SEPA
EPA/600/R-10/056 | September 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Standard Analytical Protocol
for Escherichia coli 0157:H7
in Water
Office of Research and Development
National Homeland Security Research Center

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Standard Analytical Protocol
for Escherichia coli 0157:H7
in Water
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HOMELAND SECURITY RESEARCH
CENTER
CINCINNATI, OH 45268
Office of Research and Development
National Homeland Security Research Center

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Acknowledgments
This Standard Analytical Protocol is based on procedures evaluated by BioVir Laboratories. Inc.. under direction of
the National Homeland Security Research Center within the U.S. Environmental Protection Agency's (EPAs) Office
of Research and Development under the direction of Sanjiv R. Shah for analysis of E. coli 0157:H7 in water samples.
Technical support and data evaluation were provided by Computer Sciences Corporation under EPA Contract EP-C-
05-045.
The contributions of the following persons and organizations arc gratefully acknowledged:
Study Workgroup Participants
•	Ayaad Assaad (EPA, Office of Pesticide Programs)
•	Michele Burgess and Marissa Mull ins (EPA, Office of Emergency Management)
•	Stephanie Harris (EPA, Region 10)
•	Malik Ray nor (EPA, Office of Water, Water Security Division)
•	James Sinclair (EPA, Office of Water, Office of Ground Water and Drinking Water)
Subject Matter Experts
•	Cheryl Bopp (Centers for Disease Control and Prevention)
•	Diana Cantrell and Robert Mandrell (U.S. Department of Agriculture)
•	Victor Cook, Bill Cray, Mike Grant, and Steve Weagant (U.S. Food and Drug Administration)
Volunteer Participant Laboratory
•	Rick Danielson. Rosie Newton, and JimTruscott (BioVir Laboratories, Inc.)

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Disclaimer
This document has been reviewed in accordance with EPA policy and approved for publication. Mention of trade
names or commercial products docs 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 arc intended for use in laboratories when analyzing environmental
samples in support of remediation efforts following a homeland security incident. The (culture-based) procedures
provide viability determination and identification as either qualitative or quantitative results. The sample preparation
procedures arc deemed the most appropriate for the wide variety of matrices to be examined. To the extent possible,
these procedures were developed to be consistent with other federal agency procedures. These procedures do
not include the sample collection, rapid screening, field techniques, or molecular techniques that may accompany
laboratory analysis.
Questions concerning this document or its application should be addressed to:
Sanjiv R. Shah
National Homeland Security Research Center
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NVV
USEPA-8801RR
Washington. DC 20460
(202) 564-9522
shah. saniiv@epa. gov
If you have difficulty accessing this PDF document, please contact Kathy Nickel (Nickel.Kathv@epa.gov) or Amelia
McCall (McCall.Amelia@epa.gov) for assistance.

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Contents
Acknowledgments	iii
Disclaimer	iv
I.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	19
9.0 Quality Control	21
9.1	General	21
9.2	Negative Controls	21
9.3	Positive Controls	21
9.4	Method Blank	21
9.5	Media Sterility Check	22
10.0 Procedures	23
10.1	Qualitative Sample Analysis	23
10.2	Quantitative Sample Analyses	23
10.3	Immunomagnetic Separation and Concentration	23
10.4	Isolation on Selective Agars	24
10.5	Serological Analyses	24
10.6	Isolation on TSA Plates	24
10.7	Biochemical Analyses	24
10.8	Summary of Positive and Negative Control, and /•'. coli 0157:H7 Results	25
II.0	Data Analy sis and Bacterial Density Calculation	27
11.1	Most Probable Number (MPN) Technique	27
11.2	Calculation of MPN	27
12.0 Protocol Performance	33
13.0 Pollution Prevention	35
14.0 Waste Management	37
V

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15.0 References	
16.0 Flowcharts and Diagrams.
vi

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1.0
Scope and Application
1.1
This Standard Analytical Protocol (SAP) is for the identification, confirmation, and enumeration of Escherichia
coli 0157:H7 (E. coli 0157:H7) 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 journal article "Evaluation of Techniques for Enrichment and Isolation of
Escherichia coli 0157:H7 from Artificially Contaminated Sprouts" by Weagant and Bound (Reference 15.1), and is
for use by laboratories when analyzing samples in support of U.S. Environmental Protection Agency (EPA) homeland
security efforts.
1.3
E. coli 0157:H7 is the causative agent of entcroheinorrhagic diarrhea. 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 (Reference 15.2). Use of a biological safety cabinet (BSC) is
recommended for any aerosol-generating procedures.
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 procedii
1.5
This protocol is not intended for use as a test for microorganisms other than E. coli 0157:H7 and the matrices
described.
1

