Single-Laboratory Verification of Culture-Based Procedure ...

Office of Research and DevelopmentNational Homeland Security Research Center

Single-Laboratory Verificationof Culture-Based Procedurefor Detection of SalmonellaTyphi in Drinking Water andSurface Water

STUDY REPORT

EPA/600/R-10/132 | October 2010 | www.epa.gov/ord

Single-Laboratory Verification of Culture-Based Procedure for Detection of Salmonella Typhi in Drinking Water and Surface WaterSTUDY REPORT

Office of Research and DevelopmentNational Homeland Security Research Center

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Acknowledgments

This report presents results of a single-laboratory study (Study) funded by the National Homeland Security Research Center (NHSRC) within the U.S. Environmental Protection Agency (EPA) Office of Research and Development to verify culture-based analytical procedures for Salmonella enterica subspecies (spp.) enterica serotype Typhi (S. Typhi) in water. The Study was designed under the direction under the direction of EPA technical lead Sanjiv R. Shah of the NHSRC within EPA’s Office of Research and Development, with consultation and input provided by workgroup members and subject matter experts. CSC provided coordination of activities for the Study, technical support and data evaluation under EPA contract EP-C-05-045.

The contributions of the following persons and organizations are acknowledged:

Study Workgroup Participants • Michele Burgess, Marissa Mullins (EPA, Office of Emergency Management)• Stephanie Harris (EPA, Region 10)• Sarah Perkins, Gene Rice (EPA, NHSRC)• Malik Raynor, James Sinclair, Ouida Holmes (EPA, Office of Ground Water and Drinking

Water)• Matthew Mikoleit (Centers for Disease Control and Prevention)

Subject Matter Experts • Cheryl Bopp (Centers for Disease Control and Prevention)• Nancy Hall (University of Iowa Hygienic Laboratory)• Steve Weagant (U.S. Food and Drug Administration)

Volunteer Participant Laboratory• Fu-Chih Hsu, Rebecca Wong (Scientific Methods, Inc.)

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Disclaimer

This document has been reviewed in accordance with EPA policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Neither the United States Government nor any of its employees, contractors, or their employees make and warranty, expressed or implied, or assume any legal liability or responsibility for any third party’s use of, or the results of such use of, any information, apparatus, product, or process discussed in this document, or represent that its use by such party would not infringe on privately owned rights. Mention of trade names or commercial products in this document or in the methods referenced in this document does not constitute endorsement or recommendation for use.

Questions concerning this document or its application should be addressed to:

Sanjiv R. ShahNational Homeland Security Research Center

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue, NW

USEPA-8801RR

Washington, DC 20460

(202) 564-9522

[email protected]

If you have difficulty assessing these PDF documents, please contact [email protected] or [email protected] for assistance.

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Foreword

The mission of the U.S. Environmental Protection Agency (EPA) is to protect human health and to safeguard the natural environment – the air, water, and land upon which life depends. After the 2001 terrorist attacks including the anthrax bioterrorism event, the EPA’s mission was expanded to address critical needs related to homeland security. Presidential directives identified EPA as the primary federal agency responsible for the protection and decontamination of indoor-outdoor structures and water infrastructure vulnerable to chemical, biological, or radiological (CBR) terror attacks.

The National Homeland Security Research Center (NHSRC) within the Office of Research and Development (ORD) is EPA’s focal point for providing expertise, and for conducting and reporting research to meet its homeland security mission needs. One specific focus area of the NHSRC’s research is to support the Environmental Response Laboratory Network (ERLN), a nationwide association of federal, state, local, and commercial environmental laboratories, established by EPA. The ERLN can be deployed in response to a large-scale environmental disaster to provide consistent analytical capabilities, capacities, and quality data in a systematic and coordinated manner. To this end, the NHSRC has worked with experts across EPA and other federal agencies to develop standard analytical protocols (SAPs) to be used in support of the response to national homeland security related incidents.

This report documents single-laboratory development and verification of quantitative procedures included in a draft “Standard Analytical Protocol for Salmonella enterica subsp. enterica serotype Typhi (Salmonella Typhi) in Water Samples.”

NHSRC has made this publication available to assist in preparing for and recovering from disasters involving Salmonella Typhi contamination. This work specifically represents an important step in EPA’s support for the ERLN and moves the agency closer to achieving its mission to support homeland security and its overall mission to protect human health and the environment.

Gregory D. Sayles, Ph.D., Acting Director

National Homeland Security Research Center

Office of Research and Development

U.S. Environmental Protection Agency

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Table of Contents

Acknowledgments ................................................................................................................................. ii

Disclaimer ............................................................................................................................................iii

Foreword .............................................................................................................................................. iv

Tables .................................................................................................................................................viii

Acronyms ............................................................................................................................................. ix

Executive Summary ............................................................................................................................. xi

1.0 – Background .................................................................................................................................. 1

2.0 – Study Objectives and Design ....................................................................................................... 3

2.1 – Study Objectives ................................................................................................................... 3

2.2 – Data Quality Objective ......................................................................................................... 3

2.3 – Study Preparation .................................................................................................................. 32.3.1 – Identification of Laboratory ........................................................................................ 32.3.2 – Spiking Approach ....................................................................................................... 3

2.4 – Sample Matrices ................................................................................................................... 4

2.5 – Sample Analyses ................................................................................................................... 4

2.6 – Quality Control Analyses ...................................................................................................... 5

3.0 – Study Schedule ............................................................................................................................. 7

4.0 – Data Reporting, Validation, and Censoring ................................................................................. 9

4.1 – Data Reporting ...................................................................................................................... 9

4.2 – Data Validation ..................................................................................................................... 9

4.3 – Censored Data ....................................................................................................................... 9

5.0 – Results ........................................................................................................................................ 11

5.1 – Preliminary Analyses of S. Typhi in PBS, Drinking Water, and Surface Water ................. 11

5.2 – Optimization Analyses of S. Typhi in PBS, Drinking Water, and Surface Water ............... 12

5.3 – Verification Analyses of S. Typhi in PBS, Drinking Water, and Surface Water.................. 14

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5.4 – Optimization Analyses for Surface Water........................................................................... 155.4.1 – Evaluation of Commercially-Available Immunomagnetic Separation (IMS) Beads .............................................................................................................. 155.4.2 – Evaluation of Surface Water Matrix Inhibition of Immunomagnetic Separation (IMS) Protocol ........................................................................................ 155.4.3 – Evaluation of Laboratory-Conjugated Immunomagnetic Separation (IMS) Beads .............................................................................................................. 15

6.0 – Discussion .................................................................................................................................. 17

7.0 – Conclusions ................................................................................................................................ 19

8.0 – References .................................................................................................................................. 21

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Appendices

Note: Appendices are attached to the final report in the final form that was distributed to the workgroup and laboratory and may contain minor differences in language in comparison to the Study Report.

Appendix A – Study Plan …………………………………………………………………………..A-1

Appendix B – Study-Specific Instructions …………………………………………………………B-1

Appendix C – Spiking Protocol …………………………………………………………………….C-1

Appendix D – Data Reporting Forms………………………………………………………………D-1

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Tables

Table 1. Summary of Sample Analyses ............................................................................................... 4

Table 2. Schedule for Laboratory Analyses of S. Typhi Culture-Based Procedures in Water Matrices .................................................................................................................................. 7

Table 3. Preliminary Analyses, Results: PBS, Drinking Water, and Surface Water Spiked with S. Typhi ...................................................................................................................................... 11

Table 4. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water (without Pre-Enrichment in UPD Broth) Spiked with S. Typhi .......................................................... 12

Table 5. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water (with Pre-Enrichment in UPD Broth) Spiked with S. Typhi ............................................................... 13

Table 6. Verification Analyses, Results: PBS, Drinking Water, and Surface Water Spiked with S. Typhi ....................................................................................................................................... 14

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AcronymsASTM American Society for Testing and Materials

ATCC® American Type Culture Collection

BSD Bismuth sulfite agar with DHB

°C Degrees Celsius

CFU(s) Colony forming unit(s)

CDC Centers for Disease Control and Prevention

CVD Centers for Vaccine Development

DHB 2,3-dihydroxybenzoate (indicated by subscript “D” when used with media acronym)

EPA U.S. Environmental Protection Agency

HEPA High efficiency particulate air

IMS Immunomagnetic separation

MMD Miller-Mallinson agar with DHB

MPN Most probable number

NA Not applicable

NHSRC National Homeland Security Research Center

OEM Office of Emergency Management

ORD Office of Research and Development

OW Office of Water

OGWDW Office of Ground Water and Drinking Water

PBS Phosphate buffered saline

PCR Polymerase chain reaction

QA Quality assurance

QC Quality control

RSD Relative standard deviation

SAM Standardized Analytical Methods for Environmental Restoration Following Homeland Security Events

SAP Standard Analytical Protocol

SD Standard deviation

SCBD Selenite cystine broth with DHB

TNTC Too numerous to count

TSAD Tryptic soy agar with DHB

TSB Tryptic soy broth

UPD Universal pre-enrichment with DHB

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Executive Summary

This report presents the results of the U.S. Environmental Protection Agency’s (EPA’s) single-laboratory verification study (the “Study”) to evaluate a draft culture-based Standard Analytical Protocol (SAP) for the identification and quantitation of Salmonella Typhi in drinking water and surface water samples. The original procedure evaluated during the Study was adapted from Section 9260B, “General Qualitative Isolation and Identification Procedures for Salmonella,” in Standard Methods for the Examination of Water and Wastewater. After workgroup review, the procedure, as is, was determined to be unacceptable and was revised. The procedure evaluated during the Study was adapted from the Manual of Clinical Microbiology, “Escherichia, Shigella, and Salmonella” and the U.S. Food and Drug Administration’s Bacteriological Analytical Manual [online], “Salmonella.” The purposes of the Study were to: (1) evaluate draft SAP performance (recovery and precision) in a reference matrix (phosphate buffered saline [PBS]), (2) evaluate draft SAP performance (recovery and precision) in environmental matrices of interest (drinking water, surface water), and (3) determine whether the draft SAP requires revision prior to multi-laboratory validation.

During the Study, the analytical laboratory analyzed unspiked and spiked PBS, drinking water, and surface water samples. Vaccine strains were used to reduce health risk to laboratory personnel. This strain has an engineered nutritional defect that requires a supplement (2,3-dihydroxybenzoate [DHB]) be added to all media to ensure proper growth. The Study was conducted during September 2008 through January 2010.

Some issues were observed during the Study, including (1) all Salmonella O Group D antiserum tests were negative, and (2) S. Typhi was not recovered effectively recovered from surface water samples. The Salmonella O Group D antiserum tests were likely negative because the strain selected for the study constitutively produces Vi, a surface antigen that can mask other surface antigens. To address this issue,

testing with Salmonella O Group D antiserum was discontinued, and serological typing with Salmonella Vi antiserum combined with biochemical analyses was used for confirmation. It was concluded that the laboratory was not able to effectively recover S. Typhi from surface water samples due to background organisms. Based on workgroup recommendations, the laboratory evaluated

a number of potential procedural modifications, such as the use of immunomagnetic separation (IMS) beads. Unfortunately, none of the procedures provided acceptable results for this matrix. The final optimized procedure included two plating media, bismuth sulfite (BSD) and Miller-Mallinson (MMD) agars. For spiked PBS samples, mean recoveries were 71% and 94% for BSD and MMD, respectively. For spiked drinking water samples, mean recoveries were 307% for both plating media (BSD and MMD). Mean recoveries of S. Typhi CVD 909 in surface water samples were 0% for both plating media.

Results of the single-laboratory validation study indicate that these procedures merit multi-laboratory validation to assess method performance, and set quantitative QC criteria for PBS (reference matrix) and drinking water. However, for surface water, the culture-based procedure evaluated during this study is not appropriate and development and inclusion of molecular procedures (e.g., PCR) for detection of S. Typhi in surface water samples is recommended.

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1.0 Background

Subsequent to the anthrax attacks in the fall of 2001, federal and state personnel were tasked with a mission to provide response, recovery, and remediation for biological incidents. However, it was recognized that no U.S. Environmental Protection Agency (EPA) verified or validated protocol exists for collection, isolation, and analysis of Salmonella enterica subsp. enterica serotype Typhi (S. Typhi), a potential bioterrorism agent and the causative agent of typhoid fever, in water samples.

This report presents results of the single-laboratory verification study (Study) to evaluate EPA’s draft Standard Analytical Protocol (SAP) (EPA, National Homeland Security Research Center) culture-based procedure for the identification and quantitation of S. Typhi in water samples. The original SAP was based on Section 9260B, “General Qualitative Isolation and Identification Procedures for Salmonella,” in Standard Methods for the Examination of Water and Wastewater (Reference 8.1). After workgroup review, the procedure, as is, was determined to be unacceptable and was revised. The procedure evaluated during the Study was adapted from the Manual of Clinical Microbiology, “Escherichia, Shigella, and Salmonella” (Reference 8.2) and the U.S. Food and Drug Administration’s Bacteriological Analytical Manual [online], “Salmonella” (Reference 8.3). To reduce the potential health risk to laboratory personnel, an attenuated (vaccine) strain of S. Typhi (CVD 909) was obtained from Centers for Vaccine Development (CVD), University of Maryland, Baltimore, Maryland, for the Study. This strain has an engineered nutritional defect that requires a supplement (2,3-dihydroxybenzoate [DHB]) be added to all media to ensure proper growth (indicated by a subscript “D” after the medium acronym).

The procedure recommended by the workgroup for initial evaluation included: (1) enriching in universal pre-enrichment broth supplemented with DHB (UPD) at 35.0°C ± 0.5°C for 24 ± 2 hours, (2) transferring 1 mL to selenite cystine broth with DHB (SCBD) and incubating at 35.0°C ± 0.5°C for 24 ± 2 hours, (3) streaking growth from SCBD onto bismuth sulfite agar with DHB (BSD) and incubating at 35.0°C ± 0.5°C for 24 ± 2 hours, (4) sub-culturing typical colonies onto tryptic soy agar with DHB (TSAD) and incubating at 35.0°C ± 0.5°C for 24 ± 2 hours, and (5) verifying isolates using serological analyses against Salmonella Vi and O Group D antigens.

Due to issues with BSD (e.g., tiny colonies, no growth at 24 hours) observed during preliminary analyses, the workgroup recommended adding an additional selective plating medium (Miller-Mallinson with DHB [MMD]) to the procedure.

Quantitation of S. Typhi was determined using the most probable number (MPN) technique. Tubes that confirmed positive for S. Typhi were used to determine the MPN (Reference 8.4). It should be noted that a MPN value is not considered absolute quantitation, as a direct plate count would be, because values are based on the probability of a tube being positive for a given organism concentration.

Based on workgroup discussion, a nine-tube MPN was utilized for the Study to reduce the burden (306 tubes and 612 plates for a nine-tube MPN versus 510 tubes and 1020 plates for a fifteen-tube MPN) on the participant laboratory. All of the media required the addition of DHB after autoclaving, resulting in the laboratory having to sterilize tubes prior to manually dispensing the media into each tube using aseptic technique in a biological safety cabinet. A 15-tube MPN will be considered for the multi-laboratory validation study.

