EPA 600/R-10/139 | October 2010 | www.epa.

gov/ord

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
 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
ii
 Acknowledgments
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 O1 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 are gratefully acknowledged:
Study Workgroup Participants
• Cheryl Bopp, Michele Parsons (Centers for Disease Control and Prevention)
• Michele Burgess, Marissa Mullins (EPA, Office of Emergency Management)
• Rita Colwell, Anwar Huq (Maryland Pathogen Research Institute, University of Maryland)
• Stephanie Harris (EPA, Region 10)
• Malik Raynor (EPA, Office of 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 Drug Administration)

Volunteer Participant Laboratory

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

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 Disclaimer
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 warranty, expressed or implied,
or assume any legal liability or responsibility for any third party’s use of or the results of such use of
any information, apparatus, product, or process discussed in this document, or represent that its use
by such party would not infringe on privately owned rights.
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.

iv
 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) O1 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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vi
 Contents
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 O1 and O139 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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 Tables
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 O1 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 are Used and Sample Inoculation Volumes are 10.0, 1.0, and 0.1 mL .................. 29

Table 7. Summary of Results for V. cholerae O1 and O139 in Spiked PBS, Drinking Water, and
Surface Water Samples ....................................................................................................................... 33

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x
 1.0
Scope and Application

1.1
This Standard Analytical Protocol (SAP) is for the identification, confirmation, and quantitation of Vibrio cholerae
(V. cholerae) O1 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 O1 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 O1 and O139 and the matrices
described.

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2
 2.0
Summary of Protocol
2.1
V. cholerae O1 and O139 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 are analyzed using a 15-tube MPN. Inoculated
APW tubes are incubated at 36.0°C ± 1.0°C for 6 – 8 or 24 ± 2 hours.
Note: It may be necessary to incubate APW for 24 ± 2 hours for some sample types and target organisms (e.g., V.
cholerae O1).

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 O1 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 O1 or O139 antiserum (as appropriate), followed
by biochemical characterization using commercially available test strips (e.g., API 20E® [bioMérieux or equivalent) or
with selected individual biochemical tests.

2.6
APW tubes (MPN and qualitative analysis) may be subjected to real-time polymerase chain reaction (PCR)
confirmation in place of biochemical and serological confirmation.

2.7
Quantitation of V. cholerae O1 and O139 is determined using the MPN technique (Flowchart 16.1). Tubes that are
confirmed positive for V. cholerae O1 or O139 are used to determine the MPN.

3
4
 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)
µL Microliter(s)
mL Milliliter(s)
mm Millimeter(s)
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
Interferences and Contamination

4.1
Low recoveries of V. cholerae O1 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 O1 or O139 may also account for lower recoveries (Reference
15.4).

4.3
Sample refrigeration or transport on ice may result in V. cholerae O1 or O139 becoming VBNC (Reference 15.4).

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 5.0
Safety
5.1 Laboratory Hazards
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 Recommended Precautions

5.2.1
V. cholerae O1 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.
5.2.6.3
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.

9
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 6.0
Equipment and Supplies

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
Borosilicate glass culture tubes, with autoclavable screw or snap caps (25 × 150 mm)

6.7
Borosilicate glass or plastic screw-cap, wide-mouth bottles, sterile (e.g., 250 mL)

6.8
Erlenmeyer 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
Waterbath, maintained at 45°C – 50°C for tempering agar

6.23
Weigh paper and boats

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 7.0
Reagents and Standards
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 Phosphate Buffered Saline (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 (Na2HPO4) 2.50 g
Sodium chloride 8.50 g
Reagent-grade water 1.0 L
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 Alkaline Peptone Water (APW)

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 1X, 2X, and 5X APW according to Sections
7.5.1 and 7.5.2.
7.5.1 Composition:
1X 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 1.0 L 1.0 L 1.0 L
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 × 150 mm tubes and autoclave at 121°C (15 psi) for 15 minutes. Cool to room temperature. Warm medium to
36°C ± 1.0°C prior to inoculation.

13
 7.6 Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar
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 5.0 g
Peptone 10.0 g
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 1.0 g
Bromothymol blue 0.04 g
Thymol blue 0.04 g
Agar 14.0 g
Reagent-grade water 1.0 L
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 × 100 mm
sterile Petri plate. Warm medium to room temperature prior to use.

