EPA 600/R-10/139 | October 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Standard Analytical Protocol
for Vibrio cholerae O1 and
O139 in Drinking Water and
Surface Water
Office of Research and Development
National Homeland Security Research Center
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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 Vibrio cholerae Ol and O139 in water samples. Technical support and data evaluation were
provided by Computer Sciences Corporation (CSC) under EPA Contract No. EP-C-05-045.
The contributions of the following persons and organizations arc gratefully acknowledged:
Study Workgroup Participants
* Cheryl Bopp, Michele Parsons (Centers for Disease Control and Prevention)
• Michele Burgess, Marissa Mullins (EPA, Office of Emergency Management)
• Rita Colwell, Anwar Huq (Maryland Pathogen Research Institute, University of Maryland)
* Stephanie Harris (EPA, Region 10)
* Malik Raynor (EPA, Office of Ground Water and Drinking Water)
* Gene Rice (EPA, National Homeland Security Research Center)
Subject Matter Experts
• Cheryl Bopp (Centers for Disease Control and Prevention)
« Nancy Hall (University of Iowa Hygienic Laboratory)
* Steve Weagant (U.S. Food and Drag 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 but
does not necessarily reflect the Agency's views. 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 part)" 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 event. The culture-based
procedures provide viability determination, identification, and either qualitative or quantitative
results. The sample preparation procedures are deemed the 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 analysis.
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.sanjiv@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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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 Vibrio cholerae (V. cholerae) Ol and O139 in 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 V. cholerae contamination. This work specifically represents an important step in EPA's
support for the ERLN and moves the agency closer to achieving its mission to support homeland
security and its overall mission to protect human health and the environment.
Gregory D. Sayles, Ph.D., Acting Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
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Acknowledgments iii
Disclaimer iv
Foreword v
1.0 Scope and Application 1
2.0 Summary of Protocol 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 17
9.0 Quality Control 19
9.1 General 19
9.2 Negative Controls 19
9.3 Positive Controls 19
9.4 Method Blank 20
9.5 Media Sterility Check 20
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10.0 Procedures 21
10.1 Qualitative Sample Analyses 21
10.2 Quantitative Sample Analyses 21
10.3 Isolation on TCBS Plates 21
10.4 Isolation on TSA Plates 22
10.5 Serological Analyses 22
10.6 Biochemical Analyses 22
10.7 Description of Quality Control and V. cholerae Ol andO139 Results 23
11.0 Data Analysis and Bacterial Density Calculation 25
11.1 Most Probable Number (MPN) Technique 25
11.2 Calculation of MPN 25
12.0 Protocol Performance 33
13.0 Pollution Prevention 35
14.0 Waste Management 37
15.0 References 39
16.0 Flowcharts 41
16.1 Quantitative Analysis Dilution Scheme 41
16.2 Identification Flowchart 42
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Table 1. Positive and Negative Control Cultures 15
Table 2. Storage Temperatures and Times for Prepared Media and Reagents 15
Table 3. Positive and Negative Result Descriptions and Vibrio cholerae Ol and O139 Results 23
Table 4. Examples of Appropriate Tube Selection and MPN/100 mL 25
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 26
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 29
Table 7. Summary of Results for V. cholerae Ol and O139 in Spiked PBS, Drinking Water, and
Surface Water Samples 33
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1.0
and
1.1
This Standard Analytical Protocol (SAP) is for the identification, confirmation, and quantitation of Vibrio cholerae
(V. cholerae) Ol and O139 in water samples, using selective and non-selective media followed by biochemical
characterization and serological confirmation.
1.2
This protocol has been adapted from the Centers for Disease Control and Prevention's Laboratory Methods for
the Diagnosis of Epidemic Dysentery and Cholera (Reference 15.1) and the U.S. Food and Drug Administration's
Bacteriological Analytical Manual Online (Reference 15.2), and is for use by laboratories when analyzing samples in
support of U.S. Environmental Protection Agency (EPA) homeland security efforts.