Initially, all tests with Salmonella O Group D antiserum were negative, likely because the strain selected for the study constitutively produces Vi, a surface antigen that can mask other surface antigens. Based on workgroup recommendation, the laboratory boiled the culture prior to serological testing with O Group D. However, this approach also did not provide acceptable results. Based on workgroup recommendation, testing with Salmonella O Group D antiserum was discontinued and serological typing with Salmonella Vi antiserum combined with biochemical analyses was used for confirmation.

Due to background organisms that may have overgrown or inhibited the target organism, the laboratory was not able to effectively recover S. Typhi from surface water samples. Based on workgroup recommendations, the laboratory evaluated a number of potential procedural modifications, such as the use of immunomagnetic separation (IMS) beads. Unfortunately, none of the procedures provided acceptable results for this matrix.

For drinking water, the final verified procedure included: (1) enriching in UPD at 35.0°C ± 0.5°C for 24 ± 2 hours, (2) transferring 1 mL to SCBD and incubating at 35.0°C ± 0.5°C for 18 ± 2 hours, (3) streaking growth from

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SCBD cultures onto BSD and MMD agars and incubating at 35.0°C ± 0.5°C for 24 − 48 hours, (4) sub-culturing typical colonies onto TSAD, and (5) verifying isolates using serological (Salmonella Vi) and biochemical (API® 20E test strips) analyses. Time to results, as verified, is approximately 120 – 144 hours from receipt of samples.

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2.0Study Objectives and Design

The primary objective of the Study was to evaluate the performance of the culture-based procedure included in the draft SAP for identification and quantitation of S. Typhi in drinking water and surface water. As indicated in the Study Plan (Appendix A), the Study was originally designed to verify the procedure for water, solid, and particulate matrices. However, during the Study, only water matrices were evaluated, and only these are included in this report. Additional matrices may be evaluated in the future. In support of the primary objective of evaluating the draft SAP, both study objectives (Section 2.1) and data quality objectives (Section 2.2) were set for the Study. The Study preparation, sample matrices, and sample analysis selected to meet these objectives is described in Sections 2.3 – 2.5, below.

2.1 – Study Objectives • Evaluate draft culture-based SAP performance

(recovery and precision) for a reference matrix (phosphate buffered saline [PBS])

• Evaluate draft culture-based SAP performance (recovery and precision) for water matrices of interest (drinking water, surface water)

• Determine whether the draft culture-based SAP requires revision prior to multi-laboratory validation

To accomplish these objectives, the Study was conducted in five phases, as described below:

• Phase 1.Planning and Preparation: Identification of a qualified laboratory to participate in the Study, identification of an appropriate strain of S. Typhi, and evaluation of the Spiking Protocol (Appendix C)

• Phase 2. Preliminary Analyses: Assessment of modified procedure in three matrices (PBS [reference matrix], drinking water, surface water) and identification and resolution of any analytical problems

• Phase 3. Optimization Analyses for all Matrices: Evaluation of the procedure with and without pre-enrichment (UPD broth), an additional plating medium (MMD), and boiling prior to O Group D serological analyses

• Phase 4. Verification Analyses: Analyses of PBS, drinking water, and surface water samples using the optimized procedure

• Phase 5. Optimization Analyses for Surface Water: Evaluation of commercially-available and laboratory-conjugated IMS beads and matrix inhibition

2.2 – Data Quality ObjectiveData produced under this Study were generated according to the analytical and quality assurance/quality control (QA/QC) procedures specified in the Study-specific instructions (Appendix B) and the draft SAP. This ensured data integrity and validity for all matrices evaluated and allowed the Study workgroup to use the results to identify any necessary revisions of the draft SAP.

2.3 – Study PreparationPrior to the Study, the following activities were completed, including identification of an appropriate laboratory and identification of an appropriate strain of S. Typhi and development and evaluation of the Spiking Protocol (Appendix C).

2.3.1 – Identification of Laboratory A laboratory was identified that was representative of the general user community, had experience analyzing environmental samples for Salmonella spp., and had access to representative matrices. To reduce Study costs, a volunteer laboratory (Scientific Methods, Inc.) was recruited. To reduce the burden on the laboratory and encourage participation, National Homeland Security Research Center (NHSRC) provided the media, reagents, and supplies needed for the Study. The requirements and responsibilities of the laboratory are detailed in Study-specific instructions (Appendix B) and the draft SAP.

2.3.2 – Spiking Approach The Study Plan (Appendix A) included the use of two spike types: BioBalls™ (precise pre-enumerated spikes) commercially-developed from S. Typhi CVD 909 and laboratory-prepared spiking suspensions. However, due to manufacturer development issues, BioBalls were not available during the Study.

During each week of the Study, laboratory-prepared spiking suspensions were propagated according to the Spiking Protocol (Appendix C) using S. Typhi CVD 909. To determine spike level, the cells were enumerated in triplicate on TSAD using the spread plate technique described in the Spiking Protocol (Appendix C). Spikes were enumerated on the same day that samples were spiked and analyzed. Results were reported as colony forming units (CFU) and used to determine the actual spike level.

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2.4 – Sample MatricesThe procedure was evaluated and verified using three water matrices (one reference and two matrices of interest). The reference matrix provided a means for assessing the performance of the draft SAP using a standard matrix that could be duplicated during routine use of the procedure in the future. The following matrices were analyzed during the Study:

Reference Matrix• PBS

Water Matrices (Matrices of Interest)• Drinking water (laboratory tap, dechlorinated with

sodium thiosulfate)• Surface water (source water for drinking water

treatment plants)

2.5 – Sample Analyses For preliminary analyses, one, 100-mL unspiked and one, 100-mL spiked samples were evaluated per matrix. For optimization and verification analyses, one, 100-mL unspiked PBS or two, 100-mL unspiked drinking or surface water samples, as appropriate, were evaluated by the procedure to determine background S. Typhi concentrations. In addition, four, 100-mL spiked samples were evaluated per matrix. Table 1 summarizes the number and type of samples that were evaluated to meet the objectives listed in Section 2.1.

Table 1. Summary of Sample Analyses

Analysis Matrix Spiking Description Procedure No. of Analyses

Phase 2 Analyses

Preliminary Analyses Sterile PBS (Reference Matrix)

Unspiked Assessment of workgroup-modified procedure

1

S. Typhi CVD 909 1

Drinking Water Unspiked 1

S. Typhi CVD 909 1

Surface Water Unspiked 1

S. Typhi CVD 909 1

Phase 3 Analyses

Optimization Analyses Sterile PBS (Reference Matrix)

Unspiked Evaluation with and without pre-enrichment (UP broth) D

1

S. Typhi CVD 909 4


S. Typhi CVD 909 4


S. Typhi CVD 909 4

Phase 4 Analyses

Verification Analyses Sterile PBS (Reference Matrix)

Unspiked Analyses of PBS, drinking water, and surface water samples using the optimized procedure

1

S. Typhi CVD 909 4


S. Typhi CVD 909 4


S. Typhi CVD 909 4

5

Analysis Matrix Spiking Description Procedure No. of Analyses

Phase 5 Analyses

Optimization Analyses for Surface Water

Sterile PBS (Reference Matrix)

Unspiked Evaluation of commercially-available IMS beads

1 – 4(1)

S. Typhi CVD 909

Surface Water Unspiked

S. Typhi CVD 909

Surface Water S. Typhi CVD 909 Determination of matrix inhibition

1 – 2(1)

Sterile PBS (Reference Matrix)

Unspiked Evaluation of laboratory-conjugated IMS beads

1 – 4 (1)

S. Typhi CVD 909

Surface Water Unspiked

S. Typhi CVD 909

(1) Evaluation of IMS technique was conducted with variable numbers of analyses

CVD – Centers for Vaccine Development PBS – Phosphate buffered saline

IMS – Immunomagnetic separation; UPD – Universal pre-enrichment with 2,3-dihydroxybenzoate

2.6 – Quality Control Analyses The participant laboratory performed the following QC analyses:

• Method Blank: The laboratory analyzed a sterile unspiked PBS method blank during each week of analyses to verify the sterility of equipment, materials, and supplies.

• Sterility Checks: To evaluate the sterility of media and buffer, the laboratory incubated a representative portion of each batch at 35 ± 0.5 (PBS, UPD broth, SCBD, BSD, MMD, and TSAD) for 24 ± 2 hours or 48 ± 3 hours (as appropriate) and observed for growth. In addition, sterility checks were conducted each day samples were run.

• Positive and Negative Controls: For the purpose of the Study, positive controls for selective agars and broths are those organisms that provide the characteristic growth and/or colony morphology of the target organism. Negative controls are those organisms that do not provide the characteristic target organism growth or morphology. For biochemical and serological analyses, positive and negative controls are defined by their reaction (e.g., Pseudomonas aeruginosa is oxidase positive and S. Typhi CVD 909 is oxidase negative). The following positive and negative controls were evaluated during each week of the Study:• S. Typhi CVD 909: Positive controls (target

organisms)• Enterococcus faecalis (ATCC® 29212™): Negative

control (non-target organism) • Pseudomonas aeruginosa (ATCC® 27853™):

Positive control for oxidase test and negative control for biochemical tests

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3.0Study Schedule

The duration of the Study was September 2008 through January 2010. Due to laboratory and workgroup scheduling confl icts, availability of commercial products, and development and evaluation of IMS procedures, the Study time frame was extended approximately six months beyond the anticipated schedule. The Study schedule is provided in Table 2. Analyses of additional matrices may be conducted at a later date.

Table 2. Schedule for Laboratory Analyses of S. Typhi Culture-Based Procedures in Water Matrices

Date Analysis Phase

September – October 2008 Phase 1 – Spiking Protocol Evaluation

November 2008 Phase 2 – Preliminary Analyses

December 2008 Phase 3 – Optimization Analyses for all Matrices

December 2008 – January 2009 Phase 4 – Verifi cation Analyses

May – December 2009 Phase 5 – Optimization Analyses for Surface Water

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4.0Data Reporting, Validation, and Censoring

4.1 – Data ReportingThe laboratory submitted the following data to CSC for review and validation:

• Completed cover sheet with sample collection and QC information

• Completed sample-specific data reporting forms• Documentation of any additional information that

would assist in evaluating the data

4.2 – Data ValidationCSC used data review checklists to ensure that each data package was complete and that each sample result met the Study-specific and method-specific requirements. The review for each sample confirmed the following:

• Original forms were submitted• Incubation times were met• Incubation temperatures were met• Media sterility checks were performed and

acceptable• Positive and negative controls were performed and

exhibited the appropriate response• Samples were spiked with the appropriate dilution• All procedures were performed according to study-

specific instructions and analytical procedures • Calculations were correct

This process was performed independently by two data reviewers, each of whom entered the results into separate spreadsheets designed for data review and validation for this Study. The results spreadsheets were evaluated using the compare function in Microsoft® Excel® to verify consistency and identify potential data entry errors.

All verification data generated during the Study were considered valid and were included in subsequent data analyses, as appropriate.

4.3 – Censored DataIn addition to the numerical sample results generated during this Study, low censored (“less than”) results were generated for all unspiked samples (i.e., negative for S. Typhi CVD 909). The censoring limit (the “less than” value itself [1.08]) was used in data analysis for these samples.

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5.0Results

This section includes results from preliminary analyses (Section 5.1), optimization analyses for all matrices (Section 5.2), verification analyses for all matrices (Section 5.3), and optimization analyses for surface water (Section 5.4). Only valid results are included in this section.

5.1 – Preliminary Analyses of S. Typhi in PBS, Drinking Water, and Surface WaterDuring preliminary analyses, the procedure evaluated included: (1) pre-enriching in UPD broth for 24 ± 2 hours at 35.0°C ± 0.5°C, (2) transferring 1 mL to SCBD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (3) streaking growth onto BSD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (4) sub-culturing onto TSAD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, and (5) serological confirmation with Salmonella Vi and O Group D antisera (biochemical analyses were not conducted during preliminary analyses). Results of preliminary analyses are provided in Table 3.

Table 3. Preliminary Analyses, Results: PBS, Drinking Water, and Surface Water Spiked with S. Typhi(1)

Sample ID Spike Level(CFU/100 mL)

MPN Combo S. Typhi(MPN/100 mL)

Recovery (%)

PBS Samples

Unspiked NA 0-0-0 <1.08 NA

Spiked 49.7 3-3-0 23.98 46

Drinking Water Samples


Spiked 49.7 2-3-1 12.88 24

Surface Water Samples


Spiked 49.7 0-0-3 3.27 4(1) For unspiked samples, the “<” values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08)

CFU – Colony forming units NA – Not applicable

MPN – Most probable number PBS – Phosphate buffered saline

During preliminary analyses four issues were identified:

• Serology. Typical colonies were positive for Vi antiserum but negative for O Group D antiserum. The vaccine strain used for analyses (S. Typhi CVD 909) constitutively produces the Vi capsular antigen. This may mask the O Group D antigen, producing a false-negative response.

• BSD Incubation. No growth was observed on the BSD plates at 24 hours; a minimum incubation of 40 hours was required before typical (green-black with metallic sheen, surrounded by blackish halo) colonies were observed by the laboratory.

• Recoveries of S. Typhi in Surface Water. S. Typhi recoveries were poor, likely due to overgrowth of non-target organisms.

• SCBD Incubation. During preliminary analyses, selective enrichment (SCBD) was for 24 ± 2 hours. The selective components in the medium may degrade if incubation is longer than 24 hours, possibly contributing to overgrowth of non-target organisms in the surface water samples.

These issues were discussed by the workgroup and addressed, based on workgroup recommendations, as described in Section 5.2.

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5.2 – Optimization Analyses of S. Typhi in PBS, Drinking Water, and Surface WaterIn an effort to address the issues identified during preliminary analyses, the workgroup made the following recommendations: (1) add a boiling step to the procedure to disrupt the Vi capsular antigen, (2) decrease the incubation time for SCBD to 18 ± 2 hours, (3) increase the incubation time for BSD to 24 – 48 hours, 4) add an additional selective plating medium, MMD, and (4) evaluate the procedure with and without pre-enrichment in UPD broth.

