EPA 600/R-10/133 | October 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Standard Analytical Protocol for
Salmonella Typhi in Drinking
Water
Office of Research and Development
National Homeland Security
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for
in
Office of Research and Development
National Homeland Security Research Center
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This Standard Analytical Protocol (SAP) is based on procedures evaluated by Scientific Methods,
Inc. under direction of Sanjiv R. Shah at the National Homeland Security Research Center within the
U.S. Environmental Protection Agency's (EPA's) Office of Research and Development for analysis
of Salmonella enterica subsp. enterica serotype Typhi (Salmonella Typhi) in drinking water samples.
Technical support and data evaluation were provided by Computer Sciences Corporation under EPA
Contract No. EP-C-05-045.
The contributions of the following persons and organizations are acknowledged:
Study Workgroup Participants
• Michclc Burgess, Marissa Mullins (EPA, Office of Emergency Management)
« Stephanie Harris (EPA, Region 10)
• Sarah Perkins, Gene Rice (EPA. National Homeland Security Research Center)
* Ouida Holmes, Malik Raynor, James Sinclair (EPA, Office of Ground Water and Drinking
Waier)
• Matthew Mikolcit (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 is a draft and is undergoing review. 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 any warrant}", 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 parr}' would not infringe on privately owned rights.
The procedures described in this document are intended for use in laboratories when analyzing
water samples in support of remediation efforts following a homeland security incident. The
culture-based procedures provide viability determination, identification, and either qualitative
or quantitative results. The sample preparation procedures are deemed Hie most appropriate
for the wide variety of water matrices to be examined. To the extent possible, these procedures
were developed to be consistent with other federal agency procedures. These procedures do
not include the sample collection, rapid screening, field techniques, or molecular techniques
that may accompany laboratory analysis.
Questions concerning this document or its application should be addressed to:
Sanjiv R. Shall
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
ff you have difficult}" assessing these PDF documents, please contact Nickel.Kathyi@epa.gov or
McCall.Amelia@epa.gov for assistance.
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Foreword
The mission of the U.S. Environmental Protection Agency (EPA) is to protect human health and to
safeguard the natural environment - the air, water, and land upon which life depends. After the 2001
terrorist attacks including the anthrax bioterrorism event, the EPA's mission was expanded to address
critical needs related to homeland security. Presidential directives identified EPA as the primary
federal agency responsible for the protection and decontamination of indoor-outdoor structures and
water infrastructure vulnerable to chemical, biological, or radiological (CBR) terror attacks.
The National Homeland Security Research Center (NHSRC) within the Office of Research and
Development (ORD) is EPA's focal point for providing expertise, and for conducting and reporting
research to meet its homeland security mission needs. One specific focus area of the NHSRC's
research is to support the Environmental Response Laboratory Network (ERLN), a nationwide
association of federal, state, local, and commercial environmental laboratories, established by EPA.
The ERLN can be deployed in response to a large-scale environmental disaster to provide consistent
analytical capabilities, capacities, and quality data in a systematic and coordinated manner. To this
end, the NHSRC has worked with experts across EPA and other federal agencies to develop standard
analytical protocols (SAPs) to be used in support of the response to national homeland security
related incidents.
This Standard Analytical Protocol (SAP) is for the identification, confirmation, and quantitation of
Salmonella Typhi in drinking water samples, using selective and non-selective media followed by
biochemical characterization and serological confirmation.
NHSRC has made this publication available to assist in preparing for and recovering from disasters
involving Salmonella Typhi contamination. This work specifically represents an important step
in EPA's support for the ERLN and moves the agency closer to achieving its mission to support
homeland security and its overall mission to protect human health and the environment.
Gregory D. Sayles, Ph.D., Acting Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
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Acknowledgments iii
Disclaimer iv
Foreword v
1.0 Scope and Application 1
2.0 Summary of Method 3
3.0 Acronyms and Abbreviations 5
4.0 Interferences and Contamination 7
5.0 Safety 9
5.1 Laboratory Hazards 9
5.2 Recommended Precautions 9
6.0 Equipment and Supplies 11
7.0 Reagents and Standards 13
8.0 Calibration and Standardization 19
9.0 Quality Control 21
9.1 General 21
9.2 Negative Controls 21
9.3 Positive Controls 21
9.4 Method Blank 22
9.5 Media Sterility Check 22
10.0 Procedures 23
10.1 Qualitative Sample Analysis 23
10.2 Quantitative Sample Analyses 23
10.3 Selenite Cvstine Broth Culture 23
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10.4 Isolation on Bismuth Sulfite and Miller-Mallinson Agar Plates 23
10.5 Isolation on Tryptic Soy Agar Plates 24
10.6 Biochemical Analyses 24
10.7 Serological Analyses 24
10.8 Description of Control and Salmonella Typhi Results 25
11.0 Data Analysis and Bacterial Density Calculation 27
11.1 Most Probable Number (MPN) Technique 27
11.2 Calculation of MPN 27
12.0 Protocol Performance 33
13.0 Pollution Prevention 37
14.0 Waste Management 39
15.0 References 41
16.0 Flowcharts and Diagrams 43
16.1 Quantitative Analysis Dilution Scheme 43
16.2 Identification Flowchart 44
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Table 1. Positive and Negative Control Cultures for Described Tests 16
Table 2. Storage Temperatures and Times for Prepared Media and Reagents 17
Table 3. Positive and Negative Result Descriptions and Salmonella Typhi Results 25
Table 4. Examples of Appropriate Tube Selection and MPN/100 mL 28
Table 5. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes are Used and Sample Inoculation Volumes are 20.0, 10.0, and 1.0 mL 29
Table 6. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes arc Used and Sample Inoculation Volumes arc 10.0, 1.0, and 0.1 mL 31
Table 7. Salmonella Typhi Results for PBS, Drinking Water, and Surface Water Verification
Analyses 34
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1.0
and
1.1
This Standard Analytical Protocol (SAP) is for the identification, confirmation, and quantitation of Salmonella enterica
subspecies enterica serotype Typhi (referred to as "S. Typhi" in this document) in drinking water samples, using
selective and non-selective media followed by biochemical and serological confirmation.
1.2
This method has been adapted from the American Society for Microbiology's Manual of Clinical Microbiology
(Reference 15.1) and the U.S. Food and Drug Administration's Bacteriological Analytical Manual (Reference 15.2)
for use by laboratories when analyzing samples in support of U.S. Environmental Protection Agency (EPA) homeland
security efforts.
1.3
S. Typhi is the causative agent of typhoid fever. Due to the infectious nature of the bacterium and the potential for
transmission to humans, all procedures should be performed in laboratories that use, at a minimum, biological safety
level (BSL)-2. Use of a biological safety cabinet is recommended for any aerosol-generating procedures (Reference
15.3).
1.4
All sample handling, analysis, and reporting of results must be performed in accordance with guidelines established in
the SAP. Laboratories must have requisite resources in place prior to use of these procedures.
1.5
This method is not intended for analysis of microorganisms other than S. Typhi in drinking water samples.
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2.0
of
2.1
S. Typhi can be identified in drinking water samples using selective media and biochemical and serological analyses.
Bacterial densities can be estimated using the most probable number (MPN) approach (see Section 11.0).
2.2
For qualitative results, samples are diluted 1:1 in double-strength universal pre-enrichment (UP) broth. Samples are
incubated at 35.0°C ± 0.5°C for 24 ±2 hours.
2.3
For quantitative results, samples are analyzed as received. Samples are analyzed using a 15-tube MPN. Inoculated UP
broth tubes are incubated at 35.0°C ± 0.5°C for 24 ±2 hours.