1.3
V. cholerae Ol and O139 are the causative agents of cholera. 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 practices. 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 established guidelines.
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 V. cholerae Ol and O139 and the matrices
described.
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2.0
of
2.1
V. cholerae Ol and Ol 39 can be identified in water samples using selective media and biochemical and serological
analyses. Bacterial densities can be estimated using the most probable number (MPN) technique.
2.2
For qualitative results, samples are diluted 1:1 in double-strength alkaline peptone water (APW) and incubated at
36.0°C ± 1.0°C for either 6 - 8 or 24 ± 2 hours.
2.3
For quantitative results, samples are analyzed as received. Samples arc analyzed using a 15-tubc MPN. Inoculated
APW tubes are incubated at 36.0°C ± 1,0°C for 6 - 8 or 24 ± 2 hours.
2: It may be necessary to incubate APW for 24 -r 2 hours for some sample types and target organisms (e.g., V.
cholerae Ol).
2.4
APW tubes (MPN and qualitative analyses tubes) are streaked from the top of the tube (pellicle) onto thiosulfate citrate
bile salts sucrose (TCBS) agar and incubated at 36.0°C ± 1.0°C for 24 ± 2 hours.
2.5
TCBS plates are examined for large yellow colonies typical of V. cholerae Ol and O139. Isolated, presumptive
colonies are sub-cultured onto tryptic soy agar (TSA) and submitted to biochemical and serological confirmation.
Serological confirmation is by agglutination using either V. cholerae Ol or O139 antiserum (as appropriate), followed
by biochemical characterization using commercially available test strips (e.g., API 20E® [bioMerieux or equivalent) or
with selected individual biochemical tests.
2.6
APW tubes (MPN and qualitative analysis) may be subjected to real-time polymcrase chain reaction (PCR)
confirmation in place of biochemical and serological confirmation.
2.7
Quantitalion of V. cholerae Ol and O139 is determined using the MPN technique (Flowchart 16.1). Tubes that are
confirmed positive for V. cholerae Ol or 0139 are used to determine the MPN.
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3.0
Acronyms and Abbreviations
APW Alkaline peptone water
ATCC* American Type Culture Collection
BSL Biological safety level
°C Degrees Celsius
CVD Center for Vaccine Development
EPA U.S. Environmental Protection Agency
g Gram(s)
L Liter(s)
uL Microliter(s)
mL Milliliter(s)
mm Millimeters)
MPN Most probable number
N Normal - one equivalent weight per liter
NIST National Institute of Standards and Technology
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PPE Personal protective equipment
psi Pounds per square inch
QA Quality assurance
QC Quality control
SAP Standard Analytical Protocol
TCBS Thiosulfate citrate bile salts sucrose (agar)
TSA Tryptic soy agar
VBNC Viable but non-culturable
w/v Weight to volume ratio
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4.0
and
4.1
Low recoveries of V. cholerae Ol and O139 may be caused by the presence of either high numbers of competing or
inhibitory organisms, or toxic substances (e.g., metals, organic compounds).
4.2
A viable but non-culturable (VBNC) state of V. cholerae Ol or O139 may also account for lower recoveries (Reference
15.4).
4.3
Sample refrigeration or transport on ice may result in V. cholerae Ol or O139 becoming VBNC (Reference 15.4).
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5.0
Safety
5.1 Laboratory
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 parentcral 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 Recommended Precautions
5.2,1
V. cholerae Ol and O139 are BSL-2 pathogens and all procedures should be performed in laboratories that use. at a
minimum. BSL-2 practices. This includes prohibiting eating, drinking, smoking, handling contact lenses, applying
cosmetics, and storing food and drink in the laboratory (Reference 15.3).
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 safety 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 are 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.
5.2.43
Laboratory coats covering arms and clothing and closed in the front should be worn at all times. Laboratory coats that
become soiled should be changed.
