oEPA Method 1200: Analytical Protocol
for Non-Typhoidal Salmonella in
Drinking Water and Surface Water
May 2012
-------
Office of Water (4608T)
EPA 817-R-12-004
www. epa.gov/safewater
May 2012 Printed on Recycled Paper
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Acknowledgments
This Analytical Protocol is based on procedures single-laboratory verified by the County Sanitation
Districts of Los Angeles County, Joint Water Pollution Control Plant (JWPCP) Water Quality
Laboratory, under direction of Sanjiv R. Shah at the National Homeland Security Research Center
(NHSRC) within the U.S. Environmental Protection Agency's (EPA's) Office of Research and
Development for analysis of non-typhoidal Salmonella in drinking water and surface water samples.
This protocol was multi-laboratory validated by 10 volunteer laboratories. Technical support and data
evaluation were provided by Computer Sciences Corporation under EPA Contract No. EP-C-05-045.
The contributions of the following persons and organizations are gratefully acknowledged:
Single-Laboratory Verification Study Workgroup Participants
Michele Burgess, Marissa Mullins (EPA, Office of Emergency Management)
Stephanie Harris (EPA, Region 10)
Mark Meckes (EPA, National Risk Management Research Laboratory)
Sarah Perkins, Gene Rice (EPA, NHSRC)
Malik Raynor, Ouida Holmes (EPA, Office of Water)
Multi-Laboratory Validation Study Volunteer Participant Laboratories:
American Interplex
Analytical Laboratories, Inc.
City of Los Angeles Bureau of Sanitation
County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant (JWPCP)
County Sanitation Districts of Los Angeles County, San Jose Creek (SJC)
HML
King County Environmental Laboratory
New York State Department of Health
Texas A&M University - College Station
Wisconsin State Laboratory of Hygiene
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Disclaimer
This document has been reviewed in accordance with EPA policy and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or recommendation for use.
Neither the United States Government nor any of its employees, contractors, or their employees make
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 response and remediation efforts following a homeland security incident. The
culture-based procedures provide viability determination, identification, and either qualitative or
quantitative results. The sample preparation procedures are deemed 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 rapid screening,
field techniques, or molecular techniques that may accompany laboratory analysis.
Questions concerning this document or its application should be addressed to:
Latisha Mapp
Office of Ground Water and Drinking Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, DC 20460
(202) 564-1390
www.epa.gov/safewater
iii May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table of Contents
1.0 SCOPE AND APPLICATION 1
2.0 SUMMARY OF METHOD 1
3.0 ACRONYMS AND ABBREVIATIONS 2
4.0 INTERFERENCES AND CONTAMINATION 3
5.0 SAFETY 3
5.1 Laboratory Hazards 3
5.2 Recommended Precautions 4
6.0 EQUIPMENT AND SUPPLIES 4
7.0 REAGENTS AND STANDARDS 5
8.0 SAMPLE COLLECTION, STORAGE, AND HOLDING TIME REQUIREMENTS 10
9.0 QUALITY CONTROL (QC) 12
9.1 General 12
9.2 Initial precision and recovery (IPR) 12
9.3 Ongoing precision and recovery (OPR) 13
9.4 Matrix Spikes (MS) 14
9.5 Negative Controls 15
9.6 Positive Controls 15
9.7 Method Blank 16
9.8 Media Sterility Check 16
10.0 CALIBRATION AND STANDARDIZATION 16
11.0 PROCEDURES 17
11.1 Qualitative Sample Analyses 17
11.2 Quantitative Sample Analyses 17
11.3 Isolation on MRSV Plates 17
11.4 Isolation on XLD Plates 18
11.5 Biochemical and Serological Analyses 18
11.6 Description of Quality Control and Salmonella Results 19
12.0 DATA ANALYSIS AND BACTERIAL DENSITY CALCULATION 20
12.1 Most Probable Number (MPN) Technique 20
12.2 Calculation of MPN 20
13.0 SAMPLE SPIKING AND PERCENT RECOVERY CALCULATION 26
13.1 Sample Spiking 26
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
13.2 Calculation of BioBallฎ Spike Percent Recovery 26
14.0 PROTOCOL PERFORMANCE 27
14.1 Single-Laboratory Verification Study 27
14.2 Multi-Laboratory Validation Study 28
15.0 POLLUTION PREVENTION 30
16.0 WASTE MANAGEMENT 30
17.0 REFERENCES 30
18.0 FLOWCHARTS 32
18.1 Quantitative Analysis Dilution Scheme 32
18.2 Identification Flowchart 33
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Appendix
Appendix A: Part II (General Operations), Section A (Sample Collection, Preservation, and Storage)
(Taken from Microbiological Methods for Monitoring the Environment: Water and Wastes [Reference
17.14]).
vi May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Analytical Protocol for Non-Typhoidal Salmonella in
Drinking Water and Surface Water
May 2012
1.0 SCOPE AND APPLICATION
1.1 This Analytical Protocol is for the identification, confirmation, and quantitation of non-typhoidal
Salmonella (referred to as "Salmonella" in this document) in drinking water and surface water
samples, using selective and non-selective media followed by biochemical and serological
confirmation.
1.2 This protocol has been adapted from U.S. Environmental Protection Agency's (EPA's) Method
1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis
(MSRV) Medium (Reference 17.1) and is for use by laboratories when analyzing samples in
support of EPA homeland security efforts.
1.3 Salmonella are the causative agents of salmonellosis. Due to the infectious nature of these
bacteria and the potential for transmission to humans, all procedures should be performed in
laboratories that use, at a minimum, biological safety level (BSL)-2. Use of a biological safety
cabinet is recommended for any aerosol-generating procedures (Reference 17.2).
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 analyses of microorganisms other than Salmonella spp. and the
matrices described.
2.0 SUMMARY OF METHOD
2.1 Salmonella can be identified in a variety of water samples using selective media, morphological,
biochemical, and serological analyses. Bacterial densities can be estimated by the Most Probable
Number (MPN) technique.
2.2 For qualitative results, samples are diluted 1:1 in double-strength tryptic soy broth (TSB) and
incubated at 36.0ฐC ฑ 1.5ฐC for 24 ฑ 2 hours.
2.3 For quantitative results, samples are analyzed as received. All samples are analyzed using a 15-
tube MPN. Inoculated TSB tubes are incubated at 36.0ฐC ฑ 1.5ฐC for 24 ฑ 2 hours.
2.4 TSB tubes (MPN and qualitative analysis) exhibiting growth (turbidity) are spotted onto MSRV
plates and incubated at 42.0ฐC ฑ 0.5ฐC for 16 - 18 hours.
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
2.5 MSRV plates are examined for a whitish halo around the colony, which is evidence of motility.
Presumptive colonies are sub-cultured onto xylose lysine desoxycholate (XLD) agar and
incubated at 36.0ฐC ฑ 1.5ฐC for 18-24 hours. Presumptive positive Salmonella colonies are
confirmed using lysine iron agar (LIA), triple sugar iron (TSI) agar, and urea broth, followed by
positive serological typing using Salmonella O antiserum, polyvalent A-I and Vi.
2.6 TSB tubes (MPN and qualitative analysis) exhibiting growth (turbidity) or growth from MSRV
plates may be confirmed by real-time polymerase chain reaction (PCR) in place of biochemical
and serological confirmation.
2.7 Quantitation of Salmonella is determined using the MPN technique (Flowchart 18.1). Tubes that
are confirmed positive for Salmonella are used to determine MPN.
3.0 ACRONYMS AND ABBREVIATIONS
ATCCฎ American Type Culture Collection
BSL Biological safety level
cm Centimeter
ฐC Degrees Celsius
EPA U.S. Environmental Protection Agency
g Gram
IPR Initial precision and recovery
JWPCP Joint Water Pollution Control Plant
LACSD Los Angeles County Sanitation District
L Liter
LIA Lysine iron agar
(iL Microliter
(im Micrometer
mg Milligram
mL Milliliter
mm Millimeter
MPN Most probable number
MS Matrix spike
MSRV Modified semisolid Rappaport-Vassiliadis (agar)
N Normal - one equivalent weight per liter
NCTC National Collection of Type Cultures
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
OPR Ongoing precision and recovery
2
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PPE Personal protective equipment
psi Pounds per square inch
QA Quality assurance
QC Quality control
RSD Relative standard deviation
SD Standard deviation of the percent recoveries
TSB Tryptic soy broth
TSI Triple sugar iron
w/v Weight to volume ratio
XLD Xylose lysine desoxycholate
4.0 INTERFERENCES AND CONTAMINATION
4.1 During the multi-laboratory validation study (Study Report, Reference 17.3), differences in mean
MPN/100 mL were observed in surface water matrices. This may be due to varying degrees of
laboratory proficiency with the method, as at least one laboratory retrospectively suggested that,
they should have submitted more "questionable colonies" from MSRV to confirmation. In
addition, as indicated in Section 6 of the Study Report, another laboratory indicated that
background bacteria from the TSB enrichment made it very difficult to identify presumptively
positive colonies on MSRV. As a result, it is critical that laboratories become proficient with
this protocol prior to analyzing samples from complex surface water matrices, as inappropriately
low results may be reported.
4.2 Salmonella recoveries may be impacted by the presence of high numbers of competing or
inhibitory organisms (e.g., other Enterobacteriaceae), or toxic substances (e.g., metals or organic
compounds).
4.3 A viable but non-culturable state of Salmonella may also account for low recoveries (Reference
17.4).
5.0 SAFETY
5.1 Laboratory Hazards
In order 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 and broken skin with cultures and 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.
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
5.2 Recommended Precautions
5.2.1 Salmonella are BSL-2 pathogens and all procedures should be performed in laboratories
that use, at a minimum, BSL-2 practices (Reference 17.2). This includes prohibiting
eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food
and drink in the laboratory.
5.2.2 A Class 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 protocol does not address all safety issues associated with its use. Please refer to
Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (Reference 17.2)
for additional safety information. A reference file of Material Safety Data Sheets should
be available to all personnel involved in analyses.
6.0 EQUIPMENT AND SUPPLIES
6.1 Autoclave or steam sterilizer, capable of achieving 121ฐC (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
100gฑ0.1g
6.4 Beakers, glass or plastic (assorted sizes)
6.5 Biological safety cabinet, Class II (optional but recommended)
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
6.6 Borosilicate glass or plastic screw-cap, wide-mouth bottles, sterile (e.g., 250 mL)
6.7 Borosilicate glass culture tubes, with autoclavable screw or snap caps (25 x 150 mm)
6.8 Borosilicate glass culture tubes, with autoclavable screw-caps (16 x 100 mm)
6.9 Erlenmeyer flasks (500 mL, 1 L, 2 L)
6.10 Filters and filter syringes, sterile, for reagent sterilization (0.22 (im pore size)
6.11 Graduated cylinders (assorted sizes)
6.12 Gloves, sterile, nitrile, or equivalent
6.13 Incubators, microbiological type, maintained at 36.0ฐC ฑ 1.0ฐC and 42.0ฐC ฑ 1.0ฐC
6.14 Inoculation loops, sterile, disposable
6.15 Micropipettor
6.16 Parafilmฎ or equivalent
6.17 Petri dishes, sterile, plastic (15 x 100 mm)
6.18 pH meter
6.19 Pipettes, standard tip, sterile, plastic, disposable (assorted sizes)
6.20 Pipetting device (automatic or equivalent)
6.21 Pipette tips (e.g., 2 (iL - 100 (iL), sterile
6.22 Stirring hotplates and stir bars
6.23 Test tube racks
6.24 Thermometer, National Institute of Standards and Technology (NIST)-traceable
6.25 Tissues, lint-free (Kimwipesฎ or equivalent)
6.26 Waterbath maintained at 45 ฐC - 50ฐC for tempering agar
6.27 Weigh paper and boats
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 17.5). For suggestions regarding the testing of reagents not listed
by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals (Reference
17.6) and United States Pharmacopeia and National Formulary 24 (Reference 17.7).