2.4
One mL of each UP broth culture (MPN and qualitative analyses tubes) with positive growth (turbidity) is transferred
to selenite cystine broth (SCB). Tubes are incubated at 35.0°C ± 0.5°C for 18 ± 2 hours. Tubes with growth are
streaked onto bismuth sulfite (BS) and Miller-Mallinson (MM) agars. Plates are incubated at 35.0°C ± 0.5°C for 24 -
48 hours.
2.5
Isolated, typical colonies are sub-cultured onto tryptic soy agar (TS A) and submitted to serological and biochemical
confirmation. Serological typing is by agglutination using Vi antiserum. followed by biochemical characterization
using commercially available test strips (e.g., API 20E® or equivalent) or with a group of selected individual
biochemical tests.
2.6
UP broth tubes (MPN and qualitative analysis) exhibiting growth (turbidity) or growth from agar plates (BS or MM),
may be confirmed by real-time polymerase chain reaction (PCR) in place of serological and biochemical confirmation.
2.7
Quantitation of S. Typhi is determined using the MPN technique (Flowchart 16.1). Tubes that are confirmed positive
for S. Typhi are used to determine MPN.
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3.0
Acronyms and Abbreviations
ATCC® American Type Culture Collection
BS Bismuth sulfite
BSL Biological safety level
°C Degrees Celsius
DHB 2,3-diliydroxybenzoate
EPA U.S. Environmental Protection Agency
g Gram(s)
L Liter(s)
jiL Microliter
mL Milliliter(s)
mm Millimeter(s)
MM Miller-Mallinson
MPN Most probable number
N Normal - one equivalent weight per liter
NA Not applicable
NIST National Institute of Standards and Technology
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PPE Personal protective equipment
psi Pounds per square inch
SAP Standard Analytical Protocol
SCB Selenite cystine broth
TSA Tryptic soy agar
UP Universal pre-enrichmenl
w/v Weight to volume ratio
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4.0
and
4.1
Low recoveries of S. Typhi may be caused by the presence of high numbers of competing or inhibitory organisms (e.g.,
other Enterobacteriaceae), or toxic substances (e.g., metals or organic compounds).
4.2
A viable but non-culturable state of S. Typhi may also account for low recoveries (Reference 15.4).
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5.0
Safety
5.1
To prevent transmission, disposable gloves should be worn when working with this organism. Hands should be
washed immediately following removal of gloves. Direct and indirect contact of intact or broken skin with cultures
and/or contaminated laboratory surfaces and accidental parenteral inoculation, are the primary hazards to laboratory
personnel. Rarely, exposure to infectious aerosols may occur. Staff should apply safety procedures used for pathogens
when handling all samples.
5.2
5.2.1
S. Typhi is a BSL-2 pathogen and all procedures should be performed in laboratories that use, at a minimum, BSL-2
practices (Reference 15.3). This includes prohibiting eating, drinking, smoking, handling contact lenses, applying
cosmetics, and storing food and drink in the laboratory.
5.2.2
A Class II biological safety cabinet is recommended for sample manipulations where the risk of aerosol production is
high. Production of aerosols should be avoided.
5.2,3
Disposable materials are recommended for sample manipulation.
5.2.4
Mouth-pipetting is prohibited.
5.2.5
The analyst must know and observe normal safely procedures required in a microbiology laboratory while preparing,
using, and disposing of media, cultures, reagents, and materials, including operation of sterilization equipment.
5.2.6
Personal Protective Equipment (PPE)
5.2.6.1
Disposable nitrile gloves should be worn at all times to prevent contact with infectious materials. Gloves
should be changed whenever they arc visibly soiled. Aseptic technique should be used when removing gloves
and other protective clothing.
5.2.6.2
Protective goggles and/or non-breakable, chemical-resistant glasses should be worn, as appropriate.
5.2.6.3
Laboratory coats covering amis and clothing, and closed in the front, should be worn at all dines. Laboratory
coats that become soiled should be changed.
5.2.7
This protocol docs not address all safety issues associated with its use. Please refer to Biosafety in Microbiological
andBiomedical Laboratories. 5th Edition (Reference 15.3) for additional safety information. A reference file of
Material Safety Data Sheets should be available to all personnel involved in analyses.
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6.0
and
6.1
Autoclave or steam sterilizer, capable of achieving 121°C (15 pounds per square inch [psi]) for 15 minutes
6.2
Autoclave bags, aluminum foil, or kraft paper
6.3
Balance, top loading, with ASTM International Class S reference weights, capable of weighing 100 g ± O.lg
6.4
Beakers, glass or plastic (assorted sizes)
6.5
Biological safety cabinet. Class II (optional)
6.6
Borosilicatc glass or plastic screw-cap, wide-mouth bottles, sterile (e.g., 250 inL)
6.7
Borosilicate glass culture tubes, with autoclavable screw or snap caps (25 x 150 mm)
6.8
Borosilicatc glass culture tubes, with autoclavable screw or snap caps (16 x 150 mm)
6.9
Erlenmeyer flasks (500 mL, 1 L, 2 L)
6.10
Graduated cylinders (assorted sizes)
6.11
Gloves, sterile, nitrile, or equivalent
6.12
Incubators, microbiological type, maintained at 35.0°C ± 0.5°C
6.13
Inoculation loops, sterile, disposable
6.14
Parafilm® or equivalent
6.15
Pelri dishes, sterile, plastic (15 * 100 mm)
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6.16
pH meter
6.17
Pipettes, standard tip, sterile, plastic, disposable (assorted sizes)
6.18
Pipetting device (automatic or equivalent)
6.19
Stirring hotplates and stir bars
6.20
Test tube racks
6.21
Thermometer, National Institute of Standards and Technology (NTST)-lraceable
6.22
Tissues, lint-free (Kimwipes® or equivalent)
6.23
Weigh paper and boats
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7.0
and
7.1
Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical Society (Reference 15.5). For
suggestions regarding the testing of reagents not listed by the American Chemical Society, see AnalaR Standards for
Laboratory Chemicals (Reference 15.6) and United States Pharmacopeia and National Formulary 24 (Reference
15.7).
7.2
Whenever possible, use commercially available culture media. The agar used in the preparation of culture media must
be microbiological grade. Storage requirements for prepared media and reagents are provided in Table 2 (Section
7.16).
7.3
Reagent-grade water must conform to specifications in Standard Methods for the Examination of Water and
Wastewater, 21st Edition, Section 9020 (Reference 15.8).
7.4
If & Typhi CVD 909 (non-virulent vaccine strain) is used as the positive control, all media must be supplemented with
2,3-dihydroxybenzoate (DHB) to obtain appropriate growth.
7.5 (PBS)
Prepare reagent according to the procedure below.
7,5.1 Composition:
Monosodium phosphate (NaH,PO4) 0.58 g
Disodium phosphate (Na,HPO4) 2.50 g
Sodium chloride 8.50 g
Reagent-grade water l.OL
7.5.2
Dissolve reagents in 1 L reagent-grade water, adjust pH to 7.4 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide, and dispense appropriate volumes in screw-cap bottles or tubes and autoclave at 121°C (15 psi) for 15
minutes.
7.6 Pre-enrichment (UP)
Commercially prepared media is recommended. Dehydrated medium (Difco™ 223510 or equivalent) may be used. If
commercially prepared media are not available, prepare IX, 2X, and 3X UP broth according to the following.