The procedure evaluated during optimization analyses included: (1) pre-enriching in UPD broth for 24 ± 2 hours at 35.0°C ± 0.5°C, (2) transferring 1 mL to SCBD and incubating for 18 ± 2 hours at 35.0°C ± 0.5°C, (3) streaking growth onto BSD and MMD agars and incubating for 24 − 48 hours at 35.0°C ± 0.5°C, (4) sub-culturing onto TSAD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (5) serological testing with Salmonella Vi and O Group D antisera (cells boiled prior to testing), and (6) biochemical testing with API 20E® test strips. UPD broth was omitted for samples evaluated without pre-enrichment. Results are provided in Table 4 (without pre-enrichment) and Table 5 (with pre-enrichment)

Table 4. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water (without Pre-Enrichment in UPD Broth) Spiked with S. Typhi (1)

Sample ID Medium Spike Level(CFU/100 mL)

MPN Combo S. Typhi PercentRecovery

PBS

Unspiked BSD NA 0-0-0 <1.08 NA

MMD NA 0-0-0 <1.08 NA

Spiked #1 BSD 32 0-3-0 3.895 9

MMD 32 0-3-0 3.895 9

Spiked #2 BSD 32 0-3-0 3.895 9

MMD 32 0-3-0 3.895 9

Drinking Water


MMD NA 0-0-0 <1.081 NA

Spiked #1 BSD 32 0-0-0 <1.08 0

MMD 32 0-2-0 2.421 4

Spiked #2 BSD 32 0-2-0 2.421 4

MMD 32 0-3-0 3.895 9

Surface Water


MMD NA 1-0-0 1.257 NA

Spiked #1 BSD 32 1-1-1 3.955 9

MMD 32 0-0-1 1.08 -1

Spiked #2 BSD 32 1-1-0 2.6 5

MMD 32 0-0-0 <1.08 -1(1) For unspiked samples, the “<” values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08)

BSD – Bismuth sulfite agar with DHB; MPN – Most probable number


DHB – 2,3-dihydroxybenzoate PBS – Phosphate buffered saline

MMD – Miller-Mallinson agar with DHB,

13

Table 5. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water (with Pre-Enrichment in UPD Broth) Spiked with S. Typhi (1)

Sample ID Medium Spike Level(CFU/100 mL)

MPN Combo S. Typhi PercentRecovery

PBS


MMD NA 0-0-0 <1.08 NA

Spiked #1 BSD 32 2-3-1 12.88 37

MMD 32 2-3-1 12.88 37

Spiked #2 BSD 32 2-3-3 19.33 57

MMD 32 2-3-3 19.33 57

Drinking Water


MMD NA 0-0-0 <1.08 NA

Spiked #1 BSD 32 3-3-2 109.9 340

MMD 32 3-3-2 109.9 340

Spiked #2 BSD 32 3-3-2 109.9 340

MMD 32 3-3-2 109.9 340

Surface Water


MMD NA 0-0-0 <1.08 NA

Spiked #1 BSD 32 0-0-0 <1.08 0

MMD 32 0-0-0 <1.08 0

Spiked #2 BSD 32 0-0-0 <1.08 0

MMD 32 0-0-0 <1.08 0(1) For unspiked samples, the “<” values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08)

BSD – Bismuth sulfite agar with DHB; MPN – Most probable number


DHB – 2,3-dihydroxybenzoate PBS – Phosphate buffered saline

MMD – Miller-Mallinson agar with DHB,

Originally, optimization analyses were to be conducted in two runs. Recoveries of S. Typhi in samples evaluated without pre-enrichment were poor (≤ 9%), during the first run for all three matrices. Therefore, the second optimization run was not conducted and all subsequent analyses included pre-enrichment and the modifications to the method noted above (e.g., decreased SCBD incubation period) with the exception of boiling cells prior to serological typing with O Group D antiserum. The boiling step was eliminated following optimization analyses because it did not provide acceptable results. Based on CVD input, the workgroup requested that the laboratory use a growth medium with 0.5 M salt concentration to decrease Vi production prior to serological analyses with O Group D antiserum. The

modified growth medium did not provide acceptable results; isolates were also negative for O Group D antiserum.

14

5.3 – Verification Analyses of S. Typhi in PBS, Drinking Water, and Surface WaterVerification analyses were conducted in two runs. These runs resulted in the analyses of four spiked samples for each matrix and selective plating medium (BSD and MMD). Verification results are provided in Table 6.

Table 6. Verification Analyses, Results: PBS, Drinking Water, and Surface Water Spiked with S. Typhi (1)

Date Sample ID Spike Level(CFU/100 mL)

Medium MPN Combo

S. Typhi(MPN/100 mL)

Recovery (%)

Mean Recovery (%)

SD b(%)

RSD c (%)

PBS Samples

12/16/08 Unspiked NA NA 0-0-0 <1.08

1/09/09 NA NA 0-0-0 <1.08

12/16/08 Spiked 32 BSD 2-3-1 12.88 37.00 71.18 29.16 40.97

2-3-3 19.33 57.00

1/09/09 24 3-3-0 23.98 95.41

3-3-0 23.98 95.41

12/16/08 Spiked 32 MMD 2-3-1 12.88 37.00 94.35 67.04 71.06

2-3-3 19.33 57.00

1/09/09 24 3-3-1 46.22 188.08

3-3-0 23.98 95.41

Drinking Water Samples

12/16/08 Unspiked NA NA 0-0-0 <1.08

0-0-0 <1.08

1/9/09 Unspiked NA NA 0-0-0 <1.08

0-0-0 <1.08

12/16/08 Spiked 32 BSD 3-3-2 109.9 340.00 307.24 150.99 49.14

3-3-2 109.9 340.00

1/9/09 24 3-3-0 23.98 95.41

3-3-2 109.9 453.41

12/16/08 Spiked 32 MMD 3-3-2 109.9 340.00 307.24 150.99 49.14

3-3-2 109.9 340.00

1/9/09 24 3-3-0 23.98 95.41

3-3-2 109.9 453.41

Surface Water Samples

12/16/08 Unspiked NA NA 0-0-0 <1.08

0-0-0 <1.08

1/9/09 Unspiked NA NA 0-0-0 <1.08

0-0-0 <1.08

12/16/08 Spiked 32 BSD 0-0-0 <1.08 0.00 0.00 0.00 NA

0-0-0 <1.08 0.00

1/9/09 24 0-0-0 <1.08 0.00

0-0-0 <1.08 0.00

12/16/08 Spiked 32 MMD 0-0-0 <1.08 0.00 0.00 0.00 NA

0-0-0 <1.08 0.00

1/9/09 24 0-0-0 <1.08 0.00

0-0-0 <1.08 0.00(1) For unspiked samples, the “<” values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08) for calculation of summary statistics

tion RSD – Relative standard deviation SD – Standard devia

15

Results of verification analyses for PBS and drinking water were deemed acceptable by the workgroup; however, results for surface water were unacceptable. Therefore, the workgroup recommended additional optimization of the procedure for surface water samples.

5.4 – Optimization Analyses for Surface Water To improve S. Typhi recoveries in surface water, the workgroup recommended that the laboratory evaluate the use of commercially-available Salmonella spp. IMS beads (described in Section 5.4.1, below). When these results were unacceptable, the laboratory evaluated surface water matrices to determine if matrix inhibition, independent of background organisms, could be affecting recoveries (Section 5.4.2); the laboratory then repeated the analyses using laboratory-conjugated IMS beads (Section 5.4.3).

5.4.1 – Evaluation of Commercially-Available Im-munomagnetic Separation (IMS) Beads Commercially available IMS beads specific for Salmonella spp. (Dynabeads® anti-Salmonella product No. 710.02) were evaluated for isolation and concentration of S. Typhi CVD 909 from surface water samples. The manufacturer’s instructions developed for samples with high background flora was used to evaluate samples. The procedure included: (1) pre-enriching in UPD broth for 24 ± 2 hours at 35.0°C ± 0.5°C, (2) processing 1 mL of UPD broth from each tube for IMS according to manufacturer’s instructions, (3) transferring bead complex to SCBD and incubating for 18 ± 2 hours at 35.0°C ± 0.5°C, (4) streaking growth from SCBD onto BSD and incubating for 24 – 48 hours at 35.0°C ± 0.5°C, (5) sub-culturing onto TSAD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (6) serological testing with Salmonella Vi antiserum, and (7) biochemical testing with API 20E® test strips on serology-positive isolates.

• May 2009 Evaluation. Recoveries of S. Typhi CVD 909 in PBS samples spiked with approximately 50 CFU were 92%. S. Typhi CVD 909 was not recovered from surface water samples spiked at the same level.

• June 2009 Evaluation. Analyses were repeated with the following procedural modifications: (1) increasing bead-bacteria complex washes from one to two, (2) omitting selective enrichment in SCBD and plating beads onto BSD, (3) analyzing an additional sample volume (0.1 mL) to dilute out possible inhibitors, and (4) analyses of two surface water samples, each from a different source. S. Typhi was recovered from spiked PBS samples but was not recovered from spiked surface water samples.

5.4.2 – Evaluation of Surface Water Matrix In-hibition of Immunomagnetic Separation (IMS) ProtocolAnalyses were conducted in July 2009 to determine if the matrix (surface water samples), independent of background organisms, may be inhibiting IMS, resulting in poor recoveries of S. Typhi CVD 909. Matrix inhibition analyses included: (1) centrifuging samples to remove background organisms, (2) spiking supernatant with S. Typhi CVD 909 approximately 50 CFU, (3) pre-enriching in UPD broth for 24 ± 2 hours at 35.0°C ± 0.5°C, (4) processing 1 mL of UPD broth from each tube with commercially available IMS beads, (5) plating bead complex onto BSD and incubating for 24 – 48 hours at 35.0°C ± 0.5°C, (6) sub-culturing onto TSAD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (7) serological testing with Salmonella Vi antiserum, and (8) biochemical testing with API 20E® test strips if Vi serology-positive.

S. Typhi CVD 909 was recovered from these spiked surface water samples. These results suggest that the background organisms, which were removed by centrifugation, may have caused the poor recoveries in surface water. However, it is difficult to say for certain because other potentially inhibitory components (e.g., organics, chemicals) in the surface water may have also been removed during the centrifugation process.

5.4.3 – Evaluation of Laboratory-Conjugated Im-munomagnetic Separation (IMS) BeadsLaboratory-conjugated, tosylactivated IMS beads (Dynabeads® MyOne™ product No. 655-01) were evaluated for isolation and concentration of S. Typhi CVD 909 from surface water samples. The manufacturer’s instructions were followed for conjugation of Salmonella Vi antiserum to the beads. Analyses were conducted using the same procedure that was used for IMS analyses conducted in June 2009 (see Section 5.4.1). Surface water analyses were conducted twice during December 2009, using samples from two different sources. The laboratory continued to observe background organisms from surface water samples but was not able to recover S. Typhi CVD 909. The target organism was successfully recovered from PBS processed using the same approach.

17

6.0Discussion

The culture-based procedure in the draft SAP was evaluated for identification and quantitation of S. Typhi in water matrices. As indicated in Section 2, solid and particulate matrices were not evaluated during this phase of the Study.

Following preliminary analyses, the procedure was modified based on workgroup recommendations. Optimization analyses were conducted to evaluate these modifications and to determine if pre-enrichment was necessary. During optimization analyses, spiked samples evaluated using pre-enrichment resulted in recoveries of 37% – 57%, 340%, and 0% for PBS, drinking water, and surface water, respectively. Although it may appear that the drinking water matrix may be enhancing recoveries, it should be noted that a total of eight tubes were positive for both PBS (57%) and drinking water (340%). The difference in recoveries were due to the actual MPN combinations, 2-3-3 versus 3-3-2, resulting in MPN values of 19.33 and 109.9 for PBS and drinking water, respectively. The resulting combos may be indicative of the variability associated with MPN. It should also be noted that an MPN value is not considered absolute quantitation, as a direct plate count would be, because values are based on the probability of a tube being positive. For spiked samples evaluated without pre-enrichment recoveries were less than 10% for all three matrices. The workgroup agreed that pre-enrichment was necessary; all subsequent analyses included pre-enrichment in UPD broth.

Based on the results of optimization analyses for drinking water and PBS, the workgroup gave the laboratory approval to move forward with verification analyses. For spiked PBS samples, mean recoveries were 71% and 94% for BSD and MMD, respectively. For spiked drinking water samples, mean recoveries were 307% for both plating media (BSD and MMD). Mean recoveries of S. Typhi CVD 909 in surface water samples were 0% for both plating media. In addition to the laboratory’s inability to recover S. Typhi from spiked surface water samples, all isolates were negative for O Group D antigen during serological testing. CVD was contacted to discuss the serological results and confirm that CVD 909 does produce O Group D antigen. It was recommended that the laboratory use growth medium with 0.5 M salt concentration to decrease Vi production prior to serological analyses with O Group D antiserum. The salt concentration did not decrease

Vi production sufficiently; isolates were also negative for O Group D antiserum. Since clinical isolates are considered positive for S. Typhi if they confirm by Vi antiserum and biochemical analyses, the workgroup decided to eliminate serological analyses using O Group D antiserum from the procedure. Based on verification results, the workgroup considered the procedure appropriate for only PBS and drinking water samples and recommended additional optimization for surface water samples.

To address high background levels and enhance recoveries of S. Typhi in spiked surface water samples, the workgroup recommended the evaluation of IMS beads. Recoveries of S. Typhi CVD 909 in spiked surface water samples using Salmonella spp. beads and beads conjugated in the laboratory with Vi antisera were poor (<5%). Based on these results and the matrix inhibition evaluation, the workgroup decided that molecular procedures (e.g., polymerase chain reaction [PCR]) would be more appropriate for the detection of S. Typhi in surface water samples. The workgroup decided to discontinue any additional optimization analyses for surface water samples and considered the Study complete.

19

7.0Conclusions

The original draft SAP culture-based procedure was not appropriate for analyses of water matrices for S. Typhi. The procedure was modified, optimized, and verified during the Study. The optimized procedure is considered acceptable by the workgroup for analyses of S. Typhi in PBS and drinking water samples. The verified procedure includes the following: (1) pre-enriching in UPD broth for 24 ± 2 hours at 35.0°C ± 0.5°C, (2) transferring 1 mL to SCBD and incubating for 18 ± 2 hours at 35.0°C ± 0.5°C, (3) streaking growth onto BSD and MMD agars and incubating for 24 − 48 hours at 35.0°C ± 0.5°C, (4) sub-culturing onto TSAD and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (5) serological testing with Salmonella Vi antiserum, and (6) biochemical testing with API 20E® test strips. The culture-based procedure evaluated during this study is not appropriate for identification and quantitation of S. Typhi in surface water matrices.

The revised culture-based procedure merits multi-laboratory validation to assess method performance and set quantitative QC criteria for PBS (reference matrix) and drinking water. However, for surface water, the workgroup recommended development and inclusion of molecular procedures (e.g., PCR) for the detection of S. Typhi in surface water samples.

The draft SAP has been revised based on the Study to include the verified procedure. Procedures for surface water are no longer included.

21

8.0References

8.1 Moyer, N.P. 2005. “Section 9260B – “General Qualitative Isolation and Identification Procedures for Salmonella,” In Standard Methods for the Examination of Water and Wastewater, 21st Edition. A.D. Eaton, L.S. Clesceri, E.W. Rice, A.E. Greenberg, and M.A.H. Franson (eds.). Washington, DC: American Public Health Association, American Water Works Association, and Water Environment Federation.

8.2 Bopp, C.A., Brenner, F.W., Fields, P.I., Wells, J.G., and Strockbine, N.A. 2003. Chapter 28, “Escherichia, Shigella, and Salmonella.” In Manual of Clinical Microbiology, 8th Edition. P.R. Murray, E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R.H. Yolken (eds.). Herndon, Virginia: American Society for Microbiology.

8.3 Andrews, W.H., and Hammack, T.S. December 2007. Chapter 5, Salmonella. Bacteriological Analytical Manual [online]. U.S. Food and Drug Administration. http://www.cfsan.fda.gov/~ebam/bam-5.html

8.4 Klee, A. J. 1993. “Computer Program for the Determination of Most Probable Number and its Confidence Limits.” Journal of Microbiological Methods. 18(2): 91 – 98.