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2

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2.0
Summary of Protocol
2.1
E. coli 0157:H7 can be identified in a variety of water samples using selective media, and biochemical and serological
analyses. Bacterial densities can be estimated using the most probable number (MPN) approach.
2.2
For qualitative results, samples arc diluted 1:1 in double-strength modified buffered peptone water (mBPVV). Samples
arc incubated at 36.0°C ± 1.0°C for 2 - 2.5 hours followed by incubation at 42.0°C ± 0.5°C for a total of 20 - 24
hours.
2.3
For quantitative results, samples arc analy/cd as received. All samples arc analy/cd using the MPN technique in
mBPVV and incubated at 36.0°C ± 1.0°C for 2 - 2.5 hours followed by incubation at 42.0°C ± 0.5°C for a total of 20 -
24 hours.
2.4
Broth cultures (MPN and qualitative analyses tubes) with positive growth (turbidity) arc submitted to
immunomagnetic separation (IMS) and sub-cultured onto tellurite cefixime sorbitol MacConkcy (TC-SMAC) and
Rainbow® agars.
2.5
After growth at 42.0°C ± 0.5°C for 18 - 24 hours, TC-SMAC plates arc examined for 2 - 3 mm colorlcss/gray
colonics typical of E. coli 0157:H7. On Rainbow® plates, typical colonics arc black/gray. Isolated typical colonics
arc submitted to biochemical and serological confirmation. Serological confirmation is by agglutination using E. coli
0157 and H7 antiserum, followed by biochemical characterization using commercially available test strips (e.g., API
20E® or equivalent) or with a group of selected individual biochemical tests.
2.6
Broth cultures (MPN and qualitative analysis tubes) concentrated by IMS may be subjected to real-time polymerase
chain reaction (PCR) confirmation in place of biochemical and serological confirmation.
2.7
Quantification of E. coli 0157:H7 is determined using the MPN technique (Flowchart 16.1). Tubes that arc confirmed
positive for /•'. coli 0157:H7 arc used to determine MPN.
Note: Typing by serology for the H7 antigen or PCR for the H7 gene was not conducted during the single-laboratory
verification study. To confirm E. coli 0157:H7, additional serological or PCR analyses would be necessary.
3

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3.0
Acronyms and Abbreviations
ASTM
ASTM International (formerly American Society for Testing and Materials)
ATCC®
American Type Culture Collection
BSC
Biological safety cabinet
BSL
Biological safety level
°C
Degrees Celsius
CT
Cefixime potassium tellurite
EPA
U.S. Environmental Protection Agency
g
Gram(s)
IMS
Immunomagnetic separation
L
Liter(s)
liL
Microlitcr(s)
\im
Micrometer(s)
mBPW
Modified buffered peptone water
mg
Milligrain(s)
mL
Milliliter! s)
mm
Millimcter(s)
MPN
Most probable number
N
Normal
NCTC
National Collection of Type Cultures
NHSRC
National Homeland Security Research Center
NIST
National Institute of Standards and Technology
PBS
Phosphate buffered saline
PGR
Polymerase chain reaction
PPE
Personal protective equipment, i.e., gloves, goggles, laboratory coat, etc.
psi
Pounds per square inch
QC
Quality control
SAP
Standard Analytical Protocol
TC-SMAC
Tellurite cefixime sorbitol MacConkey agar
TSA
Tryptic soy agar
w/v
Weight to volume ratio
5

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6

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4.0
Interferences and Contamination
4.1
Low recoveries of E. coli 0157:H7 may be caused by the presence of either high numbers of competing or inhibitory
organisms (e.g., other Entcrobactcriaceac). or toxic substances (e.g., metals, organic compounds).
4.2
A viable but non-culturable state of E. coli 0157:H7 may also account for lower recoveries (Reference 15.3).

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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, accidental parenteral inoculation, and rarely, exposure to infectious aerosols
are the primary hazards to laboratory personnel. Staff should apply safety procedures used for pathogens when
handling all samples.
5.2	Recommended Precautions
5.2.1
E. coli 0157:H7 is a BSL-2 pathogen and all procedures should be performed in laboratories that use, at a minimum,
BSL-2 practices (Reference 15.2). This includes prohibiting eating, drinking, smoking, handling contact lenses,
applying cosmetics, and storing food and drink in the laboratory.
5.2.2
A Class 11 BSC is recommended for sample manipulations where the risk of aerosol production is high. Production of
aerosols should be avoided.
5.2.3
Disposable materials arc 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. These
procedures must be followed while preparing, using, and disposing of media, cultures, reagents, and materials, and
while operating 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
Protective 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 protocol docs not address all safety issues associated with its use. Please refer to Biosafety in Microbiological
and Biomedical Laboratories (BMBL), 5th Edition (Reference 15.2) 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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10

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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, analytical, with ASTM Class S reference weights, capable of weighing 20 g ± 0.001 g
6.4
Balance, top loading, with Class S reference weights, capable of weighing 100 g ± 0.1 g
6.5
Bead rotator (Dynabeads® Sample Mixer or equivalent), optional
6.6
Beakers, glass or plastic (assorted sizes)
6.7
Biological safety cabinet. Class 11 (optional)
6.8
Borosilicate glass or plastic screw-cap, wide-mouth bottles, sterile (e.g., 250 inL)
6.9
Borosilicate glass culture tubes, with autoclavable screw or snap caps (25 x 150 mm)
6.10
Erlcnincyer flasks (500 inL. I L, 2 L)
6.11
Filters, for reagent sterilization, sterile (0.22 jim pore size), and filter syringes, sterile
6.12
Graduated cylinders (assorted sizes)
6.13
Gloves, sterile, nitrile or equivalent
6.14
lininunomagnetic separator (Dynal® Bead Separator or equivalent)
6.15
Incubators, microbiological type, maintained at 36.0°C ± 1.0°C and 42.0°C ± 0.5°C
6.16
Inoculation loops, sterile, disposable

