otherwise 

specified (5). The methods are technique sensitive and technique-oriented, thus exact adherence 

to the method, good laboratory practices, and quality control (QC) are required for proficiency 

and validity of the results. 

A. Media and Reagents 

(a) Media.–(1) Nutrient broth (NB).–Dehydrated NB. For use in re-hydrating test organism and 

preparing nutrient agar. (2) Nutrient agar (NA).–For stock cultures slants and plating. Add 1.5% 

(w/v) Bacto-agar to un-sterilized nutrient broth. Boil mixture until agar is dissolved. If 

necessary, adjust pH to 7.2 ± 0.2. Dispense 5 mL portions into 16 × 100 mm screw cap tubes. 

Larger tubes may be used as well. Autoclave for 20 min at 121oC. Remove from autoclave and 

slant tubes to form agar slopes. Dehydrated nutrient agar may be substituted – suspend 23 g 

nutrient agar per L water, dissolve by boiling. If necessary, adjust pH to 6.8 ± 0.2. Autoclave for 

15 min at 121oC. (3) Nutrient agar with 5μg/mL MnSO

production. Suspend 11.5 g nutrient agar in 495 mL water, add 5 mL 500 ppm MnSO

4:H20 (amended nutrient agar).–For spore 

4:H20. 

Dissolve by boiling. If necessary, adjust pH to 6.8 ± 0.2. Autoclave for 15 min at 121oC. Pour 

agar into plates. (4) Trypticase soy agar (TSA).–Poured in plates for microbe isolation and spread 

23 
plating. (5) Luria-Bertani broth (LB broth).-Dehydrated LB broth (e.g., Difco); suspend 25 g LB 

broth in 1 L water, mix well, if necessary adjust pH to 7.0 ± 0.2, dispense in bottles and 

autoclave for 15 min at 121°C; use as neutralizer. (6) Modified Luria-Bertani broth.-neutralizer 

in HCl resistance test, add 20 mL 1M NaOH to 1 L LB broth, mix well, dispense in bottles and 

autoclave for 15 min at 121°C. 

(b) Manganese sulfate monohydrate.–500 ppm. Add 0.25 g of manganese sulfate to 500 mL 

water. Filter sterilize for use. 

(c) Sterile water.–Use sterile reagent-grade water. Reagent-grade water should be free of 

substances that interfere with analytical methods. Any method of preparation of reagent-grade 

water is acceptable provided that the requisite quality can be met. Reverse osmosis, distillation, 

and deionization in various combinations all can produce reagent-grade water when used in the 

proper arrangement. See Standard Methods for the Examination of Water and Wastewater for 

details on reagent-grade water (5). 

(d) Test organism.-Bacillus subtilis (ATCC No. 19659) obtained directly from a commercial 

supplier (e.g., ATCC). 

B. Apparatus 

(a) Certified biosafety cabinet (Class I or II).–Recommended to maintain an aseptic work 

environment. 

(b) Glass coupon.-Hard surface carrier, 5 × 5 × 1 mm, Erie Scientific Company, Portsmouth, 

NH; custom order part number EPA-1101 (minimum order of 1000 pieces), single use. 

(c) Sterile 1.5 mL microcentrifuge tubes.-For exposing carrier to disinfectant, Fisherbrand cat. 

#05-408-129. 

(d) Sterile centrifuge tubes.–For preparation of spore suspension, polypropylene, 15 mL conical 

tubes with conical bottoms, Fisher, cat. #05-538-53D. 

(e) Dissecting forceps.-For the transfer of carriers, sterile, VWR cat. #25607-195 or Fisher cat. 

#13-812-42. 

(f) Micropipette.-Used to make serial dilutions, calibrated. 

(g) Positive displacement pipette.-For carrier inoculation. 

(h) Desiccator.–For carrier storage. 

24 
 

(i) Water bath/chiller unit.-Constant temperature for test chemical and controls, capable of 

maintaining 20 ± 1oC temperature or specified temperature; e.g., Neslab RTE-221 or Nalgene 

Labtop Cooler. 

(j) Orbital shaker. 

(k) Microcentrifuge. 

(l) Microcentrifuge tube lid openers.-USA Scientific #1400-1508. 

(m) Sonicator.-Ultrasonic cleaner (Branson Model 1510 Bath Sonicator, or equivalent). 

(n) Floating microcentrifuge tube holder.-For sonication, VWR: #60986-099. 

(o) Hematology rotator.-For fraction C recovery, Hematology Chemistry Mixer 346–Fisher 

Scientific; or a suitable mixer/shaker to provide gentle agitation during incubation. 

(p) Vortex mixer. (also an option for fraction C recovery using a vortex adapter). 

(q) Vortex adapters.-Fisher Scientific cat. #1281161 and 1281211. 

(r) Certified timer.-For managing timed activities, any certified timer that can display time in 

seconds. 

(s) Test tubes.-For sterilizing carriers, 25 × 150 mm. 

(t) 95% ethyl alcohol.- For cleaning carriers. 

C. Operating Technique 

(a) Culture initiation.–Initiate B. subtilis culture (e.g., use nutrient broth to rehydrate a 

lyophilized culture, and incubate the broth culture for 24 ± 2 hours at 36 ± 1oC prior to streak 

inoculation). Streak inoculate a set (e.g., six) nutrient agar slopes and incubate 24 ± 2 hours at 

36 ± 1oC. Perform purity and identification confirmation testing for QC (e.g., colony 

morphology on TSA, Gram stain, or use of other identification systems). Following incubation, 

store at 2-5oC. Maintain stock culture on nutrient agar slants by monthly (30 ± 2 days) transfers. 

(b) Production of B. subtilis spore suspension.–Using growth from a stock culture tube, inoculate 

10 mL tubes (e.g., 2 tubes, depending on the amount of spore preparation desired) of nutrient 

broth and incubate tubes 24 ± 2 hr on an orbital shaker at approximately 150 rpm at 36 ± 1oC. 

Use this culture to inoculate amended nutrient agar plates. Inoculate each plate with 500 μl of 

broth culture and spread the inoculum with a sterile bent glass rod or suitable spreading device. 

25 
 

Wrap each plate with parafilm or place in plastic bags. Incubate plates inverted for 12-14 days at 

36 ± 1oC. Following incubation, harvest the spores by adding 10 mL cold sterile water to each 

plate. Using a spreader (e.g., bent glass rod), remove growth from plates and pipet suspensions 

into 15 mL sterile conical tubes (10 plates = 14 tubes, ~10 mL each). Centrifuge tubes at 5000 

rpm for approximately 10 min at room temperature. Remove and discard supernatant. 

Resuspend pellet in each tube with 10 mL cold sterile water and centrifuge at 5000 rpm for 10 ± 

1 min. Remove and discard supernatant. Repeat twice. Resuspend the pellet in each tube with 

10 mL sterile water. Store the spore suspension at 2-5oC. Examine spore suspension with a 

phase contrast microscope or by staining to assess quality of the spores. Examine a minimum of 

5 fields and determine ratio of spores to vegetative cells (or sporangia). Percentage of spores 

versus vegetative cells should be at least 95%. Spore suspension harvested from multiple plates 

can be combined and re-aliquoted into tubes for uniformity. Prior to inoculation of carriers, 

determine spore titer of the concentrated spore suspension by plating serial dilutions (e.g., 1.0 × 

10-6 through 1.0 × 10-8) on TSA or NA. Incubate plates for 24 ± 2 hr at 36 ± 1oC and determine 

titer. Note: When harvested and processed, 10 plates of amended nutrient agar should provide 

80 - 100 mL of concentrated spore suspension. Diluting the suspension prior to carrier 

inoculation will be necessary; a spore titer of approx. 1.0 × 109

 CFU/mL in the suspension 

should be adequate to achieve the target carrier count. 

(c) Carrier Preparation.-Visually screen glass coupons (carriers) for scratches, chips, or cracks. 

Discard those which are damaged or defective. Rinse carriers once with water, rinse 3 times with 

95% ethyl alcohol, and finally rinse 3 times with water. Allow carriers to dry. Place in glass 

tubes (25 × 150 mm) 40 carriers per tube. Steam sterilize 45 min at 121oC with a 30 min dry 

cycle or sterilize for 2 hr in hot air oven at 180o C. Cool. Transfer carriers to sterile plastic Petri 

dishes for inoculation (approx. 40 carriers per dish). 

(d) Carrier Inoculation.-Transfer 10 μL of spore suspension with a micropipette using aerosol 

barrier tips or positive displacement pipette onto a 5 × 5 × 1 mm sterile, dry glass coupon. 

Apply to one central spot on each carrier. Inoculate the necessary number of carries to complete 

the validation study (approx. 140). Allow carriers to dry for minimum of 1 hour in open Petri 

dish in a biosafety cabinet, then for a minimum of 12 ± 2 hr in a desiccator. Store inoculated 

carriers under desiccation for up to 30 days. Glass carriers must be discarded after use. Note: 

During carrier inoculation, vortex mix inoculum frequently to ensure uniform distribution of 

spores. Recommend verification of carrier counts (per the method for control carriers) prior to 

test day; mean counts must be 5.0 × 106

 to 5.0 × 107 spores/carrier. 

(e) Disinfectant Sample Preparation.-Aseptically prepare disinfectant samples as directed. 

Prepare all dilutions with sterile standardized volumetric glassware. For diluted products, use 

1.0 mL or 1.0 g of sample disinfectant to prepare the use-dilution to be tested. Use v/v dilutions 

for liquid products and w/v dilutions for solids. Place approximately 1.5 mL of each disinfectant 

or control (sterile water) in microcentrifuge tubes. Allow to equilibrate to appropriate 

temperature for 15-30 min. 

(f) Test Procedure Overview.-A minimum of 3 carriers per disinfectant and 3 carriers for 

26 
the water control (control carriers) are required per product test. Three carriers per treatment will 

be required for the validation study. Use 1 pair of sterile forceps per fraction for each 

disinfectant. Fractions may be refrigerated briefly to allow for processing of other fractions. If 

possible, it is recommended that two analysts perform this method so that dilution and plating of 

the multiple fractions may be conducted as soon as possible. See Appendices TSM-1, TSM-2 

and TSM-3 for additional guidance. Note: It is recommended that no more than 3 disinfectant 

treatments (9 test carriers) plus the water control (3 control carriers) should be tested during the 

same test period. 