7.7 Tryptic Soy Agar (TSA)

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 1.0 L
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 O1 (Difco™ 210603 or equivalent) and O139 antisera (Difco™ 210431 or equivalent)

14
7.10
Biochemical test strip (bioMérieux 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 Positive Control(1) Negative Control

APW V. cholerae O1 or O139 E. coli (ATCC® 25922™)

TCBS V. cholerae O1 or O139 E. coli (ATCC®25922™)

O1 serum agglutination V. cholerae O1 E. coli (ATCC® 25922™)

O139 serum agglutination V. cholerae O139 E. coli (ATCC® 25922™)

Biochemical test strip V. cholerae O1 or O139 E. coli (ATCC® 25922™)

Oxidase V. cholerae O1 or O139 E. coli (ATCC® 25922™)

(1)
Either JBK 70 for V. cholerae O1, 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 Storage Temperature(1) Storage Time

PBS, saline
Room temperature 3 months
(in screw-cap bottles or tubes)
2 weeks in loose cap tubes
Tubes: APW Room temperature
3 weeks in screw-cap tubes
Plates: TCBS, TSA <10°C and above freezing 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
Calibration and Standardization

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 NIST-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 General
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.
Note: 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 Negative 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 every 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 Method Blank
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 Media Sterility Check

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.

20
 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 O1 and O139 are pathogens,
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 Analyses

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 O1 and O139.
Note: It may be necessary to incubate APW for 24 ± 2 hours for some sample types and target organisms (e.g., V.
cholerae O1).

10.2 Quantitative Sample Analyses

A multiple-tube assay incorporating differential sample volumes is used to estimate V. cholerae O1 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 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. 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 1X) of five tubes each. Inoculate the first row
of tubes (5 mL of 5X APW) with 20 mL of the undiluted sample. Inoculate 10 mL 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 1X APW). 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 V. cholerae. If analyzing serially diluted
samples, 1.0 mL of each dilution will be used to inoculate each tube of 1X APW, as appropriate.
10.2.3 Growth
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: It may be necessary to incubate APW for 24 hours for some sample types and target organisms (e.g., V.
cholerae O1).

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 μL).
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.

21
 10.4 Isolation on TSA Plates
10.4.1
Streak a single typical colony for isolation onto a TSA plate from each TCBS plate. Incubate the plates at 36.0°C ±
1.0°C for 24 ± 2 hours.
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 Serological Analyses

10.5.1
V. cholerae O1 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 O1 or O139 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 O1 is
agglutination-positive for O1 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 Biochemical Analyses

Use a single, isolated, large colony (2 – 3 mm diameter) from each TSA plate for biochemical and oxidase analyses.
10.6.1 Oxidase Test (BBL™ DrySlide™ 231746 or equivalent)
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 O1
and O139:
• Arginine
• Glucose fermentation
• H2S
• Lysine
• Ornithine
• Oxidase
• String test
• Sucrose fermentation
• Voges-Proskauer
V. cholerae O1 and O139 are positive for oxidase, glucose, lysine, ornithine, string test, and sucrose, and negative for
arginine and H2S. Voges-Proskauer test results are variable, but generally positive.

22
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 O1 and O139 Results
V. cholerae O1 and Positive Control Result and Negative Control Result and
Medium/Test
O139 Results Description Description

TCBS Positive Large, yellow colonies No growth

Growth at alkaline pH
APW Positive No growth
(pellicle or turbidity)
Purple to violet color change within Colorless or very light pink color change
Oxidase Positive
20 seconds over time

Biochemical test strip Consult manufacturer’s instructions

O1/O139 antiserum
Positive Agglutination No agglutination
(as appropriate)

23
24
 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 O1 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 mL(1)

Significant
Example 20 mL 10 mL 1.0 mL 0.1 mL Table MPN Index MPN/100 mL
Dilutions

A 5/5 5/5 3/5 0/5 5-3-0 6 0.792 79.2

B 4/5 5/5 1/5 0/5 4-5-1 5 0.1524 15.24

C 0/5 1/5 0/5 0/5 0-1-0 5 0.0067 0.67

D 5/5 3/5 1/5 1/5 3-1-1 6 0.137 13.7

E 4/5 4/5 0/5 1/5 4-4-1 5 0.1181 11.81

F 5/5 5/5 5/5 2/5 5-5-2 6 5.422 542.2

(1)
Appropriate volumes are underlined and the largest sample volumes analyzed are highlighted.

