STUDY REPORT - U.S EPA Web Server

EPA/600/R-10/141 | October 2010 | www.epa.gov/ord

United StatesEnvironmental ProtectionAgency

Single-Laboratory Verification of Culture-Based Analytical Procedures for Vibrio cholerae O1 and O139 in Drinking Water and Surface WaterSTUDY REPORT

Office of Research and DevelopmentNational Homeland Security Research Center

Study Report Single-Laboratory Verification of Culture-Based Analytical Procedures for Vibrio cholerae O1 and O139 in Drinking Water and Surface Water

October 2010

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY Office of Research and Development, National Homeland Security Research Center Cincinnati, OH 45268

Office of Research and Development National Homeland Security Research Center, Threat and Consequence Assessment Division

Acknowledgments This report presents results of a single-laboratory study funded by the National Homeland Security Research Center (NHSRC) within the U.S. Environmental Protection Agency (EPA’s) Office of Research and Development under the direction of Sanjiv R. Shah to verify culture-based analytical procedures for Vibrio cholerae O1 and O139 in water. Computer Sciences Corporation (CSC) provided technical support and data evaluation under EPA contract EP-C-05-045. The contributions of the following persons and organizations are gratefully acknowledged:

Study Workgroup Participants • Cheryl Bopp, Michele Parsons (Centers for Disease Control and Prevention) • Michele Burgess, Marissa Mullins (EPA, Office of Emergency Management) • Rita Colwell, Anwar Huq (Maryland Pathogen Research Institute, University of Maryland) • Stephanie Harris (EPA, Region 10) • Malik Raynor (EPA, Office of Water) • Gene Rice (EPA, NHSRC)

Subject Matter Experts • Steve Weagant (U.S. Food and Drug Administration) • Nancy Hall (University of Iowa Hygienic Laboratory)

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

i

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. Note that approval does not signify that the contents necessarily reflect the views of the Agency. Mention of trade names, products, or services does not convey EPA approval, endorsement, or recommendation. Questions concerning this document or its application should be addressed to:

Sanjiv R. Shah National Homeland Security Research Center U.S. Environmental Protection Agency 1200 Pennsylvania Avenue, NW USEPA-8801RR Washington, DC 20460 (202) 564-9522 [email protected]

ii

http://nhsrc.intra.epa.gov/Lists/Documents%20and%20Settings/Msinclai/Local%20Settings/Temp/notesFCBCEE/[email protected]

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 verification of procedures included in the draft “Standard Analytical Protocol for Vibrio cholerae O1 and O139 in Drinking Water and Surface Water.” NHSRC has made this publication available to assist in preparing for and recovering from disasters involving V. cholerae 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

iii

Table of Contents Acknowledgments.......................................................................................................................... i

Disclaimer ..................................................................................................................................... ii

Foreword.......................................................................................................................................iii

Table of Contents ........................................................................................................................ iv

Tables .......................................................................................................................................... vi

Acronyms.....................................................................................................................................vii

Executive Summary....................................................................................................................viii

Section 1.0: Background...............................................................................................................1

Section 2.0: Study Objectives and Design....................................................................................2 2.1 – Study Preparation..........................................................................................................2 2.2 – Sample Matrices............................................................................................................3 2.3 – Sample Analyses...........................................................................................................3 2.4 – Quality Control Analyses ...............................................................................................4

Section 3.0: Study Schedule.........................................................................................................5

Section 4.0: Data Reporting, Validation, and Censoring...............................................................6 4.1 – Data Reporting ..............................................................................................................6 4.2 – Data Validation ..............................................................................................................6 4.3 – Censored Data ..............................................................................................................6

Section 5.0: Results ......................................................................................................................7 5.1 – Phase 2: Preliminary Analyses......................................................................................7 5.2 – Phase 3: Assessment of Procedure Modification..........................................................7 5.3 – Phase 4: Verification Analyses......................................................................................7

Section 6.0: Results and Discussion...........................................................................................10

Section 7.0: Conclusion ..............................................................................................................11

Section 8.0: References..............................................................................................................12

iv

Table of Contents (cont.) 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

v

Tables Table 1. Summary of Sample Analyses........................................................................................3

Table 2. Schedule for Verification of V. cholerae O1/O139 Culture-Based Procedures in Water Matrices ........................................................................................................................................5

Table 3. Verification Analyses, Summary Results: PBS, Drinking Water, and Surface Water Spiked with V. cholerae O1 or O139 (Conducted the Week of September 28, 2009) ..................8

Table 4. Verification Analyses, Summary Results: Surface Water Spiked with V. cholerae O1 or O139 (Conducted the Week of October 18, 2009) .......................................................................9

vi

Acronyms APW Alkaline peptone water

ASTM American Society for Testing and Materials

ATCC® American Type Culture Collection

°C Degrees Celsius

CDC Centers for Disease Control and Prevention

CFU Colony forming unit

CSC Computer Sciences Corporation

CVD Center for Vaccine Development

EMMC Environmental Monitoring Management Council

EPA U.S. Environmental Protection Agency

MPN Most probable number

MTA Material Transfer Agreement

NHSRC National Homeland Security Research Center

OEM Office of Emergency Management

OGWDW Office of Ground Water and Drinking Water

ORD Office of Research and Development

OW Office of Water

PBS Phosphate buffered saline

PSI Pounds per square inch

QA Quality assurance

QAPP Quality assurance project plan

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

SME Subject matter expert

TCBS Thiosulfate citrate bile salts sucrose (agar)

TNTC Too numerous to count

TSA Tryptic soy agar

TSB Tryptic soy broth

VBNC Viable but not culturable

vii

viii

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 V. cholerae O1 and O139 in drinking water and surface water samples. The procedure evaluated during the Study was adapted from the Centers for Disease Control and Prevention’s Laboratory Methods for the Diagnosis of Epidemic Dysentery and Cholera and the U.S. Food and Drug Administration Bacteriological Analytical Manual [online], Chapter 9, Vibrio. 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. The laboratory spiked samples each week during the Study using attenuated (vaccine) strains of V. cholerae O1 (JBK 70) or V. cholerae O139 (CVD 112) from the University of Maryland, School of Medicine, Center for Vaccine Development. Vaccine strains were used to reduce health risk to laboratory personnel. To assess potential surface water matrix issues, four different surface water samples were analyzed. The Study was conducted during June 2009 through October 2009. During the Study some issues were observed, including (1) variable performance associated with strains and matrices (e.g., recovery differences between serotype/matrix combinations) and (2) potential for viable but not culturable (VBNC) state due to low sample temperatures (e.g., poor recoveries when samples and medium were not warmed prior to spiking and inoculation). To address the strain and matrix issues observed during the Study, extended alkaline peptone water (APW) incubation (24 ± 2 hours) was evaluated. To reduce the potential for spikes to become VBNC due to thermal shock, samples and APW were put in a 35°C incubator for 15 minutes prior to sample analyses. With the exception of V. cholerae O139 in surface water samples, which had mean site-specific recoveries ranging from 89% to 119% after only 6 – 8 hours incubation in APW, it appears that some V. cholerae strains may require 24 ± 2 hours incubation in APW for some drinking water and surface water matrices. In drinking water, mean recoveries were 85% for O1 and 163% for O139 with 24 ± 2 hours selective enrichment in APW. For surface water, mean recoveries ranged from 14% to 26% and from 53% to 152% for V. cholerae O1 after 6 – 8 and 24 ± 2 hours incubation in APW, respectively. In PBS, mean recoveries ranged from 122% to 139% when plated onto TCBS after 6 – 8 hours incubation in APW and 131% to 139% when plated on TCBS after 24 ± 2 hours. Results of the single-laboratory validation study indicate that these procedures merit multi-laboratory validation to assess performance, evaluate workgroup recommendations (e.g., evaluate the SAP using a panel of different V. cholerae O1 and O139), and develop quantitative QC criteria.

