&EPA
United States
Environmental Protection
Agency
EPA/600/R-10/141 | October 2010 | www.epa.gov/ord
Single-Laboratory Verification
of Culture-Based Analytical
Procedures for Vibrio cholerae
01 and 0139 in Drinking
Water and Surface Water
STUDY REPORT
Office of Research and Development
National Homeland Security Research Center
-------
-------
Study Report
Single-Laboratory Verification of
Culture-Based Analytical Procedures for
Vibrio cholerae 01 and 0139 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 01 and 0139 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.)
-------
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
shall. sani iv@et>a. gov
-------
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 01 and 0139 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
-------
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
-------
Tables
Table 1. Summary of Sample Analyses 3
Table 2. Schedule for Verification of V. cholerae 01/0139 Culture-Based Procedures in Water
Matrices 5
Table 3. Verification Analyses, Summary Results: PBS, Drinking Water, and Surface Water
Spiked with V. cholerae 01 or 0139 (Conducted the Week of September 28, 2009) 8
Table 4. Verification Analyses, Summary Results: Surface Water Spiked with V. cholerae 01 or
0139 (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
-------
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 01 and 0139 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 01 (JBK 70) or V. cholerae 0139 (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
0139 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 01 and 163% for 0139 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 01 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 01 and 0139), and develop quantitative QC criteria.
viii
-------
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) 01 and 0139, 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 01 or 0139. In modern times, these
two serotypes have been responsible for up to
eight cholera pandemics. Non-01 and non-
0139 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 01 and 0139 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 01).
Quantitation of V cholerae was determined
using the most probable number (MPN)
technique. Tubes that confirmed positive for V.
cholerae 01 or 0139 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 01 and 0139. 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
-------
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
01 (JBK 70) or V cholerae 0139 (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 01 or 0139 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/T reatment
No. of
Analyses
Phase 2 Analyses
Sterile PBS
Method Blank 01
1
(Phosphate
buffered saline)
(Reference
Matrix)
Method Blank 0139
1
V. cholerae 01
1
V. cholerae 0139
1
Preliminary
Analyses
Unspiked 01
Original SAP (Section 1.0)
1
Drinking Water
Unspiked 0139
Selective enrichment in APW
for 6 - 8 hours at 36.0°C ±
1.0°C
1
V. cholerae 01
1
V. cholerae 0139
1
Unspiked 01
1
Surface Water
Unspiked 0139
1
V. cholerae 01
1
V. cholerae 0139
1
3
-------
(1) "Unspiked 01" and "Unspiked 0139" refer to unspiked samples analyzed forOI and 0139 background organisms,
respectively
(2) For surface water and PBS samples analyzed for V. cholerae 01, 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 0139, growth from APW tubes was plated
onto TCBS after 6-8 hours of incubation; extended incubation was unnecessary (see Table 4)
Analysis
Matrix
Spiking
Description(1)
Procedure/T reatment
No. of
Analyses
Phase 3 Analyses
Sterile PBS
(Reference
Matrix)
Method Blank 01
1
Method Blank 0139
1
V. cholerae 01
4
V. cholerae 0139
4
Assessment of
Unspiked 01
Evaluation of extended
2
Unspiked 0139
incubation in APW at
o
Procedure
Modification
Drinking Water
36.0°C ± 1,0°C for
6-8 and/or 24 ± 2 hours (2)'(3)
V. cholerae 01
4
V. cholerae 0139
4
Unspiked 01
2
Surface Water
Unspiked 0139
2
V. cholerae 01
4
V. cholerae 0139
4
Phase 4 Analyses
Sterile PBS
(Reference
Matrix)
Method Blank 01
1
Method Blank 0139
1
V. cholerae 01
4
V. cholerae 0139
4
Unspiked 01
Incubation in APW at
2
Verification
Drinking Water
Unspiked 0139
36.0°C ± 1,0°C for
2
Analyses
V. cholerae 01
6-8 and/or 24 ± 2 hours (2)'(3)
4
V. cholerae 0139
4
Unspiked 01
2
Surface Water
Unspiked 0139 (4)
2
V. cholerae 01
4
V. cholerae 0139(4)
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 01 (JBK 70) and V cholerae 0139
(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 01 and 0139 strains and in all
matrices [PBS, drinking water, surface water])
Table 2. Schedule for Verification of V. cholerae 01/0139 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
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.