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6.17
Parafilm™ or equivalent
6.18
Petri dishes, sterile, plastic (15 x 100 mm)
6.19
pH meter
6.20
Pipettes, standard tip. sterile, plastic, disposable (assorted si/cs)
6.21
Pipetting device (automatic or equivalent)
6.22
Stirring hotplates and stir bars
6.23
Test tube racks
6.24
Thermometer, National Institute of Standards and Technology (NIST)-traccable
6.25
Tissues, lint-free (Kimwipes® or equivalent)
6.26
Vortex
6.27
Waterbath, maintained at 45°C - 50°C
6.28
Weigh paper and boats

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7.0
Reagents and Standards
7.1
Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical Society (Reference 15.4). For
suggestions regarding the testing of reagents not listed by the American Chemical Society, see AnalaR Standards for
Laboratory Chemicals (Reference 15.5) and United States Pharmacopeia and National Formulary 24 (Reference
15.6).
7.2
Whenever possible, use commercially available culture media. The agar used in the preparation of culture media must
be of microbiological grade. Storage requirements for prepared media and reagents are provided in Table 2 (Section
7.18).
7.3
Reagent-grade water must conform to specifications in Standard Methods for the Examination of Water and
Wastewater, 21" Edition, Section 9020 (Reference 15.7).
7.4	Dilution Water
Dilution water is a phosphate buffer and is also commonly referred to as Butterfield buffer or phosphate buffered
dilution water. Commercially prepared reagents are recommended (Hardy Diagnostics D699 or equivalent).
7.4.1
Composition of stock phosphate buffer solution:
Monopotassium phosphate (KH2P04)
Reagent-grade water
7.4.2
Prepare stock phosphate buffer by dissolving 34.0 g monopotassium phosphate in 0.5 L of reagent-grade water. Adjust
pH to 7.2 ± 0.5 with 1 N sodium hydroxide and dilute to 1.0 L with reagent-grade water. Sterilize by filtration or
autoclave at 121 °C (15 psi) for 15 minutes.
7.4.3
Composition of stock magnesium chloride solution:
Magnesium chloride hcxahydrate (MgCl;«6H,0)
Reagent-grade water
7.4.4
Prepare stock magnesium chloride solution by dissolving 81.1 g of magnesium chloride hcxahydrate in 1.0 L of
reagent-grade water. Sterilize by filtration or autoclave at 121 °C (15 psi) for 15 minutes.
7.4.5
After sterilization, store the stock solutions in the refrigerator. Handle aseptically. If evidence of mold or other
contamination appears, the affected stock solution should be discarded and a fresh solution should be prepared.
7.4.6
Prepare phosphate buffered dilution water by adding 1.25 inL stock phosphate buffer solution and 5.0 inL stock
magnesium chloride solution to 1.0 L of reagent-grade water. Autoclave at 121 °C (15 psi) for 15 minutes.
7.5	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.
13
34.0 g
0.5 L
81.1 g
1.0 L