5.2.7
This method does not address all safety issues associated with its use. Please refer to Biosafety in Microbiological and
Biomedical 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
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 ± 0.1 g
6.4
Beakers, glass or plastic (assorted sizes)
6.5
Biological safety cabinet. Class II (optional)
6.6
Borosilicatc glass culture tubes, with autoclavablc screw or snap caps (25 x 150 mm)
6.7
Borosilicate glass or plastic screw-cap, wide-mouth bottles, sterile (e.g.. 250 mL)
6.8
Erlcnmcyer flasks (500 mL, 1 L, 2 L)
6.9
Gloves, sterile, nitrile, or equivalent
6.10
Graduated cylinders (assorted sizes)
6.11
Incubator, microbiological type, maintained at 36.0°C ± 1.0°C
6.12
Inoculation loops, sterile, disposable
6.13
Parafilm® or equivalent
6.14
Petri dishes, sterile, plastic (15 * 100 mm)
6.15
pH meter
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6.16
Pipettes, standard tip, sterile, plastic, disposable (assorted sizes)
6.17
Pipetting device (automatic or equivalent)
6.18
Stirring hotplates and stir bars
6.19
Test tube racks
6.20
Thermometer, National Institute of Standards and Technology (NIST)-traceable
6.21
Tissues, lint-free (Kimwipes® or equivalent)
6.22
Watcrbath, maintained at 45°C - 50°C for tempering agar
6.23
Weigh paper and boats
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7.0
and
7.1
Reagent-grade chemicals must be used in all analyses. 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 of microbiological grade.
7.3
Reagent-grade water must conform to specifications in Standard Methods for the Examination of Water and
Wastewater, 21st Edition (Reference 15.8).
7.4 (PBS)
Prepare reagent according to the following and store at <10°C and above freezing for a maximum of two weeks in
tubes with loose caps or three months in screw-cap tubes.
7.4.1 Composition:
Monosodium phosphate (NaH2PO4) 0.58 g
Disodium phosphate (Na2HPO^) 2.50 g
Sodium chloride 8.50 g
Reagent-grade water l.OL
7.4.2
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.
7.5
Commercially prepared medium is recommended. Dehydrated medium (EMD Chemicals 1.01800.0500 or equivalent)
may be used. If commercially prepared medium is not available prepare IX, 2X, and 5X APW according to Sections
7.5" land 7.5.2.
7.5.1 Composition:
IX 2X 5X
Peptone 10.0 g 20.0 g 50.0 g
Sodium chloride 10.0 g 20.0 g 50.0 g
Reagent-grade water l.OL l.OL l.OL
7.5.2
Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar. Adjust pH to 8.5 ± 0.2 with 1.0 N
hydrochloric acid or 1.0 N sodium hydroxide and bring up to 1 L. Dispense 10 mL (1X and 2X) or 5 mL (5X) aliquots
in 25 x 150 mm tubes and autoclave at 121°C (15 psi) for 15 minutes. Cool to room temperature. Wann medium to
36°C ± 1.0°C prior to inoculation.
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7.6 Thiosulfate Bile (TCBS)
Commercially prepared medium is recommended. Dehydrated medium (Oxoid CM0333 or equivalent) may be used.
If commercially prepared medium is not available, prepare medium according to Sections 7.6.1 and 7.6.2.
7.6.1 Composition:
Yeast extract 5.0g
Peptone lO.Og
Sodium thiosulfate 10.0 g
Sodium citrate 10.0 g
Ox bile 8.0 g
Sucrose 20.0 g
Sodium chloride 10.0 g
Ferric citrate l.Og
Bromothymol blue 0.04 g
Thymol blue 0.04 g
Agar 14.0 g
Reagent-grade water l.OL
7,6.2
Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar and hot plate. Adjust pH to 8.6 ±
0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide and bring up to 1 L. Boil for one minute with rapid stir
bar agitation to dissolve completely. DO NOT AUTOCLAVE. Aseptically pour 12-15 mL into each 15 x 100 mm
sterile Petri plate. Warm medium to room temperature prior to use.