25
 11.2.2
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 = × 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 = × 100 = 3.29 × 105
10-4

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)

95% Confidence Limits 95% Confidence Limits

Combination of Combination of
MPN Index MPN Index
Positives Positives
Lower Upper Lower Upper

0-0-0 <0.006473 ---- 0.0223 1-3-0 0.0312 0.0055 0.0678

0-0-1 0.0065 0.0012 0.0223 1-3-1 0.0393 0.0092 0.0821
0-0-2 0.0130 0.0012 0.0352 1-3-2 0.0475 0.0132 0.0967
0-0-3 0.0195 0.0012 0.0472 1-3-3 0.0559 0.0173 0.1119
0-0-4 0.0262 0.0033 0.0589 1-3-4 0.0644 0.0216 0.1277
0-0-5 0.0328 0.0062 0.0706 1-3-5 0.0730 0.0260 0.1444
0-1-0 0.0067 0.0012 0.0228 1-4-0 0.0409 0.0099 0.0849
0-1-1 0.0134 0.0012 0.0360 1-4-1 0.0495 0.0141 0.1002
0-1-2 0.0202 0.0012 0.0483 1-4-2 0.0583 0.0185 0.1163
0-1-3 0.0270 0.0037 0.0604 1-4-3 0.0672 0.0231 0.1331
0-1-4 0.0339 0.0067 0.0725 1-4-4 0.0763 0.0277 0.1509
0-1-5 0.0408 0.0099 0.0847 1-4-5 0.0855 0.0324 0.1700
0-2-0 0.0138 0.0012 0.0367 1-5-0 0.0517 0.0152 0.1042
0-2-1 0.0208 0.0012 0.0495 1-5-1 0.0609 0.0199 0.1212
0-2-2 0.0279 0.0040 0.0619 1-5-2 0.0703 0.0247 0.1391
0-2-3 0.0350 0.0072 0.0745 1-5-3 0.0799 0.0296 0.1583
0-2-4 0.0422 0.0106 0.0871 1-5-4 0.0897 0.0346 0.1790
0-2-5 0.0494 0.0141 0.1001 1-5-5 0.0998 0.0397 0.2015
0-3-0 0.0215 0.0012 0.0507 2-0-0 0.0155 0.0012 0.0404
0-3-1 0.0288 0.0044 0.0636 2-0-1 0.0226 0.0018 0.0526
0-3-2 0.0362 0.0077 0.0766 2-0-2 0.0303 0.0051 0.0662
0-3-3 0.0437 0.0113 0.0898 2-0-3 0.0382 0.0087 0.0801
0-3-4 0.0512 0.0051 0.1243 2-0-4 0.0462 0.0125 0.0943
0-3-5 0.0588 0.0095 0.1428 2-0-5 0.0543 0.0165 0.1090

26
 95% Confidence Limits 95% Confidence Limits
Combination of Combination of
MPN Index MPN Index
Positives Positives
Lower Upper Lower Upper