Section 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 Vibrio cholerae (V. cholerae) O1 and O139, a potential bioterrorism agent, in environmental samples. Cholera has been chronicled as a disease in ancient Greek and Hebrew documents, with the causative agent first described by Filippo Pacini in 1854. The microorganism produces an intermittent watery diarrhea via expression of the cholera toxin gene, which is present in more than 95% of isolates confirmed as either serotype O1 or O139. In modern times, these two serotypes have been responsible for up to eight cholera pandemics. Non-O1 and non-O139 serotypes, which are those predominantly found in estuarine and other environmental samples, generally do not produce disease (Reference 8.1). This document presents results of the single-laboratory verification study (Study) to evaluate the draft Standard Analytical Protocol (SAP), a culture-based procedure for the identification and quantitation of V. cholerae O1 and O139 in water samples (Reference 8.2). The procedure evaluated during the Study was adapted from the Centers for Disease Control and Prevention’s Laboratory Methods for the Diagnosis of Epidemic Dysentery and Cholera (Reference 8.1) and the U.S. Food and Drug Administration Bacteriological Analytical Manual [online], Chapter 9, Vibrio (Reference 8.3). The culture-based procedure that was verified during the Study was identical to the procedure in the draft SAP, with the exception of an

extended alkaline peptone water (APW) incubation (24 ± 2 hours) option for problematic water matrices. The procedure evaluated and verified during the study included the following: (1) enriching in APW for 6 – 8 hours or 24 ± 2 hours at 36.0°C ± 1.0°C, (2) streaking growth from APW for isolation onto thiosulfate citrate bile salts sucrose (TCBS) agar plates and incubating for 24 ± 2 hours at 36.0°C ± 1.0°C, (3) sub-culturing onto tryptic soy agar (TSA), and (4) verifying isolates using serological and biochemical tests. Time to results as verified is approximately 72 – 86 hours or 88 – 104 hours from receipt of samples, for 6 – 8 or 24 ± 2 hours APW incubation, respectively. Although initial incubation of samples in APW was for 6 – 8 hours, it was determined during the Study that it may be necessary to increase the incubation of APW to 24 ± 2 hours at 36°C ± 1.0°C for some sample types (e.g., drinking water) and target analytes (e.g., V. cholerae O1). Quantitation of V. cholerae was determined using the most probable number (MPN) technique. Tubes that confirmed positive for V. cholerae O1 or O139 were used to determine 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. This can result in highly variable results (Reference 8.5). Although the draft SAP includes a standard 15-tube MPN, based on workgroup discussion, a 9-tube MPN was utilized for the Study to reduce the burden (e.g., work hours required) on the participant laboratory. The use of the 9-tube tube MPN versus a 15-tube MPN reduced the number of tubes from 795 to 475 and the number of plates from 1590 to 795. A 15-tube MPN may be considered during multi-laboratory validation.

1

Section 2.0 Study Objectives and Design The primary objective of this Study was to verify the culture-based procedure included in the draft SAP for identification and quantitation of V. cholerae O1 and O139. As indicated in the Study Plan (Appendix A), this study was originally designed to verify the procedure in water, solid, and particulate matrices. However, during this Study, only water matrices were evaluated and included in this report. Additional matrices (e.g., soil) may be evaluated in the future. In support of the primary objective of evaluating the draft SAP, both study objectives and data quality objectives were set for the Study (Appendix A).

Study Objectives • Evaluate draft SAP performance (recovery

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

• Evaluate draft SAP performance (recovery and precision) in environmental matrices of interest (drinking water, surface water)

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

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

• Phase 1. Planning and Preparation: Identification of a qualified laboratory to participate in the Study and evaluation of the spiking protocol (Appendix C)

• Phase 2. Preliminary Analyses: Assessment of original draft SAP in three matrices (PBS [reference matrix], drinking water, surface water), and identification of analytical problems

• Phase 3. Assessment of Procedure Modification: After consultation with subject matter experts (SMEs), evaluation of extended selective enrichment incubation (APW for 24 ± 2 hours) for PBS, drinking water, and surface water samples

• Phase 4. Verification Analyses: Analyses of PBS, drinking water, and surface water samples

Data Quality Objective Data 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.

2.1 – Study Preparation Identification of an appropriate laboratory and development and evaluation of a Study-specific spiking protocol (Appendix C) were completed prior to the study.

2.1.1 – Identification of Laboratory A laboratory was identified that was (1) representative of the general user community, (2) had experience analyzing environmental samples for Vibrio spp., and (3) 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, the 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.

2.1.2 – Preparation of Spiking Suspensions

The Study Plan (Appendix A) included the use of two spike types: BioBalls™ and laboratory-prepared spiking suspensions. However, due to production difficulties, BioBalls were not used during the Study. In addition, subsequent to Study Plan development, the use of flow cytometry sorted and enumerated spikes from Wisconsin State Laboratory of Hygiene was considered as an alternative to laboratory-prepared spikes. However, due to issues including variability of spike levels from run to

2

3

run, potential difficulty recovering cells from tubes, and stability of spikes, the workgroup decided not to pursue the use of flow-sorted spikes. In addition, there was concern that if V. cholerae spikes were shipped on ice, then cells may enter a viable but not culturable (VBNC) state due to the low temperatures (Reference 8.6); if shipped at ambient temperatures cells may replicate and increase spike levels. As a result, only laboratory-prepared spiking suspensions were used.

Laboratory-prepared Spikes During each week of the Study, laboratory-prepared spiking suspensions were propagated according to the spiking protocol (Appendix C) using attenuated (vaccine) strains of V. cholerae O1 (JBK 70) or V. cholerae O139 (CVD 112) from the Center for Vaccine Development (CVD) (University of Maryland, School of Medicine) to reduce health risk to laboratory personnel. Cultures were propagated in 1% APW (1 mL APW in 99 mL PBS) and incubated at 36.0°C ± 1.0°C for 18 – 24 hours. These suspensions were serially diluted and used to spike samples. The laboratory enumerated spiking suspensions on the same day that samples were spiked and analyzed using the spread plate technique (in triplicate) on TSA according to the spiking protocol.

2.2 – Sample Matrices Three water matrices (one reference and two matrices of interest) were analyzed to provide a

means for assessing the performance of the draft SAP. During the Study, PBS served as the reference matrix. The reference matrix served as a standard matrix that could be duplicated during routine use of the method. The following water matrices were evaluated 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)

2.3 – Sample Analyses Unspiked samples of sterile PBS, drinking water, and surface water, were analyzed during each phase, to determine background V. cholerae O1 or O139 concentrations. For preliminary analyses (Phase 2), a single unspiked and spiked sample was analyzed per matrix and strain. Results of the preliminary analyses were used to identify issues. Additional analyses were conducted to assess a procedure modification (extended incubation of APW) during Phase 3. During Phase 4, analyses were conducted to generate data to assess procedure performance. Table 1 summarizes the number and type of samples that were evaluated to meet the objectives listed in Section 2.

Table 1. Summary of Sample Analyses

Analysis Matrix Spiking Description (1) Procedure/Treatment No. of

Analyses Phase 2 Analyses

Method Blank O1 1 Method Blank O139 1 V. cholerae O1 1

Sterile PBS (Phosphate buffered saline) (Reference Matrix) V. cholerae O139 1

Unspiked O1 1 Unspiked O139 1 V. cholerae O1 1

Drinking Water

V. cholerae O139 1 Unspiked O1 1 Unspiked O139 1 V. cholerae O1 1

Preliminary Analyses

Surface Water

V. cholerae O139

Original SAP (Section 1.0) Selective enrichment in APW for 6 – 8 hours at 36.0°C ± 1.0°C

1

4

Analysis Matrix Spiking Description (1) Procedure/Treatment No. of

Analyses Phase 3 Analyses


Sterile PBS (Reference Matrix)


Drinking Water


Assessment of Procedure Modification

Surface Water

V. cholerae O139

Evaluation of extended incubation in APW at 36.0°C ± 1.0°C for 6 – 8 and/or 24 ± 2 hours (2), (3)

4 Phase 4 Analyses


Sterile PBS (Reference Matrix)


Drinking Water

V. cholerae O139 4 Unspiked O1 2 Unspiked O139 (4) 2 V. cholerae O1 4

Verification Analyses

Surface Water

V. cholerae O139 (4)

Incubation in APW at 36.0°C ± 1.0°C for 6 – 8 and/or 24 ± 2 hours (2), (3)

4 (1) “Unspiked O1” and “Unspiked O139” refer to unspiked samples analyzed for O1 and 0139 background organisms, respectively (2) For surface water and PBS samples analyzed for V. cholerae O1, growth from APW (alkaline peptone water) tubes was plated onto TCBS (thiosulfate citrate bile salts sucrose) agar at both 6 – 8 hours and 24 ± 2 hours (3) For drinking water samples growth from APW tubes was plated onto TCBS only at 24 ± 2 hours (4) For repeat analyses of surface water samples analyzed for V. cholerae O139, growth from APW tubes was plated onto TCBS after 6 – 8 hours of incubation; extended incubation was unnecessary (see Table 4)

2.4 – 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 36°C ± 1.0°C (PBS, APW, TCBS, and TSA) for 24 ± 2 hours and observed for growth. In addition, sterility checks were conducted each day samples were analyzed.