Using sterile forceps, carefully transfer 1 inoculated carrier into each microcentrifuge 

tube labeled fraction A. Avoid touching inoculated area of carrier and sides of microcentrifuge 

tube. Discard carrier and tube if carrier touches sides of tube. Place fraction A tubes containing 

carriers and tubes containing disinfectant(s) and sterile water (control) into chiller water bath at 

20 ± 1oC, or use a labtop cooler to maintain temperature of the tubes. Equilibrate approximately 

10 min. Add 400 μL disinfectant (test carriers) or 400 μL sterile water (control carriers) at 15 or 

30 ± 5 sec intervals to appropriate microcentrifuge tube (in triplicate). Allow contact of the 

carriers to the disinfectant or water in fraction A tubes for the appropriate exposure period. 

Following the exposure period, add 600 μL of appropriate ice-cold neutralizer (e.g., LB 

broth) to each disinfectant fraction A tube. Add 600 μL LB broth as neutralizer for water control 

fraction A tubes. Slightly agitate tubes to thoroughly mix liquid components. Transfer each 

carrier using 1 pair sterile forceps per carrier set (i.e., 3 carriers) from fraction A tube to 

corresponding fraction B tube. Fraction B tubes contain 400 μL ice-cold (0-5oC) sterile water. 

Place fraction A tubes in microcentrifuge, centrifuge for 6 min ± 30 sec at 13,000 rpm 

(15,500 × g). Remove 900 μL from each tube without disturbing pellet. Discard supernatant. 

Carefully add 900 μL ice-cold LB broth to each tube. Repeat 2 additional times. After third 

centrifugation, remove 900 μL from each tube. Carefully add 100 μL ice-cold LB Broth to each 

fraction A tube and resuspend pellet by vortex mixing 5 min ± 30 sec (use the vortex adapter) at 

midrange speed. Add 800 μL ice-cold LB Broth to each fraction A tube. Proceed to dilution and 

plating if another analyst is available, or store fraction A tubes in refrigerator. Note: Fluid 

remaining in the fraction A tubes contains spores dislodged from carrier by exposure to 

disinfectant or water control. Consistent orientation of the microcentrifuge tubes in the 

microcentrifuge is important in locating the pellet. The pellet may range in size and be difficult 

to visualize depending on the treatment. Fraction B and fraction C tubes can be evaluated while 

fraction A tubes are being centrifuged.

 Sonicate fraction B tubes 5 min ± 30 sec using a floating microcentrifuge tube holder 

placed inside an ultrasonic cleaner. After sonication is complete, add 600 μL ice-cold LB Broth 

to fraction B tubes. Vortex approx. 1 minute. Transfer each carrier using 1 pair sterile forceps 

per carrier set from fraction B tube to corresponding fraction C tube (fraction C tubes contain 

400 μL ice-cold LB broth). Proceed to dilution and plating if another analyst is available, or 

store fraction B tubes in refrigerator; however, storage should be limited to 2 hrs. Note: Fluid 

remaining in the fraction B tubes contains spores dislodged from the carrier by sonication. 

27 
 Place fraction C tubes in a hematology rotator inside incubator for 30 ± 2 min at 36 ± 

1oC. Remove fraction C tubes after 30 ± 2 min rotation/incubation from incubator. Add 600 μL 

ice-cold LB Broth to each tube. The carriers remain in the fraction C tubes. Proceed to dilution 

and plating if another analyst is available, or store fraction C tubes in refrigerator. Note: Fluid 

remaining in fraction C tubes contains spores dislodged from the carrier by gentle agitation for 

30 min. 

Vortex mix each microcentrifuge tube thoroughly prior to making dilutions. For each 

fraction and control tube, remove 100 μL and serially dilute 10-fold in 900 μL ice-cold LB broth. 

For each carrier, direct plate 100 μL of the sufficient dilutions onto TSA or NA to ensure 

obtaining counts within the target range of 30-300 CFU/plate. Incubate plates a minimum of 24 

± 2 hr at 36 ± 1oC. Record control counts at 24 ± 2 hr. Record treated carrier counts at 24 ± 2 hr 

and at 48 ± 2 hr. Confirm the identity of a minimum of one representative colony taken from at 

least one plate per treatment level (if available) using Gram staining, general growth media (e.g., 

TSA or NA) or other confirmation procedure. B. subtilis is a large Gram positive rod. On 

general growth media B. subtilis colonies are opaque, rough, round low convex colonies with 

irregular margins. Notes: After plating, dilution tubes may be stored at 2-5 oC until the results 

are recorded; the tubes may be used for additional plating if initial plate counts are beyond the 

recommended target range. 

Use counts which fall within 0-300 CFU/plate for calculations. Obtain the total number 

of spores per fraction by dividing the number of colonies counted in each fraction by its dilution, 

and account for volume plated. Obtain the total number of spores per carrier by adding the total 

number of viable spores per fraction for fractions A, B, and C. Determine log density (LD) of 

total number of viable spores per carrier by taking Log10 (total number of spores per carrier). 

Determine log reduction (LR) of test carriers by subtracting log density of test carriers from log 

density of control carriers. Determine average LD and LR for each disinfectant. 

(g) Neutralization Confirmation (EPA Laboratory only).-Prepare 12 microcentrifuge tubes. Add 

400 μL sterile water to tubes 1-6 and 400 μL of disinfectant to tubes 7-12. Allow tubes to 

equilibrate approximately 10 min at 20 ± 1oC (or other specified temperature). Add 600 μL 

neutralizer in ice-cold Luria-Bertani (LB) broth to tubes 4-6 (neutralizer controls). Add 600 μL 

neutralizer in ice-cold LB broth to tubes 7-9 (ability of neutralizer to inactivate the disinfectant). 

Gently mix. Add 10 μL of B. subtilis spore suspension (approx. 109

 spores/mL) to each tube and 

vortex mix for approximately 15 sec. Incubate tubes for 30 min ± 2 min at 20 ± 1oC (or 

temperature specified by disinfectant manufacturer). After incubation, add 600 μL ice-cold LB 

broth to tubes 1-3 (survival controls). Add 600 μL ice-cold LB Broth to tubes 10-12 (disinfectant 

controls). Serially dilute each tube (10 μL into 990 μL ice-cold LB broth) to achieve plate counts 

of 30-300 CFU/plate. Plate 100 μL of each dilution onto NA or TSA. Incubate 24 ± 2 hr at 36 ± 

1oC. Count colonies on each plate. Log densities in tubes 1-3 and 4-6 reflect the spore suspension 

titer and should be within one log of each other. If log densities between tubes 1-3 and 4-6 are 

greater than one log, then the neutralizer has a sporicidal effect. If the disinfectant is highly 

effective, log densities in tubes 10-12 should be approximately 6 logs lower than log densities in 

tubes 1-6. To be an effective neutralizer, log densities in tubes 7-9 should be within 1 log of the 

28 
log densities in tubes 1-6. Note: The lead laboratory, the OPP Microbiology Laboratory, will 

perform this assay on each of the high treatments prior to the initiation of the study to verify the 

effectiveness of the chosen neutralizers. For this assay, produce a spore preparation according to 

the procedure for amended nutrient agar. Harvest growth from plates (e.g., five plates) per the 

method, except re-suspend pellet after final centrifugation step in approximately 100 ml aqueous 

(40%) ethanol. 

(h) HCl resistance.– Perform on each preparation of inoculated carriers. Conduct TSM 

procedure on 2.5 M HCl. Follow procedure as specified in part (f) with 2 and 5 min exposure 

periods with three inoculated carriers per time period. Include three control (sterile water) 

carriers to determine control carrier counts. Use modified LB broth (addition of NaOH) as the 

neutralizer instead of LB broth for HCl treatments. Perform test at 20 ± 1oC. Calculate log 

reduction. Spores should resist HCl for ≥ 2 min (i.e., based on presence of viable spores after 

2 min) to be qualified as resistant test spores. Discard carriers if not resistant and repeat 

preparation of carriers as previously described. Note: Compared to the water control, anticipate 

1-2 log reduction of viable spores at 2 min exposure and 3-5 log reduction following the 5 min 

exposure. 

References Associated with the Three Step Method 

(1) Sagripanti, J.L. & Bonifacino, A. (1996) Am. J. Infect. Control 24, 364 – 371 

(2) Tomasino, S.F. & Hamilton, M.A. (2006) Unpublished Report. Comparative Evaluation 

of Two Quantitative Test Methods for Determining the Efficacy of Liquid Sporicides and 

Sterilants on a Hard Surface: A Pre-Collaborative Study 

(3) Standard Test Method for Quantitative Sporicidal Three-Step Method (TSM) to 

Determine Sporicidal Efficacy of Liquids, Liquid Sprays, and Vapor or Gases on 

Contaminated Surfaces. (2005) ASTM Designation E 2414 – 05 

(4) Biosafety in Microbiological and Biomedical Laboratories (1999) 4th Ed. U.S. 

Department of Health and Human Services, Public Health Service, Centers for Disease 

Control and Prevention and National Institutes of Health 

(5) Standard Methods for the Examination of Water and Wastewater. 21st Ed. American 

Public Health Association, 1015 15th Street, NW, Washington, DC 

29 
 

 

 

 

 

 

Appendix: TSM-1 

Three Step Method Processing Sheet 

Analyst(s): ______________________________ Test Date: ___/_____/_____ 

Test Chemical(s): _________________________________________________________ 

NOTE: Carriers exposed to the disinfectant(s) and water control will be tested in triplicate. It is 

recommended that no more than three disinfectants plus the water control be tested during the 

same test period. 

The contents of microcentrifuge tubes prior to processing fractions for 1 disinfectant and 

control. 

Treatment Tube Contents 

A1-A3 400 μL disinfectant + 600 μL LB broth w/neutralizer (after exposure period) 

Disinfectant B1-B3 400 μL sterile water + 600 μL LB broth (after sonication) 

C1-C3 400 μL LB broth + 600 μL LB broth (after incubation/rotation) 

A4-A6 400 μL sterile water + 600 μL LB broth (after exposure period) 

Control B4-B6 400 μL sterile water + 600 μL LB broth (after sonication) 

C4-C6 400 μL LB broth + 600 μL LB broth (after incubation/rotation) 

Prior to Testing (i.e. day before test); as you proceed, add initials to each step in the space 

provided. 

____ Label fraction A, B and C microcentrifuge tubes with fraction letter (e.g. A, B, or C) and 

carrier number (e.g., 1, 2, 3, etc.). 

• Fraction tube examples: A1, A2, A3, B1, B2, B3, C1, C2, C3 etc. 

____ In advance of testing, prepare fraction B and fraction C microcentrifuge tubes: 

• Add 400 μL of ice-cold sterile water to fraction B tubes. 

• Add 400 μL of ice-cold LB broth to fraction C tubes. 

• Store these tubes in a refrigerator (2-5°C) until ready for use. 