Appendix A

Study Plan

Study Plan – Do not Cite, Circulate, or Copy November 21, 2008 A-ii

Study Plan – Do not Cite, Circulate, or Copy November 21, 2008 A-iii

Study Plan for

Single Laboratory Verification of

Standardized Analytical Procedure for Salmonella Typhi in Environmental Samples

Note: Study plan appendices are provided as attachments to the study report.

November 21, 2008

Study Plan – Do not Cite, Circulate, or Copy November 21, 2008 A-iv

This document has been formatted according to U.S. EPA’s Environmental Monitoring Management Council (EMMC) formatting guidance (http://www.epa.gov/ttn/emc/guidlnd/gd-045.pdf). EPA uses EMMC format for the EPA microbiology 1600-method series. This document is undergoing review and is to be considered a draft. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. The procedures described in this document are intended for use in laboratories when analyzing environmental samples in support of remediation efforts following a homeland security incident. The procedures provide viability (culture based) determination, identification, and quantitation. To the extent possible, these procedures were developed to be consistent with other federal agency procedures. These procedures do not include sample collection or molecular techniques.

Study Plan – Do not Cite, Circulate, or Copy November 21, 2008 A-v

TABLE OF CONTENTS

1.0 INTRODUCTION AND BACKGROUND .............................................................................. 1

2.0 STUDY MANAGEMENT ........................................................................................................ 1

2.1 ROLES AND RESPONSIBILITIES ....................................................................................... 1

2.2 STUDY SCHEDULE .......................................................................................................... 4

3.0 OBJECTIVES ........................................................................................................................... 6

3.1 STUDY OBJECTIVES ........................................................................................................ 6

3.2 DATA QUALITY OBJECTIVES .......................................................................................... 6

4.0 STUDY IMPLEMENTATION AND TECHNICAL APPROACH ......................................... 7

4.1 PHASE 1 – IDENTIFICATION OF QUALIFIED ANALYTICAL LABORATORY ....................... 7

4.2 PHASE 2 – PREPARATION OF SPIKES ............................................................................... 7

4.2.1 Laboratory-Prepared Spiking Suspensions .................................................. 7

4.2.2 S. Typhi BioBall™ Spikes ........................................................................... 8

4.3 PHASE 3 – SAMPLE ANALYSIS ........................................................................................ 8

4.3.1 Preliminary Analyses. .................................................................................. 8

4.3.2 Assessment of Single Laboratory Method Precision and Recovery. ........... 8

4.3.3 Quality Control (QC) Analyses. ................................................................... 8

4.4 STUDY SUMMARY ........................................................................................................... 9

5.0 REPORTING AND VALIDATION OF STUDY RESULTS ................................................ 11

6.0 DATA ANALYSIS ................................................................................................................. 12

7.0 LIMITATIONS ....................................................................................................................... 12

Study Plan – Do not Cite, Circulate, or Copy A-1 July 29, 2008

1.0 INTRODUCTION AND BACKGROUND Subsequent to the anthrax attacks in the Fall of 2001, federal and state personnel were tasked with a mission to provide response, recovery, and remediation for biological incidents. It was quickly recognized, however, that standardized methods do not exist for isolation and analysis of biological agents in environmental samples. To address these gaps and to ensure confidence in results through development of quality controls, the U.S. Environmental Protection Agency’s (EPA’s) National Homeland Security Research Center (NHSRC) is developing, evaluating, and validating a set of draft protocols. The protocols are based on pathogens and analytical procedures listed in EPA’s Standardized Analytical Methods for Environmental Restoration following Homeland Security Events (SAM), which identifies Salmonella Typhi as an EPA target pathogen during environmental remediation following a homeland security incident. This study plan describes single-laboratory verification of a draft Standardized Analytical Procedure for Salmonella Typhi in Environmental Samples (SAP) for determination and quantification of S. Typhi using selective and non-selective media followed by biochemical and serological characterization. The draft SAP is included as Attachment A to this study plan, and is based on CDC 1 and FDA 2 methods. Sample processing and handling for solids is based on EPA Method 1682. 3 Particulate sample processing and handling is based on the journal article “Swab Materials and Bacillus anthracis Spore Recovery from Nonporous Surfaces” by Rose et al. 4 During this study, the draft procedures will be evaluated in multiple matrices, including water (phosphate buffered saline [PBS], surface water, and drinking water), solids (Milorganite®, American Society for Testing and Materials [ASTM] Lean Clay, ASTM Poorly Graded Sand, local soil), and particulates (swab with Arizona Test Dust, wipe with Arizona Test Dust, high-efficiency particulate air (HEPA) filter with Arizona Test Dust, swab with local particulate, wipe with local particulate, HEPA filter with local particulate). A vaccine strain of S. Typhi (CVD909) will be used. Results of the study will be used to revise the draft SAP, if necessary, prior to further validation in multiple laboratories. Results of this single laboratory verification will also be used to confirm appropriate spike levels, spiking procedure, and solid reference matrix for use during a multi-laboratory study. 2.0 STUDY MANAGEMENT 2.1 Roles and Responsibilities The study will be managed by NHSRC in EPA’s Office of Research and Development (ORD) with support from the study workgroup (Office of Emergency Management [OEM], Office of Water [OW], Office of Ground Water and Drinking Water [OGWDW], and Centers for Disease Control and Prevention [CDC]). Coordination of study activities will be performed by Computer Sciences Corporation (CSC) under NHSRC guidance. CSC will direct sample processing and analyses, conduct data tracking and review, and coordinate study activities. CSC will provide study updates to the study workgroup including but not limited to study status, study/data issues, and preliminary results. CSC will prepare a condensed study report based on the results of the study. 1 Bopp, C.A., F.W. Brenner, P. I. Fields, J. G. Wells, and N. A. Strockbine. 2007. Escherichia, Shigella, and Salmonella. 2003. In: Manual of Clinical Microbiology, 8th Edition. Editors P.R. Murray, E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken. (Washington, D.C.: American Society for Microbiology. 2U.S. Food and Drug Administration. Salmonella. December 2007. Chapter 5 in Bacteriological Analytical Manual [online]. 3 U.S. Environmental Protection Agency. Office of Water. 2006. Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium. Washington, D.C.: U.S. Environmental Protection Agency, July 2006. (EPA-821-R-06-14). 4 Rose, L., B. Jensen, A. Peterson, S. N. Banerjee, and M. J. Arduino. 2004. Swab Materials and Bacillus anthracis Spore Recovery from Nonporous Surfaces. , Emerg. Infec. Diseases. 10(6): 1023 – 1029.

Study Plan – Do not Cite, Circulate, or Copy November 21, 2008 A-2

The study workgroup will evaluate study results and work with CSC to identify next steps including revising the SAP to include any procedural modifications based on study results. The study will use a single laboratory for verification of SAP procedures and will include preparation of spiking suspensions, and preparation and analysis of study samples. CSC will be responsible for study coordination including but not limited to development of study-specific instructions and data reporting forms, procurement of supplies and spiking materials (BioBalls), and technical support. Specific responsibilities of study participants are presented in Table 1. The flow of responsibilities is presented in Figure 1. Table 1. Roles and Responsibilities

Organization Role

EPA National Homeland Security Research Center (NHSRC)

• •

Direct contractor activities Ensure that study goals and deadlines are met

Study Workgroup (EPA [NHSRC, OEM, OW and OGWDW] and CDC)

• • • •

• •• •

Provide subject matter expertise Review study schedule Review study plan Make final decisions regarding study plan and study schedule Provide support for resolution of issues, as necessary

Provide recommendations and conclusions to EPA Review study results Review study report

Computer Sciences Corporation (CSC) ••• •

••

•

• • •

•

•

Develop study schedule Develop study plan

Recruit volunteer analytical laboratory support Develop study materials (spiking protocol, laboratory instructions, data reporting forms, data review checklists)

Revise study materials, as necessary Procure and provide laboratory with spikes, reagents and media Coordinate study activities (conference calls, resolution of issues, data receipt) and provide technical support Serve as point of contact for the laboratory Coordinate resolution of issues Receive, compile, maintain, review, and analyze study data Prepare condensed study report with recommendations and conclusions Revise SAP, as necessary, to address study results and laboratory feedback

Laboratory • • •

• •

• • ••

Confirm receipt of written study materials and supplies Collect appropriate matrix samples, as necessary Provide input to CSC study coordinator to help determine sample matrices and spiking levels that would be appropriate for the multi-laboratory validation study Optimize the spiking protocol for S. Typhi CVD909 Analyze samples according to study-specific instructions and SAP Perform quality control (QC) according to SAP Maintain general laboratory QA/QC

Provide recommendations on SAP for potential revisions Provide preliminary and final analytical results to CSC study coordinator


Figure 1: Flowchart of Study Activities

Study Workgroup / CSCPrepare and review study schedule Prepare and review study plan Provide subject matter expertiseProvide support for resolution of issuesProvide recommendations and conclusions

CSCCoordinate study

activities and provide technical support

CSC Develop laboratory study instructions and reporting formsRecruit laboratory Procure and provide laboratory with reagents, media, and spiking materials

LaboratoryProvide sample matricesAnalyze samples according to study-specific instructionsProvide recommendations on SAP for potential revisionsProvide preliminary and final analytical results to CSC study coordinator

CSC Receive, compile, disseminate, validate, and analyze study data

CSC Prepare draft report

Study WorkgroupReview study results

Study WorkgroupReview draft report

CSC Revise SAP

Study WorkgroupReview revised SAP


2.2 Study Schedule The study schedule is dependent on receipt of bacterial spiking standards, media, and reagents by the laboratory. The following tentative study schedule (Table 2) is provided under the assumption that these materials will be received by the laboratory no later than August 15, 2008. This schedule also assumes that the laboratory will participate in several periodic conference calls with the study workgroup and CSC throughout the study. For this study, S. Typhi will be quantified in water, solid, and particulate matrices. Specifically, water matrices will include drinking water from the laboratory tap and surface water as well as a reference matrix (PBS). For solid matrices, a single local soil sample will be analyzed. The reference matrices for solids will include one or more of the following based on results of the E. coli O157:H7 single laboratory verification study: Milorganite®; ASTM Lean Clay; and ASTM Poorly Graded Sand. Particulate matrices will include one or more of the following based on results of the E. coli O157:H7 single laboratory verification study: swab, wipe, and HEPA filters with dust collected from laboratory surfaces. Reference matrices for particulates will include one or more of the following based on results of the E. coli O157:H7 single laboratory verification study: swab, wipe, and HEPA filters with Arizona Test Dust. Ideally matrices can be limited, pending E. coli O157:H7 study results.


Table 2. Tentative Schedule for Single Laboratory Verification of Draft Procedures for S. Typhi in Water, Solid, and Particulate Matrices

Month Activity

Planning and Preparation

July 2008 • Draft study plan

June – August 2008

• Identify and recruit laboratory • Finalize study plan • Draft study-specific instructions, spiking protocol, and data reporting

forms (focus on water)

August – September 2008 • Finalize study-specific instructions, spiking protocol, and data reporting

forms (focus on water) • Procure supplies, reagents, and media

October 2008

• Draft study-specific instructions, spiking protocol, and data reporting forms (focus on solids)

• Finalize study-specific instructions, spiking protocol, and data reporting forms (focus on solids)

November 2008

• Draft study-specific instructions, spiking protocol, and data reporting forms (focus on particulates)

• Finalize study-specific instructions, spiking protocol, and data reporting forms (focus on particulates)

Water (PBS, Surface Water, Drinking Water)

August / September 2008 • Optimize spiking protocol for S. Typhi CVD909 to determine

appropriate concentration of medium • Revise spiking protocol, as necessary

September / October 2008

• Perform preliminary analysis at laboratory (~ 1 week) • Review data / resolve issues (~ 2 weeks) • Conduct analyses at laboratory (~ 1 week) • Revise procedures (if necessary)

Solids (Milorganite®, ASTM Clay, ASTM Sand, Local Soil)

November / December 2008

• Modify study-specific instructions to address solids • Perform preliminary analysis at laboratory (~ 1 week) • Review data / resolve issues (~ 2 weeks) • Conduct analyses at laboratory (~ 2 weeks) • Revise procedures (if necessary)

Particulates (Swabs, Wipes, HEPA Filters; Each with Arizona Test Dust and a Local Particulate)

December 2008 / January 2009

• Modify study-specific instructions to address particulates • Perform preliminary analysis at laboratory (~ 1 week) • Review data / resolve issues (~ 2 weeks) • Conduct analyses at laboratory (~ 2 weeks) • Revise procedures (if necessary)

Study Report and SAP Revisions

January / February 2009 • Review data • Prepare condensed study report • Revise SAP to reflect study results


3.0 OBJECTIVES The primary objective of this study is to verify the procedures included in the draft SAP (Appendix A), for identification and enumeration of S. Typhi in water, solid, and particulate matrices. This includes ensuring that estimated counts of the spikes are reliable and that the analytical data generated by the participant laboratory are reliable. Two sets of objectives were identified for the study: study objectives and data quality objectives. 3.1 Study Objectives Results and information from this study will be used to: •

Characterize SAP performance (recovery and precision) in multiple reference matrices (water [PBS], solid [Milorganite® and two ASTM soils], and particulate [swabs, wipes, and HEPA filters with Arizona Test Dust])

• Characterize SAP performance (recovery and precision) in multiple environmental matrices of interest (water, solid, and particulate)

• Determine whether the SAP requires revision prior to multi-laboratory validation To ensure study objectives are met, the laboratory will be required to: • Follow all analytical, quality assurance (QA), and quality control (QC) procedures in the study-

specific instructions and draft SAP •

Obtain approval from study workgroup and CSC before using any modifications to the procedures described in the SAP, and provide a written description of the modification(s)

• Provide all original data reporting forms and any associated information in a format that can be verified by an independent person reviewing study results

• Respond to data and study related inquiries from the CSC study coordinator • Provide recommended revisions to the draft SAP 3.2 Data Quality Objectives Data produced under this study must be generated according to the analytical and QA/QC procedures specified in the study-specific instructions (Appendix B) and SAP (Appendix A). This will ensure data integrity and validity for all matrices evaluated and allow the study workgroup to use the study results to identify any necessary revisions of the SAP (Appendix A). To ensure that data quality objectives are met, the laboratory will be required to: •

Prepare spiking suspensions in accordance with the spiking protocol (Appendix C). This will allow for determination of recoveries and identification of potential matrix interferences.

• Analyze the following QC samples: -

Media sterility checks - Dilution water sterility checks - Method blanks (e.g., sterile unspiked phosphate buffered saline [PBS]) - Positive and negative controls


4.0 STUDY IMPLEMENTATION AND TECHNICAL APPROACH During this study, non-selective and selective media, followed by biochemical characterization and serological confirmation tests, will be used to analyze water, solid, and particulate samples spiked with S. Typhi (see draft SAP, Appendix A). One or more reference matrices for each sample type (water, solid, and particulate) will also be analyzed. 4.1 Phase 1 – Identification of Qualified Analytical Laboratory CSC will identify and recruit a laboratory that is 1) representative of the general user community and 2) has access to representative matrices. Ideally, the laboratory will have experience analyzing environmental samples for S. Typhi. To reduce study costs, a volunteer laboratory will be recruited. To reduce the burden on the laboratory and encourage participation, NHSRC will provide the media, reagents, and supplies needed for the study. The requirements and responsibilities of the laboratory will be detailed in study-specific instructions (Appendix B) and the draft SAP (Appendix A). The primary analyst and laboratory management will be asked to participate in periodic conference calls, to ensure the laboratory is aware of schedules, has the proper reagents and media, and understands data reporting requirements. 4.2 Phase 2 – Preparation of Spikes The study will use the following two sources of enumerated spiking suspensions: 1) laboratory-prepared S. Typhi spiking suspensions and 2) S. Typhi BioBalls. Prior to any study analyses, the laboratory will optimize the spiking protocol for the laboratory-prepared S. Typhi CVD909 spiking suspension to determine the appropriate concentration of medium (e.g., 1% TSB, 10% TSB). The laboratory will evaluate a maximum of four medium (TSB) concentrations to determine which concentration will provide a stable spiking suspension.