• Positive and Negative Controls: For the purpose of the Study, positive controls for

selective agars and broths are those organisms that produce the characteristic growth and/or colony morphology of the target organism. Negative controls are those organisms that do not produce the characteristic target organism growth or morphology. For biochemical and serological analyses, positive and negative controls are defined by their reaction (e.g., V. cholerae is oxidase positive and E. coli is oxidase negative). The following positive and negative controls were evaluated during each week of the Study:

• V. cholerae O1 (JBK 70) and V. cholerae O139 (CVD 112): Positive controls (target organisms)

• E. coli (ATCC® 25922™): Negative control (non-target organism)

Section 3.0 Study Schedule

The duration of the Study was March 2009 through October 2009. Development and evaluation of the modified procedure during Phase 3 (extended incubation in APW [24 ± 2 hours] for both O1 and O139 strains and in all matrices [PBS, drinking water, surface water])

increased the study time frame by approximately two months. The Study schedule is provided in Table 2. Analyses of additional matrices may be conducted at a later date.

Table 2. Schedule for Verification of V. cholerae O1/O139 Culture-Based Procedures in Water

Matrices Date Analysis Phase

March – April 2009 Phase 1 – Spiking Protocol Evaluation

June – July 2009 Phase 2 – Preliminary Analyses

August – September 2009 Phase 3 – Assessment of Procedure Modification: Extended APW(1) Incubation

September – October 2009 Phase 4 – Verification Analyses (1)APW, alkaline peptone water

5

Section 4.0 Data Reporting, Validation, and Censoring

4.1 – Data Reporting The 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 (Appendix D) • Documentation of any additional

information that would assist in evaluating the data

4.2 – Data Validation CSC used data review checklists to ensure that each data package was complete and that each sample result met the Study-specific and draft SAP-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

analyzed and they exhibited the appropriate response

• Samples were spiked with the appropriate dilution

• All procedures were performed according to Study-specific instructions (Appendix B)

• 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 were compared to verify consistency and identify potential data entry errors.

4.3 – Censored Data In 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 V. cholerae O1 or O139). The censoring limit of “less than” value (1.081) was used in data analysis for these samples. In addition to low censored values, a single high censored (or “greater than”) result was observed for one PBS sample. The censoring limit of the “greater than” value (e.g., >109.9) was used for data analysis.

6

Section 5.0 Results

This section is organized by Study phase: Phase 2, preliminary analyses (Section 5.1); Phase 3, assessment of procedure modification (Section 5.2); and Phase 4, verification analyses (Section 5.3). A summary of the number and type of sample analyses is included in Section 2.3. Only valid results are included in this section. All data generated during verification analyses were considered valid, and are therefore included in this section.

5.1 – Phase 2: Preliminary Analyses During Phase 2, V. cholerae O1 and O139 recoveries for all three matrices (PBS, drinking water, surface water) were low (<30%) using the original procedure listed in the draft SAP (selective enrichment in APW for 6 – 8 hours, plating on TCBS, followed by serological and biochemical confirmation). During these analyses, the laboratory reported that for spiked drinking water samples no growth was observed in APW tubes incubated for 6 – 8 hours. However, after being left on the bench overnight these tubes exhibited growth and were subsequently confirmed by biochemical and serological analyses as V. cholerae O1 or O139. Due to low recoveries observed for selective enrichment in APW incubated for 6 – 8 hours for all three matrices and observations provided by the laboratory, it was determined by the workgroup that evaluation of the modified procedure utilizing extended selective enrichment incubation (24 ± 2 hours in APW) should be conducted (Phase 3).

5.2 – Phase 3: Assessment of Procedure Modification The laboratory analyzed PBS and surface water samples after incubation in APW at 6 – 8 hours, and then at 24 ± 2 hours. However, drinking water samples were only evaluated after incubation in APW for 24 ± 2 hours because the laboratory did not observe growth after 6 – 8

hour incubation in APW during preliminary analyses of samples from multiple drinking water sources. Analyses with the modified procedure indicated that extended selective enrichment (24 ± 2 hours in APW) did increase V. cholerae O1 and O139 recoveries for drinking water samples (99% and 56%, respectively), as compared to preliminary analyses (<10%). Surface water sample results were variable with regard to APW incubation period, indicating that either incubation length may be suitable for samples from this matrix. Based on these results, the laboratory proceeded with verification analyses, as described in Section 5.3, below.

5.3 – Phase 4: Verification Analyses Verification analyses conducted by the laboratory during the week of September 28, 2009 included analyses of unspiked and spiked PBS, drinking water, and surface water samples. For drinking water samples, APW was incubated for 24 ± 2 hours prior to streaking growth onto TCBS plates. For PBS and surface water samples, growth from APW tubes was transferred both at 6 – 8 hours and 24 ± 2 hours to compare recoveries. Recoveries were only slightly increased for PBS samples between 6 – 8 and 24 ± 2 hours. Recoveries of V. cholerae O1 and O139 in surface water samples were poor at both 6 – 8 and 24 ± 2 hours. Due to the low recoveries in surface water samples (<15%), the laboratory repeated surface water analyses on October 18, 2009 using samples collected from four different sites (discussed below). Results of the September 28, 2009 study analyses are provided in Table 3. As indicated in Section 4.3, all unspiked samples were negative for V. cholerae O1 or O139.

7

Table 3. Verification Analyses, Summary Results: PBS, Drinking Water, and Surface Water Spiked with V. cholerae O1 or O139 (Conducted the Week of September 28, 2009 (1))

Analyte n Spike Level (CFU/100 mL)

Selective Enrichment in

APW

Mean Recovery

(%)

Minimum Recovery

(%)

Maximum Recovery

(%) SD (%)

RSD (%)

Phosphate Buffered Saline (PBS)

O1 4 6 hours 138.72 63.61 302.28 112.86 81.36

O1 4 36

24 hours 138.72 63.61 302.28 112.86 81.36

O139 4 6 hours 122.22 34.11 320.06 132.82 108.68

O139 4 34

24 hours 130.53 67.35 320.06 126.35 96.80

Drinking Water

O1 4 36 24 hours 84.91 25.25 125.39 49.30 58.06

O139 4 34 24 hours 163.23 67.35 320.06 109.00 66.78

Surface Water

O1 4 6 hours 0.00 0.00 0.00 0.00 NA

O1 4 36

24 hours 0.16 0.00 0.49 0.23 142.44

O139 4 6 hours 8.53 0.00 34.12 17.06 200.00

O139 4 34

24 hours 14.19 0.00 34.12 17.77 125.21 (1) For all unspiked samples the "<" values were replaced with the censoring limit (e.g., <1.081 was replaced with 1.081) for calculation of summary statistics APW – Alkaline peptone water PBS – Phosphate buffered saline CFU/100 mL – Colony forming unit per 100 milliliter RSD – Relative standard deviation n – Number of replicates SD – Standard deviation NA – Not applicable To address recovery issues, the laboratory was asked to repeat the surface water analyses with minor procedure modifications during the week of October 18, 2009. To reduce the potential for the temperature-sensitive spikes to become VBNC, repeat surface water analyses included warming the samples and medium (APW) in a 35°C incubator for 15 minutes prior to sample analyses (spiking and inoculation of APW). V. cholerae O1 analyses were conducted using surface water samples obtained from Sites 1(Port Huron, Michigan) and 2 (Newark, Ohio) and V. cholerae O139 analyses were conducted using surface water from Sites 3 (City of Redford, Michigan) and 4 (Eden, North Carolina). All samples were obtained as source waters for drinking water treatment plants. In

addition, the spike level was increased slightly and two different surface water samples were analyzed per analyte. For V. cholerae O1 repeat analyses, growth from the APW tubes was transferred at 6 – 8 and again at 24 ± 2 hours. For serotype O139, growth was transferred only at 6 – 8 hours, because the number of positive tubes (based on typical colonies on TCBS) at that point was consistent with recoveries of >80%. These repeat analyses resulted in improved recoveries. Summary results from October 18, 2009 analyses are provided in Table 4. As indicated in Section 4.3, all unspiked samples were negative for V. cholerae O1 or O139.