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7, §, 1 Composition:
IX 2X 3X
Pancreatic digest of casein 5.0 g 10.0 g 15.0g
Proteose peptone 5.0g 10.0 g 15.0 g
Monopotassium phosphate 15.0g 30.0 g 45.0 g
Disodium phosphate 7.0 g 14.0 g 21.Og
Sodium chloride 5.0 g 10.0 g 15.0 g
Dextrose 0.5 g l.Og 1.5g
Magnesium sulfate 0.25 g 0.5 g 0.75 g
Ferric ammonium citrate O.lg 0.2 g 0.3g
Sodium pyruvate 0.2 g 0.4 g 0.6 g
Reagent-grade water l.OL l.OL l.OL
7,6.2
Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar. Adjust pH to 6.3 ± 0.2 with 1.0 N
hydrochloric acid or 1.0 N sodium hydroxide and bring to 1 L. For 1X UP broth, aseptically dispense 10 mL volumes
into 25 x 150 mm tubes. For 2X UP broth, dispense in appropriate volumes (e.g., 100 mL). For 3X UP broth,
aseptically dispense 5 and 10 mL volumes into 25 x 150 mm tubes. Autoclave at 121 °C (15 psi) for 15 minutes.
7.7 Cystine (SCO)
Commercially prepared media is recommended. Dehydrated medium (Difco™ 268740 or equivalent) may be used. If
commercially prepared media are not available, prepare media using procedures in Sections 7.7.1 and 7.7.2.
7.7.1 Composition:
Pancreatic digest of casein 5.0 g
Lactose 4.0 g
Sodium phosphate 10.0 g
Sodium selenite 4.0 g
L-Cystine 0.01 g
Reagent-grade water l.OL
7,7.2
Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar. Adjust pH to 7.0 ± 0.2 with 1.0
N hydrochloric acid or 1.0 N sodium hydroxide and bring to 1 L. Boil for one minute with rapid stir bar agitation to
dissolve completely. DO NOT AUTOCLAVE. Aseptically dispense 10 mL-volumes into 16 x 150 mm tubes. Use
medium 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, the medium should be discarded and a new batch prepared.
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7.8
Commercially prepared media is recommended. Dehydrated medium (Difco™ 273300 or equivalent) may be used. If
commercially prepared media are not available, prepare medium using procedures in Sections 7.8.1 and 7.8.2.
7.8.1 Composition:
Beef extract 5.0 g
Peptone 10.0 g
Dextrose 5.0 g
Disodium phosphate 4.0 g
Ferrous sulfate 0.3 g
Bismuth sulfitc indicator 8.0 g
Brilliant green 0.025 g
Agar 20.0 g
Reagent-grade water 1.0 L
7.8.2
Add reagents to 1 L of reagent-grade water and mix thoroughly using a stir bar and hot plate. Boil for one minute with
rapid stir bar agitation to dissolve completely. DO NOT AUTOCLAVE. The pH should be 7.7 ± 0.2. Aseptically pour
12 - 15 mL into each 15 * 100 mm sterile Petri plate. Use plates within 48 hours of preparation.
7.9 Miller-Mallinson (MM) Agar
Commercially prepared plates (e.g., Northeast Laboratory Sendees or equivalent) should be used.
Note: Formulation is provided only to ensure that the appropriate medium is used for analyses; laboratories should
use commercially prepared plates.
7,9.1 Composition:
Sodium thiosulfate 6.8 g
a-Lactose 10.0 g
D(+) Cellobiose 5.0 g
Dipeptone 3.5 g
Yeast extract 3.0g
Sodium chloride 3.0 g
Trizma*HCl 2.3 g
Beef extract 2.0 g
D(+) Trehalose dehydrate 1.33 g
D-Mannitol 1.2 g
Ferric ammonium citrate 0.8 g
Trizma® base 0.7 g
X-Gal O.lg
Agar 15.0 g
Tergitol™ (Niaproof®) 4.5 mL
Reagent-grade water 1.0 L
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7.10 Tryptic Soy (ISA)
Commercially prepared media is recommended. Dehydrated medium (Difco™ 236950 or equivalent) may be used. If
commercially prepared media are not available, prepare media using procedures in Sections 7.10.1 and 7.10.2.
7.10.1 Composition:
Pancreatic digest of casein 15.0 g
Enzymatic digest of soybean meal 5.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Reagent-grade water 1.0 L
7.10.2
Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar and hot plate. Heat to dissolve
completely. Adjust pH to 7.3 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide and bring up to 1 L.
Autoclave at 121°C (15 psi) for 15 minutes. Aseptically pour 12 - 15 mL into each 15 x 100 mm sterile Petri plate.
7.11
Saline, physiological (0.85% w/v): Dissolve 0.85 g NaCl in 100 mL of reagent-grade water. Autoclave at 121 °C (15
psi) for 15 minutes. Cool to room temperature.
7.12 Vi (BD™ 228271 or equivalent)
7.13 (bioMerieux API 20E® or equivalent)
7.14 (BD™ DrySIide™ 231746 or
7.15
Positive and negative control cultures that are to be used with this protocol are listed in Table 2. Use of these controls
is discussed in Section 9.0.
Table 1. Positive and Negative Control Cultures for Described Tests
Media/Tests
SCB
BS
MM
Vi serum agglutination
Biochemical test strip
Oxidase
Positive Control Other strains of & Typhi may be used in place of CVD 909.
BS - Bismuth sulfite MM - Miller-Mallinson SCB - Selenite cvstine broth
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7.16
Storage temperatures and times for prepared media and reagents are provided in Table 2 Follow manufacturers'
guidelines for storage and expiration of all commercially prepared reagents.
Table 2. Storage Temperatures and Times for Prepared Media and Reagents(l)
Media/Reagents
PBS, saline
(in screw-cap containers)
Tubes: UP broth
Plates: ISA, MM
Plates: BS
Tubes: SCB
Storage Temperature
<10°C and above freezing
<10°C and above freezing
<10°C and above freezing
<1 0°C and above freezing
Storage Time
3 months
2 weeks in loose-cap tubes
3 weeks in screw-cap tubes
2 weeks
48 hours
"'> If media/reagent is refrigerated, remove from refrigerator 1 1.5 hours prior to inoculation to ensure that it reaches room temperature prior to use
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8.0
and
8.1
Check temperature in incubators twice daily, a minimum of four hours apart, to ensure operation is within stated limits
of the method. Record daily measurements in an incubator log book.
8.2
Check temperature in refrigerators/freezers at least once daily to ensure operation is within stated limits of the method.
Record daily measurements in a refrigerator/freezer log book.
8.3
Calibrate thermometers and incubators semi-annually against a NlST-certified thermometer or against a thermometer
that meets the requirements of NIST Monograph SP 250-23 (Reference 15.9). Check mercury columns for breaks.
8.4
Calibrate pH meter prior to each use with two of three standards (e.g., pH 4.0, 7.0, or 10.0) closest to the range being
tested.
8.5
Calibrate analytical and top-loading balances with ASTM International Class S reference weights once per month, at a
minimum. Check each day prior to use with Class S weights.
8.6
Re-certify biological safety cabinets once per year. Re-certification must be performed by a qualified technician.
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9.0
Quality Control
9.1
Each laboratory that uses this method is required to operate a formal quality assurance program that addresses and
documents instrument and equipment maintenance and performance, reagent quality and performance, analyst training
and certification, and records storage and retrieval. Specific quality control procedures for use with this method are
discussed below.
Following testing and validation, this method may be updated to include quality control criteria for initial and
ongoing demonstration of capability as well as matrix spike/matrix spike duplicates.
9.2 Controls
9.2.1
The laboratory should analyze negative controls to ensure that all media and reagents are performing properly.
Negative controls should be analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the
laboratory should analyze a negative control every day that samples are analyzed. Recommended negative control
organisms are provided in Table 1 (Section 7.16), and descriptions of negative results are provided in Table 3 (Section
10.8).