0-4-0 0.0299 0.0049 0.0654 2-1-0 0.0234 0.0022 0.0540

0-4-1 0.0375 0.0084 0.0789 2-1-1 0.0315 0.0056 0.0683
0-4-2 0.0453 0.0121 0.0927 2-1-2 0.0397 0.0094 0.0827
0-4-3 0.0531 0.0160 0.1069 2-1-3 0.0480 0.0134 0.0976
0-4-4 0.0611 0.0200 0.1216 2-1-4 0.0565 0.0177 0.1131
0-4-5 0.0691 0.0241 0.1369 2-1-5 0.0652 0.0221 0.1293
0-5-0 0.0390 0.0090 0.0814 2-2-0 0.0327 0.0062 0.0705
0-5-1 0.0470 0.0129 0.0958 2-2-1 0.0413 0.0101 0.0856
0-5-2 0.0553 0.0170 0.1107 2-2-2 0.0501 0.0144 0.1013
0-5-3 0.0636 0.0212 0.1262 2-2-3 0.0590 0.0189 0.1176
0-5-4 0.0720 0.0255 0.1425 2-2-4 0.0681 0.0236 0.1349
0-5-5 0.0806 0.0299 0.1596 2-2-5 0.0774 0.0283 0.1533
1-0-0 0.0072 0.0012 0.0241 2-3-0 0.0431 0.0110 0.0887
1-0-1 0.0139 0.0012 0.0369 2-3-1 0.0523 0.0155 0.1053
1-0-2 0.0209 0.0012 0.0497 2-3-2 0.0617 0.0203 0.1227
1-0-3 0.0281 0.0041 0.0623 2-3-3 0.0714 0.0252 0.1412
1-0-4 0.0353 0.0073 0.0749 2-3-4 0.0813 0.0303 0.1611
1-0-5 0.0425 0.0107 0.0878 2-3-5 0.0914 0.0354 0.1826
1-1-0 0.0144 0.0012 0.0377 2-4-0 0.0547 0.0168 0.1098
1-1-1 0.0217 0.0013 0.0509 2-4-1 0.0647 0.0218 0.1284
1-1-2 0.0290 0.0045 0.0640 2-4-2 0.0750 0.0271 0.1484
1-1-3 0.0365 0.0079 0.0771 2-4-3 0.0855 0.0325 0.1700
1-1-4 0.0441 0.0115 0.0905 2-4-4 0.0964 0.0380 0.1937
1-1-5 0.0517 0.0153 0.1043 2-4-5 0.1076 0.0436 0.2201
1-2-0 0.0224 0.0017 0.0523 2-5-0 0.0681 0.0235 0.1349
1-2-1 0.0301 0.0050 0.0658 2-5-1 0.0791 0.0292 0.1566
1-2-2 0.0379 0.0085 0.0795 2-5-2 0.0904 0.0349 0.1805
1-2-3 0.0457 0.0123 0.0935 2-5-3 0.1021 0.0409 0.2070
1-2-4 0.0537 0.0162 0.1079 2-5-4 0.1143 0.0469 0.2372
1-2-5 0.0618 0.0203 0.1229 2-5-5 0.1268 0.0531 0.2725
3-0-0 0.0255 0.0028 0.0585 4-3-0 0.0797 0.0295 0.1579
3-0-1 0.0330 0.0063 0.0710 4-3-1 0.0937 0.0366 0.1877
3-0-2 0.0417 0.0103 0.0863 4-3-2 0.1086 0.0441 0.2228
3-0-3 0.0506 0.0147 0.1023 4-3-3 0.1245 0.0520 0.2656
3-0-4 0.0598 0.0193 0.1191 4-3-4 0.1414 0.0602 0.3218
3-0-5 0.0691 0.0241 0.1368 4-3-5 0.1595 0.0686 0.4067
3-1-0 0.0344 0.0069 0.0734 4-4-0 0.1012 0.0404 0.2049
3-1-1 0.0435 0.0112 0.0896 4-4-1 0.1181 0.0489 0.2476
3-1-2 0.0529 0.0159 0.1065 4-4-2 0.1364 0.0578 0.3038
3-1-3 0.0626 0.0207 0.1244 4-4-3 0.1563 0.0672 0.3890
3-1-4 0.0725 0.0258 0.1434 4-4-4 0.1780 0.0770 0.5273
3-1-5 0.0827 0.0310 0.1640 4-4-5 0.2015 0.0873 0.6411
27
 95% Confidence Limits 95% Confidence Limits
Combination of Combination of
MPN Index MPN Index
Positives Positives
Lower Upper Lower Upper