7.2 Whenever possible, use commercially available culture media. The agar used in the preparation
of culture media must be of microbiological grade. Storage temperatures and times for prepared
media and reagents are provided in Table 2 (Section 7.15).
7.3 Reagent-grade water must conform to specifications in Standard Methods for the Examination of
Water and Wastewater, 21st Edition (Reference 17.8).
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
7.4 Phosphate Buffered Saline (PBS)
Prepare reagent according to the following and store at <10ฐC 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 8.5 0 g
Reagent-grade water 1.0 L
7.4.2 Dissolve reagents in 1 L reagent-grade water and dispense appropriate volumes in screw-
cap bottles or tubes and autoclave at 121ฐC (15 psi) for 15 minutes. Final pH should be
7.4 ฑ0.2.
7.5 Tryptic Soy Broth (TSB)
Commercially prepared medium is recommended. Dehydrated medium (Bacto 211824 or
equivalent) may be used. If commercially prepared medium is not available, prepare according
to Sections 7.5.1 and 7.5.2.
7.5.1 Composition:
IX 2X 3X
Pancreatic digest of casein 17.Og 34.0 g 51.Og
Enzymatic digest of soybean meal 3.0g 6.0 g 9.0 g
Sodium chloride 5.0 g 10.0 g 15.0 g
Dipotassium phosphate (K2HPO4) 2.5 g 5.0g 7.5 g
Dextrose 2.5 g 5.0g 7.5 g
Reagent-grade water l.OL l.OL l.OL
7.5.2 Add reagents to 950 mL reagent-grade water and mix thoroughly using a stir bar and heat
to dissolve completely. Adjust pHto 7.3 ฑ 0.2 with 1.0 N hydrochloric acid or 1.0 N
sodium hydroxide and bring to 1 L. For IX TSB, aseptically dispense 10 mL into 25 x
150 mm tubes. For 3X TSB, aseptically dispense 5 and 10 mL volumes into 25 x 150
mm tubes. For 2X TSB, dispense in appropriate volumes (e.g., 100 mL). Autoclave at
121ฐC (15 psi) for 15 minutes.
7.6 Modified Semisolid Rappaport-Vassiliadis (MSRV) Agar with Novobiocin
Commercially prepared medium is recommended. Dehydrated medium (Difco 218681 or
equivalent), with the addition of novobiocin supplement (Difco 231971 or equivalent), may be
used. If commercially prepared medium is not available, prepare according to Sections 7.6.1
through 7.6.4.
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
7.6.1 Composition:
Tryptose 4.59 g
Casein hydrolysate (acid) 4.59 g
Sodium chloride 7.34 g
Monopotassium phosphate (KH2PO4) 1.47 g
Magnesium chloride (anhydrous - MgCl2) 10.93 g
Malachite green oxalate 0.037 g
Agar 2.70 g
Reagent-grade water l.OL
7.6.2 Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar and
hot plate. Boil for one minute with rapid stir bar agitation to dissolve completely. Do
not autoclave. Adjust pH to 5.2 ฑ 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide and bring to 1 L. Cool to 45ฐC - 50ฐC in a waterbath.
7.6.3 Prepare 2% novobiocin stock solution by dissolving 500 mg of sodium novobiocin in 25
mL of reagent-grade water and filter sterilizing, using a 0.22-(im pore-size filter.
Dispense 1.1 mL of the stock solution into 2.0 mL cryogenic vials and freeze at -20ฐC.
Note: If using a commercially prepared novobiocin antimicrobic supplement, add
sufficient volume to achieve a concentration of 0.002% per liter.
7.6.4 Add 1.0 mL of the 2% solution of novobiocin per liter of medium. Mix well by swirling
the medium. Immediately pour approximately 25 mL into each 15 x 100 mm sterile Petri
plate. Do not invert plates to store. MSRV plates must be used within 48 hours of
preparation.
Note: If using a commercially prepared novobiocin antibiotic solution that is not 2%,
add sufficient volume of the novobiocin to achieve a final concentration of 0.002%.
7.7 Xylose Lysine Desoxycholate (XLD) Agar
Commercially prepared medium is recommended. Dehydrated medium (Difco 278850 or
equivalent) may be used. If commercially prepared medium is not available, prepare according
to Sections 7.7.1 and 7.7.2.
7.7.1 Composition:
Xylose 3.75g
L-lysine 5.0 g
Lactose 7.5 g
Saccharose 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Phenol red 0.08 g
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Sodium desoxycholate 2.5 g
Sodium thiosulfate 6.8 g
Ferric ammonium citrate 0.8 g
Agar 15.0 g
Reagent-grade water l.OL
7.7.2 Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar and
hot plate. Heat with agitation just until the medium boils. Do not overheat, as
overheating causes precipitation. Do not autoclave. Adjust pH to 7.4 ฑ 0.2 with 1.0 N
hydrochloric acid or 1.0 N sodium hydroxide and bring to 1 L. Cool to 45ฐC - 50ฐC in a
waterbath. Aseptically pour 12 - 15 mL into each 15 x 100 mm sterile Petri plate.
Note: Heating medium to boiling sterilizes the medium; overheating or autoclaving may
cause precipitation.
7.8 Triple Sugar Iron (TSI) Agar
Commercially prepared medium is recommended. Dehydrated medium (Difco 226540 or
equivalent) may be used. If commercially prepared medium is not available, prepare according
to Sections 7.8.1 and 7.8.2.
7.8.1 Composition:
Beef extract 3.0 g
Yeast extract 3.0 g
Pancreatic digest of casein 15.0 g
Proteose peptone no. 3 5.0 g
Dextrose 1.0 g
Lactose 10.0 g
Sucrose 10.0 g
Ferrous sulfate 0.2 g
Sodium chloride 5.0 g
Sodium thiosulfate 0.3 g
Phenol red 0.024 g
Agar 12.0 g
Reagent-grade water 1.0 L
7.8.2 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 7.4 ฑ 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide and
bring to 1 L. Aseptically dispense 5-7 mL aliquots into 16 x 100 mm screw-cap tubes,
cap, and autoclave at 121ฐC (15 psi) for 15 minutes. Cool in a slanted position so that
the surface area is equally divided between the slant and butt. Let medium warm to
room temperature prior to inoculation.
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
7.9 Lysine Iron Agar (LIA)
Commercially prepared medium is recommended. Dehydrated medium (Difco 284920 or
equivalent) may be used. If commercially prepared medium is not available, prepare according
to Sections 7.9.1 and 7.9.2.
7.9.1 Composition:
Peptone 5.0 g
Yeast extract 3.0 g
Dextrose 1.0 g
L-lysine hydrochloride 10.0 g
Ferric ammonium citrate 0.5 g
Sodium thiosulfate 0.04 g
Bromcresol purple 0.02 g
Agar 15.0g
Reagent-grade water l.OL
7.9.2 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 6.7 ฑ 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide and
bring to 1 L. Aseptically dispense 5-7 mL aliquots into 16 x 100 mm screw-cap tubes,
cap, and autoclave at 121ฐC (15 psi) for 12 minutes. Cool in a slanted position so that
the surface area is equally divided between the slant and butt. Let medium warm to
room temperature prior to inoculation.
7.10 Urea Broth
Commercially prepared medium is recommended. Dehydrated medium (Difco 227210 or
equivalent) may be used. If commercially prepared medium is not available, prepare according
to Sections 7.10. land 7.10.2.
7.10.1 Composition:
Yeast extract 0.1 g
Monopotassium phosphate (KH2PO4) 9.1 g
Dipotassium phosphate (K2HPO4) 9.5 g
Urea 20.0 g
Phenol red 0.01 g
Reagent-grade water l.OL
7.10.2 Add reagents to 950 mL of reagent-grade water and mix thoroughly using a stir bar. Do
not boil or autoclave. Adjust pH to 6.8 ฑ 0.1 with 1.0 N hydrochloric acid or 1.0 N
sodium hydroxide and bring to 1 L. Filter sterilize. Aseptically dispense 3.0 mL
aliquots into sterile 16 x 100 mm screw-cap tubes. Let medium warm to room
temperature prior to inoculation.
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
7.11 Saline, physiological (0.85% w/v): Dissolve 0.85 g NaCl in 100 mL of reagent-grade water.
Autoclave at 121ฐC (15 psi) for 15 minutes. Cool to room temperature.
7.12 Salmonella O antiserum Polyvalent Groups A-I and Vi (BD 222641 or equivalent).
7.13
BioBallฎ (30 CPU) Salmonella typhimurium (ATCCฎ 14028/NCTC 12023) BTF, Pty (a
bioMerieux Company) in North Ryde, Australia.
7.14 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.
Table 1. Positive and Negative Control Cultures
Media/Reagents
MSRV
XLD
TSI
LIA
Urea broth
Salmonella O antiserum
polyvalent A-I and Vi
Positive Control
Salmonella typhimurium
(ATCCฎ 14028)
Salmonella typhimurium
(ATCCฎ 14028)
Salmonella typhimurium
(ATCCฎ 14028)
Salmonella typhimurium
(ATCCฎ 14028)
Proteus hauseri
(formerly vulgaris) (ATCCฎ 13315)
Salmonella typhimurium
(ATCCฎ 14028)
Negative Control
Escherichia coli
(ATCCฎ 25922)
Escherichia coli
(ATCCฎ 25922)
Escherichia coli
(ATCCฎ 25922)
Escherichia coli
(ATCCฎ 25922)
Salmonella typhimurium
(ATCCฎ 14028)
Escherichia coli
(ATCCฎ 25922)
7.15 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 1
Media/Reagents
PBS, saline
in screw-cap containers
Tubes: TSB, TSI, and LIA slants,
urea broth
Plates: XLD
Plates: MSRV
Storage Temperature
Room temperature
<10ฐC and above freezing
<10ฐC and above freezing
<10ฐC and above freezing
Storage Time
3 months
2 weeks in loose cap tubes
3 months in screw-cap tubes
2 weeks
48 hours
If media/reagent is refrigerated, remove from refrigerator 1-1.5 hours prior to inoculation to ensure it
reaches room temperature prior to use.