____	 Label dilution microcentrifuge tubes (serial dilution blanks) with fraction letter (e.g. A, B, or C), 

carrier number (e.g. 1, 2, 3, etc.), and dilution (e.g., 10-1, 10-2, 10-3, etc.). 

• Serial dilution blank examples: A1-10-1, A1-10-2, A1-10-3, etc. 

____ Prepare serial dilution blanks for A, B, and C fractions for all test carriers. 

• Add 900 μL of ice-cold LB broth to each dilution blank for test carriers. 

• Store these tubes in a refrigerator (2-5°C) until ready for use. 

30 
____ 

•	 Add 900 μL of ice-cold LB broth to each dilution blank for control carriers. 

•	 Store these tubes in a refrigerator (2-5°C) until ready for use. 

____ Sterilize forceps (two pair of forceps for each disinfectant/control tested plus 3 extra). 

____ Prepare a 50 mL conical tube with the appropriate neutralizer (place it on ice on test day). 

____ Turn on the recirculating chiller and water bath and allow them to reach 20 ± 1°C or the 

temperature specified. 

____ On test day, prepare the disinfectant(s) and place ~1.5 mL of each disinfectant and water 

control into a microcentrifuge tube 

•	 Be sure to prepare and use the disinfectant within its specified period. 

•	 If the disinfectant requires a dilution, a minimum of 1 mL of the product must be used. 

•	 Ready-to-use disinfectants are tested as received; no dilution is required. 

Prepare serial dilution blanks for A, B, and C fractions for all control carriers. 

..................................................................................................................... 

3.1.1.1.1.1.1 Fraction A 

____ Using sterile forceps, carefully place one inoculated carrier inside each microcentrifuge 

tube labeled fraction A. 

•	 Avoid touching the inoculated area of the carrier and the sides of the microcentrifuge 

tube. If the carrier touches the sides of the microcentrifuge tube, the carrier and tube 

must be discarded. 

•	 These forceps should not be used for any additional transfers, unless sterilized appropriately. 

____ Place the fraction A tubes containing the carriers and the tubes containing the 

disinfectant(s) and water control into a chiller water bath equilibrated at 20 ± 1°C. 

• Allow the carriers, disinfectant(s), and water control to equilibrate for approx. 10 min. 

____ Using a 1000 μL micropipette, add 400 μL of the disinfectant or 400 μL sterile water 

control at 15 or 30 ± 5 sec intervals to the appropriate microcentrifuge tubes (in 

triplicate). 

____ Expose fraction A tubes in the chiller water bath for the appropriate exposure period. 

____	 After exposure is complete add 600 μL of the appropriate ice-cold neutralizer to each disinfectant 

and water control fraction A tube. 

• Slight agitation (e.g., light tapping) may be necessary to thoroughly mix the liquid components. 

____ Using one pair of sterile forceps per set of 3 carriers (e.g., per disinfectant), transfer each 

carrier from its fraction A tube to its corresponding fraction B tube. 

•	 Avoid touching the sides of the tube if possible. 

•	 These forceps should not be used for any additional transfers. 

31 
 

 

 

 

 

 

 

Note: Processing of the fraction B and fraction C tubes can be completed by a second 

analyst while the fraction A tubes are being centrifuged (e.g., sonication of the fraction B 

tubes, transfer of the carrier to the fraction C tubes, rotation of the fraction C tubes. 

____ Place the fraction A tubes into the microcentrifuge for 6 min ± 30 sec at 13,000 rpm 

(15,500 × g). 

•	 Centrifugation #1 

____ After fraction A tubes complete centrifugation #1, carefully remove 900 μL from each fraction A 

tube without disturbing the pellet. Discard the supernatant. 

____	 Carefully add 900 μL of ice-cold LB broth into each fraction A tube and place the fraction A 

tubes into the microcentrifuge for 6 min ± 30 sec at 13,000 rpm (15,500 × g). 

•	 Centrifugation #2 

Note: The final volume in each fraction A tube is 1000 μL. 

____ After fraction A tubes complete centrifugation #2, carefully remove 900 μL from each fraction A 

tube without disturbing the pellet. Discard the supernatant. 

____	 Carefully add 900 μL of ice-cold LB broth into each fraction A tube and place the fraction A 

tubes into the microcentrifuge for 6 min at 13,000 rpm (15,500 × g). 

• Centrifugation #3 

____ After fraction A tubes complete centrifugation #3, carefully remove 900 μL from each fraction A 

tube without disturbing the pellet. Discard the supernatant. 

____ Carefully add 100 μL of ice-cold LB broth into each fraction A tube. 

____ Resuspend the pellet in the fraction A tubes by vortex mixing for 5 min ± 30 sec at a 

 midrange speed. 

• The use of a vortex adapter is recommended. 

____ Add 800 μL of ice-cold LB broth into each fraction A tube. 

____ Proceed to the Dilutions and Plating section. 

•	 Place fraction A tubes in a refrigerator if dilutions are not made immediately. Processing should 

proceed as quickly as possible. 

.........................................................................................................................................................
 

Fraction B 

____ Sonicate fraction B tubes for 5 min ± 30 sec using a floating microcentrifuge tube holder. 

____	 After sonication is complete, add 600 μL of ice-cold LB broth to the fraction B tubes and vortex 

for one minute. 

32 
 

 

 

 

____ Using one pair of sterile forceps per set of 3 carriers (e.g., per disinfectant), transfer each 

carrier from its fraction B tube to its corresponding fraction C tube. 

• These forceps should not be used for any additional transfers, unless sterilized appropriately. 

____ Proceed to the Dilutions and Plating section. 

•	 Place fraction B tubes in a refrigerator if dilutions are not made immediately. Processing should 

proceed as quickly as possible. 

..................................................................................................................... 

Fraction C 

____ Place the fraction C tubes in a hematology rotator inside a 36 ± 1°C incubator for 30 ± 2 

min. 

____	 After the 30 min ± 2 min rotation/incubation, remove the fraction C tubes from the incubator and 

add 600 μL of ice-cold LB broth to each tube. 

• The carriers can remain in the fraction C tubes. 

____ Proceed to the Dilutions and Plating section. 

•	 Place fraction C tubes in a refrigerator if dilutions are not made immediately. Processing 

should proceed as quickly as possible. 

...................................................................................................................... 

3.1.1.1.1.1.2 Dilutions and Plating 

____ Remove dilution blanks from the refrigerator immediately prior to making dilutions. 

____ Vortex mix each microcentrifuge tube thoroughly prior to making any dilutions. 

____ Serially dilute fractions A, B, and C for test carriers. 

•	 Vortex mix each tube prior to making the next dilution. 

• Appropriate dilutions for test carriers will be determined via a screen prior to testing. 

____ Serially dilute fractions A, B, and C for control carriers. 

•	 Vortex mix each tube prior to making the next dilution. 

•	 Appropriate dilutions for control carriers will be determined via a screen prior to testing. 

____ Directly plate 100 μL of the dilutions that will yield counts within the target range (30­

300) for each carrier. 

• Spread using sterile spreader. 

____ Incubate all plates at 36 ± 1°C for a minimum of 24 ± 2 hr (see text for details) 

33 
Appendix: TSM-2 

Three Step Method Schematic 

Resuspend pellet 

Dilute and plate Dilute and plate 

Enumerate Enumerate

pellet 

Fraction A Fraction B 

Expose carrier 

(x min) 

Fraction C 

Transfer carriers to 

B tubes 

Centrifuge/wash Sonciate carrier 

pellet 3 times 5 min ± 30 sec 

Centrifuge 6 min ± Vortex approx 1 min 

30 sec each 

Transfer carriers to 

C tubes 

Dilute and plate Incubate tubes with 

carrier at 36±1°C in a 

Enumerate hematology rotator 

30 min. ± 2 min 

Nutrient agar plate 

with representative 

B. subtilis growth 

34 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time Fraction A

2 

4 

6 

8 

10 

12 

14 

16 

18 

20 

22 

24 

26 

28 

30 

32 

34 

36 

38 

40 

42 

44 

46 

48 

50 

52 

Fraction B Fraction C 

Exposure 

(x minutes) 

Add 600 μL ice-cold neutralizer 

Transfer carriers to B tubes 

Centrifugation 1 

(6 min ± 30 sec) 

Sonication 

(5 min ± 30 sec) 

Add 600 μL ice-cold LB broth 

Remove 900 μL supernatant Vortex (approx. 1 min) 

Add 900 μL ice-cold LB broth Transfer carriers to C tubes 

Centrifugation 2 

(6 min ± 30 sec) 

Dilutions and plating 

Remove 900 μL supernatant 

Add 900 μL ice-cold LB broth 

Centrifugation 3 

(6 min ± 30 sec) 

Remove 900 μL supernatant 

Add 100 μL ice-cold LB broth 

Vortex 

(5 min ± 30 sec) 

Add 800 μL ice-cold LB broth 

Dilutions and plating 

Incubate tubes at 36±1°C in a 

hematology rotator 

(30 ± 2 min) 

Add 600 μL ice-cold LB broth 

Dilutions and plating 

*Time in minutes 

Appendix: TSM-3 

TSM Time Line 

35 
Section 8. REPORTING RAW DATA 

Information and raw data will be recorded on the test forms and data sheets provided by 

the Study Director (Appendices B and C). Electronic spreadsheets, provided by the Study 

Director, will be populated with the data from the hard copy data sheets, peer-reviewed for 

accuracy, and forwarded to the statistician for analysis. The preparation of media and reagents 

will be recorded on Media Preparation Sheets provided by the Study Director (Appendix D). 

Section 9. STATISTICS AND ANALYZING RAW DATA 

For statistical analysis, the Study Director will utilize the services of Dr. Martin Hamilton 

at the Center for Biofilm Engineering, Montana State University – Bozeman. The statistical 

analysis will produce estimates of the repeatability standard deviation, denoted by Sr, and the 

reproducibility standard deviation, denoted by SR, for each treatment (disinfectant × efficacy 

level combination) and for each quantitative response (log reduction value and the control carrier 

log spores per carrier). 

In studies such as this, it is not unusual for the dilution series to miss occasionally the 

counting range of dilutions, thereby providing anomalous counts, either all zeros or all “too 

numerous to count” (TNTC). For such anomalous data, artificial counts will be substituted. If 

all dilutions produce TNTC, 300 will be substituted at the last dilution for the fraction (A, B, or 

C). If all dilutions produce zeros, 0.5 will be substituted at the first dilution. 

Overview 

The statistical analyses will provide the following information. More detailed 

descriptions of the statistical methods are presented in the next section. 

Χ	 Raw data plots – the individual data points will be plotted for visual inspection to see 

trends and effects and to detect outliers or influential observations. 