7.7 Tryptic Soy (ISA)
Commercially prepared medium is recommended. Prepared plates (BBL™ 221803 or equivalent) or dehydrated
medium (BBL™ 211043 or equivalent) may be used. If commercially prepared medium is not available, prepare
medium according to Sections 7.7.1 and 7.7.2.
7,7.1 Composition:
Pancreatic digest of casein 15.0 g
Papaic digest of soybean meal 5.0 g
Sodium chloride 5.0 g
Agar 15.0 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 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 * 100 mm sterile Petri plate.
Warm medium to room temperature prior to use.
7.8
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.
7.9
V. cholerae Ol (Difco™ 210603 or equivalent) and O139 antisera (Difco™ 210431 or equivalent)
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7.10
Biochemical test strip (bioMerieux API 20E® or equivalent)
7.11
Oxidase reagent (BBL™ DrySlide™ 231746 or equivalent)
7.12
Positive and negative control cultures that are to be used with this protocol are listed in Table 1. Use of these controls
is discussed in Section 9.0.
Table 1. Positive and Negative Control Cultures
Media/Reagents
APW
TCBS
Ol serum agglutination
O139 serum agglutination
Biochemical test strip
Oxidase
Positive Control*1'
V.choleraeOl orO139
V.choleraeOl orO139
V. cholerae Ol
V. cholerae O139
V.choleraeOl orO139
V.choleraeOl orO139
Negative Control
E. coli (ATCC® 25922™)
E. coli (ATCC®25922™)
E. coli (ATCC® 25922™)
E. coli (ATCC® 25922™)
E. coli (ATCC® 25922™)
E. coli (ATCC® 25922™)
(1)Either JBK 70 for V. cholerae Ol, CVD 112 for V. cholerae O139, Center for Vaccine Development (CVD), University of Maryland School of
Medicine, or equivalent strains may be used, as appropriate.
7.13
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
Media/Reagents
PBS, saline
(in screw-cap bottles or tubes)
Tubes: APW
Plates: TCBS, TSA
Storage Temperature11'
Room temperature
Room temperature
<10°C and above freezing
Storage Time
3 months
2 weeks in loose cap tubes
3 weeks in screw-cap tubes
2 weeks
(1'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.
APW should be brought to 36.0°C ± 1.0°C prior to inoculation.
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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 annually against a NlST-ceitified 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 (QA) 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 (QC) procedures for use with this
method are discussed below.
e: Following testing and validation, this method will be updated to include QC 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 7.12) and descriptions of negative results are provided in Table 3 (Section
10.7).
9.2.2
Analysis of negative controls is conducted by inoculating media and performing biochemical and serological tests
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
medium (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 Table 1 (Section 7.12) and descriptions of positive results are provided in Table 3 (Section 10.7).
9.3.2
Analysis of positive controls is conducted by inoculating media and performing biochemical and serological tests 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 positive control organism, and re-analyze the appropriate positive control and
corresponding sample(s).
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9.4
To verify the sterility of equipment materials, and supplies, the laboratory should analyze a method blank each day
that samples are analyzed using sterile PBS (Section 7.4). 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
Test sterility of PBS, and media (APW, TCBS, TSA) by incubating one unit (tube or plate) from each batch at 36.0°C
± 1.0°C for 24 ±2 hours and observing for growth. Absence of growth indicates the media are sterile. On an ongoing
basis, media sterility checks should be done every day that samples are analyzed.
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10.0
Procedures
Process samples promptly upon receipt, allowing no more than six hours to elapse from the time of sample collection
to sample analysis. Samples should not be placed on ice or held at <10°C. V. cholerae Ol andO139 are pathogens.
and all samples should be handled with caution, using appropriate BSL-2 procedures and PPE. A Class II biological
safety7 cabinet is recommended for sample manipulations where the risk of aerosol production is high.
