## Method Validation of U.S. Environmental Protection Agency Microbiological Methods of Analysis

Prepared for:



## The EPA Forum on Environm ental 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 so lely to provide internal guidance to U.S. Environmental Protection Agency (EPA) pers onnel. 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 us e of mandatory language such as “must” and “require” in this guidance document reflects sound scientific pract ice and should not be construed to create any legal rights or requireme nts. The use of non-mandatory language such as “may,” “can,” or “should” in this guidance doe s not connote a requireme nt but does indicate EPA’s strongly preferred approach for validati ng and peer reviewing EPA methods prior to publication for general use.

References within this document to any sp ecific commercial product, process, or service by trade name, trademark, manufacturer, or otherw ise 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, ap paratus, product, or process disclosed in this manual, nor represents that its use by such thir d 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 fo r 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 validati ng and peer reviewing EPA methods prior to publication for general use. This document cont ains guidance for micr obiological 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 a nd 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 a nd Development, USEPA
Contract Support

The authors would also like to thank Vers ar, 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

iii1.0 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 GENE RAL GUIDELINES FOR ME THOD VAL IDATION

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

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

................................................................................................. 2-1
2.3 Method Optim i zation

........................................................................................... 2-2
2.4 Developm ent of Operational Lim its a nd W ithin-L aboratory Method Perform a nce 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 Lim it of Det ection (LOD)

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

................................................................................................... 2-7
2.4.6 Calibration and Other P e rform a nce Attr ibutes of Absolute Quantitative
Test Methods

........................................................................................................ 2-8
2.4.7 Special Considerations for Culture-b ased Microbiological Test Methods 2­
9
2.4.8 Other Operational Lim its and Quality Control Measures

........................ 2-9
2.5 W r iting 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-laborator y Validation Studies

....................................... 2-12
2.6.5 Participating Laboratories

...................................................................... 2-13 TABLE OF CONTENTS

iv2.6.6 Test Materials ......................................................................................... 2-15
2.6.7 Replication of Test Materials................................................................. 2-16
2.7 Development of a Study Plan a nd Study Implementation ................................. 2-17
2.8 Reporting Results ............................................................................................... 2-17
3.0 REFERENCES ................................................................................................................ 3-1

vAppendix 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 a nd 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 Standard s 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

vi6.0 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 Va lidation of Efficacy Test Methods Introduction 1.0 Selection of Test Method to Generate Product Efficacy Data 2.0 Validation of Efficacy Test Me thods and Study Plan Preparation 3.0 Pre-Validation Considera tions: 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

viiFigure 1-1: Overall Method Valida tion 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

viii1.0 INTRODUCTION

1.1 Microbiology at EPA Microorganisms are ubiquitous in the environm ent. As such, the study of these organisms and their impact on human health through enviro nmental exposures is an important part of EPA’s mission. Consequently, the Agency is extens ively 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 pa thogenic organisms in the environment, EPA is heavily invested in determining the impact of various tr eatment and decontamination procedures on microorganisms. The data generated by these effo rts help inform EPA in the development of regulations and improved pat hogen management approaches. With specific regard to drinking and source waters, the Agency develops regulations designed to address the issue of microbial contamination. The agen cy 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 fo r compliance but to provide advisory
information/data. Consensus methods are of ten developed through collaboration between industry and EPA to assist the industry in m onitoring un-regulated parameters. A good example of the Consensus Method process is the Micr oscopic Particulate Anal ysis (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 infl uence of surface water. For both implementation of regulations and a dvisory purposes, EPA publishes microbiological methods used by public water utilities, academia , industries and other government agencies. Regardless of the purpose, reliable, and accurate met hods 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 suitab ility of an analytic method for its intended purpose (Green 1996). This document is intended to provide general guidance for the validation of micr obiological methods likely to be used in future EPA methods. This document is designed to be applicable to all methods pert aining to assaying environmental microorganisms as well as to efficacy testing of antimicrobial agents. In exploring requirements to validate a specific method, the EPA Foru m on Environmental Measurements (FEM) Microbiology Action Team concluded that the provision of specific guidance on how to conduct

1-1validation studies for every method of anal ysis 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 wi th some emphasis on certain technologies. Wherever appropriate, validati on protocols developed by organi zations and used by EPA are referenced; e.g. International St andards Organization (ISO), Amer ican Society for Testing and Materials (ASTM) International, and Association of Analytical Ch emists (AOAC) International. As new microbiological methods of analysis are devel oped, this document may be revised. This text, therefore, shoul d 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 mi crobiological methods of analysis used in environmental testing. These methods may be pu blished as serially numbered EPA methods, or incorporated by reference in regu lations or could be used purely for research efforts by EPA’s Office of Research and Developm ent and the EPA Regions. It is recognized that this document may also be used by clients, contractors or ot her interested parties who, upon reviewing an EPA method for potential use, are interested in th e Agency’s process for validation, approval, and acceptance of EPA methods.

1.4 Scope of the Guidance This guidance is intended for new EPA mi crobiological methods that have not yet undergone validation prior to being published as EPA methods or adapted as Agency-accepted regulatory standards. This guidan ce includes validation principles fo r 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) proce dures, and analy tical detection

1-2of 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 valid ation guidelines for microbiological methods. Emphasis is on issues associated with PCR meth ods and efficacy test methods for antimicrobials in Appendices C and D, respectively. The guidan ce also provides readers with an understanding of EPA’s method approval proc ess (Appendix E) after the met hods are validated. The general flow depicting overall method validation a nd approval process is shown in Figure 1.

1-3Method 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

1-42.0 GENERAL GUIDELINES FOR METHOD VALIDATION

2.1 Introduction Method validation is the process of determin ing 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 es tablishing the operational limits and performance characteristics of a new, modified or otherwis e inadequately characterized method. Secondary validation on th e other hand, is the process of gathering evidence that a laboratory can meet the specifications esta blished in primary validation (ISO 2000).
This section outlines a series of steps and procedures that ar e recognized as being integral to the selection and validation of an analyti cal method for an intended use. These steps are normally performed sequentially an d procedural details may vary for different types of methods.
It should be noted that successful validati on of any analytical method is not possible without thorough and systematic planning and pr eparation. 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 involve d in each step of a validation process will have a Quality System or quality assurance (QA) program in place to ensure standardization of laboratory operati ons, as well as adequate quality control (QC)
activities.

2.2 Method Selection Some methods may not require formal vali dation. 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 re gulatory requirement. It will also require a judgment about the expected capability of th e 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 pur suing validation include
whether a method:

Χ Is based on sound underlying scientific principles

Χ Is applicable for routin e analysis of samples

2-1Χ Can detect analytes in the c oncentration 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 a ssurance (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 me thods identified in the selection process may require optimization prior to their valida tion. 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 commonl y used to characterize the stability of a method’s results in the pr esence of variable f actors are ruggedness and robustness. Ruggedness and robustness are define d 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 unavoidabl e sources of variability can include different analysts, lots or preparations of reagents, and ca libration 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.

2-2For 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 give n 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 resu lts caused by interferences from different sample matrices. When matrix effects are a cause of signi ficant variability and/or when quantification of the analyte is sought, use of an internal cont rol 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 subs equent steps of method validation, particularly in the fi nal 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 a nd 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 at tributes within these
limits that are relevant to the in tended use. As with optimization, th is process is often carried out in the laboratory where the method was deve loped, but it may also be conducted by the organization intending to implement the method. Th is process, often referred to as primary validation, should provide preliminary baseline speci fications (numerical a nd descriptive) of the method’s performance within the laboratory pe rforming the tests. The performance attributes requiring determination may differ depending on th e nature and applications of the method (e.g., culture- vs. microscopy-based, molecular- or chem istry-based, and qualitative vs. quantitative) as are the experimental designs that are best suit ed to make these determinations. A guideline for determining performance attributes of several different broad categorie s of method types are available (ISO 1994b, ISO 1994c, ISO 2000, US FDA 2001). To obtain examples of relevant

2-3experimental designs that might be employed, new method devel opers should also consult the
scientific literature for descri ptions of similar previously va lidated methods. In many instances,
different terms and definitions ha ve 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 specifie d 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 dem onstrated 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 Laborat ories 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 appr opriate target and non-target
control cultures or other standards for use in bo th validation and routine QC would be expected in the development of any new microbial met hod. In a robust method, a si ngle target organism should be discernable in complex matrices containing potentially millions of non-target organisms.

2-4Sensitivity 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 inde pendent test results obtained under stipulated conditions (ISO 1994a). This term is usually expressed as the variance, standard deviation, or coefficien t of variation of a series of test results. Precision is often expressed as the percent coeffici ent 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 bact eria 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 e xpected levels of precision. Precision may be considered at four leve ls: within-lab repeatability, within-lab reproducibility, between-lab repeatability, and be tween-lab reproducibility. The first two levels

2-5should be addressed in the primar y 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 anal yte carried out under th e same conditions of measurement over a short interval of time. Repeat ability is also termed intra-assay precision. Suppose the within la boratory precision is S r and the between laboratory precision is S L . Then the precision S R (including within and between) amon g laboratories is expressed as

S R = 

### S 2

r 

### + S 2 L Equation (4)

Reproducibility . Reprod ucibility 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 incl ude different time intervals betw een analyses, analysts, lots or preparations of reagents, instrume nts, 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 bi as component (related to total systematic error) associated with the met hod (ISO 1994a). As with precision, this bias component should be
first characterized at the within -laboratory level as a primary at tribute of most methods. By the above definition, the determination of bias in th e analysis of a material by a new method requires the knowledge of either the true value for the an alyte 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 refe rence materials, analysis by another presumably unbiased method, or internal contro ls. When analyzing fortified or spiked samples, bias is often

2-6expressed by the terms recovery or percent reco very, 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 result s of an accepted reference method and expressed as relative recovery (ISO 2000) . If no such reference method is avai lable, 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 sa mples with unknown quan tities 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 thes e unknown samples as a reference, the relative recovery of the analyte from any number of a dditional simultaneously processed and analyzed unknown samples can be determined and compared to each other base d on their respective recovery ratios with th is reference sample. For methods with high variability, this technique is
still useful since the variation can be quantified through measurem ent of standard deviation. In addition, if the reference sample can be considered to be the same in diffe rent test runs of the method, i.e., a consistent material containing th e 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 thes e different sets of analyses.