Χ	 Analysis of control carrier spore titers – the log transformed spores per control carrier 

will be analyzed using an analysis of variance (ANOVA) two factor, nested, random 

effects model (details below). These results will describe the “normal range” of control 

carrier titers for each test method as well as estimates of the Sr and the SR and the withintest, intra-laboratory, and inter-laboratory sources of variability. 

Χ	 log reduction (LR) value – LR is the primary quantitative response and most of the 

statistical work will focus on the LR data. The LR value will be calculated using the 

formulas appropriate for each laboratory test (details below). For each combination of 

test method and test treatment, a one factor, random effects model ANOVA will be 

conducted to estimate the Sr, the SR, and the intra-laboratory versus inter-laboratory 

sources of variability. 

36 
 

Χ	 Mean LR – for each chemical × efficacy level combination, the mean LR will be 

calculated along with the associated standard error and confidence interval. For each 

chemical treatment, a statistical trend test will determine whether the mean LR values 

increase significantly with increasing efficacy level. 

Χ	 Diagnostic plots and tests – performed routinely to check whether the observations 

conform to the mathematical assumptions underlying the ANOVA calculations. 

Χ	 Presentation of results – tables and figures will be created to present the results. 

•	 LR for the AOAC test – To calculate an LR value that is consistent with each AOAC 

test result, the P/N formula will be applied (1, 4). Those LR values will be used to 

calculate SR for the AOAC test. 

•	 Percent of the Total Counts by Fraction – An assessment of the percentage of the total 

spore counts for each fraction of the TSM will be calculated – the contribution of fraction 

C will be assessed for importance. 

Statistical analysis details 

•	 Analysis of control carrier spore titers – The ANOVA will be based on a nested, twofactor, random effects model, similar to the models used in (5, 6). For each combination 

of test method, the response variable is the log spores per control carrier, the main effect 

is laboratory (a random effect), the nested effect is replication (a random effect) and the 

“chance error” is due to the variation among the carriers within the replication. The 

laboratories taking part in this collaborative study are assumed to be statistically 

representative of all laboratories that will be conducting these types of sporicide tests in 

the future. Because the ten participating laboratories were not in fact randomly selected 

from a population of testing laboratories, the assumption is inaccurate; nevertheless, it is 

required for the conventional approach to analyzing collaborative studies. 

In standard statistical notation [see for example (7)], let Yijk denote the log spores 

for the kth carrier in the jth trial (replication) in the ith laboratory, k=1,2, or 3, j=1,2, or 3 

and i = 1,2, ..., 10. Then the model is Yij = : + Li + Tj(i) + γijk , where : denotes the true 

mean log spores per carrier for that test method and treatment, Li denotes the effect of the 

i

th laboratory, Tj(i) denotes the effect of the jth trial (replication) in laboratory i, and γijk 

denotes the chance error for the kth carrier in the jth replicate test in the ith laboratory. 

The parameter : is deterministic; it is a specific, but unknown, numerical value. The 

quantities Li, Tj(i), and γij are random variables. According to conventional assumptions, 

Li follows a normal probability distribution with mean zero and variance ΦL

2

, Tj(i) follows 

a normal probability distribution with mean zero and variance ΦT

2

, and γijk follows a 

normal probability distribution with mean zero and variance Φ2

 . This model implies that 

Yij is a random variable following a normal probability distribution with mean : and 

37 
 

variance Φ 2 2 2

L + ΦT + Φ2

. Let ΦR (= Φ 2

L + Φ 2T + Φ2

) denote the total variance of Yijk. 

Conventionally, ΦR = /Φ 2

R is called the reproducibility standard deviation and Φr = [Φ 2 T

+ Φ2]

1⁄2 is called the repeatability standard deviation (5, 8). The ANOVA will provide

numerical estimates of the parameters :, Φ 2 2	 2

L , ΦT , Φ2

, Φ 2

r , and ΦR . 

•	 Log reduction (LR) values – For the TSM, the LR is the mean of log10 spores per 

control carrier minus the mean of log10 spores per treated carrier. Formulas for 

calculating the LR and associated within-test standard error are presented in Zelver et al. 

(9). For AOAC 966.04, the LR value will be calculated using the P/N formula presented 

in the report to AOAC by Tomasino and Hamilton (4). 

The ANOVA will be based on a one-way, random effects linear statistical model. For 

each combination of test method and chemical, the LR is the response variable, the main 

effect is laboratory (a random effect) and the “chance error” is due to the variation among 

independent repeats of the test within laboratories. The laboratories taking part in this 

collaborative study are assumed to be statistically representative of all laboratories that 

will be conducting these types of sporicide tests in the future. 

In standard statistical notation [see for example (7)], let Yij denote the LR value for a 

specific combination of test method and chemical at the jth replication in the ith 

laboratory, j=1, 2, or 3 and i = 1, 2, or 3. Then the model is Yij = : + Li + γij , where :

denotes the true mean LR for that test method and chemical, Li denotes the effect of the 

i

th laboratory, and γij denotes the chance error for the jth replicate test in the ith laboratory. 

The parameter : is deterministic; it is a specific, but unknown, numerical value. The 

quantities Li and γij are random variables. According to conventional assumptions, Li 

follows a normal probability distribution with mean zero and variance ΦL

2 and γij 

follows a normal probability distribution with mean zero and variance Φr

2

 . This model 

implies that Yij is a random variable following a normal probability distribution with 

mean : and variance ΦL

2

 + Φr

2

 . Let ΦR

2

 (= ΦL

2

 + Φr

2

 ) denote the variance of Yij ; ΦR

2

 is 

called the “total variance” in ANOVA textbooks. In the context of germicide tests, ΦR = 

/ΦR

2

 is called the reproducibility standard deviation and Φr = /Φr

2

 is called the 

repeatability standard deviation (5, 8). The ANOVA will provide numerical estimates of 

the parameters :, ΦL

2

, Φr

2

, and ΦR

2

. If the estimates of the variances differ 

insignificantly among test chemicals, the data may be combined across chemicals for 

purposes of running one ANOVA for that test method. If it is appropriate to do so, 

combining the data will produce more reliable estimates of the parameters. For the TSM, 

by using the within-test standard error associated with each LR, it will be possible to 

partition out the within-test component of variance from Φr

2

. 

Χ	 Mean LR – The mean for each test chemical and test method combination will be 

estimated by the ANOVA. The formula for the standard error of the mean depends on 

the results of the ANOVA and the equations will be derived by the statistician, as in (10). 

The confidence intervals will be based on normal distribution theory. 

It is of interest to determine whether each method is sensitive enough to correctly order 

38 
treatments known to have low, medium, and high efficacy. For each method and 

disinfectant, a trend test will be conducted to determine whether the log spores per treated 

carrier increases significantly with known efficacy. The trend test will be a test for a 

significant mean slope based on a simple linear regression model. The efficacy levels 

will be coded as 1, 2, and 3, going from low to high efficacy. 

•	 Sr and SR of LR values for the TSM – It is desirable for the standard deviations to be 

small. For disinfectant tests, the AOAC has no specifications for concluding that a 

standard deviation is acceptably small. Some guidance is provided by a recent literature 

review which showed that, for established suspension and dried surface disinfectant tests, 

S

median of 0.8 (6). It would be reasonable to claim that the S

r ranged from 0.2 to 1.2 with a median of 0.4 and SR ranged from 0.3 to 1.5 with a 

r and SR are acceptably small 

if they fall within these ranges. 

Χ	 Diagnostic plots and tests – conventional plots and tests of residuals will be used to check 

the homogeneous variance and normality assumptions underlying the ANOVA. The 

Anderson-Darling test will be used to check normality. 

Χ	 The % of the total spore counts for each fraction of the TSM will be evaluated – the 

contribution of fraction C will be assessed for importance. 

Section 10. REFERENCES ASSOCIATED WITH THE VALIDATION 

PROTOCOL 

1. Tomasino, S.F. & Hamilton, M.A. (2006) Unpublished Report. Comparative 

 Evaluation of Two Quantitative Test Methods for Determining the Efficacy of Liquid 

Sporicides and Sterilants on a Hard Surface: A Pre-Collaborative Study 

2. Sagripanti, J.L. & Bonifacino, A. (1996) Am. J. Infect. Control 24, 364 – 371 

3. Standard Test Method for Quantitative Sporicidal Three-Step Method (TSM) to 

Determine Sporicidal Efficacy of Liquids, Liquid Sprays, and Vapor or Gases on 

Contaminated Surfaces. (2005) ASTM Designation E 2414 – 05 

4. Tomasino, S.F. & Hamilton, M.A. (2006) Unpublished Report. Modifications to the 

AOAC Sporicidal Activity of Disinfectants Test, Method 966.04: Collaborative Study 

5. Youden, W.J. and Steiner, E.H. (1975) Statistical Manual of the AOAC. AOAC: 

Arlington, VA 

6. Tilt, N. and Hamilton, M.A. (1999) JAOAC Int., 82, 384 – 389 

7. Neter et al., (1996) Applied Linear Statistical Models – 4th edition, McGraw-Hill:Boston 

8. Helrich, K., editor, (1990) Official Methods of the AOAC, AOAC: Arlington, VA, p. 

681, 1990 

9. Zelver, N. et al. (2001) Methods in Enzymology - Biofilms II, R.J. Doyle, editor, 337:363 

– 376 

10. Johnson, K., Lundman, R., and Hamilton, M. (1993) Efficient Sampling Designs for 

Microbial Processes: A Case Study. Journal of Microbiological Methods, 18:69 – 81 

39 
Section 11. APPENDICES 

A. EPA Quality Assurance Project Plan 

B. AOAC Method Data Sheets 

C. TSM Data Sheets 

D. Media and Reagent Preparation Sheets 

E. Parameters for Testing Chemicals 

F. TSM Log Reduction Data 

G. Material Safety Data Sheets

 H. Safety Checklist 

40 
B-1 
Appendix C – Guidelines for the Development and Validation of 

Nucleic Acid Amplification (PCR) Based Microbiological Methods 
Introduction 

This section provides initial guidance that specifically considers how to develop and 

validate nucleic acid amplification-based methods for environmental sample analyses. This 

guidance specifically refers to methods incorporating the polymerase chain reaction (PCR) 

amplification technique; however, this guidance should also be applicable to methods 

incorporating other nucleic acid amplification techniques. 

1.0 The PCR Method: General Background 

A PCR method encompasses a series of molecular procedures including 1) sample 

preparation, 2) nucleic acid amplification with a select PCR assay, 3) visualization of results, and 

4) interpretation of data. PCR amplification is an in vitro enzymatic technique for rapidly 

increasing the quantities of specific nucleic acid segments present in small or complex samples 

to sufficiently high levels to allow their detection by optical, physical and other methods. PCR 

has been routinely used in clinical and food microbiology for many years. For a detailed review 

of PCR methods, please see (Sambrook and Russell 2001 McPherson and Moller 2006). 