8

Table 4. Verification Analyses, Summary Results: Surface Water Spiked with V. cholerae O1 or

O139 (Conducted the Week of October 18, 2009 (1))

Analyte n Sample Location

Spike Level (CFU/100 mL)

Selective Enrichment

in APW

Mean Recovery

(%)

Minimum Recovery

(%)

Maximum Recovery

(%) SD (%)

RSD (%)

Phosphate Buffered Saline

O1 1 NA 129 6 hours 57.20 57.20 57.20 NA NA O139 1 NA 253 6 hours 94.18 94.18 94.18 NA NA

Surface Water

O1 4 129 6 hours 25.52 16.26 30.79 6.36 24.94

O1 4 Site 1

129 24 hours 152.33 69.97 355.93 137.27 90.11

O1 4 129 6 hours 13.53 4.89 30.79 12.21 90.27

O1 4 Site 2

129 24 hours 52.86 13.99 113.47 48.26 91.31

O139 4 Site 3 253 6 hours 88.62 35.62 181.17 66.75 75.32

O139 4 Site 4 253 6 hours 119.46 57.76 181.17 71.25 59.64 (1) For all unspiked samples the "<" values were replaced with the censoring limit (e.g., <1.081 was replaced with 1.081) for calculation of summary statistics APW – Alkaline peptone water PBS – Phosphate buffered saline CFU/100 mL – Colony forming unit per 100 milliliter RSD – Relative standard deviation n – Number of replicates SD – Standard deviation NA – Not applicable Results of the verification analyses indicate that serotype O139 may grow more rapidly than O1 in APW, thus not requiring overnight incubation. Warming media and samples to room temperature prior to inoculation may also have contributed to acceptable recoveries for V. cholerae O139 at 6 – 8 hours and V. cholerae

O1 at 24 ± 2 hours, in comparison to the results observed during the week of September 28, 2009. Results indicate that 24 ± 2 hours selective enrichment in APW may be required for some drinking water matrices.

9

Section 6.0 Results and Discussion The culture-based procedure in the draft SAP was evaluated for identification and quantitation of V. cholerae O1 and O139 in water matrices. The procedure required modification for some sample types and target strains. As indicated in Section 2, solid and particulate matrices were not evaluated during this phase of the Study. With the exception of V. cholerae O139 in surface water samples, which had mean site-specific recoveries ranging from 89% to 119% after only 6 – 8 hours incubation in APW, it appears that some V. cholerae strains may require 24 ± 2 hours incubation in APW for some drinking water (O1 and O139) and surface water matrices (O1). In drinking water, mean recoveries were 85% for O1 and 163% for O139 with 24 ± 2 hours selective enrichment in APW (Table 3). For surface water, mean recoveries ranged from 14% to 26% and from 53% to 152% for V. cholerae O1 after 6 – 8 and 24 ± 2 hours incubation in APW, respectively (Table 4). Again, it should be noted that the surface water sample results provided in Table 4 were from samples and APW that were put in a 35°C incubator for 15 minutes prior to sample analyses (spiking and inoculation of APW) to reduce the potential for spikes to become VBNC due to thermal shock. In PBS, mean recoveries ranged from 122% to 139% when plated onto TCBS after 6 – 8 hours incubation in APW and 131% to 139% when plated on TCBS after 24 ± 2 hours (Table 3). While either incubation length provides acceptable results, it should be noted that PBS samples should be incubated for the same length of time that samples from the matrix of interest are incubated, since PBS samples generally serve as quality controls. Thus, if all matrix samples are being incubated for a full 24 ± 2 hours in APW; PBS samples would not need to

be processed after 6 – 8 hours incubation (and vice versa, if all matrix samples are only incubated for 6 – 8 hours). However, if samples from the matrix of interest are being processed at both 6 – 8 and 24 ± 2 hours, then PBS samples should also be processed at both times. During the Study some issues were observed that could impact implementation of the SAP on a national scale, including variable performance associated with strains and matrices (e.g., recovery differences between serotype/matrix combinations) and potential for VBNC state due to low sample temperatures (e.g., poor recoveries when samples and APW were not warmed prior to spiking and inoculation). To address the strain and matrix issues observed during the Study, the workgroup recommended an extended APW incubation (24 ± 2 hours). The extended APW incubation was verified and incorporated, as an option, into the draft SAP. To minimize the potential of V. cholerae going into a VBNC state due to exposure to low temperatures, the following recommendations were also incorporated into the draft SAP: (1) samples should not be transported on ice or stored at <10°C and (2) APW should be brought to 36.0°C ± 1.0°C prior to inoculation. In addition, to enhance the recovery for samples at ≤15°C when collected, it was recommended by the workgroup that APW tubes be incubated for 24 ± 2 hours, instead of 6 – 8 hours. Failure to implement these workgroup recommendations could result in false negative results. In addition, while not evaluated during this study, workgroup members also noted that water quality parameters (e.g., pH) could impact recoveries. The workgroup recommended that water quality data (e.g., pH) be collected prior to sample analyses.

10

Section 7.0 Conclusion

The original draft SAP, which included incubation of APW for 6 – 8 hours, provided results that the workgroup deemed acceptable for the identification and quantitation of V. cholerae O139 in surface water samples; and the modified procedure (extended [24 ± 2 hours] APW incubation) provided acceptable results for identification and quantitation of V. cholerae O1 and O139 in drinking water and V. cholerae O1 in surface water. These procedures merit multi-laboratory validation to assess performance, evaluate workgroup recommendations, and set quantitative QC criteria. The workgroup also

recommended that the SAP be evaluated using a panel of different V. cholerae O1 and O139 strains. It should also be noted that the draft SAP has been revised based on the Study to include an extended APW incubation (24 ± 2 hours) option and the following recommendations , (1) samples should not be transported on ice or stored at <10°C, and (2) APW should be brought to 36.0°C ± 1.0°C prior to inoculation.

11

Section 8.0 References 8.1 National Center for Infectious Diseases and Centers for Disease Control and Prevention. 1999.

Isolation and Identification of Vibrio cholerae Serogroups O1 and O139. In Laboratory Methods for the Diagnosis of Epidemic Dysentery and Cholera. J.P. Koplan, J.M. Hughes, M.L. Cohen, E.B. Samba, and A.B. Kabore (ed.), (pp. 41 – 54). Atlanta, Georgia: National Center for Infectious Disease and Centers for Disease Control and Prevention. http://www.cdc.gov/ncidod/DBMD/diseaseinfo/cholera_lab_manual.htm (accessed November 20, 2009)

8.2 U.S. Environmental Protection Agency. Standard Analytical Protocol for Vibrio cholerae O1 and

O139 in Drinking Water and Surface Water. Publication forthcoming; date and number to be determined.

8.3 Kaysner C.A. and Depaola, A. Jr. May 2004. Vibrio. Chapter 9 of Bacteriological Analytical Manual [online]. G.J. Jackson, R.I. Merker, and R. Bandler (eds.). U.S. Food and Drug Administration. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070830.htm

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. 8.5 Francy, D.S., Bushon, R.N., Brady, A.M.G., Bertke, E.E., Kephart, C.M., Likirdopulos, C.A.,

Mailot, B.E., Schaefer, F.W. III, and Lindquest, H.D.A. 2009. Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water. U.S. Geological Survey Scientific Investigations Report 2009–5097. http://pubs.usgs.gov/sir/2009/5097/

8.6 Huq, A., Grim, C., Colwell, R., and Nair, G.B. September 2006. “Detection, Isolation, and

Identification of Vibrio cholerae from the Environment.” Current Protocols in Microbiology, Unit 6A.5.

12

http://www.cdc.gov/ncidod/DBMD/diseaseinfo/cholera_lab_manual.htm

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070830.htm

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070830.htm

http://pubs.usgs.gov/sir/2009/5097/

Appendix A

Study Plan

Study Plan for Single Laboratory Verification

of the Standardized Analytical Procedure for

Vibrio cholerae O1 and O139 in Environmental Samples

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

December 30, 2008

Study Plan December 30, 2008

A – ii

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.

http://www.epa.gov/ttn/emc/guidlnd/gd-045.pdf�


A – iii

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 ........................................................................................................................... 5 3.1 STUDY OBJECTIVES ........................................................................................................ 5 3.2 DATA QUALITY OBJECTIVES .......................................................................................... 6

4.0 STUDY IMPLEMENTATION AND TECHNICAL APPROACH ......................................... 6 4.1 PHASE 1 – IDENTIFICATION OF QUALIFIED ANALYTICAL LABORATORY ....................... 6 4.2 PHASE 2 – PREPARATION OF SPIKES ............................................................................... 7 4.3 PHASE 3 – SAMPLE ANALYSIS ........................................................................................ 7

4.3.1 Preliminary Analyses ................................................................................... 7

4.3.2 Assessment of Single Laboratory Method Precision and Recovery ............ 7

4.3.3 Quality Control (QC) Analyses .................................................................... 7

4.4 STUDY SUMMARY ........................................................................................................... 7

5.0 REPORTING AND VALIDATION OF STUDY RESULTS ................................................ 10

6.0 DATA ANALYSIS ................................................................................................................. 10

7.0 LIMITATIONS ....................................................................................................................... 10

Study Plan A – 1 December 30, 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 Vibrio cholerae O1 and O139 as EPA target pathogens during environmental remediation following a homeland security incident. This study plan describes the single-laboratory verification of the draft Standardized Analytical Procedure for Vibrio cholerae O1 and O139 in Environmental Samples (SAP) for determination and quantification of Vibrio cholerae (V. cholerae) O1 and O139 using selective and non-selective media followed by biochemical and serological characterization. The draft SAP for V. cholerae O1 and O139 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, dust-sampling sock with Arizona Test Dust, swab with local particulate, wipe with local particulate, dust-sampling sock with local particulate). Vaccine strains of V. cholerae O1 and O139 (JBK70 and CVD112, respectively) 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 the contractor 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. CSC will be responsible for study 1 Centers for Disease Control and Prevention. 1999. Laboratory Methods for the Diagnosis of Epidemic Dysentery and Cholera.

