United States Office of Water EPA-821-R-14-012
Environmental Protection Agency (4303-T) September 2014
xvEPA
United States
Environmental Protection
Agency
Method 1682: Salmonella in Sewage
Sludge (Biosolids) by Modified
Semisolid Rappaport-Vassiliadis
(MSRV) Medium
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
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Method 1682
Acknowledgments
This method was prepared under the direction of Mark C. Meckes of the National Risk Management
Research Laboratory within the U.S. Environmental Protection Agency's (EPA's) Office of Research and
Development, and Robin K. Oshiro of the Office of Science and Technology within EPA's Office of Water.
The contributions of the following persons and organizations to this study are gratefully acknowledged:
Method Development
• Richard Danielson and Robert Cooper, BioVir Laboratory, 685 Stone Road Unit # 6, Benicia, CA
94510
• William Yanko, Private Consultant, 1111 El Sur Ave, Arcadia, CA 91006
Referee Laboratory
EPA Office of Research and Development, National Risk Management Research Laboratory: Mark C.
Meckes and Karen M. White
Volunteer Participant Laboratories
• American Interplex: John Overbey and Steve Bradford
BioVir Laboratories: Rick Danielson and Jim Truscott
• City of Los Angeles Bureau of Sanitation Environmental Monitoring Division: Farhana Mohamed and
Genevieve Espineda
• County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant (JWPCP):
Kathy Walker and Debbie Leachman
County Sanitation Districts of Los Angeles County, San Jose Creek Water Quality Laboratory (SJC):
Shawn Thompson and Julie Millenbach
• Environmental Associates: Susan Boutros and John Chandler
Hoosier Microbiological Laboratories: Don Hendrickson, Keri Nixon, Katy Bilger, and Lindsey
Shelton
King County Environmental Laboratory: Greg Ma and Bobbie Anderson
Texas A&M University: Suresh Pillai and Jessica Cardenas
• University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord
Utah Department of Health: Sanwat Chaudhuri and Devon Cole
Wisconsin State Laboratory of Hygiene: Jon Standridge and Linda Peterson
The following facilities provided biosolid matrices for the study
• Compost Facility, Columbus, OH: Angela Bianco
• Hyperion Treatment Plant, Playa del Rey, CA: Steve Fan
• N-Viro Treatment Facility, Toledo, OH: Cindy Drill
• Wastewater Treatment Facility, Sturgeon Bay, WI: Todd Maurina
• Wastewater Treatment Facility, Fairfield, OH: Drew Young
Wastewater Treatment Facility, Mason, OH: Ernie Stickler
Media Photographs
• Mark C. Meckes, NRMRL, US EPA
September 2014
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Method 1682
Disclaimer
The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and
approved this report for publication. Neither the United States Government nor any of its employees,
contractors, or their employees make any warranty, expressed or implied, or assumes 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 report, or represents that its use by such party would not infringe on privately
owned rights. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov
202-566-1075
202-566-1054 (facsimile)
September 2014
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Method 1682
Introduction
Application of treated biosolids to land can be helpful as a crop nutrient and soil conditioner but may pose
the risk of releasing pathogens into the environment if proper disinfection and use criteria are not met.
Among these organisms are Salmonella, which are pathogenic enteric bacteria that can cause salmonellosis
in animals and humans, if concentrations able to give rise to infections are present.
The density of Salmonella in Class A biosolids for unrestricted use is to be less than three most probable
number (MPN) per four grams of total solids (dry weight basis) at the time the biosolids are used or
disposed.
Method 1682 is a performance-based method for detecting Salmonella in biosolids. Method 1682 requires
calculation of the MPN via enrichment, with selection and biochemical confirmation for determination of
Salmonella. The enrichment step utilizes tryptic soy broth (TSB). After incubation, TSB is spotted onto
selective modified semisolid Rappaport-Vassiliadis (MSRV) medium. Presumptively identified colonies
are isolated on xylose-lysine desoxycholate agar (XLD). Biochemical confirmation includes lysine-iron
agar (LIA), triple sugar iron agar (TSI), and urea broth, followed by serological typing using polyvalent O
antisera. Calculations for concentration are based on dry weight.
iii September 2014
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Method 1682
Table of Contents
Acknowledgments i
Disclaimer ii
Introduction iii
1.0 Scope and Application 1
2.0 Summary of Method 2
3.0 Definitions 2
4.0 Interferences 2
5.0 Safety 2
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 5
8.0 Sample Collection, Handling, and Storage 10
9.0 Quality Control 12
10.0 Equipment Calibration and Standardization 17
11.0 Sample Preparation 17
12.0 Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium Procedure 19
13.0 Data Analysis and Calculations 23
14.0 Sample Spiking Procedure 26
15.0 Method Performance 33
16.0 Pollution Prevention 34
17.0 Waste Management 34
18.0 References 35
19.0 Figures 36
20.0 Glossary of Definitions and Purposes 38
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Method 1682
Method 1682: Salmonella in Sewage Sludge (Biosolids) by
Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium
September 2014
1.0 Scope and Application
1.1 This method is for the detection and enumeration of Salmonella (CAS registry number 68583-35-7)
in treated biosolids by enrichment, selection, and characterization. It is intended to enumerate
Salmonella to help determine the suitability of biosolids for land application in compliance with 40
Code of Federal Regulations (CFR) Part 503. Although Method 1682 is similar to existing
recognized procedures using separate media for enrichment, selection, and confirmation of the
organism, it is intended to be more specific and have greater recovery.
1.2 This method is designed to meet monitoring requirements for Salmonella under 40 CFR Part 503
Subpart D. Subpart D of the Part 503 regulation defines the requirements for biosolids to be
classified as either Class A or B with respect to pathogens. Classification of biosolids prior to land
application provides a means to protect public health and the environment. Following appropriate
treatment, a biosolid sample is classified as Class A if Salmonella densities are below 3 MPN / 4
grams of total solids (dry weight basis).
1.3 Although the Part 503 regulation does not specify the total number of samples for Class A
biosolids, it suggests that a sampling event extend over two weeks, and that at least seven samples
be tested to confirm that the mean bacterial density of the samples is below 3 MPN / 4 g of total
solids (dry weight basis). The analysis of seven samples increases the method precision by
reducing the standard error caused by inherent variations in biosolid quality.
1.4 Although Method 1682 is selective for Salmonella bacteria, it does not differentiate among
Salmonella species.
Note: H2S negative Salmonella will be missed if translucent pink to red colonies are not
submitted to biochemical and serological confirmation.
1.5 Method 1682 was submitted to interlaboratory validation in Class A biosolid matrices. A
comprehensive evaluation of the study results is presented in the validation study report (Reference
18.2). For method application please refer to Title 40 Code of Federal Regulations Part 136 (40
CFR Part 136).
1.6 This method is not intended for use in water samples or as a test for microorganisms other than
Salmonella. Use of this method and appropriate validation for matrices other than Class A
biosolids is the responsibility of the user.
1.7 Any modification of the method beyond those expressly permitted is subject to the application and
approval of alternative test procedures under 40 CFR Parts 136.4 and 136.5.
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Method 1682
2.0 Summary of Method
2.1 The modified semisolid Rappaport-Vassiliadis (MSRV) medium protocol presented in Method
1682 provides enumeration of Salmonella in biosolids based on the most probable number (MPN)
technique. The determination of Salmonella involves inoculating the enrichment medium, tryptic
soy broth (TSB), with a measured amount of sample and incubating for 24 hours. After
incubation, TSB is spotted onto the selective MSRV medium. The MSRV medium uses
novobiocin and malachite green to inhibit non-Salmonella species, while allowing most
Salmonella species to grow. Presumptively identified colonies are isolated on xylose-lysine
desoxycholate agar (XLD), and confirmed using lysine-iron agar (LIA), triple sugar iron agar
(TSI), and urea broth, followed by positive serological typing using polyvalent O antisera. A total
solids (% dry weight) determination is performed on a representative biosolids sample and is used
to calculate MPN / g dry weight. Salmonella density is reported as MPN / 4 g dry weight.
3.0 Definitions
3.1 Salmonella are gram-negative, predominately motile, facultatively-anaerobic, rod-shaped bacteria
that comprise about 2,000 serovars.
3.2 Class A biosolids are biosolids that meet bacteriological and treatment requirements stipulated in
the 40 CFR 503 Subpart D.