-------
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 01 or
0139). 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 01 and 0139
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 01 or 0139.
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 01 and 0139 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
01 and 0139 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 01 or 0139.
7
-------
Table 3. Verification Analyses, Summary Results: PBS, Drinking Water, and Surface Water Spiked
Oh
Analyte
n
Spike Level
(CFU/100 mL)
Selective
Enrichment in
APW
Mean
Recovery
(%)
Minimum
Recovery
(%)
Maximum
Recovery
(%)
SD
(%)
RSD
(%)
Phosphate Buffered Saline (PBS)
01
4
36
6 hours
138.72
63.61
302.28
112.86
81.36
01
4
24 hours
138.72
63.61
302.28
112.86
81.36
0139
4
34
6 hours
122.22
34.11
320.06
132.82
108.68
0139
4
24 hours
130.53
67.35
320.06
126.35
96.80
Drinking Water
01
4
36
24 hours
84.91
25.25
125.39
49.30
58.06
0139
4
34
24 hours
163.23
67.35
320.06
109.00
66.78
Surface Water
01
4
36
6 hours
0.00
0.00
0.00
0.00
NA
01
4
24 hours
0.16
0.00
0.49
0.23
142.44
0139
4
34
6 hours
8.53
0.00
34.12
17.06
200.00
0139
4
24 hours
14.19
0.00
34.12
17.77
125.21
a)
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
CFU/100 mL - Colony forming unit per 100 milliliter
n - Number of replicates
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 01 analyses were conducted using
surface water samples obtained from Sites
l(Port Huron, Michigan) and 2 (Newark, Ohio)
and V. cholerae 0139 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
PBS - Phosphate buffered saline
RSD - Relative standard deviation
SD - Standard deviation
addition, the spike level was increased slightly
and two different surface water samples were
analyzed per analyte. For V. cholerae 01 repeat
analyses, growth from the APW tubes was
transferred at 6 - 8 and again at 24 ± 2 hours.
For serotype 0139, 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 01 or
0139.
8
-------
Table 4. Verification Analyses, Summary Results: Surface Water Spiked with V. cholerae 01 or
Oh
Analyte
n
Sample
Location
Spike Level
(CFU/100 mL)
Selective
Enrichment
in APW
Mean
Recovery
(%)
Minimum
Recovery
(%)
Maximum
Recovery
(%)
SD
(%)
RSD
(%)
Phosphate Buffered Saline
01
1
NA
129
6 hours
57.20
57.20
57.20
NA
NA
0139
1
NA
253
6 hours
94.18
94.18
94.18
NA
NA
Surface Water
01
4
Site 1
129
6 hours
25.52
16.26
30.79
6.36
24.94
01
4
129
24 hours
152.33
69.97
355.93
137.27
90.11
01
4
Site 2
129
6 hours
13.53
4.89
30.79
12.21
90.27
01
4
129
24 hours
52.86
13.99
113.47
48.26
91.31
0139
4
Site 3
253
6 hours
88.62
35.62
181.17
66.75
75.32
0139
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
CFU/100 mL - Colony forming unit per 100 milliliter
n - Number of replicates
NA- Not applicable
PBS - Phosphate buffered saline
RSD - Relative standard deviation
SD - Standard deviation
Results of the verification analyses indicate that
serotype 0139 may grow more rapidly than 01
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 0139 at 6-8 hours and V. cholerae
01 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 01 and 0139 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 0139 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 (01 and 0139) and surface
water matrices (01). In drinking water, mean
recoveries were 85% for 01 and 163% for 0139
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 01 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 0139 in surface water samples; and the
modified procedure (extended [24 ± 2 hours]
APW incubation) provided acceptable results for
identification and quantitation of V. cholerae 01
and 0139 in drinking water and V. cholerae 01
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 01 and 0139
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 01 and 0139. 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 01 and
0139 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/LaboratorvMethods/BacteriologicalAnalvticalManualB
AM/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. Ill, 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
-------
Appendix A
Study Plan
-------
-------
Study Plan
for Single Laboratory Verification
of the
Standardized Analytical Procedure for
Vibrio cholerae 01 and 0139 in Environmental
Samples
Note: Study plan appendices are provided as attachments to the study report.