9.2.2
Analysis of negative controls is conducted by inoculating media and performing biochemical and serological analyses
with known negative control organisms as described in Section 10.0. The negative control is treated as a sample and
submitted to the same analytical procedures.
9.2.3
If a negative control fails to exhibit the appropriate response, check and/or replace the associated media, reagents, and/
or negative control organism, and re-analyze the appropriate negative control and corresponding sample(s).
9.2.4
Viability of the negative controls should be demonstrated on a monthly basis, at a minimum, using a non-selective
media (e.g., TSA).
9.3 Positive Controls
9.3.1
The laboratory should analyze positive controls to ensure that all media and reagents are performing properly. Positive
controls should be analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory
should analyze a positive control even- day that samples are analyzed. Recommended positive control organisms are
provided in (Section 7.16), and descriptions of positive results are provided in Table 3 (Section 10.8).
9.3.2
Analysis of positive controls is conducted by inoculating media and performing biochemical and serological analyses
with known positive organisms as described in Section 10.0. The positive control is treated as a sample and submitted
to the same analytical procedures.
9.3.3
If a positive control fails to exhibit the appropriate response, invalidate the sample results: check and/or replace the
associated media, reagents, and/or the positive control organism, and re-analyze the appropriate positive control and
corresponding sample(s).
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9.4
On an ongoing basis, the laboratory should perform a method blank every day that samples are analyzed using sterile
PBS (Section 7.5) to verify the sterility of equipment, materials, and supplies. The method blank is treated as a sample
and submitted to the same analytical procedures. Absence of growth indicates freedom from contamination by the
target organisms.
9.5 Sterility Check
Test sterility of PBS and media (UP broth, SCB, BS, MM, TSA) by incubating one unit (tube or plate) either from each
batch at 35.0°C ± 0.5°C for 24 ±2 hours or 48 ± 3 hours, as appropriate. Absence of growth indicates the media are
sterile. On an ongoing basis, a media sterility check should be done every day that samples are analyzed.
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10.0
Procedures
Process samples promptly upon receipt. S. Typfai is a pathogea and all samples should be handled with caution, using
appropriate BSL-2 procedures and PPE. A Class II biological safety cabinet is recommended for sample manipulations
where the risk of aerosol production is high.
10.1 Qualitative Sample Analysis
Add a sample volume (e.g., 100 niL) to an equal volume of double-strength UP broth (Section 7.6). Incubate at
35.0°C ± 0.5°C for 24 ±2 hours. After incubation, proceed to Section 10.3 for selective enrichment of & Typhi.
10.2
A multiple-tube assay incorporating differential sample volumes is used to estimate S. Typhi densities in undiluted or
diluted samples. If low levels of & Typhi are suspected, larger sample volumes (20.0 mL of original sample) should be
used to inoculate the first row of tubes in the series. If high levels of S. Typhi are suspected, additional serial dilutions
should be used. See Flowchart 16.1 for an overview of the sample dilution and inoculation scheme. A minimum
sample volume of 156 mL is required if 20 mL volumes are used to inoculate the first row of tubes.
10.2.1 Sample inoculation
Arrange UP broth tubes in three rows (10 mL of 3X, 5 mL of 3X, and 10 iriL of IX) of five tubes each. Inoculate
the first row of tubes (10 mL of 3X UP broth) with 20 mL of the undiluted sample. Inoculate 10 mL of the undiluted
sample into each of the tubes in the second row (5 mL of 3X UP broth). Inoculate 1 mL from the initial sample
into each of the tubes in the third row (10 mLof 1 UP broth). See Flowchart 16.1 for an overview of the sample
inoculation scheme.
10.2.2 Sample dilutions
Samples may require serial dilution prior to inoculation due to high levels of S. Typhi. If analyzing serially diluted
samples, 1.0 mL of each dilution will be used to inoculate each tube of IX UP broth, as appropriate.
10.2.3 Growth
Incubate tubes at 35.0°C ± 0.5°C for 24 ± 2 hours. Proceed to Section 10.3 for selective enrichment of S Typhi.
10.3 Cystine Culture
10.3.1
For each tube with growth, gently swirl the tube to mix and transfer 1.0 mL to a set of tubes with 10 mL of SCB.
Incubate at 35.0°C ± 0.5°C for 18± 2 hours.
10.3.2
Proceed to Section 10.4 for isolation on BS and MM agars.
10.4 on Miller-Mallinson Agar
See Flowchart 16.2 for an overview of the colony identification procedures.
10.4.1
Select all SCB tubes with growth (qualitative and quantitative) and streak for isolation onto BS and MM plates using a
sterile inoculation loop (20 uL).
10.4.2
Incubate plates for 24 - 48 hours at 35.0°C ± 0.5°C. Typical S. Typhi colonies are green-black with metallic sheen on
BS agar and black on MM agar.
Note: // is important to streak for isolation. In areas of heavy growth on BS agar, S. Typhi colonies appear light green
and may he misinterpreted as negative for S. Typhi.
-------
10.5 on Tryptic Agar
10.5.1
Examine plates at 24 ± 2 hours. For each BS and MM plate with typical colonies, streak a single typical colony onto
TS A and incubate at 35.0°C ± 0.5°C. If no typical colonies are observed, continue incubation at 35.0°C ± 0.5°C for a
total of 48 ± 3 hours. Re-examine plates, streak typical colonies onto TSA, and incubate at 35.0°C ± 0.5°C.
10.5.2
Seal the BS and MM plates with Parafilm® and store at <10°C and above freezing for use as backup plates. Use the
TSA plates for serological and biochemical analyses.
10.6
10.6.1 DrySlide™ 231746 or
Following manufacturer's instructions, transfer a small amount of growth from an isolated colony to the slide.
Oxidase-positive bacteria turn the reagent dark purple within 20 seconds. S. Typhi is oxidase-negative. Results should
be compared with those for positive and negative controls (Table 1) analyzed at the same lime.
10.6.2 Biochemical Test Strips (API 20£® or equiwalent)
Emulsify the remainder of the colony in sterile physiological saline (Section 7.11). Follow manufacturer's instructions
to inoculate wells and add appropriate reagents. Incubate test strip according to manufacturer's instructions. Add
additional reagents, read, and record results.
f 0.6.3 Alternative Biochemical Tests
The following individual biochemical tests may be used instead of biochemical test strips to identify S. Typhi:
• Citrate
« Glucose fermentation
• H,S
• Indolc
* Lysine
• Motility
* Ornithine
* Urease
* Voges-Proskauer
S. Typhi ferments glucose without the production of gas; produces trace amounts of H,S, is lysine decarboxylase
positive, motile and produces negative reactions for citrate, indole, ornithine, urease, and Voges-Prokauer.
10.7
Use a single, isolated, large colony (2-3 mm diameter) from each TSA plate for serological and biochemical test strip
and oxidase analyses.
10.7.1
Take a portion of growth from a typical colony from each of the TSA plates and emulsify growth using sterile
physiological saline (Section 7.11). Place two discrete drops of emulsified growth onto a slide. To the first drop of
emulsified growth, add one drop of Salmonella Vi antiserum (Section 7.12). To the second drop of emulsified growth.
add one drop of sterile saline (as a visual comparison).
10.7.2
Observe under magnification for an agglutination reaction, which indicates a positive result. S. Typhi is agglutination-
positive for Vi antiserum. Results should be compared with those for positive and negative controls (Table 1, Section
7.15 ) analyzed at the same time.
-------
10.8 of Control Typhi
Typical results are provided in Table 3.