3.3 Definitions for other terms are provided in the glossary at the end of the method (Section 20.0).
4.0 Interferences
4.1 Low estimates of Salmonella may be caused by the presence of high numbers of competing or
inhibitory organisms, or toxic substances such as metals or organic compounds.
5.0 Safety
5.1 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 Field and laboratory staff collecting and analyzing environmental samples are under some risk of
exposure to pathogenic microorganisms. Staff should apply safety procedures used for pathogens
to handle all samples.
5.3 This method does not address all safety issues associated with its use. It is the responsibility of the
laboratory to establish appropriate safety and health practices prior to use of this method. A
reference file of material safety data sheets (MSDSs) should be available to all personnel involved
in Method 1682 analyses.
5.4 Mouth-pipetting is prohibited.
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Method 1682
6.0 Equipment and Supplies
6.1 Sterile plastic bags, 1-gallon
6.2 Sterile plastic or glass jars with lids, 1-L
6.3 Sterile auger
6.4 Sterile scoops (do not use curved scoops)
6.5 Ice chest
6.6 Wet ice
6.7 Ice packs, blue ice
6.8 Bubble wrap
6.9 Sterile trowels
6.10 Sterile aluminum foil or kraft paper
6.11 Sterile container, such as a stainless steel or plastic bucket suitable for sample collection
6.12 Flat shovel
6.13 Tubes, 25 x 150 mm, borosilicate glass, with loose-fitting aluminum, stainless steel or autoclavable
caps
6.14 Tubes, 16 x 100 mm, screw cap, borosilicate glass, with autoclavable plastic caps
6.15 Test tube racks to hold sterile culture tubes
6.16 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets
6.17 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, wide-tip of appropriate volume
6.18 Pipet bulbs, or automatic pipettor
6.19 Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic loops
6.20 Sterile disposable applicator sticks
6.21 Bunsen burner or alcohol burner
6.22 Cornwall syringe, sterile, to deliver at least 5 mL
6.23 Media dispensing pump
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Method 1682
6.24 Incubators, water- or air-jacketed, humidity-controlled, microbiological type to hold temperatures
at 36°C ± 1.5°C and 42°C ± 0.5°C
6.25 Plastic sterile petri dishes, microbiological grade, 15 mm x 100 mm
6.26 Glass slides for agglutination test
6.27 Erlenmeyer flasks, 1-L and 2-L
6.28 Stir bar
6.29 Stir plate
6.30 Sterile blender j ars and base
6.31 Water bath maintained at 50°C for tempering agar
6.32 Media filtration equipment, sterile, 0.22-(im pore size syringe filters
6.33 Magnifying glass or dissection scope
6.34 Latex gloves for handling samples and extraction equipment
6.35 pH meter
6.36 Vortex mixer
6.37 Micro pipettor
6.38 Pipet tips to deliver 30 (iL
6.39 Autoclave
6.40 Drying oven, maintained at 103°C to 105°C for tempering agar
6.41 Beakers, glass or plastic, assorted sizes
6.42 Lint-free tissues
6.43 Steel pan of water, 30" x 26" x 10"
6.44 Autoclave or steam sterilizer capable of achieving 121°C (15 Ib pressure per square inch [PSI]) for
15 minutes
6.45 Crucible or aluminum evaporating dish
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Method 1682
7.0 Reagents and Standards
7.1 Purity of reagents: Reagent-grade chemicals must be used in all tests. Unless otherwise indicated,
reagents shall conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society (Reference 18.4). The agar used for preparation of culture media
must be microbiological grade.
7.2 Whenever possible, use commercial culture media as a means of quality control. Storage
temperatures and times for prepared media and reagents are provided in Table 1 in Section 7.16
below.
7.3 Purity of reagent water: Reagent-grade water conforming to specifications in Standard Methods for
the Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or
141, as applicable), Section 9020 (Reference 18.1).
7.4 Phosphate Buffered Dilution Water
7.4.1 Composition of stock phosphate buffer solution:
Monopotassium phosphate (KH2PO4) 34.0 g
Reagent-grade water 500.0 mL
Preparation: Dissolve KH2PO4 in 500 mL reagent-grade water. Adjust the pH of the
solution to 7.2 with 1 N NaOH, and bring the volume to 1 L with reagent-grade water.
Sterilize by filtration or autoclave at 121°C (15 PSI) for 15 minutes.
7.4.2 Preparation of stock magnesium chloride (MgCl2) solution: Add 38 g anhydrous MgCl2 or
81.1 g magnesium chloride hexahydrate (MgCl2 • 6H2O) to 1 L reagent-grade water.
Sterilize by filtration or autoclave at 121°C (15 PSI) for 15 minutes.
7.4.3 After sterilization, store the stock solutions in the refrigerator until used. If evidence of
mold or other contamination appears, the affected stock solution should be discarded and a
fresh solution should be prepared.
7.4.4 Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer
and 5 mL of the MgCl2 stock per liter of reagent-grade water. Dispense in appropriate
amounts for dilutions and/or for use as rinse buffer. Autoclave at 121°C (15 PSI) for 15
minutes. Final pH should be 7.0 ± 0.2. The amount of time in the autoclave must be
adjusted for the volume of buffer in the containers and the size of the load.
Note: When test tube racks containing 9.0 mL sterile dilution water are prepared, they
are placed into an autoclavable pan with a small amount of water to contain
breakage and minimize evaporation from the tubes.
7.5 Sterile physiological saline (0.85% w/v)
7.5.1 Dissolve 8.5 g NaCl in 1 L reagent-grade water. Dispense 5-10 mL into 16 x 100 mm
screw cap test tubes, cap and autoclave for 15 minutes at 121°C (15 PSI). Store at room
temperature.
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Method 1682
7.6 Tryptic soy broth (TSB)
7.6.1 Composition:
Pancreatic digest of casein 17.Og
Enzymatic digest of soybean meal 3.0 g
Sodium chloride 5.0g
Dipotassium phosphate (K2HPO4) 2.5 g
Dextrose 2.5 g
Reagent-grade water l.OL
7.6.2 For single strength (IX) TSB, add reagents to 1 L of reagent-grade water, mix thoroughly,
and heat to dissolve. Adjust pH to 7.3 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide. Dispense 10 mL volumes into 25 x 150 mm culture tubes. The IX TSB will
be used for inoculation volumes <1 mL. Autoclave for 15 minutes at 121°C (15 PSI).
7.6.3 For triple strength (3X) TSB, prepare as in Section 7.6.2 but use 333 mL of reagent-grade
water instead of 1 L. Dispense 10 mL and 5 mL volumes into 25 x 150 mm culture tubes.
The 3X TSB tubes containing 10 mL of media will be inoculated with 20 mL of
homogenized sample. The 3X TSB tubes containing 5 mL of media will be inoculated
with 10 mL of homogenized sample. Autoclave for 15 minutes at 121°C (15 PSI). Let
the media warm to room temperature prior to analysis.
Note: 3X TSB is necessary for 20- and 10-mL inoculations to ensure that the inoculation
volume does not excessively dilute the media.
7.7 Modified Semisolid Rappaport-Vassiliadis (MSRV) medium
7.7.1 Basal medium 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 (37 mg)
Agar 2.7 g
Reagent-grade water l.OL
7.7.2 Novobiocin (2%) stock solution:
Sodium novobiocin 500 mg
Reagent-grade water 25 mL
7.7.2.1 Dissolve 500 mg of sodium novobiocin into 25 mL of reagent-grade water and
filter sterilize by passing solution through a sterile, 0.22(im pore-size filter into a
sterile container. Aliquot 1.1 mL of the stock solution into 2.0 mL cryovials
and freeze at -20°C.
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Method 1682
7.7.3 Add reagents for basal medium to 1 L of reagent-grade water, mix thoroughly, and heat to
boiling to dissolve completely (do not autoclave). Adjust pH to 5.2 ± 0.2 with l.ON
hydrochloric acid or 1.0 N sodium hydroxide, cool to 50°C, and add 1.0 mL of a 2% stock
solution of novobiocin per liter of medium. Mix well by swirling the medium.
Immediately pour approximately 25 mL into 15 x 100 mm petri plates. Do not invert
plates to store.
Note: If using a commercially prepared novobiocin antimicrobic supplement add
sufficient volume to achieve a concentration of 0.002% per liter.