10.1
Add a sample volume (e.g., 100 mL) to an equal volume of double-strength APW (Section 7.5) and incubate at 36.0°C
± 1.0°C for 6 - 8 hours or 24 ± 2 hours. Based on the single-laboratory verification study, analysis of drinking water
matrices may require a 24 ± 2 hour incubation period. After incubation, proceed to Section 10.3 for selective isolation
of V. cholerae Ol and O139.
Note: // may be necessary to incubate APW for 24 ±2 hours for some sample types and target organisms (e.g., V.
cholerae Ol).
10.2
A multiple-tube assay incorporating differential sample volumes is used to estimate V. cholerae Ol and O139 densities
in undiluted or diluted samples. If low levels of V. cholerae are suspected, larger sample volumes (20 mL of original
sample) should be used to inoculate the first row of tubes in the series. If high levels of V. cholerae arc 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. Bring APW to 36.0°C ± 1.0°C prior to inoculation.
10.2.1 Sample inoculation
Arrange APW tubes in three rows (5 mL of 5X, 10 mL of 2X, 10 mL of IX) of five tubes each. Inoculate the first row
of tubes (5 mL of 5X APW) with 20 inL of the undiluted sample. Inoculate 10 inL of the undiluted sample into each of
the tubes in the second row (10 mL of 2X APW). Inoculate 1 mL from the initial sample into each of the tubes in the
third row (10 mL of IX APW). See Flowchart 16.1 for an overview of the sample inoculation scheme.
10.2.2 dilutions
Samples may require serial dilution prior to inoculation due to high levels of V. cholerae. If analyzing serially diluted
samples, 1.0 mL of each dilution will be used to inoculate each tube of IX APW, as appropriate.
10.2.3
Incubate tubes at 36.0°C ± 1.0°C for 6 - 8 hours or 24 ± 2 hours. Based on the single-laboratory verification study,
analysis of drinking water matrices may require a 24 ± 2 hour incubation period. After incubation, proceed to Section
10.3 for isolation of V. cholerae.
Note: // may be necessary to incubate APW for 24 hours for some sample types and target organisms (e.g., V.
cholerae Ol).
10.3 Isolation on TCBS Plates
10.3.1
Perform primary isolation on all APW tubes incubated 6-8 hours and on all tubes incubated 24 ± 2 hours exhibiting
growth. Do not shake or mix tubes. Obtain inoculum 2-5 mm from the top of the tube (pellicle) and streak for
isolation onto TCBS plates using a sterile inoculation loop (20 uL).
10.3.2
Incubate TCBS plates at 36.0°C ± 1.0°C for 24 ± 2 hours. Typical V. cholerae colonies are large and yellow.
Note: Cultures grown on TCBS should be examined quickly after removal from an incubator because the yellow
colonies may revert to a green color when left at room temperature.
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10.4 on ISA
10.4.1
Streak a single typical colony for isolation onto a TSA plate from cadi TCBS plate. Incubate the plates at 36.0°C ±
1.0°Cfor24±2hours.
10.4.2
Seal the TCBS plates with Parafilm* and store at room temperature for use as backup plates. Use the TSA plates for
serological and biochemical analyses.
10.5
10.5.1
V. cholerae Ol or O139 antiserum: Choose a well-isolated colony from each of the TSA plates and emulsify growth
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 Ol or Ol 39 antiserum (as appropriate). To the second drop of emulsified growth,
add one drop of sterile saline (as a visual comparison).
10.5.2
Observe under magnification for an agglutination reaction, which indicates a positive result. V. cholerae Ol is
agglutination-positive for Ol antiserum and V. cholerae O139 is agglutination-positive for O139 antiserum. Results
should be compared with those for positive and negative controls (Table 1) analyzed at the same time.