Atlanta, Georgia. 2Kaysner, C.A. and Depaola, A. Jr. May 2004. Vibrio. Chapter 9 of Bacteriological Analytical Manual [Online],. U.S. Food and Drug Administration. 3 U.S. Environmantal Protection Agency, Office of Water. July 2006. Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, , U.S. Environmental Protection Agency. 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. Infect. Dis. 10(6): 1023 – 1029.


A – 2

coordination, including but not limited to, development of study-specific instructions and data reporting forms, procurement of supplies and spiking materials (V. cholerae vaccine strains), and technical support. 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. 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 • 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 • Receive, compile, maintain, review, validate, 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 V. cholerae vaccine strains

• 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 analytical results to CSC study

coordinator


A – 3

Figure 1: Flowchart of Study Activities


A – 4

2.2 Study Schedule The study schedule is dependent on receipt of bacterial spiking standards, media, and reagents by the participating laboratory. The following tentative study schedule (Table 2) is provided under the assumption that these materials will be received by laboratories no later than the end of January 2009. This schedule also assumes that the laboratory will participate in several periodic conference calls with study workgroup and CSC throughout the study. For this study, V. cholerae O1 and O139 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 and/or Salmonella Typhi single laboratory verification studies: 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 and/or Salmonella Typhi single laboratory verification studies: swab, wipe, and dust-sampling socks 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 and/or Salmonella Typhi single laboratory verification studies: swab, wipe, and dust-sampling socks with Arizona Test Dust. Ideally, matrices would be reduced, pending other study results. Table 2. Tentative Schedule for Single Laboratory Verification of Draft Procedures for V. cholerae O1 and O139 in Water, Solid, and Particulate Matrices

Month Activity

Planning and Preparation

November – December 2008

• Draft study plan • Identify and recruit laboratory • Identify and recruit workgroup participants • Initiate Materials Transfer Agreement (MTA) with CVD

January – February 2009

• Revise and finalize study plan based on workgroup comments • Draft study-specific instructions, spiking protocol, data reporting forms

(focus on water) • Revise study-specific instructions, spiking protocol and data reporting

forms for water based on workgroup comments • Procure supplies, reagents, and media

March – April 2009

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

• Revise study-specific instructions, spiking protocol, and data reporting forms for solids based on workgroup comments

June - August 2009

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

• Revise study-specific instructions, spiking protocol, and data reporting forms for particulates base on workgroup comments

Water (PBS, Surface Water, Drinking Water)

January – April 2009

• Verify spiking protocol for V. cholerae O1 and O139 vaccine strains • Revise spiking protocol, as necessary • Conduct preliminary analysis of V. cholerae O1 and O139 (SAP

evaluation) at laboratory (~ 2 weeks) • Review data / resolve issues (~ 2 weeks) • Update study-specific instructions and other study documentation, as

necessary • Conduct study analyses of V. cholerae O1 and O139 (SAP evaluation) at


A – 5

laboratory (~ 3 weeks) and revise procedures, as necessary Solids (Milorganite®, ASTM Clay, ASTM Sand, Local Soil)

April – May 2009

• Conduct preliminary analysis of V. cholerae O1 and O139 (SAP evaluation) at laboratory (~ 4 weeks)

• Review data / resolve issues (~ 2 weeks) • Update study-specific instructions and other study documentation, as

necessary

June – July 2009

• Conduct study analyses of V. cholerae O1 (SAP evaluation) at laboratory (~ 3 weeks)


Particulates (Swabs, Wipes, Dust-sampling Socks; Each with Arizona Test Dust and a Local Particulate)

August – September 2009

• Conduct preliminary analysis of V. cholerae O1 and O139 (SAP evaluation) at laboratory (~ 4 weeks)

• Review data / resolve issues (~ 2 weeks) • Update study-specific instructions and other study documentation, as

necessary

October – November 2009



Study Report and SAP Revisions

December 2009 • Review data • Prepare condensed study report • Revise SAP to reflect study results

3.0 OBJECTIVES The primary objective of this study is to evaluate the procedures included in the draft SAP (Appendix A), for identification and enumeration of V. cholerae O1 and O139 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®, ASTM Sand, and ASTM Clay], and particulate [swabs, wipes, and dust-sampling socks 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 the study workgroup and CSC before making any modifications to the

procedures described in the SAP, and provide a written description of the modification(s)


A – 6

• 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, under development) and SAP (Appendix A). This will ensure data integrity and validity for all matrices evaluated and allows 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, under

development). 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 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 V. cholerae O1. A second identical set of samples will be analyzed for V. cholerae O139 (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 V. cholerae. 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, under development) and the draft SAP (Appendix A).

This section provides a summary of the activities that will be performed during the study. Detailed study-specific instructions (Appendix B, under development) 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.


A – 7

4.2 Phase 2 – Preparation of Spikes The study will use laboratory-prepared V. cholerae O1 and O139 spiking suspensions to evaluate method performance. 4.2.1 Laboratory-Prepared Spiking Suspensions

Laboratory-prepared spiking suspensions will be prepared from V. cholerae O1 and O139 (strains JBK70 and CVD112, respectively) according to the spiking protocol (Appendix C, under development). A stock culture will be propagated in 20% tryptic soy broth (TSB) with 3% NaCl and incubated at 35°C ± 5°C for 20 ± 4 hours. Serially diluted spiking suspensions (~50 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).

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, under development). Sample matrices will be prepared as described in Appendix B. V. cholerae 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 using laboratory-prepared spikes approximately 1 – 2 weeks prior to start of the study analyses. The time between preliminary 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 V. cholerae O1 and O139 spikes. 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. V. cholerae O1 and O139 (strains JBK70 and CVD112, respectively) will serve as the positive control and Escherichia 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.


A – 8

Table 3. Summary of Sample Analyses

Matrix Spiking Description

No. of Samples

Purpose of Analysis Preliminary Analyses

Study Analyses

Water (Drinking Water and Liquid / Water Samples)

Sterile PBS (Reference

Matrix)

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

V. cholerae O1 1 4 Confirmation that approach is appropriate for the multi-laboratory validation study V. cholerae O139 1 4

Surface Water

Unspiked 1 2 Evaluation of background V. cholerae concentrations


Drinking Water



Solids (Soil, Powder Samples)

Milorganite® (Reference Matrix #1)



ASTM Lean Clay (Reference

Matrix #2)



ASTM Sand (Reference Matrix #3)



Local Soil




A – 9

Matrix Spiking Description

No. of Samples

Purpose of Analysis Preliminary Analyses

Study Analyses

Particulates (Swabs, Wipes, Filters)

Swab With Reference

Matrix (Arizona Test Dust)



Wipe With Reference




Dust-sampling Sock With Reference




Swab With Local Particulate



Wipe With Local Particulate



Dust-sampling Sock With Local

Particulate




A – 10

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, under development), 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:

Yildiz Chambers, Computer Sciences Corporation (CSC) [email protected] Phone: (703) 461-2165 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 • 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 will include the relative standard deviation (RSD) between results of replicate samples. Mean, median, and range of individual recoveries also may be included. Recoveries will be assessed based on enumeration of spiking suspension by the participant laboratory, using spread plate technique (see Spiking Protocol [Appendix C, under development]). 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.

mailto:[email protected]�


A – 11

Appendix B

Study-Specific Instructions

B – 1 August 14, 2009

Laboratory Instructions for Vibrio cholerae O1 and O139 SAP Single Laboratory Verification Study: Water Analyses

August 14, 2009

The purpose of this study is to evaluate procedures that will be included in the revised draft “Standardized Analytical Procedure for Vibrio cholerae O1 and O139 in Environmental Samples” (referred to as the draft SAP). Results of this preliminary study will be used to revise the draft SAP, if necessary, prior to further validation in multiple laboratories.

All Quality Assurance/Quality Control (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 quality assurance project plan (QAPP).