Table 3. Positive and Negative Result Descriptions and Salmonella Typhi Results
Medium/Test
SCB
BS
MM
Oxidasc
Vi antiscrum '
Biochemical test strip
S. Typhi Results'1'
Positive
Positive
Positive
Negative
Positive
Positive Control Result and
Description
Growth
Green-black colonies with
metallic sheen
Black colonies
Purple to violet color change
within 20 seconds
Agglutination
Negative Control Result and
Description
No growth
No growth
No growth
Colorless or very light pink color
change over time
No agglutination
Consult manufacturer's instructions
('! Most wild type S. Typhi strains will be Vi antiserum positive.
BS - Bismuth sulfite SCB - Selenite cystine broth
MM - Miller-Mallinson
-------
-------
11.0
and
11.1 Number (MPN) Technique
Estimation of bacterial densities may be determined based on the number of tubes positive for S. Typhi either by
morphological, biochemical, and serological results or PCR.
11.2
If only three rows of tubes were analyzed, identify appropriate MPN value using either Table 5 or 6, depending on
volumes assayed. If more than three rows of tubes were analyzed, the appropriate rows must be selected and MPN
value calculated as described in Sections 11.2.1 and 11.2.2. Table 5 should only be used for volumes of 20.0 mL, 10.0
mL, and 1.0 mL. To select MPN values for volumes of 10.0 mL or less, use Table 6.
11.2.1 Selection of Tubes
If more than three rows of tubes are inoculated with sample (e.g., volumes/dilutions), select the most appropriate rows
of tubes according to the criteria provided below. Examples of row selections and MPN/100 mL values are provided
in Table 4.
11.2.1.1
Choose the smallest volume or the highest dilution giving positive results in all five tubes inoculated plus the two
succeeding lower concentrations. In Example A from Table 4, 10 mL is a smaller volume than 20 mL and is the lowest
volume giving positive results in all five tubes.
11.2.1.2
If the largest volume tested has less than five tubes with positive results, select it and the next two volumes (Table 4,
Examples B and C).
11.2.1.3
When a positive result occurs in a smaller volume than the three rows selected according to the rules above, change
the selection to the largest volume that has less than five positive results and the next two smaller volumes (Table 4,
Example D).
11.2.1.4
When the selection rules above have left unselected any smaller volumes with positive results, add those positive tubes
to the row of tubes for the smallest volume selected (Table 4, Example E).
11.2.1.5
If there were not enough lower volumes analyzed to select three dilutions using the rales above, then select the three
smallest volumes (Table 4. Example F).
-------
Table 4. Examples of Appropriate Tube Selection and MPN/100 mL(1)
Example
A
B
C
D
E
F
20mL
5/5
4/5
Q/5
5/5
4/5
5/5
10 mL
5/5_
5/5_
1/5
3/5
4/5
5J5
1.0 mL
3/5
1/5
Q/5
1/5
Q/5
5/5
0.1 mL
Q/5_
0/5
0/5
1/5
1/5
2/5
Significant
Dilutions
5-3-0
4-5-1
0-1-0
3-1-1
4-4-1
5-5-2
Table
6
5
5
6
5
6
MPN Index
0.792
0.1524
0.0067
0.137
0.1181
5.422
MPN/100 mL
79.2
15.24
0.67
13.7
11.81
542.2
(1) Appropriate volumes are underlined and the largest sample volumes analyzed are highlighted.
77.2.2
For calculation of MPN/100 mL when additional dilutions are analyzed (e.g., 10~2, 10~3), obtain the MPN index value
from Table 6 using the number of positive tubes in the three selected dilutions. Calculate MPN/100 mL using the
equation below.
MPN/100mL
MPN Index from Table 6
100
Middle volume analyzed in the series used for MPN determination
For example, a dilution series of 10~3, 10~4, 10~5, with the following positive tubes 5, 1,0, respectively, would be:
0.329
MPN/100 mL
10-4
*100 = 3.29x10=
-------
Table 5. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five Tubes are
Used and Sample Inoculation Volumes are 20.0, 10.0, and 1.0 mL(1)
Combination of
Positives
0-0-0
0-0-1
0-0-2
0-0-3
0-0-4
0-0-5
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
0-2-0
0-2-1
0-2-2
0-2-3
0-2-4
0-2-5
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
0-4-0
0-4-1
0-4-2
0-4-3
0-4-4
0-4-5
0-5-0
0-5-1
0-5-2
0-5-3
0-5-4
0-5-5
1-0-0
1-0-1
1-0-2
1-0-3
1-0-4
1-0-5
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
1-2-0
1-2-1
1-2-2
1-2-3
1-2-4
1-2-5
MPN Index
<0.006473
0.0065
0.0130
0.0195
0.0262
0.0328
0.0067
0.0134
0.0202
0.0270
0.0339
0.0408
0.0138
0.0208
0.0279
0.0350
0.0422
0.0494
0.0215
0.0288
0.0362
0.0437
0.0512
0.0588
0.0299
0.0375
0.0453
0.0531
0.0611
0.0691
0.0390
0.0470
0.0553
0.0636
0.0720
0.0806
0.0072
0.0139
0.0209
0.0281
0.0353
0.0425
0.0144
0.0217
0.0290
0.0365
0.0441
0.0517
0.0224
0.0301
0.0379
0.0457
0.0537
0.0618
95% Confidence Limits
Lower
....
0.0012
0.0012
0.0012
0.0033
0.0062
0.0012
0.0012
0.0012
0.0037
0.0067
0.0099
0.0012
0.0012
0.0040
0.0072
0.0106
0.0141
0.0012
0.0044
0.0077
0.0113
0.0051
0.0095
0.0049
0.0084
0.0121
0.0160
0.0200
0.0241
0.0090
0.0129
0.0170
0.0212
0.0255
0.0299
0.0012
0.0012
0.0012
0.0041
0.0073
0.0107
0.0012
0.0013
0.0045
0.0079
0.0115
0.0153
0.0017
0.0050
0.0085
0.0123
0.0162
0.0203
Upper
0.0223
0.0223
0.0352
0.0472
0.0589
0.0706
0.0228