4.2.1 Laboratory-Prepared Spiking Suspensions

Laboratory-prepared spiking suspensions will be prepared from S. Typhi (Center for Vaccine Development [CVD] 909) according to the spiking protocol (Appendix C). A stock culture will be propagated in TSB with 0.0001% 2, 3 dihydrobenzoic acid (DHB) and incubated at 35.0°C ± 0.5°C for 24 ± 2 hours. Serially diluted spiking suspensions (~30 colony forming units [CFU] per mL) will be used to spike samples. The laboratory will enumerate spiking suspensions on the same day samples are spiked and analyzed, using the spread plate technique (in triplicate) on tryptic soy agar (TSA) with 0.0001% DHB.

This section provides a summary of the activities that will be performed during the study. Detailed study-specific instructions (Appendix B) and data reporting forms (Appendix D) will be provided to the participant laboratory. Activities and schedules described in this study plan may change as the study progresses and additional data and information become available.


4.2.2 S. Typhi BioBall™ Spikes

S. Typhi BioBalls (BTF, Pty in North Ryde, Australia) prepared from CVD 909 will be procured by CSC and provided to the laboratory. BioBalls are commercially available flow-sorted pre-enumerated QC spikes. A single BioBall™ (~30 CFU) will be used to spike individual samples. The laboratory will also enumerate three BioBalls to verify the manufacturer’s lot mean value using the spread plate technique on the first day of sample spiking for each matrix (water, solids, and particulates).

4.3 Phase 3 – Sample Analysis Samples will be analyzed as described in the draft SAP (Appendix A) and the study-specific instructions (Appendix B). Sample matrices will be prepared as described in Appendix B (Study-Specific Instructions). S. Typhi is considered a BSL-2 pathogen. The laboratory is required to process all samples according to the safety requirements included in Section 4 of the draft SAP (Appendix A). Table 3 provides a detailed summary of the preliminary and study analyses.

4.3.1 Preliminary Analyses

The laboratory will conduct preliminary analyses for each matrix and spike type (unspiked, laboratory-prepared and BioBall™) approximately 1 – 2 weeks prior to start of the study analyses. The time between preliminary analyses and study analyses will be used to resolve any issues that arise.

4.3.2 Assessment of Single Laboratory Method Precision and Recovery For each matrix, method precision and recovery will be evaluated through the analysis of four replicates (of each matrix) spiked with laboratory-prepared spikes and four replicates (of each matrix) spiked with S .Typhi BioBalls. 4.3.3 Quality Control (QC) Analyses During assessment of precision and recovery in replicate samples, the laboratory will complete the following QC requirements: media sterility checks, dilution water sterility checks, method blanks, filtration blanks, positive controls, and negative controls. S. Typhi (CVD 909) and Pseudomonas aeruginosa (ATCC ® 27853™) will serve as the positive controls and E. coli (ATCC ® 25922™) as the negative control.


4.4 Study Summary The following table summarizes the sample matrices and number of samples that will be analyzed, the spiking description, and purpose of the analysis. Table 3. Summary of Sample Analyses

Matrix Spiking

Description

No. of Samples

Purpose of Analysis Preliminary Analyses

Study Analyses

Water

Sterile PBS (Reference

Unspiked 1 1 Confirmation of freedom from contamination (Method Blank)

BioBall™ Spike 1 4 Assessment of method precision Matrix) Lab-Prepared

Spike 1 4

Confirmation that approach is appropriate for the multi-laboratory validation study

Surface Water

Unspiked 1 2 Evaluation of background S. Typhi concentrations

BioBall™ Spike 1 4 Assessment of method precision and recovery

Lab-Prepared Spike

1 4 Confirmation that approach is appropriate for the multi-laboratory validation study

Drinking Water



Lab-Prepared Spike


Solids

Milorganite® Unspiked 1 1

Confirmation of freedom from contamination (Method Blank)

(Reference BioBall™ Spike 1 4 Assessment of method precision Matrix #1) Lab-Prepared

Spike 1 4

Confirmation that approach is appropriate for the multi-laboratory validation study

ASTM Lean Unspiked 1 1 Evaluation of background S. Typhi concentrations

Clay (Reference Matrix #2)

BioBall™ Spike 1 4 Assessment of method precision

Lab-Prepared Spike


ASTM Sand Unspiked 1 1

Evaluation of background S. Typhi concentrations

(Reference BioBall™ Spike 1 4 Assessment of method precision Matrix #3)

Lab-Prepared Spike


Local Soil



Lab-Prepared Spike



Matrix Spiking

Description

No. of Samples

Purpose of Analysis Preliminary Analyses

Study Analyses

Particulates

Swab with Reference


Matrix (Arizona

Test Dust)


Lab-Prepared Spike


Wipe with Reference

Matrix (Arizona

Test Dust)



Lab-Prepared Spike


HEPA Filter with

Reference Matrix

(Arizona Test Dust)



Lab-Prepared Spike


Swab with Local

Particulate



Lab-Prepared Spike


Wipe with Local

Particulate



Lab-Prepared Spike


HEPA Filter with Local Particulate



Lab-Prepared Spike


Note: Solid and particulate matrices may be limited pending E. coli O157:H7 single-laboratory verification study results.


5.0 REPORTING AND VALIDATION OF STUDY RESULTS After analyses are complete, the laboratory will be required to submit data on standardized data reporting forms (Appendix D), designed specifically for the study. In addition, the laboratory will be required to submit detailed explanations of any deviations from the study-specific instructions (Appendix B), as well as any recommended revisions to the draft SAP (Appendix A). Laboratories will submit all study results, data packages, descriptive information, and SAP revision recommendations to the CSC study coordinator:

Laura Jones, Computer Sciences Corporation (CSC) [email protected] 6101 Stevenson Avenue Alexandria, VA 22304 Phone: (703) 461-2007 Fax: (703) 461-8056

Data received will include: •

Volume/weight of all samples and dates of preparation and processing of study matrices • Inoculation volumes and plate counts for spike enumeration (laboratory-prepared and BioBalls) • Plate counts for preliminary and study analyses, including plate counts for any atypical organisms • Results for all QC analyses (as listed in Section 4.3.3) • Date of preparation, pH, lot number and expiration dates for all media and reagents Upon receipt of the laboratory data package, CSC will review the data to ensure that they were generated in accordance with the SAP (Appendix A) and study-specific requirements. Items that will be reviewed for each sample include the following: •

Confirmation that original forms are submitted and complete • Confirmation that all calculations are correct • Confirmation that QC checks were performed and exhibit the appropriate response • Confirmation that media and reagents were used within expiration dates • Confirmation that method-specific incubation times and temperatures were met • Confirmation that all required data elements are reported


6.0 DATA ANALYSIS Descriptive statistics of sample recoveries will be calculated using spiked sample data from each matrix and spike type and will include the relative standard deviation (RSD) between results of replicate samples. Mean, median, and range of individual recoveries also may be included. For samples spiked with BioBalls, recoveries will be assessed by comparing spike recovery (concentration in the spiked samples minus the unspiked concentration) to the manufacturer-provided lot mean value with each set of analyses. If BioBalls are not used for spiking, recoveries will be assessed based on enumeration of spiking suspension by the participant laboratory, using spread plate technique (Spiking Protocol [Appendix C]). 7.0 LIMITATIONS Because this study will be performed by a single laboratory analyzing the limited number of samples and matrices specified in Table 3, the results of this study may not represent all potential variability that could arise in real-world implementation of this SAP (Appendix A). This study is designed, however, to ensure that the laboratory and matrices included in this study are as representative as possible of real-world conditions.

Draft – Do Not Cite, Circulate, or Copy

Appendix B

Study-Specific Instructions

Draft – Do Not Cite, Circulate, or Copy B-1 October 31, 2008

Instructions for Salmonella Typhi SAP Single Laboratory Verification Study: Preliminary Water Analyses

October 31, 2008

The purpose of this study is to evaluate procedures that are included in the “Draft Standardized Analytical Procedure for Salmonella enterica subsp. enterica serotype Typhi (Salmonella Typhi) in Environmental Samples” (referred to as the “draft SAP”). Results of the study will be used to revise the draft SAP, if necessary, prior to further validation in multiple laboratories. Media/Reagents/Supplies • Phosphate buffered saline (PBS) • 10% sodium thiosulfate • 2,3-dihydroxybenzoate (DHB) • 1X, 2X, and 5X universal pre-enrichment broth with DHB (UPD) • Selenite cystine broth with DHB (SCBD) • Bismuth sulfite with DHB (BSD) agar • Tryptic soy agar with DHB (TSAD) • Tryptic soy broth (TSB) • 1% Tryptic soy broth with DHB (TSBD) • Physiological saline (0.85%) • Salmonella Vi and O group D antisera Control Cultures

o

S. Typhi (CVD 909)

o Enterococcus faecalis (ATCC® 29212™) Water Matrices

Water matrices will include a reference matrix (PBS), drinking water from the laboratory tap, and surface water from a lake, reservoir, or other open body of water. Preliminary Sample Analyses

• The following unspiked and spiked PBS, drinking water, and surface water samples will be enumerated using the most probable number (MPN) technique. Following pre-enrichment in UPD, samples will be sub-cultured in a selective medium (SCBD), followed by plating on a selective agar (BSD). Isolated, typical colonies with a green-black metallic sheen will then be submitted to serological confirmation according to study-specific instructions.

• PBS Samples (Reference Matrix) o

1, 100-mL unspiked

o 1, 100-mL spiked

• Drinking Water Samples o

1, 100-mL unspiked

o 1, 100-mL spiked


• Surface Water Samples o

1, 100-mL unspiked

o 1, 100-mL spiked

Prior to Study Reagent and Media Preparation

It is recommended that the following reagents and media be prepared either prior to the week of preliminary study analyses (PBS, DHB, UPD, TSAD, TSBD) or the day before analyses (SCBD, BSD):

• Phosphate Buffered Saline (PBS): Prepare 1.0 L of PBS according to the following instructions and store at room temperature for a maximum of three months in screw cap bottles.

Monosodium phosphate (NaH2PO4) 0.58 g

Disodium phosphate (Na2HPO4) 2.50 g

Sodium chloride 8.50 g

Reagent-grade water 1.0 L

Dissolve reagents in 1.0 L of reagent-grade water in a flask and dispense in appropriate volumes in screw cap bottles and autoclave at 121°C (15 lb pressure per square inch [PSI]) for 15 minutes. Final pH should be 7.4 ± 0.2.

Note: Assuming all analyses are conducted within the same week a total of 695 mL of PBS will be required. As a result, it is recommended that the laboratory prepare 1.0 L of PBS [5, 99-mL dilution blanks; 2, 100-mL samples].

•

2,3-dihydroxybenzoate (DHB): Prepare 100 mL of 0.1% (w/v) DHB stock solution by dissolving 0.1 g of DHB powder in 100 mL of reagent-grade water and filter sterilize.

• Universal Pre-enrichment Broth with DHB (UPD): Prepare 500 mL of 1X, 2X, and 5X UPD according to the following and store at room temperature for a maximum of three months in screw cap bottles.

Reagents 1X 2X 5XPancreatic digest of casein 2.5 g 5.0 g 12.5 g

Proteose peptone 2.5 g 5.0 g 12.5 g

Monopotassium phosphate (KH2PO4) 7.5 g 15.0 g 37.5 g

Disodium phosphate (Na2HPO4) 3.5 g 7.0 g 17.5 g

Sodium chloride 2.5 g 5.0 g 12.5 g

Dextrose 0.25 g 0.5 g 1.25 g

Magnesium sulfate (MgSO4) 0.125 g 0.25 g 0.625 g

Ferric ammonium citrate 0.05 g 0.1 g 0.25 g

Sodium pyruvate 0.1 g 0.2 g 0.5 g

Reagent-grade water 500 mL 500 mL 500 mL

For 1X, 2X and 5X UPD, dissolve reagents in 450 mL of reagent-grade water in a flask and mix thoroughly. Adjust pH to 7.2 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide and bring volume to 500 mL. Autoclave at 121°C (15 PSI) for 15 minutes. Cool to 45EC – 50EC in a waterbath. For 1X UPD, add 0.5 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0 mL aliquots in 20 × 150 mm tubes. For 2X UPD, add 1.0 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0 mL aliquots in 20 × 150 mm tubes. For 5X UPD, add 2.5 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 5.0 mL aliquots in 20 × 150 mm tubes. Store at <10°C and above freezing for a maximum of 2 weeks in tubes with loose caps or three months in screw cap tubes.


• Selenite Cystine Broth with DHB (SCBD): Prepare 1.0 L of SCBD according to the following instructions and use within 48 hours of preparation.

Pancreatic digest of casein 5.0 g

Lactose 4.0 g

Sodium phosphate 10.0 g

Sodium selenite 4.0 g

L-Cystine 0.01 g


Add reagents to 1.0 L of reagent-grade water and mix thoroughly using a stir bar and hot plate.

Heat to boiling. Avoid overheating. Do not autoclave. Final pH should be 7.0 ± 0.2. Cool to 45EC – 50EC in a waterbath and add 1.0 mL of DHB [54, 9-mL tubes].

Note: A brick red precipitate may appear if the medium is overheated during preparation or exposed to excessive moisture during storage. If this occurs medium should be discarded and anew batch prepared.

•

Bismuth Sulfite with DHB (BSD): Prepare 1.0 L of BSD agar (the recommended volume assumes that 80% of spiked MPN tubes are presumptive positive) according to manufacturer’s instructions the day prior to analyses. Cool to 45°C – 50°C in a waterbath and add 1.0 mL of the 0.1% DHB solution.

Evenly disperse the precipitate while aseptically pouring 12 – 15 mL into 100 × 15 mm sterile Petri plates. Store at room temperature [54, 15-mL, 100 × 15-mm plates].

• Tryptic Soy Agar with DHB (TSAD): Prepare 30 TSAD plates according to manufacturer’s instructions. After autoclaving, cool to 45°C – 50°C in a waterbath and add 0.1 mL of the 0.1% DHBsolution per 100 mL of medium. Aseptically pour 12 – 15 mL into each 100 × 15 mm sterile Petri plate. Store at <10°C and above freezing for a maximum of 2 weeks.

• Tryptic Soy Broth with DHB (TSB): Prepare TSB by preparing TSB according manufacturer’s instructions. Store at <10°C and above freezing for a maximum of three months in a screw cap bottle.