Two common approaches for PCR amplification are end-point and real-time detection. 

End-point detection is the classical approach where the presence of the target sequence is 

determined by analysis for the amplified copies by an independent technique (e.g., gel 

electrophoresis) after the reaction has been completed. This approach is most commonly used as 

a presence/absence test. Real-time PCR monitors production of target sequence copies 

throughout the amplification process, either by use of a specific fluorescently-labeled probe 

sequence or with a nonspecific intercalating dye, and yields both qualitative and quantitative 

data. Detailed explanations of real-time PCR detection processes are available elsewhere 

(Sambrook and Russell 2001, Bustin 2004). Both end-point and real-time PCR approaches can 

easily detect a specific target sequence in a complex mixture of nucleic acids and often with a 

limit of detection of as little as one copy per reaction, yet the potential for poor DNA purification 

efficiency and/or potential inhibition of amplification from environmental samples often means 

that many target microbes are required in the original sample. Nonetheless, various advantages 

of PCR methods have led many researchers to develop applications of this technology for 

environmental science. 

C-1 
2.0 Method Selection 

When considering a particular PCR method for validation, it is necessary to clearly define 

the intended use of the method, manner of data analysis and the environmental matrix or 

matrices of interest. The decision to select a PCR-based analytical method for validation should 

also take into account the criteria specified in Section 2.2 of the main report. Criteria that may be 

particularly important in deciding between a PCR or culture-based method for an intended 

purpose are accuracy, precision, and the relative cost and level of training required for each 

approach. In general, this justification for PCR methods requires instrumentation, with expected 

advantages in sensitivity, specificity, rapidity, and throughput; or by the unavailability of a 

comparable culture method for the target microorganism(s) of interest. Another consideration 

may be the objective of detecting viable organisms. PCR methods generally will be unable to 

make this distinction although more recent modifications have shown promise in this regard, 

e.g., reverse transcription PCR for detection of labile messenger RNA molecules or 

intermediates of ribosomal RNA processing and pretreatment of samples with intercalating dyes 

(such as propidium monoazide) that may only permeate cellular membranes of nonviable 

organisms (Nocker et al. 2007). 

3.0 Method Development and Optimization 

A sound method development and optimization process is critical for a successful 

validation. The following section briefly describes some of the key elements to consider during 

the development and optimization of a PCR method. 

3.1 Design of Primers and Probes 

PCR method development normally begins with the design of PCR assay primers and, in 

the case of real-time PCR, probes. A primer is a strand of nucleic acid that serves as a starting 

point for DNA replication. PCR assays typically require two primers to target a specific genomic 

region. PCR primers are short, chemically synthesized oligonucleotides, with a length of about 

20 bases. They are designed to hybridize to a DNA target, which is then copied by a DNA 

polymerase. Probes are another type of oligonucleotide used in many quantitative real-time PCR 

applications. Probes are typically designed to anneal within a genomic region amplified by a 

specific set of PCR primers, which can be used to increase PCR assay specificity, Probes 

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normally contain both fluorophore and quencher molecules. The quencher molecule quenches 

the fluorescence emitted by the fluorophore during excitation when in close proximity. This 

predictable fluorescence trend can then be used to estimate the concentration of target DNA 

molecules after each step of amplification. 

After determining the intended use of a particular PCR method, the first consideration 

may be the choice of the genomic region that will be targeted by the assay. For example, a PCR 

method intended to detect and enumerate fecal pollution may target genomic regions that are 

unique to bacteria associated with fecal contamination. The prediction of specificity and 

sensitivity of the primers and/or probe for the intended genetic target will typically be the next 

consideration and is determined from the available database of sequences from both target and 

non-target organisms. Several publicly (such as GenBank) and commercially available computer 

software programs are available that can aid in this process. 

Concurrent with this process should be the evaluation of the candidate primer and/or 

probe sequences for their abilities to satisfy a number of basic requirements for PCR 

amplification. Some of these requirements include primer sequence lengths and melting 

temperatures, G + C content, secondary structure, and hybridization stringency, as well as other 

features. Despite the increasing sophistication of PCR assay primer and probe design programs, 

different primer pairs for the same target sequence region can exhibit significant differences in 

performance (He et al 1994). For this reason, there is no substitute for experimental testing of a 

candidate PCR assay. Part of this process may also include the optimization of conditions under 

which the assay is performed. 

3.2 Optimization of Reaction Conditions 

Variable conditions that can be examined include thermal cycling times and 

temperatures, salt and polymerase co-factor (e.g., magnesium) concentrations, primer and probe 

concentrations, and nucleic acid quantity in the reaction. These variables may also be 

characterized within the context of a single PCR instrument and reagent system or with multiple 

combinations of commercially available instruments and PCR reagents. With so many conditions 

and their interactions, a factorial design works very well for PCR optimization. 

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3.3 Sample Preparation 

A PCR method consists of not only the target sequence PCR assay, but also the 

procedures for preparing samples for these analyses. A requisite component in developing a PCR 

method is therefore the selection and optimization of the sample preparation procedure. At a 

minimum, this procedure includes the isolation and recovery of nucleic acids across a range of 

environmental sample quantities determined by the intended use of a particular PCR method. In 

some cases, this procedure may also include sample concentration and purification of the 

extracted nucleic acids. The primary variables that must be considered in the selection and 

optimization of a sample preparation procedure are target sequence recovery efficiency and 

inhibition of target sequence amplification by co-extracted substances originating from the 

environmental sample matrix. Hence, controls should be designed to estimate sample preparation 

efficiency as well as PCR assay inhibition. 

3.4 PCR Inhibition Control 

Partial or complete inhibition of PCR amplification can be caused by a number of 

contaminants most often resulting from insufficient purification during sample preparation. 

There are a number of inhibitory substances that can co-extract with nucleic acids recovered 

from environmental samples due to similarities in solubility, charge and/or molecule size 

(Wilson 1997). The resulting PCR inhibition can completely prevent the amplification of target 

nucleic acids or reduce sensitivity resulting in false negatives or incorrect real-time quantitative 

PCR measurements. Thus, the inadvertent presence of PCR inhibitors can confound even the 

best designed and optimized PCR method. The addition of an amplification control sequence to 

each reaction can be used to distinguish between a false negative, true negative, or PCR 

inhibition. Amplification controls can be designed to amplify either simultaneously with the 

target nucleic acid or in a separate reaction. The amplification control signal should always be 

produced even when there is no target nucleic acid present. There are many strategies to 

construct, detect, quantify, and store IAC templates. Some design approaches require substantial 

technical skills, while others rely on more basic techniques. It is important to consider the 

advantages and drawbacks, as well as the intended use of the PCR method when selecting a 

particular IAC strategy. For a detailed discussion, see Hoorfar et al. 2004. At this stage, most 

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IAC are not suited to also check for nucleic acid extraction performance, hence additional 

sample processing controls may also be required. 

3.5 Sample Processing Control 

Controls that can be used to monitor the efficiency of DNA recovery from the 

sample are commonly recommended and are often referred to as sample processing controls 

(SPCs). In addition to their use for estimating sample preparation efficiencies, data resulting 

from these analyses can also be used to adjust results for variability in sample preparation 

efficiency in a particular environmental matrix compared to standard laboratory conditions. 

Depending on the sample preparation method and the target organism involved, an SPC can 

consist of a nucleic acid target sequence or whole organism that is added to the sample prior to 

processing. Whole organisms that are as similar as possible to the true target organism(s) in their 

physical properties are preferable. 

3.6 Preparation and Use of Standards for Quantitative Real-time PCR Applications 

Estimates of absolute target DNA concentration in environmental samples depends upon 

the quality of the nucleic acid standards that are employed. For microbial gene targets, DNA 

standards can consist of purified preparations of genomic DNA, PCR amplicons, or synthetically 

prepared DNA molecules. Each of these types of standards has advantages and disadvantages. 

Genomic DNA standards have the advantage of most closely representing the actual template of 

a particular PCR assay and any possible effects they may have on amplification efficiency. The 

potential disadvantages of these standards lie in the difficulty that they create for accurately 

determining the concentration of target sequence copies present and in obtaining reproducible 

DNA preparations. Estimates of the target gene copy concentrations in these preparations should 

be verified by limiting dilution PCR analysis or by some other comparable means. Amplicons 

and synthetic DNA standards can be used in PCR assays for which genomic DNA is not 

available and are also more amenable to accurate quantitative estimation of target sequence copy 

concentrations. However, the amplification efficiency of these templates should be compared, 

when possible, with genomic DNA templates to determine equivalency. For target nucleic acids 

of major importance, internationally accepted standard reference materials should be established. 

For example the World Health Organization has established quantitative standards for HCV, 

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HBV, and HIV viruses. Similar approaches for the establishment of reference standards for other 

real-time PCR applications should be possible through certification by national or international 

organizations such as NIST or NIBSC. Analysis results of a reputable commercial or other noncommercial organization may be sufficient for the certification of quantitative reference 

standards for some real-time PCR applications. 

3.7 Real-Time Quantitative PCR Data Analysis 

For most real-time quantitative PCR applications, a cycle threshold (CT) measurement is 

used to estimate the DNA target concentration in a particular environmental sample. Because 

real-time quantitative PCR is based on the theoretical premise that there is a log-linear 

relationship between the starting amount of DNA target in the reaction and the CT value that is 

obtained, the CT value can then be used to estimate the initial concentration of a DNA target 

from an unknown sample. Two general strategies are often used to make these estimates, 

including relative and absolute approaches (ABI: Essentials of Real Time PCR. Applied 

Biosystems 2006). A relative quantification approach measures the change in target DNA 

concentration relative to another reference sample. In contrast, absolute quantification approach 

is achieved by using a standard curve, constructed by amplifying known amounts of target DNA 

in a parallel set of reactions (ABI: Absolute Quantitation Using Standard Curve Getting Started 

Guide. Applied Biosystems 2006). The approach selected should be clearly described and should 

adequately address uncertainties associated with a particular PCR method. For example, 

uncertainty can arise within and between experiments from numerous sources such as 

inconsistencies in quality of reagents, pipet calibration, as well as dilution preparation and 

storage of standards. Any of these factors could significantly alter CT measurements from 

experiment to experiment. Therefore, estimation and propagation of uncertainty throughout data 

analysis becomes critical to account for sources of variability and make reasonable estimates of 

environmental sample DNA target concentrations. 