0.0360
0.0483
0.0604
0.0725
0.0847
0.0367
0.0495
0.0619
0.0745
0.0871
0.1001
0.0507
0.0636
0.0766
0.0898
0.1243
0.1428
0.0654
0.0789
0.0927
0.1069
0.1216
0.1369
0.0814
0.0958
0.1107
0.1262
0.1425
0.1596
0.0241
0.0369
0.0497
0.0623
0.0749
0.0878
0.0377
0.0509
0.0640
0.0771
0.0905
0.1043
0.0523
0.0658
0.0795
0.0935
0.1079
0.1229
Combination of
Positives
1-3-0
1-3-1
1-3-2
1-3-3
1-3-4
1-3-5
1-4-0
1-4-1
1-4-2
1-4-3
1-4-4
1-4-5
1-5-0
1-5-1
1-5-2
1-5-3
1-5-4
1-5-5
2-0-0
2-0-1
2-0-2
2-0-3
2-0-4
2-0-5
2-1-0
2-1-1
2-1-2
2-1-3
2-1-4
2-1-5
2-2-0
2-2-1
2-2-2
2-2-3
2-2-4
2-2-5
2-3-0
2-3-1
2-3-2
2-3-3
2-3-4
2-3-5
2-4-0
2-4-1
2-4-2
2-4-3
2-4-4
2-4-5
2-5-0
2-5-1
2-5-2
2-5-3
2-5-4
2-5-5
MPN Index
0.0312
0.0393
0.0475
0.0559
0.0644
0.0730
0.0409
0.0495
0.0583
0.0672
0.0763
0.0855
0.0517
0.0609
0.0703
0.0799
0.0897
0.0998
0.0155
0.0226
0.0303
0.0382
0.0462
0.0543
0.0234
0.0315
0.0397
0.0480
0.0565
0.0652
0.0327
0.0413
0.0501
0.0590
0.0681
0.0774
0.0431
0.0523
0.0617
0.0714
0.0813
0.0914
0.0547
0.0647
0.0750
0.0855
0.0964
0.1076
0.0681
0.0791
0.0904
0.1021
0.1143
0.1268
95% Confidence Limits
Lower
0.0055
0.0092
0.0132
0.0173
0.0216
0.0260
0.0099
0.0141
0.0185
0.0231
0.0277
0.0324
0.0152
0.0199
0.0247
0.0296
0.0346
0.0397
0.0012
0.0018
0.0051
0.0087
0.0125
0.0165
0.0022
0.0056
0.0094
0.0134
0.0177
0.0221
0.0062
0.0101
0.0144
0.0189
0.0236
0.0283
0.0110
0.0155
0.0203
0.0252
0.0303
0.0354
0.0168
0.0218
0.0271
0.0325
0.0380
0.0436
0.0235
0.0292
0.0349
0.0409
0.0469
0.0531
Upper
0.0678
0.0821
0.0967
0.1119
0.1277
0.1444
0.0849
0.1002
0.1163
0.1331
0.1509
0.1700
0.1042
0.1212
0.1391
0.1583
0.1790
0.2015
0.0404
0.0526
0.0662
0.0801
0.0943
0.1090
0.0540
0.0683
0.0827
0.0976
0.1131
0.1293
0.0705
0.0856
0.1013
0.1176
0.1349
0.1533
0.0887
0.1053
0.1227
0.1412
0.1611
0.1826
0.1098
0.1284
0.1484
0.1700
0.1937
0.2201
0.1349
0.1566
0.1805
0.2070
0.2372
0.2725
-------
Combination of
Positives
3-0-0
3-0-1
3-0-2
3-0-3
3-0-4
3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
3-1-5
3-2-0
3-2-1
3-2-2
3-2-3
3-2-4
3-2-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-0
3-4-1
3-4-2
3-4-3
3-4-4
3-4-5
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
4-0-1
4-0-2
4-0-3
4-0-4
4-0-5
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0
4-2-1
4-2-2
4-2-3
4-2-4
4-2-5
MPN Index
0.0255
0.0330
0.0417
0.0506
0.0598
0.0691
0.0344
0.0435
0.0529
0.0626
0.0725
0.0827
0.0456
0.0555
0.0657
0.0763
0.0872
0.0984
0.0583
0.0693
0.0806
0.0924
0.1046
0.1173
0.0733
0.0856
0.0984
0.1118
0.1258
0.1405
0.0913
0.1055
0.1204
0.1362
0.1529
0.1707
0.0381
0.0461
0.0563
0.0668
0.0777
0.0890
0.0484
0.0592
0.0705
0.0822
0.0945
0.1072
0.0626
0.0748
0.0875
0.1009
0.1150
0.1299
95% Confidence Limits
Lower
0.0028
0.0063
0.0103
0.0147
0.0193
0.0241
0.0069
0.0112
0.0159
0.0207
0.0258
0.0310
0.0122
0.0171
0.0223
0.0277
0.0333
0.0390
0.0186
0.0241
0.0299
0.0359
0.0421
0.0484
0.0262
0.0325
0.0390
0.0457
0.0526
0.0597
0.0354
0.0426
0.0500
0.0577
0.0656
0.0738
0.0082
0.0125
0.0175
0.0229
0.0284
0.0342
0.0136
0.0190
0.0248
0.0308
0.0370
0.0434
0.0207
0.0269
0.0335
0.0403
0.0473
0.0546
Upper
0.0585
0.0710
0.0863
0.1023
0.1191
0.1368
0.0734
0.0896
0.1065
0.1244
0.1434
0.1640
0.0932
0.1112
0.1303
0.1510
0.1735
0.1984
0.1164
0.1371
0.1597
0.1847
0.2128
0.2452
0.1450
0.1700
0.1982
0.2307
0.2695
0.3184
0.1825
0.2150
0.2538
0.3029
0.3715
0.4795
0.0809
0.0942
0.1126
0.1323
0.1537
0.1773
0.0983
0.1181
0.1395
0.1631
0.1894
0.2193
0.1244
0.1479
0.1742
0.2041
0.2392
0.2820
Combination of
Positives
4-3-0
4-3-1
4-3-2
4-3-3
4-3-4
4-3-5
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-0
4-5-1
4-5-2
4-5-3
4-5-4
4-5-5
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-1-0
5-1-1
5-1-2
5-1-3
5-1-4
5-1-5
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0
5-3-1
5-3-2
5-3-3
5-3-4
5-3-5
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
0.0797
0.0937
0.1086
0.1245
0.1414
0.1595
0.1012
0.1181
0.1364
0.1563
0.1780
0.2015
0.1304
0.1524
0.1769
0.2046
0.2357
0.2708
0.0549
0.0637
0.0763
0.0896
0.1037
0.0953
0.0678
0.0816
0.0963
0.1121
0.1291
0.1293
0.0879
0.1046
0.1227
0.1427
0.1646
0.1767
0.1151
0.1368
0.1614
0.1895
0.2216
0.2527
0.1571
0.1907
0.2319
0.2834
0.3475
0.4256
0.2398
0.3477
0.5422
0.9178
1.6090
>1.6090
95% Confidence Limits
Lower
0.0295
0.0366
0.0441
0.0520
0.0602
0.0686
0.0404
0.0489
0.0578
0.0672
0.0770
0.0873
0.0549
0.0653
0.0766
0.0886
0.1015
0.1150
0.0162
0.0213
0.0277
0.0345
0.0417
0.0165
0.0234
0.0304
0.0379
0.0459
0.0542
0.0304
0.0337
0.0421
0.0511
0.0608
0.0710
0.0503
0.0474
0.0580
0.0695
0.0821
0.0957
0.0814
0.0676
0.0826
0.0999
0.1196
0.1417
0.1437
0.0762
0.1172
0.1791
0.2672
0.3837
0.3837
Upper
0.1579
0.1877
0.2228
0.2656
0.3218
0.4067
0.2049
0.2476
0.3038
0.3890
0.5273
0.6411
0.2836
0.3687
0.5210
0.6528
0.7516
0.8426
0.1116
0.1265
0.1510
0.1787
0.2107
0.2234
0.1344
0.1618
0.1936
0.2316
0.2796
0.3090
0.1751
0.2128
0.2605
0.3267
0.4385
0.5230
0.2394
0.3050
0.4183
0.5899
0.7101
0.7971
0.3935
0.5954
0.7409
0.8726
1.0160
1.1800
0.7629
1.0160
1.4190
2.2010
4.1030
— -
(1) Table was developed using the MPN calculator developed by Albert Klee (Reference 15.10).