10.6
Use a single, isolated, large colony (2-3 mm diameter) from each TSA plate for biochemical and oxidase analyses.
10.6,1 Test or
Following manufacturer's instructions, transfer a small amount of cells from the selected colony to the slide. Oxidase-
positive bacteria turn the reagent dark purple within 20 seconds. V. cholerae is oxidase-positive. Results should be
compared with those for positive and negative controls (Table 1) analyzed at the same time.
10.6.2 Biochemical Test Strips (API 20E® or equivalent)
Emulsify the remainder of the colony in 0.85% NaCl. 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.
10.6.3 Alternative Biochemical Tests
The following individual biochemical tests may be used instead of biochemical test strips to identify V. cholerae Ol
andO139:
* Arginine
• Glucose fermentation
* H2S
• Lysine
* Ornithine
* Oxidase
* String test
* Sucrose fermentation
• Voges-Proskauer
V. cholerae Ol and O139 are positive for oxidase. glucose, lysine, ornithine, string test, and sucrose, and negative for
arginine and H0S. Voges-Proskauer test results are variable, but generally positive.
-------
10.7 Description of Quality Control and V. cholerae O1 and O139 Results
Typical results are provided in Table 3.
Table 3. Positive and Negative Result Descriptions and Vibrio cholerae Ol and O139 Results
Medium/Test
TCBS
APW
Oxidase
Biochemical test strip
Ol/O139antiserum
(as appropriate)
V. cholerae Ol and
O139 Results
Positive
Positive
Positive
Positive Control Result and
Description
Large, yellow colonies
Growth at alkaline pH
(pellicle or turbidity)
Purple to violet color change within
20 seconds
Negative Control Result and
Description
Pink to red colonies
No growth
Colorless or very light pink color change
over time
Consult manufacturer's instructions
Positive
Agglutination
No agglutination
-------
-------
11.0
Data Analysis and Bacterial Density Calculation
11.1 Most Probable Number (MPN) Technique
Estimation of bacterial densities may be determined based on the number of tubes positive for V. cholerae Ol or O139
either by biochemical and serological confirmation or PCR.
11.2 Calculation of MPN
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 (i.e., volumes and/or 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 Table 4, Example A, 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 rules above, then select the three
smallest volumes (Table 4, Example F).
Table 4. Examples of Appropriate Tube Selection and MPN/100
Example
A
B
C
D
E
F
20 mL
5/5
4/5
0/5
5/5
4/5
5/5
10 mL
5/5
5/5
1/5
3/5
4/5
5/5
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
'''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 5 using the number of positive tubes in the three selected dilutions. Calculate MPN/100 mL using the
equation below:
MPN Index from Table 6
MPN/100 ml
Middle volume analyzed in the series used for MPN determination
x100
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
x100
3.29 x105
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
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
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
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
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
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
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
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
-------
Combination of
Positives
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
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
MPN Index
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
0.0255
0.0330
0.0417
0.0506
0.0598
0.0691
0.0344
0.0435
0.0529
0.0626
0.0725
0.0827
95% Confidence Limits
Lower
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
0.0028
0.0063
0.0103
0.0147
0.0193
0.0241
0.0069
0.0112
0.0159
0.0207
0.0258
0.0310
Upper
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
0.0585
0.0710
0.0863
0.1023
0.1191
0.1368
0.0734
0.0896
0.1065
0.1244
0.1434
0.1640
Combination of
Positives
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
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
MPN Index
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
0.0797
0.0937
0.1086
0.1245
0.1414
0.1595
0.1012
0.1181
0.1364
0.1563
0.1780
0.2015
95% Confidence Limits
Lower
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
0.0295
0.0366
0.0441
0.0520
0.0602
0.0686
0.0404
0.0489
0.0578
0.0672
0.0770
0.0873
Upper
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
0.1579
0.1877
0.2228
0.2656
0.3218
0.4067
0.2049
0.2476
0.3038
0.3890
0.5273
0.6411
-------
Combination of