7.8 Xylose-lysine desoxycholate agar (XLD)
7.8.1 Composition:
Yeast extract 3.0 g
L-lysine 5.0g
Xylose 3.75g
Lactose 7.5 g
Saccharose 7.5 g
Sodium desoxycholate 2.5 g
Ferric ammonium citrate 0.8g
Sodium thiosulfate 6.8 g
Sodium chloride 5.0 g
Agar 15. Og
Phenol red 0.08 g
Reagent-grade water l.OL
7.8.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to boiling to dissolve
completely, avoid overheating (do not autoclave). Adjust pH to 7.4 ± 0.2 with l.ON
hydrochloric acid or 1.0 N sodium hydroxide. Cool to 45°C - 50°C and immediately pour
approximately 12 mL into 15 x 100 mm sterile petri plates. Let the media warm to room
temperature prior to inoculation.
Note: Heating media to boiling sterilizes the media, overheating or autoclaving may
cause precipitation.
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Method 1682
7.9 Triple sugar iron agar (TSI)
7.9.1 Composition:
Beef extract 3.0g
Yeast extract 3.0 g
Pancreatic digest of casein 15.0 g
Proteose peptone No. 3 5.0 g
Dextrose l.Og
Lactose 10.0 g
Sucrose 10.0 g
Ferrous sulfate 0.2 g
Sodium chloride 5.0g
Sodium thiosulfate 0.3 g
Agar 12.0 g
Phenol red 0.024 g
Reagent-grade water l.OL
7.9.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Adjust pH to 7.4 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide. Dispense 5-7 mL aliquots into 16 x 100 mm screw cap test tubes, cap and
autoclave at 121°C (15 PSI) for 15 minutes. Allow medium to solidify in a slant rack or
rack that is tilted in such a manner that the surface area is equally divided between the slant
and butt. Let the media warm to room temperature prior to inoculation.
7.10 Lysine iron agar (LIA)
7.10.1 Composition:
Peptone 5.0g
Yeast extract 3.0g
Dextrose l.Og
L-lysine hydrochloride lO.Og
Ferric ammonium citrate 0.5 g
Sodium thiosulfate 0.04 g
Bromcresol purple 0.02 g
Agar 15.Og
Reagent-grade water l.OL
7.10.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
completely. Adjust pH to 6.7 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide. Dispense 5-7 mL aliquots into 16 x 100 mm screw cap test tubes, cap and
autoclave at 121°C (15 PSI) for 12 minutes. Allow medium to solidify in a slant rack or
rack that is tilted in such a manner that the surface area is equally divided between the slant
and butt. Let the media warm to room temperature prior to inoculation.
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Method 1682
7.11 Urea broth
7.11.1 Composition:
Yeast extract O.lg
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
7.11.2 Add reagents to 1 L of reagent-grade water, mix thoroughly to dissolve (do not boil or
autoclave). Adjust pH to 6.8 ± 0.1 with 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide. Filter sterilize by passing solution through a sterile, 0.22 (im filter into a
sterile flask. Aseptically dispense 3 mL into sterile 16 x 100 mm screw cap test tubes
using a sterile pipet or sterile dispensing syringe. Let the media warm to room
temperature prior to inoculation.
7.12 Heart infusion agar (HIA)
7.12.1 Composition:
Beef heart, infusion from 500 g 10.Og
Bacto tryptose 10.0 g
Sodium chloride 5.0g
Bacto agar 15.0g
Reagent-grade water l.OL
7.12.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve. Adjust
pH to 7.4 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide. Stir well and
autoclave at 121°C for 15 minutes. Pour into 15 x 100 mm sterile petri plates. Let the
media warm to room temperature prior to inoculation. Other general growth media may
be used for quality assurance (QA) (Section 9.0) purposes.
7.13 Salmonella O antiserum Polyvalent Groups A-I and Vi
7.14 Positive controls
7.14.1 Obtain a stock culture of Salmonella typhimurium ATCC 14028 as a positive control for
MSRV, XLD, TSI, LIA, and polyvalent O antiserum.
Note: ATCC recommends that no more than 5 transfers be made before returning to the
original culture. This will minimize the chance of contamination during transfers
and genetic shift of the culture. One suggestion is to make your own frozen seed
stock upon receipt of the organism that can be used for future work. For
additional information go to http://www.atcc.org.
7.14.2 Obtain a stock culture of Proteus vulgaris ATCC 13315 as a positive control for urease.
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Method 1682
7.15 Negative controls
7.15.1 Obtain a stock culture of Escherichia coll ATCC 25922 as a negative control for MSRV,
XLD, TSI, LIA, and polyvalent O antiserum.
7.15.2 Obtain a stock culture of Salmonella typhimurium ATCC 14028 as a negative control for
urease.
7.16 Storage temperatures and times for prepared media and reagents are provided in Table 1, below:
Table 1. Storage Temperatures and Times for Prepared Media and Reagents1
Media
Sterile physiological saline (0.85% w/v)
TSB: loose-capped tubes
MSRV: poured-plates (do not store inverted)
XLD: poured plates (store inverted)
TSI, LIA, Urea broth: tight-capped tubes
HIA: poured plates (store inverted)
2% novobiocin
Polyvalent O antiserum
Storage Temperature
room temperature
room temperature
room temperature
1°Cto5°C
1°Cto5°C
1°Cto5°C
-20°Cto-10°C
2° to 8°C lyophilized
Storage Time
<3 months
<2 weeks
<48 hours
<2 weeks
<3 months
<2 weeks
<1 year
<3 years
If media is refrigerated, remove from refrigerator 1-1.5 hours prior to inoculation to ensure that it reaches room
temperature prior to use.
7.17 Milorganite® (CAS 8049-99-8) or equivalent
Milorganite® (heat-dried Class A biosolid) is produced by Milwaukee Metropolitan Sewerage
District. It is available in many home gardening centers. Obtain Milorganite® as the reference
matrix for initial precision and recovery (IPR) and ongoing precision and recovery (OPR) analyses.
Milorganite® is used as the reference matrix because it is easily accessible, inexpensive, generally
does not contain the analyte of interest, and is of consistent quality.
8.0 Sample Collection, Handling, and Storage
8.1 The most appropriate location for biosolid sample collection is the point prior to leaving the
wastewater treatment plant. Samples may be taken from pipes, conveyor belts, bins, compost
heaps, drying beds, and stockpiles.
8.2 Collect samples in sterile, non-toxic glass or plastic containers with leak-proof lids. All sampling
containers and equipment must be clean and sterile.
8.3 Equipment and container cleaning procedure
8.3.1 Wash sample collection apparatus with laboratory-grade detergent and water
8.3.2 Rinse with tap water
8.3.3 Rinse with 10% HC1 acid wash
10
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Method 1682
8.3.4 Rinse with distilled water
8.3.5 Allow to air dry
8.3.6 Cover with foil and autoclave for 15 minutes at 121°C (15 PSI)
8.4 Digester biosolids sampling procedure
8.4.1 Collect digester biosolids sample from the discharge pipe.
8.4.2 Purge the discharge pipe of old biosolids and warm to the digester temperature by allowing
biosolids to flow through the pipe into a container or waste collection device.
8.4.3 Position a 1-gallon sterile bag under the flow so that only the sample touches the inside of
the bag. Fill the bag, leaving 0.5 inches of head space in the bag for gas production.
Leaving head room is extremely important when taking samples of biosolids that have
been anaerobically digested.
8.5 Procedure for sampling conveyor belt biosolid output
8.5.1 Using a sterile scoop, transfer the pressed biosolids directly from the conveyer into a sterile
container, without mixing or transferring to another area.
8.5.2 Pack sample into sterile container. Leaving additional head space is not as important as in
Section 8.4 because there is less gas formation.
8.6 Procedure for sampling from a bin, drying bed, truck bed, or similar container
8.6.1 Remove surface material (upper six inches) and set it aside. Divide the underlying
material to be sampled into four quadrants.
8.6.2 Use a scoop or core the sample with the auger if material is deep.
8.6.3 Take a sample from each of the quadrants and combine in a sterile container.
8.6.4 After all the samples have been taken, pour the contents of the container out onto a sterile
surface and mix by folding the sample back onto itself several times.
8.6.5 Reduce the sample size by "coning and quartering." Divide the container contents into
four even piles. If sample size is still too large, divide each quarter into quarters and
discard half. Put into a glass or plastic sampling container.
8.6.6 An alternate method to "coning and quartering" is to randomly take a flat shovel full of
biosolids from the contents of the container that has been placed on a sterile surface and put
samples into a sampling container. (Curved scoops have been shown to favor a certain
size particle and should not be used.)