8.0 SAMPLE COLLECTION, STORAGE, AND HOLDING TIME REQUIREMENTS
8.1 Drinking Water Sample Collection (200 mL)
8.1.1 Select a cold water line faucet and remove aerator, if present.
10
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
8.1.2 Clean the faucet exterior with disinfection solution (e.g., 10% household bleach).
8.1.3 Open the tap to obtain a smooth-flowing stream at moderate pressure without splashing.
8.1.4 Allow water to run at least 2-3 minutes.
8.1.5 Remove the cap from a sterile bottle containing 1 mL of a 10% sodium thiosulfate
solution (dechlorinating agent).
8.1.6 Avoid contaminating the sample bottle lip or inside the cap.
8.1.7 Reduce the water flow to fill the bottle without splashing and fill to within 2.5 cm - 5 cm
(1" - 2") of the top for proper mixing before analyses.
8.1.8 Do not rinse dechlorinating agent out of the bottle.
8.1.9 Tightly cap the container and transport to the laboratory on ice (do not freeze).
8.2 Surface Water Sample Collection (200 mL)
8.2.1 Collect samples by hand or with a sampling pole if the sampling site has difficult access
such as a dock, bridge, or bank adjacent to surface water.
8.2.2 The sampling depth should be 6" - 12" below the water surface.
8.2.3 Sample containers should be positioned such that the mouth of the container is pointed
away from the sampler or sample point.
8.2.4 After removal of the container from the water, a small portion of the sample should be
discarded to leave a headspace of 2.5 cm - 5 cm (1" - 2") for proper mixing before
analyses.
8.2.5 Transport to the laboratory on ice (do not freeze).
8.3 Sample handling: Maintain bacteriological samples at <10ฐC during transit to the laboratory. Do
not allow the sample to freeze. Use insulated containers to ensure proper maintenance of storage
temperature. Sample bottles should be placed inside waterproof bags, excess air purged, and
bags sealed to ensure that bottles remain dry during transit or storage. Refrigerate samples upon
arrival in the laboratory and analyze as soon as possible after collection. Bring samples to room
temperature before analysis.
8.4 Holding time and temperature limitations: Analyses should begin immediately, preferably, within
2 hours of collection. If it is impossible to examine samples within 2 hours, samples must be
maintained at <10ฐC until analysis. Samples must not be frozen. Sample analysis must begin
within 31 hours of sample collection. Note: Adherence to sample handling procedures and
holding time limits is critical to the production of valid data. Sample results will be considered
invalid if these conditions are not met.
11 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
9.0 QUALITY CONTROL (QC)
9.1 General
Each laboratory that uses this method is required to operate a formal quality assurance program
that addresses and documents instrument and equipment maintenance and performance, reagent
quality and performance, analyst training and certification, and records storage and retrieval.
Specific quality control (QC) procedures for use with this method are discussed below.
The minimum analytical QC requirements for the analysis of samples using this protocol include
an initial demonstration of laboratory capability through performance of the initial precision and
recovery (IPR) analyses (Section 9.2), ongoing demonstration of laboratory capability through
performance of the ongoing precision and recovery (OPR) analysis (Section 9.3), matrix spike
(MS) analysis (Section 9.4), and the routine analysis of negative and positive controls (Sections
9.5 and 9.6), method blanks (Section 9.7), and media sterility checks (Section 9.8). For the IPR,
OPR and MS analyses, it is necessary to spike samples with BioBalls as described in Section
13.1.
9.2 Initial precision and recovery (IPR)
IPR analyses are used to demonstrate acceptable method performance (recovery and precision)
and should be performed by each laboratory before the method is used for monitoring field
samples. If field samples will be analyzed on the day of sample collection (ideally within 6
hours), all IPR sample analyses should begin within 30 minutes of spiking and results compared
to the 0-Hour IPR Criteria provided in Table 3, below. If field samples are shipped overnight for
analyses (e.g., samples analyzed within 30 ฑ 1 hours of sample collection); IPRs should be
spiked and refrigerated at <10ฐC and above freezing for 30 ฑ 1 hours prior to analyses, and
results compared to the 30-Hour IPR Criteria provided in Table 3.
EPA recommends but does not require that an IPR be performed by each analyst. IPR samples
should be accompanied by an acceptable method blank (Section 9.7) and appropriate media
sterility checks (Section 9.8). The IPR analyses are performed as follows:
9.2.1 Prepare four, 200-mL PBS samples and spike each sample with a single Salmonella
typhimurium (ATCCฎ 14028/NCTC 12023) BioBallฎ according to the spiking
procedure in Section 13 and analyze according to Section 11.
9.2.2 Calculate the percent recovery (R) for each IPR sample using the equation in Section 13.
9.2.3 Using the percent recoveries of the four analyses, calculate the mean percent recovery
and the relative standard deviation (RSD) of the recoveries. The RSD is the standard
deviation divided by the mean, multiplied by 100.
9.2.4 Compare the mean percent recovery and RSD with the corresponding IPR criteria in
Table 3. If the mean percent recovery and RSD meet acceptance criteria, system
performance is acceptable and analysis of field samples may begin. If the mean percent
recovery or the RSD fall outside of the IPR criteria, system performance is unacceptable.
In this event, identify the problem by evaluating each step of the analytical process,
media, reagents, and controls, correct the problem and repeat the IPR analyses.
12 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 3. Calculated IPR and OPR Criteria Based on 95% Prediction Interval
Holding
Time
0-Hour
30-Hour
IPR/OPR
IPR
(4 PBS samples)
OPR
(1 PBS sample)
IPR
(4 PBS samples)
OPR
(1 PBS sample)
Performance Test
Mean percent recovery
Precision a
Percent recovery
Mean percent recovery
Precision a
Percent recovery
Analytical Method
Acceptance Criteria
for 200-mL PBS Samples
61% -151%
67%
20% -191 %
31% -171%
75%
detect - 204%
Precision as maximum RSD
9.3 Ongoing precision and recovery (OPR)
To demonstrate ongoing control of the analytical system, the laboratory should routinely process
and analyze spiked PBS samples. The laboratory should analyze one OPR sample after every 20
field and MS samples or one per week that samples are analyzed, whichever occurs more
frequently. If field samples will be analyzed on the day of sample collection (ideally within 6
hours), OPR samples should be analyzed within 30 minutes of spiking and results compared to the
0-Hour OPR Criteria provided in Table 3, above. If field samples are shipped overnight for
analyses (e.g., samples analyzed within 30 ฑ 1 hours of sample collection); OPRs should be spiked
and refrigerated at <10ฐC and above freezing for 30 ฑ 1 hours prior to analyses, and results
compared to the 30-Hour OPR Criteria provided in Table 3.
EPA recommends but does not require that an OPR be performed by each analyst. OPR samples
must be accompanied by an acceptable method blank (Section 9.7) and appropriate media sterility
checks (Section 9.8). OPR analyses are performed as follows:
9.3.1 Prepare one, 200-mL PBS sample and spike sample with a single Salmonella
typhimurium (ATCCฎ 14028/NCTC 12023) BioBallฎ according to the spiking
procedure in Section 13.
9.3.2 Calculate the percent recovery (R) for the OPR sample using the equation in Section 13.
9.3.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery
criteria in Table 3, above. If the OPR result meets the acceptance criteria for recovery,
method performance is acceptable and analysis of field samples may continue. If the
OPR result falls outside of the acceptance criteria, system performance is unacceptable
and all associated field data should be flagged or considered invalid. In this event,
identify the problem by evaluating each step of the analytical process (media, reagents,
and controls), correct the problem and repeat the OPR analysis.
9.3.4 As part of the laboratory quality assurance (QA) program, results for OPR and IPR
samples should be charted and updated records maintained in order to monitor ongoing
13
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
method performance. The laboratory should also develop a statement of accuracy for
this protocol by calculating the average mean percent recovery (R) and the standard
deviation of the percent recoveries (sr). Express the accuracy as a recovery interval from
R - 2sr to R + 2sr.
9.4 Matrix Spikes (MS)
MS analyses are performed to determine the effect of a particular matrix on Salmonella recoveries.
The laboratory should analyze one MS sample when drinking water (DW) or surface water (SW)
samples are first received from a source from which the laboratory has not previously analyzed
samples. Subsequently, 5% of field samples (1 per 20) from a given source should include a MS
sample. MS samples must be accompanied by the analysis of an unspiked field sample
sequentially collected from the same sampling site, an acceptable method blank (Section 9.7), and
appropriate media sterility checks (Section 9.8). When possible, MS analyses should also be
accompanied by an OPR sample (Section 9.3). The MS analysis is performed as follows:
9.4.1 Prepare two, 200-mL drinking water or surface water samples that were sequentially
collected from the same site. One sample will remain unspiked and will be analyzed to
determine the background or ambient concentration of Salmonella for calculating MS
recoveries. The other sample will serve as the MS sample and will be spiked with a
single Salmonella typhimurium (ATCCฎ 14028/NCTC 12023) BioBallฎ according to
the spiking procedure in Section 13. If field samples will be analyzed on the day of
sample collection (ideally within 6 hours), process and analyze both the unspiked and
spiked matrix samples immediately according to the procedures in Section 11 and
calculate the Salmonella MPN / 100 mL according to Section 12. If field samples will be
shipped overnight (analyzed within 30 ฑ 1 hours of sample collection), refrigerate both
the unspiked matrix and the MS samples for 30 ฑ 1 hours prior to analyses.
9.4.2 For the MS sample, calculate the Salmonella MPN / 100 mL according, including
adjusting the density based on the ambient concentration of Salmonella observed in the
unspiked matrix sample, as described in Section 13.
9.4.3 Calculate the percent recovery (R) for the MS sample according to Section 13.
9.4.4 Compare the MS result (percent recovery) with the appropriate method performance
criteria in Table 4. If the MS recovery meets the acceptance criteria, system
performance is acceptable and analysis of field samples from this source may continue.
If the MS recovery is unacceptable and the OPR sample result associated with this batch
of samples is acceptable, matrix interference may be causing the poor results. If the MS
recovery is unacceptable, all associated field data should be flagged.
14 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 4. Matrix Spike (MS) Criteria Based on 95% Prediction Interval
Matrix
Drinking Water
Surface Water
Holding Time
0-Hour
30-Hour
0-Hour
30-Hour
Performance Test
Percent recovery
Percent recovery
Percent recovery
Percent recovery
Analytical Method
Acceptance Criteria 200-mL
Samples
11% -206%
Detect - 320%
Detect - 1 50%
Detect - 1 50%
9.4.5 Laboratories should record and maintain a control chart comparing MS recoveries for all
matrices to batch-specific and cumulative OPR sample results analyzed using this
protocol. These comparisons should help laboratories recognize matrix effects on
recovery and may also help to recognize inconsistent or sporadic matrix effects from a
particular source.
9.5 Negative Controls
9.5.1 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 7.14), and descriptions of negative results are provided
in Table 5 (Section 11.6).
9.5.2 Analysis of negative controls is conducted by inoculating media and conducting
biochemical and serological analyses with known negative control organisms as
described in Section 11. The negative control is treated as a sample and submitted to the
same analytical procedures.
9.5.3 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).