Positives
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
0.0456
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
0.0122
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
0.0932
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
4-5-0
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
0.1304
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
0.0549
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
0.2836
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
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
MPN Index
<0.018
0.018
0.036
0.054
0.072
0.091
0.018
0.036
0.055
0.073
0.091
0.110
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
0.190
0.020
0.040
0.060
0.081
0.101
0.122
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
Upper
0.063
0.063
0.101
0.137
0.174
0.212
0.063
0.101
0.138
0.175
0.214
0.256
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
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
MPN Index
0.083
0.104
0.125
0.147
0.169
0.191
0.105
0.127
0.148
0.170
0.193
0.215
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
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
0.067
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
0.069
0.026
0.037
0.048
0.059
0.070
0.082
Upper
0.196
0.243
0.296
0.364
0.460
0.566
0.245
0.300
0.370
0.468
0.575
0.657
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
0.675
0.279
0.355
0.467
0.591
0.683
0.759
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Combination of
Positives
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
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
MPN Index
0.040
0.061
0.081
0.102
0.123
0.144
0.061
0.082
0.103
0.124
0.146
0.167
0.079
0.106
0.135
0.165
0.196
0.229
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
95% Confidence Limits
Lower
0.003
0.003
0.011
0.019
0.028
0.037
0.003
0.012
0.020
0.029
0.038
0.047
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
Upper
0.109
0.150
0.192
0.238
0.290
0.354
0.151
0.194
0.240
0.293
0.359
0.451
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
Combination of
Positives
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
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
MPN Index
0.146
0.172
0.199
0.226
0.254
0.282
0.174
0.201
0.229
0.257
0.286
0.315
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
95% Confidence Limits
Lower
0.038
0.049
0.060
0.072
0.083
0.094
0.050
0.061
0.073
0.084
0.095
0.107
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
Upper
0.361
0.477
0.600
0.692
0.768
0.836
0.488
0.610
0.700
0.776
0.845
0.910
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
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Combination of
Positives
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
0.248
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
0.080
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
0.753
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
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
0.493
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
>16.090
95% Confidence Limits
Lower
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
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
''Table was developed using the MPN calculator developed by Albert Klee (Reference 15.10).
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12.0
Protocol Performance
Culture-based procedures were evaluated for V. cholerae Ol and O139 in a reference matrix (PBS) and two matrices
of interest (drinking water, surface water) during a single-laboratory verification study. Details regarding procedure
performance are provided in the study report (Reference 15.11). Summary results from this evaluation are listed in
Table 7.
Table 7. Summary of Results for V. cholerae Ol and O139 in Spiked PBS, Drinking Water, and Surface Water
Samples*1'
Analyte
Sample Location
Spike
level
Pre-
Enrichment
inAPW
Mean
Recovery
(%)
Minimum
Recovery
(%)
Maximum
Recovery
(%)
SD (%)
RSD (%)
Phosphate Buffered Saline
01
O139
NA
NA
129
253
6 hours
6 hours
57.20
94.18
57.20
94.18
57.20
94.18
NA
NA
NA
NA
Drinking Water
Ol
O139
NA
NA
36
34
24 hours
24 hours
84.91
163.23
25.25
67.35
125.39
320.06
49.30
109.00
58.06
66.78
Surface Water
Ol
01
01
01
0139
O139
Site 1
Site 2
Site 3
Site 4
129
129
129
129
253
253
6 hours
24 hours
6 hours
24 hours
6 hours
6 hours
25.52
152.33
13.53
52.86
88.62
119.46
16.26
69.97
4.89
13.99
35.62
57.76
30.79
355.93
30.79
113.47
181.17
181.17
6.36
137.27
12.21
48.26
66.75
71.25
24.94
90.11
90.27
91.31
75.32
59.64
(1)These values are based on a 9-tube, as opposed to a 15-tube, MPN.