3-2-0 0.0456 0.0122 0.0932 4-5-0 0.1304 0.0549 0.2836

3-2-1 0.0555 0.0171 0.1112 4-5-1 0.1524 0.0653 0.3687
3-2-2 0.0657 0.0223 0.1303 4-5-2 0.1769 0.0766 0.5210
3-2-3 0.0763 0.0277 0.1510 4-5-3 0.2046 0.0886 0.6528
3-2-4 0.0872 0.0333 0.1735 4-5-4 0.2357 0.1015 0.7516
3-2-5 0.0984 0.0390 0.1984 4-5-5 0.2708 0.1150 0.8426
3-3-0 0.0583 0.0186 0.1164 5-0-0 0.0549 0.0162 0.1116
3-3-1 0.0693 0.0241 0.1371 5-0-1 0.0637 0.0213 0.1265
3-3-2 0.0806 0.0299 0.1597 5-0-2 0.0763 0.0277 0.1510
3-3-3 0.0924 0.0359 0.1847 5-0-3 0.0896 0.0345 0.1787
3-3-4 0.1046 0.0421 0.2128 5-0-4 0.1037 0.0417 0.2107
3-3-5 0.1173 0.0484 0.2452 5-0-5 0.0953 0.0165 0.2234
3-4-0 0.0733 0.0262 0.1450 5-1-0 0.0678 0.0234 0.1344
3-4-1 0.0856 0.0325 0.1700 5-1-1 0.0816 0.0304 0.1618
3-4-2 0.0984 0.0390 0.1982 5-1-2 0.0963 0.0379 0.1936
3-4-3 0.1118 0.0457 0.2307 5-1-3 0.1121 0.0459 0.2316
3-4-4 0.1258 0.0526 0.2695 5-1-4 0.1291 0.0542 0.2796
3-4-5 0.1405 0.0597 0.3184 5-1-5 0.1293 0.0304 0.3090
3-5-0 0.0913 0.0354 0.1825 5-2-0 0.0879 0.0337 0.1751
3-5-1 0.1055 0.0426 0.2150 5-2-1 0.1046 0.0421 0.2128
3-5-2 0.1204 0.0500 0.2538 5-2-2 0.1227 0.0511 0.2605
3-5-3 0.1362 0.0577 0.3029 5-2-3 0.1427 0.0608 0.3267
3-5-4 0.1529 0.0656 0.3715 5-2-4 0.1646 0.0710 0.4385
3-5-5 0.1707 0.0738 0.4795 5-2-5 0.1767 0.0503 0.5230
4-0-0 0.0381 0.0082 0.0809 5-3-0 0.1151 0.0474 0.2394
4-0-1 0.0461 0.0125 0.0942 5-3-1 0.1368 0.0580 0.3050
4-0-2 0.0563 0.0175 0.1126 5-3-2 0.1614 0.0695 0.4183
4-0-3 0.0668 0.0229 0.1323 5-3-3 0.1895 0.0821 0.5899
4-0-4 0.0777 0.0284 0.1537 5-3-4 0.2216 0.0957 0.7101
4-0-5 0.0890 0.0342 0.1773 5-3-5 0.2527 0.0814 0.7971
4-1-0 0.0484 0.0136 0.0983 5-4-0 0.1571 0.0676 0.3935
4-1-1 0.0592 0.0190 0.1181 5-4-1 0.1907 0.0826 0.5954
4-1-2 0.0705 0.0248 0.1395 5-4-2 0.2319 0.0999 0.7409
4-1-3 0.0822 0.0308 0.1631 5-4-3 0.2834 0.1196 0.8726
4-1-4 0.0945 0.0370 0.1894 5-4-4 0.3475 0.1417 1.0160
4-1-5 0.1072 0.0434 0.2193 5-4-5 0.4256 0.1437 1.1800
4-2-0 0.0626 0.0207 0.1244 5-5-0 0.2398 0.0762 0.7629
4-2-1 0.0748 0.0269 0.1479 5-5-1 0.3477 0.1172 1.0160
4-2-2 0.0875 0.0335 0.1742 5-5-2 0.5422 0.1791 1.4190
4-2-3 0.1009 0.0403 0.2041 5-5-3 0.9178 0.2672 2.2010
4-2-4 0.1150 0.0473 0.2392 5-5-4 1.6090 0.3837 4.1030
4-2-5 0.1299 0.0546 0.2820 5-5-5 >1.6090 0.3837 ----
28 (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 95% Confidence Limits Combination of 95% Confidence Limits