• 1%Tryptic Soy Broth with DHB (TSBD): Prepare a 1% solution of TSB by combining 99 mL of sterile phosphate buffered dilution water, 1 mL of sterile single-strength TSB (prepared according to manufacturer’s instructions), and 0.1 mL of 2,3-dihydroxybenzoate (DHB) solution in a sterile screw cap bottle. Shake to mix.

Week of Analyses: Day One

Laboratory-prepared spiking suspension: Inoculate 1% TSBD with S. Typhi according to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 2 hours.


Day Two Enumeration of Spiking Suspensions

•

Dilute and plate laboratory-prepared spiking suspension on TSAD according to spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 2 hours.

o Sample Collection – PBS, Drinking Water, and Surface Water Preliminary Analyses

• Aliquot the following volumes of sterile PBS and label the samples as follows:

o 1, 100-mL unspiked – PBS unspiked

o 1, 100-mL spiked – PBS spiked

• Collect a 1.0-L bulk drinking water sample as follows:

o

Select a cold water line faucet and remove aerator, if present.

o Clean the faucet exterior with disinfection solution (e.g., 10% household bleach).

o Open the tap to obtain a smooth-flowing stream at moderate pressure without splashing.

o Allow water to run at least 2 – 3 minutes.

o Remove the cap from a sterile bottle containing 1 mL of a 10% sodium thiosulfate solution (dechlorinating agent).

o Avoid contaminating the sample bottle lip or inside the cap.

o Reduce the water flow to fill the bottle without splashing and fill to within 2.5 cm – 5 cm (1" – 2") of the top for proper mixing before analyses.

o Do not rinse dechlorinating agent out of the bottle.

o Tightly cap the container.

o Mix thoroughly and split the bulk sample into aliquots and label the samples as follows.

1, 100-mL – Drinking water unspiked

1, 100-mL – Drinking water spiked

• Collect a 1.0-L bulk surface water sample as follows:

o

Collect samples by hand or with a sampling pole if the sampling site has difficult access such as a dock, bridge, or bank adjacent to surface water.

o The sampling depth should be 6" – 12" below the water surface.

o Sample containers should be positioned such that the mouth of the container is pointed away from the sampler or sample point.

o After removal of the container from the water, a small portion of the sample should be discarded to leave a headspace of 2.5 cm – 5 cm (1" – 2") for proper mixing before analyses.

o Transport to the laboratory on ice (do not freeze).

o In the laboratory, split the bulk sample into aliquots and label the samples as follows:

1, 100-mL – Surface water unspiked

1, 100-mL – Surface water spiked


Sample Spiking

• Spike 1, 100-mL sample each of PBS, drinking water, and surface water with laboratory-prepared spiking suspension, according to the spiking protocol.

Most Probable Number (MPN)

•

Mix unspiked and spiked samples by shaking 25 times.

• Inoculate UPD tubes with the following volumes for each unspiked or spiked sample:

o

For each of three tubes, add 20 mL of undiluted sample to 5 mL (5X) UPD

o For each of three tubes, add 10 mL of undiluted sample to 10 mL (2X) UPD


• Incubate inoculated UPD tubes at 35.0°C ± 0.5°C for 24 ± 2 hours.

Day Three

TSA Enumeration – Laboratory-Prepared Suspensions

•

Count colonies on TSAD plates prepared on Day Two and record results of colony forming units (CFU) enumeration on provided data reporting forms.

• Fax TSAD enumeration results to Yildiz Chambers at (703) 461-8056. MPN

•

Examine UPD tubes at 24 ± 2 hours; record results.

• From each UPD tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes containing 9.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 24 ± 2 hours.

Day Four

MPN

•

Examine SCBD tubes at 24 hours; record results.

• From each SCBD tube with growth, gently swirl the tube to mix and streak for isolation on BSD plates using a sterile inoculation loop (20 µL). Incubate at 35.0°C ± 0.5°C for 24 – 48 hours.

Day Five

BSD Plates

• Examine BSD plates at 24 hours; record results. If no typical (green-black metallic sheen) colonies are observed on BSD plates, incubate for an additional 24 ± 2 hours at 35.0°C ± 0.5°C. For BSD plates with typical colonies, streak a typical colony onto TSA and incubate at 35°C ± 0.5°C for 24 ± 2 hours.


Day Six

BSD Plates

• Examine BSD plates at 48 hours; record results. For BSD plates with typical colonies, streak a typical colony onto TSA and incubate at 35°C ± 0.5°C for 24 ± 2 hours.

TSAD Plates

• Serological Analyses: A single colony from each TSAD plate will be submitted to serological confirmation, as described below.

o Emulsify a portion of a single typical colony from each TSAD plate using sterile physiological saline and test for agglutination with Salmonella antiserum (Vi and O Group D) as follows:

Place three discrete drops of emulsified growth onto a slide. To the first drop of emulsified growth, add one drop of Salmonella O antiserum Group D. To the second drop of emulsified growth, add one drop of Vi antiserum. To the third drop of emulsified growth, add one drop of sterile saline (as a visual comparison). Observe under magnification for an agglutination reaction, which indicates a positive result. S. Typhi is agglutination-positive for both O Group D and Vi antisera. Results should be compared with those for positive and negative controls analyzed at the same time.

o If results are not consistent with S. Typhi (e.g., negative serology), go back to the TSAD plate and pick another isolated colony and repeat serological analyses. Note: If neither of the two isolates from the plate provides a positive serology result, please contact CSC immediately for additional instructions; Yildiz Chambers (703) 461-2165, [email protected]).

Note: Additional serological confirmation will be required on Day Seven only if there are no typical colonies observed and/or confirmed on Day Six.

Day Seven

TSAD Plates

•

Serological Analyses: A single colony from each plate with typical colonies will be submitted to serological confirmation, as described above (Day 6).

• Please fax results to Yildiz Chambers at (703) 461-8056.

Note: Please contact Yildiz Chambers (703) 461-2165, [email protected]) if you have any questions.

Do Not Cite, Circulate, or Copy B-7 December 10, 2008

Method Optimization Instructions for Salmonella Typhi SAP Single Laboratory Verification Study: Water Matrices

December 10, 2008

The instructions provided below are to be repeated for a total of two runs, resulting in the analysis of 2 unspiked and 4 spiked samples for each matrix (PBS, drinking water, and surface water). Each sample will be processed with pre-enrichment in UPD broth (direct inoculation into SCBD) and without pre-enrichment (Sets A and B, respectively) and plated on 2 selective agars (BSD and MMD). Please contact Yildiz Chambers (703) 461-2165, [email protected]) if you have any questions regarding these instructions.

All QA/QC analyses specified in the study-specific instructions and draft SAP must be conducted in conjunction with sample analyses described below. In addition, the laboratory must follow all QA/QC procedures outlined in their QAPP.

Media/Reagents/Supplies • Phosphate buffered saline (PBS)

• 10% sodium thiosulfate

• 2,3-dihydroxybenzoate (DHB)

• 1X, 2X, and 5X universal pre-enrichment broth with DHB (UPD)

• 1X, 2X, and 5X selenite cystine broth with DHB (SCBD)

• Bismuth sulfite with DHB (BSD) agar

• Miller-Mallinson with DHB (MMD) agar

• Tryptic soy agar with DHB (TSAD)

• Tryptic soy broth (TSB)

• 1% tryptic soy broth with DHB (TSBD)

• Physiological saline (0.85%)

• API® 20E Test Strips

• Oxidase reagent

• Salmonella Vi and O group D antisera Control Cultures

o

S. Typhi (CVD 909)

o Enterococcus faecalis (ATCC® 29212™)

o Pseudomonas aeruginosa (ATCC® 27853™)


Water Matrices

Water matrices will include a reference matrix (PBS), drinking water from the laboratory tap, and surface water from a lake, reservoir, or other open body of water.

Quality Control (QC) Analyses

QC analyses listed on the batch specific coversheet should be analyzed with each run.

Sample Analyses

• Set A will be pre-enriched in UPD broth, with samples sub-cultured into a selective broth (SCBD), followed by plating on selective agars (BSD and MMD).

• Set B will be cultured in SCBD without pre-enrichment, followed by plating on BSD and MMD.

• Isolated, typical colonies from BSD and MMD plates from both Set A and Set B will then be sub-cultured onto TSAD and submitted to serological and biochemical confirmation according to study-specific instructions.

• Two separate analytical runs, consisting of the following samples will be conducted resulting in 2 unspiked and 4 spiked samples for each matrix (PBS, drinking water, and surface water).for a total of 18 samples. See flowchart in Appendix A to this Appendix (see page B-16, below) for an overview of the analyses.

Run 1 Analyses:

o PBS Samples (Reference Matrix)

o 1, 200-mL unspiked

o 2, 200-mL spiked

o Drinking Water Samples


o 2, 200-mL spiked

o Surface Water Samples


o 2, 200-mL spiked

Run 2 Analyses:

o PBS Samples (Reference Matrix)


o 2, 200-mL spiked

o Drinking Water Samples


o 2, 200-mL spiked

o Surface Water Samples


o 2, 200-mL spiked


Media and Reagent Preparation Reagent Preparation

Assuming all analyses are conducted in 2 separate 1-week runs, a total of 2190 mL of PBS will be required. As a result, it is recommended that the laboratory prepare 2.5 L of PBS prior to the first week of study analyses [10, 99-mL dilution blanks and 6, 200-mL samples].

• Phosphate Buffered Saline (PBS): Prepare 2.5 L of PBS according to the following instructions and store at room temperature for a maximum of three months in screw cap bottles. Note: The following instructions are for preparation of 1.0 L; please adjust for preparation of appropriate volumes.





Dissolve reagents in 2.5 L of reagent-grade water in a flask and dispense in appropriate volumes in screw cap bottles and autoclave at 121°C (15 lb pressure per square inch [PSI]) for 15 minutes. Final pH should be 7.4 ± 0.2.

Media Preparation

It is recommended that the following be prepared, either prior to the week of study analyses (DHB, UPD, TSAD, TSB, 1% TSBD), or the day before analyses (SCBD, BSD), so that appropriate QC analyses can be conducted on the media prior to use. MMD medium will be provided as prepared plates.

• 2,3-dihydroxybenzoate (DHB): Prepare 100 mL of 0.1% (w/v) DHB stock solution by dissolving 0.1 g of DHB powder in 100 mL of reagent-grade water and filter sterilize.

• Universal Pre-enrichment Broth with DHB (UPD):

o Prepare UPD according to the following:

For 300 mL of 1X UPD, add 11.4 g dehydrated medium to 300 mL reagent-grade water; mix thoroughly.



o Autoclave all solutions at 121°C (15 PSI) for 15 minutes. Final pH should be 6.3 ± 0.2. Cool to 45EC – 50EC in a waterbath.

For 1X UPD, add 0.3 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0 mL aliquots in 25 × 150 mm tubes.



o Store at <10°C and above freezing for a maximum of 2 weeks.


• Selenite Cystine Broth with DHB (SCBD):

o Prepare SCBD according to the following:

For 1.2 L of 1X SCBD, add 27.6 g dehydrated medium to 1.2 L reagent-grade water; mix thoroughly.

For 300 mL of 2X SCBD, add 13.8 g dehydrated medium to 300 mL reagent-grade water; mix thoroughly.

For 200 mL of 5X SCBD, add 23.0 g dehydrated medium to 200 mL reagent-grade water; mix thoroughly.

o Prepare SCBD according to the manufacturer’s instructions. Do not autoclave. Final pH should be 7.0 ± 0.2. Cool to 45EC – 50EC in a waterbath.

For 1X SCBD, add 1.2 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0 mL aliquots in 25 × 150 mm tubes.



o Use within 48 hours of preparation. Note: A brick red precipitate may appear if the medium is overheated during preparation or exposed to excessive moisture during storage. If this occurs medium should be discarded and a new batch prepared.

• Bismuth Sulfite with DHB (BSD): Prepare 2.0 L of BSD agar (the recommended volume assumes that 80% of spiked and 20% of unspiked MPN tubes are presumptive positive) according to manufacturer’s instructions the day prior to analyses. Cool to 45°C – 50°C in a waterbath and add 1.5 mL of the 0.1% DHB solution. Evenly disperse the precipitate while aseptically pouring 12 – 15 mL into 100 × 15 mm sterile Petri plates. Use within 48 hours of preparation. Store at room temperature [130, 15-mL, 100 × 15-mm plates].

• Miller-Mallinson with DHB (MMD): Agar will be received as prepared plates. Store at <10°C and above freezing [130, 100 × 15-mm plates].

• Tryptic Soy Agar with DHB (TSAD): Prepare 278 TSAD plates according to manufacturer’s instructions. After autoclaving, cool to 45°C – 50°C in a waterbath and add 0.1 mL of the 0.1% DHB solution per 100 mL of medium. Aseptically pour 12 – 15 mL into each 100 × 15 mm sterile Petri plate. Store at <10°C and above freezing for a maximum of 2 weeks.

• Tryptic Soy Broth (TSB): Prepare 100 mL TSB according manufacturer’s instructions. Store at <10°C and above freezing for a maximum of three months in a screw cap bottle.

• 1%Tryptic Soy Broth with DHB (TSBD): Prepare a 1% solution of TSBD by combining 99 mL of sterile PBS, 1 mL of sterile single-strength TSB, and 0.1 mL of DHB solution in a sterile screw cap bottle. Shake to mix.


Study Analyses Instructions provided below are for a single week of analyses (1 run). These instructions are to be repeated on a subsequent week for a total of 2 runs. Day One (Sunday) Propagation of Spiking Suspension

Inoculate 1% TSBD with S. Typhi according to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 2 hours.

Day Two (Monday) TSA Enumeration of Spiking Suspensions

• Dilute and plate laboratory-prepared spiking suspension on TSAD according to spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 2 hours.

Preparation and Collection of Samples

o Sample Collection and/or Preparation – PBS, Drinking Water, and Surface Water

• PBS (Reference Matrix) Samples

o Aliquot the following volumes of sterile PBS and label the samples as follows:

1, 200-mL unspiked sample – PBS unspiked

2, 200-mL spiked samples – PBS spiked

• Collect a 1.0-L bulk drinking water sample as follows:

o Select a cold water line faucet and remove aerator, if present.

o Clean the faucet exterior with disinfection solution (e.g., 10% household bleach).

o Open the tap to obtain a smooth-flowing stream at moderate pressure without splashing.

o Allow water to run at least 2 – 3 minutes.

o Remove the cap from a sterile bottle containing 1 mL of a 10% sodium thiosulfate solution (dechlorinating agent).

o Avoid contaminating the sample bottle lip or inside the cap.

o Reduce the water flow to fill the bottle without splashing and fill to within 2.5 cm – 5 cm (1" – 2") of the top for proper mixing before analyses.

o Do not rinse dechlorinating agent out of the bottle.

o Tightly cap the container.

o Mix thoroughly and split the bulk sample into aliquots and label the samples as follows.

1, 200-mL unspiked sample – Drinking water unspiked

2, 200-mL spiked samples – Drinking water spiked


o Collect samples by hand or with a sampling pole if the sampling site has difficult access such as a dock, bridge, or bank adjacent to surface water.






o In the laboratory, split the bulk sample into aliquots and label the samples as follows.