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7.5.1
Composition:
Monosodium phosphate (NaH2P04)	0.58 g
Diosodium phosphate (Na2HP04)	2.50 g
Sodium chloride	8.50 g
Reagent-grade water	1.0 L
7.5.2
Dissolve reagents in 1.0 L reagent-grade water, adjust pH to 7.4 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide, and dispense appropriate volumes in screw-cap bottles or tubes and autoclave at 121 °C (15 psi) for 15
minutes.
7.6 Modified Buffered Peptone Water (mBPW)
Prepare IX, 2X, and 5X mBPW 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.
7.6.1
Composition:
IX	2X	5X
Peptone	10.0 g	20.0 g	50.0 g
Sodium chloride	5.0 g	10.0 g	25.0 g
Disodium phosphate (Na2HP04)	3.6 g	7.2 g	18.0 g
Monopotassium phosphate	1.5 g	3.0 g	7.5 g
(KH2P04)
Casamino acids	5.0 g	10.0 g	25.0 g
Yeast extract	6.0 g	12.0 g	30.0 g
Lactose	10.0 g	20.0 g	50.0 g
Pyruvate	2.0 g	4.0 g	10.0 g
Reagent-grade water	1.0 L	1.0 L	1.0 L
7.6.2
Add reagents to 950 niL of reagent-grade water and mix thoroughly using a stir bar. Adjust pH to 7.2 ± 0.2 with 1.0
N hydrochloric acid or 1.0 N sodium hydroxide and bring up to 1.0 L. Dispense 10 niL (IX and 2X) or 5 niL (5X)
aliquots in 25 x 150 mm tubes and autoclave at 121 °C for 15 minutes. Cool to room temperature.
7.7 Tellurite Cefixime Sorbitol MacConkey Agar (TC-SMAC)
Commercially prepared media is recommended. Dehydrated medium (Oxoid CM0813 or equivalent), with cefixime
and potassium tellurite (CT) supplement (Oxoid SR172 or equivalent), or prepared plates (Oxoid P00702 or
equivalent) may be used. If commercially prepared media are not available, prepare medium using procedures in
Sections 7.7.1 through 7.7.4.
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7.7.1
Composition:
Peptone
20.0 g
D-Sorbitol
10.0 g
Bile salts No. 3
1.5 g
Sodium chloride
5.0 g
Agar
15.0 g
Neutral red
0.03 g
Crystal violet
0.001 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. Boil for one minute
with rapid stir bar agitation to dissolve completely. Adjust pH to 7.1 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N
sodium hydroxide and bring up to 1.0 L. Autoclave at 121 °C (15 psi) for 15 minutes. Cool to 45°C - 50°C in a
waterbath.
7.7.3
Prepare CT supplement according to manufacturer's instructions and filter stcrili/c.
7.7.4
Prepare TC-SMAC plates by adding two reconstituted CT supplement vials to the cooled medium, swirling to mix.
Final concentrations for cefixime and tellurite are 0.05 mg/L and 2.5 mg/L, respectively. Aseptically pour 12- 15 niL
into each 15 x 100 mm sterile Petri plate.
7.8	Rainbow® Agar
Commercially prepared dehydrated medium (Biolog 80101 or equivalent) is recommended.
7.8.1
Add 60.0 g dehydrated reagent to 1.0 L of reagent-grade water and mix thoroughly using a stir bar and hot plate. Boil
gently to dissolve and autoclave at 121 °C (15 psi) for 15 minutes. Cool to 45°C - 50°C in a waterbath.
7.8.2
Prepare stock potassium tellurite (Sigma-Aldrich® P0677 or equivalent) solution by dissolving 0.008 g in 10.0
inL reagent-grade water and filter sterilizing. Prepare stock novobiocin (Sigma-Aldrich® N1628 or equivalent) by
dissolving 0.010 g in 10 niL reagent-grade water and filter sterilizing.
7.8.3
Prepare plates by adding 1.0 inL each of tellurite and novobiocin stock solutions to the cooled medium, swirling to
mix. Final concentrations should be 0.8mg/L and 10.0 mg/L, respectively, with medium pH of 7.9 - 8.3. Aseptically
pour 12- 15 inL into each 15 x 100 mm sterile Petri plate.
7.9	Tryptic Soy Agar (ISA)
Commercially prepared inediuin is recommended. Prepared plates (BBL™ 221803 or equivalent) or dehydrated
media (BBL™ 211043 or equivalent) may be used. If coininercially prepared media are not available, prepare media
using procedures in Sections 7.9.1 and 7.9.2.
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7.9.1
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
7.9.2
Add reagents to 950 niL 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 1.0 L.
Autoclave at 121 °C (15 psi) for 15 minutes and cool to 45°C - 50°C in a waterbath. Ascptically pour 12 - 15 niL into
each 15 x 100 mm sterile Petri plate.
7.10
E. coli 0157 IMS beads (Invitogen™ 71004 or equivalent)
7.11
IMS wash buffer: Add 50 jiL Tween® 20 to 100 niL of PBS. ensuring all of the Tween® 20 is expelled from the pipette
tip; filter stcrili/c or autoclave at 121 °C (15 psi) for 15 minutes.
7.12
Saline, physiological (0.85% w/v): Dissolve 0.85 g NaCl in 100 niL of reagent-grade water. Autoclave at 121 °C (15
psi) for 15 minutes. Cool to room temperature.
7.13
E. coli 0157 latex agglutination reagent (Oxoid DR0620M or equivalent)
7.14
E. coli H7 latex agglutination reagent (Wellcolex® E. coli 0157:H7 Rapid Latex Agglutination Test or equivalent)
7.15
Biochemical test strip (bioMcrieux API 20E® or equivalent)
7.16
Oxidase reagent (BD™ DrySlide™ 231746 or equivalent)
7.17
Positive and negative control cultures that are to be used in the procedures in Section 10.0 are listed in Table 1, below.
Use of these controls is discussed in Section 9.0.

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Table 1. Positive and Negative Control Cultures for Described Tests
Media/Tests
Positive Control
Negative Control
TC-SMAC
E. coli 0157:H7
(ATCC® 700728™ /NCTC 12900)
E. coli
(ATCC® 25922™)
Rainbow®
E. coli 0157:H7
(ATCC® 700728™ / NCTC 12900)
E. coli
(ATCC® 25922™)
0157 latex agglutination
E. coli 0157:H7
(ATCC® 700728™/NCTC 12900)
E. coli
(ATCC® 25922™)
H7 serum agglutination (Optional)
E. coli 0157:H7
(ATCC® 700728™ / NCTC 12900)
E. coli
(ATCC® 25 922™)
Biochemical test strip
E. coli 0157:H7
(ATCC® 700728™ / NCTC 12900)
Pseudomonas aeruginosa
(ATCC® 27853™)
Oxidase
Pseudomonas aeruginosa
(ATCC® 27853™)
E. coli 0157:H7
(ATCC® 700728™ / NCTC 12900)
7.18
Storage temperatures and times for prepared media and reagents arc 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 Reagents1
Media/Reagents
Storage Temperature
Storage Time
Dilution water, PBS, saline
(in screw-cap containers)
<10°C and above freezing
3 months
Tubes: mBPW
<10°C and above freezing
2	weeks in loose-cap tubes
3	weeks in screw-cap tubes
Plates: TC-SMAC, Rainbow®2, 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
2	Dehydrated medium is hygroscopic and light-sensitive; store tightly sealed and protected from light

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18

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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 protocol. Record daily measurements in an incubator log book.
8.2
Check temperature in rcfrigcrators/frcc/ers at least once daily to ensure operation is within stated limits of the
protocol. Record daily measurements in a refrigcrator/frcc/cr log book.
8.3
Calibrate thermometers and incubators annually against a NIST-ccrtified thermometer or against a thermometer that
meets the requirements of NIST Monograph SP 250-23 (Reference 15.8). 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 Class S reference weights once per month, at a minimum.
Check each day prior to use with Class S weights.
8.6
Calibrate micropipettors once per year. Spot-check micropipettor accuracy once per month by weighing a measured
amount of reagent-grade water (1 jj.L = 1 mg).
8.7
Re-certify BSCs once per year. Re-certification must be performed by a qualified technician.
19