APW - Alkaline peptone water NA - Not applicable
SD - Standard deviation RSD - Relative standard deviation
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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.
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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 minimi/ing 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 have viable V. cholerae Ol or O139 attached or
contained 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.
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-------
15.0
References
15.1 National Center for Infectious Diseases, Centers for Disease Control and Prevention. 1999. "Isolation and
Identification of Vibrio cholerae Serogroups Ol and O139." In Laboratory Methods for the Diagnosis of
Epidemic Dysentery and Cholera. J.P Koplan, J.M. Hughes, M.L. Cohen, E.M.B. Samba, and A.B. Kabore
(eds.). 41-54. Atlanta, GA: National Center for Infectious Diseases, Centers for Disease Control and
Prevention. http://www.cdc.gov/ncidod/DBMD/diseaseinfo/cholera_lab_manual.htm
15.2 Kaysner, C.A. andDepaola, A., Jr. May 2004. "Vibrio." In Bacteriological Analytical Manual Online. G.J.
Jackson, R.I. Merker, and R. Handler, (eds.) U.S. Food and Drug Administration. http://www.fda.gov/Food/
ScienceResearch/LaboratoryMemods/BacteriologicalAnalyticalManualBAM/ucm070830.htm
15.3 U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National
Institutes of Health. 2007. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition.
Washington, D.C.: U.S. Government Printing Office. http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.
htm
15.4 Alam, M, Sultana, M, Nair, G.B., Siddique, A.K., Hasan, N.A., Sack, R.B., Sack, D.A. Ahmed, K.U.,
Sadique, A., Watanabe, H., Grim, C.J., Huq, A., and Colwell, R.R. 2007. "Viable But Nonculturable Vibrio
cholerae Ol in Biofilms in the Aquatic Environment and Their Role in Cholera Transmission." Proceedings
of the National Academy of Sciences. 104(45): 17801-17806.
15.5 American Chemical Society. 2000. Reagent Chemicals, American Chemical Society Specifications. New
York, NY: American Chemical Society.
15.6 British Drug Houses, 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,
MD: United States Pharmacopeia! 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,
andM.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.
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. "A 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. Results of Single-Laboratory Verification of Culture-based
Analytical Procedures for Vibrio Cholerae Ol and O139 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.
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16.1 Quantitative Analysis Dilution Scheme
Distribution of Sample to APW
(Sections 10.2.1 and 10.2.2)
Incubation at 36.0" ± 1.0°C for 6 - 8 hours or 24 ± 2 hours
(Section 10,2.3)
Undiluted
grab sample
tube of 2X APW (10 ml)
Serial dilutions as
necessary
1,0 ml to each tube of
T\T\T\
••*.—--> v—-r ---—-f
:TI
rr
1X APW (10 ml) j— |— —|
Incubate at
36°C±1.0°C
6-8 hours
OR
24 + 2 hours
Analysis of
positive tubes
o
o
-------
16.2 Identification Flowchart
Enrichment in APW and Isolation on TCBS
Plates (Section 10.3) or PCR Confirmation
Qualitative and
Quantitative (MPN)
analyses in APW tubes
TCBS
Sub-culture on TSA Plates
Section 10.4
Seroiogical and Biochemical Testing
Sections 10.5 and 10.6
o o o
1 2 3
TSA
O1/O139 serum
agglutination
incubate at
36.0°C ± 1 .0°C for
18-24 hours
Test strip with
oxidase test:
Incubate at
36.0°C ± 1.0°C for
24 ±2 hours
Incubate test strip
at36.0°C± 1.0°C
for18-24hrs
Individual
biochemical
tests:
Arginine
Glucose
HZS
Lysine
Ornithine
Oxidase
String test
Sucrose
Voges-
Proskauer
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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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