1, 200-mL unspiked sample – Surface water unspiked

2, 200-mL spiked samples – Surface water spiked Sample Spiking

• Spike 2, 200-mL samples each of PBS, drinking water, and surface water with laboratory-prepared spiking suspension using twice the volume indicated in the spiking protocol to accommodate the larger 200 mL sample volume.


• Mix unspiked and spiked samples by shaking 25 times. Each 200 mL sample will be used to inoculate tubes for both sets (A and B).

Set A (with Pre-enrichment)

• For Set A tubes (with pre-enrichment), inoculate UPD tubes with the following volumes for each unspiked or spiked sample:





Set B (without Pre-enrichment)

• For Set B tubes (without pre-enrichment), inoculate SCBD tubes with the following volumes for each unspiked or spiked sample:

o For each of three tubes, add 20 mL of undiluted sample to 5 mL (5X) SCBD



• Incubate inoculated SCBD tubes at 35.0°C ± 0.5°C for 18 ± 2 hours.

Day Three (Tuesday) TSA Enumeration of Spiking Suspension

• Count colonies on TSA plates prepared on Day Two and record results of CFU enumeration on provided data reporting forms

• Fax TSA enumeration results to Yildiz Chambers at (703) 461-8056.


MPN


• Examine UPD tubes at 24 hours; record results.

• For each UPD tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes with 10.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 18 ± 2 hours.


• For each SCBD tube with growth, gently swirl the tube to mix and streak for isolation on BSD and MMD plates using a sterile inoculation loop (20 µL). Incubate at 35.0°C ± 0.5°C for 48 ± 3 hours, examining plates at 24 ± 2 hours and 48 ± 3 hours.

Day Four (Wednesday) MPN


• Examine SCBD tubes at 18 ± 2 hours; record results.



BSD and MMD Plates

• Examine BSD and MMD plates at 24 ± 2 hours; record results.

• If no typical (presumptive positive) S. Typhi (green-black with metallic sheen for BSD or black on MMD) colonies are observed on plates, re-incubate for a total of 48 ± 3 hours at 35.0°C ± 0.5°C.

• For each BSD and MMD plate with typical colonies, streak a single typical colony onto TSAD and incubate 24 ± 2 hours at 35.0°C ± 0.5°C. Note: Unspiked samples are not expected to have presumptive-positive colonies, however, if typical S. Typhi colonies are observed, these (up to 5) should be streaked onto TSAD for biochemical (API 20E) and serological analyses.

Day Five (Thursday)


BSD and MMD Plates


• If no typical (green-black with metallic sheen for BSD or black on MMD) colonies are observed on plates, re-incubate for a total of 48 ± 3 hours at 35.0°C ± 0.5°C.

• For each BSD and MMD with typical colonies, streak a single typical colony onto TSAD and incubate 24 ± 2 hours at 35.0°C ± 0.5°C. Note: Unspiked samples are not expected to have presumptive-positive colonies, however, if typical S. Typhi colonies are observed, these should be streaked onto TSAD for biochemical (API 20E) and serological analyses.



BSD and MMD Plates



TSAD Plates

• Examine TSAD plates at 24 ± 2 hours. Note: TSAD plates may be refrigerated following incubation prior to serological and biochemical analyses.

• Serological Analyses with Salmonella antiserum (Vi and O Group D):

Vi

o Emulsify a portion of a single presumptive positive colony from each TSA plate using sterile physiological saline. Place two discrete drops of emulsified growth onto a slide. To the first drop of emulsified growth, add one drop of Vi antiserum. To the second drop of emulsified growth, add one drop of sterile saline (as a visual comparison). Observe under magnification for an agglutination reaction and record results.

O Group D

o Prepare a heavy suspension of cells (3 – 4 McFarland tube concentration) in 500 µL of physiological saline and place in a boiling waterbath for 20 – 30 minutes.

o Place two discrete drops of boiled cell suspension onto a slide. To the first drop of cell suspension, add one drop of Salmonella O antiserum Group D. To the second drop of cell suspension, add one drop of sterile saline (as a visual comparison). Observe under magnification for an agglutination reaction, which indicates a positive result.

Results should be compared with those for positive (S. Typhi) and negative (E. faecalis) controls analyzed at the same time. S. Typhi is agglutination-positive for both O Group D and Vi antisera.

• Biochemical Analyses:

o Oxidase Test: Transfer a small amount of cells from a single colony to the slide and follow manufacturers’ instructions for analysis. Oxidase-positive bacteria turn the reagent dark purple within 20 seconds. S. Typhi is oxidase-negative.

o API 20E Test Strips: Emulsify a large colony (2 – 3 mm) in 5.0 mL physiological saline supplemented with 5 µL DHB. Follow manufacturer’s instructions to inoculate wells. Incubate test strip at 36.0°C ± 2.0°C for 18 – 24 hours.

Day Six (Friday)


BSD and MMD Plates



• For each Set A BSD and MMD plate with typical colonies, streak a single typical colony onto TSAD and incubate 24 ± 2 hours at 35.0°C ± 0.5°C.

TSAD Plates

• Conduct serological analyses (Vi and O Group D) and biochemical (oxidase and API 20E) tests as described above (Day Five) using TSAD plates inoculated on Day Five.


TSAD Plates

• Conduct serological analyses (Vi and O Group D) and biochemical (oxidase and API 20E) tests as described above (Day Five) using TSAD plates inoculated on Day Five.

Biochemical Analyses

• API 20E Test Strips: Add appropriate reagents according to manufacturer’s instructions, observe and record results to strips inoculated on Day Five. If results are not consistent with S. Typhi, please contact CSC (Yildiz Chambers) immediately for additional instructions.

Day Seven (Saturday)


TSAD Plates

• If additional TSAD plates were streaked on Day Six, conduct serological analyses (Vi and O Group D) and biochemical (oxidase and API 20E) tests as described above (Day Five).


• If additional API 20E Test Strips were inoculated on Day Six, add appropriate reagents as described above (Day Six).


• Set B was completed on Day Six

Day Eight (Sunday)



• If additional API 20E Test Strips were inoculated on Day Seven, add appropriate reagents as described above (Day Six).

Reporting Results

• Please fax results to Yildiz Chambers at (703) 461-8056 at the end of each week.

• Within one week of optimization study analysis completion (runs 1 and 2), please FedEx all original hard copy data to CSC.


Appendix A

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Do Not Cite, Circulate, or Copy B-18 April 22, 2008

Method Optimization Instructions for Salmonella Typhi using Immunomagnetic Separation (IMS): Water Matrices

April 22, 2008

The instructions provided below are for analysis of unspiked and spiked samples for two matrices (PBS and surface water). Surface water samples will be processed without immunomagnetic separation (IMS) and with IMS (Sets A and B, respectively) and plated on two selective agars (BSD and MMD). The reference matrix, PBS, will be processed only with IMS. Please contact Yildiz Chambers (703) 461-2165, [email protected]) if you have any questions regarding these instructions.

All QA/QC analyses specified in the study-specific instructions and draft SAP must be conducted in conjunction with sample analyses described below. In addition, the laboratory must follow all QA/QC procedures outlined in their QAPP.

Media/Reagents/Supplies • Phosphate buffered saline (PBS)

• 10% sodium thiosulfate

• 2,3-dihydroxybenzoate (DHB)

• 1X, 2X, and 5X universal pre-enrichment broth with DHB (UPD)

• 1X, 2X, and 5X selenite cystine broth with DHB (SCBD)

• Bismuth sulfite with DHB (BSD) agar

• Miller-Mallinson with DHB (MMD) agar

• Tryptic soy agar with DHB (TSAD)

• Tryptic soy broth (TSB)

• 1% tryptic soy broth with DHB (TSBD)

• Salmonella spp. immunomagnetic separation (IMS) beads

• Physiological saline (0.85%)

• API 20E Test Strips

• Oxidase reagent

• Salmonella Vi antisera Control Cultures

o S. Typhi (CVD 909)

o Enterococcus faecalis (ATCC® 29212™)

o Pseudomonas aeruginosa (ATCC® 27853™)


Water Matrices

Water matrices will include a reference matrix (PBS) and surface water from a lake, reservoir, or other open body of water.

Quality Control (QC) Analyses

QC analyses listed on the batch specific coversheet should be analyzed with each run.

Sample Analyses

• Set A (without IMS) will be pre-enriched in UPD broth, with samples sub-cultured into a selective broth (SCBD), followed by plating on selective agars (BSD and MMD).

• Set B (with IMS) will be pre-enriched in UPD broth, concentrated by IMS, IMS beads transferred to selective broth (SCBD), followed by plating on selective agars (BSD and MMD).

• Isolated, typical colonies from BSD and MMD plates from both Set A and Set B will then be sub-cultured onto TSAD and submitted to serological and biochemical confirmation according to study-specific instructions.

Analyses:

o Set A: Surface Water (without IMS)


o 2, 100-mL spiked

o Set B: PBS (with IMS)


o 1, 100-mL spiked

o Set B: Surface Water (with IMS)


o 4, 100-mL spiked

Media and Reagent Preparation Reagent Preparation

• Phosphate Buffered Saline (PBS): Prepare 1.0 L of PBS according to the following instructions and store at room temperature for a maximum of three months in screw cap bottles [4, 99-mL dilution blank, 2, 100-mL samples; and 100-mL IMS wash buffer].





Dissolve reagents in 1 L of reagent-grade water in a flask and dispense in appropriate volumes in screw cap bottles and autoclave at 121°C (15 lb pressure per square inch [PSI]) for 15 minutes. Final pH should be 7.4 ± 0.2.


• Prepare IMS wash buffer by adding 50 µL Tween® 20 to 100 mL of PBS, ensuring all of the Tween® 20 is expelled from the pipette tip; filter sterilize or autoclave at 121°C (15 PSI) for 15 minutes.

Media Preparation

It is recommended that the following be prepared, either prior to the week of study analyses (DHB, UPD, TSAD, TSB, 1% TSBD), or the day before analyses (SCBD, BSD), so that appropriate QC analyses can be conducted on the media prior to use. MMD medium will be provided as prepared plates.

• 2,3-dihydroxybenzoate (DHB): Prepare 100 mL of 0.1% (w/v) DHB stock solution by dissolving 0.1 g of DHB powder in 100 mL of reagent-grade water and filter sterilize.

• Universal Pre-enrichment Broth with DHB (UPD):

o Prepare UPD according to the following:




o Autoclave all solutions at 121°C (15 PSI) for 15 minutes. Final pH should be 6.3 ± 0.2. Cool to 45EC – 50EC in a waterbath.

For 1X UPD, add 0.35 mL of 0.1% DHB stock solution, mix thoroughly, and dispense 10.0 mL aliquots in 25 × 150 mm tubes.



o Store at <10°C and above freezing for a maximum of 2 weeks.

• Selenite Cystine Broth with DHB (SCBD):

o Prepare 1.0 L SCBD according to the manufacturer’s instructions. Do not autoclave. Final pH should be 7.0 ± 0.2. Cool to 45EC – 50EC in a waterbath.


o Use within 48 hours of preparation. Note: A brick red precipitate may appear if the medium is overheated during preparation or exposed to excessive moisture during storage. If this occurs medium should be discarded and a new batch prepared.

• Bismuth Sulfite with DHB (BSD): Prepare 1.5 L of BSD agar according to manufacturer’s instructions the day prior to analyses. Cool to 45°C – 50°C in a waterbath and add 1.5 mL of the 0.1% DHB solution. Evenly disperse the precipitate while aseptically pouring 12 – 15 mL into 100 × 15 mm sterile Petri plates. Use within 48 hours of preparation. Store at room temperature and protected from light [100, 15-mL, 100 × 15 mm plates].

• Miller-Mallinson with DHB (MMD): Agar will be received as prepared plates. Store at <10°C and above freezing [100, 100 × 15 mm plates].

• Tryptic Soy Agar with DHB (TSAD): Prepare 200 TSAD plates according to manufacturer’s instructions. After autoclaving, cool to 45°C – 50°C in a waterbath and add 0.1 mL of the 0.1% DHB


solution per 100 mL of medium. Aseptically pour 12 – 15 mL into each 100 × 15 mm sterile Petri plate. Store at <10°C and above freezing for a maximum of 2 weeks.

• Tryptic Soy Broth (TSB): Prepare 100 mL TSB according manufacturer’s instructions. Store at <10EC and above freezing for a maximum of three months in a screw cap bottle.

• 1% Tryptic Soy Broth with DHB (TSBD): Prepare a 1% solution of TSBD by combining 99 mL of sterile PBS, 1 mL of sterile single-strength TSB, and 0.1 mL of DHB solution in a sterile screw cap bottle. Shake to mix.

Study Analyses Instructions provided below are for a single week of analyses (1 run). Day One (Sunday) Propagation of Spiking Suspension

Inoculate 1% TSBD with S. Typhi according to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 2 hours.

Day Two (Monday) TSA Enumeration of Spiking Suspensions

• Dilute and plate laboratory-prepared spiking suspension on TSAD according to spiking protocol and incubate at 35EC ± 0.5EC for 24 ± 2 hours.

Preparation and Collection of Samples

o Sample Collection and/or Preparation – PBS and Surface Water

• PBS (Reference Matrix) Samples

o Aliquot the following volumes of sterile PBS and label the samples as follows:

1, 100-mL unspiked sample – PBS unspiked, with IMS (Set B)

1, 100-mL spiked samples – PBS spiked, with IMS (Set B)


o Collect samples by hand or with a sampling pole if the sampling site has difficult access such as a dock, bridge, or bank adjacent to surface water.





o In the laboratory, split the bulk sample into aliquots and label the samples as follows:

Set A:

- 1, 100-mL unspiked sample – Surface water unspiked, without IMS


- 2, 100-mL spiked sample – Surface water spiked, without IMS

Set B:

- 1, 100-mL unspiked sample – Surface water unspiked, with IMS (Set B)

- 4, 100-mL spiked samples – Surface water spiked, with IMS (Set B) Sample Spiking

• Spike 1, 100-mL sample of PBS and 6, 100-mL surface water samples with laboratory-prepared spiking suspension.


• Mix unspiked and spiked samples (Sets A and B) by shaking 25 times. Each 100 mL sample will be used to inoculate tubes.

• For each Set A and Set B samples, inoculate UPD tubes with the following volumes for each unspiked or spiked sample:





Day Three (Tuesday) TSA Enumeration of Spiking Suspension

• Count colonies on TSA plates prepared on Day Two and record results of CFU enumeration on provided data reporting forms.

• Fax TSA enumeration results to Yildiz Chambers at (703) 461-8056.

MPN

Set A (without IMS)

• Examine UPD tubes at 24 hours; record results.

• For each UPD tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes with 10.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 18 ± 2 hours.

Set B (with IMS)

• For each UPD tube with growth, conduct IMS as follows:

o Suspend Salmonella beads by vortexing and add 20 µL of bead suspension to a sterile, 1.5 – 2.0 mL tube.

o Gently swirl contents of UPD tubes to mix and remove 1.0 mL of enrichment culture; add to tube with beads and tightly cap.

o Mix enrichment culture with beads for 10 minutes at room temperature using tube rotator.


o Place tubes in magnetic holder, inverting tubes several times to ensure that beads are concentrated in a pellet on the side of the tube; allow beads to settle for 3 minutes.

o Carefully aspirate liquid and discard; resuspend beads in 1.0 mL IMS wash buffer by inverting the tube several times; place tubes in magnetic holder and allow beads to settle for 3 minutes; aspirate wash buffer.

o Resuspend the beads in 100 µL of IMS buffer.