4.0 Quality Assurance and Quality Control (QA/QC) 

Proper laboratory QA/QC procedures are essential to a successful PCR method. The 

sensitivity required for the synthesis of billions of target nucleic acid molecules make PCR 

methods prone to contamination from extraneous DNA, which can lead to false-positive results. 

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Laboratories performing PCR methods should establish a sufficient number of controls for the 

detection of contaminating DNA molecules that can be introduced during sample preparation and 

PCR amplification. Thus, strict protocols must be followed to assure that personnel, facilities, 

workflow, equipment, disposables, negative controls, and laboratory cleaning practices are 

adequate to avoid contamination of results. It is also important that instrument QC procedures be 

followed. Laboratory QA/QC guidance for PCR methods are discussed in “Quality 

Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on 

Environmental Samples” (USEPA 2004). As part of a lab’s QA program the fact that QA 

procedures have been followed should be clearly documented. 

5.0 PCR Method Performance Criteria 

Although the general definitions of the performance criteria described in Section 2.4 of 

the main document remain the same, this section provides examples of approaches for deriving 

the required performance criteria for methods utilizing PCR. It should be noted that these 

examples are not all-inclusive and that performance criteria should be measured across the entire 

method from sample collection through sample preparation to PCR amplification and 

interpretation of results. In addition, performance criteria that require the use of a standard or 

control spike may utilize DNA targets reported as cell equivalents, genome equivalents, copies 

of DNA and/or mass of DNA depending on the intended use of the PCR method. Metrics for 

report values should be detailed in the method. 

5.1 Specificity and Sensitivity 

Specificity is the ability of a PCR method to discriminate between target sequences. 

There are many factors that can impact the specificity of a PCR method such as primer design; 

degeneracy of the primers; presence of heterologous nucleic acids originating from a sample of 

interest; total amount of nucleic acids in the PCR experiment; quality of extracted nucleic acids; 

PCR amplification conditions such as buffer composition, primer concentration, and thermal 

cycling parameters; matrix effects such as co-extracted impurities that can cause inhibition; nonspecific fragment amplification, and quality of reference samples available. It is important to 

note that specificity values are determined from a collection of reference samples, and therefore 

this estimate is only as good as the available reference standards. Reference collections such as 

C-7 
DNA sequence databases can be riddled with errors and are routinely updated making it even 

more challenging to standardize specificity testing. Poor specificity can be identified in 

numerous ways including amplification of incorrect sized target sequences, background smears 

in samples that contain fragments of the correct size, unexpected bands in controls that are not 

attributable to cross-contamination, and correct sized fragments that fail to be confirmed by 

another technique (e.g. restriction analysis, DNA sequencing, hybridization). However, some 

PCR methods are designed to detect target sequences in complex mixtures of heterologous 

nucleic acid preparations that can significantly vary in composition and amount from one sample 

to the next. For these methods, mixture studies that measure the ability of a given PCR method to 

obtain reliable results from mixed source samples may be necessary. Mixture ratios should 

represent the range of conditions that may be encountered when implementing the method 

(SWGDAM 2004). 

Sensitivity is the probability that a PCR method will classify a test sample as positive, 

given that the test sample is a “known” positive. Sensitivity can be affected by characteristics of 

the matrix and can be measured under laboratory conditions with a target spike into a 

characterized matrix such as molecular grade water (laboratory sensitivity) or uncharacterized 

matrix such as an environmental sample (field sensitivity). 

5.2 Precision 

For end-point PCR detection methods that generate qualitative data, measures of 

precision, such as repeatability and reproducibility have little value. Quantitative real-time PCR 

applications allow for more refined measurements of precision in which the amount of variability 

observed from a series of repeated measurements of a reference standard can be determined. 

Precision is often expressed as the relative standard deviation (RSD), which is the absolute value 

of the percent coefficient of where: 

RSD = (standard deviation of measurements x 100) / mean Equation (6) 

Precision can be reported for the amplification assay or for the entire method. Figure C-1 

illustrates an example of calculating precision for a quantitative real-time PCR assay. 

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Figure C-1. Example Calculation of precision expressed as RSD for raw CT data from a 

fitted curve for quantitative real-time PCR. Data points in circle represent 

independent measurements of the same DNA standard concentration used to 

calculate a mean and standard deviation. 

For many chemical and culture-based methods, reference standards are typically defined 

by the International System of Units and are maintained by national or international 

organizations. However, for most quantitative real-time PCR methods, nationally or 

internationally recognized reference standards are not available, making it more challenging to 

establish precision within and between laboratories. To date, there is no commonly accepted 

practice for determining precision for a quantitative real-time PCR method. This is, in part, due 

to a lack of standardized reference samples, but also because a quantitative real-time PCR 

method includes multiple steps such as sample preparation, amplification, and data analysis that 

can each introduce uncertainty and error. Ideally, a standardized reference sample should be 

included through the entire quantitative real-time PCR method, resulting in an estimated 

concentration measurement. 

Precision is sometimes classified into repeatability and reproducibility (Section 2.4.2 of 

the main document). Repeated measurements generated on the same day, with the same lot of 

reagents, on the same instruments, by the same technician can be used to calculate quantitative 

C-9 
real-time PCR method repeatability. In contrast, repeated measurements generated from the same 

process among different lots of reagents, instruments, and technicians over longer periods of 

time can be used to estimate the reproducibility of the method. 

5.3 Accuracy and Bias 

Accuracy is defined as the ability of a quantitative real-time PCR method to correctly 

enumerate a “known” number of DNA targets. Accuracy should be measured with blind spikes 

of DNA targets and blind spikes of the whole organism. Known DNA targets would be spiked 

into molecular grade water and also a characterized matrix with known quantities of potential 

chemical and microbial interferences as well as inert substances typical of the intended sample 

substrate (laboratory accuracy) or uncharacterized matrix such as an environmental sample (field 

accuracy). Samples of uncharacterized matrices containing blind spike material should be split 

for analysis in different laboratories. Careful preparation and handling of standards for accuracy 

measurements is critical for the estimation of PCR method accuracy. Bias is the difference 

between the observed measurement/estimate of DNA copy number and the known standard 

concentration. 

5.4 Limit of Detection (LOD) and Limit of Quantification (LOQ) 

LOD is the minimum amount of target sequence that can be detected with a given level of 

confidence in a well characterized background matrix (e.g., distilled water or a buffer solution). 

LOQ is similar to LOD except it is a range of the upper and lower bounds that can be quantified 

with a predetermined acceptance level of precision, accuracy and specificity. Acceptance levels 

are subjectively determined based on the intended use of a particular method. For example, 40 

CFR Part 136 Appendix B contains equations for calculating detection limits for methods 

promulgated by EPA and defines the detection limit as the minimum concentration of a 

substance that can be measured and reported with 99% confidence that the analyte concentration 

is greater than zero. It should be noted that a 99% confidence may not be necessary for all 

methods. It is important to establish the LOD and/or LOQ for a PCR method to generate a 

baseline performance level to which a researcher can compare performance in uncharacterized 

matrices where there could be potential amplification inhibitors. LOD/LOQ measurements are 

easily determined for both end-point detection and real-time PCR approaches. However, endC-10 
point detection is typically based on visual detection of PCR products and can vary somewhat 

among analysts, types of electrophoresis gels, and nucleic acid staining agents. 

5.5 Sample Limit of Detection (SLOD) 

SLOD is the minimum amount of target sequence that can be detected with a given level 

of confidence in an uncharacterized background matrix (e.g., environmental sample). SLOD 

estimates are specific to a particular matrix background and can vary from one sample to the 

next. SLOD experiments are performed in the same manner as LOD experiments (Section 2.4.4), 

however, reference standards are spiked into the environmental sample matrix rather than 

laboratory grade water or buffer. SLOD can be expressed as the minimum number of cell 

equivalents, genome equivalents, target sequence copies, or mass of target sequence with a 

confidence interval that can be detected (end-point PCR) or enumerated (quantitative real-time 

PCR). 

5.6 Linearity, Range of Quantification (ROQ), and Amplification Efficiency 

A standard curve, constructed by testing a series of serial dilutions of known 

concentrations or copy numbers, provides important information for validating qPCR methods. 

Due to the stochastic nature of nucleic acid amplification, especially at low nucleic acid target 

concentrations, five to eight dilutions (e.g., 10-fold) bracketing the range of concentrations (for 

which the PCR method will be used) are used to characterize the relationship between nucleic 

acid concentration and response (FDA 2001). From this data a linear plot of CT vs. the logarithm 

of the target copy number is generated (Figure C-1). Linearity, expressed as the coefficient of 

determination R-squared (R2

); (Moore and McCabe 1989), is a measure of the range of target 

nucleic acid concentrations for which a quantitative PCR test result is directly proportional to the 

nucleic acid target concentration. ROQ is defined as the range of nucleic acid target 

concentrations that are detectable with an acceptable level of precision, accuracy, and specificity. 

Linearity and ROQ are determined by testing different concentrations of standard nucleic acid 

control samples to generate a plotted curve. For real-time PCR, R2 is a statistical measure of how 

well a regression line approximates CT values obtained from repeated testing of nucleic acid 

standards (Figure C-2). An R2

 value of 1.0 (100%) indicates a perfect fit. The degree to which 

the plotted curve conforms to a straight line indicates the PCR method linearity. ROQ can then 

C-11 
be calculated by determining the difference between upper and lower (LOQ) bound 

concentrations where quantitative measurements are linear and within acceptable levels of 

precision, accuracy, and specificity. 

Amplification efficiency is calculated from the slope of a qPCR standard curve and is 

expressed as: 

Amplification Efficiency = (10 (1/-slope) )-1 Equation (7) 

Under ideal conditions, the PCR product doubles after each cycle during exponential 

growth. This relationship can be numerically expressed as an exponential amplification of 2.0, 

which is equivalent to amplification efficiency of 100%. Amplification efficiency can be 

influenced by factors such as length, G/C content, and secondary structure of the amplification 

product. To assure accurate and reproducible estimates of DNA target concentration, the slope of 

the standard curve should indicate an amplification efficiency as close to 100% as possible. 

Figure C-2: Example of determining the linearity and ROQ for a quantitative real-time 

PCR assay. The fitted curve line with a 95% confidence interval represents the best 

fit line based on CT measurement from three independent experiments testing a 

broad range of target nucleic acid standard concentrations. The linearity of the 

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assay is reported as an R2 value. The ROQ is depicted as the range of nucleic acid 

standard concentrations detectable within a 95% confidence interval. 