-------
Table 6. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five Tubes are
Used and Sample Inoculation Volumes are 10.0, 1.0, and 0.1 mL(1)
Combination of
Positives
0-0-0
0-0-1
0-0-2
0-0-3
0-0-4
0-0-5
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
0-2-0
0-2-1
0-2-2
0-2-3
0-2-4
0-2-5
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
0-4-0
0-4-1
0-4-2
0-4-3
h 0-4-4
0-4-5
0-5-0
0-5-1
0-5-2
0-5-3
0-5-4
0-5-5
1-0-0
1-0-1
1-0-2
1-0-3
1-0-4
1-0-5
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
1-2-0
1-2-1
1-2-2
1-2-3
1-2-4
1-2-5
MPN Index
<0.018
0.018
0.036
0.054
0.072
0.091
0.018
0.036
0.055
0.073
0.091
(1110
0.037
0.055
0.074
0.092
0.111
0.129
0.056
0.074
0.093
0.112
0.130
0.149
0.075
0.094
0.112
0.131
0.150
0.169
0.094
0.113
0.133
0.152
0.171
(1190
0.020
0.040
0.060
0.081
0.101
0.122
0.040
0.061
0.081
0.102
0.123
0.144
0.061
0.082
0.103
0.124
0.146
0.167
95% Confidence Limits
Lower
—
0.003
0.003
0.003
0.008
0.015
0.003
0.003
0.003
0.008
0.015
0.023
0.003
0.003
0.008
0.015
0.023
0.031
0.003
0.009
0.016
0.023
0.031
0.039
0.009
0.016
0.024
0.032
0.040
0.048
0.016
0.024
0.032
0.040
0.048
0.056
0.003
0.003
0.003
0.011
0.019
0.028
0.003
0.003
0.011
0.019
0.028
0.037
0.003
0.012
0.020
0.029
0.038
0.047
Upper
0.063
0.063
0.101
0.137
0.174
0.212
0.063
0.101
0.138
0.175
0.214
^(X256
0.102
0.139
0.176
0.215
0.258
0.307
0.140
0.177
0.217
0.260
0.310
0.372
0.179
0.219
0.263
0.313
0.377
0.462
0.221
0.265
0.317
0.382
0.470
0.563
0.068
0.108
0.149
0.191
0.236
0.287
0.109
0.150
0.192
0.238
0.290
0.354
0.151
0.194
0.240
0.293
0.359
0.451
Combination of
Positives
1-3-0
1-3-1
1-3-2
1-3-3
1-3-4
1-3-5
1-4-0
1-4-1
1-4-2
1-4-3
1-4-4
^^1-4-5
1-5-0
1-5-1
1-5-2
1-5-3
1-5-4
1-5-5
2-0-0
2-0-1
2-0-2
2-0-3
2-0-4
2-0-5
2-1-0
2-1-1
2-1-2
2-1-3
2-1-4
2-1-5
2-2-0
2-2-1
2-2-2
2-2-3
2-2-4
2-2-5
2-3-0
2-3-1
2-3-2
2-3-3
2-3-4
2-3-5
2-4-0
2-4-1
2-4-2
2-4-3
2-4-4
^^2-4-5
2-5-0
2-5-1
2-5-2
2-5-3
2-5-4
2-5-5
MPN Index
0.083
0.104
0.125
0.147
0.169
0.191
0.105
0.127
0.148
0.170
0.193
0215
0.128
0.150
0.172
0.195
0.217
0.240
0.045
0.068
0.091
0.115
0.139
0.164
0.068
0.092
0.116
0.141
0.166
0.192
0.093
0.118
0.143
0.168
0.194
0.221
0.119
0.144
0.170
0.197
0.223
0.251
0.146
0.172
0.199
0.226
0.254
0.282
0.174
0.201
0.229
0.257
0.286
0.315
95% Confidence Limits
Lower
0.012
0.020
0.029
0.038
0.048
0.057
0.021
0.030
0.039
0.048
0.058
^(1067
0.030
0.040
0.049
0.058
0.068
0.077
0.003
0.006
0.015
0.025
0.035
0.046
0.006
0.015
0.025
0.036
0.046
0.057
0.016
0.026
0.036
0.047
0.058
^(1069
0.026
0.037
0.048
0.059
0.070
0.082
0.038
0.049
0.060
0.072
0.083
^(1094
0.050
0.061
0.073
0.084
0.095
0.107
Upper
0.196
0.243
0.296
0.364
0.460
0.566
0.245
0.300
0.370
0.468
0.575
^^0657
0.303
0.375
0.477
0.583
0.664
0.731
0.119
0.164
0.213
0.269
0.338
0.437
0.166
0.216
0.272
0.343
0.447
0.571
0.218
0.276
0.349
0.456
0.581
^^0675
0.279
0.355
0.467
0.591
0.683
0.759
0.361
0.477
0.600
0.692
0.768
^^0836
0.488
0.610
0.700
0.776
0.845
0.910
-------
Combination of
Positives
3-0-0
3-0-1
3-0-2
3-0-3
3-0-4
3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
3-1-5
3-2-0
3-2-1
3-2-2
3-2-3
3-2-4
3-2-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-0
3-4-1
3-4-2
3-4-3
3-4-4
3-4-5
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
4-0-1
4-0-2
4-0-3
4-0-4
4-0-5
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0
4-2-1
4-2-2
4-2-3
4-2-4
4-2-5
MPN Index
0.079
0.106
0.135
0.165
0.196
0229
0.107
0.137
0.167
0.199
0.232
0.267
0.138
0.170
0.202
0.236
0.271
0.308
0.172
0.205
0.240
0.276
0.313
0.352
0.209
0.244
0.281
0.319
0.358
0.399
0.248
0.286
0.325
0.365
0.407
0.450
0.130
0.166
0.207
0.253
0.302
0.355
0.169
0.212
0.258
0.310
0.365
0.425
0.216
0.264
0.317
0.375
0.438
0.504
95% Confidence Limits
Lower
0.010
0.021
0.033
0.046
0.059
0.073
0.022
0.034
0.047
0.060
0.074
0.088
0.035
0.048
0.062
0.076
0.090
0.104
0.049
0.063
0.077
0.092
0.106
0.120
0.064
0.079
0.093
0.108
0.123
0.137
0.080
0.095
0.110
0.125
0.140
0.154
0.031
0.046
0.064
0.082
0.102
0.121
0.048
0.066
0.085
0.105
0.125
0.145
0.067
0.087
0.108
0.129
0.150
0.171
Upper
0.188
0.246
0.323
0.440
0.589
0.699
0.250
0.329
0.452
0.601
0.710
0.800
0.335
0.464
0.613
0.720
0.810
0.894
0.477
0.624
0.731
0.821
0.906
0.989
0.636
0.742
0.833
0.918
1.002
1.086
0.753
0.844
0.931
1.017
1.103
1.189
0.311
0.445
0.631
0.764
0.881
0.996
0.460
0.646
0.779
0.898
1.016
1.138
0.661
0.794
0.915
1.037
1.164
1.297
Combination of
Positives
4-3-0
4-3-1
4-3-2
4-3-3
4-3-4
4-3-5
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-0
4-5-1
4-5-2
4-5-3
4-5-4
4-5-5
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-1-0
5-1-1
5-1-2
5-1-3
5-1-4
5-1-5
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0
5-3-1
5-3-2
5-3-3
5-3-4
5-3-5
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
0.271
0.326
0.386
0.451
0.521
0.593
0.335
0.398
0.466
0.539
0.615
0.693
0.411
0.483
0.559
0.639
0.722
0.806
0.240
0.314
0.427
0.578
0.759
0.953
0.329
0.456
0.631
0.839
1.062
1.293
0.493
0.700
0.944
1.205
1.479
1.767
0.792
1.086
1.406
1.750
2.122
2.527
1.299
1.724
2.212
2.781
3.454
4.256
2.398
3.477
5.422
9.178
16.090
>1 6.090
95% Confidence Limits
Lower
0.090
0.111
0.132
0.154
0.176
0.196
0.114
0.137
0.159
0.181
0.202
0.223
0.141
0.164
0.187
0.209
0.230
0.250
0.076
0.106
0.146
0.192
0.239
0.165
0.112
0.156
0.207
0.257
0.304
0.304
0.167
0.224
0.280
0.331
0.381
0.503
0.247
0.308
0.368
0.434
0.529
0.814
0.348
0.429
0.563
0.882
1.159
1.437
0.762
1.172
1.791
2.672
3.837
3.837
Upper
0.809
0.934
1.060
1.192
1.331
1.477
0.953
1.084
1.223
1.368
1.521
1.681
1.111
1.256
1.409
1.570
1.739
1.916
0.763
0.908
1.142
1.446
1.816
2.234
0.940
1.202
1.553
1.985
2.485
3.090
1.276
1.694
2.213
2.843
3.714
5.230
1.886
2.544
3.445
5.131
6.798
7.971
3.108
4.975
7.087
8.600
10.110
11.800
7.629
10.160
14.190
22.010
41.030
(1) Table was developed using the MPN calculator developed by Albert Klee (Reference 15.10).