8.7 Record the following in your log book:
8.7.1 Facility name and location
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Method 1682
8.7.2 Date
8.7.3 Arrival time
8.7.4 Name of facility and contact
8.8 Record the following onto sample container and in log book when known:
8.8.1 Sample number
8.8.2 Date and time
8.8.3 Sampler name
8.8.4 Sample location
8.8.5 Parameters (e.g., type of analysis, field measurements- pH and temperature)
8.8.6 Volume
8.8.7 Observations
8.9 Ensure that the chain-of-custody form is filled out.
8.10 Sample handling: Maintain bacteriological samples at < 10°C 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.
8.11 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 <10°C until analysis. Samples must not be frozen. Sample analysis must begin
within 6 hours unless otherwise specified in the Code of Federal Regulations Part 503.
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.
9.0 Quality Control
9.1 Each laboratory that uses Method 1682 is required to operate a formal quality assurance (QA)
program that addresses and documents instrument and equipment maintenance and performance,
reagent quality and performance, analyst training and certification, and records storage and
retrieval. General requirements and recommendations for QA and quality control (QC)
procedures for microbiological laboratories are provided in Reference 18.3.
12 September 2014
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Method 1682
9.2 The minimum analytical QC requirements for the analysis of samples using Method 1682 include
an initial demonstration of laboratory capability through performance of the initial precision and
recovery (IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through
performance of the ongoing precision and recovery (OPR) analysis (Section 9.4) and matrix spike
(MS) analysis (Section 9.5), and the routine analysis of positive and negative controls (Section 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 either laboratory-prepared spiking suspensions or
BioBalls as described in Section 14.0.
9.3 Initial precision and recovery (IPR): The 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. 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:
9.3.1 Prepare four, 30-g samples of Milorganite® and spike each sample with Salmonella
typhimurium ATCC 14028 according to the spiking procedure in Section 14.0. Spiking
with laboratory-prepared suspensions is described in Section 14.3 and spiking with
BioBalls is described in Section 14.6. Process and analyze each IPR sample according to
the procedures in Sections 11 and 12 and calculate the Salmonella MPN / 4 g dry weight
according to Section 13.0.
9.3.2 Calculate the percent recovery (R) for each IPR sample using the appropriate equation in
Sections 14.5 or 14.8 for laboratory-prepared and BioBall™ spikes, respectively.
9.3.3 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.
9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 2. If the
mean and RSD for recover of Salmonella meet acceptance criteria, system performance is
acceptable and analysis of field samples may begin. If the mean recovery or the RSD fall
outside of the required range for recovery, 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.
Table 2. Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria
Performance test
Initial precision and recovery (IPR)
• Mean percent recovery
• Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
BioBall™
acceptance criteria
22% -126%
69%
1%-147%
Lab-prepared spike
acceptance criteria
Detect - 254%
92%
Detect - 287%
9.4 Ongoing precision and recovery (OPR): To demonstrate ongoing control of the analytical
system, the laboratory should routinely process and analyze spiked Milorganite® samples. The
laboratory should analyze one OPR sample after every 20 field and matrix spike samples or one per
week that samples are analyzed, whichever occurs more frequently. OPR samples must be
13 September 2014
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Method 1682
accompanied by an acceptable method blank (Section 9.7) and appropriate media sterility checks
(Section 9.8). The OPR analysis is performed as follows:
9.4.1 Spike a 30-g sample of Milorganite® with Salmonella typhimurium ATCC 14028
according to the spiking procedure in Section 14.0. Spiking with laboratory-prepared
suspensions is described in Section 14.3 and spiking with BioBalls is described in Section
14.6. Process and analyze each OPR sample according to the procedures in Sections 11.0
and 12.0 and calculate the number of Salmonella MPN / 4 g dry weight according to
Section 13.0.
9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in
Section 14.5 or 14.8 for samples spiked with laboratory-prepared spiking suspensions or
BioBalls, respectively.
9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria
in Table 2, 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. 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.
9.4.4 As part of the laboratory QA program, results for OPR and IPR samples should be charted
and updated records maintained in order to monitor ongoing method performance. The
laboratory should also develop a statement of accuracy for Method 1682 by calculating the
average percent recovery (R) and the standard deviation of the percent recovery (sr).
Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
9.5 Matrix spikes (MS): MS analysis are performed to determine the effect of a particular matrix on
Salmonella recoveries. The laboratory should analyze one MS sample when biosolid 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 biosolids 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 9.4), using the same spiking procedure
(laboratory-prepared spiking suspension or BioBalls). The MS analysis is performed as follows:
9.5.1 Prepare two, 30-g field 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 Salmonella typhimurium ATCC 14028 according
to the spiking procedure in Section 14.0.
9.5.2 Select dilutions based on previous analytical results or anticipated levels of Salmonella in
the field sample in order to accurately estimate Salmonella density. Neither above or
below the detection limit of the method.
9.5.3 Spike the MS sample with a laboratory-prepared suspension as described in Section 14.3 or
with BioBalls as described in Section 14.6. Process and analyze the unspiked and spiked
field samples according to the procedures in Sections 11.0 and 12.0.
14 September 2014
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Method 1682
9.5.4 For the MS sample, calculate the Salmonella MPN / 4 g dry weight according to Section
13.0 and adjust the density (MPN / 4 g dry weight) based on the ambient concentration of
Salmonella observed in the unspiked matrix sample.
9.5.5 Calculate the percent recovery (R) for the MS sample (adjust based on ambient Salmonella
in the unspiked sample) using the appropriate equations Section 14.5 or 14.8 for samples
spiked with laboratory-prepared spiking suspensions or BioBalls, respectively.
9.5.6 Compare the MS result (percent recovery) with the appropriate method performance
criteria in Table 3. If the MS recovery meets the acceptance criteria, system performance
is acceptable and analysis of field samples from this biosolid source may continue. If the
MS recovery is unacceptable and the OPR sample result associated with this batch of
samples is acceptable, a matrix interference may be causing the poor results. If the MS
recovery is unacceptable, all associated field data should be flagged.
Table 3. Matrix Spike Precision and Recovery Acceptance Criteria
Performance test
Percent recovery for MS or MS/MSD
Precision (as maximum relative percent difference of MS/MSD)
BioBall™ acceptance
criteria
Detect -158%
177%
Lab-prepared
acceptance criteria
Detect - 246%
172%
9.5.7 Laboratories should record and maintain a control chart comparing MS recoveries for all
matrices to batch-specific and cumulative OPR sample results analyzed using Method
1682. These comparisons should help laboratories recognize matrix effects on method
recovery and may also help to recognize inconsistent or sporadic matrix effects from a
particular source.
9.6 Culture Controls
9.6.1 Negative controls: The laboratory should analyze negative controls to ensure that the
MSRV, XLD, TSI, LIA, urea broth, and polyvalent O antiserum are performing properly.
Negative controls should be analyzed whenever a new batch of media or reagent is used.
On an ongoing basis, the laboratory should perform a negative control every day that
samples are analyzed. Positive and negative control results are provided in Table 4.
9.6.1.1 Negative controls are conducted by inoculating MSRV, XLD, TSI, LIA, and
polyvalent O antiserum with a known negative control (e.g., E. coll ATCC
25922) and analyzing as described in Section 12.0.
9.6.1.2 Negative controls are conducted by inoculating urea broth with a known
negative control (e.g., Salmonella typhimurium ATCC 14028) and analyzing as
described in Section 12.0.
9.6.1.3 If a negative control fails to exhibit the appropriate response, check and/or
replace the associated media or reagents, and/or the negative control, and
reanalyze the appropriate negative control.
9.6.2 Positive controls: The laboratory should analyze positive controls to ensure that the
MSRV, XLD, TSI, LIA, urea broth, and polyvalent O antiserum are performing properly.
Positive controls should be analyzed whenever a new batch of media or reagent is used.
On an ongoing basis, the laboratory should perform a positive control every day that
15
September 2014
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Method 1682
samples are analyzed. An OPR sample (Section 9.4) may take the place of a positive
control. Positive and negative control results are provided in Table 4.
9.6.2.1 Positive controls are conducted by inoculating MSRV, XLD, TSI, LIA, and
polyvalent O antiserum with a known positive culture (e.g., Salmonella
typhimurium ATCC 14028) and analyzing as described in Section 12.0.
9.6.2.2 Positive controls are conducted by inoculating urea broth with a known positive
culture (e.g., Proteus vulgaris ATCC 13315) and analyzing as described in
Section 12.0.