• Phosphate buffered saline (PBS)

Media/Reagents/Supplies

• 10% sodium thiosulfate

• 1X, 2X, and 5X alkaline peptone water (APW)

• 1% APW

• Thiosulfate citrate bile salts sucrose (TCBS) agar

• Tryptic soy agar (TSA)

• Physiological saline (0.85% NaCl)

• Polyvalent V. cholerae O1 antiserum

• Polyvalent V. cholerae O139 antiserum

• API® 20E test strips

V. cholerae O1 and O139 (strains JBK70 and CVD112, respectively)

Control Cultures

E. coli (ATCC® 25922™)

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.

Water Matrices

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

Quality Control Analyses

B – 2 August 14, 2009

The following unspiked and spiked PBS, drinking water, and surface water samples will be analyzed using the most probable number (MPN) technique for each V. cholerae O1 and O139 strain (JBK70 and CVD112, respectively). Following enrichment in APW, samples will be plated on a selective agar (TCBS). Since the two matrices, surface water and drinking water, will be incubated differently, 6-8 hours and 24 ± 2 hours, respectively, growth from PBS samples will be streaked onto TCBS after 6 – 8 hour and 24 ± 2 hour incubation of APW. Isolated, typical, large yellow colonies will be sub-cultured on a non-selective medium (TSA) and subsequently submitted to serological and biochemical confirmation. The first week of analyses will be conducted with V. cholerae O1 (JBK70). The second week of analyses will be conducted with V. cholerae O139 (CVD112). Analyses for each week will include the following samples:

Preliminary Sample Analyses

• PBS Samples (Reference Matrix) o 1, 100-mL Unspiked o 4, 100-mL Spiked

• Surface Water Samples o 2, 100-mL Unspiked o 4, 100-mL Spiked

• Drinking Water Samples o 2, 100-mL Unspiked o 4, 100-mL Spiked

Prior to Analyses

It is recommended that the following reagents and media be prepared prior to the first week of preliminary study analyses (PBS, APW, TCBS, TSA) so that appropriate QC analyses can be conducted on the media. Volumes provided are sufficient for the two consecutive weeks of analyses.

Reagent and Media Preparation

• PBS: Prepare 2.0 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 2.0 liters.

Sodium dihydrogen phosphate (NaH2PO4) 0.58 g

Disodium hydrogen 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 appropriate volumes into 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: For both weeks, a total of 1990 mL of PBS will be required. As a result, it is recommended that the laboratory prepare 2.0 L of PBS [10, 99-mL dilution blanks, 10, 100-mL samples].

• Alkaline Peptone Water (APW): Prepare 1X, 2X and 5X APW according to the following.

Reagents 1X 2X 5X Dehydrated medium 22.0 g 44.0 g 60.0 g Reagent-grade water 1100 mL 1100 mL 600 mL

B – 3 August 14, 2009

For 1X and 2X APW, dissolve reagents in 1000 mL of reagent-grade water in a flask and mix thoroughly. For 5X APW, dissolve reagents in 500 mL of reagent-grade water in a flask and mix thoroughly. Adjust pH of medium to 8.5 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide, add remaining 100 mL of reagent-grade water, and mix thoroughly. Dispense 10 mL (1X and 2X) or 5 mL (5 X) aliquots in 25 × 150 mm tubes and autoclave at 121°C (15 PSI) for 15 minutes. 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.

• 1% APW: Prepare a 1% solution of APW by combining 99 mL of sterile PBS and 1 mL of sterile single-strength APW (prepared according to instructions above). A total of 2, 100 mL volumes of 1% APW will be required for V. cholerae O1 and O139 analyses.

• TCBS: Prepare 5.0 L of TCBS agar according to manufacturer’s instructions. Aseptically pour approximately 12 mL into 100 × 15 mm sterile plates. Store prepared plates at <10°C and above freezing for a maximum of 2 weeks [400, 12-mL, 100 × 15 mm plates].

• TSA: Prepare a minimum of 400 TSA plates according to manufacturer’s instructions. Aseptically pour approximately 12 mL into 100 × 15 mm sterile plates. Store at <10°C and above freezing for a maximum of 2 weeks.

Analyses

Instructions provided below are repeated for each week of analyses: • Week One: V. cholerae O1 • Week Two: V. cholerae O139 Day One (Monday) Inoculate 1% APW with V. cholerae (O1 during Week One and O139 during Week Two) according to the spiking protocol and incubate at 36.0°C ± 1.0°C for 18 – 24 hours. Day Two (Tuesday)

• Dilute and plate laboratory-prepared spiking suspension on TSA according to spiking protocol and incubate at 36.0°C ± 1.0°C for 18 – 24 hours.

Enumeration of Spiking Suspensions

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

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

o 1, 100-mL Unspiked o 4, 100-mL 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.

B – 4 August 14, 2009

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.

2, 100-mL – Surface water Unspiked

4, 100-mL – Surface water 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.

2, 100-mL – Drinking water Unspiked

4, 100-mL – Drinking water Spiked

• Spike 4, 100-mL samples each of PBS, drinking water, and surface water with laboratory-prepared spiking suspension, according to the spiking protocol.

Sample Spiking

• Mix unspiked and spiked samples by shaking each sample 25 times.

Most Probable Number (MPN)

• Inoculate APW 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) APW



•

o Incubate APW tubes for 6 – 8 hours at 36°C ± 1.0°C.

PBS and Surface Water Samples

•

o Incubate APW tubes for 24 ± 2 hours at 36°C ± 1.0°C.

Drinking Water Samples

•

o Examine APW tubes after 6 – 8 hour incubation and record results. Do not shake or mix tubes.

PBS and Surface Water Samples

o From each tube, use a sterile inoculation loop (20 μL) to transfer inoculum from the top 2 – 5 mm of each APW tube and streak for colony isolation onto TCBS plates. Incubate at 36°C ± 1.0°C for 24 ± 2 hours.

B – 5 August 14, 2009

Note: Not all positive tubes will exhibit growth (i.e., turbidity) at 6 – 8 hours; therefore, primary isolation must be performed on all tubes.

o Place PBS tubes back into the incubator and incubate for a total of 24 ± 2 hours at 36°C ± 1.0°C. Day Three (Wednesday) TSA Enumeration – Laboratory-Prepared Spiking Suspensions

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

Most Probable Number (MPN)

• PBS (6 – 8 hr) and Surface Water Samples

o Examine TCBS agar plates inoculated on Day Two; record results on data reporting forms.

o For TCBS plates with typical large yellow colonies, streak a typical colony onto TSA and incubate at 36°C ± 1.0°C for 18 – 24 hours. Note: Cultures grown on TCBS should be examined immediately after removal from an incubator as the yellow colonies of Vibrio cultures may revert to a green color when left at room temperature.

• PBS (24 ± 2 hr) and Drinking Water Samples

o Examine APW tubes after 24 ± 2 hour incubation and record results. Do not shake or mix tubes.

o From each tube, use a sterile inoculation loop (20 μL) to transfer inoculum from the top 2 – 5 mm of each APW tube and streak for colony isolation onto TCBS plates. Incubate at 36°C ± 1.0°C for 24 ± 2 hours.

Day Four (Thursday) PBS (6 – 8 hr) and Surface Water Samples

• Examine TSA plates inoculated on Day Three.

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

o Emulsify a portion of a single typical colony from each TSA plate using sterile physiological saline and test for agglutination with the appropriate antisera as follows:

Place two discrete drops of emulsified growth onto a slide. To the first drop of emulsified growth, add one drop of V. cholerae O1 (Week One) or O139 (Week Two) antiserum. To the second drop of emulsified growth, add one drop of sterile physiological saline (for a visual comparison).

Observe under magnification for an agglutination reaction, which indicates a positive result. Results should be compared with those for positive and negative controls analyzed at the same time.

o If results are not consistent with V. cholerae O1 or O139, go back to the TSA plate and pick another isolated colony and repeat serological analyses. Note: If neither of the two isolates provides a positive serology result, please contact Yildiz Chambers (703.461.2165, [email protected]) for additional instructions.

• Biochemical Analyses: A single colony from one TSA plate for each volume (3 colonies per sample) of the spiked PBS sample will be submitted to biochemical confirmation, as described below.

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

mailto:[email protected]�

B – 6 August 14, 2009

o API® 20E Test Strips: Emulsify a large colony (2 – 3 mm) in 5.0 mL physiological saline. Follow manufacturer’s instructions to inoculate wells. Incubate test strip at 36.0°C ± 1.0°C for 18 – 24 hours.

• Examine TCBS agar plates at inoculated on Day Three; record results on data reporting forms.

PBS (24 ± 2 hr) and Drinking Water Samples

• For TCBS plates with typical large yellow colonies, streak a typical colony onto TSA and incubate at 36°C ± 1.0°C for 18 – 24 hours.