December 30, 2008
-------
This document has been formatted according to U.S. EPA's Environmental Monitoring Management
Council (EMMC) formatting guidance (http://www.epa. gov/ttn/emc/guidlnd/gd-045 .pdf). EPA uses
EMMC format for the EPA microbiology 1600-method series. This document is undergoing review
and is to be considered a draft. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
The procedures described in this document are intended for use in laboratories when analyzing
environmental samples in support of remediation efforts following a homeland security incident. The
procedures provide viability (culture based) determination, identification, and quantitation. To the
extent possible, these procedures were developed to be consistent with other federal agency
procedures. These procedures do not include sample collection or molecular techniques.
Study Plan
A-ii
December 30, 2008
-------
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-iii
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 01 and 0139 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 01 and 0139 in Environmental Samples (SAP) for determination and
quantification of Vibrio cholerae (V. cholerae) 01 and 0139 using selective and non-selective media
followed by biochemical and serological characterization.
The draft SAP for V cholerae 01 and 0139 is based on CDC 1 and FDA2 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 01 and 0139 (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.
Study Plan
A- 1
December 30, 2008
-------
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
Study Plan
A-2
December 30, 2008
-------
Figure 1: Flowchart of Study Activities
Study Plan
A - 3
December 30, 2008
-------
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 01 and 0139 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
0157: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 0157: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 0157: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 01 and 0139 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 01 and 0139 vaccine strains
• Revise spiking protocol, as necessary
• Conduct preliminary analysis of V. cholerae 01 and 0139 (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 01 and 0139 (SAP evaluation) at
Study Plan
A-4
December 30, 2008
-------
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 01 and 0139 (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 01 (SAP evaluation) at laboratory
(~ 3 weeks)
• Conduct study analyses of V. cholerae 0139 (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 01 and 0139 (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
• Conduct study analyses of V. cholerae 01 (SAP evaluation) at laboratory
(~ 3 weeks)
• Conduct study analyses of V. cholerae 0139 (SAP evaluation) at
laboratory (~ 3 weeks)
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 01 and 0139 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)
Study Plan
A- 5
December 30, 2008
-------
• 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 01. A second identical set of samples will be analyzed for V cholerae 0139 (see draft SAP,
Appendix A). One or more reference matrices for each sample type (water, solid, and particulate) will
also be analyzed.
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.
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).
Study Plan
A-6
December 30, 2008
-------
4.2 Phase 2 - Preparation of Spikes
The study will use laboratory-prepared V. cholerae 01 and 0139 spiking suspensions to evaluate method
performance.
4.2.1 Laboratory-Prepared Spiking Suspensions
Laboratory-prepared spiking suspensions will be prepared from V. cholerae 01 and 0139 (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 01 and 0139 spikes.
4.3.3 Quality Control (OC) 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 01 and 0139
(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.
Study Plan
A-7
December 30, 2008
-------
Table 3. Summary of Sample Analyses
No. of Samples
Matrix
Spiking Description
Purpose of Analysis
Preliminary
Analyses
Study
Analyses
Water (Drinking Water and Liquid / Water Samples)
Sterile PBS
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
(Reference
Matrix)
V. cholerae 01
1
4
Confirmation that approach is
V. cholerae 0139
1
4
appropriate for the multi-laboratory
validation study
Surface
Water
Unspiked
1
2
Evaluation of background V. cholerae
concentrations
V. cholerae 01
1
4
Confirmation that approach is
V. cholerae 0139
1
4
appropriate for the multi-laboratory
validation study
Drinking
Unspiked
1
2
Evaluation of background V. cholerae
concentrations
Water
V. cholerae 01
1
4
Confirmation that approach is
V. cholerae 0139
1
4
appropriate for the multi-laboratory
validation study
Solids (Soil, Powder Samples)
Milorganite®
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
(Reference
Matrix #1)
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
ASTM Lean
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
Clay (Reference
Matrix #2)
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
ASTM Sand
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
(Reference
Matrix #3)
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Unspiked
1
2
Evaluation of background V. cholerae
concentrations
Local Soil
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Study Plan
A- 8
December 30, 2008
-------
Matrix
Spiking Description
No. of Samples
Purpose of Analysis
Preliminary
Analyses
Study
Analyses
Particulates (Swabs, Wipes, Filters)
Swab With
Reference
Matrix (Arizona
Test Dust)
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Wipe With
Reference
Matrix (Arizona
Test Dust)
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Dust-sampling
Sock With
Reference
Matrix (Arizona
Test Dust)
Unspiked
1
1
Confirmation of freedom from
contamination (Method Blank)
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Swab With
Local Particulate
Unspiked
1
2
Evaluation of background V. cholerae
concentrations
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Wipe With Local
Particulate
Unspiked
1
2
Evaluation of background V. cholerae
concentrations
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Dust-sampling
Sock With Local
Particulate
Unspiked
1
2
Evaluation of background V. cholerae
concentrations
V. cholerae 01
1
4
Confirmation that approach is
appropriate for the multi-laboratory
validation study
V. cholerae 0139
1
4
Study Plan
A- 9
December 30, 2008
-------
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)
vchambers@csc.com
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.