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20

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9.0
Quality Control
9.1	General
Each laboratory that uses this protocol is required to operate a formal quality assurance program that addresses and
documents instrument and equipment maintenance and performance, reagent quality and performance, analyst training
and certification, and records storage and retrieval. Specific quality control (QC) procedures for use with this protocol
arc discussed below.
Note: Following testing and validation, this protocol 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 arc 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 arc analyzed. Recommended negative control
organisms arc provided in Table 1 (Section 7.17), and descriptions of negative results arc provided in Table 3 (Section
10.8).
9.2.2
Analysis of negative controls is conducted by inoculating media and performing biochemical 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 the respective negative control organism, and re-analyze the appropriate negative control and corresponding
sample(s).
9.2.4
Viability of the negative controls should be demonstrated on a monthly basis, at a minimum, using a non-selective
media (e.g., TSA).
9.3	Positive Controls
9.3.1
The laboratory should analyze positive controls to ensure that all media and reagents arc 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 arc
provided in Table 1 (Section 7.17), and descriptions of positive results arc provided in Table 3 (Section 10.8).
9.3.2
Analysis of positive controls is conducted by inoculating media and performing biochemical 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, check and/or replace the associated media, reagents, and/
or the positive control organism, and rc-analy/c the appropriate positive control and corresponding sample(s).
9.4	Method Blank
On an ongoing basis, every day that samples arc analyzed, the laboratory should analyze a method blank using sterile
dilution water or PBS (Sections 7.4 and 7.5, respectively) to verify the sterility of equipment, materials, and supplies.
The method blank is treated as a sample and submitted to the same analytical procedures. Absence of growth indicates
freedom from contamination.
21

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9.5 Media Sterility Check
Test sterility of dilution water, PBS. mBPW, and TSA by incubating one unit (tube or plate) from each batch at 35.0°C
- 37.0°C for 24 ± 2 hours and observing for growth. Test sterility of TC-SMAC and Rainbow* by incubating one
unit (tube or plate) from each batch at 42.0°C ± 0.5°C for 24 ± 2 hours and observing for growth. Absence of growth
indicates the media arc sterile. On an ongoing basis, a media sterility check should be done every day that samples arc
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 the start of sample processing, which should be completed within two hours. E. coli 0157:H7 is a pathogen,
and all samples should be handled with caution, using appropriate BSL-2 procedures and PPE. A Class 11 BSC is
recommended for sample manipulations where the risk of aerosol production is high, such as vigorous shaking.
10.1	Qualitative Sample Analysis
Add a sample volume (e.g., 200 inL) to an equal volume of double-strength mBPVV (Section 7.6). Incubate at 36.0°C
± 1.0°C for 2.0 - 2.5 hours, non-shaking. Transfer samples to 42.0°C ± 0.5°C and continue incubation, non-shaking,
for a total of 20 - 24 hours. After incubation, proceed to Section 10.4 for selective isolation of E. coli 0157:H7.
10.2	Quantitative Sample Analyses
A multiple-tube assay incorporating differential sample volumes is used to estimate E. coli 0157:H7 densities in
undiluted or diluted samples. If low levels of E. coli 0157:H7 are suspected, larger sample volumes (20.0 inL of
original sample) should be used to inoculate the first row of tubes in the series. If high levels of E. coli 0157:H7 are
suspected, additional serial dilutions should be used. See Flowchart 16.1 for an overv iew of the sample dilution and
inoculation scheme. A minimum sample volume of 156 inL is required if 20 inL volumes arc used to inoculate the
first row of tubes.
10.2.1	Sample inoculation
Arrange mBPVV tubes in three rows (5 inL of 5X, 10 inL of 2X, and 10 inL of IX) of five tubes each. Inoculate the
first row of tubes (5 inL of 5X mBPVV) with 20 inL of the undiluted sample. Inoculate 10 inL of the undiluted sample
into each of the tubes in the second row (5 inL of 3X mBPVV). Inoculate 1 inL from the initial sample into each of the
tubes in the third row (IX mBPVV). 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 E. coli 0157:H7. If analyzing serially
diluted samples, 1.0 inL of each dilution will be used to inoculate each tube of IX mBPVV, as appropriate.
10.2.3	Growth
Incubate tubes at 36.0°C ± 1.0°C for 2.0 - 2.5 hours, non-shaking. Transfer samples to 42.0°C ± 0.5°C and continue
incubation, non-shaking, for a total of 20 - 24 hours. After incubation, proceed to Section 10.3 for selective separation
and concentration of E. coli 0157:H7.
10.3	Immunomagnetic Separation and Concentration
See Flowchart 16.2 for an overview of the colony identification procedures. From each tube with growth, conduct
IMS as follows:
10.3.1
Suspend E. coli 0157:H7 beads by vortexing and add 20 pL of the bead suspension to a sterile. 1.5 inL - 2.0 inL tube.
10.3.2
Gently swirl contents of mBPVV tubes to mix, remove 1.0 inL of mBPVV culture, add to tube with beads, and tightly
cap. Mix for 10 minutes at room temperature using the tube rotator.
10.3.3
Place tubes in magnetic holder, inverting tubes several times to ensure that beads arc concentrated in a pellet on the
side of the tube; allow beads to settle for three minutes. Carefully aspirate liquid and discard.
10.3.4
Resuspcnd beads in l.O inL IMS wash buffer (Section 7.11) by inverting the tube several times. Place tubes in
magnetic holder and allow beads to settle for three minutes. Aspirate wash buffer and resuspcnd the beads in 100 pL
of IMS buffer.
23