MPN Index MPN Index
Positives Lower Upper Positives Lower Upper
0-0-0 <0.018 --- 0.063 1-3-0 0.083 0.012 0.196
0-0-1 0.018 0.003 0.063 1-3-1 0.104 0.020 0.243
0-0-2 0.036 0.003 0.101 1-3-2 0.125 0.029 0.296
0-0-3 0.054 0.003 0.137 1-3-3 0.147 0.038 0.364
0-0-4 0.072 0.008 0.174 1-3-4 0.169 0.048 0.460
0-0-5 0.091 0.015 0.212 1-3-5 0.191 0.057 0.566
0-1-0 0.018 0.003 0.063 1-4-0 0.105 0.021 0.245
0-1-1 0.036 0.003 0.101 1-4-1 0.127 0.030 0.300
0-1-2 0.055 0.003 0.138 1-4-2 0.148 0.039 0.370
0-1-3 0.073 0.008 0.175 1-4-3 0.170 0.048 0.468
0-1-4 0.091 0.015 0.214 1-4-4 0.193 0.058 0.575
0-1-5 0.110 0.023 0.256 1-4-5 0.215 0.067 0.657
0-2-0 0.037 0.003 0.102 1-5-0 0.128 0.030 0.303
0-2-1 0.055 0.003 0.139 1-5-1 0.150 0.040 0.375
0-2-2 0.074 0.008 0.176 1-5-2 0.172 0.049 0.477
0-2-3 0.092 0.015 0.215 1-5-3 0.195 0.058 0.583
0-2-4 0.111 0.023 0.258 1-5-4 0.217 0.068 0.664
0-2-5 0.129 0.031 0.307 1-5-5 0.240 0.077 0.731
0-3-0 0.056 0.003 0.140 2-0-0 0.045 0.003 0.119
0-3-1 0.074 0.009 0.177 2-0-1 0.068 0.006 0.164
0-3-2 0.093 0.016 0.217 2-0-2 0.091 0.015 0.213
0-3-3 0.112 0.023 0.260 2-0-3 0.115 0.025 0.269
0-3-4 0.130 0.031 0.310 2-0-4 0.139 0.035 0.338
0-3-5 0.149 0.039 0.372 2-0-5 0.164 0.046 0.437
0-4-0 0.075 0.009 0.179 2-1-0 0.068 0.006 0.166
0-4-1 0.094 0.016 0.219 2-1-1 0.092 0.015 0.216
0-4-2 0.112 0.024 0.263 2-1-2 0.116 0.025 0.272
0-4-3 0.131 0.032 0.313 2-1-3 0.141 0.036 0.343
0-4-4 0.150 0.040 0.377 2-1-4 0.166 0.046 0.447
0-4-5 0.169 0.048 0.462 2-1-5 0.192 0.057 0.571
0-5-0 0.094 0.016 0.221 2-2-0 0.093 0.016 0.218
0-5-1 0.113 0.024 0.265 2-2-1 0.118 0.026 0.276
0-5-2 0.133 0.032 0.317 2-2-2 0.143 0.036 0.349
0-5-3 0.152 0.040 0.382 2-2-3 0.168 0.047 0.456
0-5-4 0.171 0.048 0.470 2-2-4 0.194 0.058 0.581
0-5-5 0.190 0.056 0.563 2-2-5 0.221 0.069 0.675
1-0-0 0.020 0.003 0.068 2-3-0 0.119 0.026 0.279
1-0-1 0.040 0.003 0.108 2-3-1 0.144 0.037 0.355
1-0-2 0.060 0.003 0.149 2-3-2 0.170 0.048 0.467
1-0-3 0.081 0.011 0.191 2-3-3 0.197 0.059 0.591
1-0-4 0.101 0.019 0.236 2-3-4 0.223 0.070 0.683
29
1-0-5 0.122 0.028 0.287 2-3-5 0.251 0.082 0.759
 Combination of 95% Confidence Limits Combination of 95% Confidence Limits
MPN Index MPN Index
Positives Lower Upper Positives Lower Upper
1-1-0 0.040 0.003 0.109 2-4-0 0.146 0.038 0.361
1-1-1 0.061 0.003 0.150 2-4-1 0.172 0.049 0.477
1-1-2 0.081 0.011 0.192 2-4-2 0.199 0.060 0.600
1-1-3 0.102 0.019 0.238 2-4-3 0.226 0.072 0.692
1-1-4 0.123 0.028 0.290 2-4-4 0.254 0.083 0.768
1-1-5 0.144 0.037 0.354 2-4-5 0.282 0.094 0.836
1-2-0 0.061 0.003 0.151 2-5-0 0.174 0.050 0.488
1-2-1 0.082 0.012 0.194 2-5-1 0.201 0.061 0.610
1-2-2 0.103 0.020 0.240 2-5-2 0.229 0.073 0.700
1-2-3 0.124 0.029 0.293 2-5-3 0.257 0.084 0.776
1-2-4 0.146 0.038 0.359 2-5-4 0.286 0.095 0.845
1-2-5 0.167 0.047 0.451 2-5-5 0.315 0.107 0.910
3-0-0 0.079 0.010 0.188 4-3-0 0.271 0.090 0.809
3-0-1 0.106 0.021 0.246 4-3-1 0.326 0.111 0.934
3-0-2 0.135 0.033 0.323 4-3-2 0.386 0.132 1.060
3-0-3 0.165 0.046 0.440 4-3-3 0.451 0.154 1.192
3-0-4 0.196 0.059 0.589 4-3-4 0.521 0.176 1.331
3-0-5 0.229 0.073 0.699 4-3-5 0.593 0.196 1.477
3-1-0 0.107 0.022 0.250 4-4-0 0.335 0.114 0.953
3-1-1 0.137 0.034 0.329 4-4-1 0.398 0.137 1.084
3-1-2 0.167 0.047 0.452 4-4-2 0.466 0.159 1.223
3-1-3 0.199 0.060 0.601 4-4-3 0.539 0.181 1.368
3-1-4 0.232 0.074 0.710 4-4-4 0.615 0.202 1.521
3-1-5 0.267 0.088 0.800 4-4-5 0.693 0.223 1.681
3-2-0 0.138 0.035 0.335 4-5-0 0.411 0.141 1.111
3-2-1 0.170 0.048 0.464 4-5-1 0.483 0.164 1.256
3-2-2 0.202 0.062 0.613 4-5-2 0.559 0.187 1.409
3-2-3 0.236 0.076 0.720 4-5-3 0.639 0.209 1.570
3-2-4 0.271 0.090 0.810 4-5-4 0.722 0.230 1.739
3-2-5 0.308 0.104 0.894 4-5-5 0.806 0.250 1.916
3-3-0 0.172 0.049 0.477 5-0-0 0.240 0.076 0.763
3-3-1 0.205 0.063 0.624 5-0-1 0.314 0.106 0.908
3-3-2 0.240 0.077 0.731 5-0-2 0.427 0.146 1.142
3-3-3 0.276 0.092 0.821 5-0-3 0.578 0.192 1.446
3-3-4 0.313 0.106 0.906 5-0-4 0.759 0.239 1.816
3-3-5 0.352 0.120 0.989 5-0-5 0.953 0.165 2.234
3-4-0 0.209 0.064 0.636 5-1-0 0.329 0.112 0.940
3-4-1 0.244 0.079 0.742 5-1-1 0.456 0.156 1.202
3-4-2 0.281 0.093 0.833 5-1-2 0.631 0.207 1.553
3-4-3 0.319 0.108 0.918 5-1-3 0.839 0.257 1.985
3-4-4 0.358 0.123 1.002 5-1-4 1.062 0.304 2.485
3-4-5 0.399 0.137 1.086 5-1-5 1.293 0.304 3.090