Study Plan
A - 10
December 30, 2008
-------
Study Plan A-11 December 30, 2008
-------
Appendix B
Study-Specific Instructions
-------
-------
Laboratory Instructions for Vibrio cholerae 01 and 0139 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 01 and 0139 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).
Media/Reagents/Supplies
• Phosphate buffered saline (PBS)
• 10% sodium thiosulfate
• IX, 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 01 antiserum
• Polyvalent V cholerae 0139 antiserum
• API® 20E test strips
Control Cultures
V cholerae 01 and 0139 (strains JBK70 and CVD112, respectively)
E. coli (ATCC® 25922™)
Water Matrices
Water matrices will include a reference matrix (PBS), drinking water from the laboratory tap, and surface water
from a lake, reservoir, or other open body of water.
Quality Control Analyses
QC analyses listed on the batch-specific coversheet should be analyzed with each run.
B- 1
August 14, 2009
-------
Preliminary Sample Analyses
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 01 and 0139 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 01 (JBK70). The second week of analyses will be conducted with V. cholerae 0139
(CVD112). Analyses for each week will include the following samples:
• 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
Reagent and Media Preparation
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.
• 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 (Nah^PC^)
0.58 g
Disodium hydrogen phosphate (Na2HPC>4)
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 IX, 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-2
August 14, 2009
-------
For IX 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 (IX and 2X) or 5 mL (5 X) aliquots in
25 x 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 01 and 0139 analyses.
• TCBS: Prepare 5.0 L of TCBS agar according to manufacturer's instructions. Aseptically pour
approximately 12 mL into 100 x 15 mm sterile plates. Store prepared plates at <10°C and above freezing for
a maximum of 2 weeks [400, 12-mL, 100 x 15 mm plates],
• TSA: Prepare a minimum of 400 TSA plates according to manufacturer's instructions. Aseptically pour
approximately 12 mL into 100 x 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 01
• Week Two: V cholerae 0139
Day One (Monday)
Inoculate 1% APW with V. cholerae (01 during Week One and 0139 during Week Two) according to the spiking
protocol and incubate at 36.0°C ± 1.0°C for 18 - 24 hours.
Day Two (Tuesday)
Enumeration of Spiking Suspensions
• 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.
Sample Collection - PBS. Drinking Water, and Surface Water Preliminary Analyses
• Aliquot the following volumes of sterile PBS and label the samples as follows:
o 1, 100-mL Unspiked
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 - 3
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
Sample Spiking
• Spike 4, 100-mL samples each of PBS, drinking water, and surface water with laboratory-prepared spiking
suspension, according to the spiking protocol.
Most Probable Number (MPN)
• Mix unspiked and spiked samples by shaking each sample 25 times.
• 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 For each of three tubes, add 10 mL of undiluted sample to 10 mL (2X) APW
o For each of three tubes, add 1 mL of undiluted sample to 10 mL (IX) APW
• PBS and Surface Water Samples
o Incubate APW tubes for 6 - 8 hours at 36°C ± 1.0°C.
• Drinking Water Samples
o Incubate APW tubes for 24 ± 2 hours at 36°C ± 1.0°C.
• PBS and Surface Water Samples
o Examine APW tubes after 6-8 hour incubation and record results. Do not shake or mix tubes.
o From each tube, use a sterile inoculation loop (20 |iL) 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 - 4
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 |iL) 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 01 (Week One) or 0139 (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 01 or 0139, 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, vchambers@csc.com) 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.
B - 5
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.
PBS (24 ± 2 hr) and Drinking Water Samples
• Examine TCBS agar plates at inoculated on Day Three; record results on data reporting forms.