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Note: IMS bead complex may be subjected to real-time PCR confirmation in place of, or in addition to, culture on
selective media followed by biochemical and serological confirmation.
10.4	Isolation on Selective Agars
10.4.1
Pipette 50 pL (half the beads) onto Rainbow® agar; use a sterile loop to spread the 50-^iL aliquot over one-third of the
plate then streak for isolation on the remaining portion of the plate. Repeat this procedure with the remaining 50 pL
onto TC-SMAC.
10.4.2
Incubate Rainbow® and TC-SMAC plates for 18 - 24 hours at 42.0°C ± 0.5°C. On TC-SMAC, typical E. coli
0157:H7 colonics arc colorless and 2-3 mm in diameter. On Rainbow® agar, typical E. coli 0157:H7 colonics arc
black/gray.
Note: Colonies on Rainbow® agar tend to be much smaller and run together making it difficult to obtain well-isolated
colonies for serological and biochemical confirmation. Prolonged incubation of TC-SMAC plates may result in
colonies ofE. coli 0157:H7 losing their characteristic colorless appearance. Also, the color of sorbitol-positive
colonies can fade, making them difficult to distinguish from sorbitol-negative colonies. Appropriate negative and
positive controls should be analyzed to avoid confusion (Sections 9.2 and 9.3, respectively).
10.5	Serological Analyses
The procedures for the use of latex agglutination kits arc provided in Sections 10.5.1 and 10.5.2. Section 10.5.3
provides an optional procedure for the use of antiserum for serological confirmation.
10.5.1
E. coli 0157 latex agglutination kit: Choose a well-isolated typical colony from each of the positive TC-SMAC and/
or Rainbow® plates and emulsify growth using sterile physiological saline (Section 7.12). Evaluate according to
manufacturer's instructions.
10.5.2
Repeat Section 10.5.1 using H7 latex agglutination reagent.
Note: Identification of the H7flagellar antigen is usually conducted by reference laboratories and may be difficult
since isolates often require multiple passages in motility medium before the antigen is detected. PCR identification of
the gene for the H7 antigen may be conducted in place of serology. Neither of these procedures was verified during
the single-laboratory study.
10.5.3
E. coli 0157 or H7 antiserum (optional): Choose a well-isolated typical colony from each of the positive TC-SMAC
and/or Rainbow® 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 0157 antiserum. To the second drop of
emulsified growth, add one drop of sterile saline (as a visual comparison).
Observe under magnification for an agglutination reaction, which indicates a positive result. E. coli 0157:H7 is
agglutination-positive for 0157 antiserum. Results should be compared with those for positive and negative controls
(Table 1) analyzed at the same time.
10.6	Isolation on TSA Plates
10.6.1
Streak one 0157 scralogical-positivc isolate onto a TSA plate from each serology-positive TC-SMAC or Rainbow®
plate. For spiked samples with no background, a single scralogical-positivc isolate from each dilution should be
streaked onto a TSA plate. Incubate the plates at 36.0°C ± 1.0°C for 24 ± 2 hours.
10.6.2
Seal the TC-SMAC and Rainbow® plates with Parafilm™ and store at <10°C and above freezing for use as backup/
archive plates. Use the TSA plates for biochemical analyses.
10.7	Biochemical Analyses
Use a single, isolated, large colony (2-3 mm diameter) from each TSA plate for biochemical test strip and oxidase
analyses.

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10.7.1	Oxidase Test(BD™ DrySlide™ 231746 or equivalent)
Following manufacturer's instructions, transfer a small amount of growth from an isolated colony to the slide.
Oxidasc-positivc bacteria turn the reagent dark purple within 20 seconds. E. coli 0157:H7 is oxidasc-ncgativc.
Results should be compared with those for positive and negative controls (Table 3) analyzed at the same time.
10.7.2	Biochemical Test Strips (API 20E® or equivalent)
Emulsify the remainder of the colony in 0.85% NaCl. Follow manufacturers' instructions to inoculate wells and add
appropriate reagents. Incubate test strip according to manufacturers' instructions. Add additional reagents, read, and
record results.
Note: Individual biochemical tests may be used instead of biochemical test strips to identify E. coli 0157 :H7.
10.8 Summary of Positive and Negative Control, and E. coli 0157:H7 Results
Typical results are provided in Table 3.
Table 3. Positive and Negative Result Descriptions and K coli 0157:H7 Results
Medium/Test
K. coli 0157:H7
Results
Positive Control Result and
Description
Negative Control Result and
Description
TC-SMAC
Positive
Colorless colonies
(sorbitol not fermented)
Pink to red colonies (sorbitol
fermented)
Rainbow®
Positive
Black to gray colonies
(glucoronidase-negative)
Pink to magenta colonies
(glucoronidase-positive)
Oxidase
Negative
Purple to violet color change
within 20 seconds
Colorless or very light pink color
change over time
Biochemical test strip
Consult manufacturers' instructions
0157 antiserum
Positive
Agglutination
No agglutination
H7 antiserum (Optional)
Positive
Agglutination
No agglutination
25