9.6.2.3 If the positive control fails to exhibit the appropriate response, check and/or
replace the associated media or reagents, and/or the positive control, and
reanalyze the appropriate positive control.
9.7 Method blank. Test a 20-mL sterile dilution water sample in the analytical scheme to verify the
sterility of equipment, materials, and supplies. Absence of growth indicates freedom of
contamination from the target organism. On an ongoing basis, the laboratory should perform a
method blank every day that samples are analyzed.
9.8 Media sterility check. To test sterility of media, incubate a representative portion of each batch
at 36°C ± 1.5°C (TSB, XLD, TSI, LIA, HIA, and urea broth) or 42°C ± 0.5°C (MSRV) for 24 ± 2
hours and observe for growth. A batch is defined as one tube/plate out of 50 in each lot, or one
tube/plate if the lot contains less than 50 tubes/plates.
Table 4. Positive and Negative Control Results
Medium
Tryptic Soy Broth (TSB)
Modified Semisolid
Rappaport-Vassiliadis
(MSRV) medium
Xylose lysine
desoxycholate agar
(XLD)
Triple sugar iron agar
(TSI)
Lysine iron agar (LIA)
Urea broth
Polyvalent O
Salmonella
result
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Positive Method 1682 reaction
Turbidity
Migrated cells visible as a
gray-white turbid zone (halo)
extending out from inoculations
Pink to red colonies with black
centers
Good growth with alkaline slant
(red) with acid butt (yellow) with or
without hbS production (which may
result in a black butt)
Alkaline slant (purple) with alkaline
butt (purple) with or without h^S
production (which may result in a
black butt)
Pink
Agglutination
Negative Method 1682 reaction
No turbidity
Medium remains blue-green
around inoculations with no
gray-white turbid zone (halo) (£.
co// has marked inhibition)
Other colors with or without black
centers (e.g. £ co// is yellow
without black center)
Other color combinations (e.g., £
co// is yellow slant and butt)
Other color combinations (e.g., £.
co// is red to red/purple slant and
butt without hhS production)
No color change (Salmonella is
urease negative)
No agglutination
16
September 2014
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Method 1682
10.0 Equipment Calibration and Standardization
10.1 Check temperatures in incubators/waterbaths twice daily, a minimum of four hours apart, to ensure
operation is within stated limits of the method and record daily measurements in incubator log
book.
10.2 Check temperatures in refrigerators/freezers at least once daily to ensure operation is within stated
limits of the method. Record daily measurements in refrigerator/freezer log book.
10.3 Calibrate thermometers and incubators semiannually against a NIST certified thermometer or one
that meets the requirements of NIST Monograph SP 250-23 (Reference 18.1). Check mercury
columns for breaks.
10.4 Calibrate pH meter prior to each use with two standards (pH 4.0, 7.0, and 10.0) closest to the range
being tested.
10.5 Calibrate top-loading balances once per month with reference weights of ASTM Class 2.
11.0 Sample Preparation
11.1 Homogenization
Sample homogenization procedures are based on whether the sample is a liquid or a solid. If
sample is alkaline-stabilized (liquid or solid), adjust the pH as described in Section 11.1.3. Liquid
samples are generally defined as samples containing <7% total solids (dry weight).
11.1.1 Liquid samples: Homogenize 300 mL of sample in a sterile blender on high speed for one
to two minutes. Adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid or 1.0 N
sodium hydroxide, if necessary. This is the "homogenized" sample. When adjusting pH
do not exceed the homogenized sample volume by greater than 5% (15 mL).
11.1.2 Solid samples: Weigh out 30.0 g ± 0.1 g of well-mixed sample in a sterile dish. Whenever
possible, the sample tested should contain all materials that will be included in the biosolid.
For example, if wood chips are part of the biosolid compost, some mixing or grinding may
be needed to achieve homogeneity before testing. Large pieces of wood that are not easily
ground may be discarded before homogenizing. Transfer the sample to a sterile blender.
Use 270 mL of sterile buffered dilution water (Section 7.4) to rinse any remaining sample
into the blender. Alternatively, the sample may be directly weighed in the sterile blender
jar. Cover and blend on high speed for one to two minutes. This is the "homogenized"
sample. A volume of 10-mL of the "homogenized" sample contains 1.0 g of the original
sample. Adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide, if necessary. When adjusting pH do not exceed the homogenized sample
volume by greater than 5% (15 mL).
Note: Do not suspend bacteria in dilution water for more than 30 minutes at room
temperature. Chill on wet ice or at 4°C ± 1 °C to slow replication between spiking
samples.
17 September 2014
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Method 1682
11.1.3 Alkaline -stabilized: The alkaline-stabilized biosolid samples generally have a pH of
approximately 12. Prior to analysis, the alkaline-stabilized biosolid sample must be
neutralized to a pH of approximately 7.5. Do not add lab-prepared spikes or BioBalls to
the samples prior to pH adjustment.
11.1.3.1 Adjustment of pH should be done in a fume hood. Prior to adjusting the pH of
the sample, calibrate/standardize the pH meter with pH buffers 7.0 and 10.0.
Weigh out 30 g of sample into a sterile 600 mL beaker, add 250 mL of sterile
buffered dilution water and a sterile magnetic stir bar. Place beaker on a mixing
plate, insert pH probe into mixture, begin stirring, and take an initial pH reading.
To minimize the amount of volume added to each sample, pH should be adjusted
using 10NHC1.
Note: The addition of the 10 N HC1 will produce fumes, do not be alarmed.
The addition of the acid should be done incrementally to ensure that the
pH does not drop instantaneously below 5.0. It is recommended that
the pH adjustment be completed within 10-15 minutes and monitored
for an additional 15 minutes to ensure that the sample is able to maintain
a constant pH of around 7.5. Pour pH adjusted sample into blender jar,
use the remaining sterile buffered dilution water (15 mL) to rinse the
beaker twice and pour rinse water into the blender jar.
11.2 Inoculation
Media inoculation procedures are based on whether the original sample was a liquid or a solid.
For some transfers, it may be convenient to use a sterile, wide-mouth pipette, capable of
transferring particulate matter. If samples are being spiked, a maximum of 1 hour may elapse
between initial unspiked sample homogenization and analysis of spiked samples.
11.2.1 Liquid samples: For unspiked and spiked samples, three series of five tubes will be used for
the analysis with 20-, 10-, and 1.0-mL of the original sample. See Figure 1 in Section
19.0 for an overview of the inoculation scheme.
11.2.1.1 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of five tubes
(containing 10 mL of 3X TSB) with 20.0 mL of the original "homogenized"
sample per tube.
(B) Use a sterile pipette to inoculate each of the second series of tubes
(containing 5 mL of 3X TSB) with 10.0 mL of the original "homogenized"
sample per tube.
(C) Use a sterile pipette to inoculate each of the third series of tubes (containing
10 mL of IX TSB) with 1.0 mL of the original "homogenized" sample per
tube.
11.2.1.2 Repeat Section 11.2.1.1 for the remaining liquid samples. When inoculations
are complete, go to Section 12.0 to continue the analyses.
18 September 2014
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Method 1682
11.2.2 Solid samples: For unspiked and spiked samples, three series of five tubes will be used for
the analysis with 2.0-, 1.0-, and 0.1-g of the original sample (20.0, 10.0, and 1.0 mL of the
homogenized sample). The first two series of tubes must contain 3X TSB. See Figure 2
in Section 19.0 for an overview of the inoculation scheme.
11.2.2.1 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of tubes (containing
10 mL of 3X TSB) with 20.0 mL of the "homogenized" sample per tube.
This is 2.0 g of the original sample. Solids that will not separate easily
and/or may float should be submerged into the broth with a sterile loop.
(B) Use a sterile pipette to inoculate each of the second series of tubes
(containing 5 mL of 3X TSB) with 10.0 mL of the "homogenized" sample
per tube. This is 1.0 g of the original sample. Solids that will not separate
easily and/or may float should be submerged into the broth with a sterile
loop.
(C) Use a sterile pipette to inoculate each of the third series of tubes (containing
10 mL of IX TSB) with 1.0 mL of the "homogenized" sample per tube.
This is 0.1 g of the original sample.
11.2.2.2 Repeat Section 11.2.2.1 for remaining solid samples. When inoculations are
complete, go to Section 12.0 to continue the analysis.
Note: For regulatory purposes, Salmonella monitoring is required only for
Class A biosolids in 40 CFR Part 503. If this procedure is being used to
enumerate Salmonella in samples other than Class A biosolid samples,
additional dilutions may be required prior to analysis. When
attempting to quantify samples containing higher Salmonella
concentrations than can be evaluated using the 20-10-1 scheme, it may
be necessary to evaluate additional dilution volumes of 0.1-, 0.01-, and
0.001-mL. The MPN table in Standard Methods would then be used,
instead of Table 5 in Section 13.0.
Although other dilution and inoculation schemes may be used for the analysis of
samples with higher Salmonella concentrations, the first transfer from the
"homogenized" sample should always be 11 mL of homogenized sample to 99
mL dilution water or 10 mL of homogenized sample to 90 mL dilution water.
This will ensure that a sufficient amount of the original biosolid sample is
transferred at the beginning of the dilution scheme.
12.0 Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium
Procedure
12.1 In this protocol, the modified semisolid Rappaport-Vassiliadis (MSRV) medium MPN technique is
used to determine Salmonella densities in Class A biosolid samples. Although the Part 503
regulation does not specify the total number of samples for Class A biosolids, it suggests that a
sampling event extend over two weeks, and that at least seven samples be tested to confirm that the
19 September 2014
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Method 1682
mean bacterial density of the samples is below 3 MPN / 4 g of total solids (dry weight basis). The
analysis of seven samples increases the method precision by reducing the standard error caused by
inherent variations in biosolid quality. The precision of the test increases with increasing numbers
of replicates per sample tested. For an overview of the MPN procedure refer to Figure 3 in
Section 19.0.
12.2 Enrichment phase
12.2.1 Prepare TSB media and dispense into tubes as directed in Section 7.6.
Note: If media is refrigerated, remove from refrigerator 1-1.5 hours priorto inoculation,
so that it reaches room temperature prior to use.
12.2.2 For each sample, arrange test tubes in three rows of five tubes each. When 20 mL of
homogenized sample is inoculated, tubes should contain 10 mL of 3X TSB media, when 10
mL of homogenized sample is inoculated, tubes should contain 5 mL of 3X TSB media,
and when 1.0 mL of homogenized sample is inoculated, tubes should contain 10 mL of IX
TSB media.
Note: 3X TSB is necessary for 20- and 10-mL inoculation volumes, to ensure that the
TSB is not excessively diluted.
12.2.3 Inoculate samples according to Section 11.2, based on whether the original sample was
liquid or solid.
12.2.4 Incubate the TSB tubes and controls for 24 ± 2 hours at 36°C ± 1.5°C.
12.2.5 Record all turbid tubes as positive. Because of the non-inhibitory nature of the
enrichment medium, all tubes will be positive in most instances. If none of the tubes
appear to be positive, then this may indicate the presence of a toxic substance or that the
tubes were not inoculated.
12.3 Selection phase
12.3.1 Apply six discrete, 30-(iL drops from each TSB tube onto a corresponding MSRV plate
that has been labeled with sample ID, date, and original inoculation volume (e.g., 20.0,
10.0, or 1.0 mL). Space the drops evenly over the entire plate. In addition, inoculate an
MSRV plate with positive and negative controls. Do not invert the plates. Allow the
drops to absorb into the agar for approximately 1 hour at room temperature and incubate
plates at 42°C ± 0.5°C for 16 to 18 hours in a humidity-controlled hot air incubator. If a
humidity-controlled hot air incubator is not available, an open pan of water placed in the
bottom of the incubator will suffice.
12.3.2 Examine plates for the appearance of motility surrounding the inoculations, as evidenced
by a "whitish halo" of growth approximately 2 cm from the center of the spot.
12.3.3 Using a sterile inoculating loop, stab into a halo from the outer edge of a target colony on
the MSRV plate and streak onto an XLD plate. Since Salmonella are predominately
located within the MSRV media, the loop should penetrate the MSRV at least half-way.
20 September 2014
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Method 1682
Repeat this step using another target colony from the MSRV plate. In addition, inoculate
XLD positive and negative controls. (See Photo 1)
Photo 1. Salmonella spp. produce halos indicating motility on MSRV plates.
12.3.4 Incubate XLD plates for 18 to 24 hours at 36°C ± 1.5°C. After incubation, refrigerate one
of the XLD plates at 1°C to 5°C and submit the other plate to biochemical confirmation.
(If issues arise in subsequent steps of the method, the laboratory may wish to return to the
refrigerated XLD plate.)
Note: XLD plates may be refrigerated over the weekend prior to submitting to
biochemical confirmation.
12.3.5 Black and pink to red colonies with black centers are considered Salmonella. (See Photo 2)
Photo 2. Salmonella spp. produce pink to red colonies with black centers on XLD plates.
12.4 Biochemical confirmation phase
12.4.1 Label all tubes with inoculation date, sample identification, and original inoculation
volume (e.g., 20.0, 10.0, or 1.0 mL). Pick isolated colonies exhibiting Salmonella
morphology (pink to red colonies with black centers) and inoculate triple sugar iron agar
(TSI) slants, lysine iron agar (LIA) slants, and urea broth. Inoculate slants by stabbing the
butt and streaking the slant. Use the same XLD colony to inoculate all three media. This
will require going back to the same XLD colony multiple times to ensure sufficient
21
September 2014
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Method 1682
inoculum for each medium. In addition, inoculate each medium with the appropriate
positive and negative controls. Incubate for 24 ± 2 hours at 36°C ± 1.5°C. If only
atypical colony morphology is observed on XLD plates, pick from an atypical colony and
inoculate TSI slants, LIA slants, and urea broth and incubate as described above.
12.4.1.1 TSI: A positive TSI reaction is an acid butt (yellow in color) and an alkaline slant
(red in color) with or without H2S gas production. When H2S gas production is
present, the butts of both the LIA and TSI may be black, which would be
considered a positive reaction for Salmonella. H2S is more likely but acid butt
is also possible (but rare).
12.4.1.2 LIA: A positive LIA reaction is an alkaline butt (purple in color) and an alkaline
slant (purple in color) with or without H2S gas production. When H2S gas
production is present, the butts of both the LIA and TSI may be black, which
would be considered a positive reaction for Salmonella.
12.4.1.3 Urea broth: Urea is an orange medium and will change to pink or deep
purplish-red if positive. A negative urease test is one that exhibits no color
change after inoculation. Salmonella are negative for urease.
Note: H2S negative Salmonella will be missed if translucent pink to red
colonies are not submitted to biochemical and serological confirmation.
12.4.2 To confirm cultures via polyvalent O antiserum:
Emulsify growth on the slant portion of TSI (regardless of whether TSI is positive or
negative) using sterile physiological saline (Section 7.5). Place two discrete drops of
emulsified growth onto slide. To the first drop of emulsified growth, add one drop of
polyvalent O antiserum. To the second drop of emulsified growth, add one drop of sterile
saline (as a visual comparison). Observe under magnification for an agglutination
reaction which indicates a positive result. Appropriate positive and negative controls
from TSI must be analyzed for each batch of samples.
12.4.3 In order for the original TSB tube to be considered positive for Salmonella, the associated
inoculations must be MSRV positive, XLD positive, either TSI or LIA positive, urease
negative, and polyvalent-O positive (Table 4). Correlate all positive plates and tubes to
original TSB tube and record results. Determine the MPN from this information (see
Section 13.0). Record all data clearly into a laboratory notebook.
12.5 Total solids determination
12.5.1 Determination of percent dry weight -When sample results are to be calculated on a dry
weight basis, a second portion of sample should be weighed at the same time as the portion
used for analytical determination.
WARNING: The drying oven should be contained in a hood or be vented. Significant
laboratory contamination may result from drying a heavily contaminated
sample.
12.5.2 Immediately after weighing the sample for microbiological examination, weigh 10-30 g of
sample into a tarred crucible or aluminum evaporating dish. Dry this aliquot overnight at
22 September 2014
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Method 1682
103°C to 105°C Allow to cool in a desiccator before weighing.
weight as follows:
g dry sample
% dry weight = g sample x 100
Calculate the % dry
13.0 Data Analysis and Calculations
The estimated density of Salmonella bacteria is calculated as the most probable number (MPN). Salmonella
results from biosolid samples are reported as MPN / 4 g total solids (dry weight basis), which is calculated
according to the following steps:
1. Selection of MPN / mL (wet weight)
2. Conversion to MPN / g (dry weight) and calculation of MPN / 4 g total solids (dry weight)
13.1 Step 1: Obtain MPN / mL (wet weight)
Obtain the MPN index value from Table 5 using the number of positive tubes in the three
significant dilutions series. Since Table 5 assumes that 20.0, 10.0, and 1.0 mL of homogenized
sample were inoculated into TSB and because liquid samples are not diluted in the homogenization
step (Section 11.0), the MPN index = MPN / mL for liquid samples.
Since solid samples were diluted in the homogenization step (Section 11.1), the dilution must be
taken into account when calculating MPN / mL (wet weight). As a result the MPN index value
from Table 5 is divided by 0.1 to account for diluting the sample during the homogenization step.
Table 5. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five
Tubes are used per 20.0,10.0, and 1.0 mL Homogenized Sample Inoculation Volumes a
Combination of
Positives
0-0-0
0-0-1
0-0-2
0-0-3
0-0-4
0-0-5
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
0-2-0
\J-£.-\
0-2-2
0-2-3
0-2-4
r\ ry c
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
MPN Index
<0.006473
0.0065
0.0130
0.0195
0.0262
0.0328
0.0067
0.0134
0.0202
0.0270
0.0339
0.0408
0.0138
0.0208
0.0279
0.0350
0.0422
0.0494
0.0215
0.0288
0.0362
0.0437
0.0512
0.0588
95% Confidence Limits Combination of
Lower | Upper
0.0223
0.0012 0.0223
0.0012 0.0352
0.0012 0.0472
0.0033 0.0589
0.0062 0.0706
0.0012 0.0228
0.0012 0.0360
0.0012 0.0483
0.0037 0.0604
0.0067 0.0725
0.0099 0.0847
0.0012 0.0367
0.0012 0.0495
0.0040 0.0619
0.0072 0.0745
0.0106 0.0871
0.0141 0.1001
0.0012 0.0507
0.0044 0.0636
0.0077 0.0766
0.0113 0.0898
0.0051 0.1243
0.0095 0.1428
Positives
1-3-0
1-3-1
1-3-2
1-3-3
1-3-4
1-3-5
1-4-0
1-4-1
1-4-2
1-4-3
1-4-4
1-4-5
1-5-0
1-5-1
1-5-2
1-5-3
1-5-4
1-5-5
2-0-0
2-0-1
2-0-2
2-0-3
2-0-4
2-0-5
MPN Index
0.0312
0.0393
0.0475
0.0559
0.0644
0.0730
0.0409
0.0495
0.0583
0.0672
0.0763
0.0855
0.0517
0.0609
0.0703
0.0799
0.0897
0.0998
0.0155
0.0226
0.0303
0.0382
0.0462
0.0543
95% Confidence Limits
Lower | Upper
0.0055 0.0678
0.0092 0.0821
0.0132 0.0967
0.0173 0.1119
0.0216 0.1277
0.0260 0.1444
0.0099 0.0849
0.0141 0.1002
0.0185 0.1163
0.0231 0.1331
0.0277 0.1509
0.0324 0.1700
0.0152 0.1042
0.0199 0.1212
0.0247 0.1391
0.0296 0.1583
0.0346 0.1790
0.0397 0.2015
0.0012 0.0404
0.0018 0.0526
0.0051 0.0662
0.0087 0.0801
0.0125 0.0943
0.0165 0.1090
23
September 2014
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Method 1682
Table 5. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five
Tubes are used per 20.0,10.0, and 1.0 mL Homogenized Sample Inoculation Volumes a
Combination of
Positives
0-4-0
0-4-1
0-4-2
U 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 n 4
1-0-5
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
1-2-0
1-2-1
1-2-2
1-2-3
1-2-4
1-2-5
3-0-0
3-0-1
3-0-2
3-0-3
3-0-4
3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
315
3-2-0
3-2-1
3-2-2
3-2-3
3-2-4
3-2-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-0
3-4-1
3-4-2
3-4-3
3-4-4
3-4-5
MPN Index
0.0299
0.0375
0.0453
0.0531
0.0611
0.0691
0.0390
0.0470
0.0553
0.0636
0.0720
0.0806
0.0072
0.0139
0.0209
0.0281
0.0353
0.0425
0.0144
0.0217
0.0290
0.0365
0.0441
0.0517
0.0224
0.0301
0.0379
0.0457
0.0537
0.0618
0.0255
0.0330
0.0417
0.0506
0.0598
0.0691
0.0344
0.0435
0.0529
0.0626
0.0725
0.0456
0.0555
0.0657
0.0763
0.0872
0.0984
0.0583
0.0693
0.0806
0.0924
0.1046
0.1173
0.0733
0.0856
0.0984
0.1118
0.1258
0.1405
95% Confidence Limits Combination of
Lower | Upper
0.0049 0.0654
0.0084 0.0789
0.0121 0.0927
0.0160 0.1069
0.0200 0.1216
0.0241 0.1369
0.0090 0.0814
0.0129 0.0958
0.0170 0.1107
0.0212 0.1262
0.0255 0.1425
0.0299 0.1596
0.0012 0.0369
0.0012 0.0497
0.0041 0.0623
0.0073 0.0749
0.0107 0.0878
0.0012 0.0377
0.0013 0.0509
0.0045 0.0640
0.0079 0.0771
0.0115 0.0905
0.0153 0.1043
0.0017 0.0523
0.0050 0.0658
0.0085 0.0795
0.0123 0.0935
0.0162 0.1079
0.0203 0.1229
0.0028 0.0585
0.0063 0.0710
0.0103 0.0863
0.0147 0.1023
0.0193 0.1191
0.0241 0.1368
0.0069 0.0734
0.0112 0.0896
0.0159 0.1065
0.0207 0.1244
0.0258 0.1434
0.0310 0.1640
0.0122 0.0932
0.0171 0.1112
0.0223 0.1303
0.0277 0.1510
0.0333 0.1735
0.0390 0.1984
0.0186 0.1164
0.0241 0.1371
0.0299 0.1597
0.0359 0.1847
0.0421 0.2128
0.0484 0.2452
0.0262 0.1450
0.0325 0.1700
0.0390 0.1982
0.0457 0.2307
0.0526 0.2695
0.0597 0.3184
Positives
2-1-0
2-1-1
2-1-2
2-1-3
2-1-4
2-1-5
2-2-0
2-2-1
2-2-2
2-2-3
2-2-4
2-2-5
2-3-1
2-3-2
2-3-3
2-3-4
2-3-5
2-4-0
2-4-1
2-4-2
2-4-3
2-4-4
2-4-5
2-5-0
2-5-1
2-5-2
2-5-3
2-5-4
2-5-5
4-3-0
4-3-1
4-3-2
4-3-3
4-3-4
4-3-5
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-0
4-5-1
4-5-2
4-5-3
4-5-4
4-5-5
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-1-0
5-1-1
5-1-2
5-1-3
5-1-4
5-1-5
MPN Index
0.0234
0.0315
0.0397
0.0480
0.0565
0.0652
0.0327
0.0413
0.0501
0.0590
0.0681
0.0774
0.0523
0.0617
0.0714
0.0813
0.0914
0.0547
0.0647
0.0750
0.0855
0.0964
0.1076
0.0681
0.0791
0.0904
0.1021
0.1143
0.1268
0.0797
0.0937
0.1086
0.1245
0.1414
0.1595
0.1012
0.1181
0.1364
0.1563
0.1780
0 2015
0.1304
0.1524
0.1769
0.2046
0.2357
0.2708
0.0549
0.0637
0.0763
0.0896
0.1037
0.0953
0.0678
0.0816
0.0963
0.1121
0.1291
0.1293
95% Confidence Limits
Lower | Upper
0.0022 0.0540
0.0056 0.0683
0.0094 0.0827
0.0134 0.0976
0.0177 0.1131
0.0221 0.1293
0.0062 0.0705
0.0101 0.0856
0.0144 0.1013
0.0189 0.1176
0.0236 0.1349
0.0283 0.1533
0.0155 0.1053
0.0203 0.1227
0.0252 0.1412
0.0303 0.1611
0.0354 0.1826
0.0168 0.1098
0.0218 0.1284
0.0271 0.1484
0.0325 0.1700
0.0380 0.1937
0.0436 0.2201
0.0235 0.1349
0.0292 0.1566
0.0349 0.1805
0.0409 0.2070
0.0469 0.2372
0.0531 0.2725
0.0295 0.1579
0.0366 0.1877
0.0441 0.2228
0.0520 0.2656
0.0602 0.3218
0.0686 0.4067
0.0404 0.2049
0.0489 0.2476
0.0578 0.3038
0.0672 0.3890
0.0770 0.5273
0.0873 0.641 1
0.0549 0.2836
0.0653 0.3687
0.0766 0.5210
0.0886 0.6528
0.1015 0.7516
0.1150 0.8426
0.0162 0.1116
0.0213 0.1265
0.0277 0.1510
0.0345 0.1787
0.0417 0.2107
0.0165 0.2234
0.0234 0.1344
0.0304 0.1618
0.0379 0.1936
0.0459 0.2316
0.0542 0.2796
0.0304 0.3090
24
September 2014
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Method 1682
Table 5. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five
Tubes are used per 20.0,10.0, and 1.0 mL Homogenized Sample Inoculation Volumes a
Combination of
Positives
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
4-0-1
4-0-2
4-0-3
4-0-4
4-0-5
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0
4-2-1
4-2-2
4-2-3
4-2-4
4-2-5
MPN Index
0.0913
0.1055
0.1204
0.1362
0.1529
0.1707
0.0381
0.0461
0.0563
0.0668
0.0777
0.0890
0.0484
0.0592
0.0705
0.0822
0.0945
0.1072
0.0626
0.0748
0.0875
0.1009
0.1150
0.1299
95% Confidence Limits Combination of
Lower | Upper
0.0354 0.1825
0.0426 0.2150