• 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)
PBS (6-8 hr) and Surface Water Samples
• 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 (24 ± 2 hr) and Drinking Water Samples
• Examine TSA plates inoculated on Day Four. Conducted serological and biochemical tests as described
above.
Day Six (Saturday)
PBS (24 ± 2 hr) and Drinking Water Samples
• Examine API® 20E test strips inoculated on Day Five; process as described above.
• Fax results to Yildiz Chambers at 703.461.8056.
B - 6
August 14, 2009
-------
Appendix C
Spiking Protocol
-------
-------
Water Matrices Spiking Protocol for the Draft Vibrio cholerae 01 and
0139 SAP Verification Study
For Discussion - March 2009
The purpose of this protocol is to provide laboratories with Vibrio cholerae spiking procedures for Vibrio
cholerae 01 (strain # JBK70) and 0139 (strain # CVD 112) for the verification and validation studies to
evaluate the draft "Standardized Analytical Procedure for Vibrio cholerae 01 and 0139 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 (01 [JBK70] and 0139
[CVD 112]) by inoculating a tryptic soy agar (TSA) slant. Incubate at 36°C ± 1.0°C 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 -80 °C.
1.3 Spiking Suspension (Undiluted). From the stock culture of Vibrio cholerae 01 or 0139 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 36°C ± 1.0°C
for 18-24 hours.
• The resulting spiking suspension contains approximately l.Ox 106 to l.Ox 107 V. cholerae
colony forming units (CFU) per mL. This is referred to as the "undiluted spiking
suspension." Note. During the V. cholerae 01 and 0139 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 01 or 0139 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.
C-l
March 2009
-------
2.1.1 Dilute spiking suspension
Perform steps 2.1.1.1 through 2.2.6 using laboratory-prepared undiluted spiking suspension
(Section 1.3).
2.1.1.1 Mix 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 x 10"2 of the undiluted spiking
suspension and is equal to 1 x 104to 1 / 1 (f 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 lxl02to lxlO3 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 x 101 to 1 x 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 x 10"6 of the undiluted spiking suspension and is equal
to 1x10°to lxlO1 CFU/mL.
2.1.2 Spike samplers)
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 x 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.
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 x 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.
C-2
March 2009
-------
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 01 or 0139 (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 = (CFUl + CFU2 + ... + CFUn) / (V1 + V2 + ... + Vn)
C-3
March 2009
-------
Where,
V. cholerae undiluted spike = V. cholerae 01 or 0139 (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
Table 1. Example Calculations of V cholerae Spiking Suspension Concentration
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 x 10"5) = 32.3 x 105
= 3.2 x 10s CFU/mL
Example 2
45, 56, 61
4, 3, 8
0, 2, 0
(45+56+61 )/(10"5+10"5+10"5) =
162/(3 x 10"5) = 54.0 x 105
= 5.4 x 10s 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.
1 Spiked V. cholerae
(V. cholerae undiluted spike) x (V spiked per 100 mL sample)
Where,
T
1 Spiked V. cholerae
V. cholerae undiluted spike
V
spiked per 100 mL sample
= Number of spiked V. cholerae (CFU/100 mL)
= V. cholerae (CFU/mL) in undiluted spiking suspension
(calculated in Section 3.1.3)
= mL of undiluted spiking suspension per 100 mL sample
(Section 2.1.2.1)
C-4
March 2009
-------
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 x 10s CFU/mL
3.0x10"5 mL per 100 mL of sample
(3.2 x 10s CFU/mL) x (3.0 x 10"5 mL/100 mL) =
96 CFUs/100 mL
5.4 x 10s CFU/mL
3.0x10"5 mL per 100 mL of sample
(5.4 x 10s CFU/mL) x (3.0 x 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.