5.7 Ruggedness 

Ruggedness (unavoidable changes) is the ability for a PCR method to perform within 

acceptable precision and accuracy performance levels under normal but unavoidable variable 

conditions; determined by testing identical samples under variable conditions. Factors such as 

reagent stock stability, analyst to analyst variation, use of different thermal cycling instruments, 

laboratory to laboratory variation, nucleic acid target stability, and optimal performance over 

time can all contribute to the ruggedness of a PCR method (FDA 2001). Ruggedness can be 

expressed as the change in precision. For qualitative end-point PCR methods, ruggedness can be 

determined by measuring changes in false positives/false negatives and LOD. For real-time 

quantitative PCR methods, ruggedness can be evaluated using the Horwitz equation (Horwitz 

1995) where the relative standard deviation of reproducibility (RSDR) is given as: 

RSDR (predicted) = 2 (1-0.5logC); and Equation (8) 

HORRAT = RSDR (observed) / RSDR (predicted) Equation (9) 

Where: 

C is the observed concentration expressed as a decimal fraction and log is base 10. The 

ratio between observed and predicted RSDR values is designated HORRAT and can be 

used as an indication of acceptability (HORRAT ratios < 1.0 indicates acceptable 

precision). 

6.0 Multilaboratory Validation Studies 

A document describing the PCR method should be prepared as described in Section 2.5 

of the main document. General guidance for the performance of collaborative validation studies, 

as described in Section 2.6 of the main document should be applicable for these methods. 

6.1 Influence of PCR Platform and Reagent System 

It is contrary to general policies within EPA that discourage endorsement or 

recommendation of specific commercial products; however, it may be necessary to treat assays 

that utilize different platforms as different methods for the purpose of validation. The popularity 

C-13 
of the PCR technique has given rise to the availability of a wide choice of thermal cycling 

instrument and amplification reagent systems from different commercial vendors. Since PCR 

methods are typically developed and optimized with only one such system, the validation of 

these methods would be limited to that system under ideal circumstances. Particularly with 

respect to different instruments, however, this practice would limit the number of participants 

that may be available for multiple laboratory studies, as well as the general acceptance of a 

method by its anticipated end-user community. 

Prior to performing a collaborative validation study involving multiple instrument and/or 

reagent systems, it is highly desirable to experimentally assess the equivalence of method 

performance with all systems being considered. Particular attention should be paid to any 

significant differences in specificity and sensitivity with these systems. Assay specificity may be 

affected by different amplification reagents (Siefring et al. 2008). Preliminary assessments of 

specificity can be performed through the analysis of common DNA standards from one or more 

non-target controls that are closely related to the target of interest. If variability in specificity is 

observed, it may be possible to adjust the annealing temperature of the thermal cycling protocol 

for each respective reagent and instrument to bring it within acceptable levels. It should be noted, 

however, that comparable specificity may be difficult to demonstrate for all possible target and 

non-target organisms. 

The sensitivity of an assay on different instruments can be predetermined by the analysis 

of common DNA standards. Large differences in sensitivity with a given reagent may require 

instrument-specific adjustments in thermal cycling parameters or the exclusion of less sensitive 

instruments from being utilized in a validation study with that reagent. Due to differences in 

optics, some variability in sensitivity may be unavoidable with different instruments but this may 

not necessarily exclude the use of the less sensitive instruments. Since such differences in 

sensitivity should apply equally to the analysis results of test samples and DNA standards on 

each instrument, the comparability of quantitative measurements should not be affected within 

each instrument’s respective ROQ. If acceptable minor differences in sensitivity and LOQ are 

observed, these characteristics may be defined beforehand for each platform if economically 

feasible, and the differences taken into consideration in the design of a validation study. 

C-14 
 

7.0 Potential Differences between PCR Chemistries 

Several different probe and non-probe based PCR product detection chemistries are also 

available for real-time PCR assays (Wittwer and Kusukawa 2004). Moreover, product detection 

reagents employing these chemistries are often available from numerous commercial vendors. 

Because of the high potential for the performance of a PCR assay to be altered by these different 

chemistries, as well as by different commercial sources of the product detection reagents, it is 

recommended that the chemistry and commercial source of reagents used for method 

development be specified for each real-time PCR method. Alternative sources of reagents can be 

designated if their products can be experimentally demonstrated to provide equivalent 

performance in the method to that of the reagents from the originally specified source. 

8.0 References 

1. Bustin, S.A. 2004. Quantification of Nucleic Acids by PCR. In S.A. Bustin, A-Z of 

Quantitative PCR. La Jolla, CA. International University Line. 

2. FDA. 2001. Guidance for Industry: Bioanalytical Method Validation. U.S. Food and 

Drug Administration. 

3. He, Q, M. Marjamäki, H. Soini, J. Mertsola, and M.K. Viljanen. 1994. Primers are 

decisive for sensitivity of PCR. Biotechniques 17: 82, 84, 86-7. 

4. Hoorfar, J., B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach. 2004. 

Practical Considerations in Design of Internal Amplification Controls for Diagnostic PCR 

Assays. Journal of Clinical Microbiology 42:1863-1868. 

5. Horwitz, W. 1995. Protocol for the design, conduct and interpretation of method 

performance studies. Pure Appl. Chem. 67:331-343. 

6. McPherson, M.J. and S.G. Moller. 2006 PCR (The Basics). Second Edition. Taylor and 

Francis, New York, NY. 

7. Moore and McCabe 1989, Introduction to the Practice of Statistics. W.H. Freeman & 

Company, New York, NY. 

8. Nocker, A., Sossa, K.E. and Camper, A.K. (2007) Molecular monitoring of disinfection 

efficacy using propidium monoazide in combination with quantitative PCR. Journal of 

Microbiological Methods 70, 252-260. 

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9. Sambrook, J., and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 4th 

ed, vol. 1-3. Cold Spring Harbor Laboratory Press, New York. 

10. Siefring, S., M. Varma, E. Atikovic, L. Wymer and R.A. Haugland R.A. 2008. Improved 

real-time PCR assays for the detection of fecal indicator bacteria in surface waters with 

different instrument and reagent systems. J. Water and Health. 6, 225-237. 

11. SWGDAM. 2004. Revised Validation Guidelines: Scientific Working Group on DNA 

Analysis Methods. Forensic Science Communications 6. 

12. Wilson, I. G. 1997. Inhibition and facilitation of nucleic acid amplification. Applied and 

Environmental Microbiology 63:3741-3751. 

13. Wittwer, C.T., and N. Kusukawa. 2004. Real-time PCR, p. 71-84. In D.L. Persing, F.C. 

Tenover, J. Versalovic, Y-W. Tang, E.R. Unger, D.A. Relman, and T.J. White (ed.), 

Molecular Microbiology, Diagnostic Principles and Practice. ASM Press, Washington 

D.C. 

C-16 
Appendix D – Guidelines for the Validation of Efficacy Test Methods 
Introduction 

EPA regulates antimicrobial pesticides under the Federal Insecticide, Fungicide, and 

Rodenticide Act (FIFRA). Antimicrobial pesticides are substances used to kill or suppress the 

growth of harmful microorganisms on inanimate objects and surfaces. They are divided into two 

categories: Non-public health and public health products. 

The Agency has waived requirements to submit efficacy data for registration of nonpublic health antimicrobial products. However, each applicant and registrant must ensure 

through laboratory testing that products are efficacious, and maintain the data on file if the 

Agency requests its submission. Registration of public health antimicrobial products, on the 

other hand, requires submission of efficacy data to support each label claim and use pattern. 

1.0 Selection of Test Method to Generate Product Efficacy Data 

Efficacy requirements of antimicrobials are set forth in the 40 Code of Federal 

Regulations (CFR) Part 158. Guidance documents specific for efficacy testing of antimicrobials 

are found in Registration Policy Documents and Disinfectant Technical Science Section 

(DIS/TSS) and are posted on the Agency’s Web site (www.epa.gov/oppad001). The guidance 

documents recommend specific test methods and performance standards (e.g., pass/fail criteria) 

for each efficacy claim (e.g., disinfectant, sanitizer, sterilant, etc.). Use of standardized, validated 

test methods are preferred by the Agency, but not required. Validated methods are preferred 

because the method’s performance was evaluated and deemed suitable for its use (e.g., suitable 

for efficacy evaluation of liquid products on hard surfaces). Most of the efficacy test methods are 

archived and managed by AOAC International, a standard-setting organization. In most cases, an 

AOAC method is validated according to strict guidance provided by AOAC, and official changes 

or modifications to a method can only be approved under AOAC purview. AOAC methods have 

been in place for several decades and are primarily qualitative in nature (i.e., presence or absence 

of viable test organism following exposure to test chemical). For example, data generated using 

the AOAC Use Dilution Methods, the AOAC Germicidal Spray Products Method, and the 

AOAC Sporicidal Activity of Disinfectants Method support certain claims for public health 

pesticides and are considered critical to the Agency’s decision making process in registering 

antimicrobial pesticides. Other currently-recommended methods are those that the Agency 

believes have historically proven to be well-developed and suitable for their intended use (e.g., 

D-1 
ASTM standards) but not necessarily subjected to multi-laboratory validation studies. Currently, 

the Agency is promoting the development of quantitative (i.e., kill measured as a log reduction) 

rather than qualitative efficacy tests and is spearheading improvements to existing qualitative and 

quantitative methods to further enhance method performance. In addition, the Agency is 

currently working to harmonize antimicrobial efficacy test methods internationally. For efficacy 

claims for which no recommended/standard test method exists (e.g., biofilm disinfection) or for 

instances in which a registrant believes a new protocol may better demonstrate efficacy of a 

product than the standard method, registrants may submit protocols to the Agency for review 

prior to data collection. The review process includes an extensive in-house and optional expert 

panel protocol review, requirements for independent verification of the protocol in three separate 

laboratories and demonstration of statistical validity 

(www.epa.gov/oppad001/efficacyproto.htm). For all new methodology, the registrant, through 

EPA’s protocol review process, will have to provide historical evidence (i.e., data) that the 

method is reproducible and relevant for its intended use. 

2.0 Validation of Efficacy Test Methods and Study Plan Preparation 

Although EPA does not currently require validated methodology, the user community 

(i.e., registrant, government agencies) may determine that there is a need for determining method 

performance across laboratories and may seek validation. EPA is interested in rigorous method 

validation to ensure that the Agency and its stakeholders have the means to generate data that are 

accurate and reliable. The conventional approach of validating a method through collaborative 

study under the auspices of a third party, standard setting organization such as AOAC 

International is highly desirable, but is not required. 

Historically, EPA recognizes and recommends the use of many AOAC International 

methods because of the standard setting organization’s validation process. AOAC International 

has a well-structured validation program (Official Methods Program) that evaluates methods 

through interlaboratory collaborative studies (minimum of 8-10 labs), and provides a benchmark 

for the method validation process. Multi-lab collaborative studies are used to determine key 

performance indicators of the method, including between and within-lab variability and 

ruggedness. In advance of submitting a protocol for validation, it is advisable to generate inhouse validation data or arrange for an independent lab to validate the method. For more 

D-2 
information on AOAC International’s method validation program, consult their Web site at 

www.aoac.org. 