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26

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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 /•'. coli 0157:H7 by
biochemical and serological or by PGR confirmation.
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 analy/cd. 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 (Sections 11.2.1 and 11.2.2).
11.2.1 Selection of Tubes
If more than three rows of tubes arc inoculated with sample (e.g., volumes/dilutions), select the most appropriate rows
of tubes according to the criteria listed in Sections 11.2. l .l to 11.2.1.5. Examples of row selections and MPN/1(H) mL
values arc provided in Table 4.
11.2.1.1
Choose the smallest volume or the highest dilution giving positive results in all five tubes inoculated plus the
two succeeding lower concentrations. In Example A from Table 4, 10 mL is a smaller volume than 20 mL and
is the lowest volume giving positive results in all five tubes.
11.2.1.2
If the largest volume tested has less than five tubes with positive results, select it and the next two smaller
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 unsclected 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 mL1
Example
20 mL
10 mL
1.0 mL
0.1 mL
Significant
Dilutions
Table
MPN Index
MPN/100 mL
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
2/5
5-5-2
6
5.422
542.2
1 Appropriate volumes are underlined and the largest sample volumes analyzed are highlighted
27

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11.2.2
For calculation of MPN/100 mL when additional dilutions arc analyzed (e.g., 102, 103), obtain the MPN index value
from Table 6 using the number of positive tubes in the three selected dilutions. Calculate MPN/100 mL using the
equation below.
MPN Index from Table 6
MPN/100 mL = 	 x 100
Middle volume analyzed in the scries used for MPN
determination
For example, a dilution series of 103, 10 4, 10 5, with the following positive tubes 5, 1, 0, respectively would be:
0.329
MPN /100 mL = 	 x 100 = 3.29 x 105
10"4
28

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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 mL1
Combination of
Positives
MPN Index
95% Confidence Limits
Combination of
Positives
MPN Index
95% Confidence Limits
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
] -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
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
0.0/89
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
0.079:
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

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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 (cont.)1
Combination of
Positives
MPN Index
95% Confidence Limits
Combination of
Positives
MPN Index
95% Confidence Limits
Lower
Upper
Lower
Upper
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
.1-0-2
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
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
0.11 50
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. i 450
5-1-0
0.0678
0.0234
0.1344
3-4-1
0.0856
0.0325
0.1 700
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-i
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.-!
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

1.6090
0.3837
....
^able was developed using the MPN calculator developed by Albert Klee (Reference 15.9)

-------
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 mL1
Combination of
Positives
MPN Index
95% Confidence Limits
Combination of
Positives
MPN Index
95% Confidence Limits
Lower
Upper
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.: 72
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
0.08 i
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
1 -0-5
0.1 22
0.028
0.287
2-3-5
0.251
0.082
0.759
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

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

-------
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 (cont.)1
Combination of
Positives
MPN Index
95% Confidence Limits
Combination of
Positives
MPN Index
95% Confidence Limits
Lower
Upper
Lower
Upper
3-0-0
0.079
0.010
0.1 88
4-3-0
0.2.71
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
i .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. i 64
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
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
i 6.090
3.837
	
^able was developed using the MPN calculator developed by Albert Klee (Reference 15.9)

-------
12.0
Protocol Performance
Culture-based procedures were evaluated for /•'. coli 0157 in a reference matrix (PBS) and two matrices of interest
(drinking water, surface water) during a single-laboratory study. Based on workgroup discussion, a nine-tube MPN
as opposed to the standard fifteen-tube MPN was utilized for study analyses. It is expected that the SAP will be
implemented using a standard 15-tube MPN. Details regarding laboratory method performance arc provided in the
study report (Reference 15.10). Summary results using the optimized (i.e., final) procedure from this study arc
provided in Table 7. Additional method performance data will be provided following testing in multiple laboratories.
Table 7. K. coli 0157 Verification Results for Drinking Water and Surface Water Analyses using the "Optimized" Procedure
Date
Sample ID
Spike Level
(CFU/100 mL)
MPN Combo
K. coli 0157:H7
(MPN/100 mL)
Percent Recovery
(corrected for ambient
concentrations')
PBS Samples
3/16/2009
Unspiked
NA
0-0-0
<1.08

Spiked
438
3-3-1
462.2
105
3-3-1
462.2
105
Drinking Water Samples
3/16/2009
Unspiked
NA
0-0-0
<1.08

0-0-0
<1.08
Spiked
438
3-3-3
>1099
250
3-3-2
1099
250
3-3-2
1099
250
3-3-1
462.2
105
Surface Water Samples
12/1/2008
Unspiked
NA
u-u-u
<3.008

0-0-0
<3.008
Spiked
863
3-3-2
1099
127
3-1-3
158.8
18
12/3/2008
Unspiked
NA
0-0-1
3.008

0-0-1
3.008
Spiked
963
3-3-2
1099
114
3-3-3
>1099
114
12/8/2008
Unspiked
NA
3-1-0
42.73

1-0-0
3.61
Spiked
1047
3-3-2
1099
103
3-3-2
1099
103

-------
Date
Sample ID
Spike Level
(CFU/100 mL)
MPN Combo
K. coli 0157:H7
(MPN/KM) mL)
Percent Recovery
(corrected for ambient
concentrations')
12/15/2008
Unspiked
NA
2-1-0
14.69

1-2-2
19.5
Spiked
462
3-3-1
462.2
96
3-3-1
462.2
96
12/22/2008
Unspiked
NA
3-1-0
42.73