9.5.4 Viability of the negative controls should be demonstrated on a monthly basis, at a
minimum, using a non-selective medium (e.g., trypticase soy agar).
9.6 Positive Controls
9.6.1 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 7.14), and descriptions of positive results are provided
in Table 5 (Section 11.6).
15
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
9.6.2 Analysis of positive controls is conducted by inoculating media and conducting
biochemical and serological tests with known positive organisms as described in Section
11. The positive control is treated as a sample and submitted to the same analytical
procedures.
9.6.3 If a positive control fails to exhibit the appropriate response, check and/or replace the
associated media, reagents, and/or positive control organism, and re-analyze the
appropriate positive control and corresponding sample(s).
9.7 Method Blank
On an ongoing basis, the laboratory should analyze a method blank every day that samples are
analyzed using sterile PBS (Section 7.4) to verify the sterility of equipment, materials, and
supplies. The method blank is treated as a sample, and subjected to the same analytical
procedures. Absence of growth indicates freedom from contamination by the target organisms.
9.8 Media Sterility Check
Test sterility of PBS and media (TSB, MSRV, XLD, TSI, LIA, urea broth) by incubating one
unit (tube or plate) from each batch at 36.0ฐC ฑ 1.5ฐC for 24 hours and observe 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.
10.0 CALIBRATION AND STANDARDIZATION
10.1 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.
10.2 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.
10.3 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 17.9).
Check mercury columns for breaks.
10.4 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.
10.5 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.
10.6 Calibrate micropipettors once per year. Spot-check micropipettor accuracy once per month by
weighing a measured amount of reagent-grade water (1 \\L = 1 mg).
10.7 Re-certify biological safety cabinets once per year. Re-certification must be performed by a
qualified technician.
16 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
11.0 PROCEDURES
Salmonella is a pathogen and all samples should be handled with caution, using BSL-2 procedures and
PPE. A Class II biological safety cabinet is recommended for sample manipulation where the risk of
aerosol production is high.
11.1 Qualitative Sample Analyses
Add a sample volume (e.g., 100 mL) to an equal volume of double-strength TSB (Section 7.5)
and incubate at 36ฐC ฑ 1.5ฐ C for 24 ฑ 2 hours. After incubation, proceed to Section 11.3 for
isolation of Salmonella.
11.2 Quantitative Sample Analyses
A multiple-tube assay incorporating differential sample volumes is used to estimate Salmonella
densities in undiluted or diluted samples. If low levels of Salmonella 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 Salmonella are suspected, additional serial dilutions should be used.
See Flowchart 18.1 for an overview of the sample dilution and inoculation scheme. A minimum
sample volume of 155 mL is required if 20 mL volumes are used to inoculate the first row of
tubes.
11.2.1 Sample inoculation
Arrange TSB tubes in three rows (10 mL of 3X, 5 mL of 3X, and 10 mL of IX) of five
tubes each. Inoculate the first row of tubes (10 mL of 3X TSB) with 20 mL of the
undiluted sample. Inoculate 10 mL of the undiluted sample into each of the tubes in the
second row (5 mL of 3X TSB). Inoculate 1 mL from the undiluted sample into each of
the tubes in the third row (10 mL of IX TSB). See Flowchart 18.1 for an overview of
the sample inoculation scheme.
11.2.2 Sample dilutions
Samples may require serial dilution prior to inoculation due to high levels of Salmonella.
If analyzing serially diluted samples, 1.0 mL of each dilution will be used to inoculate
each tube of IX TSB, as appropriate.
11.2.3 Growth
Incubate tubes at 36.0ฐC ฑ 1.5ฐC for 24 ฑ 2 hours. After incubation, proceed to Section
11.3 for isolation of Salmonella.
11.3 Isolation on M RSV Plates
See Flowchart 18.2 for an overview of the colony identification procedures.
11.3.1 Select all TSB (qualitative and quantitative) tubes exhibiting growth within 24 ฑ 2 hours.
Apply six discrete, 30 (iL drops from each TSB tube onto separate MSRV plates. Space
the drops evenly over the entire plate. Do not invert the plates, as the semisolid
17 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
medium will not remain intact. Allow the drops to absorb into the agar for
approximately one hour at room temperature before incubating the plates at 42.0ฐC ฑ
0.5ฐCfor 16- 18 hours.
11.3.2 On MSRV plates, presumptive Salmonella colonies produce a whitish halo around the
colony, an indication of motility.
11.4 Isolation on XLD Plates
11.4.1 Examine plates for the appearance of motility surrounding the inoculations, as evidenced
by a whitish halo.
11.4.2 Using a sterile inoculating loop, stab into a halo at the outer edge of a target colony on
the MSRV plate and streak for isolation onto an XLD plate. Since Salmonella are
predominately located within the MSRV medium, the loop should penetrate the MSRV
at least half-way. Incubate the plates at 36.0ฐC ฑ 1.5ฐC for 18 - 24 hours.
11.4.3 Seal the MSRV plates with Parafilmฎ and store at <10ฐC and above freezing as backup,
for a maximum of one week.
11.4.4 Use isolated colonies from the XLD plates for biochemical and serological analyses.
11.5 Biochemical and Serological Analyses
Salmonella produce black and/or pink to red colonies with black centers on XLD plates. Use a
single well-isolated colony exhibiting Salmonella morphology from the XLD plate to inoculate
all three media (TSI, LIA, and urea broth) used for biochemical confirmation.
11.5.1 TSI Agar
Using a sterile inoculating needle, stab the butt of a slant of TSI agar with a portion of
the isolated colony and streak the slant. Loosen the cap to prevent anaerobic conditions
and incubate at 36.0ฐC ฑ 1.5ฐC for 24 ฑ 2 hours. Salmonella have an acid (yellow) butt
and alkaline (red) slant, with or without (rare) H2S gas production. When H2S gas
production is present, the butt may appear black.
11.5.2 LIA
Using a sterile inoculating needle, stab the butt of a slant of LIA agar with a portion of
the isolated colony and streak the slant. Loosen the cap to prevent anaerobic conditions
and incubate at 36.0ฐC ฑ 1.5ฐC for 24 ฑ 2 hours. Salmonella have an alkaline (purple)
butt and alkaline (purple) slant, with or without (rare) H2S gas production. When H2S
gas production is present, the butt may appear black.
11.5.3 Urea Broth
Using a sterile loop, inoculate a urea broth tube with a portion of the isolated colony.
Loosen the cap to prevent anaerobic conditions and incubate at 36.0ฐC ฑ 1.5ฐC for 24 ฑ
2 hours. If the bacterium is urease-positive, it will hydrolyze the urea in the broth,
producing ammonia, and making the broth alkaline. This will turn the broth from orange
18 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
to pink or deep purplish-red. A negative urease test will exhibit no color change.
Salmonella are urease-negative.
11.5.4 Serological Analyses
Emulsify growth from the TSI slant using sterile physiological saline (Section 7.11).
Place two discrete drops of emulsified growth onto a slide. To the first drop of
emulsified growth, add one drop of Salmonella O antiserum, polyvalent A-I and Vi
(Section 7.12). 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. Salmonella are agglutination-positive for O, polyvalent
A-I and Vi. Results should be compared with those for positive and negative controls
(Table 1) analyzed at the same time.
Note: If using antiserum equivalent to BD 222641 (Section 7.12), the laboratory
should verify each lot of the antiserum with control organisms.
11.6 Description of Quality Control and Salmonella Results
Typical results are provided in Table 5.
Table 5. Positive and Negative Result Descriptions and Salmonella Results
Medium/Test
MSRV
XLD
TSI
(slant/butt)
LIA
(slant/butt)
Urea broth
Salmonella O antiserum
polyvalent A-I and Vi
Salmonella Results
Positive
Positive
Alkaline slant (red)
with acid butt (yellow),
with or without H2S
production
Alkaline slant (purple)
with alkaline butt
(purple), with or
without H2S production
Negative
Positive
Positive Control Result
and Description
Growth with whitish halo
around the colony
Black and/or pink to red
colonies with black
centers
Alkaline slant (red) with
acid butt (yellow), with or
without H2S production
Alkaline slant (purple) with
alkaline butt (purple), with
or without H2S production
Pink to purplish red color
change
Agglutination
Negative Control Result
and Description
No growth or growth
without halos
Yellow colonies
Acid slant (yellow) with acid
butt (yellow), without H2S
production
Alkaline slant (red) with
acid butt (yellow), without
H2S production
No color change
No agglutination
19
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
12.0 DATA ANALYSIS AND BACTERIAL DENSITY CALCULATION
12.1 Most Probable Number (MPN) Technique
Estimation of bacterial densities may be determined based on the number of tubes positive for
Salmonella either by morphological, biochemical, and serological results or PCR.
12.2 Calculation of MPN
If only three rows of tubes were analyzed, identify appropriate MPN value using either Table 7
or 8, 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 12.2.1 and
12.2.2. Table 7 should only be used for volumes of 20.0 mL, 10.0 mL, and 1.0 mL. If volumes
of 10.0 mL or less were analyzed, refer to Table 8 for the MPN values (Sections 12.2.1 and
12.2.2).
12.2.1 Selection of Tubes
If more than three rows of tubes are inoculated with sample (e.g., volumes/dilutions),
select the most appropriate rows of tubes according to the criteria provided below.
Examples of row selections and MPN/100 mL values are provided in Table 6.
12.2.1.1 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 6, 10 mL is a smaller volume than 20 mL, and is the
lowest volume giving positive results in all five tubes.
12.2.1.2 If the largest volume tested has less than five tubes with positive results, select
it and the next two volumes (Table 6, Examples B and C).
12.2.1.3 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 6,
Example D).
12.2.1.4 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 6, Example E).
12.2.1.5 If there were not enough lower volumes analyzed to select three dilutions using
the rules above, then select the three smallest volumes (Table 6, Example F).
20 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 6. Examples of Appropriate Tube Selection and MPN/100 mL 1
Example
A
B
C
D
E
F
20 mL
5/5
4/5
0/5
5/5
4/5
5/5
10 mL
.. 5J>
5/5
1/5
3/5
4/5
5J5
1.0 mL
3/5
1/5
0/5
1/5
0/5
5/5
0.1 mL
0/5
0/5
0/5
1/5
1/5
2/5
Significant
Dilutions
5-3-0
4-5-1
0-1-0
3-1-1
4-4-1
5-5-2
Table
8
7
7
8
7
8
MPN Index
0.792
0.1524
0.0067
0.137
0.1181
5.422
MPN/100 mL
79.2
15.24
0.67
13.7
11.81
542.2
1 Appropriate volumes are underlined and the largest sample volumes analyzed are highlighted.
12.2.2 For calculation of MPN/100 mL when additional dilutions are analyzed (e.g., 10"2, 10"3),
obtain the MPN index value from Table 8 using the number of positive tubes in the three
selected dilutions. Calculate MPN/100 mL using the equation below:
MPN Index from Table 8
MPN/100mL =
Middle volume analyzed in the series used for MPN determination
100
For example, a dilution series of 10"3, 10"4, 10"5, with the following positive tubes 5, 1, 0,
respectively, would be:
MPN/100mL =
0.329
100 = 3.29 x 103
10"
21
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 7. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes are Used per Volume and Inoculation Volumes are 20.0,10.0, and 1.0 mL 1
Combination of
Positives
0-0-0
' ".0-0-1
: 0*0-2 '
: " '0-0-3
: o-0r4 '
: " o-o-ei ",
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
0-2-0
0-2-1
0-2-2
: 0^2-3 '
: . 0-2-4
: ' ' 0-2-5 .