-------
12.0
Protocol Performance
Culture-based procedures were evaluated for S. Typhi in a reference matrix (PBS) and two matrices of interest
(drinking water and surface water) during a single-laboratory verification study. Results for surface water analyses
were unacceptable; therefore, surface water procedures were not included in this SAP. Details regarding procedure
performance are provided in the study report (Reference 15.11). Verification results are provided in Table 7.
-------
Table 7. Salmonella Typhi Results for PBS, Drinking Water, and Surface Water Verification Analyses (1)
Date
Sample
Spike Level
(CFU/100 mL)
Medium
MPN
Combo
S. Typhi
(MPN/100
mL)
Recovery
Mean
Recovery
SD (%)
RSD
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
BV>
-
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
MMD<2>
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 (3)
307.24
150.99
150.99
49.14
49.14
-------
Date
Sample
Spike Level
(CFU/100 mL)
Medium
MPN
Combo
S. Typhi
(MPN/100
mL)
Recovery
(%)
Mean
Recovery
(%)
SD (%)
RSD
(%)
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(2)
MM^
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) These values are based on a 9-tube, as opposed to a 15-tube, MPN
(2) Supplemented with 2,3-dihydroxybenzoate
(3) It should be noted that while 307% recovery may seem high, the variability in the 109.9 MPN value ranges from 22.5 - 307.5 for the 95%
confidence internal.
BS - Bismuth sulfite
CFU/lOOmL - Colony forming unit per 100 milliliter
MM - Miller-Mallinson
NA - Not applicable
RSD - Relative standard deviation
SCB - Selenite cystine broth
SD - Standard deviation
-------
-------
13.0
13.1
The solutions and reagents used in this method pose little threat to the environment when recycled and managed
properly.
13.2
Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the volume of
expired materials to be disposed.
-------
-------
14.0
14.1
It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste
management, particularly the biohazard and hazardous waste identification rules and land disposal restrictions, and
to protect the air. water, and land by minimizing and controlling all releases from fume hoods and bench operations.
Compliance with all sewage discharge permits and regulations is also required.
14.2
Samples, reference materials, and equipment known or suspected to be contaminated with viable S. Typhi must be
sterilized prior to disposal.
14.3
For further information on waste management, consult The Waste Management Manual for Laboratory Personnel
(Reference 15.12) and Less Is Better: Laboratory Chemical Management for Waste Reduction (Reference 15.13), both
available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th
Street NW, Washington, DC 20036.
-------
-------
15.0
References
15.1
Bopp, C.A., Brenner, F.W., Fields, P.I., Wells, J.G., and Strockbine, N.A. 2003. Escherichia, Shigella, and Salmonella.
Chapter 28 in Manual of Clinical Microbiology, 8th Edition. PR. Murray, EJ. Baron, J.H. Jorgensen, M.A. Pfaller, and
R.H. Yolken (eds.). Washington, D.C.: American Society for Microbiology.
15.2
Andrews, W H. and Hammack, T. 2007. Salmonella. Bacteriological Analytical Manual [Online], Chapter 5. Silver
Spring, Maryland: U.S. Food and Drug Administration. http://www.cfsan.fda.gov/~ebanVbam-5.html
15.3
U.S. Department of Health and Human Services: Centers for Disease Control and Prevention, and National Institutes
of Health. 2009. Biosafety in Microbiological and Biomedical Laboratories (BMBL). 5th Edition. Washington, D.C.:
U.S. Government Printing Office, http://www.cdc.gov/biosafety/pubrications/bmbl5/index.htm
15.4
Cho, J.C., and Kim, S.J. 1999. "Viable, But Non-culturable, State of a Green Fluorescence Protein-tagged
Environmental Isolate of Salmonella typhi in Groundwater and Pond Water." FEMSMicrobiology Letters. 170(1): 257
-264.
15.5
American Chemical Society. 2000. Reagent Chemicals, American Chemical Society Specifications. 9th Edition. New
York: Oxford University Press, Inc.
15.6
British Drug Houses, Ltd., and Hopkin and Williams, Ltd. 1957. AnalaR Standards for Laboratory Chemicals. 5th
Edition. Poole, Dorset, U.K.: BDH, Ltd.
15.7
United States Pharmacopeia. 2005. United States Pharmacopeia and National Formulary 24. Rockville, Maryland:
United States Pharmacopeial Convention.
15.8
Bordner, R.H. 2005. Section 9020 - Quality Assurance/Quality Control. In Standard Methods for the Examination of
Water and Wastewater, 21st Edition. A.D. Eaton, L.S. Clesceri, E.W. Rice, A.E. Greenberg, and M.A.H. Franson (eds.).
Washington, D.C.: American Public Health Association, American Water Works Association, and Water Environment
Federation.
15.9
Wise, J. 1988. NISTMeasurement Services: Liquid-In-Glass Thermometer Calibration Service, SP 250-23. SP
250, Series on NIST Measurement Services. Washington, D.C.: U.S. Department of Commerce, National Institute of
Standards and Technology. http://ts.nist.gov/MeasurementServices/Calibrations/upload/SP250-23.pdf
15.10
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.
-------
15.11
U.S. Environmental Protection Agency. Single-Laboratory Verification of Culture-Based Procedure for the Detection
of Salmonella Typhi in Drinking Water and Surface Water. Publication forthcoming; date and number to be
determined.
15.12
American Chemical Society (ACS). 1990. The Waste Management Manual for Laboratory Personnel. Washington,
D.C.: American Chemical Society, Department of Government Relations and Science Policy.
15.13
American Chemical Society (ACS). 1985. Less Is Better: Laboratory Chemical Management for Waste Reduction.
Washington, D.C.: American Chemical Society, Department of Government Relations and Science Policy.
-------
16.0
Flowcharts and Diagrams
16.1 Quantitative Analysis Dilution Scheme
Distribution of Sample to UP Broth
Sections 10.2.1 and 10.2.2
Serial dilutions as
necessary
1.0 ml to each
Tl
IT
m
m
nn
Tube of 1X UP broth
m
m
Incubation at 35.0° ± 0.5°C for 24 ± 2 hours
Section 10.2.3
^ Incubate at 35°C ± 0.5°C Analysis of
for 24 ±2 hours *" positive tubes
-------
16.2 Identification Flowchart
Enrichment in UP Broth and
SCD Enrichment
Sections 10.2 and 10.3
Isolation on
BSand
MM Plates
Section
10.4
UP broth after
35°C ± 0.5°C for
24 ± 2 hours
Incubate at
35°C ± 0.5°C for
18 ±2 hours
BS agar
Incubate at
35°C ± 0.5°C for
24 - 48 hours
Streak onto
plates BS
and MM
agars
Incubate at
35°C ± 0.5°C for
24 - 48 hours
Subculture on TSA and
Serological and Biochemical Testing
Sections 10.5, 10.6, and 10.7
Incubate at
35°C ± 0.5°C for
24 ± 2 hours
Incubate test strip
according to
manufacturer's instructions
Citrate
Glucose
fermentation
H2S
Indole
Lysine
Motility
Ornithine
Urease
Voges-
Proskauer
-------
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
------- |