0-0-1
3.008
Spiked
320
3-2-1
149.4
40
3-3-2
1099
336
1/6/2009
Unspiked
NA
3-1-3
158.8

3-1-0
42.73
Spiked
713
3-3-2
1099
140
3-3-2
1099
140
1 Background levels of E. coli 0157 in unspiked surface water samples were subtracted from spiked surface water levels to
calculate percent recovery

-------
13.0
Pollution Prevention
13.1
The solutions and reagents used in this protocol 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

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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 E. coli 0157:H7 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 15.11) and Less Is Better: Laboratory Chemical Management for Waste Reduction (Reference 15.12), both
available from the American Chemical Society's Department of Government Relations and Science Policy. 1155 16th
Street NVV, Washington. DC 20036.
37

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38

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15.0
References
15.1	Weagant. S.D. and Bound. A.J. 2001. "Evaluation of Techniques for Enrichment and Isolation of Escherichia
coli 0157:H7 from Artificially Contaminated Sprouts." International Journal ofFood Microbiology. 71(1):
87-92.
15.2	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/biosftv/bmbl5/bmbl5toc.htm
15.3	Makino. S., Kii. T., Asakura. H.. Shirahata. T., Ikeda. T., Takeshi. K.„ and I toll. K. 2000. "Docs
Enterohemorrhagic Escherichia coli 0157:H7 Enter the Viable but Nonculturable State in Salted Salmon
RoeT' Applied and Environmental Microbiology. 66(12): 5536-5539.
15.4	American Chemical Society. 2000. Reagent Chemicals, American Chemical Society Specifications. 9th edition.
New York. NY: American Chemical Society.
15.5	British Drug Houses. Ltd. 1957. AnalaR Standards for Laboratory Chemicals. 5th Edition. Poole. Dorset. U.K.:
BDH. Ltd.
15.6	U nited States Pharmacopeia. 2005. United States Pharmacopeia and National Formulary 24. Rockville. MD:
United States Pharmacopcial Convention.
15.7	Bordner, R.H. 2005. "Section 9020 - Quality Assurance/Quality Control." In Standard Methods for the
Examination of Water and Wastewater. 21" Edition. A.D. Eaton, L.S. Clcsceri, E.VV. Rice. A.E. Greenbcrg.
and M.A.H. Franson (eds.). Washington, D C.: American Public Health Association. American Water Works
Association, and Water Environment Federation.
15.8	Wise, J. 1988. NISTMeasurement Services: Liquid-In-Glass Thermometer Calibration Service. SP 250 - 23.
U.S. Department of Commerce, National Institute of Standards and Technology.
http://ts.iiist.gov/MeasurementServices/Calibrations/upload/SP250-23.pdf
15.9	Klee, A. J. 1993. "A Computer Program for the Determination of the Most Probable Number and its
Confidence Limits." Journal of Microbiological Methods. 18(2): 91-98.
15.10	U.S. Environmental Protection Agency. Single-Laboratory Verification of Culture-Based Analytical Procedures
for Escherichia coli 0157:H7 in Water. Publication forthcoming; date and number to be determined.
15.11	American Chemical Society (ACS). 1990. The Waste Management Manual for Laboratory Personnel.
Washington. DC: American Chemical Society Department of Government Relations and Science Policy.
15.12	American Chemical Society (ACS). 1985. Less Is Better: Laboratory Chemical Management for Waste
Reduction. Washington. DC: American Chemical Society Department of Government Relations and Science
Policy.
39

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40

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Distribution of Sample to mBPW
(Sections 10.2.1 and 10.2.2)
1.0 mL to each
m
tube of 1X mBPW
Serial dilutions as
necessary
10.0 mL to each tube
of 2X mBPW (10 mL)
p
Incubation at 36.0° ± 1.0°C and 42.0° ± 0.5°C
(Section 10.2.3)
"\
r

>
Incubate at
36.0°C ± 1.0°C
for 2.0-2.5
hours
Transfer tubes
and incubate at
42.0°C ± 0.5°C^
for total 20 -
hours
24
I I
Analysis of
positive tubes
D
c
&
3
B)
<
CD
3>
3
as.
<
OS
W
o
o
3
0>
O
3"
CD
3
CD
J
o
£D
ST
SD
Q_
5
0)
CO
S
5
C/)

-------
p>
ks
Enrichment in mBPW (Sections 10.1
and 10.2) and IMS Concentration
(Section 10.3) or PCR Confirmation
Isolation on TC-SMAC and
Rainbow® Plates
(Section 10.4)
Qualitative and Quantitative
mBPW analyses after 18-24
hours incubation

Conduct IMS on
1 mL aliquot
Biochemical and Serological Testing
(Sections 10.5, 10.5, and 10.7)
TC-SMAC
0	o o
1	2 3
E, coli 0157 latex
agglutination
0	o o
1	2 3
Incubate at
42.0'C ± 0.5°C for
18-24 hours
Rainbow® agar
Incubate at
42.0°C ± 0.5°C for
18-24 hours
Streak
agglutination-
positive colony
onto TSA
Optional H7
antiserum test
Incubate at
36.0eC ±
1.0°C for 24 ±
2 hours
a
©
3
O
fii
O
3
S3
3.
Biochemical test strip
oooooot
)000
000(
100000
Incubate test strip at 36°C ± 1°C
for 18 - 24 hrs
o
o
o
o
Oxidase test

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