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
0-4-0
0-4-1
0-4-2
: 0-4-3
: . 0-4-4
: ' ' 0-4-5 '
0-5-0
0-5-1
0-5-2
0-5-3
0-5-4
0-5-5
1-0-0
1-0-1
1-0-2
: 1-0-3
: . 1-0-4
: ' '1-0-5
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
1-2-0
1-2-1
1-2-2 - -
: 1-2-3 -
: . '1-2-4
: ' '1-2-5
MPN Index
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 7. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes are Used per Volume and Inoculation Volumes are 20.0,10.0, and 1.0 mL (cont)1
Combination of
Positives
3-0-0 - - -
'' ' '. 3.0-1
: 3-0-2 "
: " 3-0-3 ' '
: '3-0-4' -
: 3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
3-1-5
3-2-0
" ' 3-2-1
: 3-2-2 '
: " 3-2-3 ' '
: 3-2-4 .
: 3-2-S ' :
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-0 - - -
'' "-3-4-1
: 3-4-2 .
: " .3-4-3
: 3-4-4 ,- -
: .'3-4-5
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
" ' 4-0-1
: 4-0-2
: 4-0-3 ' .
: 4-0-4
: 4-0-5
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0 - - -
' ".4-2-1
: . 4-2-2 '
: " 4-2-3
: '4-2-4
: . 4-2-5\.
MPN Index
0.0255
' 0,0330
0.0417' .
0.0506
0.0598
0.0631
0.0344
0.0435
0.0529
0.0626
0.0725
0.0827
0.0456
0.0555. ".
0,'Q657
00763
0.0872 .
'0,0984
0.0583
0.0693
0.0806
0.0924
0.1046
0.1173
0.0733
0.0858 '
0.0984
0,1118
0.1258
0.1405'
0.0913
0.1055
0.1204
0.1362
0.1529
0.1707
0.0381 ,
0.0461
0,'0563 '
. OQ6S8
0.0777
'0,0890
0.0484
0.0592
0.0705
0.0822
0.0945
0.1072
0.0626----
' 0.0748 '
0.0875
0,1009
0.1150 '
0.1299 .
95% Confidence Limits
Lower
0.0028-
0,0063 .
0.0103 .'
0.0147
0.0193'
0.0241
0.0069
0.0112
0.0159
0.0207
0.0258
0.0310
0.0122...
0.0171 '
0.0223
0.0277
0.0333- -
0.0390 "',
0.0186
0.0241
0.0299
0.0359
0.0421
0.0484
0.0262
0.0325 '
0,0390
0.0457
Q.Q526
0.0597.
0.0354
0.0426
0.0500
0.0577
0.0656
0.0738
0.0082
0.0125 -'
0.0175
0.0229
0.0284 '
0.0342
0.0136
0.0190
0.0248
0.0308
0.0370
0.0434
0.0207
0.0269
0.0335
0.0403
0.0473
0.0546
Upper
0.0585
0,0710
0,0863
0.1023
0.1191
0.1368' ' '
0.0734
0.0896
0.1065
0.1244
0.1434
0.1640
-0.0932 - - -
0,1112
0,1303 .
0.1510
0,1735 -,
0.1984
0.1164
0.1371
0.1597
0.1847
0.2128
0.2452
0.1450
0.1700
0,1982 >
0,2307 '
.
.0,3184
0.1825
0.2150
0.2538
0.3029
0.3715
0.4795
-0.0809
0,0942 '
0,1126
0.1323. :
0.1537'
0.1773 -' ,
0.0983
0.1181
0.1395
0.1631
0.1894
0.2193
0.1244
0.1479
0,1742
0,2041 '
0.2392 .
.0.2820 , -
Combination of
Positives
4-3-0
473-1 '..'
' .4-3-2. .
. 4-3-3 : : '
4-3-4" '.
4-3-5 -, - -
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-0
4-5-1 . ' : '
'' .4-5-2 ' '
4-5-3 '..
. 4-5-4 -
-. 4-s-s :
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-1-0
5-1-1,'-..'
\',5-1-2' ,
' 5-1-3 '
,5-1-4
5-1-5 '
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0
5-3-1.'-.: '
' '.5-3-2' '
5-3-3. . "
,5-3-4 '
' 5-3-5
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
5-5-0
5-5-1,' .'
''.',5-5-2,,. '
. 5-5-3- .
,5-5-4 .-' '
5-5-5 ' '
MPN Index
0.0797
" 0.0937 .
:- 0:1086 .
: 0,1245
: ''0,1414
: 0.1595
0.1012
0.1181
0.1364
0.1563
0.1780
0.2015
---0.1304
' 01524
:. 0.1769
: 0.2046 -.
: 0.2357 .
: 0.2708
0.0549
0.0637
0.0763
0.0896
0.1037
0.0953
0.0678----
" 0.0816'
:- 0.0963 '-
: 0.1121
: '0.1291
: 0.1293
0.0879
0.1046
0.1227
0.1427
0.1646
0.1767
---0.1151 ---
' 01368 '
:. 0.1614 '
: 0.1895
: 0.2216
: 0.2527
0.1571
0.1907
0.2319
0.2834
0.3475
0.4256
0,2398----
" 0.3477'
:- 0.5422
: 0.9178
: '1,8090
: >'1,8Q9G-
95% Confidence Limits
Lower
---0.0295
' 0,0366" ' ' ..
:. 0.0441 '
: 00520
: .0,0602 ;
: 0,0686
0.0404
0.0489
0.0578
0.0672
0.0770
0.0873
0.0549 - -
" 0.0653
:- 0,0766""--
: 0,0886 -
: -.0,1.015 '
: -0.1150'
0.0162
0.0213
0.0277
0.0345
0.0417
0.0165
---0.0234
' 0.0304. '
: 0.0379 '
: 0,0459
: .-0,0542
: -0.0304
0.0337
0.0421
0.0511
0.0608
0.0710
0.0503
0.0474 ----
" 0.0680'
:- 0,0695 -
: 0,0821
: '.0.0957
: -0.0814 -
0.0676
0.0826
0.0999
0.1196
0.1417
0.1437
---0.0762 - -
' 0.1172 -
: 0,1791 ' '
: 0.2672
: ..Q.3837 '
: -0,3837
Upper
0.1579 - -
0.1877
0,2228
. -
0.3218 .
0.4067
0.2049
0.2476
0.3038
0.3890
0.5273
0.641 1
..0,2836
0.3687 '
05210
0.8528
-0,7518 .
0.8426
0.1116
0.1265
0.1510
0.1787
0.2107
0.2234
0.1344
0.1618
0.1936
0.2316
0.2796 -
0.3090
0.1751
0.2128
0.2605
0.3267
0.4385
0.5230
..0,2394
0.3050
0:4183 -
0,5899
-0.710-1
0.7971 ,
0.3935
0.5954
0.7409
0.8726
1.0160
1.1800
0.7829 - -
1.0180"
1,4190
2,2010
4.1030 .
^_..
Table was developed using the MPN calculator developed by Albert Klee (Reference 17'. 10).
23
May 2012
-------
Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 8. MPN Index and 95% Confidence Limits for Various Combinations
When Five Tubes are Used per Volume and Inoculation Volumes are 10.0
Combination
of Positives
:----;:::::OiO-0/"-"""?
;x.\;0-b-i;;';'::n:
':::^^&.':
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
S'^^S
:;.;- :... 0-2-2;; .X;/;;;
,-. :.':-'-:0-2-3<. : v-:.
;; -r^:4- ..;:; V:.:;:
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
|-y * f\
i.-:''-':'..- -. ~=J,"-'A.'' ''.'''
'_ .,-: ;;. p-4-4/. ' . /;
'"' "- ri'2[ ^ " "''
- '- ' VJ-*t--v? .. / ... ./.
/ , U,.' '.' '-,:' .'.'.- ' "'
'. ' Q-4-4 ' ' '' '' '" '
A;: XtM-si-; :;
0-5-0
0-5-1
0-5-2
0-5-3
0-5-4
0-5-5
.;;-.-.-.-.-.-.-.-;f.iQ.Q ;;..-.....;
v;.'".v: :\-QA-'- ^''-":':
:y:;^;-1;0*2.;y;;oV:'
''''' "i n *-E
.. . '. .' .[ -lJ-ซ3 . .-.-
:','..',;.'. -].j(ji4-:i;'; ';"'.:'.
'-.!: .':.-.-i^i6.:'-v--.;;;.--.:;
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
;-;;.;-.-.-.-.-.-.^.2.o;;;--;-----:
;v:-',;':;;1-2-i: .' :
:;;-, 1-2-2 ;'::::.v
v/xXl-2-3 rr;,;.
: .... '; .. "\ _2-4 "' ' '"v
'' ' '. >''-. -!,' ' ' '
1-2-5
MPN Index
^O.DIS;:.;,;
'::.':'-0:dl8P:; '. '
:-:"' -vddse'Jvv".
;'. "^0.054': '0'.:"
;.:;?;d:W2;-...;.;;
0.018
0.036
0.055
0.073
0.091
0.110
'v.'.d.OS5>'/.;'
:--:'. 0:074-':'. A
;;x 0;092 :>,
:'-:^K--;[
0.056
0.074
0.093
0.112
0.130
0.149
r\ n-jc
-;:doi44'0
l--:;0:1|2^;v
/;>-r'd;i'90; ',';;
:;::;b:169.'v-;..
0.094
0.113
0.133
0.152
0.171
0.190
"/'' 0.020,.,.
':':. 0.040 '::
"-.' :o:Q6b:X
;-':. OOSI. ;".'.-
. ;^;dibi >
':. '.'' d.'t22'-'-''-':.-:
0.040
0.061
0.081
0.102
0.123
0.144
./: -::0.P51. :....,,
;-... '.Q.p82.:,;.. .;
. ; ;:Q:1bS;>:: :
" '" "''~C\ -"\fAf\ ' '
. .- . \J. -\ HrD :
95% Confidence Limits
Lower
:-;:::::,--;::::::::';
;0;b03 '
::/: 0,003" X,
..; '.o,OCJ8-'.:.'y.-
0.003
0.003
0.003
0.008
0.015
0.023
Y,^o:o63-;^:;;
.-.^."ddos":.::-.^
.... .'.vdOl $.;.. :. "''..
'.''.' ' U,U-Z-V . "
!. '' ' ' ' '' : '
0^03
0.009
0.016
0.023
0.031
0.039
A:..f"urU3
v;?p;pid::i.;
/;:': b,d24V /;
': ' '".. . " -.'._'.'
:;xOK048- ;.:,:
0.016
0.024
0.032
0.040
0.048
0.056
r;;;..0;OQ3.'.::^3
v,; OJOOS 3: : :
-.;". 0,003 'y{.y
,-. ;/b.011.. : ::;..
:X>."djdis '',.'''
;:,. o..oi28;: : .
0^03
0.003
0.011
0.019
0.028
0.037
:";;.;--0:OQ3.'.:':;-;-.
. * - '.
: ;:o,02o:.:;,::
;;,.;vbQ29,::g-
:;>:' 0,047 ;;:f
Upper
;.VrvO;063'.:-"""v
' 0 063 :'''<
'"'' ^ ' i >,_ . " "
01 37
, "-.., ; .. ' '.". ' ......
0.063
0.101
0.138
0.175
0.214
0.256
">: "'. 0l'39-;' .'.''
"': *;'&\76:-':.:-'
:':-;:."b:215>;x.V
:;- :;:^M-.:'/:;^
0.140
0.177
0.217
0.260
0.310
0.372
A'l TQ
'v ^0.210%'' /;:'
':;.";: b,263;, ';;.
'v'.';,d37y^.-':v
^^.'r^dfe :.;:':'
0.221
0.265
0.317
0.382
0.470
0.563
: ,.0.068 ....
v -; 0:,1'pS:>.v:V.
:>'" :d'.149--- ''.'.'":
''I ':'0:tSi.T ..'.'';''
;.',;:. ;:,o;236: '---v
:.; :v':/d.- 287 'v''''
0.109
0.150
0.192
0.238
0.290
0.354
,.. 0.151 ...
":'> a:i94i$i'
:-'.-.'';6.246-'.::r;.v
;';:;:;b;293?;r;
"' 'WjBH ':':- "''
. . : ...0.451 x
Combination
of Positives
;;.: :.:.:;Tr3>0:;:-.;.":::::
;i .-.,. '1^34'}:'. ;"''-..
5S?}HXI
;'-H--::^.-V.-;^
1-4-0
1-4-1
1-4-2
1-4-3
1-4-4
1-4-5
.,.' ' ' "*1"f^'- *| ' '''
;/;. ' ','.- V- .' : ... ' '
.[ "' . .' . . ,-: ....
'' '' ' ''' ' A C '*!5'; " ' '
;/.;?: -i'Ji5-4\-'V^
':'.',.-.. /. '.? ' '
..' '. '.i^Kig'.- '.- ''
2-0-0
2-0-1
2-0-2
2-0-3
2-0-4
2-0-5
O"t n
ฃ$ฃ*; "&
]>/.::\-?^~?'-y-:'\-'f
:.-v;':.^*l"3.';,:<:;:
:,...;. ..' 2-T.-4 ,' ../,
:'.".:'-2ilซi5>i: '.-';
2-2-0
2-2-1
2-2-2
2-2-3
2-2-4
2-2-5
O o r\
^"*O-\J-
.f.'..::-;'vl2^3r1-! '..- ^'.;
'.."': 2H3i2;";.-"':'.
; :\;-;.2?3^3' ::;;; .::
;:::-.:;-'2-3-4.':?;.'
:'. "' '2-3^5 : '.;:;.;.;:
2-4-0
2-4-1
2-4-2
2-4-3
2-4-4
2-4-5
.2-5-Q. : .'
.f.;.Vvl2^i\;.,,-'''.;
':,". '.,-.2-^-2- '";'/
V;-;<-:^^-:;1.';
V;:V2ii;-'^
MPN Index
;,: 0.083 :
: ' 0 104
-;Ug;^
.'". ' .''.
' ' 'n ^''Qi '' " ' '
0.105
0.127
0.148
0.170
0.193
0.215
-.'dfsd^l
:-":-.d-i'72 ;v-
'" V.-.: ,O1 95, .'.':'..;:
0/>rO:2f7:,r;-
0.045
0.068
0.091
0.115
0.139
0.164
. n ACXJ .
/; .U.UHDO .
.' ;' '". ":-' ':'''
/:i;.'d-;i^'' .'
' . -,- /._ : -_^i ' ' .'".
-'01 32 '
0.093
0.118
0.143
0.168
0.194
0.221
; ;.; ::0.119.
V. .0.144
: :- ::0/t7o:: ;V-
;K:.0.197,:.".- '/
;:^r-0:223 ; ^'.
: d251 :,
0.146
0.172
0.199
0.226
0.254
0.282
'/:'::0.'1.74- .,.,;
:;.. 0.201 v^,>.
x"';:0;229:. .;.!
'v^^.Vfl.
'^Mws'-y
of Positive Results
1.0, and 0.1 mL 1
95% Confidence Limits
Lower
::::Q.Q12. :::
'i :0 '020 ':' '' '.'''
':-:- ';-d029;;-:xr;:
;..?.' ';d048:; ':',;
0.021
0.030
0.039
0.048
0.058
0.067
v^UQ4d'.-;:;
: : ; 0,049 .C:;
,'.-. :.':-'d;o58'----;',\.'-.
'-': 0,.068'.' - .'
.- \ . 'A fV7.~7-' '. ' .
0.003
0.006
0.015
0.025
0.035
0.046
r. AAR
v^rolbis;;^;-
.-:yo;(j25.^.-;-..-.
;U.v' do46.;i::.;-
:.:: :o,b57;' ::/:;;'
0.016
0.026
0.036
0.047
0.058
0.069
: "iiV ''0,026 : ;
' '' ' '' ri A*3i*' ' " ' '
. ''',. UtUOt*- ' . '
: : ;0>048l:; .';
... .:.':.d;069".''-.:'.:;
C--':.' 0,070-' ;';';'.
'v-'.6j682::.V::
0.038
0.049
0.060
0.072
0.083
0.094
.::.: 0,050 :
'' ' '' f\ /"ICM
. . . UtUO 1 .': '. ..'
: : 0!073:,: .
,;'.. -0-OS4;;;: -:
:-:i ,o;:i07-:? ;
Upper
:: ::::0.1.p6 ::::::
0 243
..-. .!''-. ' -- '"' .'' ''' '."
; Q,296 .. .
0 460
-.' '. ;' : ; .: -.-:'.''.= ':
0.245
0.300
0.370
0.468
0.575
0.657
:''''/ '" .-.''. . '.
; ; . '.0/477"-:.-'.; .
,-. v-ttSQS/v'O.
-.'../f.-'o.aw.-.'-^1
'.' ' '.':'-- :. - '' ' ."
0.119
0.164
0.213
0.269
0.338
0.437
r. j &*ฃ* .
'. .. 0.21.6 :
':';;.. ^^-y"
?;:;/--:b;44t::^v.
:' '. O.Sri '. v;>:
0.218
0.276
0.349
0.456
0.581
0.675
;;;---0/279 :
;;:;;: 0:355;'.;;.;;:
.... v-0.591'. - ;,'
;..,;. 0,683
'' '0.759-r V-.;
0.361
0.477
0.600
0.692
0.768
0.836
r;;;---o, 48S'.; ;;-
;:;-',;'d6id;%:-;:
: :' 0;7,0p;...::; ;
;;,.:.;.o.776-;;;.;:
^'pigtct^y
24
May 2012
-------
Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Table 8. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes are Used per Volume and Inoculation Volumes are 10.0,1.0, and 0.1 mL (cont.)1
Combination
of Positives
MPN Index
95% Confidence Levels
Lower
Upper
Combination
of Positives
MPN Index
95% Confidence Limits
Lower
Upper
3-0-0-
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
3-1-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
;3-4-p;-
3-4^1
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
: 0.229;:
0.107
0.137
0.167
0.199
0.232
0.267
,0:1.38.:
0.172
0.205
0.240
0.276
0.313
0.352
,0209
,0.319 -
0.248
0.286
0.325
0.365
0.407
0.450
,0:130.
0.169
0.212
0.258
0.310
0.365
0.425
.0.046
0.022
0.034
0.047
0.060
0.074
0.088
0.035
;o648:
-..090"-
0.049
0.063
0.077
0.092
0.106
0.120
OOS4*
-0.093'.
0.080
0.095
0.110
0.125
0.140
0.154
; 0.046:
-0064;
0.048
0.066
0.085
0.105
0.125
0.145
:-""0;188
A;'0.246.:.
0.250
0.329
0.452
0.601
0.710
0.800
0,335;
0.477
0.624
0.731
0.821
0.906
0.989
0.636
. 0.833;
0.753
0.844
0.931
1.017
1.103
1.189
0,311;
. -0.631";
;.' 0:881-
0.460
0.646
0.779
0.898
1.016
1.138
0.661;
0,915:
4-3-0
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-0,
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0.
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
"5-5-0;
,,: 0.271
:.: 0-451.
0.335
0.398
0.466
0.539
0.615
0.693
: 6,483 ;
: 0,559;"
0,639^
0.240
0.314
0.427
0.578
0.759
0.953
:: 0,329
0,631;;
0,839;,
0.493
0.700
0.944
1.205
1.479
1.767
:: 0,792
2.122
1.299
1.724
2.212
2.781
3.454
4.256
: 16:090. '
0.114
0.137
0.159
0.181
0.202
0.223
/-'OL187':
0.076
0.106
0.146
0.192
0.239
0.165
-. 0:304 :
0.167
0.224
0.280
0.331
0.381
0.503
-.529 :
0.348
0.429
0.563
0.882
1.159
1.437
-1.060.
.;1:;477:
0.953
1.084
1.223
1.368
1.521
1.681
0.763
0.908
1.142
1.446
1.816
2.234
:0.940.::
1.276
1.694
2.213
2.843
3.714
5.230
3.108
4.975
7.087
8.600
10.110
11.800
1 Table was developed using the MPN calculator developed by Albert Klee (Reference 17.10).
25
May 2012
-------
Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
13.0 SAMPLE SPIKING AND PERCENT RECOVERY CALCULATION
QC requirements (Section 9) include the preparation and analysis of spiked reference (PBS) samples in
order to assess initial and ongoing method performance and matrix effects. For IPR (Section 9.2), OPR
(Section 9.3), and MS (Section 9.4) analyses, it is necessary to spike samples with Salmonella
typhimurium (ATCCฎ 14028/NCTC 12023) BioBallฎas described below.
13.1 Sample Spiking
13.1.1 Open vial and aseptically add 1 BioBallฎ to 200 ml of sample. Mix by vigorously
shaking the bottles a minimum of 25 times.
13.1.2 Analyze samples according to Section 11.
13.2 Calculation of BioBallฎ Spike Percent Recovery
Since one BioBallฎ is spiked per 200 mL sample, use the S. typhimurium lot mean value provided by the
manufacturer as the "true" spiked S. typhimurium. That is:
T spiked s. typhimurium (CFU/200 mL) = BioBallฎ lot mean value
Calculate percent recovery (R) using the following equation:
_ 100x(Ns- Nu)
K ~ T
1 Spiked S. typhimurium
Where,
R = Percent recovery
Ns = S. typhimurium (CFU/200 mL) in the spiked sample)
Nu = S. typhimurium (CFU/200 mL) in the unspiked sample
Note: For recovery calculations, when no background is observed in the unspiked sample, the
detection limit should be used for Nu in the calculations, instead of zero. See first example in
Table 9, below.
Percent recovery example calculations are provided in Table 9.
Table 9. Percent Recovery Example Calculations
Ns (CFU/200 mL)
25
39
Nu (CFU/200 mL)
<1
10
T Spiked S. typhimurium
(CFU/200 mL)
27
27
Percent recovery (R)
100 x(25-1)/27 = 89%
100 x (39 -10) 727 = 107%
26
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
14.0 PROTOCOL PERFORMANCE
A summary of protocol performance based on the single-laboratory verification and multi-laboratory
validation studies are provided in Sections 14.1 and 14.2, respectively.
14.1 Single-Laboratory Verification Study
Culture-based procedures were evaluated for S. typhimurium in a reference matrix (PBS) and two
matrices of interest (drinking water, surface water) during a single-laboratory verification study.
Results are based upon recovery of a single strain laboratory strain of Salmonella (S.
typhimurium ATCCฎ 14028); results may vary when assaying for environmental strains.
Details regarding procedure performance are provided in the study report (Reference 17.11).
Single laboratory verification results are provided in Table 10.
Table 10. S. typhimurium Results for PBS, Drinking Water, and Surface Water Samples
Sample
ID
Spike Level
(CFU/100mL)a
MPN
Combo
S. typhimurium
(MPN/100 mL)
Percent
Recovery
Mean
Recovery
(%)
SDb
(%)
RSDC
(%)
PBS Samples
Unspiked
Spiked
NA
15.25
0-0-0
5-3-1
5-4-1
5-4-0
5-4-0
< 0.6473
13.68
19.07
15.71
15.71
NA
85.46
120.80
98.77
98.77
100.95
14.65
14.51
Drinking Water Samples
Unspiked
Spiked
NA
15.25
0-0-0
0-0-0
4-3-1
5-4-0
4-3-3
4-5-0
< 0.6473
< 0.6473
9.37
15.71
12.45
13.04
NA
NA
57.20
98.77
77.39
81.26
78.66
17.06
21.69
Surface Water Samples
Unspiked
Spiked
NA
15.25
0-0-0
0-0-0
5-5-0
5-5-0
4-3-1
5-5-0
< 0.6473
< 0.6473
23.98
23.98
9.37
23.98
NA
NA
153.00
153.00
57.20
153.00
129.05
47.90
37.12
Colony forming unit per 100 milliliter
b Standard deviation
c Relative standard deviation
27
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
14.2 Multi-Laboratory Validation Study
Ten volunteer laboratories participated in EPA's multi-laboratory validation study of evaluating
this protocol. A detailed description of the study and results are provided in the validation study
report (Reference 17.3). The Study included the analysis of PBS, drinking water, and surface
water samples spiked with S. typhimurium BioBallฎ spikes. The results of this Study were used
to assess method performance (i.e., recovery and precision) across multiple laboratories and
matrices, compare effect of holding time (0-8 hours after sample spiking, compared to 30 ฑ 1
hours after sample spiking), assess reproducibility of results from analyses of "blind" samples
collected from each of two sites per matrix (drinking water and surface water), and develop QC
acceptance criteria. Results submitted by laboratories were validated using a standardized data
review process to confirm that results were generated in accordance with study-specific
instructions and the April 2010 draft version of the protocol.
14.2.1 Validation and Holding Time Analyses - Recovery and Precision
For this assessment, Laboratory 7's data were excluded even though the 0-Hour data
were valid, because including 0-Hour results when there were no 30-hour results (30-
Hour analyses were not conducted) would bias the analysis.
14.2.1.1 PBS
For PBS, the mean recovery measured at Time-0 did not differ significantly
from the mean recovery measured at the 30-Hour holding time (p-
value=0.0797). The overall mean recovery for Time-0 in PBS, excluding
Laboratory 7, was 137.41% with a pooled within-laboratory relative standard
deviation (RSD) of 57.62%; laboratory-specific mean recoveries ranged from
95.05% to 270.56%. The overall mean recovery for 30-hour PBS was
101.06% with a pooled within-laboratory RSD of 37.13%; and laboratory
specific mean recoveries ranged from 60.87% to 162.39%.
14.2.1.2 Drinking Water
For drinking water, the mean recovery measured at Time-0 did not differ
significantly from the mean recovery measured at the 30-Hour holding time (p-
value=0.7915). The overall mean recovery for Time-0 drinking water,
excluding Laboratory 7, was 117.65% with a pooled within-laboratory RSD of
47.71%; laboratory-specific mean recoveries ranged from 69.62% to 162.77%.
For the 30-hour holding time, drinking water had an overall mean recovery of
122.71% and a pooled within-laboratory RSD of 61.79%; laboratory-specific
mean recoveries ranged from 41.12% to 182.92%.
14.2.1.3 Surface Water
For surface water, the mean recovery measured at Time-0 differed
significantly from the mean recovery measured at the 30-Hour holding time (p-
value=0.0423). The overall mean recovery for Time-0 surface water,
excluding Laboratory 7, was 79.60% with a pooled within-laboratory RSD of
79.25%; laboratory-specific mean recoveries ranged from 1.14% to 199.82%.
For the 30-Hour holding time, there was an overall recovery of 58.10% and a
pooled within-laboratory RSD of 66.01%; laboratory-specific mean recoveries
ranged from 0.00% to 95.17%. It should be noted that the significant
28 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
difference in holding time was strongly influenced by the Laboratory 10,
Time-0 recoveries which were 71.23%, 223.76%, 351.3%, and 153.00%
resulting in a mean laboratory-specific recovery of 199.82% - more than twice
as high as the other laboratories. When data from Laboratory 10 are removed,
there is not a significant difference in recoveries between Time-0 and 30-hour
results (p-value=0.2221).
For PBS and drinking water matrices, recoveries at Time-0 did not differ significantly compared
to the 30-Hour holding time, indicating that these samples can be held at <10ฐC, but above
freezing for up to 30 hours without detrimental effect (i.e., samples can be shipped overnight).
While a significant difference was observed between Time-0 and 30-Hour results for surface
water, this was largely driven by unusual results from a single laboratory. In most cases, holding
surface water samples for 30 hours did not appear to have a detrimental effect on recovery.
14.2.2 Assessment of Reproducibility
Reproducibility data were analyzed for each matrix (drinking water sample 1, drinking
water sample 2, surface water sample 1, and surface water sample 2). The main goal of
this analysis was to assess whether any laboratory yielded significantly different mean
MPN/100 mL results from any other laboratory, thereby indicating that results were not
fully reproducible at different laboratories when the same "blind" sample was being
analyzed across all laboratories.
14.2.2.1 Drinking Water
Laboratory-specific mean MPN/100 mL for drinking water sample 1 ranged
from 11.39 to 34.52 with laboratory-specific RSDs ranging from 13.15% to
110.96%. For drinking water sample 2, laboratory specific mean MPN/100
mL ranged from 11.59 to 24.05 with laboratory-specific RSDs ranging from
1.29% to 86.41%.
There was not a significant difference between laboratories for either drinking
water matrix (F-test p-values of 0.465 and 0.516 for drinking water samples 1
and 2, respectively).
14.2.2.2 Surf ace Water
Laboratory-specific mean MPN/100 mL for surface water sample 1 one ranged
from 0.69 to 21.09 with laboratory-specific RSDs ranging from 5.16% to
82.53%. For surface water sample 2, laboratory-specific mean MPN/100 mL
ranged from 1.09 to 16.23 with laboratory-specific RSDs ranging from 16.42%
to 107.79.
Significant differences in mean MPN/100 mL were observed between at least
two laboratories for surface water sample 1 and also for surface water sample
2 (F-test p-values of 0.013 and 0.017 for surface water samples 1 and 2,
respectively).
Because there were no significant differences in mean MPN/100 mL values between
laboratories for drinking water matrices, it appears that laboratory results are
reproducible. The differences in mean MPN/100 mL values observed in the surface
water matrices may be due to varying degrees of laboratory proficiency with the method,
29 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
as at least one laboratory retrospectively suggested that, they should have submitted
more "questionable colonies" from MSRV to confirmation. In addition, another
laboratory indicated that background bacteria from the TSB enrichment made it very
difficult to identify presumptively positive colonies on MSRV. As a result, it is critical
that laboratories become proficient with this protocol prior to analyzing samples from
complex surface water matrices, as inappropriately low results may be reported.
15.0 POLLUTION PREVENTION
15.1 The solutions and reagents used in this procedure pose little threat to the environment when
recycled and managed properly.
15.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
16.0 WASTE MANAGEMENT
16.1 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.
16.2 Samples, reference materials, and equipment known or suspected to be contaminated with viable
Salmonella must be sterilized prior to disposal.
16.3 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel (Reference 17.12) and Less Is Better: Laboratory Chemical Management
for Waste Reduction (Reference 17.13), both available from the American Chemical Society's
Department of Government Relations and Science Policy, 1155 16th Street NW, Washington, DC
20036.
17.0 REFERENCES
17.1 U.S. Environmental Protection Agency. July 2006. Method 1682: Salmonella in
Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium. EPA-821-
R-06-14. Washington, DC: U.S. Environmental Protection Agency, Office of Water.
http://www.epa.gov/waterscience/methods/method/biological/1682.pdf
17.2 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Office of
Health and Safety. 2007. Biosafety in Microbiological and Biomedical Laboratories, 5th
Edition. Atlanta, GA: National Center for Infectious Diseases, Centers for Disease Control and
Prevention. http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm
30 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
17.3 U.S. Environmental Protection Agency. Multi-Laboratory Validation of the Analytical Protocol
for Non-Typhoidal Salmonella in Drinking Water and Surface Water. Publication forthcoming;
date and number to be determined.
17.4 Gupte, A.R., de Rezende, C.L.E., and Joseph, S.W. 2003. Induction and Resuscitation of Viable
but Nonculturable Salmonella enterica Serovar Typhimurium DTI 04. Applied and
Environmental Microbiology. 69(11): 6669 - 6675.
17.5 American Chemical Society. 2000. Reagent Chemicals, American Chemical Society
Specifications. New York, NY: American Chemical Society.
17.6 British Drug Houses, Ltd. 1957. AnalaR Standards for Laboratory Chemicals. 5th Edition.
Poole, Dorset, U.K.: BDH, Ltd.
17.7 United States Pharmacopeia. 2005. United States Pharmacopeia and National Formulary 24.
Rockville, MD: United States Pharmacopeial Convention.
17.8 Bordner, RH. 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, DC: American Public
Health Association, American Water Works Association, and Water Environment Federation.
17.9 Wise, J. 1988. NISTMeasurement Services: Liquid-In-Glass Thermometer Calibration Service,
SP 250-23. Washington, DC: U.S. Department of Commerce, National Institute of Standards
and Technology. http://ts.nist.gov/MeasurementServices/Calibrations/upload/SP250-23.pdf
17.10 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.
17.11 U.S. Environmental Protection Agency. Single-Laboratory Verification of Culture-Based
Analytical Procedure for Non-Typhoidal Salmonella in Drinking Water and Surface Water.
Publication forthcoming; date and number to be determined.
17.12 American Chemical Society (ACS). 1990. The Waste Management Manual for Laboratory
Personnel. Washington, DC: American Chemical Society Department of Government Relations
and Science Policy.
17.13 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.
17.14 Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). Microbiological Methods for Monitoring the
Environment: Water and Wastes, EPA-600/8-78-017. Cincinnati, OH: U.S. Environmental
Protection Agency, 1978.
31 May 2012
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_ Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
18.0 FLOWCHARTS
18.1 Quantitative^Analysis DiIution Scheme
Distribution of Sample to TSB
Section 11.2.1
Incubation at 36.CTC ฑ 1 5"C for 24 ฑ 2 hours
Section 11.2,3.
10.0 mLo each
. tubeofSXTSS (5 ml)
Serial dilutions as
necessary
! I 0 ml to each lube of
r
1X TSB (10 ml)
1H
at ฑ
1.5ฐCfor24ฑ2hours
Analysis of
positive tubes
32
May 2071
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
18.2 Identification Flowchart
Enrichment fn TSB and Isolation on MSRV
Plates (Section 11 3) or PCR Confirmation
Qualitative and
Quantitative (MPN)
analyses in TSB
tubes
___SpotซTte_
\ ' '
MSRV
Sub-culture on XLD Plates
Section 11.4
XLD
. \ Streak colony /
\
'-~-^^'
Incubate at 42.CTC ฑ
0.5ฐCfor16-18hours
Incubate at 36-Gf?C ฑ
1,5ฐCfor 18-24 hours
Biochemical and
Serological Testing
Section 11,5
Urea broth
LIA
fMBL
TSi
Sa/monelfe serum
agglutination
*Procedure not included rn SAP
0
1
0
2
33
May 2011
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Appendix A:
Part II (General Operations), Section A (Sample Collection,
Preservation, and Storage)
The following is an excerpt from Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). Microbiological
Methods for Monitoring the Environment: Water and Wastes, EPA-600/8-78-017. Cincinnati, OH: U.S.
Environmental Protection Agency, 1978 [Reference 17.14].
34 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Sample Collection1
1.0 Sample Containers
1.1 Sample Bottles: Bottles must be resistant to sterilizing conditions and the solvent action
of water. Wide-mouth borosilicate glass bottles with screw-cap or ground-glass stopper
or heat-resistant plastic bottles may be used if they can be sterilized without producing
toxic materials (see examples A and C in Figure 1). Screw-caps must not produce
bacteriostatic or nutritive compounds upon sterilization.
u.
Figure 1. Suggested sample containers.
1.2 Selection and Cleaning of Bottles: Samples bottles should be at least 125 mL volume for
adequate sampling and for good mixing. Bottles of 250 mL, 500 mL, and 1000 mL volume are often used
for multiple analyses. Discard bottles which have chips, cracks, and etched surfaces. Bottle closures must
be water-tight. Before use, thoroughly cleanse bottles and closures with detergent and hot water,
followed by a hot water rinse to remove all trace of detergent. Then rinse them three times with
laboratory-pure water.
1.3 Dechlorinating Agent: The agent must be placed in the bottle when water and wastewater
samples containing residual chlorine are anticipated. Add sodium thiosulfate to the bottle before
sterilization at a concentration of 0.1 mL of a 10% solution for each 125 mL sample volume. This
concentration will neutralize approximately 15 mg/L of residue chlorine.
1.4 Chelating Agent: A chelating agent should be added to sample bottles used to collect samples
suspected of containing >0.01 mg/L concentrations of heavy metals such as copper, nickel or zinc, etc.
35
May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Add 0.3 mL of a 15% solution of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt, for each
125 mL sample volume prior to sterilization.
1.5 Wrapping Bottles: Protect the tops and necks of glass stoppered bottles from contamination by
covering them before sterilization with aluminum foil or kraft paper.
1.6 Sterilization of Bottles: Autoclave glass or heat-resistant plastic bottles at 121ฐC for 15 minutes.
Alternatively, dry glassware may be sterilized in a hot oven at 170ฐC for not less than two hours.
Ethylene oxide gas sterilization is acceptable for plastic containers that are not heat-resistant. Sample
bottles sterilized by gas should be stored overnight before being used to allow the last traces of gas to
dissipate.
1.7 Plastic Bags: The commercially available bags (Whirl-pak) (see example B in Figure 1) are a
practical substitute for plastic or glass samples bottles in sampling soil, sediment, or biosolids. The bags
are sealed in manufacture and opened only at time of sampling. The manufacturer states that such bags
are sterilized.
2.0 Sampling Techniques
Samples are collected by hand or with a sampling device if the sampling site has difficult access such as
a bridge or bank adjacent to a surface water.
2.1 Chlorinated Samples: When samples such as treated waters, chlorinated wastewaters or
recreational waters are collected, the sample bottle must contain a dechlorinating agent (see section 1.3
above).
2.2 Composite Sampling: In no case should a composite sample be collected for bacteriologic
examination. Data from individual samples show a range of values. A composite sample will not display
this range. Individual results will give information about industrial process variations in flow and
composition. Also, one or more portions that make up a composite sample may contain toxic or nutritive
materials and cause erroneous results.
2.3 Surface Sampling by Hand: A grab sample is obtained using a sample bottle prepared as
described in (1) above. Identify the sampling site on the bottle label and on a field log sheet. Remove the
bottle covering and closure and protect from contamination. Grasp the bottle at the base with one hand
and plunge the bottle mouth down into the water to avoid introducing surface scum (Figure 2). Position
the mouth of the bottle into the current away from the hand of the collector and, if applicable, away from
the side of the sampling platform. The sampling depth should be 15-30 cm (6-12 inches) below the water
surface. If the water body is static, an artificial current can be created, by moving the bottle horizontally
in the direction it is pointed and away from the sampler. Tip the bottle slightly upwards to allow air to
exit and the bottle to fill. After removal of the bottle from the stream, pour out a small portion of the
sample to allow an air space of 2.5-5 cm (1-2 inches) above each sample for proper mixing of the sample
before analyses. Tightly stopper the bottle and place on ice (do not freeze) for transport to the laboratory.
36 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
Figure 2. Grab sampling technique for surface waters.
3.0 Selection of Sampling Sites and Frequency
These will be described for streams, rivers, estuarine, marine, and recreational waters as well as
domestic and industrial wastewaters.
3.1 Stream Sampling: The objectives of the initial survey dictate the location, frequency and
number of samples to be collected.
3.1.1 Selection of Sampling Sites: A typical stream sampling program includes sampling locations
upstream of the area of concern, upstream and downstream of waste discharges, upstream and
downstream from tributary entrances to the river and upstream of the mouth of the tributary. For more
complex situations, where several waste discharges are involved, sampling includes sites upstream and
downstream from the combined discharge area and samples taken directly from each industrial or
municipal waste discharge. Using available bacteriological, chemical and discharge rate data, the
contribution of each pollution source can be determined.
3.1.2 Small Streams: Small streams should be sampled at background stations upstream of the
pollution sources and at stations downstream from pollution sources. Additional sampling sites should be
located downstream to delineate the zones of pollution. Avoid sampling areas where stagnation may
occur (e.g., backwater of a tributary) and areas located near the inside bank of a curve in the stream
which may not be representative of the main channel.
37 May 2012
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
3.1.3 Large Streams and Rivers: Large streams are usually not well mixed laterally for long
distances downstream from the pollution sources. Sampling sites below point source pollution
should be established to provide desired downstream travel time and dispersal as determined by
flow rate measurements. Particular care must be taken to establish the proper sampling points.
Occasionally, depth samples are necessary to determine vertical mixing patterns.
3.2 Estuarine and Marine Sampling: Sampling estuarine and marine waters requires the
consideration of other factors in addition to those usually recognized in fresh water sampling. They
include tidal cycles, current patterns, bottom currents and counter-currents, stratification, seasonal
fluctuations, dispersion of discharges and multi-depth samplings. The frequency of sampling varies with
the objectives. When a sampling program is started, it may be necessary to sample every hour around the
clock to establish pollution loads and dispersion patterns. The sewage discharges may occur continuously
or intermittently.
When the sampling strategy for a survey is planned, data may be available from previous hydrological
studies done by the Coast Guard, Corps of Engineers, National Oceanic and Atmospheric Administration
(NOAA), U.S. Geological Survey, or university and private research investigations. In a survey, float
studies and dye studies are often carried out to determine surface and undercurrents. Initially depth
samples are taken on the bottom and at five feet increments between surface and bottom. A random grid
pattern for selecting sampling sites is established statistically.
3.2.1 Estuarine Sampling: When a survey is made on an estuary, samples are often taken from a boat,
usually making an end to end traverse of the estuary. Another method involves taking samples throughout
a tidal cycle, every hour or two hours from a bridge or from an anchored boat at a number of fixed points.
In a large bay or estuary where many square miles of area are involved, a grid or series of stations may be
necessary. Two sets of samples are usually taken from an area on a given day, one at ebb or flood slack
water, and the other three hours earlier, or later, at the half tidal interval. Sampling is scheduled so that
the mid-sampling time of each run coincides with the calculated occurrence of the tidal condition. In
location sampling sites, one must consider points at which tributary waters enter the main stream or
estuary, location of shellfish beds and bathing beaches. The sampling stations can be adjusted as data
accumulate. For example, if a series of stations half mile apart consistently show similar values, some of
these stations may be dropped and other stations added in areas where data shows more variability.
Considerable stratification can occur between the salt water from the sea and the fresh water supplied by
a river. It is essential when starting a survey of an unknown estuary to find out whether there is any
marked stratification. This can be done by chloride determinations at different locations and depths. It is
possible for stratification to occur in one part of an estuary and not in another.
On a flood tide, the more dense salt water pushing up into the less dense fresh river water will cause an
overlapping with the fresh water flowing on top. A phenomenon called a salt water wedge can form. As a
result, stratification occurs. If the discharge of pollution is in the salt water layer, the contamination will
be concentrated near the bottom at the flood tide. The flow or velocity of the fresh water will influence
the degree of stratification which occurs. If one is sampling only at the surface, it is possible that the data
will not show the polluted underflowing water which was contaminated at the point below the fresh
water river. Therefore, where stratification is suspected, samples at different depths will be needed to
measure vertical distribution.
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Method 1200: Analytical Protocol for Non-Typhoidal Salmonella in Drinking Water and Surface Water
3.3 Recreational Waters (Bathing Beaches): Sampling sites at bathing beaches or other
recreational areas should include upstream or peripheral areas and locations adjacent to natural drains
that would discharge stormwater, or run-off areas draining septic wastes from restaurants, boat marinas,
or garbage collection areas. Samples of bathing beach water should be collected at locations and times of
heaviest use. Daily sampling, preferably in the afternoon, is the optimum frequency during the season.
Weekends and holidays which are periods of highest use must be included in the sampling program.
Samples of estuarine bathing waters should be obtained at high tide, ebb tide and low tide in order to
determine the cyclic water quality and deterioration that must be monitored during the swimming season.
39 May 2012
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