30
 Combination of 95% Confidence Limits Combination of 95% Confidence Limits
MPN Index MPN Index
Positives Lower Upper Positives Lower Upper
3-5-0 0.248 0.080 0.753 5-2-0 0.493 0.167 1.276
3-5-1 0.286 0.095 0.844 5-2-1 0.700 0.224 1.694
3-5-2 0.325 0.110 0.931 5-2-2 0.944 0.280 2.213
3-5-3 0.365 0.125 1.017 5-2-3 1.205 0.331 2.843
3-5-4 0.407 0.140 1.103 5-2-4 1.479 0.381 3.714
3-5-5 0.450 0.154 1.189 5-2-5 1.767 0.503 5.230
4-0-0 0.130 0.031 0.311 5-3-0 0.792 0.247 1.886
4-0-1 0.166 0.046 0.445 5-3-1 1.086 0.308 2.544
4-0-2 0.207 0.064 0.631 5-3-2 1.406 0.368 3.445
4-0-3 0.253 0.082 0.764 5-3-3 1.750 0.434 5.131
4-0-4 0.302 0.102 0.881 5-3-4 2.122 0.529 6.798
4-0-5 0.355 0.121 0.996 5-3-5 2.527 0.814 7.971
4-1-0 0.169 0.048 0.460 5-4-0 1.299 0.348 3.108
4-1-1 0.212 0.066 0.646 5-4-1 1.724 0.429 4.975
4-1-2 0.258 0.085 0.779 5-4-2 2.212 0.563 7.087
4-1-3 0.310 0.105 0.898 5-4-3 2.781 0.882 8.600
4-1-4 0.365 0.125 1.016 5-4-4 3.454 1.159 10.110
4-1-5 0.425 0.145 1.138 5-4-5 4.256 1.437 11.800
4-2-0 0.216 0.067 0.661 5-5-0 2.398 0.762 7.629
4-2-1 0.264 0.087 0.794 5-5-1 3.477 1.172 10.160
4-2-2 0.317 0.108 0.915 5-5-2 5.422 1.791 14.190
4-2-3 0.375 0.129 1.037 5-5-3 9.178 2.672 22.010
4-2-4 0.438 0.150 1.164 5-5-4 16.090 3.837 41.030
4-2-5 0.504 0.171 1.297 5-5-5 >16.090 3.837 -----
(1)
Table was developed using the MPN calculator developed by Albert Klee (Reference 15.10).