N -N
R = 100 x
/;
Spiked V.cholerae
Where,
R =
Ns =
Nu =
T =
1 Spiked V cholerae
Percent recovery
V cholerae (CFU/100 mL) in the spiked sample
V cholerae (CFU/100 mL) in the unspiked sample
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. Exam
pie Percent Recovery Calculations
Ns (CFU/100 mL)
Nu (CFU/100 mL)
T spiked . o v; (CFU/100 mL)
Percent recovery (R)
42
<1
96
100 x (42-1)/96 = 43%
34
10
96
100 x (34 - 10)/96 = 25%
153
2
162
100 x (153-2)/162 = 93%
142
1
162
100 x (142- 1)/162 = 87%
C-5
March 2009
-------
March 2009
-------
Appendix D
Data Reporting Forms
-------
-------
V. cholerae 01
Preliminary Verification Study: V. cholerae 01 Analyses Batch-Specific Cover Sheet
Laboratory: Date:
Section 1. Media and Sample Preparation
Media
Analyst Initials
Lot#
pH
Expiration Date
1
Date of APW preparation:
2
Date of TCBS plate preparation:
3
Date of TSA preparation:
4
V. cholerae 01 antiserum
Section 2. Quality Control (please circle yes or no); if no, please explain in Section 4.
5
Did APW broth media sterility check exhibit the appropriate response?
yes no
6
Did TCBS media sterility check exhibit the appropriate response?
yes no
7
Did TSA media sterility check exhibit the appropriate response?
yes no
8
Did the method blank exhibit the appropriate response?
yes no
9
Did the V. cholerae 01 positive control for APW, TCBS, and V. cholerae 01 antiserum agglutination
exhibit the appropriate response?
yes no
10
Did the E. coll (ATCC® 25922™) negative control for APW, TCBS, V. cholerae 01 antiserum
agglutination, API® 20E test strips exhibit the appropriate response?
yes no
Section 3. Sample Collection (Drinking water, surface water)
11
Sample collection date:
Time:
Sampler's initials:
12
Sample location:
13
Sample collection date:
Time:
Sampler's initials:
14
Sample location:
Section 4. Comments
D - 1
August 14, 2009
-------
V. cholerae 01
V. cholerae 01 Water Analyses
Spiking Suspension Enumeration
Laboratory:
1 % APW
Incubation
Start
Date:
1% APW
Incubation
End
Date:
TSA
Incubation
Start
Date:
TSA
Incubation
End
Date:
Time:
Time:
Time:
Time:
Temp:
Temp:
Temp:
Temp:
Initials:
Initials:
Initials:
Initials:
Step 1.
Lab-Prepared Spike Enumeration (TSA)
(Spiking Protocol, Section 4)
TSA inoculation
volume (mL)
Replicate 1
plate count
Replicate 2
plate count
Replicate 3
plate count
10"5
10"6
10"7
V. cholerae 01
undiluted spike
CFU1 + CFU2 + ... + CFUn
^ + v2+ ... + vn
Step 2.
' spiked V. cholerae 01
(V. cholerae 01
undiluted spike,
) x (V,
spiked per 100 mL sampled
D-2
August 14, 2009
-------
V. cholerae 01
V. cholerae 01 MPN Analyses - Drinking Water or PBS: 24 Hour Incubation
SPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Drinking Water PBS
Spike dilution:
Spike volume:
Spiking date and time:
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
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 01 Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
1/. cholerae 01 Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 01 Antisera
Agglutination, indicated by the formation of a white precipitate
D -3
August 14, 2009
-------
V. cholerae 01
V. cholerae 01 MPN Analyses - Surface Water or PBS: 6-8 Hour Incubation
SPIKED
Sample#:
Lab:
Date:
Matrix (circle one): Surface Water PBS
Spike dilution:
Spike volume:
Spiking date and time:
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
APW
(6 - 8 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 01 Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1XAPW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 01 Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 01 Antisera
Agglutination, indicated by the formation of a white precipitate
D -4
August 14, 2009
-------
V. cholerae 01
V. cholerae 01 MPN Analyses - Drinking Water or PBS: 24 hour Incubation
UNSPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Drinking Water PBS
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
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 01 Serum
Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 01 Results
Final Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 01 Antisera
Agglutination, indicated by the formation of a white precipitate
D -5
August 14, 2009
-------
V. cholerae 01
V. cholerae 01 MPN Analyses - Surface Water or PBS: 6-8 hour Incubation
UNSPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Surface Water PBS
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
APW
(6 - 8 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 01 Serum
Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 01 Results
Final Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 01 Antisera
Agglutination, indicated by the formation of a white precipitate
D -6
August 14, 2009
-------
V. cholerae 0139
Preliminary Verification Study: V. cholerae 01 Analyses Batch-Specific Cover Sheet
Laboratory: Date:
Section 1. Media and Sample Preparation
Media
Analyst Initials
Lot#
pH
Expiration Date
1
Date of APW preparation:
2
Date of TCBS plate preparation:
3
Date of TSA preparation:
4
V. cholerae 0139 antiserum
Section 2. Quality Control (please circle yes or no); if no, please explain in Section 4.