Day Five (Friday)

• API® 20E Test Strips: Add appropriate reagents according to manufacturer’s instructions; observe and record results. If results are not consistent with V. cholerae, please contact CSC (Yildiz Chambers) immediately for additional instructions.

PBS (6 – 8 hr) and Surface Water Samples

• Examine TSA plates inoculated on Day Four. Conducted serological and biochemical tests as described above.


Day Six (Saturday)

• Examine API® 20E test strips inoculated on Day Five; process as described above.


• Fax results to Yildiz Chambers at 703.461.8056.

Appendix C

Spiking Protocol

March 2009

C-1

Water Matrices Spiking Protocol for the Draft Vibrio cholerae O1 and O139 SAP Verification Study

For Discussion – March 2009

The purpose of this protocol is to provide laboratories with Vibrio cholerae spiking procedures for Vibrio cholerae O1 (strain # JBK70) and O139 (strain # CVD 112) for the verification and validation studies to evaluate the draft “Standardized Analytical Procedure for Vibrio cholerae O1 and O139 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 Spiked V. cholerae Percent Recovery

1.0 Preparation of Laboratory-Prepared Spiking Suspensions 1.1 1% Alkaline Peptone Water (APW) Broth. Prepare a 1% solution of APW by combining

99 mL of sterile PBS with 1 mL of single-strength APW in a screw-cap bottle. Shake to mix. 1.2 Stock Cultures. Prepare a stock culture of each Vibrio cholerae strain (O1 [JBK70] and O139

[CVD 112]) by inoculating a tryptic soy agar (TSA) slant. Incubate at 36C ± 1.0C for 18 – 24 hours. Store working stock cultures at room temperature; refrigerating the cultures will decrease culture viability. Maintain back up stocks of each strain at -80C.

1.3 Spiking Suspension (Undiluted). From the stock culture of Vibrio cholerae O1 or O139 in

section 1.2, aseptically transfer a small loopful of growth to 1% Alkaline peptone water (APW) (Section 1.1) and vigorously shake the bottle a minimum of 25 times. Incubate at 36C ± 1.0C for 18 – 24 hours. • The resulting spiking suspension contains approximately 1.0×106 to 1.0×107 V. cholerae

colony forming units (CFU) per mL. This is referred to as the “undiluted spiking suspension.” Note: During the V. cholerae O1 and O139 SAP verification study, propagation of spiking suspensions should begin one day prior to the day samples will be spiked. For example, if samples will be spiked on Tuesday, propagation of spiking suspensions will begin on Monday.

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 V. cholerae O1 or O139 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 thoroughly homogenize the spiking suspensions in the steps below, to ensure accurate sample spiking and spiking suspension enumeration.

March 2009

C-2

2.1.1 Perform steps 2.1.1.1 through 2.2.6 using laboratory-prepared undiluted spiking suspension (Section 1.3).

Dilute spiking suspension

2.1.1.1 Mix the undiluted spiking suspension (Section 1.3) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipet to transfer 1.0 mL of this suspension to 99 mL of sterile PBS, cap, and mix by again 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 pipet to transfer 1.0 mL of spiking suspension dilution “A” 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 pipet to transfer 11.0 mL of spiking suspension dilution “B” 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 pipet to transfer 11.0 mL of spiking suspension dilution “C” 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.

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 mL of sample is 3 × 10–5 of the undiluted spiking suspension and is equal to 30 to 300 CFU/mL. This is referred to as Vspiked per 100 mL sample in section.

2.1.2.2 Analyze unspiked and spiked samples according to study instructions.

2.2 Enumeration of Spiking Suspensions (Prepared in Section 1.3)

2.2.1 Prepare TSA according to manufacturer’s and study instructions. Add 12 mL of TSA per 100 × 15 mm Petri plate 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.

March 2009

C-3

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” (Section 2.1.1.2) 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 3.1.3 and Table 1 for

calculation of spiking suspension concentration. 3.0 Calculation of Spiked V. cholerae Percent Recovery (Laboratory-Prepared

Undiluted Spiking Suspension) Spiked V. cholerae percent recovery will be calculated in three steps as indicated in Sections 3.1 through 3.3, below. Note: Figures calculated in the examples and provided in the 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 V. cholerae O1 or O139 (CFU/mL) in Undiluted

Spiking Suspension

3.1.1 The number of V. cholerae (CFU/mL) in the undiluted spiking suspension (prepared in Section 1.3) 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 V. cholerae (CFU/mL) in undiluted spiking suspension according to the following equation. Example calculations are provided in Table 1.

V. cholerae undiluted spike = (CFU1 + CFU2 + ... + CFUn) / (V1 + V2 + ... + Vn)

March 2009

C-4

Where,

V. cholerae undiluted spike = V. cholerae O1 or O139 (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 Tab le 1. Example Ca lcu la tions o f V. cholerae Spiking Sus p ens ion Concen tra tion

Examples CFU/plate (triplicate analyses) from

TSA plates in Section 2.2.2 V. cholerae CFU/mL in undiluted

spiking suspension (V. cholerae 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) =

97 / (3.0 × 10-5) = 32.3 × 105

= 3.2 × 106 CFU/mL

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

(45+56+61) / (10-5+10-5+10-5) =

162 / (3 × 10-5) = 54.0 × 105

= 5.4 × 106 CFU/mL

* V. cholerae 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 V. cholerae (CFU/100 mL) Calculate the true concentration of spiked V. cholerae (CFU/100 mL) according to the following equation. Example calculations are provided in Table 2.

TSpiked V. cholerae = (V. cholerae undiluted spike) × (V spiked per 100 mL sample)

Where, TSpiked V. cholerae = Number of spiked V. cholerae (CFU/100 mL)

V. cholerae undiluted spike = V. cholerae (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)

March 2009

C-5

Table 2. Example Calculations of “True” Spiked V. cholerae (CFU/100 mL)

V. cholerae undiluted spike (Table 1 above)

V spiked per 100 mL sample (Section 2.1.2.1 above)

TSpiked V. cholerae

3.2 × 106 CFU/mL 3.0 × 10-5 mL per 100 mL of sample (3.2 × 106 CFU/mL) × (3.0 × 10-5 mL/100 mL) = 96 CFUs/100 mL

5.4 × 106 CFU/mL 3.0 × 10-5 mL per 100 mL of sample (5.4 × 106 CFU/mL) × (3.0 × 10-5 mL/100 mL) =

162 CFUs/100 mL 3.3 Step 3: Calculate Percent Recovery

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

choleraeVSpiked

us

TNN

R.

100−

×=

Where,

R = Percent recovery Ns = V. cholerae (CFU/100 mL) in the spiked sample Nu = V. cholerae (CFU/100 mL) in the unspiked sample TSpiked V. cholerae = True spiked V. cholerae (CFU/100 mL) in spiked sample (Section 3.2)

3.3.2 Example percent recovery calculations are provided in Table 3.

Table 3. Example Percent Recovery Calculations

Ns (CFU/100 mL) Nu (CFU/100 mL) T Spiked V. cholerae (CFU/100 mL) Percent recovery (R)

42 <1 96 100 × (42 – 1) / 96 = 43%

34 10 96 100 × (34 - 10) / 96 = 25%

153 2 162 100 × (153 - 2) / 162 = 93%

142 1 162 100 × (142 - 1) / 162 = 87%

March 2009

C-6

Appendix D

Data Reporting Forms

D - 1 August 14, 2009

Laboratory: ____________________________________________ Date: ______________________________________

Analyst Initials pH Expiration Date

1 Date of APW preparation:

2 Date of TCBS plate preparation:

3 Date of TSA preparation:

4 V. cholerae O1 antiserum

5 yes no

6 yes no

7 yes no

8 yes no

9 yes no

10 yes no

11 Sample collection date: Time: Sampler's initials:

12 Sample location:


14 Sample location:

Section 4. Comments

Section 2. Quality Control (please circle yes or no); if no, please explain in Section 4.

V. cholerae O1Preliminary Verification Study: V. cholerae O1 Analyses Batch-Specific Cover Sheet

Section 1. Media and Sample Preparation

Media Lot #

Did the E. coli (ATCC® 25922™) negative control for APW, TCBS, V. cholerae O1 antiserum agglutination, API® 20E test strips exhibit the appropriate response?

Section 3. Sample Collection (Drinking water, surface water)

Did APW broth media sterility check exhibit the appropriate response?

Did the method blank exhibit the appropriate response?

Did the V. cholerae O1 positive control for APW, TCBS, and V. cholerae O1 antiserum agglutination exhibit the appropriate response?

Did TCBS media sterility check exhibit the appropriate response?

Did TSA media sterility check exhibit the appropriate response?