Method validation may also be achieved by individuals who wish to conduct a 

collaborative study and publish the results in a peer-reviewed scientific journal, without 

participation in a formal third party validation program such as the AOAC Official Methods 

Program. For scientists interested in pursuing method validation independently, the AOAC Web 

site is an excellent source of information on collaborative study design and data analysis (see the 

Official Methods Program Manual at www.aoac.org/vmeth/omamanual/omamanual.htm). 

Once a method is validated and published, it is important that it be updated on a periodic 

basis. Methods are not static. Over time, vendors and technologies change, and new formulations 

or surfaces are introduced, necessitating revisions of standard methods. 

3.0 Pre-Validation Considerations: Factors Affecting Efficacy of Antimicrobial 

Products 

Springthorpe and Sattar (2005) provide an excellent overview of factors affecting the 

antimicrobial activity of products. An understanding of these factors is crucial to the 

development of an efficacy test method that is to be subjected to validation. Below is a brief 

summary of the factors described by Springthorpe and Sattar (2005): 

Χ	 Formulation – minimum concentration of active ingredients required for efficacy; 

different inert ingredients have different affects on efficacy of the product. 

Χ	 Target organism/organic soil load – Microorganisms vary in their ability to survive 

exposure to antimicrobials. In a healthcare environment, microorganisms may be 

found in blood and other body fluids (organic soil) present on surfaces, making them 

more difficult to kill. 

Χ	 Temperature – Product label instructions regarding temperature must be provided, as 

efficacy generally increases with increase in temperature. 

Χ	 Product diluent – Hard water can decrease efficacy of a diluted product. For products 

without hard water claims, labels rarely specify the type of water to use for dilution. 

Distilled water was typically the diluent used in efficacy testing performed to support 

product registration. 

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Χ	 However, product users, without specific label guidance, are more likely to use tap 

water than distilled water to dilute the product. Tap water varies in hardness and the 

user may unintentionally reduce the efficacy of the product by using tap water. 

Χ	 Contact time – Treated surfaces must remain wet for a minimum of the labelspecified contact time for the product to be effective. 

Χ	 Carrier surface – Microorganisms must be eliminated from a variety of surfaces (e.g., 

steel, glass, wood) in the environment. Carriers used in efficacy tests must effectively 

simulate the surfaces to be treated by an end user. 

Χ	 Precleaning agents – Many antimicrobial product labels specify pre-cleaning of the 

surface to be treated. Some cleaning agents may inhibit the efficacy if not rinsed 

effectively from the surface prior to application of the antimicrobial. 

Χ	 Method of application – Different methods of application (e.g., mop, cloth, sprayers) 

result in different amounts of product applied to the surface to be treated. Amount 

applied per surface area may affect efficacy. 

Χ	 Storage and shelf life – Conditions and length of storage of a product may adversely 

affect efficacy. Once diluted, a product’s potency may decrease more rapidly than 

with the concentrated product. 

Χ	 pH/humidity – Antimicrobial products work best at specified pH and humidity levels. 

Product labels should provide guidance as to optimal pH and humidity levels for 

product use. 

In addition to the factors discussed by Springthorpe and Sattar (2005), there are the 

following factors that influence the outcome of an efficacy evaluation of an antimicrobial 

product: 

Χ	 Inoculum titer of microorganisms present in the test system – Many current EPArecommended efficacy test methods are qualitative (presence/absence of viable 

microbes after treatment) rather than quantitative. Consequently, tests may vary in the 

number of organisms present on a carrier or in the test system. Depending upon the 

sensitivity of a method, the outcome of efficacy evaluation may be affected by a 

variable population of microorganisms in the test system. 

Χ	 Quality of microorganisms present in the test system – Currently, there is no 

established standard (i.e., chemicals/disinfectants) for use in efficacy test methods. 

Consequently, for decades, scientists have relied upon AOAC efficacy test standards 

such as phenol resistance testing or HCl testing to estimate the intrinsic resistance of 

test microbes to disinfectants (indicator of suitable organism population). In 2001 (PR 

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Notice 2001-04; http://www.epa.gov/opppmsd1/PR_Notices), EPA determined that 

phenol resistance testing was an unsatisfactory standard for determining organism 

hardiness and recommended a minimum inoculum level of 104

 organisms/carrier for 

AOAC carrier based efficacy tests. HCl testing still remains a required component of 

the AOAC Sporicidal Activity Test as an indicator of spore hardiness. The quality of 

the microorganisms used in an efficacy evaluation affects the outcome of 

testing⎯use of less resistant organisms may make the product appear more 

efficacious than it actually is. 

Χ	 Technique-sensitive procedures – Many of the recommended efficacy methods may 

contain technique-sensitive steps. It is critical that scientists performing the tests be 

trained in the conduct of the method. Following the method exactly is crucial for 

proficiency and validity of test results. 

4.0 Desirable Attributes of a Validated Test Method 

In addition to the criteria for method selection described Section 2.2 of the main 

document, the following are desirable components of and recommendations for an efficacy test 

method that is to be subjected to validation: 

Χ	 Suitable for use with the test microorganisms; 

Χ	 Protocols for culturing/enumerating test microorganism; 

Χ	 Acceptable statistical profile addressing within and between lab variability (Tilt 

and Hamilton 1999); 

Χ	 Suitable for multiple active ingredients (i.e., different formulations); 

Χ	 Contains a percent recovery/minimum detection level; 

Χ	 For carrier-based tests, includes inoculation procedure, method of determining 

populations of microorganisms on a carrier, and a target range for the 

microorganism population; 

S Includes a neutralization confirmation procedure; 

S Addresses means for managing contact time and temperatures (i.e., includes 

references to calibrated timers and thermometers); and 

S	 Prior to pursuing method validation, knowledge of the target performance 

standard is essential. For example, if a 6-log10 kill of the target organism on a 

porcelain carrier is required for sporicidal decontamination products, then the 

efficacy method must be able to accommodate a minimum of a 7-log10 

challenge per carrier. 

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5.0 Method Performance Characteristics 

The method performance characteristics for efficacy testing remain the same as described 

in Section 2.4 of the main document. Accuracy defined as measure of the overall agreement 

(e.g., pass or fail efficacy) to a known value, does not apply to efficacy testing since there are 

currently no established standards (i.e., chemicals/disinfectants) available for use in efficacy 

tests. Accuracy is measured from results of repeating the test several times and by comparison to 

an existing method. 

6.0 References 

1. Springthorpe, V.S. and Sattar, S.A. (2005) Carrier Tests to Assess Microbiological 

Activities of Chemical Disinfectants for Use on Medical Devices and Environmental 

Surfaces. J. AOAC Int. 88, 182-201. 

2. Tilt, N. and Hamilton, M.A. 1999. Repeatability and Reproducibility of Germicide Tests. 

J. AOAC Int. 82, 384-389. 

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Appendix E – The Method Approval Process 
Once validated, a method can receive Agency approval and be designated as an EPA 

method. There are several ways that methods can be approved; these approaches are outlined 

below. 

Method Approval through the Rulemaking Process 

The rulemaking process begins with EPA publication of a proposed rule in which the 

agency cites a method or methods that it plans to use to implement the rule. EPA then solicits 

and receives comments during a public comment period. Once these comments are addressed 

and the Agency promulgates the final rule, the method identified in the rule is considered 

approved and ready for use. The method in the rule is then considered to be a reference method. 

Until recently, new or modified methods under the Alternate Test Procedures (ATP) 

program also followed the same approval and rule making process described above (see 

Section 1.1 of this appendix). Thus, any modified or new method may take several years before 

it becomes a final rule. 

1.0 Alternate Method Approval Processes 

After a reference method has been established, alternate methods that are easier, less 

expensive, or more accurate often become available. The possible approval processes for these 

alternate methods are described below. 

1.1 Alternate Test Procedure (ATP) Program 

The ATP program is another avenue to allow emerging technologies that reduce cost and 

enhance data quality. Under this program, all modifications that are not explicitly allowed by a 

method cited in a rule require prior EPA approval through the ATP program. An ATP can be a 

modified method or a new method. The applicant(s) of a particular ATP can only submit an 

application to the ATP program after the method has undergone performance characterization. 

2.0 Expedited Methods Approval 

Due to the lengthy time required for new or modified method approval, EPA has recently 

developed the expedited method approval notice. To use this process, EPA must have already 

promulgated at least one analytical testing method for the analyte or microorganism through the 

E-1 
rulemaking process. Section 1401 of the SDWA allows EPA to approve additional testing 

methods through this expedited approval that simply involves publishing the alternative method 

in the Federal Register (Charlton et al. 2000). Therefore, the new or modified methods submitted 

through the ATP program can now be approved by this expedited approval process. However, 

the performance equivalence of the new testing method to the reference method (2008, FR 73: 

31616) must be demonstrated before being considered for approval by the expedited process. A 

process flow diagram illustrating the approval steps for undertaking an ATP and its relationship 

to the reference method is provided in Figure E-1. 

Alternate Test 

Procedure 

(ATP) 

New Modified 

Establishment of 

Performance 

Characteristics 

Demonstration 

of Equivalency 

to reference 

method 

Expedited Methods 

approval via notice in 

Federal Register 

Additional methods 

validated outside the

 ATP Program. e.g., 

EPA, consensus body 

methods 

Final 

Rule 

EPA approved Method 

Action on public 

comments 

Public comments 

Citation in proposed rule 

Reference Methods 

Figure E-1. Methods Approval Schematic Representing Reference Methods and Alternate 

Test Procedures 

3.0 Reference Methods and Other Approved Methods 

EPA designates an approved method as the “reference method” for each combination of 

analyte and technique. Any newly developed method that contains a unique combination of 

analyte and technique is considered a new method and, when approved, can be designated as the 

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reference method for that unique combination of analyte and technique. Any approved method 

not designated as a reference method has been designated as an “other approved method.” All 

methods must contain standardized quality control (QC) tests. 

The person or organization that develops a reference method for a particular combination 

of analyte and technique is responsible for validating the method and for developing the QC 

acceptance criteria. QC acceptance criteria are based on data generated during the method 

validation study. 

4.0 References 

1. Charlton, S., R. Giroux, D. Hondred, C. Lipton, and K. Worden. 2000. PCR Validation 

and Performance Characteristics - AEIC Biotech Consensus Paper. Analytical 

Environmental Immunochemical Consortium. 

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