31
32
 12.0
Protocol Performance

Culture-based procedures were evaluated for V. cholerae O1 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 O1 and O139 in Spiked PBS, Drinking Water, and Surface Water
Samples(1)

Pre- Mean Minimum Maximum

Spike
Analyte Sample Location Enrichment Recovery Recovery Recovery SD (%) RSD (%)
level
in APW (%) (%) (%)

Phosphate Buffered Saline

O1 NA 129 6 hours 57.20 57.20 57.20 NA NA

O139 NA 253 6 hours 94.18 94.18 94.18 NA NA

Drinking Water

O1 NA 36 24 hours 84.91 25.25 125.39 49.30 58.06

O139 NA 34 24 hours 163.23 67.35 320.06 109.00 66.78

Surface Water

O1 129 6 hours 25.52 16.26 30.79 6.36 24.94

Site 1
O1 129 24 hours 152.33 69.97 355.93 137.27 90.11

O1 129 6 hours 13.53 4.89 30.79 12.21 90.27

Site 2
O1 129 24 hours 52.86 13.99 113.47 48.26 91.31

O139 Site 3 253 6 hours 88.62 35.62 181.17 66.75 75.32

O139 Site 4 253 6 hours 119.46 57.76 181.17 71.25 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

33
34
 13.0
Pollution Prevention

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.

35
36
 14.0
Waste Management

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 have viable V. cholerae O1 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.

37
38
 15.0
References

15.1 National Center for Infectious Diseases, Centers for Disease Control and Prevention. 1999. “Isolation and
Identification of Vibrio cholerae Serogroups O1 and O139.” In Laboratory Methods for the Diagnosis of
Epidemic Dysentery and Cholera. J.P. Koplan, J.M. Hughes, M.L. Cohen, E.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. and Depaola, A., Jr. May 2004. “Vibrio.” In Bacteriological Analytical Manual Online. G.J.
Jackson, R.I. Merker, and R. Bandler. (eds.) U.S. Food and Drug Administration. http://www.fda.gov/Food/
ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070830.htm
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 O1 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 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. NIST Measurement 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 O1 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.

39
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 16.1 Quantitative Analysis Dilution Scheme

Flowcharts
16.0

41
42
16.2 Identification Flowchart
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