• Pipette beads from each sample (100 µL) into a set of corresponding tubes with 10.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 18 ± 2 hours.

Day Four (Wednesday) MPN

• Examine SCBD tubes (Sets A and B) at 18 ± 2 hours; record results.


Day Five (Thursday) BSD and MMD Plates

• Examine BSD and MMD plates (Sets A and B) at 24 ± 2 hours; record results.

• If no typical (green-black with metallic sheen for BSD or black on MMD) colonies are observed on plates, re-incubate for a total of 48 ± 3 hours at 35.0°C ± 0.5°C.


Day Six (Friday)

TSAD Plates

• Examine TSAD plates (Sets A and B) at 24 ± 2 hours. Note: TSAD plates may be refrigerated following incubation prior to serological and biochemical analyses.

• Serological Analyses with Salmonella Vi antiserum:

o Emulsify a portion of a single presumptive positive colony from each TSA plate using sterile physiological saline. Place two discrete drops of emulsified growth onto a slide. To the first drop of emulsified growth, add one drop of Vi antiserum. To the second drop of emulsified growth, add one drop of sterile saline (as a visual comparison). Observe under magnification for an agglutination reaction and record results.

o Results should be compared with those for positive (S. Typhi) and negative (E. faecalis) controls analyzed at the same time. S. Typhi is agglutination-positive for Vi antisera.

• Biochemical Analyses:


o Oxidase Test: Transfer a small amount of cells from a single colony to the slide and follow manufacturers’ instructions for analysis. Oxidase-positive bacteria turn the reagent dark purple within 20 seconds. S. Typhi is oxidase-negative.

o API 20E Test Strips: Emulsify a large colony (2 – 3 mm) in 5.0 mL physiological saline supplemented with 5 µL DHB. Follow manufacturer’s instructions to inoculate wells. Incubate test strip at 36.0°C ± 2.0°C for 18 – 24 hours.

BSD and MMD Plates

• Examine BSD and MMD plates (Sets A and B) at 48 ± 3 hours; record results.

• For each BSD and MMD plate with typical colonies, streak a single typical colony onto TSAD and incubate 24 ± 2 hours at 35.0°C ± 0.5°C.

Day Seven (Saturday)

TSAD Plates

• If additional TSAD plates were streaked on Day Six, conduct serological analyses and biochemical (oxidase and API 20E) tests as described above.


• API 20E Test Strips: Add appropriate reagents according to manufacturer’s instructions, observe and record results to strips inoculated on Day Five. If results are not consistent with S. Typhi, please contact CSC (Yildiz Chambers) immediately for additional instructions.

Day Eight (Sunday)


• If additional API 20E Test Strips were inoculated on Day Seven, add appropriate reagents as described above.

Reporting Results

• Please fax results to Yildiz Chambers at (703) 461-8056 at the end of each week.

• Within one week of optimization study analysis completion (runs 1 and 2), please FedEx all original hard copy data to CSC.


Appendix C

Spiking Protocol

Draft – Do Not Cite, Circulate, or Cop C-1 November, 2008

Water Matrices Spiking Protocol for the Draft Salmonella Typhi SAP Verification Study

Draft for Discussion – November, 2008

Do Not Cite, Circulate, or Copy The purpose of this protocol is to provide laboratories with Salmonella enterica subsp. enterica serotype Typhi (S. Typhi) spiking procedures for the verification and validation study to evaluate the Draft Standardized Analytical Procedure for Salmonella enterica subsp. enterica serotype Typhi (Salmonella Typhi or S. Typhi) in Environmental Samples (referred to as the “draft SAP”). The following sections are included in this protocol: Laboratory-Prepared Spiking Solutions Section 1: Preparation of Laboratory-Prepared Spiking Suspensions Section 2: Laboratory-Prepared Sample Spiking and Enumeration

Section 3: Calculation of Laboratory-Prepared Spike Percent Recovery 1.0 Preparation of Laboratory-Prepared Spiking Suspensions 1.1 Stock Culture. Prepare a stock culture by inoculating a tryptic soy agar (TSA) slant (or other

non-selective media) containing 0.0001% DHB with S. Typhi and incubating at 36°C ± 1.5°C for 20 ± 4 hours. After incubation, the stock culture may be stored in the dark at room temperature for up to 30 days.

1.2 1% Tryptic Soy Broth (TSB). Prepare a 1% solution of TSB by combining 99 mL of sterile

PBS, 1 mL of sterile single-strength TSB (prepared according to manufacturer’s instructions), and 0.1 mL of 2,3-dihydroxybenzoate (DHB) solution (prepared according to the study instructions) in a sterile screw cap bottle. Shake to mix.

1.3 Spiking Suspension (Undiluted). From the stock culture of S. Typhi in section 1.1, aseptically

transfer a small loopful of growth to the 1% TSB solution and vigorously shake a minimum of 25 times. Incubate at 36C ± 1.5C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0×106 to 1.0×107

S. Typhi colony forming units (CFU) per mL. This is referred to as the “undiluted spiking suspension.” Note: During the S. Typhi draft SAP verification study, growth of spiking suspensions should begin one day prior to the day samples will be spiked. For example, if samples will be spiked on Tuesday, growth of spiking suspensions will begin on Monday.

1.4 Proceed to Section 2.0 for sample spiking and enumeration of spiking suspension. 2.0 Laboratory Prepared Sample Spiking and Spiking Suspension Enumeration Since the objective of spiking the sample is to establish percent recovery, it is necessary to determine the concentration of S. Typhi in laboratory prepared undiluted spiking suspensions (Section 1.3). This section provides instructions for sample spiking (2.1) and spiking suspension enumeration (2.2).


2.1 Sample spiking Please be sure to homogenize well the spiking suspensions in the steps below as homogenization is critical for accurate sample spiking and spiking suspension enumeration. 2.1.1 Dilute spiking suspension Perform steps 2.1.1.1 through 2.2.6 using laboratory-prepared undiluted spiking suspension (Section 1.3).

2.1.1.1 Mix spiking suspension by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of undiluted spiking suspension (from Section 1.3 above) to 99 mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “A.” A 1.0-mL volume of dilution “A” is equal to 1 × 10-2 of the undiluted spiking suspension and is equal to 1×104 to 1×105 CFU/mL.

2.1.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution “A” (from

Section 2.1.1.1 above) to 99 mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “B.” A 1.0 mL volume of dilution “B” is equal to 1 × 10–4 of the undiluted spiking suspension and is equal to 1×102 to 1×103 CFU/mL.

2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “B” (from Section 2.1.1.2 above) to 99 mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “C.” A 1.0-mL volume of dilution “C” is equal to 1 × 10–5 of the undiluted spiking suspension and is equal to 1×101 to 1×102 CFU/mL.

2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “C” (from Section 2.1.1.3 above) to 99 mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “D.” A 1.0-mL volume of dilution “D” is equal to 1 × 10-6 of the undiluted spiking suspension and is equal to 1×100 to 1×101 CFU/mL (i.e., 1 to 10 CFU/mL).

2.1.2 Spike sample(s)

2.1.2.1 To spike the sample, add 0.3 mL of spiking suspension dilution “B” (from Section 2.1.1.2) to 100 mL of unspiked sample and mix by vigorously shaking the bottle a minimum of 25 times. The concentration of the spiking suspension added to each 100.0 mL of sample is 3 × 10–5 of the undiluted spiking suspension. This is referred to as Vspiked per 100 mL sample. Analyze the spiked sample according to study instructions and the draft SAP.

2.1.2.2 For samples analyzed by MPN, inoculate unspiked and spiked samples

according to study instructions and the draft SAP.


2.2 Enumeration of Spiking Suspensions (Prepared in Section 1.3)

2.2.1 Prepare TSA with 0.0001% DHB according to manufacturer’s and study instructions. Add 10 – 15 mL of TSA per 100 × 15 mm petri dish, and allow the agar to solidify. Note: Agar plates must be dry and free from condensation prior to use. To ensure that agar surface is dry, plates should be made several days in advance and stored inverted at room temperature or dried using a laminar-flow hood.

2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine

spread plates:

• Mix dilution “B” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of dilution “B” (Section2.1.1.2) onto the surface of the pre-dried TSA plate. This is 10 – 5 of the undiluted spiking suspension.

• Mix dilution “C” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of dilution “C” (Section 2.1.1.3) onto the surface of the pre-dried TSA plate. This is 10 – 6 of the undiluted spiking suspension.

• Mix dilution “D” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of dilution “D” (Section 2.1.1.4) onto the surface of the pre-dried TSA plate. This is 10 – 7 of the undiluted spiking suspension.

2.2.3 For each spread plate, use a sterile bent glass rod or spreader to distribute inoculum over the surface of the medium by rotating the dish by hand or on a turntable. Note: Please ensure that inoculum is evenly distributed over entire surface of the plate.

2.2.4 Allow inoculum to absorb into the medium completely.

2.2.5 Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours. 2.2.6 Count and record number of colonies per plate. Refer to Section 5.0 for calculation of

spiking suspension concentration. 3.0 Calculation of Spiked S. Typhi Percent Recovery (Laboratory-Prepared Undiluted

Spiking Suspension) Spiked S. Typhi percent recovery will be calculated in three steps as indicated in Sections 3.1

through 3.3, below. Note: Example calculated numbers provided in tables below have been rounded at the end of each step. If your laboratory recalculates examples using a spreadsheet and rounds only after the final calculation (Section 3.3), percent recoveries may be slightly different.

3.1 Step 1: Calculate Concentration of S. Typhi (CFU/mL) in Undiluted Spiking

Suspension

3.1.1 The number of S. Typhi (CFU/mL) in the undiluted spiking suspension (prepared in Section 1.3, above) will be calculated using all TSA plates from Section 2.2 yielding counts within the ideal range of 30 to 300 CFU per plate.

3.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if colonies are not

discrete, results should be recorded as “too numerous to count” (TNTC).


3.1.3 Calculate the concentration of S. Typhi (CFU/mL) in undiluted spiking suspension

according to the following equation. Example calculations are provided in Table 2.

S. Typhi undiluted spike = (CFU1 + CFU2 + ... + CFUn) / (V1 + V2 + ... + Vn)

Where,

S. Typhi undiluted spike = S. Typhi (CFU/mL) in undiluted spiking suspension

CFU = Number of colony forming units from TSA plates yielding counts within the ideal range of 30 to 300 CFU per plate

V = Volume of undiluted sample on each TSA plate yielding counts within the ideal range of 30 to 300 CFU per plate

n = Number of plates with counts within the ideal range

T able 2. E xample C alculations of S. Typhi S piking S us pens ion C oncentration

Examples CFU/plate (triplicate analyses) from

TSA plates in Section 2.2.2 S. Typhi CFU/mL in undiluted

spiking suspension (S. Typhi undiluted spike)* 10-5 mL plates 10-6 mL plates 10-7 mL plates

Example 1 31, 34, 32 3, 0, 5 0, 1, 0

(31+34+32) / (10-5+10-5+10-5) = 597 / (3.0 x 10-5) = 32.3 × 10

6= 3.2 × 10 CFU/mL

Example 2 45, 56, 61 4, 3, 8 0, 2, 0

(45+56+61) / (10-5+10-5+10-5) = 5 162 / (3 x 10-5) = 54.0 × 10

6= 5.4 x 10 CFU/mL

* S. Typhi undiluted spike is calculated using all plates yielding counts within the ideal range of 30 – 300 CFU per plate. 3.2 Step 2: Calculate “True” Spiked S. Typhi (CFU/100 mL)

Calculate the true concentration of spiked S. Typhi (CFU/100 mL) according to the following equation. Example calculations are provided in Table 3.

TSpiked S. Typhi = (S. Typhi undiluted spike) x (V spiked per 100 mL sample)

Where,

TSpiked S. Typhi = Number of spiked S. Typhi (CFU/100 mL) S. Typhi undiluted spike = S. Typhi (CFU/mL) in undiluted spiking suspension

(calculated in Section 3.1.3) V spiked per 100 mL sample = mL of undiluted spiking suspension per 100 mL sample

(Section 2.1.2.1)


Table 3. Example Calculations of Spiked S. Typhi

S. Typhi undiluted spike (Table 2 above)

V spiked per 100 mL sample (Section 2.1.2.1 above)

T

Spiked S. Typhi

3.2 x 106 CFU/mL -53.0 x 10 mL per 100 mL of sample (3.2 6x 10 CFU/mL) x (3.0 x 10-5 96 CFU/100 mL

mL/100 mL) =

5.4 x 106 CFU/mL -53.0 x 10 mL per 100 mL of sample (5.4 6x 10 CFU/mL) x (3.0 x 10-5

162 CFU/100 mL

mL/100 mL) =

3.3 Step 3: Calculate Percent Recovery

3.3.1 Calculate percent recovery (R) using the following equation.

N −R = 100 s N

x u TSpiked S .Typhi

Where,

R = Percent recovery Ns = S. Typhi (CFU/100 mL) in the spiked sample Nu = S. Typhi (CFU/100 mL) in the unspiked sample TSpiked S. Typhi = True spiked S. Typhi (CFU/100 mL) in spiked sample (Section 3.2, above)

3.3.2 Example percent recovery calculations are provided in Table 4.

Table 4. Example Percent Recovery Calculations

Ns (CFU/100 mL) Nu (CFU/100 mL) T Spiked S. Typhi (CFU/100 mL) Percent recovery (R)

42 <1 96 100 x (42 – 1) / 96 = 43%

34 10 96 100 x (34 - 10) / 96 = 25%

153 <1 162 100 x (153 - 1) / 162 = 94%

142 <1 162 100 x (142 - 1) / 162 = 87%

Appendix D

Do Not Cite, Circulate, or Copy D-6 December 18, 2008

S. Typhi Optimization Analyses: SET B (without Pre-enrichment) SPIKED

Sample #: Lab: Date: Matrix (circle one): PBS Drinking Water Surface Water

Spike dilution: Spike volume: Spiking date and time:

Please record plate counts and + / - for biochemical and serological analyses.

Sample VolumeSCBD BSD MMD TSAD API Vi

Agglutination O Group D

Agglutination

24 ± 2 h @ 35.0°C ± 0.5°C 24 ± 2 h @ 35.0°C ± 0.5°C

48 ± 3 h @ 35.0°C ± 0.5°C

24 ± 2 h @ 35.0°C ± 0.5°C

48 ± 3 h @ 35.0°C ± 0.5°C 18 - 24 h @ 35°C ± 0.5°C (+ or -) (+ or -) (+ or -)

20 mL1

(5X SCBD) 2

3

10 mL1

(2X SCBD) 2

3

1 mL1

(1X SCBD) 2

3

Incubation Start

Date:

Time:

Temp:

Initials:

Incubation End

Date:

Time:

Temp:

Initials:Analyses S . Typhi Results Final Results

SCBD Growth, indicated by turbidity. MPN Tube Combination

BSD Black colonies surrounded by black or brownish-black zone, which may have metallic sheen (in light).

MMD Black colonies MPN / 100 mL

Salmonella Antisera Agglutination, indicated by the formation of a white precipitate

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