5
Did APW broth media sterility check exhibit the appropriate response?
yes no
6
Did TCBS media sterility check exhibit the appropriate response?
yes no
7
Did TSA media sterility check exhibit the appropriate response?
yes no
8
Did the method blank exhibit the appropriate response?
yes no
9
Did the V. cholerae 0139 positive control for APW, TCBS, and V. cholerae 0139 antiserum
agglutination exhibit the appropriate response?
yes no
10
Did the E. coll (ATCC® 25922™) negative control for APW, TCBS, V. cholerae 0139 antiserum
agglutination, API® 20E test strips exhibit the appropriate response?
yes no
Section 3. Sample Collection (Drinking water, surface water)
11
Sample collection date:
Time:
Sampler's initials:
12
Sample location:
13
Sample collection date:
Time:
Sampler's initials:
14
Sample location:
Section 4. Comments
D - 7
August 14, 2009
-------
V. cholerae 0139
V. cholerae 0139 Water Analyses
Spiking Suspension Enumeration
Laboratory:
1% APW
Incubation
Start
Date:
Time:
Temp:
Initials:
1% APW
Incubation
End
Date:
Time:
Temp:
Initials:
TSA
Incubation
Start
Date:
Time:
Temp:
Initials:
TSA
Incubation
End
Date:
Time:
Temp:
Initials:
Step 1.
Step 2.
Lab-Prepared Spike Enumeration (TSA)
(Spiking Protocol, Section 4)
TSA inoculation
volume (mL)
Replicate 1
plate count
Replicate 2
plate count
Replicate 3
plate count
10"5
10"6
10"7
Tspiked V. cholerae 0139 ( ^¦ Chol&fSG 0139 undiluted spike) X (Vgpjked per 100 mL sample)
V. cholerae 0139
undiluted spike
CFU-i +CFU2+ ... + CFUn
V! + V2+ ... + Vn
D -8
August 14, 2009
-------
V. cholerae 0139
V. cholerae 0139 MPN Analyses - Drinking Water or PBS: 24 Hour Incubation
SPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Drinking Water PBS
Spike dilution:
Spike volume:
Spiking date and time:
Please record plate counts and + / - for biochemical and serolo
gical analyses.
Sample Volume
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 01 Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 0139 Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 0139 Antisera
Agglutination, indicated by the formation of a white precipitate
D -9
August 14, 2009
-------
V. cholerae 0139
V. cholerae 0139 MPN Analyses - Surface Water or PBS: 6-8 Hour Incubation
SPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Surface Water PBS
Spike dilution:
Spike volume:
Spiking date and time:
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
APW
(6 - 8 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 01 Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 0139 Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 0139 Antisera
Agglutination, indicated by the formation of a white precipitate
D- 10
August 14, 2009
-------
V. cholerae 0139
V. cholerae 01 MPN Analyses - Drinking Water or PBS: 24 Hour Incubation
UNSPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Drinking Water PBS
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
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 01 Serum
Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 0139 Results
Final Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 0139 Antisera
Agglutination, indicated by the formation of a white precipitate
D- 11
August 14, 2009
-------
V. cholerae 0139
V. cholerae 0139 MPN Analyses - Surface Water or PBS: 6-8 Hour Incubation
UNSPIKED
Sample #:
Lab:
Date:
Matrix (circle one): Surface Water PBS
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
APW
(6 - 8 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 01 Serum
Agglutination
(+ or -)
20 mL
(5X APW)
1
2
3
10 mL
(2X APW)
1
2
3
1 mL
(1X APW)
1
2
3
Incubation
Start
Date:
Time:
Temp:
Initials:
Incubation
End
Date:
Time:
Temp:
Initials:
Analyses
V. cholerae 0139 Results
Final Results
APW
Growth, indicated by turbidity or pellicle at surface
MPN Tube Combination
TCBS
Large yellow colonies
MPN/100 mL
V. cholerae 0139 Antisera
Agglutination, indicated by the formation of a white precipitate
D- 12
August 14, 2009
-------
-------
Printed on 100% recycled/recyclable paper
with a minimum of 50% post-consumer
fiber using vegetable-based ink.
&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |