EPA/600/R-10/132 | October 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Single-Laboratory Verification
of Culture-Based Procedure
for Detection of Salmonella
Typhi in Drinking Water and
Surface Water
STUDY REPORT
Office of Research and Development
National Homeland Security Research Center
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for of
in
STUDY
Office of Research and Development
National Homeland Security Research Center
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This report presents results of a single-laboratory study (Study) funded by the National Homeland
Security Research Center (NHSRC) within the U.S. Environmental Protection Agency (EPA) Office
of Research and Development to verify culture-based analytical procedures for Salmonella enterica
subspecies (spp.) enterica serotype Typhi (S. Typhi) in water. The Study was designed under the
direction under the direction of EPA technical lead Sanjiv R. Shah of the NHSRC within EPA's
Office of Research and Development, with consultation and input provided by workgroup members
and subject matter experts. CSC provided coordination of activities for the Study, technical support
and data evaluation under EPA contract EP-C-05-045.
The contributions of the following persons and organizations are acknowledged:
Study Workgroup Participants
* Michele Burgess. Marissa Mullins (EPA. Office of Emergency Management)
* Stephanie Harris (EPA, Region 10)
• Sarah Perkins, Gene Rice (EPA, NHSRC)
« Malik Raynor, James Sinclair, Ouida Holmes (EPA, Office of Ground Water and Drinking
Water)
* Matthew Mikoleit (Centers for Disease Control and Prevention)
Subject Matter Experts
• Cheryl Bopp (Centers for Disease Control and Prevention)
« Nancy Hall (University of Iowa Hygienic Laboratory)
* Steve Weagant (U.S. Food and Drug Administration)
Volunteer Participant Laboratory
• Fu-Chih Hsu, Rebecca Wong (Scientific Methods, Inc.)
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This document has been reviewed in accordance with EPA policy and approved for publication.
Mention of trade names or commercial products does not constitute endorsement or recommendation
for use. Neither the United States Government nor any of its employees, contractors, or their
employees make and warranty, expressed or implied, or assume any legal liability or responsibility
for any third party's use of, or the results of such use of, any information, apparatus, product, or
process discussed in this document, or represent that its use by such party would not infringe on
privately owned rights. Mention of trade names or commercial products in this document or in the
methods referenced in this document does not constitute endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Sanjiv R. Shah
National Homeland Security Research Center
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue. NW
USEPA-8801RR
Washington, DC 20460
(202) 564-9522
shah, sanj iv@epa. gov
If you have difficulty assessing these PDF documents, please contact Nickel.Kathy@epa.gov or
McCall.Amelia@epa.gov for assistance.
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The mission of the U.S. Environmental Protection Agency (EPA) is to protect human health and to
safeguard the natural environment - the air, water, and land upon which life depends. After the 2001
terrorist attacks including the anthrax bioterrorism event the EPA's mission was expanded to address
critical needs related to homeland security. Presidential directives identified EPA as the primary
federal agency responsible for the protection and decontamination of indoor-outdoor structures and
water infrastructure vulnerable to chemical, biological, or radiological (CBR) terror attacks.
The National Homeland Security Research Center (NHSRC) within the Office of Research and
Development (ORD) is EPA's focal point for providing expertise, and for conducting and reporting
research to meet its homeland security mission needs. One specific focus area of the NHSRC's
research is to support the Environmental Response Laboratory Network (ERLN), a nationwide
association of federal, state, local, and commercial environmental laboratories, established by EPA.
The ERLN can be deployed in response to a large-scale environmental disaster to provide consistent
analytical capabilities, capacities, and quality data in a systematic and coordinated manner. To this
end, the NHSRC has worked with experts across EPA and other federal agencies to develop standard
analytical protocols (SAPs) to be used in support of the response to national homeland security
related incidents.
This report documents single-laboratory development and verification of quantitative procedures
included in a draft "Standard Analytical Protocol for Salmonella enterica subsp. enferica serotype
Typhi (Salmonella Typhi) in Water Samples."
NHSRC has made this publication available to assist in preparing for and recovering from disasters
involving Salmonella Typhi contamination. This work specifically represents an important step
in EPA's support for the ERLN and moves the agency closer to achieving its mission to support
homeland security and its overall mission to protect human health and the environment.
Oregon' D. Sayles, PhD., Acting Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
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of
Acknowledgments ii
Disclaimer iii
Foreword iv
Tables viii
Acronyms ix
Executive Summary xi
1.0 -Background 1
2.0 - Study Objectives and Design
2.1 - Study Objectives
2.2 - Data Quality Objective
2.3 - Study Preparation
2.3.1 - Identification of Laboratory
2.3.2 - Spiking Approach
2.4 - Sample Matrices 4
2.5 - Sample Analyses 4
2.6 - Quality Control Analyses 5
3.0-Study Schedule 7
4.0 - Data Reporting, Validation, and Censoring 9
4.1 - Data Reporting 9
4.2 - Data Validation 9
4.3 - Censored Data 9
5.0-Results 11
5.1- Preliminary Analyses of S. Typhi in PB S, Drinking Water, and Surface Water 11
5.2 - Optimization Analyses of S. Typhi inPBS, Drinking Water, and Surface Water 12
5.3 - Verification Analyses ofS. Typhi in PBS, Drinking Water, and Surface Water 14
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5.4-Optimization Analyses for Surface Water 15
5.4.1 - Evaluation of Commercially-Available Immunomagnetic Separation
(IMS) Beads 15
5.4.2 - Evaluation of Surface Water Matrix Inhibition of Immunomagnetic
Separation (IMS) Protocol 15
5.4.3 - Evaluation of Laboratory-Conjugated Immunomagnetic Separation
(IMS) Beads 15
6.0 -Discussion 17
7.0 - Conclusions 19
8.0 - References 21
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Note: Appendices are attached to the final report in the final form that was distributed to the
workgroup and laboratory and may contain minor differences in language in comparison to
the Study Report.
Appendix A - Study Plan A-l
Appendix B - Study-Specific Instructions B-l
Appendix C - Spiking Protocol C-l
Appendix D - Data Reporting Forms D-l
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Table 1. Summary of Sample Analyses .
Table 2. Schedule for Laboratory Analyses of S. Typhi Culture-Based Procedures
in Water Matrices 7
Table 3. Preliminary Analyses. Results: PBS. Drinking Water, and Surface Water Spiked
withS. Typhi 11
Table 4. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water
(without Pre-Enricfanient in UPD Broth) Spiked with S. Typhi 12
Table 5. Optimization Analyses. Results: PBS, Drinking Water, and Surface Water
(with Pre-Enrichment in UPD Broth) Spiked with S. Typhi 13
Table 6. Verification Analyses, Results: PBS, Drinking Water, and Surface Water Spiked
wit.h,S'. Typhi 14
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ASTM
ATCC®
BSD
°C
CFU(s)
CDC
CVD
DHB
EPA
HEPA
IMS
MMD
MPN
NA
NHSRC
OEM
ORD
ow
OGWDW
PBS
PCR
QA
QC
RSD
SAM
SAP
SD
SCBD
TNTC
TSAD
TSB
American Society for Testing and Materials
American Type Culture Collection
Bismuth sulfite agar with DHB
Degrees Celsius
Colony forming unit(s)
Centers for Disease Control and Prevention
Centers for Vaccine Development
2.3-dihydroxybenzoate (indicated by subscript "D" when used with media acronym)
U.S. Environmental Protection Agency
High efficiency paniculate air
Immunomagnetic separation
Miller-Mallinson agar with DHB
Most probable number
Not applicable
National Homeland Security Research Center
Office of Emergency Management
Office of Research and Development
Office of Water
Office of Ground Water and Drinking Water
Phosphate buffered saline
Polymerase chain reaction
Quality assurance
Quality control
Relative standard deviation
Standardized Analytical Methods for Environmental Restoration Following Homeland
Security Events
Standard Analytical Protocol
Standard deviation
Seleriite cystine broth with DHB
Too numerous to count
Tryptic soy agar with DHB
Tryptic soy broth
Universal prc-cnrichmcnt with DHB
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This report presents the results of the U.S.
Environmental Protection Agency's (EPA's) single-
laboratory verification study (the "Study") to evaluate a
draft culture-based Standard Analytical Protocol (SAP)
for the identification and quantitation of Salmonella
Typhi in drinking water and surface water samples.
The original procedure evaluated during the Study was
adapted from Section 9260B, "General Qualitative
Isolation and Identification Procedures for Salmonella"
in Standard Methods for the Examination of Water and
Wastewater. After workgroup review, the procedure, as
is. was determined to be unacceptable and was revised.
The procedure evaluated during the Study was adapted
from the Manual of Clinical Microbiology, "Escherichia,
Shigella, and Salmonella'' and the U.S. Food and Drag
Administration's Bacteriological Analytical Manual
[online]. "'Salmonella''' The purposes of the Study were
to: (1) evaluate draft SAP performance (recover}' and
precision) in a reference matrix (phosphate buffered
saline [PBSJ), (2) evaluate draft SAP performance
(recoveiy and precision) in environmental matrices
of interest (drinking water, surface water), and (3)
determine whether the draft SAP requires revision prior
to multi-laboratory validation.
During the Study, the analytical laboratory analyzed
unspiked and spiked PBS, drinking water, and surface
water samples. Vaccine strains were used to reduce
health risk to laboratory personnel. This strain has an
engineered nutritional defect that requires a supplement
(2,3-dihydroxybenzoate [DHB]) be added to all media to
ensure proper growth. The Study was conducted during
September 2008 through Ja.nua.ty 2010.
Some issues were observed during the Study, including
(1) all Salmonella O Group D antiserum tests were
negative, and (2) S. Typhi was not recovered effectively
recovered from surface water samples. The Salmonella
O Group D antiserum tests were likely negative because
the stain selected for the study constitutively produces
Vi, a surface antigen that can mask other surface
antigens. To address this issue,
testing with Salmonella O Group D antiserum was
discontinued, and serological typing with Salmonella Vi
antiserum combined with biochemical analyses was used
for confirmation. It was concluded that the laboratory
was not able to effectively recover S. Typhi from surface
water samples due to background organisms. Based on
workgroup recommendations, the laboratory evaluated
a number of potential procedural modifications, such
as the use of immunomagnetic separation (IMS)
beads. Unfortunately, none of the procedures provided
acceptable results for this matrix. The final optimized
procedure included two plating media, bismuth sulfite
(BSD) and Miller-Mallinson (MMD) agars. For spiked
PBS samples, mean recoveries were 71% and 94% for
BSD and MMD, respectively. For spiked drinking water
samples, mean recoveries were 307% for both plating
media (BSD and MMD). Mean recoveries of S Typhi
CVD 909 in surface water samples were 0% for both
plating media.
Results of the single-laboratory validation study indicate
that these procedures merit multi-laboratory validation
to assess method performance, and set quantitative
QC criteria for PBS (reference matrix) and drinking
water. However, for surface water, the culture-based
procedure evaluated during this study is not appropriate
and development and inclusion of molecular procedures
(e.g., PCR) for detection of S. Typhi in surface water
samples is recommended.
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1.0
Background
Subsequent to the anthrax attacks in the fall of 2001,
federal and state personnel were tasked with a mission
to provide response, recovery, and remediation for
biological incidents. However, it was recognized that no
U.S. Environmental Protection Agency (EPA) verified
or validated protocol exists for collection, isolation, and
analysis of Salmonella enterica subsp. enterica serotype
Typhi (S. Typhi). a potential bioterrorism agent and the
causative agent of typhoid fever, in water samples.
This report, presents results of llie single-laboratory
verification study (Study) to evaluate EPA's draft
Standard Analytical Protocol (SAP) (EPA, National
Homeland Security Research Center) culture-based
procedure for the identification and quantilation of 5'.
Typhi in water samples. The original SAP was based
on Section 9260B, "General Qualitative Isolation
and Identification Procedures for Salmonella,"' in
Standard Methods for the Examination of Water
and Wastewater (Reference 8.1). After workgroup
review, the procedure, as is, was determined to be
unacceptable and was revised. The procedure evaluated
during the Study was adapted from the Manual of
Clinical Microbiology, "Escherichia, Shigella, and
Salmonella*' (Reference 8.2) and the U.S. Food and Drag
Administration's Bacteriological Analytical Manual
[online], "Salmonella** (Reference 8.3). To reduce
the potential health risk to laboratory personnel, an
attenuated (vaccine) strain of S. Typhi (CVD 909) was
obtained from Centers for Vaccine Development (CVD).
University of Maryland, Baltimore, Maryland, for the
Study. This sixain has an engineered nutritional defect
that requires a supplement (2,3-dihydroxybenzoate
[DHBD be added to all media to ensure proper growth
(indicated by a subscript "D" after the medium
acronym).
The procedure recommended by the workgroup for
initial evaluation included: (1) enriching in universal
prc-cnrichment broth supplemented with DHB (UPD) at
35.0°C ± 0.5°C for 24 ±2 hours, (2) transferring 1 niL to
selenite cystine broth with DHB (SCBD) and incubating
at 35.0°C ± 0.5°C for 24 ± 2 hours, (3) streaking growth
from SCBD onto bismuth sulfitc agar with DHB (BSD)
and incubating at 35.0°C ± 0.5°C for 24 ±2 hours, (4)
sub-culturing typical colonies onto tryptic soy agar with
DHB (TSAD) and incubating at 35.0°C ± 0.5°C for 24
± 2 hours, and (5) verifying isolates using scrological
analyses against Salmonella Vi and O Group D antigens.
Due to issues withBSD (e.g., tiny colonies, no growth
at 24 hours) observed during preliminary analyses, the
workgroup recommended adding an additional selective
plating medium (Miller-Mallinson with DHB [MMJ) to
the procedure.
Quanlitation of S. Typhi was determined using the
most probable number (MPN) technique. Tubes that
confirmed positive for & Typhi were used to determine
the MPN (Reference 8.4). It should be noted that a MPN
value is not considered absolute quantilation, as a direct
plate count would be, because values are based on the
probability of a tube being positive for a given organism
concentration.
Based on workgroup discussion, a nine-tube MPN was
utilized for the Study to reduce the burden (306 tubes
and 612 plates for a nine-tube MPN versus 510 tubes and
1020 plates for a fifteen-tube MPN) on the participant
laboratory. All of the media required the addition of
DHB after autoclaving, resulting in the laboratory having
to sterilize tubes prior to manually dispensing the media
into each tube using aseptic technique in a biological
safety cabinet. A 15-tube MPN will be considered for
the multi-laboratory validation study.
Initially, all tests with Salmonella O Group D antiseram
were negative, likely because the strain selected for the
study constitutively produces Vi, a surface antigen that
can mask other surface antigens. Based on workgroup
recommendation, the laboratory boiled the culture prior
to serological testing with O Group D. However, this
approach also did not provide acceptable results. Based
on workgroup recommendation, testing with Salmonella
O Group D antiseram was discontinued and serological
typing with Salmonella Vi antiserum combined with
biochemical analyses was used for confirmation.
Due to background organisms that may have overgrown
or inhibited the target organism, the laboratory was not
able to effectively recover S. Typhi from surface water
samples. Based on workgroup recommendations, the
laboratory evaluated a number of potential procedural
modifications, such as the use of immunomagnetic
separation (IMS) beads. Unfortunately, none of Hie
procedures provided acceptable results for this matrix.
For drinking water, the final verified procedure included:
(1) enriching in UPD at 35.0°C ± 0.5°C for 24 ± 2 hours,
(2) transferring 1 mL to SCBD and incubating at 35.0°C
± 0.5°C for 18 ± 2 hours. (3) streaking growth from
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SCBD cultures onto BSD and MMD agars and incubating
at 35.0°C ± 0.5°C for 24 - 48 hours, (4) sub-culturing
typical colonies onto TSAD, and (5) verifying isolates
using serological (Salmonella Vi) and biochemical (API®
20E test strips) analyses. Time to results, as verified, is
approximately 120 - 144 hours from receipt of samples.
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2.0
Study Objectives and Design
The primary objective of the Study was to evaluate the
performance of the culture-based procedure included
in the draft SAP for identification and quantitation
of S. Typhi in drinking water and surface water. As
indicated in the Study Plan (Appendix A), the Study
was originally designed to verify the procedure for
water, solid, and paniculate matrices. However,
during the Study, only water matrices were evaluated,
and only these are included in this report. Additional
matrices may be evaluated in the future. In support
of the primary objective of evaluating the draft SAP,
both study objectives (Section 2.1) and data quality
objectives (Section 2.2) were set for the Study. The
Study preparation, sample matrices, and sample analysis
selected to meet these objectives is described in Sections
2.3 - 2.5, below.
2.1 -
• Evaluate draft culture-based SAP performance
(recovery and precision) for a reference matrix
(phosphate buffered saline |PBSJ)
* Evaluate draft culture-based SAP performance
(recovety and precision) for water matrices of
interest (drinking water, surface water)
* Determine whether the draft culture-based SAP
requires revision prior to multi-laboratory validation
To accomplish these objectives, the Study was conducted
in five phases, as described below:
• Phase 1.Planning and Preparation: Identification
of a qualified laboratory to participate in the Study,
identification of an appropriate strain of £ Typhi.
and evaluation of the Spiking Protocol (Appendix
C)
* Phase 2. Preliminary Analyses: Assessment
of modified procedure in three matrices (PBS
[reference matrix], drinking water, surface water)
and identification and resolution of any analytical
problems
• Phase 3. Optimization Analyses for all Matrices:
Evaluation of the procedure with and without
pre-enrichment (UPD broth), an additional plating
medium (MMD), and boiling prior to O Group D
serological analyses
« Phase 4. Verification Analyses: Analyses of PBS,
drinking water, and surface water samples using the
optimized procedure
* Phase 5. Optimization Analyses for Surface
Water: Evaluation of commercially-available
and laboratory-conjugated IMS beads and matrix
inhibition
2.2 - 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.
2.3 - Study
Prior to the Study, the following activities were
completed, including identification of an appropriate
laboratory and identification of an appropriate strain of
S. Typhi and development and evaluation of the Spiking
Protocol (Appendix C).
2.3.1 - Identification of Laboratory
A laboratory was identified that was representative of
the general user community, had experience analyzing
environmental samples for Salmonella spp.. and had
access to representative matrices. To reduce Study costs,
a volunteer laboratory (Scientific Methods, Inc.) was
recruited. To reduce the burden on the laboratory and
encourage participation. National Homeland Security
Research Center (NHSRC) provided the media, reagents,
and supplies needed for the Study. The requirements and
responsibilities of the laboratory are detailed in Study-
specific instructions (Appendix B) and the draft SAP.
2.3.2 - Spiking Approach
The Study Plan (Appendix A) included the use of two
spike types: BioBalls™ (precise pre-enumerated spikes)
commercially-developed from S. Typhi CVD 909 and
laboratory-prepared spiking suspensions. However, due
to manufacturer development issues, BioBalls were not
available during the Study.
During each week of the Study, laboratory-prepared
spiking suspensions were propagated according to the
Spiking Protocol (Appendix C) using S. Typhi CVD 909.
To determine spike level, the cells were enumerated
in triplicate on TS AD using Hie spread plate technique
described in the Spiking Protocol (Appendix C). Spikes
were enumerated on the same day that samples were
spiked and analyzed. Results were reported as colony
forming units (CPU) and used to determine Hie actual
spike level.
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2.4 - Sample Matrices
The procedure was evaluated and verified using three
water matrices (one reference and two matrices of
interest). The reference matrix provided a means for
assessing the performance of the draft SAP using a
standard matrix that could be duplicated during routine
use of the procedure in the future. The following
matrices were analyzed during the Study:
Reference Matrix
• PBS
Water Matrices (Matrices of Interest)
• Drinking water (laboratory tap, dechlorinated with
sodium thiosulfate)
• Surface water (source water for drinking water
treatment plants)
Table 1. Summary of Sample Analyses
2.5 - Sample Analyses
For preliminary analyses, one, 100-mL unspiked
and one, 100-mL spiked samples were evaluated per
matrix. For optimization and verification analyses,
one, 100-mL unspiked PBS or two, 100-mL unspiked
drinking or surface water samples, as appropriate, were
evaluated by the procedure to determine background S.
Typhi concentrations. In addition, four, 100-mL spiked
samples were evaluated per matrix. Table 1 summarizes
the number and type of samples that were evaluated to
meet the objectives listed in Section 2.1.
Analysis
Matrix
Spiking Description
Procedure
No. of Analyses
Phase 2 Analyses
Preliminary Analyses
Sterile PBS (Reference
Matrix)
Drinking Water
Surface Water
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Assessment of workgroup-
modified procedure
1
1
1
1
1
1
Phase 3 Analyses
Optimization Analyses
Sterile PBS (Reference
Matrix)
Drinking Water
Surface Water
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Evaluation with and
without pre-enrichment
(UPD broth)
1
4
2
4
2
4
Phase 4 Analyses
Verification Analyses
Sterile PBS (Reference
Matrix)
Drinking Water
Surface Water
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Analyses of PBS, drinking
water, and surface
water samples using the
optimized procedure
1
4
2
4
2
4
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Analysis
Matrix
Spiking Description
Procedure
No. of Analyses
Phase 5 Analyses
Optimization Analyses for
Surface Water
Sterile PBS (Reference
Matrix)
Surface Water
Surface Water
Sterile PBS (Reference
Matrix)
Surface Water
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Unspiked
S. Typhi CVD 909
Evaluation of
commercially-available
IMS beads
Determination of matrix
inhibition
Evaluation of laboratory-
conjugated IMS beads
1 - 4<«
1 - 2<«
l_4
(1) Evaluation of IMS technique was conducted with variable numbers of analyses
CVD - Centers for Vaccine Development PBS - Phosphate buffered saline
IMS - Immunomagnetic separation; UPD - Universal pre-enrichment with 2,3-dihydroxybenzoate
2.6 - Quality Control Analyses
The participant laboratory performed the following QC
analyses:
• Method Blank: The laboratory analyzed a sterile
unspiked PBS method blank during each week
of analyses to verify the sterility of equipment,
materials, and supplies.
• Sterility Checks: To evaluate the sterility of media
and buffer, the laboratory incubated a representative
portion of each batch at 35 ± 0.5 (PBS, UPD broth,
SCBD, BSD, MMD, and TSAD) for 24 ± 2 hours or 48
± 3 hours (as appropriate) and observed for growth.
In addition, sterility checks were conducted each
day samples were run.
Positive and Negative Controls: For the purpose
of the Study, positive controls for selective agars
and broths are those organisms that provide the
characteristic growth and/or colony morphology of
the target organism. Negative controls are those
organisms that do not provide the characteristic
target organism growth or morphology. For
biochemical and serological analyses, positive and
negative controls are defined by their reaction (e.g.,
Pseudomonas aeruginosa is oxidase positive and S.
Typhi CVD 909 is oxidase negative). The following
positive and negative controls were evaluated during
each week of the Study:
• S. Typhi CVD 909: Positive controls (target
organisms)
• Enterococcusfaecalis (ATCC® 29212™): Negative
control (non-target organism)
• Pseudomonas aeruginosa (ATCC® 27853™):
Positive control for oxidase test and negative
control for biochemical tests
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3.0
Study Schedule
The duration of the Study was September 2008 through
January 2010. Due to laboratory and workgroup
scheduling conflicts, availability of commercial products,
and development and evaluation of IMS procedures,
the Study time frame was extended approximately six
months beyond the anticipated schedule. The Study
schedule is provided in Table 2. Analyses of additional
matrices may be conducted at a later date.
Table 2. Schedule for Laboratory Analyses of S. Typhi Culture-Based Procedures in Water Matrices
Date
September - October 2008
November 2008
December 2008
December 2008 - January 2009
May - December 2009
Analysis Phase
Phase 1 - Spiking Protocol Evaluation
Phase 2 - Preliminary Analyses
Phase 3 - Optimization Analyses for all Matrices
Phase 4 - Verification Analyses
Phase 5 - Optimization Analyses for Surface Water
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4.0
Data Reporting, Validation, and Censoring
4.1 -
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
* Documentation of any additional information that
would assist in evaluating the data
4.2 -
CSC used data review checklists to ensure that each data
package was complete and that each sample result met
the Study-specific and method-specific requirements.
The review for each sample confirmed the following:
• Original forms were submitted
« Incubation times were met
* Incubation temperatures were met
• Media sterility checks were performed and
acceptable
• Positive and negative controls were performed and
exhibited the appropriate response
* Samples were spiked with the appropriate dilution
* All procedures were performed according to study-
specific instructions and analytical procedures
• Calculations were correct
This process was perfonned independently by two data,
reviewers, each of whom entered the results into separate
spreadsheets designed for data review and validation
for this Study. The results spreadsheets were evaluated
using the compare function in Microsoft® Excel® to
verify consistency and identify potential data entry
errors.
All verification data generated during the Study were
considered valid and were included in subsequent data
analyses, as appropriate.
4.3 -
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 S.
Typhi CVD 909). The censoring limit (the "less than"
value itself [1.08J) was used in data analysis for these
samples.
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5.0
Results
This section includes results from preliminary analyses
(Section 5.1), optimization analyses for all matrices
(Section 5.2), verification analyses for all matrices
(Section 5.3), and optimization analyses for surface
water (Section 5.4). Only valid results are included in
this section.
5.1 - Preliminary Analyses of S. Typhi in
PBS, Drinking Water, and Surface Water
During preliminary analyses, the procedure evaluated
included: (1) pre-enriching in UPD broth for 24 ± 2
hours at 35.0°C ± 0.5°C, (2) transferring 1 mL to SCBD
and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, (3)
streaking growth onto BSD and incubating for 24 ± 2
hours at 35.0°C ± 0.5°C, (4) sub-culturing onto TSAD
and incubating for 24 ± 2 hours at 35.0°C ± 0.5°C, and
(5) serological confirmation with Salmonella Vi and
O Group D antisera (biochemical analyses were not
conducted during preliminary analyses). Results of
preliminary analyses are provided in Table 3.
During preliminary analyses four issues were identified:
• Serology. Typical colonies were positive for Vi
antiserum but negative for O Group D antiserum.
The vaccine strain used for analyses (S. Typhi CVD
909) constitutively produces the Vi capsular antigen.
This may mask the O Group D antigen, producing a
false-negative response.
• BSD Incubation. No growth was observed on the
BSD plates at 24 hours; a minimum incubation of
40 hours was required before typical (green-black
with metallic sheen, surrounded by blackish halo)
colonies were observed by the laboratory.
• Recoveries of S. Typhi in Surface Water. S. Typhi
recoveries were poor, likely due to overgrowth of
non-target organisms.
• SCBD Incubation. During preliminary analyses.
selective enrichment (SCBD) was for 24 ± 2
hours. The selective components in the medium
may degrade if incubation is longer than 24 hours.
possibly contributing to overgrowth of non-target
organisms in the surface water samples.
These issues were discussed by the workgroup and
addressed, based on workgroup recommendations, as
described in Section 5.2.
Table 3. Preliminary Analyses, Results: PBS, Drinking Water, and Surface Water Spiked with S. Typhi0
Sample ID
Spike Level
(CFU/100 mL)
MPN Combo
S. Typhi
(MPN/100 mL)
Recovery (%)
PBS Samples
Unspiked
Spiked
NA
49.7
0-0-0
3-3-0
<1.08
23.98
NA
46
Drinking Water Samples
Unspiked
Spiked
NA
49.7
0-0-0
2-3-1
<1.08
12.88
NA
24
Surface Water Samples
Unspiked
Spiked
NA
49.7
0-0-0
0-0-3
<1.08
3.27
NA
4
(1) For unspiked samples, the "<" values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08)
CPU - Colony forming units NA - Not applicable
MPN - Most probable number PBS - Phosphate buffered saline
-------
5.2 - Optimization Analyses of S. Typhi
in PBS, Drinking Water, and Surface
Water
In an effort to address the issues identified during
preliminary analyses, the workgroup made the following
recommendations: (1) add a boiling step to the procedure
to disrupt the Vi capsular antigen, (2) decrease the
incubation time for SCBD to 18 ± 2 hours, (3) increase
the incubation time for BSD to 24 - 48 hours, 4) add
an additional selective plating medium, MMD, and (4)
evaluate the procedure with and without pre-enrichment
in UPD broth.
The procedure evaluated during optimization analyses
included: (1) pre-enriching in UPD broth for 24 ± 2
hours at 35.0°C ± 0.5°C, (2) transferring 1 mL to SCBD
and incubating for 18 ± 2 hours at 35.0°C ± 0.5°C,
(3) streaking growth onto BSD and MMD agars and
incubating for 24 - 48 hours at 35.0°C ± 0.5°C, (4) sub-
culturing onto TSAD and incubating for 24 ± 2 hours at
35.0°C ± 0.5°C, (5) serological testing with Salmonella
Vi and O Group D antisera (cells boiled prior to testing).
and (6) biochemical testing with API 20E® test strips.
UPD broth was omitted for samples evaluated without
pre-enrichment. Results are provided in Table 4 (without
pre-enrichment) and Table 5 (with pre-enrichment)
Table 4. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water (without Pre-Enrichment in
UPD Broth) Spiked with S. Typhi<»>
Sample ID
Medium
Spike Level
(CFU/100 mL)
MPN Combo
S. Typhi
Percent
Recovery
PBS
Unspiked
Spiked #1
Spiked #2
BSD
MMD
BSD
MMD
BSD
MMD
NA
NA
32
32
32
32
0-0-0
0-0-0
0-3-0
0-3-0
0-3-0
0-3-0
<1.08
<1.08
3.895
3.895
3.895
3.895
NA
NA
9
9
9
9
Drinking Water
Unspiked
Spiked #1
Spiked #2
BSD
MMD
BSD
MMD
BSD
MMD
NA
NA
32
32
32
32
0-0-0
0-0-0
0-0-0
0-2-0
0-2-0
0-3-0
<1.08
<1.081
<1.08
2.421
2.421
3.895
NA
NA
0
4
4
9
Surface Water
Unspiked
Spiked #1
Spiked #2
BSD
MMD
BSD
MMD
BSD
MMD
NA
NA
32
32
32
32
0-0-0
1-0-0
1-1-1
0-0-1
1-1-0
0-0-0
<1.08
1.257
3.955
1.08
2.6
<1.08
NA
NA
9
-1
5
-1
(1) For unspiked samples, the "<" values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08)
BSD - Bismuth sulfite agar with DHB; MPN - Most probable number
CPU - Colony forming units NA - Not applicable
DHB - 2,3-dihydroxybenzoate PBS - Phosphate buffered saline
MMD - Miller-Mallinson agar with DHB.
-------
Table 5. Optimization Analyses, Results: PBS, Drinking Water, and Surface Water (with Pre-Enrichment in
UPD Broth) Spiked with S. Typhi<»>
Sample ID
Medium
Spike Level
(CFU/100 mL)
MPN Combo
S. Typhi
Percent
Recovery
PBS
Unspiked
Spiked #1
Spiked #2
BSD
MMD
BSD
MMD
BSD
MMD
NA
NA
32
32
32
32
0-0-0
0-0-0
2-3-1
2-3-1
2-3-3
2-3-3
<1.08
<1.08
12.88
12.88
19.33
19.33
NA
NA
37
37
57
57
Drinking Water
Unspiked
Spiked #1
Spiked #2
BSD
MMD
BSD
MMD
BSD
MMD
NA
NA
32
32
32
32
0-0-0
0-0-0
3-3-2
3-3-2
3-3-2
3-3-2
<1.08
<1.08
109.9
109.9
109.9
109.9
NA
NA
340
340
340
340
Surface Water
Unspiked
Spiked #1
Spiked #2
BSD
MMD
BSD
MMD
BSD
MMD
NA
NA
32
32
32
32
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
NA
NA
0
0
0
0
(1) For unspiked samples, the "<" values were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08)
BSD - Bismuth sulfite agar with DHB; MPN - Most probable number
CPU - Colony forming units
DHB - 2,3-dihydroxybenzoate
MMD - Miller-Mallinson agar with DHB.
NA - Not applicable
PBS - Phosphate buffered saline
Originally, optimization analyses were to be conducted
in two runs. Recoveries of S. Typhi in samples evaluated
without pre-enrichment were poor (< 9%), during the
first run for all three matrices. Therefore, the second
optimization run was not conducted and all subsequent
analyses included pre-enrichment and the modifications
to the method noted above (e.g., decreased SCBD
incubation period) with the exception of boiling cells
prior to serological typing with O Group D antiserum.
The boiling step was eliminated following optimization
analyses because it did not provide acceptable results.
Based on CVD input, the workgroup requested that
the laboratory use a growth medium with 0.5 M salt
concentration to decrease Vi production prior to
serological analyses with O Group D antiserum. The
modified growth medium did not provide acceptable
results; isolates were also negative for O Group D
antiserum.
-------
5.3 - Verification Analyses of S. Typhi in
PBS, Drinking Water, and Surface Water
Verification analyses were conducted in two runs. These
runs resulted in the analyses of four spiked samples for
each matrix and selective plating medium (BSD and
MMD). Verification results are provided in Table 6.
Table 6. Verification Analyses, Results: PBS, Drinking Water, and Surface Water Spiked with S. Typhi*1'
Date
Sample ID
Spike Level
(CFU/100 mL)
Medium
MPN
Combo
S. Typhi
(MPN/100 mL)
Recovery
(%)
Mean
Recovery (%)
SDb
(%)
RSDc
(%)
PBS Samples
12/16/08
1/09/09
12/16/08
1/09/09
12/16/08
1/09/09
Unspiked
Spiked
Spiked
NA
NA
32
24
32
24
NA
NA
BSD
MMD
0-0-0
0-0-0
2-3-1
2-3-3
3-3-0
3-3-0
2-3-1
2-3-3
3-3-1
3-3-0
<1.08
<1.08
12.88
19.33
23.98
23.98
12.88
19.33
46.22
23.98
37.00
57.00
95.41
95.41
37.00
57.00
188.08
95.41
71.18
94.35
29.16
67.04
40.97
71.06
Drinking Water Samples
12/16/08
1/9/09
12/16/08
1/9/09
12/16/08
1/9/09
Unspiked
Unspiked
Spiked
Spiked
NA
NA
32
24
32
24
NA
NA
BSB
MMD
0-0-0
0-0-0
0-0-0
0-0-0
3-3-2
3-3-2
3-3-0
3-3-2
3-3-2
3-3-2
3-3-0
3-3-2
<1.08
<1.08
<1.08
<1.08
109.9
109.9
23.98
109.9
109.9
109.9
23.98
109.9
340.00
340.00
95.41
453.41
340.00
340.00
95.41
453.41
307.24
307.24
150.99
150.99
49.14
49.14
Surface Water Samples
12/16/08
1/9/09
12/16/08
1/9/09
12/16/08
1/9/09
Unspiked
Unspiked
Spiked
Spiked
NA
NA
32
24
32
24
NA
NA
BSD
MMD
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
0-0-0
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
<1.08
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
NA
NA
(1) For unspiked samples, the "<" values
SD - Standard deviation
were replaced with the censoring limit (e.g., <1.08 was replaced with 1.08) for calculation of summary statistics
RSD - Relative standard deviation
-------
Results of verification analyses for PBS and drinking
water were deemed acceptable by the workgroup;
however, results for surface water were unacceptable.
Therefore, the workgroup recommended additional
optimization of the procedure for surface water samples.
5.4 - Optimization for
Water
To improve S. Typhi recoveries in surface water, the
workgroup recommended that the laboratory evaluate
the use of commercially-available Salmonella spp. IMS
beads (described in Section 5.4.1, below). When these
results were unacceptable, the laboratory evaluated
surface water matrices to determine if matrix inhibition,
independent of background organisms, could be
affecting recoveries (Section 5.4.2); the laboratory then
repeated the analyses using laboratory-conjugated IMS
beads (Section 5.4.3).
5.4.1 - Ewaluation of Commercially-Available Im-
munomagnetic Separation (IMS)
Commercially available IMS beads specific for
Salmonella spp. (Dynabeads® anti-Salmonella
product No. 710.02) were evaluated for isolation and
concentration of S. Typhi CVD 909 from surface water
samples. The manufacturer's instructions developed for
samples with high background flora was used to evaluate
samples. The procedure included: (1) pre-enriching
in UPD broth for 24 ± 2 hours at 35.0°C ± 0.5°C, (2)
processing 1 mL of UPD broth from each tube for IMS
according to manufacturer's instructions, (3) transferring
bead complex to SCBD and incubating for 18 ± 2 hours
at 35.0°C ± 0.5°C, (4) streaking growth from SCBD onto
BSD and incubating for 24 - 48 hours at 35.0°C ± 0.5°C,
(5) sub-culturing onto TSAD and incubating for 24 ±
2 hours at 35.0°C ± 0.5°C, (6) serological testing with
Salmonella Vi antiserum, and (7) biochemical testing
with API 20E® test strips on serology-positive isolates.
* May 2009 Evaluation. Recoveries of S
Typhi CVD 909 in PBS samples spiked with
approximately 50 CPU were 92%. S. Typhi CVD
909 was not recovered from surface water samples
spiked at the same level.
* June 2009 Evaluation. Analyses were repeated
with the following procedural modifications: (1)
increasing bead-bacteria complex washes from
one to two, (2) omitting selective enrichment in
SCBD and plating beads onto BSD, (3) analyzing
an additional sample volume (0.1 mL) to dilute out
possible inhibitors, and (4) analyses of two surface
water samples, each from a different source. S.
Typhi was recovered from spiked PBS samples
but was not recovered from spiked surface water
samples.
5.4.2 - Evaluation of Surface Water Matrix In-
hibition of Immunomagnetic Separation (IMS)
Protocol
Analyses were conducted in July 2009 to determine
if the matrix (surface water samples), independent of
background organisms, may be inhibiting IMS, resulting
in poor recoveries of S. Typhi CVD 909. Matrix
inhibition analyses included: (1) centrifuging samples to
remove background organisms, (2) spiking supernatant
with S. Typhi CVD 909 approximately 50 CPU, (3)
pre-enriching in UPD broth for 24 ± 2 hours at 35.0°C
± 0.5°C, (4) processing 1 mL of UPD broth from each
tube with commercially available IMS beads, (5) plating
bead complex onto BSD and incubating for 24 - 48
hours at 35.0°C ± 0.5°C, (6) sub-culturing onto TSAD
and incubating for 24 ±2 hours at 35.0°C ± 0.5°C, (7)
serological testing with Salmonella Vi antiserum, and
(8) biochemical testing with API 20E® test strips if Vi
serology-positive.
S. Typhi CVD 909 was recovered from these spiked
surface water samples. These results suggest that
the background organisms, which were removed by
centrifugation, may have caused the poor recoveries in
surface water. However, it is difficult to say for certain
because other potentially inhibitory components (e.g.,
organics, chemicals) in the surface water may have also
been removed during the centrifugation process.
5.4.3 - Evaluation of Laboratory-Conjugated Im-
munomagnetic (IMS)
Laboratory-conjugated, tosylactivated IMS beads
(Dynabeads® MyOne™ product No. 655-01) were
evaluated for isolation and concentration of S.
Typhi CVD 909 from surface water samples. The
manufacturer's instructions were followed for
conjugation of Salmonella Vi antiserum to the beads.
Analyses were conducted using the same procedure that
was used for IMS analyses conducted in June 2009 (see
Section 5.4.1). Surface water analyses were conducted
twice during December 2009, using samples from two
different sources. The laboratory continued to observe
background organisms from surface water samples
but was not able to recover S. Typhi CVD 909. The
target organism was successfully recovered from PBS
processed using the same approach.
-------
-------
6.0
Discussion
The culture-based procedure in the draft SAP was
evaluated for identification and quantitation of S. Typhi
in water matrices. As indicated in Section 2, solid and
particulate matrices were not evaluated during this phase
of the Study.
Following preliminary analyses, the procedure was
modified based on workgroup recommendations.
Optimization analyses were conducted to evaluate these
modifications and to determine if pre-enrichment was
necessary. During optimization analyses, spiked samples
evaluated using pre-enrichment resulted in recoveries of
37% - 57%, 340%, and 0% for PBS, drinking water, and
surface water, respectively. Although it may appear that
the drinking water matrix may be enhancing recoveries,
it should be noted that a total of eight tubes were positive
for both PBS (57%) and drinking water (340%). The
difference in recoveries were due to Hie actual MPN
combinations, 2-3-3 versus 3-3-2, resulting in MPN
values of 19.33 and 109.9 for PBS and drinking water,
respectively. The resulting combos may be indicative
of the variability associated with MPN. It should also
be noted that an MPN value is not considered absolute
quantitation, as a direct plate count would be, because
values are based on the probability of a tube being
positive. For spiked samples evaluated without pre-
enrichment recoveries were less than 10% for all three
matrices. The workgroup agreed that pre-enrichment
was necessary; all subsequent analyses included pre-
enrichment in UPD broth.
Based on the results of optimization analyses for
drinking water and PBS, the workgroup gave the
laboratory approval to move forward with verification
analyses. For spiked PBS samples, mean recoveries
were 71% and 94% for BSD and MMD, respectively.
For spiked drinking water samples, mean recoveries
were 307% for both plating media (BSD and MMD).
Mean recoveries of S. Typhi CVD 909 in surface water
samples were 0% for both plating media. In addition
to the laboratory's inability to recover 5'. Typhi from
spiked surface water samples, all isolates were negative
for O Group D antigen during serological testing. CVD
was contacted to discuss the serological results and
confirm that CVD 909 does produce O Group D antigen.
It was recommended that the laboratory use growth
medium with 0.5 M salt concentration to decrease Vi
production prior to serological analyses with O Group
D antiserum. The salt concentration did not decrease
Vi production sufficiently: isolates were also negative
for O Group D antiserum. Since clinical isolates are
considered positive for S. Typhi if they confirm by Vi
antiserum and biochemical analyses, the workgroup
decided to eliminate serological analyses using O Group
D antiserum from the procedure. Based on verification
results, the workgroup considered the procedure
appropriate for only PBS and drinking water samples
and recommended additional optimization for surface
water samples.
To address high background levels and enhance
recoveries of & Typhi in spiked surface water samples,
the workgroup recommended the evaluation of IMS
beads. Recoveries of & Typhi CVD 909 in spiked
surface water samples using Salmonella spp. beads
and beads conjugated in the laboratory with Vi antisera
were poor (<5%). Based on these results and the matrix
inhibition evaluation, the workgroup decided that
molecular procedures (e.g., polymcrase chain reaction
[PCR]) would be more appropriate for the detection of S.
Typhi in surface water samples. The workgroup decided
to discontinue any additional optimization analyses
for surface water samples and considered the Study
complete.
-------
-------
The original draft SAP culture-based procedure was not
appropriate for analyses of water matrices for S. Typhi.
The procedure was modified, optimized, and verified
during the Study. The optimized procedure is considered
acceptable by the workgroup for analyses of S. Typhi in
PBS and drinking water samples. The verified procedure
includes the following: (1) pre-enriching in UPD broth
for 24 ± 2 hours at 35.0°C ± 0.5°C, (2) transferring 1
mL to SCBD and incubating for 18 ± 2 hours at 35.0°C
± 0.5°C. (3) streaking growth onto BSD and MMD agars
and incubating for 24 - 48 hours at 35.0°C ± 0.5°C,
(4) sub-culturing onto TSAD and incubating for 24 ±
2 hours at 35.0°C ± 0.5°C. (5) serological testing with
Salmonella Vi antiserum, and (6) biochemical testing
with API 20E® test strips. The culture-based procedure
evaluated during this study is not appropriate for
identification and quantitation of S. Typhi in surface
water matrices.
The revised culture-based procedure merits multi-
laboratory validation to assess method performance and
set quantitative QC criteria for PBS (reference matrix)
and drinking water. However, for surface water, the
workgroup recommended development and inclusion of
molecular procedures (e.g., PCR) for the detection of S.
Typhi in surface water samples.
The draft SAP has been revised based on the Study to
include the verified procedure. Procedures for surface
water are no longer included.
7.0
Conclusions
-------
-------
8.0
References
8.1 Mover. N.P. 2005. ''Section 9260B - ''General Qualitative Isolation and Identification Procedures for
Salmonella" In Standard Methods for the Examination of Water and Waste-water, 21st Edition. A.D. Eaton. L.S.
Clesceri, E.W. Rice, A.E. Greenberg, and M.A.H. Franson (eds.). Washington, DC: American Public Health
Association, American Water Works Association, and Water Environment Federation.
8.2 Bopp, C.A., Brenner, F.W., Fields, P.I., Wells, J.G., and Strockbine, N.A. 2003. Chapter 28, "Escherichia,
Shigella, and Salmonella." In. Manual of Clinical.Microbiology, 8th Edition. P.R. Murray, E.J. Baron, J.H. Jorgensen,
M.A. Pfallcr, and R.H. Yolken (eds.). Hcrndon, Virginia: American Society for Microbiology.
8.3 Andrews, W.H., andHammack, T.S. December 2007. Chapters, Salmonella. Bacteriological Analytical Manual
[online]. U.S. Food and Drug Administration. hUp://www.cfsan.fda.gov/~ebam/bam-5.html
8.4 Klee. A. J. 1993. "Computer Program for the Determination of Most Probable Number and its Confidence
Limits." Journal of Microbiological Methods. 18(2): 91 -98.
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Appendix A
Study Plan
-------
Study Plan - Do not Cite, Circulate, or Copy A-ii November 21,2008
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Study Plan
for
Single Laboratory Verification
of
Standardized Analytical Procedure for
Salmonella Typhi in Environmental Samples
Note: Study plan appendices are provided as attachments to the study report.
November 21, 2008
Study Plan - Do not Cite, Circulate, or Copy A-iii November 21,2008
-------
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 - Do not Cite, Circulate, or Copy A-iv November 21,2008
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TABLE OF CONTENTS
1.0 INTRODUCTION AND BACKGROUND 1
2.0 STUDY MANAGEMENT 1
2.1 ROLES AND RESPONSIBILITIES 1
2.2 STUDY SCHEDULE 4
3.0 OBJECTIVES 6
3.1 STUDY OBJECTIVES 6
3.2 DATA QUALITY OBJECTIVES 6
4.0 STUDY IMPLEMENTATION AND TECHNICAL APPROACH 7
4.1 PHASE 1 - IDENTIFICATION OF QUALIFIED ANALYTICAL LABORATORY 7
4.2 PHASE 2 - PREPARATION OF SPIKES 7
4.2.1 Laboratory-Prepared Spiking Suspensions 7
4.2.2 S. Typhi BioBall™ Spikes 8
4.3 PHASE 3 - SAMPLE ANALYSIS 8
4.3.1 Preliminary Analyses 8
4.3.2 Assessment of Single Laboratory Method Precision and Recovery 8
4.3.3 Quality Control (QC) Analyses 8
4.4 STUDY SUMMARY 9
5.0 REPORTING AND VALIDATION OF STUDY RESULTS 11
6.0 DATA ANALYSIS 12
7.0 LIMITATIONS 12
Study Plan - Do not Cite, Circulate, or Copy A-v November 21,2008
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1.0 INTRODUCTION AND BACKGROUND
Subsequent to the anthrax attacks in the Fall of 2001, federal and state personnel were tasked with a
mission to provide response, recovery, and remediation for biological incidents. It was quickly
recognized, however, that standardized methods do not exist for isolation and analysis of biological
agents in environmental samples. To address these gaps and to ensure confidence in results through
development of quality controls, the U.S. Environmental Protection Agency's (EPA's) National
Homeland Security Research Center (NHSRC) is developing, evaluating, and validating a set of draft
protocols. The protocols are based on pathogens and analytical procedures listed in EPA's Standardized
Analytical Methods for Environmental Restoration following Homeland Security Events (SAM), which
identifies Salmonella Typhi as an EPA target pathogen during environmental remediation following a
homeland security incident.
This study plan describes single-laboratory verification of a draft Standardized Analytical Procedure for
Salmonella Typhi in Environmental Samples (SAP) for determination and quantification of S. Typhi
using selective and non-selective media followed by biochemical and serological characterization. The
draft SAP is included as Attachment A to this study plan, and is based on CDC : 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, high-efficiency particulate air (HEPA) filter with
Arizona Test Dust, swab with local particulate, wipe with local particulate, HEPA filter with local
particulate). A vaccine strain of S. Typhi (CVD909) will be used. Results of the study will be used to
revise the draft SAP, if necessary, prior to further validation in multiple laboratories. Results of this
single laboratory verification will also be used to confirm appropriate spike levels, spiking procedure, and
solid reference matrix for use during a multi-laboratory study.
2.0 STUDY MANAGEMENT
2.1 Roles and Responsibilities
The study will be managed by NHSRC in EPA's Office of Research and Development (ORD) with
support from the study workgroup (Office of Emergency Management [OEM], Office of Water [OW],
Office of Ground Water and Drinking Water [OGWDW], and Centers for Disease Control and Prevention
[CDC]). Coordination of study activities will be performed by Computer Sciences Corporation (CSC)
under NHSRC guidance. CSC will direct sample processing and analyses, conduct data tracking and
review, and coordinate study activities. CSC will provide study updates to the study workgroup including
but not limited to study status, study/data issues, and preliminary results. CSC will prepare a condensed
study report based on the results of the study.
1 Bopp, C.A., F.W. Brenner, P. I. Fields, J. G. Wells, and N. A. Strockbine. 2007. Escherichia, Shigella, and
Salmonella. 2003. In: Manual of Clinical Microbiology, 8th Edition. Editors P.R. Murray, E. J. Baron, J.H.
Jorgensen, M. A. Pfaller, and R. H. Yolken. (Washington, D.C.: American Society for Microbiology.
2U.S. Food and Drug Administration. Salmonella. December 2007. Chapters in Bacteriological Analytical Manual
[online].
3 U.S. Environmental Protection Agency. Office of Water. 2006. Method 1682: Salmonella in Sewage Sludge (Biosolids) by
Modified SemisolidRappaport-Vassiliadis (MSRV) Medium. Washington, D.C.: U.S. Environmental Protection Agency, July
2006. (EPA-821-R-06-14).
4 Rose, L., B. Jensen, A. Peterson, S. N. Banerjee, and M. J. Arduino. 2004. Swab Materials sad Bacillus anthracis Spore
Recovery from Nonporous Surfaces. ,Emerg. Infec. Diseases. 10(6): 1023 - 1029.
Study Plan - Do not Cite, Circulate, or Copy A-l July 29, 2008
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The study workgroup will evaluate study results and work with CSC to identify next steps including
revising the SAP to include any procedural modifications based on study results. The study will use a
single laboratory for verification of SAP procedures and will include preparation of spiking suspensions,
and preparation and analysis of study samples. CSC will be responsible for study coordination including
but not limited to development of study-specific instructions and data reporting forms, procurement of
supplies and spiking materials (BioBalls), and technical support. Specific responsibilities of study
participants are presented in Table 1. The flow of responsibilities is presented in Figure 1.
Table 1. Roles and Responsibilities
Organization
Role
EPA National Homeland Security Research
Center (NHSRC)
Direct contractor activities
Ensure that study goals and deadlines are met
Study Workgroup
(EPA [NHSRC, OEM, OW and OGWDW] and
CDC)
Provide subject matter expertise
Review study schedule
Review study plan
Make final decisions regarding study plan and study
schedule
Provide support for resolution of issues, as necessary
Provide recommendations and conclusions to EPA
Review study results
Review study report
Computer Sciences Corporation (CSC)
Develop study schedule
Develop study plan
Recruit volunteer analytical laboratory support
Develop study materials (spiking protocol, laboratory
instructions, data reporting forms, data review checklists)
Revise study materials, as necessary
Procure and provide laboratory with spikes, reagents and
media
Coordinate study activities (conference calls, resolution
of issues, data receipt) and provide technical support
Serve as point of contact for the laboratory
Coordinate resolution of issues
Receive, compile, maintain, review, and analyze study
data
Prepare condensed study report with recommendations
and conclusions
Revise SAP, as necessary, to address study results and
laboratory feedback
Laboratory
Confirm receipt of written study materials and supplies
Collect appropriate matrix samples, as necessary
Provide input to CSC study coordinator to help determine
sample matrices and spiking levels that would be
appropriate for the multi-laboratory validation study
Optimize the spiking protocol for S. Typhi CVD909
Analyze samples according to study-specific instructions
and SAP
Perform quality control (QC) according to SAP
Maintain general laboratory QA/QC
Provide recommendations on SAP for potential revisions
Provide preliminary and final analytical results to CSC
study coordinator
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Figure 1: Flowchart of Study Activities
esc
Coordinate study
activities and provide
technical support
Study Workgroup / CSC
Prepare and review study schedule
Prepare and review study plan
Provide subject matter expertise
Provide support for resolution of issues
Provide recommendations and conclusions
I
CSC
Develop laboratory study instructions and
reporting forms
Recruit laboratory
Procure and provide laboratory with
reagents, media, and spiking materials
Laboratory
Provide sample matrices
Analyze samples according to study-
specific instructions
Provide recommendations on SAP for
potential revisions
Provide preliminary and final analytical
results to CSC study coordinator
CSC
Receive, compile, disseminate,
validate, and analyze study data
Study Workgroup \
Review study results I
CSC
Prepare draft report
Study Workgroup
Review draft report
CSC
Revise SAP
Study Workgroup
Review revised SAP
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2.2 Study Schedule
The study schedule is dependent on receipt of bacterial spiking standards, media, and reagents by the
laboratory. The following tentative study schedule (Table 2) is provided under the assumption that these
materials will be received by the laboratory no later than August 15, 2008. This schedule also assumes
that the laboratory will participate in several periodic conference calls with the study workgroup and CSC
throughout the study.
For this study, S. Typhi will be quantified in water, solid, and particulate matrices. Specifically, water
matrices will include drinking water from the laboratory tap and surface water as well as a reference
matrix (PBS). For solid matrices, a single local soil sample will be analyzed. The reference matrices for
solids will include one or more of the following based on results of the E. coll O157:H7 single laboratory
verification study: Milorganite®; ASTM Lean Clay; and ASTM Poorly Graded Sand. Particulate
matrices will include one or more of the following based on results of the E. coll O157:H7 single
laboratory verification study: swab, wipe, and HEPA filters with dust collected from laboratory surfaces.
Reference matrices for particulates will include one or more of the following based on results of the E.
coll O157:H7 single laboratory verification study: swab, wipe, and HEPA filters with Arizona Test Dust.
Ideally matrices can be limited, pending E. coll O157:H7 study results.
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Table 2. Tentative Schedule for Single Laboratory Verification of Draft Procedures for
S. Typhi in Water, Solid, and Particulate Matrices
Month
Activity
Planning and Preparation
July 2008
June -August 2008
August - September 2008
October 2008
November 2008
• Draft study plan
• Identify and recruit laboratory
• Finalize study plan
• Draft study-specific instructions, spiking protocol, and data reporting
forms (focus on water)
• Finalize study-specific instructions, spiking protocol, and data reporting
forms (focus on water)
• Procure supplies, reagents, and media
• Draft study-specific instructions, spiking protocol, and data reporting
forms (focus on solids)
• Finalize study-specific instructions, spiking protocol, and data reporting
forms (focus on solids)
• Draft study-specific instructions, spiking protocol, and data reporting
forms (focus on particulates)
• Finalize study-specific instructions, spiking protocol, and data reporting
forms (focus on particulates)
Water (PBS, Surface Water, Drinking Water)
August / September 2008
September / October 2008
• Optimize spiking protocol for S. Typhi CVD909 to determine
appropriate concentration of medium
• Revise spiking protocol, as necessary
• Perform preliminary analysis at laboratory (~ 1 week)
• Review data / resolve issues (~ 2 weeks)
• Conduct analyses at laboratory (~ 1 week)
• Revise procedures (if necessary)
Solids (Milorganite®, ASTM Clay, ASTM Sand, Local Soil)
November / December 2008
• Modify study-specific instructions to address solids
• Perform preliminary analysis at laboratory (~ 1 week)
• Review data / resolve issues (~ 2 weeks)
• Conduct analyses at laboratory (~ 2 weeks)
• Revise procedures (if necessary)
Participates (Swabs, Wipes, HEPA Filters; Each with Arizona Test Dust and a Local Particulate)
December 2008 / January 2009
• Modify study-specific instructions to address particulates
• Perform preliminary analysis at laboratory (~ 1 week)
• Review data / resolve issues (~ 2 weeks)
• Conduct analyses at laboratory (~ 2 weeks)
• Revise procedures (if necessary)
Study Report and SAP Revisions
January / February 2009
• Review data
• Prepare condensed study report
• Revise SAP to reflect study results
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3.0 OBJECTIVES
The primary objective of this study is to verify the procedures included in the draft SAP (Appendix A),
for identification and enumeration of S. Typhi in water, solid, and particulate matrices. This includes
ensuring that estimated counts of the spikes are reliable and that the analytical data generated by the
participant laboratory are reliable. Two sets of objectives were identified for the study: study objectives
and data quality objectives.
3.1 Study Objectives
Results and information from this study will be used to:
• Characterize SAP performance (recovery and precision) in multiple reference matrices (water [PBS],
solid [Milorganite® and two ASTM soils], and particulate [swabs, wipes, and HEPA filters with
Arizona Test Dust])
• Characterize SAP performance (recovery and precision) in multiple environmental matrices of
interest (water, solid, and particulate)
• Determine whether the SAP requires revision prior to multi-laboratory validation
To ensure study objectives are met, the laboratory will be required to:
• Follow all analytical, quality assurance (QA), and quality control (QC) procedures in the study-
specific instructions and draft SAP
• Obtain approval from study workgroup and CSC before using any modifications to the procedures
described in the SAP, and provide a written description of the modification(s)
• Provide all original data reporting forms and any associated information in a format that can be
verified by an independent person reviewing study results
• Respond to data and study related inquiries from the CSC study coordinator
• Provide recommended revisions to the draft SAP
3.2 Data Quality Objectives
Data produced under this study must be generated according to the analytical and QA/QC procedures
specified in the study-specific instructions (Appendix B) and SAP (Appendix A). This will ensure data
integrity and validity for all matrices evaluated and allow the study workgroup to use the study results to
identify any necessary revisions of the SAP (Appendix A).
To ensure that data quality objectives are met, the laboratory will be required to:
• Prepare spiking suspensions in accordance with the spiking protocol (Appendix C). This will allow
for determination of recoveries and identification of potential matrix interferences.
• Analyze the following QC samples:
Media sterility checks
- Dilution water sterility checks
Method blanks (e.g., sterile unspiked phosphate buffered saline [PBS])
Positive and negative controls
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4.0 STUDY IMPLEMENTATION AND TECHNICAL APPROACH
During this study, non-selective and selective media, followed by biochemical characterization and
serological confirmation tests, will be used to analyze water, solid, and particulate samples spiked with S.
Typhi (see draft SAP, Appendix A). One or more reference matrices for each sample type (water, solid,
and particulate) will also be analyzed.
This section provides a summary of the activities that will be performed during the study. Detailed
study-specific instructions (Appendix B) and data reporting forms (Appendix D) will be provided to
the participant laboratory. Activities and schedules described in this study plan may change as the
study progresses and additional data and information become available.
4.1 Phase 1 - Identification of Qualified Analytical Laboratory
CSC will identify and recruit a laboratory that is 1) representative of the general user community and 2)
has access to representative matrices. Ideally, the laboratory will have experience analyzing
environmental samples for S. Typhi. To reduce study costs, a volunteer laboratory will be recruited. To
reduce the burden on the laboratory and encourage participation, NHSRC will provide the media,
reagents, and supplies needed for the study. The requirements and responsibilities of the laboratory will
be detailed in study-specific instructions (Appendix B) and the draft SAP (Appendix A).
The primary analyst and laboratory management will be asked to participate in periodic conference calls,
to ensure the laboratory is aware of schedules, has the proper reagents and media, and understands data
reporting requirements.
4.2 Phase 2 - Preparation of Spikes
The study will use the following two sources of enumerated spiking suspensions: 1) laboratory-prepared
S. Typhi spiking suspensions and 2) S. Typhi BioBalls. Prior to any study analyses, the laboratory will
optimize the spiking protocol for the laboratory-prepared S. Typhi CVD909 spiking suspension to
determine the appropriate concentration of medium (e.g., 1% TSB, 10% TSB). The laboratory will
evaluate a maximum of four medium (TSB) concentrations to determine which concentration will provide
a stable spiking suspension.
4.2.1 Laboratory-Prepared Spiking Suspensions
Laboratory-prepared spiking suspensions will be prepared from S. Typhi (Center for Vaccine
Development [CVD] 909) according to the spiking protocol (Appendix C). A stock culture will
be propagated in TSB with 0.0001% 2, 3 dihydrobenzoic acid (DHB) and incubated at 35.0°C ±
0.5°C for 24 ± 2 hours. Serially diluted spiking suspensions (~30 colony forming units [CPU] per
mL) will be used to spike samples. The laboratory will enumerate spiking suspensions on the
same day samples are spiked and analyzed, using the spread plate technique (in triplicate) on
tryptic soy agar (TSA) with 0.0001% DHB.
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4.2.2 S. Typhi BioBall™ Spikes
S. Typhi BioBalls (BTF, Pty in North Ryde, Australia) prepared from CVD 909 will be procured
by CSC and provided to the laboratory. BioBalls are commercially available flow-sorted pre-
enumerated QC spikes. A single BioBall™ (-30 CPU) will be used to spike individual samples.
The laboratory will also enumerate three BioBalls to verify the manufacturer's lot mean value
using the spread plate technique on the first day of sample spiking for each matrix (water, solids,
andparticulates).
4.3 Phase 3 - Sample Analysis
Samples will be analyzed as described in the draft SAP (Appendix A) and the study-specific instructions
(Appendix B). Sample matrices will be prepared as described in Appendix B (Study-Specific
Instructions). S. Typhi is considered a BSL-2 pathogen. The laboratory is required to process all samples
according to the safety requirements included in Section 4 of the draft SAP (Appendix A). Table 3
provides a detailed summary of the preliminary and study analyses.
4.3.1 Preliminary Analyses
The laboratory will conduct preliminary analyses for each matrix and spike type (unspiked,
laboratory-prepared and BioBall™) approximately 1-2 weeks prior to start of the study
analyses. The time between preliminary analyses and study analyses will be used to resolve any
issues that arise.
4.3.2 Assessment of Single Laboratory Method Precision and Recovery
For each matrix, method precision and recovery will be evaluated through the analysis of four
replicates (of each matrix) spiked with laboratory-prepared spikes and four replicates (of each
matrix) spiked with S .Typhi BioBalls.
4.3.3 Quality Control (QC) Analyses
During assessment of precision and recovery in replicate samples, the laboratory will complete
the following QC requirements: media sterility checks, dilution water sterility checks, method
blanks, filtration blanks, positive controls, and negative controls. S. Typhi (CVD 909) and
(R)
Pseudomonas aeruginosa (ATCC 27853™) will serve as the positive controls and E. coll
(ATCC ® 25922™) as the negative control.
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4.4 Study Summary
The following table summarizes the sample matrices and number of samples that will be analyzed, the
spiking description, and purpose of the analysis.
Table 3. Summary of Sample Analyses
Matrix
Spiking
Description
No. of Samples
Preliminary
Analyses
Study
Analyses
Purpose of Analysis
Water
Sterile PBS
(Reference
Matrix)
Surface
Water
Drinking
Water
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
1
1
1
1
1
1
1
1
1
1
4
4
2
4
4
2
4
4
Confirmation of freedom from
contamination (Method Blank)
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision and
recovery
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision and
recovery
Confirmation that approach is appropriate
for the multi-laboratory validation study
Solids
Milorganite®
(Reference
Matrix #1)
ASTM Lean
Clay
(Reference
Matrix #2)
ASTM Sand
(Reference
Matrix #3)
Local Soil
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
1
1
1
1
1
1
1
1
1
1
1
1
1
4
4
1
4
4
1
4
4
2
4
4
Confirmation of freedom from
contamination (Method Blank)
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision and
recovery
Confirmation that approach is appropriate
for the multi-laboratory validation study
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Matrix
Spiking
Description
No. of Samples
Preliminary
Analyses
Study
Analyses
Purpose of Analysis
Particulates
Swab with
Reference
Matrix
(Arizona
Test Dust)
Wipe with
Reference
Matrix
(Arizona
Test Dust)
HEPA Filter
with
Reference
Matrix
(Arizona
Test Dust)
Swab with
Local
Particulate
Wipe with
Local
Particulate
HEPA Filter
with Local
Particulate
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
Unspiked
BioBall™ Spike
Lab-Prepared
Spike
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
4
1
4
4
1
4
4
2
4
4
2
4
4
2
4
4
Evaluation of background S. Typhi
concentrations
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision and
recovery
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision and
recovery
Confirmation that approach is appropriate
for the multi-laboratory validation study
Evaluation of background S. Typhi
concentrations
Assessment of method precision and
recovery
Confirmation that approach is appropriate
for the multi-laboratory validation study
Note: Solid and particulate matrices may be limited pending E.
results.
co//O157:H7 single-laboratory verification study
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5.0 REPORTING AND VALIDATION OF STUDY RESULTS
After analyses are complete, the laboratory will be required to submit data on standardized data reporting
forms (Appendix D), designed specifically for the study. In addition, the laboratory will be required to
submit detailed explanations of any deviations from the study-specific instructions (Appendix B), as well
as any recommended revisions to the draft SAP (Appendix A).
Laboratories will submit all study results, data packages, descriptive information, and SAP revision
recommendations to the CSC study coordinator:
Laura Jones, Computer Sciences Corporation (CSC)
Iiones76(g),csc.com
6101 Stevenson Avenue
Alexandria, VA 22304
Phone:(703)461-2007
Fax:(703)461-8056
Data received will include:
• Volume/weight of all samples and dates of preparation and processing of study matrices
• Inoculation volumes and plate counts for spike enumeration (laboratory-prepared and BioBalls)
• Plate counts for preliminary and study analyses, including plate counts for any atypical organisms
• Results for all QC analyses (as listed in Section 4.3.3)
• Date of preparation, pH, lot number and expiration dates for all media and reagents
Upon receipt of the laboratory data package, CSC will review the data to ensure that they were generated
in accordance with the SAP (Appendix A) and study-specific requirements. Items that will be reviewed
for each sample include the following:
• Confirmation that original forms are submitted and complete
• Confirmation that all calculations are correct
• Confirmation that QC checks were performed and exhibit the appropriate response
• Confirmation that media and reagents were used within expiration dates
• Confirmation that method-specific incubation times and temperatures were met
• Confirmation that all required data elements are reported
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6.0 DATA ANALYSIS
Descriptive statistics of sample recoveries will be calculated using spiked sample data from each matrix
and spike type and will include the relative standard deviation (RSD) between results of replicate
samples. Mean, median, and range of individual recoveries also may be included. For samples spiked
with BioBalls, recoveries will be assessed by comparing spike recovery (concentration in the spiked
samples minus the unspiked concentration) to the manufacturer-provided lot mean value with each set of
analyses. If BioBalls are not used for spiking, recoveries will be assessed based on enumeration of
spiking suspension by the participant laboratory, using spread plate technique (Spiking Protocol
[Appendix C]).
7.0 LIMITATIONS
Because this study will be performed by a single laboratory analyzing the limited number of samples and
matrices specified in Table 3, the results of this study may not represent all potential variability that could
arise in real-world implementation of this SAP (Appendix A). This study is designed, however, to ensure
that the laboratory and matrices included in this study are as representative as possible of real-world
conditions.
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Appendix B
Study-Specific Instructions
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Instructions for Salmonella Typhi SAP Single Laboratory
Verification Study: Preliminary Water Analyses
October 31, 2008
The purpose of this study is to evaluate procedures that are included in the "Draft Standardized Analytical
Procedure for Salmonella enterica subsp. enterica serotype Typhi (Salmonella Typhi) in Environmental
Samples" (referred to as the "draft SAP"). Results of the study will be used to revise the draft SAP, if
necessary, prior to further validation in multiple laboratories.
Media/Reagents/Supplies
• Phosphate buffered saline (PBS)
• 10% sodium thiosulfate
• 2,3-dihydroxybenzoate (DHB)
• IX, 2X, and 5X universal pre-enrichment broth with DHB (UPD)
• Selenite cystine broth with DHB (SCBD)
• Bismuth sulfite with DHB (BSD) agar
• Tryptic soy agar with DHB (TSAD)
• Tryptic soy broth (TSB)
• 1 % Tryptic soy broth with DHB (TSBD)
• Physiological saline (0.85%)
• Salmonella Vi and O group D antisera
Control Cultures
o S. Tvphi (CVD 909)
o Enterococcus faecalis (ATCC® 29212™)
Water Matrices
Water matrices will include a reference matrix (PBS), drinking water from the laboratory tap, and surface
water from a lake, reservoir, or other open body of water.
Preliminary Sample Analyses
• The following unspiked and spiked PBS, drinking water, and surface water samples will be
enumerated using the most probable number (MPN) technique. Following pre-enrichment in UPD,
samples will be sub-cultured in a selective medium (SCBD), followed by plating on a selective agar
(BSD). Isolated, typical colonies with a green-black metallic sheen will then be submitted to
serological confirmation according to study-specific instructions.
• PBS Samples (Reference Matrix)
o 1, 100-mL unspiked
o 1, 100-mL spiked
• Drinking Water Samples
o 1, 100-mL unspiked
o 1, 100-mL spiked
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• Surface Water Samples
o 1, 100-mLunspiked
o 1, 100-mL spiked
Prior to Study
Reagent and Media Preparation
It is recommended that the following reagents and media be prepared either prior to the week of
preliminary study analyses (PBS, DHB, UPD, TSAD, TSBD) or the day before analyses (SCBD, BSD):
• Phosphate Buffered Saline (PBS): Prepare 1.0 L of PBS according to the following instructions and
store at room temperature for a maximum of three months in screw cap bottles.
Monosodium phosphate (NahbPC^)
Disodium phosphate (Na2HPC>4)
Sodium chloride
Reagent-grade water
0.58 g
2.50 g
8.50 g
1.0L
Dissolve reagents in 1.0 L of reagent-grade water in a flask and dispense in appropriate volumes
in screw cap bottles and autoclave at 121°C (15 Ib pressure per square inch [PSI]) for 15 minutes.
Final pH should be 7.4 ± 0.2.
Note: Assuming all analyses are conducted within the same week a total of 695 mL of PBS will
be required. As a result, it is recommended that the laboratory prepare 1.0 L of PBS [5, 99-mL
dilution blanks; 2, 100-mL samples].
2,3-dihydroxybenzoate (DHB): Prepare 100 mL of 0.1% (w/v) DHB stock solution by dissolving
0.1 g of DHB powder in 100 mL of reagent-grade water and filter sterilize.
Universal Pre-enrichment Broth with DHB (UPD): Prepare 500 mL of IX, 2X, and 5X UPD
according to the following and store at room temperature for a maximum of three months in screw
cap bottles.
Reagents
Pancreatic digest of casein
Proteose peptone
Monopotassium phosphate (KhbPC^)
Disodium phosphate (Na2HPO4)
Sodium chloride
Dextrose
Magnesium sulfate (MgSO4)
Ferric ammonium citrate
Sodium pyruvate
Reagent-grade water
1X
2.5 g
2.5 g
7.5 g
3.5 g
2.5 g
0.25 g
0.125 g
0.05 g
0.1 g
500 mL
2X
5.0 g
5.0 g
15. Og
7.0 g
5.0 g
0.5 g
0.25 g
0.1 g
0.2 g
500 mL
5X
12.5 g
12.5 g
37.5 g
17.5 g
12.5 g
1.25g
0.625 g
0.25 g
0.5 g
500 mL
For IX, 2X and 5X UPD, dissolve reagents in 450 mL of reagent-grade water in a flask and mix
thoroughly. Adjust pHto 7.2 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide and
bring volume to 500 mL. Autoclave at 121°C (15 PSI) for 15 minutes. Cool to 45 °C - 50°C in a
waterbath. For IX UPD, add 0.5 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense
10.0 mL aliquots in 20 x 150 mm tubes. For 2X UPD, add 1.0 mL of 0.1 % DHB stock solution,
mix thoroughly, and dispense 10.0 mL aliquots in 20 x 150 mm tubes. For 5X UPD, add 2.5 mL
of 0.1 % DHB stock solution, mix thoroughly, and dispense 5.0 mL aliquots in 20 x 150 mm
tubes. Store at <10°C and above freezing for a maximum of 2 weeks in tubes with loose caps or
three months in screw cap tubes.
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• Selenite Cystine Broth with DHB (SCBD): Prepare 1.0 L of SCBD according to the following
instructions and use within 48 hours of preparation.
Pancreatic digest of casein
Lactose
Sodium phosphate
Sodium selenite
L-Cystine
Reagent-grade water
5.0 g
4.0 g
10.0 g
4.0 g
0.01 g
1.0L
Add reagents to 1.0 L of reagent-grade water and mix thoroughly using a stir bar and hot plate.
Heat to boiling. Avoid overheating. Do not autoclave. Final pH should be 7.0 ± 0.2. Cool to
45°C - 50°C in a waterbath and add 1.0 mL of DHB [54, 9-mL tubes].
Note: A brick red precipitate may appear if the medium is overheated during preparation or
exposed to excessive moisture during storage. If this occurs medium should be discarded and a
new batch prepared.
• Bismuth Sulfite with DHB (BSD): Prepare 1.0 L of BSD agar (the recommended volume assumes
that 80% of spiked MPN tubes are presumptive positive) according to manufacturer's instructions the
day prior to analyses. Cool to 45°C - 50°C in a waterbath and add 1.0 mL of the 0.1% DHB solution.
Evenly disperse the precipitate while aseptically pouring 12-15 mL into 100 x 15 mm sterile Petri
plates. Store at room temperature [54, 15-mL, 100 x 15-mmplates].
• Tryptic Soy Agar with DHB (TSAD): Prepare 30 TSAD plates according to manufacturer's
instructions. After autoclaving, cool to 45°C - 50°C in a waterbath and add 0.1 mL of the 0.1% DHB
solution per 100 mL of medium. Aseptically pour 12-15 mL into each 100 x 15 mm sterile Petri
plate. Store at <10°C and above freezing for a maximum of 2 weeks.
• Tryptic Soy Broth with DHB (TSB): Prepare TSB by preparing TSB according manufacturer's
instructions. Store at <10°C and above freezing for a maximum of three months in a screw cap
bottle.
• l%Tryptic Soy Broth with DHB (TSBD): Prepare a 1% solution of TSB by combining 99 mL of
sterile phosphate buffered dilution water, 1 mL of sterile single-strength TSB (prepared according to
manufacturer's instructions), and 0.1 mL of 2,3-dihydroxybenzoate (DHB) solution in a sterile screw
cap bottle. Shake to mix.
Week of Analyses:
Day One
Laboratory-prepared spiking suspension: Inoculate 1% TSBD with S. Typhi according to the spiking
protocol and incubate at 35°C ± 0.5°C for 24 ± 2 hours.
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October 31, 2008
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Day Two
Enumeration of Spiking Suspensions
• Dilute and plate laboratory-prepared spiking suspension on TSAD according to spiking protocol and
incubate at 35°C ± 0.5°C for 24 ± 2 hours.
o Sample Collection - PBS. Drinking Water, and Surface Water Preliminary Analyses
• Aliquot the following volumes of sterile PBS and label the samples as follows:
o 1, 100-mL unspiked- PBS unspiked
o 1, 100-mL spiked - PBS spiked
• Collect a 1.0-L bulk drinking water sample as follows:
o Select a cold water line faucet and remove aerator, if present.
o Clean the faucet exterior with disinfection solution (e.g., 10% household bleach).
o Open the tap to obtain a smooth-flowing stream at moderate pressure without splashing.
o Allow water to run at least 2-3 minutes.
o Remove the cap from a sterile bottle containing 1 mL of a 10% sodium thiosulfate solution
(dechlorinating agent).
o Avoid contaminating the sample bottle lip or inside the cap.
o Reduce the water flow to fill the bottle without splashing and fill to within 2.5 cm - 5 cm (1" -
2") of the top for proper mixing before analyses.
o Do not rinse dechlorinating agent out of the bottle.
o Tightly cap the container.
o Mix thoroughly and split the bulk sample into aliquots and label the samples as follows.
• 1, 100-mL-Drinking water unspiked
• 1, 100-mL-Drinking water spiked
• Collect a 1.0-L bulk surface water sample as follows:
o Collect samples by hand or with a sampling pole if the sampling site has difficult access such
as a dock, bridge, or bank adjacent to surface water.
o The sampling depth should be 6" - 12" below the water surface.
o Sample containers should be positioned such that the mouth of the container is pointed away
from the sampler or sample point.
o After removal of the container from the water, a small portion of the sample should be
discarded to leave a headspace of 2.5 cm - 5 cm (1" - 2") for proper mixing before analyses.
o Transport to the laboratory on ice (do not freeze).
o In the laboratory, split the bulk sample into aliquots and label the samples as follows:
• 1, 100-mL - Surface water unspiked
• 1, 100-mL - Surface water spiked
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Sample Spiking
• Spike 1, 100-mL sample each of PBS, drinking water, and surface water with laboratory-prepared
spiking suspension, according to the spiking protocol.
Most Probable Number (MPN)
• Mix unspiked and spiked samples by shaking 25 times.
• Inoculate UPD tubes with the following volumes for each unspiked or spiked sample:
o For each of three tubes, add 20 mL of undiluted sample to 5 mL (5X) UPD
o For each of three tubes, add 10 mL of undiluted sample to 10 mL (2X) UPD
o For each of three tubes, add 1 mL of undiluted sample to 10 mL (IX) UPD
• Incubate inoculated UPD tubes at 35.0°C ± 0.5°C for 24 ± 2 hours.
Day Three
TSA Enumeration - Laboratory-Prepared Suspensions
• Count colonies on TSAD plates prepared on Day Two and record results of colony forming units
(CPU) enumeration on provided data reporting forms.
• Fax TSAD enumeration results to Yildiz Chambers at (703) 461-8056.
MPN
• Examine UPD tubes at 24 ±2 hours; record results.
• From each UPD tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes
containing 9.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 24 ± 2 hours.
Day Four
MPN
• Examine SCBD tubes at 24 hours; record results.
• From each SCBD tube with growth, gently swirl the tube to mix and streak for isolation on BSD plates
using a sterile inoculation loop (20 (iL). Incubate at 35.0°C ± 0.5°C for 24 - 48 hours.
Day Five
BSn Plates
• Examine BSD plates at 24 hours; record results. If no typical (green-black metallic sheen) colonies
are observed on BSD plates, incubate for an additional 24 ± 2 hours at 35.0°C ± 0.5°C. For BSD
plates with typical colonies, streak a typical colony onto TSA and incubate at 35°C ± 0.5°C for 24 ± 2
hours.
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Day Six
BSn Plates
• Examine BSD plates at 48 hours; record results. For BSD plates with typical colonies, streak a typical
colony onto TSA and incubate at 35°C ± 0.5°C for 24 ± 2 hours.
TSAn Plates
• Serological Analyses: A single colony from each TSAD plate will be submitted to serological
confirmation, as described below.
o Emulsify a portion of a single typical colony from each TSAD plate using sterile physiological
saline and test for agglutination with Salmonella antiserum (Vi and O Group D) as follows:
• Place three discrete drops of emulsified growth onto a slide. To the first drop of emulsified
growth, add one drop of Salmonella O antiserum Group D. To the second drop of emulsified
growth, add one drop of Vi antiserum. To the third drop of emulsified growth, add one drop
of sterile saline (as a visual comparison). Observe under magnification for an agglutination
reaction, which indicates a positive result. S. Typhi is agglutination-positive for both O
Group D and Vi antisera. Results should be compared with those for positive and negative
controls analyzed at the same time.
o If results are not consistent with S. Typhi (e.g., negative serology), go back to the TSAD plate and
pick another isolated colony and repeat serological analyses. Note: If neither of the two isolates
from the plate provides a positive serology result, please contact CSC immediately for additional
instructions; Yildiz Chambers (703) 461-2165, vchambers@csc.com).
Note: Additional serological confirmation will be required on Day Seven only if there are no typical
colonies observed and/or confirmed on Day Six.
Day Seven
TSAn Plates
• Serological Analyses: A single colony from each plate with typical colonies will be submitted to
serological confirmation, as described above (Day 6).
• Please fax results to Yildiz Chambers at (703) 461-8056.
Note: Please contact Yildiz Chambers (703) 461-2165, ychambers@csc.com) if you have any questions.
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Method Optimization Instructions for Salmonella Typhi SAP
Single Laboratory Verification Study: Water Matrices
December 10, 2008
The instructions provided below are to be repeated for a total of two runs, resulting in the analysis of 2
unspiked and 4 spiked samples for each matrix (PBS, drinking water, and surface water). Each sample
will be processed with pre-enrichment in UPD broth (direct inoculation into SCBD) and without pre-
enrichment (Sets A and B, respectively) and plated on 2 selective agars (BSD and MMD). Please contact
Yildiz Chambers (703) 461-2165, ychambers@csc.com) if you have any questions regarding these
instructions.
All QA/QC analyses specified in the study-specific instructions and draft SAP must be conducted in
conjunction with sample analyses described below. In addition, the laboratory must follow all QA/QC
procedures outlined in their QAPP.
Media/Reagents/Supplies
• Phosphate buffered saline (PBS)
• 10% sodium thiosulfate
• 2,3-dihydroxybenzoate (DHB)
• IX, 2X, and 5X universal pre-enrichment broth with DHB (UPD)
• IX, 2X, and 5X selenite cystine broth with DHB (SCBD)
• Bismuth sulfite with DHB (BSD) agar
• Miller-Mallinson with DHB (MMD) agar
• Tryptic soy agar with DHB (TSAD)
• Tryptic soy broth (TSB)
• 1 % tryptic soy broth with DHB (TSBD)
• Physiological saline (0.85%)
• API® 20E Test Strips
• Oxidase reagent
• Salmonella Vi and O group D antisera
Control Cultures
o S. Typhi (CVD 909)
o Enterococcus faecalis (ATCC® 29212™)
o Pseudomonas aeruginosa (ATCC® 27853™)
Do Not Cite, Circulate, or Copy B-7 December 10,2008
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Water Matrices
Water matrices will include a reference matrix (PBS), drinking water from the laboratory tap, and surface
water from a lake, reservoir, or other open body of water.
Quality Control (QC) Analyses
QC analyses listed on the batch specific coversheet should be analyzed with each run.
Sample Analyses
• Set A will be pre-enriched in UPD broth, with samples sub-cultured into a selective broth (SCBD),
followed by plating on selective agars (BSD and MMD).
• Set B will be cultured in SCBD without pre-enrichment, followed by plating on BSD and MMD.
• Isolated, typical colonies from BSD and MMD plates from both Set A and Set B will then be sub-
cultured onto TSAD and submitted to serological and biochemical confirmation according to study-
specific instructions.
• Two separate analytical runs, consisting of the following samples will be conducted resulting in 2
unspiked and 4 spiked samples for each matrix (PBS, drinking water, and surface water).for a total of
18 samples. See flowchart in Appendix A to this Appendix (see page B-16, below) for an overview
of the analyses.
Run 1 Analyses:
o PBS Samples (Reference Matrix)
o 1. 200-mL unspiked
o 2, 200-mL spiked
o Drinking Water Samples
o 1. 200-mL unspiked
o 2. 200-mL spiked
o Surface Water Samples
o 1. 200-mL unspiked
o 2. 200-mL spiked
Run 2 Analyses:
o PBS Samples (Reference Matrix)
o 1. 200-mL unspiked
o 2. 200-mL spiked
o Drinking Water Samples
o 1. 200-mL unspiked
o 2. 200-mL spiked
o Surface Water Samples
o 1. 200-mL unspiked
o 2. 200-mL spiked
Do Not Cite, Circulate, or Copy B-8 December 10,2008
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Media and Reagent Preparation
Reagent Preparation
Assuming all analyses are conducted in 2 separate 1-week runs, a total of 2190 mL of PBS will be
required. As a result, it is recommended that the laboratory prepare 2.5 L of PBS prior to the first week of
study analyses [10, 99-mL dilution blanks and 6, 200-mL samples].
• Phosphate Buffered Saline (PBS): Prepare 2.5 L of PBS according to the following instructions and
store at room temperature for a maximum of three months in screw cap bottles. Note: The following
instructions are for preparation of 1.0 L; please adjust for preparation of appropriate volumes.
Monosodium phosphate (NahbPC^)
Disodium phosphate (Na2HPC>4)
Sodium chloride
Reagent-grade water
0.58 g
2.50 g
8.50 g
1.0L
Dissolve reagents in 2.5 L of reagent-grade water in a flask and dispense in appropriate volumes
in screw cap bottles and autoclave at 121°C (15 Ib pressure per square inch [PSI]) for 15 minutes.
Final pH should be 7.4 ± 0.2.
Media Preparation
It is recommended that the following be prepared, either prior to the week of study analyses (DHB, UPD,
TSAD, TSB, 1% TSBD), or the day before analyses (SCBD, BSD), so that appropriate QC analyses can be
conducted on the media prior to use. MMD medium will be provided as prepared plates.
• 2,3-dihydroxybenzoate (DHB): Prepare 100 mL of 0.1% (w/v) DHB stock solution by dissolving
0.1 g of DHB powder in 100 mL of reagent-grade water and filter sterilize.
• Universal Pre-enrichment Broth with DHB (UPD):
o Prepare UPD according to the following:
• For 300 mL of IX UPD, add 11.4 g dehydrated medium to 300 mL reagent-grade water; mix
thoroughly.
• For 300 mL of 2X UPD, add 22.8 g dehydrated medium to 300 mL reagent-grade water; mix
thoroughly.
• For 200 mL of 5X UPD, add 38.0 g dehydrated medium to 200 mL reagent-grade water; mix
thoroughly.
o Autoclave all solutions at 121°C (15 PSI) for 15 minutes. Final pH should be 6.3 ± 0.2. Cool to
45 °C - 50°C in a waterbath.
• For IX UPD, add 0.3 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
• For 2X UPD, add 0.6 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
• For 5X UPD, add 1.0 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 5.0 mL
aliquots in 25 x 150 mm tubes.
o Store at <10°C and above freezing for a maximum of 2 weeks.
Do Not Cite, Circulate, or Copy B-9 December 10,2008
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Selenite Cystine Broth with DHB (SCBD):
o Prepare SCBD according to the following:
• For 1.2 L of IX SCBD, add 27.6 g dehydrated medium to 1.2 L reagent-grade water; mix
thoroughly.
• For 300 mL of 2X SCBD, add 13.8 g dehydrated medium to 300 mL reagent-grade water;
mix thoroughly.
• For 200 mL of 5X SCBD, add 23.0 g dehydrated medium to 200 mL reagent-grade water;
mix thoroughly.
o Prepare SCBD according to the manufacturer's instructions. Do not autoclave. Final pH should
be 7.0 ± 0.2. Cool to 45 °C - 50°C in a waterbath.
• For IX SCBD, add 1.2 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
• For 2X SCBD, add 0.6 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
• For 5X SCBD, add 1.0 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 5.0
mL aliquots in 25 x 150 mm tubes.
o Use within 48 hours of preparation. Note: A brick red precipitate may appear if the medium is
overheated during preparation or exposed to excessive moisture during storage. If this occurs
medium should be discarded and a new batch prepared.
Bismuth Sulfite with DHB (BSD): Prepare 2.0 L of BSD agar (the recommended volume assumes
that 80% of spiked and 20% of unspiked MPN tubes are presumptive positive) according to
manufacturer's instructions the day prior to analyses. Cool to 45°C - 50°C in a waterbath and add
1.5 mL of the 0.1% DHB solution. Evenly disperse the precipitate while aseptically pouring 12 - 15
mL into 100 x 15 mm sterile Petri plates. Use within 48 hours of preparation. Store at room
temperature [130, 15-mL, 100 x 15-mm plates].
Miller-Mallinson with DHB (MMD): Agar will be received as prepared plates. Store at <10°C and
above freezing [130, 100 x 15-mm plates].
Tryptic Soy Agar with DHB (TSAD): Prepare 278 TSAD plates according to manufacturer's
instructions. After autoclaving, cool to 45 °C - 50°C in a waterbath and add 0.1 mL of the 0.1% DHB
solution per 100 mL of medium. Aseptically pour 12 - 15 mL into each 100 x 15 mm sterile Petri
plate. Store at <10°C and above freezing for a maximum of 2 weeks.
Tryptic Soy Broth (TSB): Prepare 100 mL TSB according manufacturer's instructions. Store at
<10°C and above freezing for a maximum of three months in a screw cap bottle.
l%Tryptic Soy Broth with DHB (TSBD): Prepare a 1% solution of TSBD by combining 99 mL of
sterile PBS, 1 mL of sterile single-strength TSB, and 0.1 mL of DHB solution in a sterile screw cap
bottle. Shake to mix.
Do Not Cite, Circulate, or Copy B-10 December 10,2008
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Study Analyses
Instructions provided below are for a single week of analyses (1 run). These instructions are to be
repeated on a subsequent week for a total of 2 runs.
Day One (Sunday)
Propagation of Spiking Suspension
Inoculate 1% TSBD with S. Typhi according to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ±
2 hours.
Day Two (Monday)
TSA Enumeration of Spiking Suspensions
• Dilute and plate laboratory-prepared spiking suspension on TSAD according to spiking protocol and
incubate at 35°C ± 0.5°C for 24 ± 2 hours.
Preparation and Collection of Samples
o Sample Collection and/or Preparation - PBS, Drinking Water, and Surface Water
• PBS (Reference Matrix) Samples
o Aliquot the following volumes of sterile PBS and label the samples as follows:
• 1, 200-mL unspiked sample - PBS unspiked
• 2, 200-mL spiked samples - PBS spiked
• Collect a 1.0-L bulk drinking water sample as follows:
o Select a cold water line faucet and remove aerator, if present.
o Clean the faucet exterior with disinfection solution (e.g., 10% household bleach).
o Open the tap to obtain a smooth-flowing stream at moderate pressure without splashing.
o Allow water to run at least 2-3 minutes.
o Remove the cap from a sterile bottle containing 1 mL of a 10% sodium thiosulfate solution
(dechlorinating agent).
o Avoid contaminating the sample bottle lip or inside the cap.
o Reduce the water flow to fill the bottle without splashing and fill to within 2.5 cm - 5 cm (1" -
2") of the top for proper mixing before analyses.
o Do not rinse dechlorinating agent out of the bottle.
o Tightly cap the container.
o Mix thoroughly and split the bulk sample into aliquots and label the samples as follows.
• 1, 200-mL unspiked sample - Drinking water unspiked
• 2, 200-mL spiked samples - Drinking water spiked
• 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.
Do Not Cite, Circulate, or Copy B-ll December 10,2008
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o The sampling depth should be 6" - 12" below the water surface.
o Sample containers should be positioned such that the mouth of the container is pointed away
from the sampler or sample point.
o After removal of the container from the water, a small portion of the sample should be discarded
to leave a headspace of 2.5 cm - 5 cm (1" - 2") for proper mixing before analyses.
o Transport to the laboratory on ice (do not freeze).
o In the laboratory, split the bulk sample into aliquots and label the samples as follows.
• 1, 200-mL unspiked sample - Surface water unspiked
• 2, 200-mL spiked samples - Surface water spiked
Sample Spiking
• Spike 2, 200-mL samples each of PBS, drinking water, and surface water with laboratory-prepared
spiking suspension using twice the volume indicated in the spiking protocol to accommodate the
larger 200 mL sample volume.
Most Probable Number (MPN)
• Mix unspiked and spiked samples by shaking 25 times. Each 200 mL sample will be used to
inoculate tubes for both sets (A and B).
Set A (with Pre-enrichment)
• For Set A tubes (with pre-enrichment), inoculate UPD tubes with the following volumes for each
unspiked or spiked sample:
o For each of three tubes, add 20 mL of undiluted sample to 5 mL (5X) UPD
o For each of three tubes, add 10 mL of undiluted sample to 10 mL (2X) UPD
o For each of three tubes, add 1 mL of undiluted sample to 10 mL (IX) UPD
• Incubate inoculated UPD tubes at 35.0°C ± 0.5°C for 24 ± 2 hours.
Set B (without Pre-enrichment)
• For Set B tubes (without pre-enrichment), inoculate SCBD tubes with the following volumes for each
unspiked or spiked sample:
o For each of three tubes, add 20 mL of undiluted sample to 5 mL (5X) SCBD
o For each of three tubes, add 10 mL of undiluted sample to 10 mL (2X) SCBD
o For each of three tubes, add 1 mL of undiluted sample to 10 mL (IX) SCBD
• Incubate inoculated SCBD tubes at 35.0°C ± 0.5°C for 18 ± 2 hours.
Day Three (Tuesday)
TSA Enumeration of Spiking Suspension
• Count colonies on TSA plates prepared on Day Two and record results of CPU enumeration on
provided data reporting forms
• Fax TSA enumeration results to Yildiz Chambers at (703) 461-8056.
Do Not Cite, Circulate, or Copy B-12 December 10,2008
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MPN
Set A (with Pre-enrichment)
• Examine UPD tubes at 24 hours; record results.
• For each UPD tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes with
10.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 18 ± 2 hours.
Set B (without Pre-enrichment)
• For each SCBD tube with growth, gently swirl the tube to mix and streak for isolation on BSD and
MMD plates using a sterile inoculation loop (20 |JL). Incubate at 35.0°C ± 0.5°C for 48 ± 3 hours,
examining plates at 24 ±2 hours and 48 ± 3 hours.
Day Four (Wednesday)
MPN
Set A (with Pre-enrichment)
• Examine SCBD tubes at 18 ± 2 hours; record results.
• For each SCBD tube with growth, gently swirl the tube to mix and streak for isolation on BSD and
MMD plates using a sterile inoculation loop (20 |JL). Incubate at 35.0°C ± 0.5°C for 48 ± 3 hours,
examining plates at 24 ±2 hours and 48 ± 3 hours.
Set B (without Pre-enrichment)
BSn and MMn Plates
• Examine BSD and MMD plates at 24 ±2 hours; record results.
• If no typical (presumptive positive) S. Typhi (green-black with metallic sheen for BSD or black on
MMD) colonies are observed on plates, re-incubate for a total of 48 ± 3 hours at 35.0°C ± 0.5°C.
• For each BSD and MMD plate with typical colonies, streak a single typical colony onto TSAD and
incubate 24 ± 2 hours at 35.0°C ± 0.5°C. Note: Unspiked samples are not expected to have
presumptive-positive colonies, however, if typical S. Typhi colonies are observed, these (up to 5)
should be streaked onto TSAD for biochemical (API 20E) and serological analyses.
Day Five (Thursday)
Set A (with Pre-enrichment)
BSn and MMn Plates
• Examine BSD and MMD plates at 24 ±2 hours; record results.
• If no typical (green-black with metallic sheen for BSD or black on MMD) colonies are observed on
plates, re-incubate for a total of 48 ± 3 hours at 35.0°C ± 0.5°C.
• For each BSD and MMD with typical colonies, streak a single typical colony onto TSAD and incubate
24 ± 2 hours at 35.0°C ± 0.5°C. Note: Unspiked samples are not expected to have presumptive-
positive colonies, however, if typical S. Typhi colonies are observed, these should be streaked onto
TSAD for biochemical (API 20E) and serological analyses.
Do Not Cite, Circulate, or Copy B-13 December 10,2008
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Set B (without Pre-enrichment)
BSn and MMn Plates
• Examine BSD and MMD plates at 48 ± 3 hours; record results.
• For each BSD and MMD with typical colonies, streak a single typical colony onto TSAD and incubate
24 ± 2 hours at 35.0°C ± 0.5°C. Note: Unspiked samples are not expected to have presumptive-
positive colonies, however, if typical S. Typhi colonies are observed, these should be streaked onto
TSAD for biochemical (API 20E) and serological analyses.
TSAn Plates
• Examine TSAD plates at 24 ± 2 hours. Note: TSAD plates may be refrigerated following incubation
prior to serological and biochemical analyses.
• Serological Analyses with Salmonella antiserum (Vi and O Group D):
Vi
o Emulsify a portion of a single presumptive positive colony from each TSA plate using sterile
physiological saline. Place two discrete drops of emulsified growth onto a slide. To the first
drop of emulsified growth, add one drop of Vi antiserum. To the second drop of emulsified
growth, add one drop of sterile saline (as a visual comparison). Observe under magnification for
an agglutination reaction and record results.
O Group D
o Prepare a heavy suspension of cells (3-4 McFarland tube concentration) in 500 (iL of
physiological saline and place in a boiling waterbath for 20-30 minutes.
o Place two discrete drops of boiled cell suspension onto a slide. To the first drop of cell
suspension, add one drop of Salmonella O antiserum Group D. To the second drop of cell
suspension, add one drop of sterile saline (as a visual comparison). Observe under magnification
for an agglutination reaction, which indicates a positive result.
Results should be compared with those for positive (S. Typhi) and negative (E. faecalis) controls analyzed
at the same time. S. Typhi is agglutination-positive for both O Group D and Vi antisera.
• Biochemical Analyses:
o Oxidase Test: Transfer a small amount of cells from a single colony to the slide and follow
manufacturers' instructions for analysis. Oxidase-positive bacteria turn the reagent dark purple
within 20 seconds. S. Typhi is oxidase-negative.
o API 20E Test Strips: Emulsify a large colony (2-3 mm) in 5.0 mL physiological saline
supplemented with 5 |JL DHB. Follow manufacturer's instructions to inoculate wells. Incubate
test strip at 36.0°C ± 2.0°C for 18 - 24 hours.
Day Six (Friday)
Set A (with Pre-enrichment)
BSn and MMn Plates
• Examine BSD and MMD plates at 48 ± 3 hours; record results.
Do Not Cite, Circulate, or Copy B-14 December 10,2008
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• For each Set A BSD and MMD plate with typical colonies, streak a single typical colony onto TSAD
and incubate 24 ± 2 hours at 35.0°C ± 0.5°C.
TSAr, Plates
• Conduct serological analyses (Vi and O Group D) and biochemical (oxidase and API 20E) tests as
described above (Day Five) using TSAD plates inoculated on Day Five.
Set B (without Pre-enrichment)
TSAr, Plates
• Conduct serological analyses (Vi and O Group D) and biochemical (oxidase and API 20E) tests as
described above (Day Five) using TSAD plates inoculated on Day Five.
Biochemical Analyses
• API 20E Test Strips: Add appropriate reagents according to manufacturer's instructions, observe and
record results to strips inoculated on Day Five. If results are not consistent with S. Typhi, please
contact CSC (Yildiz Chambers) immediately for additional instructions.
Day Seven (Saturday)
Set A (with Pre-enrichment)
TSAr, Plates
• If additional TSAD plates were streaked on Day Six, conduct serological analyses (Vi and O Group
D) and biochemical (bxidase and API 20E) tests as described above (Day Five).
Biochemical Analyses
• If additional API 20E Test Strips were inoculated on Day Six, add appropriate reagents as described
above (Day Six).
Set B (without Pre-enrichment)
• Set B was completed on Day Six
Day Eight (Sunday)
Set A (with Pre-enrichment)
Biochemical Analyses
• If additional API 20E Test Strips were inoculated on Day Seven, add appropriate reagents as
described above (Day Six).
Reporting Results
• Please fax results to Yildiz Chambers at (703) 461-8056 at the end of each week.
• Within one week of optimization study analysis completion (runs 1 and 2), please FedEx all original
hard copy data to CSC.
Do Not Cite, Circulate, or Copy B-15 December 10,2008
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Appendix A
Do Not Cite, Circulate, or Copy B-16 December 10,2008
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S. Typhi Method Optimization Analytical Flowchart
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
P re-enrichment
inUPD
Set
A
Selective
enrichment
in SCBD
EC
BS0 agar
TSA0 agar
mm.
mm
—
h
3 °-
Iflj.
-
Incubate at
35°C ± 0.5°C
for 24 ±2 hrs
Incubate at
35°C ± 0.5°C for
18 ±2 hrs
x
X X
MMD agar
X
X X
O)
co
CL
i_
(M
5.
=
I
O
Incubate at 35°C ± 0.5°C
Observe at 24 and 48 hrs
i Biochemical test strip
X X
X
X X
[ V- -S
Confirm 1 isolate
per dilution, per
medium (6 total)
Incubate at 35°C ±
0.5°Cfor24±2 hrs
Vi
sen
Co
fro
d
and O Group D
jrn agglutination
O O 0
nfirm 1 isolate
m each plate
2 total in this
example)
AND
\
BSo agar
Set
B
Selective
enrichment
in SCBD
OL
arc UK rat
F
UlL
If
<1J
y a
~ O T3
0. C
_ CO
(O Q"
re CD
a)
Incubate at
O
XI
X X
MMD agar
X
X X
o
CM
O) O
TSA0 agar
X
X X
Biochemical test strip
rjOQOOOQQOQOQQOOOOOO
Confirm 1 isolate
per dilution, per
medium (6 total)
Vi and O Group D
serum agglutination
O
O
O
Confirm 1 isolate
from each plate (12
total in this example)
Incubate at 355C ± 0.5°C Incubate at 35°C ±
35°C ± 0.5°C for observe at 24 and 48 hrs 0.5"C for 24 ± 2 hrs
18 ± 2 hrs
If necessary, at 48
hrs, streak 1 "+" —
colony per plate onto
TSAo plates
If necessary, at 48
hrs, streak 1 "+" "
colony per plate
onto TSAD plates
Conduct serological
confirmation and
begin biochemical
test as previously
described
Conduct serological
confirmation and
•-begin biochemical
test as previously
described
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December 10, 2008
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Method Optimization Instructions for Salmonella Typhi using
Immunomagnetic Separation (IMS): Water Matrices
April 22, 2008
The instructions provided below are for analysis of unspiked and spiked samples for two matrices (PBS
and surface water). Surface water samples will be processed without immunomagnetic separation (IMS)
and with IMS (Sets A and B, respectively) and plated on two selective agars (BSD and MMD). The
reference matrix, PBS, will be processed only with IMS. Please contact Yildiz Chambers (703) 461-
2165, ychambers@csc.com) if you have any questions regarding these instructions.
All QA/QC analyses specified in the study-specific instructions and draft SAP must be conducted in
conjunction with sample analyses described below. In addition, the laboratory must follow all QA/QC
procedures outlined in their QAPP.
Media/Reagents/Supplies
• Phosphate buffered saline (PBS)
• 10% sodium thiosulfate
• 2,3-dihydroxybenzoate (DHB)
• IX, 2X, and 5X universal pre-enrichment broth with DHB (UPD)
• IX, 2X, and 5X selenite cystine broth with DHB (SCBD)
• Bismuth sulfite with DHB (BSD) agar
• Miller-Mallinson with DHB (MMD) agar
• Tryptic soy agar with DHB (TSAD)
• Tryptic soy broth (TSB)
• 1 % tryptic soy broth with DHB (TSBD)
• Salmonella spp. immunomagnetic separation (IMS) beads
• Physiological saline (0.85%)
• API 20E Test Strips
• Oxidase reagent
• Salmonella Vi antisera
Control Cultures
o S. Tvphi (CVD 909)
o Enterococcus faecalis (ATCC® 29212™)
o Pseudomonas aeruginosa (ATCC® 27853™)
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Water Matrices
Water matrices will include a reference matrix (PBS) and surface water from a lake, reservoir, or other
open body of water.
Quality Control (QC) Analyses
QC analyses listed on the batch specific coversheet should be analyzed with each run.
Sample Analyses
• Set A (without IMS) will be pre-enriched in UPD broth, with samples sub-cultured into a selective
broth (SCBD), followed by plating on selective agars (BSD and MMD).
• Set B (with IMS) will be pre-enriched in UPD broth, concentrated by IMS, IMS beads transferred to
selective broth (SCBD), followed by plating on selective agars (BSD and MMD).
• Isolated, typical colonies from BSD and MMD plates from both Set A and Set B will then be sub-
cultured onto TSAD and submitted to serological and biochemical confirmation according to study-
specific instructions.
Analyses:
o Set A: Surface Water (without IMS)
o 1, 100-mLunspiked
o 2, 100-mL spiked
o Set B: PBS (with IMS)
o 1, 100-mLunspiked
o 1, 100-mL spiked
o Set B: Surface Water (with IMS)
o 1, 100-mL unspiked
o 4, 100-mL spiked
Media and Reagent Preparation
Reagent Preparation
• Phosphate Buffered Saline (PBS): Prepare 1.0 L of PBS according to the following instructions and
store at room temperature for a maximum of three months in screw cap bottles [4, 99-mL dilution
blank, 2, 100-mL samples; and 100-mL IMS wash buffer].
Monosodium phosphate (NahbPC^)
Disodium phosphate (Na2HPC>4)
Sodium chloride
Reagent-grade water
0.58 g
2.50 g
8.50 g
1.0L
Dissolve reagents in 1 L of reagent-grade water in a flask and dispense in appropriate volumes in
screw cap bottles and autoclave at 121°C (15 Ib pressure per square inch [PSI]) for 15 minutes. Final
pH should be 7.4 ± 0.2.
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April 22, 2008
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• Prepare IMS wash buffer by adding 50 (iL Tween 20 to 100 mL of PBS, ensuring all of the Tween
20 is expelled from the pipette tip; filter sterilize or autoclave at 121°C (15 PSI) for 15 minutes.
Media Preparation
It is recommended that the following be prepared, either prior to the week of study analyses (DHB, UPD,
TSAD, TSB, 1% TSBD), or the day before analyses (SCBD, BSD), so that appropriate QC analyses can be
conducted on the media prior to use. MMD medium will be provided as prepared plates.
• 2,3-dihydroxybenzoate (DHB): Prepare 100 mL of 0.1% (w/v) DHB stock solution by dissolving
0.1 g of DHB powder in 100 mL of reagent-grade water and filter sterilize.
• Universal Pre-enrichment Broth with DHB (UPD):
o Prepare UPD according to the following:
• For 350 mL of IX UPD, add 13.3 g dehydrated medium to 350 mL reagent-grade water; mix
thoroughly.
• For 350 mL of 2X UPD, add 26.6 g dehydrated medium to 350 mL reagent-grade water; mix
thoroughly.
• For 200 mL of 5X UPD, add 38.0 g dehydrated medium to 200 mL reagent-grade water; mix
thoroughly.
o Autoclave all solutions at 121°C (15 PSI) for 15 minutes. Final pH should be 6.3 ± 0.2. Cool to
45°C-50°Cinawaterbath.
• For IX UPD, add 0.35 mL of 0.1% DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
• For 2X UPD, add 0.7 mL of 0.1% DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
• For 5X UPD, add 1.0 mL of 0.1% DHB stock solution, mix thoroughly, and dispense 5.0 mL
aliquots in 25 x 150 mm tubes.
o Store at <10°C and above freezing for a maximum of 2 weeks.
• Selenite Cystine Broth with DHB (SCBD):
o Prepare 1.0 L SCBD according to the manufacturer's instructions. Do not autoclave. Final pH
should be 7.0 ± 0.2. Cool to 45 °C - 50°C in a waterbath.
• For IX SCBD, add 1.0 mL of 0.1 % DHB stock solution, mix thoroughly, and dispense 10.0
mL aliquots in 25 x 150 mm tubes.
o Use within 48 hours of preparation. Note: A brick red precipitate may appear if the medium is
overheated during preparation or exposed to excessive moisture during storage. If this occurs
medium should be discarded and a new batch prepared.
• Bismuth Sulfite with DHB (BSD): Prepare 1.5 L of BSD agar according to manufacturer's
instructions the day prior to analyses. Cool to 45°C - 50°C in a waterbath and add 1.5 mL of the
0.1% DHB solution. Evenly disperse the precipitate while aseptically pouring 12 - 15 mL into
100 x 15 mm sterile Petri plates. Use within 48 hours of preparation. Store at room temperature and
protected from light [100, 15-mL, 100 x 15 mm plates].
• Miller-Mallinson with DHB (MMD): Agar will be received as prepared plates. Store at < 10°C and
above freezing [100, 100 x 15 mm plates].
• Tryptic Soy Agar with DHB (TSAD): Prepare 200 TSAD plates according to manufacturer's
instructions. After autoclaving, cool to 45 °C - 50°C in a waterbath and add 0.1 mL of the 0.1% DHB
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solution per 100 mL of medium. Aseptically pour 12-15 mL into each 100 x 15 mm sterile Petri
plate. Store at <10°C and above freezing for a maximum of 2 weeks.
• Tryptic Soy Broth (TSB): Prepare 100 mL TSB according manufacturer's instructions. Store at
<10°C and above freezing for a maximum of three months in a screw cap bottle.
• 1% Tryptic Soy Broth with DHB (TSBD): Prepare a 1% solution of TSBD by combining 99 mL of
sterile PBS, 1 mL of sterile single-strength TSB, and 0.1 mL of DHB solution in a sterile screw cap
bottle. Shake to mix.
Study Analyses
Instructions provided below are for a single week of analyses (1 run).
Day One (Sunday)
Propagation of Spiking Suspension
Inoculate 1% TSBD with S. Typhi according to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ±
2 hours.
Day Two (Monday)
TSA Enumeration of Spiking Suspensions
• Dilute and plate laboratory-prepared spiking suspension on TSAD according to spiking protocol and
incubate at 35 °C ± 0.5 °C for 24 ± 2 hours.
Preparation and Collection of Samples
o Sample Collection and/or Preparation - PBS and Surface Water
• PBS (Reference Matrix) Samples
o Aliquot the following volumes of sterile PBS and label the samples as follows:
• 1, 100-mL unspiked sample - PBS unspiked, with IMS (Set B)
• 1, 100-mL spiked samples - PBS spiked, with IMS (Set B)
• Collect a 1.0-L bulk surface water sample as follows:
o Collect samples by hand or with a sampling pole if the sampling site has difficult access such as a
dock, bridge, or bank adjacent to surface water.
o The sampling depth should be 6" - 12" below the water surface.
o Sample containers should be positioned such that the mouth of the container is pointed away
from the sampler or sample point.
o After removal of the container from the water, a small portion of the sample should be discarded
to leave a headspace of 2.5 cm - 5 cm (1" - 2") for proper mixing before analyses.
o Transport to the laboratory on ice (do not freeze).
o In the laboratory, split the bulk sample into aliquots and label the samples as follows:
• Set A:
1, 100-mL unspiked sample - Surface water unspiked, without IMS
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2, 100-mL spiked sample - Surface water spiked, without IMS
• Set B:
1, 100-mL unspiked sample - Surface water unspiked, with IMS (Set B)
4, 100-mL spiked samples - Surface water spiked, with IMS (Set B)
Sample Spiking
• Spike 1, 100-mL sample of PBS and 6, 100-mL surface water samples with laboratory-prepared
spiking suspension.
Most Probable Number (MPN)
• Mix unspiked and spiked samples (Sets A and B) by shaking 25 times. Each 100 mL sample will be
used to inoculate tubes.
• For each Set A and Set B samples, inoculate UPD tubes with the following volumes for each unspiked
or spiked sample:
o For each of three tubes, add 20 mL of undiluted sample to 5 mL (5X) UPD
o For each of three tubes, add 10 mL of undiluted sample to 10 mL (2X) UPD
o For each of three tubes, add 1 mL of undiluted sample to 10 mL (IX) UPD
• Incubate inoculated UPD tubes at 35.0°C ± 0.5°C for 24 ± 2 hours.
Day Three (Tuesday)
TSA Enumeration of Spiking Suspension
• Count colonies on TSA plates prepared on Day Two and record results of CPU enumeration on
provided data reporting forms.
• Fax TSA enumeration results to Yildiz Chambers at (703) 461-8056.
MPN
Set A (without I MS)
• Examine UPD tubes at 24 hours; record results.
• For each UPD tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes with
10.0 mL of SCBD. Incubate at 35.0°C ± 0.5°C for 18 ± 2 hours.
Set B (with IMS)
• For each UPD tube with growth, conduct IMS as follows:
o Suspend Salmonella beads by vortexing and add 20 (iL of bead suspension to a sterile, 1.5-2.0
mL tube.
o Gently swirl contents of UPD tubes to mix and remove 1.0 mL of enrichment culture; add to tube
with beads and tightly cap.
o Mix enrichment culture with beads for 10 minutes at room temperature using tube rotator.
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o Place tubes in magnetic holder, inverting tubes several times to ensure that beads are
concentrated in a pellet on the side of the tube; allow beads to settle for 3 minutes.
o Carefully aspirate liquid and discard; resuspend beads in 1.0 mL IMS wash buffer by inverting
the tube several times; place tubes in magnetic holder and allow beads to settle for 3 minutes;
aspirate wash buffer.
o Resuspend the beads in 100 (iL of IMS buffer.
• Pipette beads from each sample (100 (iL) into a set of corresponding tubes with 10.0 mL of SCBD.
Incubate at 35.0°C ± 0.5°C for 18 ± 2 hours.
Day Four (Wednesday)
MPN
• Examine SCBD tubes (Sets A and B) at 18 ± 2 hours; record results.
• For each SCBD tube with growth, gently swirl the tube to mix and streak for isolation on BSD and
MMD plates using a sterile inoculation loop (20 |JL). Incubate at 35.0°C ± 0.5°C for 48 ± 3 hours,
examining plates at 24 ±2 hours and 48 ± 3 hours.
Day Five (Thursday)
BSn and MMn Plates
• Examine BSD and MMD plates (Sets A and B) at 24 ±2 hours; record results.
• If no typical (green-black with metallic sheen for BSD or black on MMD) colonies are observed on
plates, re-incubate for a total of 48 ± 3 hours at 35.0°C ± 0.5°C.
• For each BSD and MMD with typical colonies, streak a single typical colony onto TSAD and incubate
24 ± 2 hours at 35.0°C ± 0.5°C. Note: Unspiked samples are not expected to have presumptive-
positive colonies, however, if typical S. Typhi colonies are observed, these should be streaked onto
TSAD for biochemical (API 20E) and serological analyses.
Day Six (Friday)
TSAn Plates
• Examine TSAD plates (Sets A and B) at 24 ±2 hours. Note: TSAD plates may be refrigerated
following incubation prior to serological and biochemical analyses.
• Serological Analyses with Salmonella Vi antiserum:
o Emulsify a portion of a single presumptive positive colony from each TSA plate using sterile
physiological saline. Place two discrete drops of emulsified growth onto a slide. To the first
drop of emulsified growth, add one drop of Vi antiserum. To the second drop of emulsified
growth, add one drop of sterile saline (as a visual comparison). Observe under magnification for
an agglutination reaction and record results.
o Results should be compared with those for positive (S. Typhi) and negative (E. faecalis) controls
analyzed at the same time. S. Typhi is agglutination-positive for Vi antisera.
• Biochemical Analyses:
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o Oxidase Test: Transfer a small amount of cells from a single colony to the slide and follow
manufacturers' instructions for analysis. Oxidase-positive bacteria turn the reagent dark purple
within 20 seconds. S. Typhi is oxidase-negative.
o API 20E Test Strips: Emulsify a large colony (2-3 mm) in 5.0 mL physiological saline
supplemented with 5 |JL DHB. Follow manufacturer's instructions to inoculate wells. Incubate
test strip at 36.0°C ± 2.0°C for 18 - 24 hours.
BSn and MMn Plates
• Examine BSD and MMD plates (Sets A and B) at 48 ± 3 hours; record results.
• For each BSD and MMD plate with typical colonies, streak a single typical colony onto TSAD and
incubate 24 ± 2 hours at 35.0°C ± 0.5°C.
Day Seven (Saturday)
TSAn Plates
• If additional TSAD plates were streaked on Day Six, conduct serological analyses and biochemica/
(oxidase and API 20E) tests as described above.
Biochemical Analyses
• API 20E Test Strips: Add appropriate reagents according to manufacturer's instructions, observe and
record results to strips inoculated on Day Five. If results are not consistent with S. Typhi, please
contact CSC (Yildiz Chambers) immediately for additional instructions.
Day Eight (Sunday)
Biochemical Analyses
• If additional API 20E Test Strips were inoculated on Day Seven, add appropriate reagents as
described above.
Reporting Results
• Please fax results to Yildiz Chambers at (703) 461-8056 at the end of each week.
• Within one week of optimization study analysis completion (runs 1 and 2), please FedEx all original
hard copy data to CSC.
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Appendix C
Spiking Protocol
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Draft - Do Not Cite, Circulate, or Copy
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Water Matrices Spiking Protocol for the Draft Salmonella Typhi SAP
Verification Study
Draft for Discussion - November, 2008
Do Not Cite, Circulate, or Copy
The purpose of this protocol is to provide laboratories with Salmonella enterica subsp. enterica serotype
Typhi (S. Typhi) spiking procedures for the verification and validation study to evaluate the Draft
Standardized Analytical Procedure for Salmonella enterica subsp. enterica serotype Typhi (Salmonella
Typhi or S. Typhi) in Environmental Samples (referred to as the "draft SAP"). The following sections are
included in this protocol:
Laboratory-Prepared Spiking Solutions
Section 1: Preparation of Laboratory-Prepared Spiking Suspensions
Section 2: Laboratory-Prepared Sample Spiking and Enumeration
Section 3: Calculation of Laboratory-Prepared Spike Percent Recovery
1.0 Preparation of Laboratory-Prepared Spiking Suspensions
1.1 Stock Culture. Prepare a stock culture by inoculating a tryptic soy agar (TSA) slant (or other
non-selective media) containing 0.0001% DHB with S. Typhi and incubating at 36°C ± 1.5°C for
20 ± 4 hours. After incubation, the stock culture may be stored in the dark at room temperature
for up to 30 days.
1.2 1% Tryptic Soy Broth (TSB). Prepare a 1% solution of TSB by combining 99 mL of sterile
PBS, 1 mL of sterile single-strength TSB (prepared according to manufacturer's instructions), and
0.1 mL of 2,3-dihydroxybenzoate (DHB) solution (prepared according to the study instructions)
in a sterile screw cap bottle. Shake to mix.
1.3 Spiking Suspension (Undiluted). From the stock culture of S. Typhi in section 1.1, aseptically
transfer a small loopful of growth to the 1% TSB solution and vigorously shake a minimum of 25
times. Incubate at 36°C ± 1.5°C for 20 ± 4 hours. The resulting spiking suspension contains
approximately 1.0* 106 to 1.0* 107 S. Typhi colony forming units (CPU) per mL. This is referred
to as the "undiluted spiking suspension." Note: During the S. Typhi draft SAP verification study,
growth of spiking suspensions should begin one day prior to the day samples will be spiked. For
example, if samples will be spiked on Tuesday, growth of spiking suspensions will begin on
Monday.
1.4 Proceed to Section 2.0 for sample spiking and enumeration of spiking suspension.
2.0 Laboratory Prepared Sample Spiking and Spiking Suspension Enumeration
Since the objective of spiking the sample is to establish percent recovery, it is necessary to determine the
concentration of S. Typhi in laboratory prepared undiluted spiking suspensions (Section 1.3). This
section provides instructions for sample spiking (2.1) and spiking suspension enumeration (2.2).
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2.1 Sample spiking
Please be sure to homogenize well the spiking suspensions in the steps below as homogenization
is critical for accurate sample spiking and spiking suspension enumeration.
2.1.1 Dilute spiking suspension
Perform steps 2.1.1.1 through 2.2.6 using laboratory-prepared undiluted spiking suspension
(Section 1.3).
2.1.1.1 Mix spiking suspension by vigorously shaking the bottle a minimum of 25
times. Use a sterile pipette to transfer 1.0 mL of undiluted spiking suspension
(from Section 1.3 above) to 99 mL of sterile PBS, cap, and mix by vigorously
shaking the bottle a minimum of 25 times. This is spiking suspension dilution
"A." A 1.0-mL volume of dilution "A" is equal to 1 x 10~2 of the undiluted
spiking suspension and is equal to 1 x 104to 1 x 105 CFU/mL.
2.1.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution "A" (from
Section 2.1.1.1 above) to 99 mL of sterile PBS, cap, and mix by vigorously
shaking the bottle a minimum of 25 times. This is spiking suspension dilution
"B." A 1.0 mL volume of dilution "B" is equal to 1 x 10^ of the undiluted
spiking suspension and is equal to 1 x 102to 1 x 103 CFU/mL.
2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B"
(from Section 2.1.1.2 above) to 99 mL of sterile PBS, cap, and mix by
vigorously shaking the bottle a minimum of 25 times. This is spiking
suspension dilution "C." A 1.0-mL volume of dilution "C" is equal to 1 x 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 pipette to transfer 11.0 mL of spiking suspension dilution "C"
(from Section 2.1.1.3 above) to 99 mL of sterile PBS, cap, and mix by
vigorously shaking the bottle a minimum of 25 times. This is spiking
suspension dilution "D." A 1.0-mL volume of dilution "D" is equal to 1 x 10"6
of the undiluted spiking suspension and is equal to 1x10° to IxlO1 CFU/mL
(i.e., 1 to 10 CFU/mL).
2.1.2 Spike sample(s)
2.1.2.1 To spike the sample, add 0.3 mL of spiking suspension dilution "B" (from
Section 2.1.1.2) to 100 mL of unspiked sample and mix by vigorously shaking
the bottle a minimum of 25 times. The concentration of the spiking suspension
added to each 100.0 mL of sample is 3 x io~5 of the undiluted spiking
suspension. This is referred to as Vspiked per 100 mL sample. Analyze the
spiked sample according to study instructions and the draft SAP.
2.1.2.2 For samples analyzed by MPN, inoculate unspiked and spiked samples
according to study instructions and the draft SAP.
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2.2 Enumeration of Spiking Suspensions (Prepared in Section 1.3)
2.2.1 Prepare TSA with 0.0001% DHB according to manufacturer's and study instructions.
Add 10-15 mL of TSA per 100 x 15 mm petri dish, and allow the agar to solidify.
Note: Agar plates must be dry and free from condensation prior to use. To ensure that
agar surface is dry, plates should be made several days in advance and stored inverted at
room temperature or dried using a laminar-flow hood.
2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine
spread plates:
• Mix dilution "B" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1
mL of dilution "B" (Section2.1.1.2) onto the surface of the pre-dried TSA plate. This
is 10 ~5 of the undiluted spiking suspension.
• Mix dilution "C" by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1
mL of dilution "C" (Section 2.1.1.3) onto the surface of the pre-dried TSA plate.
This is 10 ~6of 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 at35°C±0.5°Cfor20±4 hours.
2.2.6 Count and record number of colonies per plate. Refer to Section 5.0 for calculation of
spiking suspension concentration.
3.0 Calculation of Spiked S. Typhi Percent Recovery (Laboratory-Prepared Undiluted
Spiking Suspension)
Spiked S. Typhi percent recovery will be calculated in three steps as indicated in Sections 3.1
through 3.3, below. Note: Example calculated numbers provided in tables below have been
rounded at the end of each step. If your laboratory recalculates examples using a spreadsheet and
rounds only after the final calculation (Section 3.3), percent recoveries may be slightly different.
3.1 Step 1: Calculate Concentration of S. Typhi (CFU/mL) in Undiluted Spiking
Suspension
3.1.1 The number of S. Typhi (CFU/mL) in the undiluted spiking suspension (prepared in
Section 1.3, above) will be calculated using all TSA plates from Section 2.2 yielding
counts within the ideal range of 30 to 300 CPU 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).
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3.1.3 Calculate the concentration of S. Typhi (CFU/mL) in undiluted spiking suspension
according to the following equation. Example calculations are provided in Table 2.
S. Typhi undiluted spike = (CFUi + CFU2 + ... + CFUn) / (Vi + V2 + ... + Vn)
Where,
S. Typhi undiluted spike = S. Typhi (CFU/mL) in undiluted spiking suspension
CPU = Number of colony forming units from TSA plates yielding counts within the
ideal range of 30 to 300 CPU per plate
V = Volume of undiluted sample on each TSA plate yielding counts within the
ideal range of 30 to 300 CPU per plate
n = Number of plates with counts within the ideal range
Table 2. Example Calculations of S. Typhi Spiking Suspension Concentration
Examples
Example 1
Example 2
CFU/plate (triplicate analyses) from
TSA plates in Section 2.2.2
10~5 mL plates
31, 34, 32
45, 56, 61
10~6 mL plates
3, 0, 5
4, 3, 8
10~7 mL plates
0, 1, 0
0,2, 0
S. Typhi CFU/mL in undiluted
spiking suspension
(S. Typhi undiluted spike)*
(31 +34+32) /(10'5+10'5+10'5) =
97/(3.0x10'5) = 32.3x 105
= 3.2 x 10s CFU/mL
(45+56+61 )/(10'5+10'5+10'5) =
162/(3x10'5) = 54.0 x 1Q5
= 5.4 x 10s CFU/mL
* S. Typhi undiluted spike is calculated using all plates yielding counts within the ideal range of 30 - 300 CPU per plate.
3.2 Step 2: Calculate "True" Spiked S. Typhi (CFU/100 mL)
Calculate the true concentration of spiked S. Typhi (CFU/100 mL) according to the following
equation. Example calculations are provided in Table 3.
Tspiked s Typhi = (S. Typhi undiluted spike) X (V spiked per 100 ml sample)
Where,
A Spiked S. Typhi ~~
S. Typhi undiluted spike =
» spik
.piked per 100 mL sample
Number of spiked S. Typhi (CFU/100 mL)
S. Typhi (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)
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C-4
November, 2008
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Table 3. Example Calculations of Spiked S. Typhi
S. Typhi undiluted spike
(Table 2 above)
3.2x1 0s CFU/mL
5.4x1 0s CFU/mL
V spiked per 100 mL sample
(Section 2.1.2.1 above)
3. Ox 10"5 mL per 100 mL of sample
3. Ox 10"5 mL per 100 mL of sample
Tspiked S. Typhi
(3.2 x 10s CFU/mL) x (3.0 x 10'5 mL/100 mL) =
96 CFU/100 mL
(5.4 x 10s CFU/mL) x (3.0 x 10'5 mL/100 mL) =
162 CFU/100 mL
3.3 Step 3: Calculate Percent Recovery
3.3.1 Calculate percent recovery (R) using the following equation.
N-N..
[ Spiked S.Typhi
Where,
R
Ns
Nu
TypM
Percent recovery
S. Typhi (CFU/100 mL) in the spiked sample
S. Typhi (CFU/100 mL) in the unspiked sample
True spiked S. Typhi (CFU/100 mL) in spiked sample (Section 3.2, above)
3.3.2 Example percent recovery calculations are provided in Table 4.
Table 4. Example Percent Recovery Calculations
Ns (CFU/100 mL)
42
34
153
142
Nu (CFU/100 mL)
<1
10
<1
<1
T spiked s. Typm (CFU/1 00 mL)
96
96
162
162
Percent recovery (R)
100x(42-1)/96 = 43%
100 x (34 - 10) / 96 = 25%
100 x (153 -1)/ 162 = 94%
100x(142-1)/162= 87%
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C-5
November, 2008
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Appendix D
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S. Typhi Optimization Analyses: SET B (without Pre-enrichment)
SPIKED
Sample #:
Spike dilution:
Lab:
Date:
Matrix (circle one): PBS Drinking Water Surface Water
Spike volume:
Spiking date and time:
Please record plate counts and + / - for biochemical and serological analyses.
Sample Volume
20 mL
(5X SCBD)
10 mL
(2X SCBD)
1 mL
(1XSCBD)
Incubation
Start
Incubation
End
1
2
3
1
2
3
1
2
3
Date:
Time:
Temp:
Initials:
Date:
Time:
Temp:
Initials:
Analyses
SCBD
BSD
MMD
Salmonella Antisera
SCBD
24 ± 2 h @ 35.0°C ± 0.5°C
BSD
24 ± 2 h @
35.0°C ± 0.5°C
48 ± 3 h @
35.0°C ± 0.5°C
MMD
24 ± 2 h @
35.0°C ± 0.5°C
48 ± 3 h @
35.0°C ± 0.5°C
TSAD
18-24h@35°C±0.5°C
API
(+or-)
S. Typhi Results
Growth, indicated by turbidity.
Black colonies surrounded by black or brownish-black zone, which may have metallic sheen (in light).
Black colonies
Agglutination, indicated by the formation of a white precipitate
Vi
Agglutination
(+or-)
O Group D
Agglutination
(+ or -)
Final Results
MPN Tube Combination
MPN/100mL
Do Not Cite, Circulate, or Copy
D-6
December 18, 2008
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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
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