2.4.4 Limit of Detection (LOD)

Limit of detection can be defined as the mi nimum amount of analyte that can be reliably detected (i.e., distinguishable from known and characterized b ackground with a given level of confidence). The LOD measurements establis h a baseline detection value under optimal
conditions. Culture based methods ar e typically expected to be capab le of detecting single target organisms in large samples. Where no organism s are detected, the resu lt is reported as <1 organism per sample volume or mass.

2.4.5 Linearity Linearity is defined as the ability of the analy tical 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.

2-72.4.6 Calibration and Other Performance Attri butes 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. Th ey can be characterized either directly or with minimal mathematical data tran sformations on the basis of the response values of
the detector or instrument uti lized in the method, or in the cas e of culture based methods, the
bacterial/viral counts obtained. For methods specifically designed to obtain absolute quantitative measurements of an analyte, several additional op erational limits and performance attributes may require determination and in many instances the relationships between de tector or instrument response and analyte concentratio ns must also be establishe d through the development of a calibration curve using reference standards of know n concentration. The concentration of analyte
in a reference standard can be established by several alternative a pproaches including: assignment or certification based on experiment al work by some national or international organization; consensus or certification based on collaborative experimental work under the auspices of a scientific or e ngineering group; or in some cas es, 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 developm ent, the simplest model (e.g., linearity) that adequately
describes the concentration-res ponse relationship should be used. The calibration curve is also used to establish the limit of qua ntification of the method (i.e., th e 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 con centration and limit of detection of the analyte over which the concentr ation-response model provi des suitable precision and accuracy). During this process, the statistica l dispersion of the standard measurements by the instrument or detector (instr ument 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.

2-82.4.7 Special Considerations for Culture-ba sed Microbiological Test Methods Historically, most microbial work has involved cultivation (viability testing) of microorganisms in a selective medium for enumer ation or for the determination of presence or absence of the target microorganism(s). EPA or other regulatory requirements determine whether quantitative or qualitative resu lts 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 microorgani sms in a sample, quantitative culture-based microbiological methods have performance attribut es and operational limits that are the same as, or are analogous to, most of th e terms defined above, including sp ecificity, sensitivity, precision, recovery, limit of detection, linearity and limit, and range of quantification (ISO 2000). Unlike chemical analytical methods, however, many of th e performance attributes of culture methods are based upon particle distribution (i.e., distri bution of the microorganisms) as defined by the
Poisson distribution or where ove r-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 skew ed distribution of target microbes in waters, when using methods that are based on normality of count data, it is ge nerally necessary to convert the data to log-base 10 (o r in other ways transform) to m eet 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 collec tion 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 eff ect on the test results of many analytical methods. Consequently, the determination of accept able holding times and storage conditions of samples and (if applicable) reference standards ma y 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

2-9or relative recovery of the method (see above). The objective is to id entify the limits of conditions that produce no significa nt 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 determ ine the range and/or propo rtion of these samples compared to those obtained from a specified refe rence matrix (i.e., no sign ificant difference at a specified confidence level). Many analytical methods employ the use of matr ix spike or internal control materials as surrogates of the true analyte fo r the routine determination of th e 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 determin e whether their recoveries and responsiveness to interferences is representative of the true analyte. Another impor tant consideration is to ensure
that control material does not interfere with the de tection of the true analyt e 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 wr itten in a format that includes the following components: scope and application; method summ ary; definitions; interferences; health and safety; equipment and supplies; reagents and standards; sample colle ction, preservation and storage; quality control; calibration and standardiza tion; procedural steps; calculations and data analysis; method performance; po llution prevention; and waste ma nagement. Each component of this format is described in greater detail in A ppendix 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 descrip tive specifications of the method’s operational limits and performance attributes determined from the within-laboratory testing results during primary validation. This is justified si nce for some methods the primary validation process should be sufficient for meeting the intended use of the me thod. In addition, if subse quent 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.

2-102.6 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 s upport regulatory activity. A tiered approach developed under th e streamlining initiative (U.S. EPA 1996) has typically been used by EPA for the validation of microbiol ogical 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-labo ratory and single-industry use) and instead
focuses resources on validation of methods that are intended for nationw ide use. Because QC acceptance criteria are developed from validation studies and validation requirements vary with each tier, the statistical procedures used to de velop the criteria will vary by tier as well.

2.6.1 Tier 1 Validation

Tier 1 refers to new methods or method m odifications 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 requ ire 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 me thod validation study.

2.6.2 Tier 2 Validation

Tier 2 refers to new methods or method m odifications 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 th e matrix in which the method will be used.

2.6.3 Tier 3 Validation

Tier 3 refers to new methods or method modifi cations that will be used by laboratories on a nationwide basis for all matrix types. Valid ation at Tier 3 requi res a nine-laboratory interlaboratory study on nine matrix types. Valid ation 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 par ticipating laboratories in different collaborative

2-11studies may be influenced by a number of addition al factors. Such factor s, as detailed further below, can include the specific objectives of a st udy 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 sta tistical theory and s ubjective judgments. Study design must be peer-reviewed by qualified memb ers 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 interl aboratory validation is to de monstrate 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 st eps in this process (Horwitz 1995, Holmes et al., 2001).

Primary Validation or Laboratory Performance Study . Collaborative studies have been developed into a widely used t ool for testing performance charac teristics of chemical methods, but this process is normally associated with the widespread availability of laboratories that already have extensive experien ce with these methods. This may not be the case for many newer microbiological methods (ISO 2000). For methods of th is type, the more prudent next step in the validation process may be to conduct a collabor ative laboratory perfor mance study. Studies of this type are usually conducted with the obj ective of evaluating or improving laboratory performance of the method. Reported results by diffe rent 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, we ll-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 profic iency testing, which is normally associated with the evaluation or acc reditation of laborato ries in performing a previously validated and accepted method.

2-12Secondary Validation or Method Performance Study . The type of collaborative study that is normally conducted in the validati on process is a method performance study. The objective of these studies is normally to provi de specifications for the methods performance attributes across a representative sample populati on 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 ne w 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; preparati on for; and implementation and anal yses 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 recomme ndations that generally are consistent in these different guidance documents and other recommendations that may be specifically associated with the validation of microbiological test me thods 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 interv als can be predicted base d 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 pa rticipating laboratories, the number of test samples, the acceptable margin of error (precisi on), and the desired le vel of confidence when considering the number of independent laborato ries 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 guidan ce 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 co mpared to quantitative data. Following is a mathematical model to estimate the margin of error (p 2 ) (precision) given a certain number of laboratories (L) and test samples (m) for a method th at is required to corr ectly classify at least

2-13(80%) (p 1 ) of the positive test portions (sensitivity rate), with a confidence limit of 90%. The equation is as follows:

p 2 = 100 Z α S ˆ / [L 1/2 m (p 1 /100)] Equation (5)

a

Where: L = 2, 3....., 25 m = 3, 5, 10

α = .05 ( corresponds to Z α = 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 wi th 80 % Sensitivity Rate and Confidence Interval of 90 % Because of the diminishing return in decrea sing uncertainty that is associated with increasing numbers of independe nt test results from diffe rent laboratories, a common compromise is 8-15 laboratories to participate. The expectation is that a small number of these data sets may be statistical out liers or may be obtained from in correct performance of the method
and thus will be excluded. Howeve r, there are limitations to a pplying this purely statistically based approach to microbiological methods. Ultimately decisions on sample size (i.e., number of

2-14laboratories) in a study must be based to some degree on a subjective judgme nt as to whether the results of the study will be effective in de monstrating 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 laborat ories for a method performance study not be restricted to only la boratories with extensive expe rience with the specific method under consideration. Conversely, it is highly recommended that al l 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 newe r methods, it has been suggested that the performance attribute specifications obtained in these studies be re ported as preliminary or initial estimates because they could fu rther improve as laboratories ga in 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 comb inations to which the method performance parameters apply (Horwitz 1995). Similar to with in-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. Severa l different analyte conc entrations should be tested and the range of analyt e concentrations should be with in the operational range of the method as specified from primary validation study results and should also be representative of the expected range of concentratio ns 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 in clude and bracket this value. La rger numbers of test materials are generally required for met hod equivalency studies (ISO 2004). A highly important prerequisite of these studies is that the test samples that are analyzed by the different laboratories should originat e from materials in which the analyte is homogeneously distributed. This is often challenging with mi crobes. This property can be established by the use of certified reference materi als or determined from analyses of replicate samples with materials in the laboratory that procures them or prepared by a reference

2-15laboratory. In the latter instances, precision estimat es from these analyses should be compared with the method’s primary validation study precis ion 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 microor ganisms) unlike in chemical methods. If this property can not be established w ith reasonable certainty in natural materials or
if natural materials can not be readily found that c ontain the analyte, then another commonly employed alternative is the utilization of spik ed 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 recove ry is a performance attribute that is being evalua ted, spike levels should be su fficiently high to be readily differentiable from the levels of any naturally occurring analytes in th e 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 attribut es 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 influe nce the uncertainty of estimates for repeatability and reproducibility standard deviations. The latte r uncertainty value is also shown to be a
function of the ratio of these two standard deviations. It is comm only observed with many chemical analytical methods that this ratio is relatively high, i.e ., the variability of test results obtained under repeatability c onditions is considerably sma ller than that obtained under reproducibility conditions (AOAC 1989). When this situation occurs, analys es of more than two replicate samples of the different study mate rials will provide relatively little additional information. Microbiological analyt ical methods may differ in this regard, however, and analysis of additional replicate samples is warrante d. 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 know n replicate samples is a common

2-16practice, the use of blind repli cate samples is generally recommended to eliminate the potential for any selectivity by the laboratories in thei r procedures and reporti ng 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 obje ctives 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 adhere nce to instructed validati on methodologies, accuracy, and completeness; and specific instructions for data an alysis including statistical tests and anticipated metrics for the results. Any study-specific procedur al instructions that ar e not fully explained in the written method (e.g., sample storage conditio ns, 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 c onsult study plans for previously validated EPA methods or from the ge neral scientific literatu re, if applicable. An example of study plan for a previously c onducted validation study by EPA is given in Appendix B. Additional guidance that may be us eful in the study planning and implementation process can be found in documents from se veral other organizations (AOAC 1989, ISO 2000, ASTM 2001).