D - 2 August 14, 2009

Laboratory:

Date: Date: Date: Date:

Time: Time: Time: Time:

Temp: Temp: Temp: Temp:

Initials: Initials: Initials: Initials:

Step 1. Step 2.

CFU1 + CFU2 + … + CFUn

Tspiked V. cholerae O1 = (V. cholerae O1 undiluted spike) x (Vspiked per 100 mL sample)

V. cholerae O1V. cholerae O1 Water Analyses

Spiking Suspension Enumeration

Lab-Prepared Spike Enumeration (TSA)(Spiking Protocol, Section 4)

Replicate 1plate count

TSA inoculationvolume (mL)



1% APW Incubation

Start

1% APWIncubation

End

TSAIncubation

End

TSA Incubation

Start

V. cholerae O1undiluted spike = V1 + V2 + … + Vn

10-5

10-6

10-7

D - 3 August 14, 2009

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

Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:

APW Growth, indicated by turbidity or pellicle at surfaceMPN Tube Combination

TCBS Large yellow colonies MPN / 100 mL

V. cholerae O1 Antisera Agglutination, indicated by the formation of a white precipitate

Incubation End

Analyses V. cholerae O1 Results

Incubation Start

1 mL(1X APW)

10 mL(2X APW)

APW(24 ± 2 h @ 36.0°C ± 1.0°C)

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

TSA(18 - 24 h @ 36°C ± 1.0°C)

API® 20E1

(+ or -)V. cholerae O1 Agglutination

(+ or -)

20 mL(5X APW)

V. cholerae O1V. cholerae O1 MPN Analyses - Drinking Water or PBS: 24 Hour Incubation

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

Spiking date and time:Spike dilution: Spike volume:

D - 4 August 14, 2009


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:



APW

TCBS Large yellow colonies

Growth, indicated by turbidity or pellicle at surface

V. cholerae O1 Agglutination (+ or -)

API® 20E1

(+ or -)TSA

(18 - 24 h @ 36°C ± 1.0°C)

Incubation End

10 mL(2X APW)

1 mL(1X APW)

Incubation Start

Spiking date and time:

MPN Tube Combination

MPN / 100 mL

V. cholerae O1V. cholerae O1 MPN Analyses - Surface Water or PBS: 6 – 8 Hour Incubation

SPIKED

Spike dilution:

Matrix (circle one): Surface Water PBS Date:Lab:

APW(6 – 8 h @ 36.0°C ± 1.0°C)

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

Sample #:

Spike volume:

20 mL(5X APW)

D - 5 August 14, 2009

Date:


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:




Incubation End

Analyses V. cholerae O1 Results Final Results

Incubation Start

1 mL(1X APW)

10 mL(2X APW)

V. cholerae O1 Serum Agglutination

(+ or -)

20 mL(5X APW)

APW(24 ± 2 h @ 36.0°C ± 1.0°C)

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

TSA(18 - 24 h @ 36°C ± 1.0°C)

API® 20E1

(+ or -)

V. cholerae O1V. cholerae O1 MPN Analyses - Drinking Water or PBS: 24 hour Incubation

UNSPIKED

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

D - 6 August 14, 2009

Date:


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:

V. cholerae O1V. cholerae O1 MPN Analyses - Surface Water or PBS: 6 – 8 hour Incubation

UNSPIKED

Lab:

API® 20E1

(+ or -)

Matrix (circle one): Surface Water PBS Sample #:

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

Incubation End

APW

20 mL(5X APW)

10 mL(2X APW)

1 mL(1X APW)

Incubation Start

TSA(18 - 24 h @ 36°C ± 1.0°C)



V. cholerae O1 Antisera


(+ or -)

Final ResultsMPN Tube Combination

APW(6 – 8 h @ 36.0°C ± 1.0°C)

MPN / 100 mL


Agglutination, indicated by the formation of a white precipitate

D - 7 August 14, 2009

Laboratory: ____________________________________________ Date: ______________________________________

Analyst Initials pH Expiration Date

1 Date of APW preparation:

2 Date of TCBS plate preparation:

3 Date of TSA preparation:

4 V. cholerae O139 antiserum

5 yes no

6 yes no

7 yes no

8 yes no

9 yes no

10 yes no


12 Sample location:


14 Sample location:

Section 4. Comments

Section 2. Quality Control (please circle yes or no); if no, please explain in Section 4.

V. cholerae O139Preliminary Verification Study: V. cholerae O1 Analyses Batch-Specific Cover Sheet

Section 1. Media and Sample Preparation

Media Lot #

Did the E. coli (ATCC® 25922™) negative control for APW, TCBS, V. cholerae O139 antiserum agglutination, API® 20E test strips exhibit the appropriate response?

Section 3. Sample Collection (Drinking water, surface water)

Did APW broth media sterility check exhibit the appropriate response?

Did the method blank exhibit the appropriate response?

Did the V. cholerae O139 positive control for APW, TCBS, and V. cholerae O139 antiserum agglutination exhibit the appropriate response?

Did TCBS media sterility check exhibit the appropriate response?

Did TSA media sterility check exhibit the appropriate response?

D - 8 August 14, 2009

Laboratory:

Date: Date: Date: Date:

Time: Time: Time: Time:

Temp: Temp: Temp: Temp:

Initials: Initials: Initials: Initials:

Step 1. Step 2.

CFU1 + CFU2 + … + CFUn

Tspiked V. cholerae O139 = (V. cholerae O139 undiluted spike) x (Vspiked per 100 mL sample)

V. cholerae O139V. cholerae O139 Water AnalysesSpiking Suspension Enumeration

Lab-Prepared Spike Enumeration (TSA)(Spiking Protocol, Section 4)


TSA inoculationvolume (mL)



1% APW Incubation

Start

1% APWIncubation

End

TSAIncubation

End

TSA Incubation

Start

V. cholerae O139 undiluted spike = V1 + V2 + … + Vn

10-5

10-6

10-7

D - 9 August 14, 2009


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:




Incubation End


Incubation Start

10 mL(2X APW)

1 mL(1X APW)

APW(24 ± 2 h @ 36.0°C ± 1.0°C)

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

TSA(18 - 24 h @ 36°C ± 1.0°C)

API® 20E1

(+ or -)V. cholerae O1 Agglutination

(+ or -)

20 mL(5X APW)


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

Spike dilution: Spike volume: Spiking date and time:

D - 10 August 14, 2009


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:



APW



V. cholerae O1 Agglutination (+ or -)

API® 20E1

(+ or -)TSA

(18 - 24 h @ 36°C ± 1.0°C)

Incubation End

10 mL(2X APW)

1 mL(1X APW)

Incubation Start

Spiking date and time:

MPN Tube Combination

MPN / 100 mL


SPIKED

Spike dilution:

Matrix (circle one): Surface Water PBS Date:Lab:

APW(6 – 8 h @ 36.0°C ± 1.0°C)

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

Sample #:

Spike volume:

20 mL(5X APW)

D - 11 August 14, 2009

Date:


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:




Incubation End

Analyses V. cholerae O139 Results Final Results

Incubation Start

1 mL(1X APW)

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

TSA(18 - 24 h @ 36°C ± 1.0°C)

API® 20E1

(+ or -)

10 mL(2X APW)


UNSPIKED

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


(+ or -)

20 mL(5X APW)

APW(24 ± 2 h @ 36.0°C ± 1.0°C)

D - 12 August 14, 2009

Date:


Sample Volume

1

2

3

1

2

3

1

2

3

Date:

Time:

Temp:

Initials:

Date:

Time:

Temp:

Initials:


UNSPIKED

Lab:

API® 20E1

(+ or -)

Matrix (circle one): Surface Water PBS Sample #:

TCBS Plates(24 ± 2 h @ 36.0°C ± 1.0°C)

Incubation End

APW

20 mL(5X APW)

10 mL(2X APW)

1 mL(1X APW)

Incubation Start

TSA(18 - 24 h @ 36°C ± 1.0°C)



V. cholerae O139 Antisera


(+ or -)

Final ResultsMPN Tube Combination

APW(6 – 8 h @ 36.0°C ± 1.0°C)

MPN / 100 mL


Agglutination, indicated by the formation of a white precipitate

Printed on 100% recycled/recyclable paper with a minimum of 50% post-consumer fiber using vegetable-based ink.

United StatesEnvironmental ProtectionAgency

Office of Research and Development (8101R) Washington, DC 20460

Official Business Penalty for Private Use $300

PRESORTED STANDARDPOSTAGE & FEES PAID

EPAPERMIT NO. G-35

STUDY REPORT - U.S EPA Web Server

Documents

Transcript of STUDY REPORT - U.S EPA Web Server