2.8 Reporting Results Results from the method performance study sh ould be disseminated and made available to the scientific community for peer review befo re presentation at a scientific meeting, peerreviewed publication, and/or publication of a technical manua l. 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 de scription of how they were prepared and documentation of

2-17their homogeneity. In many instances (e.g., when submitting for peer-reviewed publication), the report will also provide a summar y of the study design, procedures and statistical methods used for analyses of the results. A critical component of the re port is a presentation and di scussion of the numerical and descriptive specifications for the performance attributes of the methods that were determined from the collaborative study. These specificati ons 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 developmen t 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 availa ble reference method should be provided. Criteria and guidelines for the establishment of equivale nce between microbiological test methods have been developed (ISO 2004).

2-183.0 REFERENCES

AOAC. 1989. Guidelines for collaborative study of pro cedure to validate ch aracteristics of a method of analysis. J. Assoc. Off. Anal. Chem

. 72 , 694–704.

AOAC. 2006. AOAC Task Force Recommends Best Pr actices 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 (20 th 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, 5 th 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 Guid e to Analytical Method Valid ation. 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. Viro l. Methods 92:141-150.
ICH. 1996. Validation of analytical proce dures: Methodology ICH- Q2B. International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use.
International Organization for Standardizati on. 1994a. ISO 5725-1:1994. Accuracy (trueness and precision) of measurement methods and resu lts - Part 1: General principles and definitions. Geneva, Switzerland.

3-1International Organization for Standardiza tion. 1994b. ISO 5725-2:1994. Accuracy (trueness and precision) of measurement methods and re sults - Part 2: Basic method for the determination of repeatability and reproducib ility of a standard measurement method. Geneva, Switzerland.
International Organization for Standardizati on. 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 Standardiza tion. 2000. ISO/TR 13843:2000(E). Water quality – Guidance on validation of microbiolog ical methods. Geneva, Switzerland.
International Organization for Standardiza tion. 2004. ISO 17994:2004. Water quality - Criteria for establishing equivalence between microbiological me thods. Geneva, Switzerland.
McClure F.D. 1990. Design and analysis of Qu alitative Collaborative Studies: Minimum Collaborative Program, J. Assoc. Off. Anal. Chem. Vol. 73, No. 6.
Niemela, S. I. 2003. Uncertainty of quantitativ e determinations derived by cultivation of microorganisms. Centre for Metrolo gy and Accreditation, Helsinki, Finland.
USEPA. 1992. Consensus Method for Determining Groundwaters Under the Direct Influence of Surface Water Using Microscopic Particul ate 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 Contro l Guidance for Laboratories Performing PCR Analyses on Environmental Samples EP A 815-B-04-001. United Stat es 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/cde r/guidance/index.htm
Youden and Steiner, 1975. Statis tical Manual of the Associati on of the AOAC, Association of Official Analytical Chemists. Arlington, VA.

3-2

### Appendix A – Environmental Moni toring Management Council (EMMC) Format for Writing a MethodThe components of the Environmental Mon itoring 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 de scription 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. Th e purpose of the summary is to provide a succinct overview of the technique to aid the review er or data user in evaluating the method and the data. List sample volume, concentration, ot her preparation steps em ployed, the analytical instrumentation, and the techniques us ed for quantitative determinations.

3.0 Definitions of Method Include the definitions of all method-speci fic 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 inte rferences 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-1This section should discuss only those safe ty issues specific to the method and beyond the scope of routine laboratory practices. Reagents that pose speci fic toxicity or safety issues
should be addressed in this section.

6.0 Equipment and Supplies Use generic language wherever possible. Howe ver, for specific equipment do not assume equivalency of equipment that was not specifically evaluated, a nd 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 re agents and standards to allow the work to be duplicated, but a void lengthy discussions of common procedures.

8.0 Sample Collection, Preservation and Storage Provide information on sample collection, pres ervation, shipment, and storage conditions.

9.0 Quality Control Describe specific quality control steps, includ ing such procedures as laboratory control samples, QC check samples, instrument check s, etc., defining all terms in Section 2.0.

10.0 Calibration and Standardization Discuss initial calibration procedures here. In dicate frequency of such calibrations, refer to performance specifications , and indicate corrective actions that must be taken when performance specifications are not met. This Sect ion may also include procedures for calibration
verification or cont inuing 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 re strictive instructions.

A-212.0 Calculations and Data Analysis Describe qualitative and quantitative aspects of the method. List id entification criteria used. Provide equations used to de rive final sample results from typical instrument data. Provide discussion of estimating dete ction limits, if appropriate.

13.0 Method Performance A precision/bias statement should be in corporated in the Section, including:
Χ Detection limits
Χ Source/limitations of data Provide detailed description of method performance, includi ng data on precision, bias, detection limits (including the method by which th ey were determined and matrices to which they apply), statistical proce dures used to develop performa nce specification, etc. Where performance is tested relative to the referen ce 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 – Interlaborat ory 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)

1SECTION 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 intenti onal 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. Envi ronmental 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 chem icals used in building decontam ination. 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 producti on, the carrier materials (unglazed porcelain and silk sutu re 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 – Ameri can Society for Testing and Ma terials (ASTM) E 2111-00 and an adaptation of a quantitative micro-method (the Th ree Step Method) as reported by Sagripanti and Bonifacino, were compared (1). By conducting th e study in a comparative, standardized manner across multiple laboratories, valuable statistical information on the performance of two quantitative methods was generated. Based on th e 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 TS M for incorporating coupo n materials other than glass was considered as an advantage, and that the practice time and trai ning 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 proce ss checklist. As the development and use of quantitative test methods for sporicides and st erilants proceeds, additional studies will be necessary to develop meaningful and relevant pe rformance standards (i.e., pass/fail criteria with a minimum LR).
Although the details of the me thod have been published, th e 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 qualitati ve procedures and suspension-based tests. The TSM uses 5 × 5 × 1 mm glass coupons to deliver spores into th e 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 Log 10 reduction (LR) of spores; LR = log 10 (mean

3spores/control carrier) - log 10 (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 Collabor ative Study (CS) is to evaluate the TSM according to AOACI Official Me thods of Analysis (OMA) proc edures for official method validation. The applicability will be li mited to one spore-forming microorganism ( Bacillus subtilis ), one hard surface (glass), liquid formulations of sporicides and st erilants, and without organic burden added to the spore inoculum. The test chemicals used in the CS represent three chemical classes of sporicides: sodium hypochl orite, a combination of peracetic acid and hydrogen peroxide, and glutaradeh yde. 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 th e 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 perfor mance 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 la boratories, 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 th ree lots of product are required for an EPA registration – all carriers must show no growth to suppor t 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 compara tive 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 collabora tive effort, the AOAC INTERNATIONAL will assemble a review panel, the AOAC Sporicidal Method Expert Revi ew Panel (ERP), to evaluate the CS protocol. The ERP, along with me mbers of the AOAC IN TERNATIONAL 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 Met hods 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) laboratorie s are currently collaborating with EPA per an Interagency Agreement (IAG) and will also participate in th e study. A third FDA laboratory is expected to participate under the IAG. At least five laboratories who ha ve not been involved with the development or evaluation of the TSM will be included. The name and associated contact person for each partic ipating 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 6 th 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

55 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 ensurecompliance 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, standa rdized 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 desirabl e 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 th e 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 de sign 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, th ree 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 conc urrently on the first replicati on only for a total of nine 30-carrier AOAC tests per laboratory.

E. Testing must be initiated within 4 weeks af ter 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 te chnician team which wi ll 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 techni cian team always conducted the testing, the technician effect will not c onfound the results. Practice r uns will be encouraged in advance of testing to ensure analys t proficiency in performing each method.

7G. It is im portant to randomize any steps in which subjective decisions or unknown factors could affect the conclusions. The experiment al results could be cr iticized 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 or der 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 hydr ogen 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 chemi cals are experimental components only and are not being tested to support or verify product la bel claims. Each chemi cal will be tested at three levels: high (efficacious), medium (modera te 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 leve ls 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 chem ical from a reagent-grade product per the preparation sheets provided by the Study Direct or – HACH Test Kits will be used to verify available chlorine for diluted samples. The Study Director wi ll request the use of a single lot for each test chemical. In advanc e of testing, an analysis of formulation chemistry will be performed on each lot (one container) by OPP chem ists to confirm the percent active ingredient.

8K. The test microbe is Bacillus subtilis (ATCC # 19659) obtained di rectly 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 th e quality control standards (e.g., proper documentation, confirmation testing) – Study Dire ctor 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 tota l 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 revise d method (i.e., the use of nutrient agar amended with manganese sulfate) for generati on of spore suspensions and inoculation of carriers. No organic burden wi ll be added to the spore inoc ulum. 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 × 10 5 (log 10 density = 5.0) and a maximu m of approximately 1.0 × 10 6 spores/carrier will be required. Multiple lo ts 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 car rier load for the TSM is 1.0 × 10 7 spores/carrier or 7.0 logs per carrier – a level suitabl e for measuring a log reduction of ≥ 6 logs. No organic burden will be added to the spore inoc ulum. Carriers will be inoculated from one spore preparation per laboratory; enough carrier s 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 th e 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 laborator y with test chemicals randomized for three replications – see Table 3.

9Table 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

10Table 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 treatmen t; **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 r eagent 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% ac etic acid. pH adjusted treat ment must be used in test within approximately 120 min after prepar ation; used for the high treatment.

2. Sodium hypochlorite (Sigma Aldrich reag ent grade solution), diluted to 6000 ± 300ppm with reagent grade water, unadjusted pH (p H ~10.0). Test within approximately 120 min after preparation; used for medium treatment.

113. Sodium hypochlorite (Sigma Aldrich reag ent grade solution), diluted to 3000 ± 300ppm with reagent grade water, unadjusted pH (p H ~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 accordin g to product directions, 14-day shelf-life post activation. The activated product may us ed for testing during the 14-day period. 

### Section 5. Quality Assurance
The sponsor of this project is the EPA Offi ce of Research and Development. Document archiving will be adequate to ensure that all studies are supporte d by complete, accurate, consistent, and chronological records from initia l collection of raw data to final analysis interpretation and re porting 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 consis tent with EPA Good Laboratory Practices. Numerous guidance documents, standard methods and Standard Oper ating Procedures (SOP s) 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 th e assays must be familiar with standard microbiological techniques such as aseptic transfer, serial d ilutions, plate counts and microbe identification. Scientists and analysts involved in testing shall be familiar with each efficacy method and associated procedure (e.g., carrier c ounts, neutralization confirmation) and will be proficient in conducting each de signated efficacy test method.
In-house practice sessions will be required for each laboratory to bui ld proficiency with each method prior to official testing. The L ead laboratory (OPP Microbiology Laboratory) will conduct a series of conference call s with the participating laborator ies to discuss the details of each method protocol. Documentation of practice a nd 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 co rrection and the reason for the change. Project-specific documentation sh all be maintained in a project file, or the project file must identify where the documen ts are stored. Where possible, EPA SOPs and standard forms shall be used for those operation s 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

12performance of routine procedur es. 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 a nd reagents will be tr acked using an assigned media preparation number. Samples, test chemicals, will be maintained to ensure their integrity. Test chemicals will be stored away from sta ndards, media, and reag ents to prevent crosscontamination. No official chain of custody doc umentation will be required for test chemicals evaluated in this research; however specific information on source, id entification, 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 ensu re 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 ster ility; 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 a nd components with specific requirements such as sodium hypochlorite and inoculated carriers w ill be requested to provide verification of the desired specifications. The information and data will be maintained and ar chived 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 re port of the findings or data summary will be compiled and forwarded to each lab’s Quality A ssurance 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 c ontamination 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; Sporici dal Activity of Disinfectants Modified Method

First Action 2006
Applicable to testing sporic idal activity of li quid disinfectants using modified method 966.04 against Bacillus subtilis on a hard surface (por celain carrier). Perf ormance criteria for product efficacy are not impacted. This method has been validated for products containing sodium hypochlorite, peracetic acid/hydrogen peroxi de, 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 stipulat ed in the institutional biosafet y regulations. Use the equipment and facilities indicated for the test organi sm. For recommendations on safe handling of microorganisms refer to the Centers for Dis ease Control/National In stitutes of Health (CDC/NIH) Biosafety in Microbio logical and Biomedical Laborator ies manual. (2) Disinfectants may contain a number of different active ingredients, such as heavy metals, aldehydes, peroxides, and phenol. Personal protective cl othing 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 c ourse of action. (3) References to water mean reagent-grade wa ter, 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-o riented, 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 an d 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 102 nd St., Omaha, NE 68127) to approximately 1 L water. Boil mixture for 20 mi nutes with constant stirring. Readjust volume to 1 L with water and allow cool ing 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.

14Dehydrated nutrient broth may be substitute d – 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 dissolve d. Adjust pH to 7.2 ± 0.2 if necessary. Dispense 5 mL portions into 16 x 1 00 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 MnSO 4 :H 2 0 (amended nutrient agar) .–For spore production. Suspend 11.5 g nutrient ag ar in 495 mL water, add 5 mL 500 ppm MnSO 4 :H 2 0. 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 wate r and heat gently while stirri ng. Boil one minute or until completely dissolved. Adjust pH to 7.3 ± 0.2. Au toclave 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 th ioglycollate medium in 1 L wate r. 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 be tween 8 and 9 is typical). Di spense 10 mL into 20 × 150 mm culture tubes and autoclave for 15 minutes at 121oC. Store at room temper ature. Protect from light. Note: If after autoclaving the aerated por tion 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 manga nese 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-gr ade water should be free of substances that interfere with analytical methods. Any method of prepara tion 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.–B acillus 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 steril izing. Sterilize all glassware 2 hr in hot air oven at 180 o C or steam sterilize for a minimum of 20 min at 121 o C 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 chem ical, capable of maintaining 20 ± 1oC temperature or specified temp erature 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/transf er loop for culture transfer.
(i) Wire hook .–For carrier transfer. Make 3 mm ri ght 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 cert ified timer that can display time in seconds.

16

### C. Operating Technique

(a) Culture initiation .– Initiate B. subtilis culture (e.g., us e 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 sl opes and incubate 24 ± 2 hours at 36 ± 1oC. Concurrently, perfor m purity and identification conf irmation testing for QC (e.g., colony morphology on TSA, Gram stain, or use of other identification systems). Following incubation, store at 2-5oC. Main tain stock culture on nutrient agar slants by monthly (30 ± 2 days) transfers.
(b) Production of B. subtilis spore suspension .–Using growth from a stoc k 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 inocul ate 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, harv est 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 c onical tubes (10 plates = 14 tube s, ~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 dis card 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 determin e ratio of spores to vegetative cells (or sporangia). Percentage of spor es versus vegetative cells s hould 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 spor e titer of the concentrated spore suspension by plating serial di lutions (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 amende d nutrient agar should provide 80 - 100 mL of concentrated spore suspension (approx. 10 9 CFU/mL). Diluting the suspension prior to carrier inoculation will be nece ssary; a titer of 1.0 × 10 8 to 5.0 × 10 8 CFU/mL should be adequate to achieve the target carrier count.
(c) Preparation of porcelain carriers.– Prior to use, examine porcel ain 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 car riers with care when placing in Petri dishes. Minimize carrier m ovement 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 achiev e carrier counts between 1.0 × 10 5 and approximately 1.0 × 10 6 spores/carrier. Dispense 10 mL diluted spore su spension 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 dish es containing inoculated carriers in vac uum desiccator containing CaCl 2 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 a nd 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 se lected carriers per pr eparation. Place each inoculated carrier into a 50 mL plastic, polypr opylene conical centrifuge tube containing 10 mL of sterile water. Sonicate carriers for 5 minut es ± 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 clea ner) 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 touc h 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 transf erring 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 inc ubate 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 coun ts less than 30. Report plates with colony counts over 300 as TNTC (T oo Numerous to Count). Average spore counts per carrier should be between 1.0 × 10 5 and approximately 1.0 × 10 6 spores/carrier. Do not use carriers with counts out side 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 in side of wall of acid tube. Transfer individual carriers after 2, 5, 10, and 20 minute s of HCl exposure to a separate tube of modified FTM. Rotate each tube vigorously by hand for approximate ly 20 seconds and then transfer carrier to a second tube of modified FTM. For viability c ontrol, 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 th e use-dilution to be tested. Use v/ v dilutions for liquid products and w/v dilutions for solids. For a 30-carrier test, pl ace 10 mL product at dilution recommended for use or under investigation into each of six 25 × 150 mm or 25 × 100 mm te st tubes, or use appropriate number of tubes assumi ng 5 test carriers per tube of te st chemical. Place tubes in 20 ± 1oC water bath and let equilibrate to temperatur e. 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 t ube, neither carriers nor wire hook may touch sides of tubes. If in terior sides are touched, note tube number. Do not count carrier set if any carrier from that group of 5 yields a positiv e result; testing another set of five carriers is recommended. Carriers must be deposited into test tubes within ± 5 sec onds of the prescribed drop time. Return tubes to water bath immediat ely after adding carriers. After contact period has been achieved, transfer carriers in same se quential 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 ho ok, tap against interior si de of tube to remove excess sporicidal agent, and transf er 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 co rresponding neutralizer tubes at a ppropriate time. Carriers may touch interior sides of neutraliz er tube during transfer, but cont act should be minimized. After each carrier is deposited, recap neutralizer tube a nd gently shake to facilitate adequate mixing and efficient neutralization. Within one hour from when last carri er 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 s econdary tubes after all carriers have been transferred. Incubate primary (neutralizer) and secondar y 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 whic h medium appears clear. Shake each tube prior to recording results to determ ine 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 sec ondary subculture tube is consider ed a positive result for the carrier set.
Media sterility controls and system contro ls (check for aseptic technique during carrier transfer process) are recommended. For medi a 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 chem ical. 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 carr ier to tube of neutralizer medium. After all test carriers (last tube) have been transf erred to neutralizer tubes, transfer third sterile carrier to tube of neutralizer me dium. Transfer system control carriers to second ary subculture medium as follows: immediately pr ior to initiating tran sfer of test carrie rs into secondary subculture medium tubes, transfer first system c ontrol sterile carrier from neutralizer medium to tube of subculture medium. Afte r one half of test carriers have been transferred to secondary

19subculture medium tubes, transf er second system control sterile carrier to tube of subculture medium. After all test carriers have been transferred to seconda ry subculture medium tubes, transfer third system control ster ile 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 da ys 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 pos itive 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 with in 5-7 days of conducting the efficacy test.
(h) Neutralization Confirmation Procedure.– A neutralization confir mation 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 a ssay provides for a quantitative approach to assessing the effectiveness of the neutralizer a nd any bacteriostatic ac tion resulting from the neutralizer itself or neutralizer – disinfectant in teractions.

Produce a spore preparation according to the pr ocedure for amended nutrient agar. Harvest growth from plates (e.g., five plates) per the method, excep t re-suspend pellet after final centrifugation step in ap proximately 100 ml aqueous (40%) et hanol. Determine spore count by serial dilution and plating on TS A. Desirable target of the in itial working suspension is 1.0 × 10 8 to 1.0 × 10 9 CFU/mL. The suspension may require adjust ment 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 neutrali zer 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 thr ee dilutions in duplicate on TSA agar. Incubate plates inverted for 24-48 hours at 36 ± 1oC. Count co lonies (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, spor es sheared from inoculated carri ers may be used as a working suspension.
Use 5 sterile porcelain carriers (only 3 to be us ed 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 tube s containing the neutralizer per section (g). Th is set of tubes is

20the Neutralizer/Primary Subculture treatment. Fo llowing the transfer of the last carrier into neutralizer tube, tran sfer 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. Follow ing carrier transfer, inoculate each tube (Neutralizer/Primary and Secondary Subculture tr eatment 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 tube s 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 steri lity 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 th e disinfectant or bacteriostatic activity of the neutralizer itself may be masked when a high level of inoculum (s pores) is added to the subculture tubes.

Table 1. Neutralization confirmation procedu re – 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 10 8 spores/mL Confirm a minimum of one positive per treatm ent and control (if available) using Gram staining and colony morphology on TSA. For each treatment and control group, conduct confirmation testing on growth from t ube with fewest spores delivered. B. subtilis is a Gram positive rod and colonies on TSA are opaque, rough, dull, round, with irregul ar 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 Neutra lizer/Primary Subculture and Secondary Subculture treatment tubes is used to assess the effectiveness of the neut ralizer. No growth or growth only in tubes which receiv ed a high level of inoculum (e.g., the dilution with plate counts which are too numerous to count) indicates poor neutralization and/or presen ce of bacteriostatic properties of the neutralizer or neutralizer-disin fectant interactions. Fo r a neutralizer to be deemed effective, growth must occur in th e 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 tr eatment 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 me dia performance. Grow th in the Neutralizer21Primary inoculated Control should be comparable to the Secondary Subculture inoculated Control if the neutralizer is the same as the s econdary subculture media. There may be cases when the neutralizer is significan tly 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 mi croorganisms × 60 carriers/type = 240 carriers per batch sample; 3 product batches × 240 carriers/ba tch = total of 720 carrier s). 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 Wate r and Wastewater. 21st Ed. American Public Health Association, 1015 15th Street, NW, Washington, DC
Biosafety in Microbiological and Biomedical Labor atories. 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. Quantitati ve Three Step Method Note: Laboratories will conduct the method in its entirety.
Determining the Efficacy of Liquid Spo ricides 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 s poricide and sterilant are consid ered synonymous. The TSM is an adaptation of a quantitative micro-method as re ported by Sagripanti and Bonifacino (1). See results of the pre-collaborative st udy supporting the use of the TSM (2). A similar version of the TSM was recently accepted and publishe d by ASTM International (3).

Note: All manipulations of the test organism are required to be performed in accordance with biosafety practices stipulated in the institutio nal biosafety regulations. Use the equipment and facilities indicated for the test organism . For recommendations on safe handling of microorganisms refer to the CDC/NIH Biosaf ety in Microbiological and Biomedical Laboratories manual (4). Disinfect ants may contain a number of different active ingredients, such as heavy metals, aldehydes, peroxides, and phenol. Personal protectiv e clothing or devices are recommended during the handling of these items for purpose of activation, dilution, or efficacy testing. A chemical fu me hood or other containment e quipment may be employed when appropriate during performing tasks with concentrated products. The study analys t may wish to consult the Material Safety Data Sheet for th e specific product/active i ngredient to determine best course of action. References to water m ean reagent-grade water, except where otherwise specified (5). The methods are technique sensi tive and technique-oriente d, thus exact adherence to the method, good laboratory prac tices, 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 br oth. 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. Rem ove 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 w ith 5μg/mL MnSO production. Suspend 11.5 g nutrient agar in 495 m L water, add 5 mL 500 ppm MnSO 4 :H 2 0 (amended nutrient agar) .–For spore

4 :H 2 0. Dissolve by boiling. If necessary, adjust pH to 6.8 ± 0.2. Auto clave for 15 min at 121oC. Pour agar into plates. (4) Trypticase soy agar (TSA) .–Poured in plates for micr obe isolation and spread

23plating. (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 mangane se sulfate to 500 mL water. Filter sterilize for use.
(c) Sterile water.– Use sterile reagent-grade water. Re agent-grade water should be free of substances that interfere with analytical met hods. Any method of prepar ation of reagent-grade water is acceptable provided that the requisite quali ty can be met. Reverse osmosis, distillation, and deionization in various combinations all ca n 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 dire ctly 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, Er ie Scientific Company, Portsmouth, NH; custom order part number EPA-1101 (min imum order of 1000 pieces), single use.
(c) Sterile 1.5 mL micr ocentrifuge tubes .-For exposing carrier to disi nfectant, 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, ster ile, 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 ch emical 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 agitat ion 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 nutrien t 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 sl opes and incubate 24 ± 2 hours at 36 ± 1oC. Perform purity and identificati on 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 nutrien t agar slants by monthly (30 ± 2 days) transfers.
(b) Production of B. subtilis spore suspension .–Using growth from a stoc k 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 orbita l 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 suitab le spreading device.

25Wrap each plate with parafilm or pl ace in plastic bags. Incubate pl ates 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), re move 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 temperat ure. 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 asse ss quality of the spores. Examine a minimum of 5 fields and determine ratio of spores to vegeta tive 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 i noculation of carriers, determine spore titer of the concentrated spor e suspension by plating serial dilutions (e.g., 1.0 × 10 -6 through 1.0 × 10 -8 ) on TSA or NA. Incubate plates fo r 24 ± 2 hr at 36 ± 1oC and determine titer. Note: When harvested and processed, 10 plates of amende d nutrient agar should provide 80 - 100 mL of concentrated sp ore suspension. Diluting the suspension prior to carrier inoculation will be necessary; a spore titer of approx. 1.0 × 10 9 CFU/mL in the suspension should be adequate to achiev e the target carrier count.
(c) Carrier Preparation. -Visually screen glass c oupons (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 ri nse 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 180 o C. Cool. Transfer carriers to sterile plastic Petri dishes for inoculation (a pprox. 40 carriers per dish).
(d) Carrier Inoculation .-Transfer 10 μL of spore suspensi on with a micropipette using aerosol barrier tips or positive displacemen t 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 en sure uniform distribution of spores. Recommend verification of carrier counts (per the method for cont rol carriers) prior to test day; mean counts must be 5.0 × 10 6 to 5.0 × 10 7 spores/carrier.
(e) Disinfectant Sample Preparation .-Aseptically prepare disinfect ant sam ples as directed. Prepare all dilutions with steril e 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 di sinfectant and 3 carriers for

26the water control (control carrier s) are required per product test. Th ree 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 briefl y 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 contro l (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 ca rrier and sides of microcentrifuge tube. Discard carrier and tube if carrier touches sides of tube. Place fraction A tubes containing carriers and tubes containing disinf ectant(s) and sterile water (control) into chiller water bath at 20 ± 1oC, or use a labtop cooler to maintain te mperature of the tubes. Equilibrate approximately 10 min. Add 400 μL disinfectant (test carriers) or 400 μL sterile wate r (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 thor oughly mix liquid components. Transfer each carrier using 1 pair sterile forceps pe r carrier set (i.e., 3 carriers) from fraction A tube to corresponding fraction B tube. Fraction B tubes contain 400 μL ice-co ld (0-5oC) st erile water.

Place fraction A tubes in microcentrifuge, centrif uge for 6 min ± 30 sec at 13,000 rpm (15,500 × g). Remove 900 μL from each tube wit hout 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. Care fully 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 dislo dged 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 floa ting microcentrifuge tube holder placed inside an ultrasonic cleaner. After sonica tion 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). Pro ceed to dilution and pla ting 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 dislodge d from the carrier by sonication.

27Place fraction C tubes in a hematology rotator insi de 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 analys t 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 thor oughly prior to making dilutions. For each fraction and control tube, re move 100 μL and serially dilute 10-fold in 900 μ L 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 cont rol counts at 24 ± 2 hr. Record treated carrier counts at 24 ± 2 hr and at 48 ± 2 hr. Confirm the identity of a mini mum of one representative colony taken from at least one plate per treatment leve l (if available) using Gram st aining, 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 additio nal 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. Obta in 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 Log 10 (total number of spores per carrier). Determine log reduction (LR) of test carriers by s ubtracting log density of test carriers from log density of control carriers. Determine averag e 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 mi n at 20 ± 1oC (or other specified temperature). Add 600 μL neutralizer in ice-cold Luria-Be rtani (LB) broth to tubes 4-6 (n eutralizer contro ls). 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. 10 9 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 manufactur er). After incubation, a dd 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 s poricidal effect. If th e 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 ne utralizer, log densities in tubes 7- 9 should be within 1 log of the

28log densities in tubes 1-6. No te: The lead laboratory, the O PP Microbiology Laboratory, will perform this assay on each of the high treatments pr ior to the initiat ion of the study to verify the effectiveness of the chosen neutra lizers. For this assay, produce a spore preparation according to the procedure for amended nutrient agar. Harves t growth from plates (e.g., five plates) per the method, except re-suspend pellet after final centr ifugation step in approximately 100 ml aqueous (40%) ethanol.
( h ) HCl resistance .– Perform on each preparation of i noculated 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 ca rriers per time period. Include three control (sterile water) carriers to determine control carrier counts. Us e modified LB broth (addition of NaOH) as the neutralizer instead of LB broth for HCl treatment s. 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 resist ant test spores. Discard carriers if not resistant and repeat preparation of carriers as previous ly described. Note: Compared to the water control, anticipate 1-2 log reduction of viable s pores at 2 min exposure and 3-5 log reduction following the 5 min exposure.


### References Associated wi th 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 Determin ing 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 Biom edical Laboratories (1999) 4th Ed. U.S. Department of Health and Hu man Services, Public Health Service, Centers for Disease Control and Prevention and Nati onal Institutes of Health
(5) Standard Methods for the Examination of Water and Wastewater. 21st Ed. American Public Health Association, 1015 15t h 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 contro l will be tested in triplicate. It is recommended that no more than three disinfectant s plus the water contro l 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 μ L disinfectant + 600 μ L LB broth w/neutralizer (after exposure period) Disinfectant B1-B3 400 μ L sterile water + 600 μ L LB broth (after sonication) C1-C3 400 μ L LB broth + 600 μ L LB broth (after incubation/rotation) A4-A6 400 μ L sterile water + 600 μ L LB broth (after exposure period) Control B4-B6 400 μ L sterile water + 600 μ L LB broth (after sonication) C4-C6 400 μ L LB broth + 600 μ L 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 fractio n 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 μ L of ice-cold sterile water to fraction B tubes.

• Add 400 μ L 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 diluti on 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 μ L 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 μ L of ice-cold LB broth to each d ilution 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 chi ller and water bath and allow them to reach 20 ± 1°C or the temperature specified.

____ On test day, prepare the disinfectant(s) an d place ~1.5 mL of each disinfectant and water control into a microcentrifuge tube

• Be sure to prepare and use the disi nfectant 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, carefu lly place one inoculat ed carrier inside each microcentrifuge tube labeled fraction A

.
• Avoid touching the inoculated area of the ca rrier and the sides of the microcentrifuge tube. If the carrier touches the sides of th e microcentrifuge tube, the carrier and tube must be discarded.

• These forceps should not be used for any additio nal transfers, unless sterilized appropriately. ____ Place the fraction A tubes containing the carriers and the tubes containing the disinfectant(s) and water control into a chille r water bath equilibrated at 20 ± 1°C.

• Allow the carriers, disinfectan t(s), and water control to e quilibrate for approx. 10 min.

____ Using a 1000 μ L micropipette, add 400 μ L of the disinfectant or 400 μ L 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 fo r the appropriate exposure period.

____ After exposure is complete add 600 μ L of the appropriate ice-cold neutralizer to each disinfectant and water control fraction A tube.

• Slight agitation (e.g., light tapping) may be n ecessary to thoroughly mix the liquid components.

____ Using one pair of sterile for ceps per set of 3 carrie rs (e.g., per disinfect ant), 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.

31Note: 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 micr ocentrifuge for 6 min ± 30 sec at 13,000 rpm (15,500 × g).

• Centrifugation #1
____ After fraction A tubes complete centrifugation #1, carefully remove 900 μ L from each fraction A tube without disturbing the pellet. Discard the supernatant.

____ Carefully add 900 μ L 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 μ L.
____ After fraction A tubes complete centrifugation #2, carefully remove 900 μ L from each fraction A tube without disturbing the pellet. Discard the supernatant.

____ Carefully add 900 μ L 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 μ L from each fraction A tube without disturbing the pellet. Discard the supernatant.
____ Carefully add 100 μ L 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 μ L 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 μ L of ice-cold LB broth to the fraction B tubes and vortex for one minute.

32____ Using one pair of sterile for ceps per set of 3 carrie rs (e.g., per disinfect ant), transfer each carrier from its fraction B tube to its corresponding fraction C tube.

• These forceps should not be used for any additio nal 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 μ L 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 refriger ator 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 μ L 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 mini mum 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 hematology rotator Enumerate 30 min. ± 2 min

Nutrient agar plate with representative B. subtilis growth

34Fr act io n A Tim e 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 Fr act io n B Fr act ion C Exposure (x minutes)

Add 600 μ L ice-cold neutralize r Transfer carriers to B tubes Centrifugation 1 (6 min ± 30 sec) Sonication (5 min ± 30 sec)

Add 600 μ L ice-cold LB broth
Remove 900 μ L su p ernatan t Vortex (a pp rox. 1 min )
Add 900 μ L ice-cold LB broth Transfer carriers to C tubes Centrifugation 2 (6 min ± 30 sec) Dilutions and p latin g

Remove 900 μ L su p ernatan t
Add 900 μ L ice-cold LB broth Centrifugation 3 (6 min ± 30 sec)

Remove 900 μ L su p ernatan t
Add 100 μ L ice-cold LB broth
Vortex (5 min ± 30 sec)

Add 800 μ L ice-cold LB broth
Dilutions and p latin g Incubate tubes at 36±1°C in a hematology rotator (30 ± 2 min)

Add 600 μ L ice-cold LB broth
Dilutions and p latin g *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 spread sheets, 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 anal ysis. 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 w ill 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 repe atability standard deviation, denoted by S r , and the reproducibility standard deviation, denoted by S R, for each treatment (disinfectant × efficacy level combination) and for each quantitative res ponse (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, ther eby 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 out liers or influential observations.

Χ Analysis of control carrier spore titers – the log transformed s pores per control carrier will be analyzed using an analysis of va riance (ANOVA) two factor, nested, random effects model (details below). These results will describe the “nor mal range” of control carrier titers for each test met hod as well as estimates of the S r and the S Rand the withintest, intra-laboratory, and inter-laboratory so urces 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 f actor, random effects model ANOVA will be conducted to estimate the S r , the S R , and the intra-laboratory versus inter-laboratory sources of variability.

36Χ Mean LR – for each chemical × efficacy leve l 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 ch eck 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 S R 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 m odels used in (5, 6). For each combination of test method, the response vari able is the log spores per c ontrol carrier, the main effect is laboratory (a random effect), the nested e ffect is replication (a random effect) and the “chance error” is due to the variation among th e carriers within th e 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 labor atories 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 notat ion [see for example (7)], let Y ijk denote the log spores for the k th carrier in the j th trial (replication) in the i th laboratory, k=1,2, or 3, j=1,2, or 3 and i = 1,2, ..., 10. Then the model is Y ij = : + L i + T j(i) + γ ijk , where : denotes the true mean log spores per carrier for that test method and treatment, L i denotes the effect of the i th laboratory, T j(i) denotes the effect of the j th trial (replication) in laboratory i, and γ ijk denotes the chance error for the k th carrier in the j th replicate test in the i th laboratory. The parameter : is deterministic; it is a specifi c, but unknown, numerical value. The quantities L i , T j(i) , and γ ij are random variables. Accord ing to conventional assumptions, L i follows a normal probability distribu tion with mean zero and variance Φ L 2 , T j(i) follows a normal probability distributi on with mean zero and variance Φ T 2 , and γ ijk follows a normal probability distribution with mean zero and variance Φ 2 . This model implies that Y ij is a random variable following a normal probability distribution with mean : and

37variance Φ 2 2 2 L + Φ T + Φ 2 . Let Φ R (= Φ 2 L + Φ 2 T + Φ 2 ) denote the total variance of Y ijk . Conventionally, Φ R = /Φ 2 R is called the reproducibility st andard deviation and Φ r = [ Φ 2 T + Φ 2 ] 1⁄2 is called the repeatability standard deviation (5, 8). The ANOVA will provide numerical estimates of the parameters : , Φ 2 2 2 L , Φ T , Φ 2

, Φ 2
r , and Φ R .

• Log reduction (LR) values – For the TSM, the LR is the mean of log 10 spores per control carrier minus the mean of log 10 spores per treated car rier. Formulas for calculating the LR and associated within-tes t 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 m odel. 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 laborat ories. The laboratories taking part in this collaborative study are assumed to be statistic ally representative of all laboratories that will be conducting these types of sporicide tests in the future.

In standard statistical notati on [see for example (7)], let Y ij denote the LR value for a specific combination of test method and chemical at the j th replication in the i th laboratory, j=1, 2, or 3 and i = 1, 2, or 3. Then the model is Y ij = : + L i + γ ij , where : denotes the true mean LR for that test method and chemical, L i denotes the effect of the i th laboratory, and γ ij denotes the chance error for the j th replicate test in the i th laboratory. The parameter : is deterministic; it is a specifi c, but unknown, numerical value. The quantities L i and γ ij are random variables. Accordi ng to conventional assumptions, L i follows a normal probability distri bution with mean zero and variance Φ L 2 and γ ij follows a normal probability distribution with mean zero and variance Φ r 2 . This model implies that Y ij is a random variable following a normal probability distribution with

mean : and variance Φ L 2 + Φ r 2 . Let Φ R 2 (= Φ L 2 + Φ r 2 ) denote the variance of Y ij ; Φ R 2 is called the “total variance” in ANOVA textbooks. In the context of germicide tests, Φ R = /Φ R 2 is called the reproducibility standard deviation and Φ r = /Φ r 2 is called the repeatability standard deviation (5, 8). The ANOVA will provide numerical estimates of the parameters : , Φ L 2

, Φ r 2 , and Φ R 2 . If the estimates of the variances differ insignificantly among test chemicals, the da ta 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 co mponent of variance from Φ r 2

.
Χ Mean LR – The mean for each test chem ical 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 statis tician, 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

38treatments 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 e fficacy. 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.

• S r and S R of LR values for the TSM – It is desi rable for the standard deviations to be small. For disinfectant tests, the AOAC has no specifications for concluding that a standard deviation is acceptably small. So me guidance is provided by a recent literature review which showed that, for established su spension and dried surface disinfectant tests, S

m edian 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 S R ranged from 0.3 to 1.5 with a

r and S R are acceptably small if they fall within these ranges.

Χ Diagnostic plots and tests – conventional plots a nd 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 fr action of the TSM will be evaluated – the contribution of fraction C will be assessed for importance.



### Section 10. REFERENCES ASSOCI ATED WITH THE VALIDATION PROTOCOL

1. Tomasino, S.F. & Hamilton, M.A. (2006) Unpublished Report. Comparative Evaluation of Two Quantitati ve 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 – 4 th edition, McGraw-Hill:Boston 8. Helrich, K., editor, (19 90) Official Methods of th e AOAC, AOAC: Arlington, VA, p. 681, 1990 9. Zelver, N. et al. (2001) Methods in Enzymol ogy - 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

40B-1

### Appendix C – Guidelines for the Development and Validation of Nucleic Acid Amplification (PCR ) Based Microbiological MethodsIntroduction 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 inco rporating the polymerase chain reaction (PCR) amplification technique; howev er, this guidance should also be applicable to methods incorporating other nucleic ac id amplification techniques.

1.0 The PCR Method: General Background A PCR method encompasses a series of mol ecular procedures including 1) sample preparation, 2) nucleic acid amplif ication 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 speci fic nucleic acid segments present in small or complex samples to sufficiently high levels to allow their det ection by optical, physical and other methods. PCR has been routinely used in clin ical and food microbiology for many years. For a detailed review of PCR methods, please see (Sambrook a nd Russell 2001 McPherson and Moller 2006). Two common approaches for PCR amplifica tion 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 c opies by an independent technique (e.g., gel electrophoresis) after the reaction has been completed. This a pproach is most commonly used as a presence/absence test. Real-time PCR monito rs production of target sequence copies throughout the amplification process, either by use of a specific fluores cently-labeled probe sequence or with a nonspecific in tercalating dye, and yields bo th qualitative and quantitative data. Detailed explanations of real-time PCR detection proc esses 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 co mplex 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 inhi bition of amplification from e nvironmental samples often means that many target microbes are required in the orig inal sample. Nonetheless, various advantages of PCR methods have led many re searchers to develop applicati ons of this technology for
environmental science.

C-12.0 Method Selection When considering a particular PCR method for va lidation, 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 just ification for PCR methods requires instrumentation, with expected advantages in sensitivity, sp ecificity, 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 organism s. PCR methods generally will be unable to make this distinction although more recent modifi cations have shown promise in this regard, e.g., reverse transcription PCR for detecti on of labile messenger RNA molecules or intermediates of ribosomal RNA processing and pr etreatment of samples with intercalating dyes (such as propidium monoazide) that may onl y 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 requi re two primers to target a specific genomic region. PCR primers are short, chemically synthesi zed oligonucleotides, w ith 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 t ype 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 us ed to increase PCR assay specificity, Probes

C-2normally 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 par ticular 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 in tended genetic target will typically be the next
consideration and is determined from the availa ble 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 satis fy 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 stringen cy, as well as other features. Despite the increasing sophistication of PCR assay primer and probe design programs, different primer pairs for the same target sequen ce region can exhibit sign ificant 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 optimi zation of conditions under which the assay is performed.

3.2 Optimization of Reaction Conditions
Variable conditions that can be examin ed 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 va riables may also be characterized within the context of a single PCR instrument and reagent system or with multiple combinations of commercially available instru ments and PCR reagents. With so many conditions and their interactions, a f actorial design works very well for PCR optimization.

C-33.3 Sample Preparation A PCR method consists of not only the ta rget sequence PCR assay, but also the procedures for preparing samples for these anal yses. A requisite component in developing a PCR method is therefore the selection and optimizatio n of the sample prepar ation procedure. At a minimum, this procedure includes the isolation and recovery of nucleic acids across a range of environmental sample quantities determined by th e intended use of a particular PCR method. In some cases, this procedure may also include sample concentration a nd purification of the extracted nucleic acids. The primary variables th at 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 sh ould be designed to estimate sample preparation efficiency as well as PCR assay inhibition.

3.4 PCR Inhibition Control Partial or complete inhibition of PCR am plification can be cau sed by a number of contaminants most often resulting from insuffi cient purification during sample preparation. There are a number of inhibitory substances th at 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 comp letely prevent the amp lification of target
nucleic acids or reduce sensitivit y resulting in false negatives or incorrect real-time quantitative PCR measurements. Thus, the in advertent presence of PCR inhibitors can confound even the best designed and optimized PCR method. The add ition of an amplification control sequence to each reaction can be used to distinguish betw een a false negative, true negative, or PCR inhibition. Amplification controls can be designe d to amplify either simultaneously with the target nucleic acid or in a sepa rate reaction. The amplification c ontrol signal should always be produced even when there is no target nucleic acid present. There are many strategies to construct, detect, quantify, and st ore IAC templates. Some design approaches require substantial technical skills, while others re ly on more basic techniques. It is important to consider the advantages and drawbacks, as well as the inte nded use of the PCR method when selecting a particular IAC strategy. For a detailed discussion, see Hoorfar et al. 2004. At this stage, most

C-4IAC 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 preparati on 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 se quence or whole organism that is added to the sample prior to processing. Whole organisms that are as similar as possible to the tr ue target organism(s) in their physical properties are preferable.

3.6 Preparation and Use of Standards for Qu antitative Real-time PCR Applications Estimates of absolute target DNA concentra tion in environmental samples depends upon the quality of the nucleic acid standards that are employed. For microbi al 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 a dvantage of most closely represen ting 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 c opy concentrations in th ese 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 effi ciency of these templates should be compared, when possible, with genomic DNA templates to de termine equivalency. For target nucleic acids of major importance, internati onally accepted standard reference materials should be established. For example the World Health Organization has established quantitativ e standards for HCV,

C-5HBV, and HIV viruses. Similar approaches for th e establishment of reference standards for other real-time PCR applications shoul d be possible through certificati on 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 applicatio ns, 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 theo retical 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 estim ate 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 amplif ying 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 uncertaintie s associated with a particul ar PCR method. For example, uncertainty can arise within and between e xperiments from numerous sources such as inconsistencies in quality of reagents, pipet calibration, as well as d ilution preparation and storage of standards. Any of these factors co uld significantly alter CT measurements from experiment to experiment. Therefore, estimati on and propagation of uncertainty throughout data analysis becomes critical to ac count 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.

C-6Laboratories performing PCR method s should establish a sufficient number of controls for the detection of contaminating DNA mo lecules 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 c ontrols, and laboratory cleaning practices are adequate to avoid contamination of results. It is also important th at instrument QC procedures be followed. Laboratory QA/QC guidance for P CR methods are discussed in “Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples” (USEPA 2004). As pa rt of a lab’s QA progr am the fact that QA procedures have been followed should be clearly documented.

5.0 PCR Method Performance Criteria Although the general definitions of the perfor mance criteria describe d in Section 2.4 of the main document remain the same, this sectio n 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 a nd that performance cr iteria should be measured across the entire method from sample collection through sample preparation to PCR amplification and interpretation of results. In addi tion, performance criteria that re quire the use of a standard or control spike may utilize DNA target s 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 de tailed 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 im pact the specificity of a PCR method such as primer design; degeneracy of the primers; presen ce 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-e xtracted impurities that can cause inhibition; nonspecific fragment amplification, a nd quality of reference samples av ailable. 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-7DNA sequence databases can be riddled with e rrors and are routinely updated making it even more challenging to standardize specificity test ing. Poor specificity can be identified in numerous ways including amplification of inco rrect 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. restrict ion analysis, DNA sequencing, hybridization). However, some PCR methods are designed to detect target se quences in complex mixtures of heterologous nucleic acid preparations that ca n significantly vary in composition and amount from one sample to the next. For these methods, mixture studies th at 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 encounter ed when implementing the method (SWGDAM 2004). Sensitivity is the probability that a PCR met hod will classify a test sample as positive, given that the test sample is a “known” positive. Sensitivity can be affect ed by characteristics of
the matrix and can be measured under labora tory conditions with a target spike into a characterized matrix such as molecular grade wa ter (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 a nd reproducibility have little value. Quant itative real-time PCR applications allow for more refined measurements of precision in which th e 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 measur ements 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 precisi on for a quantitative real-time PCR assay.

C-8Figure C-1. Example Calculation of precision expressed as RSD for raw C T 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 ar e typically defined by the International System of Units and are maintained by national or international organizations. However, for most quantita tive real-time PCR methods, nationally or internationally recognized reference standards are not available, making it more challenging to establish precision within and between laborat ories. 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 re ference samples, but also becau se a quantitative real-time PCR method includes multiple steps such as sample prep aration, amplification, and data analysis that can each introduce uncertainty and error. Ideall y, a standardized reference sample should be included through the entire quantitative real-t ime PCR method, resulting in an estimated
concentration measurement.

Precis ion is sometimes classified into repe atability 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 t echnician can be used to calculate quantitative

C-9real-time PCR method repeatability. In contrast, repeated measurements generated from the same process among different lots of reagents, instru ments, and technicians over longer periods of
time can be used to estimate th e reproducibility of the method.

5.3 Accuracy and Bias Accuracy is defined as the ability of a qua ntitative real-time PCR method to correctly enumerate a “known” number of DNA targets. Accu racy 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 characte rized 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 Li mit of Quantification (LOQ) LOD is the minimum amount of target sequence th at can be detected with a given level of
confidence in a well characterized background matrix (e.g., distille d 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 preci sion, accuracy and specific ity. Acceptance levels are subjectively determined base d 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 detecti on 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% confid ence may not be necessary for all methods. It is important to establish the L OD 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 amplif ication inhibitors. LOD/LOQ measurements are easily determined for both end-point detecti on and real-time PCR approaches. However, endC-10point detection is typically ba sed on visual detection of PCR products and can vary somewhat among analysts, types of electrophoresis ge ls, 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 backgr ound 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 sa me manner as LOD experiments (Section 2.4.4), however, reference standards are spiked into th e 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 ma ss of target sequence with a confidence interval that can be detected (end-poi nt PCR) or enumerated (quantitative real-time PCR).

5.6 Linearity, Range of Quantification (ROQ), and Amplific ation 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 ampl ification, especially at low nucleic acid target concentrations, five to eight dilutions (e.g., 10-fo ld) bracketing the range of concentrations (for which the PCR method will be used) are used to characterize the re lationship between nucleic acid concentration and response (FDA 2001). From this data a linear plot of C T vs. the logarithm of the target copy number is gene rated (Figure C-1). Linearity, e xpressed as the coefficient of determination R-squared (R 2 ); (Moore and McCabe 1989), is a m easure of the range of target nucleic acid concentrations for which a quantitative PCR test result is dire ctly proportional to the nucleic acid target concentra tion. ROQ is defined as the range of nucleic acid target concentrations that are detectable with an accep table level of precision, ac curacy, and specificity. Linearity and ROQ are determined by testing differe nt concentrations of standard nucleic acid control samples to generate a plotted curve. For real-time PCR, R 2 is a statistical measure of how well a regression line approximates C T values obtained from repeat ed testing of nucleic acid standards (Figure C-2). An R 2 value of 1.0 (100%) indicates a pe rfect fit. The degree to which the plotted curve conforms to a straight line indicates the P CR method linearity. ROQ can then

C-11be 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 doubl es after each cycle during exponential growth. This relationship can be numerically expressed as an exponential amplification of 2.0, which is equivalent to amplification efficien cy of 100%. Amplification efficiency can be influenced by factors such as length, G/C content, and secondar y structure of the amplification product. To assure accurate and reproducible estim ates of DNA target con centration, the slope of
the standard curve should indicate an amplification efficiency as close to 100% as possible.

Figure C-2: Example of determining the lin earity 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 C T measurement from three inde pendent experiments testing a broad range of target nucleic acid standa rd concentrations. The linearity of the

C-12assay is reported as an R2 value. The ROQ is depicted as the ra nge 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 leve ls under normal but unavoidable variable conditions; determined by testing identical samp les under variable conditions. Factors such as reagent stock stability, analyst to analyst variation, use of differ ent thermal cycling instruments, laboratory to laboratory variati on, 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 quali tative end-point PCR methods, ruggedness can be determined by measuring changes in false posi tives/false negatives and LOD. For real-time quantitative PCR methods, ruggedness can be ev aluated using the Horwitz equation (Horwitz 1995) where the relative standard deviation of reproducibility (RSD R ) is given as:

RSD R (predicted) = 2 (1-0.5logC) ; and Equation (8)
HORRAT = RSD R (observed) / RSD R (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 RSD R values is designated HORRAT and can be used as an indication of accep tability (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 docum ent should be applicable for these methods.

6.1 Influence of PCR Platform and Reagent System It is contrary to general policies with in EPA that discourage endorsement or recommendation of specific commercial products; however, it may be necessary to treat assays that utilize different platforms as different me thods for the purpose of validation. The popularity

C-13of the PCR technique has given rise to the ava ilability of a wide choice of thermal cycling instrument and amplification reagent systems from different commerc ial 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 st udies, 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 e xperimentally assess the equivalence of method performance with all systems being considered. Particular attention s hould be paid to any significant differences in specific ity and sensitivity with these sy stems. Assay specificity may be affected by different amplif ication reagents (Siefring et al. 2008). Preliminar y assessments of specificity can be performed through the analys is of common DNA standards from one or more non-target controls that ar e closely related to the target of inte rest. If variability in specificity is observed, it may be possible to adjust the annea ling temperature of the thermal cycling protocol for each respective reagent and instrument to bri ng it within acceptable levels. It should be noted, however, that comparable specificity may be diffi cult to demonstrate for all possible target and non-target organisms. The sensitivity of an assay on different inst ruments 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 r eagent. Due to differences in optics, some variability in sensitivity may be una voidable 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 te st 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 consid eration in the design of a validation study.

C-147.0 Potential Differences between PCR Chemistries Several different probe and non-probe based PCR product dete ction chemistries are also available for real-time PCR assays (Wittwer and Kusukawa 2004). Moreover, product detection reagents employing these chemis tries 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 diffe rent commercial sources of the product detection reagents, it is recommended that the chemistry and commerc ial source of reagents used for method development be specified for each real-time PCR method. Alternativ e sources of reagents can be designated if their products can be experiment ally demonstrated to provide equivalent performance in the method to that of the reag ents 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: Bioana lytical 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. Abdulmawj ood, N. Cook, M. Wagner, and P. Fach. 2004. Practical Considerations in De sign of Internal Amplificati on Controls for Diagnostic PCR Assays. Journal of Clinical Microbiology 42:1863-1868.

5. Horwitz, W. 1995. Protocol for the design, conduct and interp retation 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 Statistic s. W.H. Freeman & Company, New York, NY.

8. Nocker, A., Sossa, K.E. and Camper, A.K. (2007) Molecular monito ring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. Journal of Microbiological Methods 70, 252-260

.

C-159. Sambrook, J., and D. W. Russell. 2001. Mo lecular Cloning: A La boratory 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 Guide lines: 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-t ime PCR, p. 71-84. In D.L. Persing, F.C. Tenover, J. Versalovic, Y-W. Tang, E.R. U nger, D.A. Relman, and T.J. White (ed.), Molecular Microbiology, Diagnostic Principl es and Practice. ASM Press, Washington D.C.

C-16

### Appendix D – Guidelines for the Va lidation of Efficacy Test MethodsIntroduction 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 s ubmit efficacy data for registration of nonpublic health antimicrobial products. However, each applicant and registrant must ensure through laboratory testing that products are efficacious, and main tain 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 Ge nerate Product Efficacy Data Efficacy requirements of antimicrobials ar e set forth in the 40 Code of Federal Regulations (CFR) Part 158. Guidance documents sp ecific for efficacy testing of antimicrobials are found in Registration Polic y 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 st andard-setting organization. In most cases, an
AOAC method is validated accordin g to strict guidance provided by AOAC, and official changes or modifications to a method can only be a pproved under AOAC purview. AOAC methods have been in place for several decades and are primaril y qualitative in nature (i.e., presence or absence of viable test organism followi ng exposure to test chemical). Fo r example, data generated using the AOAC Use Dilution Methods, the AOAC Ge rmicidal Spray Products Method, and the AOAC Sporicidal Activity of Disinfectants Me thod support certain claims for public health pesticides and are considered critical to the Agency’s decision making process in registering antimicrobial pesticides. Other currently-reco mmended methods are those that the Agency believes have historically prove n to be well-developed and suita ble for their intended use (e.g.,

D-1ASTM standards) but not necessarily subjected to multi-laboratory validation studies. Currently, the Agency is promoting the development of quan titative (i.e., kill measured as a log reduction) rather than qualitative efficacy tests and is spearheading improvements to existing qualitative and quantitative methods to furthe r enhance method performance. In addition, the Agency is currently working to harmonize antimicrobial effi cacy test methods internationally. For efficacy
claims for which no recommended/standard test me thod 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 revi ew process includes an extensiv e in-house and optional expert panel protocol review, requirements for independent verification of the protoc ol in three separate
laboratories and demonstrati on of statistical validity (www.epa.gov/oppad001/efficacyproto.htm). For al l 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 releva nt for its intended use.

2.0 Validation of Efficacy Test Met hods and Study Plan Preparation Although EPA does not currently require va lidated methodology, the user community (i.e., registrant, government agencies) may determin e that there is a need for determining method performance across laboratories and may seek va lidation. EPA is interested in rigorous method validation to ensure that the Agency and its stake holders 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 setti ng organization such as AOAC International is highly desira ble, but is not required. Historically, EPA recognizes and recommends the use of many AOAC International methods because of the standard setting organi zation’s validation proces s. AOAC International has a well-structured validation program (Offic ial Methods Program) that evaluates methods through interlaboratory collaborati ve 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, includ ing between and within-lab variability and ruggedness. In advance of submitting a protocol fo r validation, it is advisable to generate inhouse validation data or arrange for an inde pendent lab to validate the method. For more

D-2information on AOAC International’s method valid ation 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 scientif ic journal, without participation in a formal third party validati on program such as the AOAC Official Methods Program. For scientists intere sted in pursuing method valida tion independently, the AOAC Web
site is an excellent source of information on co llaborative 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, necessitati ng revisions of standard methods.

3.0 Pre-Validation Considerations: Factors Affecting Efficacy of Antimicrobial Products

Springthorpe and Sattar (2005) provide an excellent overvie w of factors affecting the antimicrobial activity of products. An understa nding of these factor s 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 a nd Sattar (2005):

Χ Formulation – minimum concentration of active ingredients required for efficacy; different inert ingredients have differe nt affects on efficacy of the product.

Χ Target organism/organic soil load – Microorg anisms vary in their ability to survive exposure to antimicrobials. In a health care 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 e fficacy 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.

D-3Χ However, product users, without specific labe l guidance, are more likely to use tap water than distilled water to dilute the product. Tap wate r varies in hardness and the user may unintentionally reduce the effi cacy of the product by using tap water.

Χ Contact time – Treated surfaces must re main wet for a minimum of the labelspecified contact time for the product to be effective.

Χ Carrier surface – Microorganisms must be e liminated from a variety of surfaces (e.g., steel, glass, wood) in the environment. Carrie rs used in efficacy tests must effectively simulate the surfaces to be treated by an end user.

Χ Precleaning agents – Many antimicrobial pr oduct 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 a pplied 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 po tency may decrease more rapidly than with the concentrated product.

Χ pH/humidity – Antimicrobial products work be st 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 Sp ringthorpe 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 presen t in the test system – Many current EPArecommended efficacy test methods are qua litative (presence/absence of viable microbes after treatment) rather than quantitat ive. 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 microor ganisms in the test system.

Χ Quality of microorganisms present in th e test system – Currently, there is no established standard (i.e., chemicals/disi nfectants) for use in efficacy test methods. Consequently, for decades, scientists have relied upon AOAC efficacy test standards such as phenol resistance testing or HCl te sting to estimate the intrinsic resistance of test microbes to disinfectants (indicator of suitable organism population). In 2001 (PR

D-4Notice 2001-04; http://www.epa.gov/opppmsd1/ PR_Notices), EPA determined that phenol resistance testing was an unsatisfact ory standard for determining organism hardiness and recommended a minimum inoculum level of 10 4 organisms/carrier for AOAC carrier based efficacy tests. HCl testing still remains a required component of the AOAC Sporicidal Activity Test as an indi cator of spore hardiness. The quality of the microorganisms used in an efficacy evaluation affects the outcome of testing

⎯ use of less resistant organisms ma y make the product appear more efficacious than it actually is.

Χ Technique-sensitive procedures – Many of the recommended efficacy methods may contain technique-sensitive step s. It is critical that scie ntists performing the tests be trained in the conduct of the method. Follo wing 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 sele ction 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 inocul ation procedure, method of determining populations of microorganisms on a car rier, 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-log 10 kill of the target organism on a porcelain carrier is required for s poricidal decontamination products, then the efficacy method must be able to accommodate a minimum of a 7-log 10 challenge per carrier.

D-55.0 Method Performance Characteristics The method performance characteristics for effi cacy 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 standard s (i.e., chemicals/disinfectants) available for use in efficacy tests. Accuracy is measured from results of re peating 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 ProcessOnce 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 public ation 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 unde r the Alternate Test Procedures (ATP) program also followed the same approval a nd 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 establishe d, 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 allo w emerging technologies that reduce cost and enhance data quality. Under this program, all mo difications that are not explicitly allowed by a method cited in a rule require prior EPA appr oval through the ATP program. An ATP can be a
modified method or a new met hod. The applicant(s) of a particular ATP can only submit an application to the ATP program after the met hod 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 a pproval notice. To use this pro cess, EPA must have already promulgated at least one analytical testing me thod for the analyte or microorganism through the

E-1rulemaking process. Section 1401 of the SDWA allows EPA to approve additional testing methods through this expedited approval that s imply 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 th is expedited approval process. However, the performance equivalence of the new testi ng method to the reference method (2008, FR 73: 31616) must be demonstrated before being cons idered for approval by the expedited process

. A process flow diagram illustrating the approval st eps 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 Rep resenting 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 me thod that contains a unique combination of analyte and technique is consid ered a new method and, when appr oved, can be designated as the

E-2reference 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 re ference 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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