0.0500 0.2538
0.0577 0.3029
0.0656 0.3715
0.0738 0.4795
0.0082 0.0809
0.0125 0.0942
0.0175 0.1126
0.0229 0.1323
0.0284 0.1537
Positives
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0
5-3-1
5-3-2
5-3-3
5-3-4
0.0342 0.1773 | 5-3-5
0.0136 0.0983 5-4-0
0.0190 0.1181
0.0248 0.1395
0.0308 0.1631
0.0370 0.1894
0.0434 0.2193
0.0207 0.1244
0.0269 0.1479
0.0335 0.1742
0.0403 0.2041
0.0473 0.2392
0.0546 0.2820
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
0.0879
0.1046
0.1227
0.1427
0.1646
0.1767
0.1151
0.1368
0.1614
0.1895
0.2216
0.2527
0.1571
0.1907
0.2319
0.2834
0.3475
0.4256
0.2398
0.3477
0.5422
0.9178
1.6090
>1. 609000
95% Confidence Limits
Lower | Upper
0.0337 0.1751
0.0421 0.2128
0.051 1 0.2605
0.0608 0.3267
0.0710 0.4385
0.0503 0.5230
0.0474 0.2394
0.0580 0.3050
0.0695 0.4183
0.0821 0.5899
0.0957 0.7101
0.0814 0.7971
0.0676 0.3935
0.0826 0.5954
0.0999 0.7409
0.1196 0.8726
0.1417 1.0160
0.1437 1.1800
0.0762 0.7629
0.1172 1.0160
0.1791 1.4190
0.2672 2.2010
0.3837 4.1030
0.3837
a Table was developed using the MPN calculator developed by Albert Klee (Reference 18.5)
13.2 Step 2: Convert to MPN / g (dry weight) and calculate MPN / 4 g total solids (dry weight):
For analysis and calculation of percent total solids, refer to Section 12.5.
For the conversion to MPN / g total solids (dry weight), we assume that,
MPN / mL wet weight = MPN / g wet weight.
Therefore, we may calculate MPN / 4 g total solids (dry weight) for liquid samples using the
following equation:
MPN / 4 g (dry
weight)
[MPN / mL wet weight from Step 1] * 4
Percent total solids expressed as a decimal
Example calculations are provided in Table 6, below.
25
September 2014
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Method 1682
Table 6. Example Calculation of Salmonella Density
Example
A (liquid)
B (liquid)
C (liquid)
D (solid)
E (solid)
F (solid)
Volume of homogenized
sample used to inoculate TSB
20.0 mL
0/5
3/5
5/5
0/5
4/5
5/5
10.0 mL
1/5
1/5
5/5
1/5
4/5
5/5
1.0 mL
0/5
1/5
2/5
0/5
4/5
2/5
Step 1 :
MPN/mL
(wet weight)
0.0067
0.0435
0.5422
0.0067 /0.11 = . 067
0.1181 /0.11 = 1.181
0.5422 /0.11 =5.422
Percent total
solids
(dry weight)
1%
2%
3%
96%
18%
43%
Step 2:
MPN/4g
(dry weight)
(0.0067) (4) / (0.01) =
2.68 MPN / 4 g
(0.0435) (4) / (0.02) =
8.7MPN/4g
(0.5422) (4) / (0.03) = 72
MPN/4g
(0.067) (4) / (0.96) =
0.28 MPN / 4 g
(1.181)(4)/(0.18) = 26
MPN/4g
(5.422) (4) / (0.43) = 50
MPN/4g
Dilution factor (1:10) for solid samples
14.0 Sample Spiking Procedure
14.1 Method 1682 QC requirements (Section 9) include the preparation and analysis of spiked reference
(Milorganite®) samples in order to monitor initial and ongoing method performance. For the IPR
(Section 9.3) and OPR (Section 9.4) analyses it is necessary to spike samples with
laboratory-prepared spiking suspensions as described below.
14.2 Preparation of Laboratory-Prepared Spiking Suspensions
14.2.1 Preparation
14.2.1.1 Stock Culture. Prepare a stock culture by inoculating a heart infusion agar
(HIA) slant (or other non-selective media) with Salmonella typhimurium ATCC
14028 and incubating at 36°C ± 1.5°C for 20 ± 4 hours. After incubation, the
stock culture may be stored in the dark at room temperature for up to 30 days.
14.2.1.2 1% Tryptic Soy Broth (TSB). Prepare a 1% solution of TSB by combining 99
mL of sterile phosphate buffered dilution water and 1 mL of sterile
single-strength tryptic soy broth in a sterile screw cap bottle or re-sealable
dilution water container. Shake to mix.
14.2.1.3 Spiking Suspension (Undiluted). From the stock culture of Salmonella
typhimurium ATCC 14028, aseptically transfer a small loopful of growth to the
1% TSB solution and vigorously shake a minimum of 25 times. Incubate at
36°C ± 1.5°C for 20 ± 4 hours. The resulting spiking suspension contains
approximately 1.0 x 107 to 1.0 x 10s Salmonella typhimurium colony forming
units (CPU) per mL. This is referred to as the "undiluted spiking suspension".
14.3 Laboratory-Prepared Sample Spiking (Class A Biosolids)
Since the objective of spiking the biosolid sample is to establish percent recovery, it is necessary to
determine the number of Salmonella typhimurium in the undiluted spiking suspension.
26
September 2014
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Method 1682
14.3.1 Sample spiking
14.3.1.1 Dilute spiking suspension
Mix the undiluted spiking suspension by vigorously shaking the bottle a
minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the undiluted
spiking suspension to 99 mL of sterile phosphate buffered dilution water
(Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25
times. This is spiking suspension dilution "A". A 1.0-mL volume of dilution
"A" is 10~2 mL of the original undiluted spiking suspension.
14.3.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution "A" to 99
mL of sterile phosphate buffered dilution water, cap, and mix by vigorously
shaking the bottle a minimum of 25 times. This is spiking suspension dilution
"B". A 1.0-mL volume of dilution "B" is 10"4 mL of the original undiluted
spiking suspension.
14.3.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" to 99
mL of sterile phosphate buffered dilution water, cap, and mix by vigorously
shaking the bottle a minimum of 25 times. This is spiking suspension dilution
"C". A 1.0-mL volume of dilution "C" is 10"5 mL of the original undiluted
spiking suspension.
14.3.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" to 99
mL of sterile phosphate buffered dilution water, cap, and mix by vigorously
shaking the bottle a minimum of 25 times. This is spiking suspension dilution
"D". A 1.0-mL volume of dilution "D" is 10"6 mL of the original undiluted
spiking suspension.
14.3.2 Spike sample(s)
Since sample homogenization procedures in Method 1682 are specific to either liquid or
solid samples, this spiking procedure is also liquid/solid-specific.
14.3.2.1 Liquid Samples: Homogenize an unspiked Class A biosolid sample (Section
11.1). To spike a liquid sample, add 0.5 mL of spiking suspension dilution "D"
to 300 mL of pH adjusted unspiked homogenized sample, cover, and blend on
high speed for 1 - 2 minutes. This is the "spiked" sample. The volume (mL) of
undiluted spiking suspension added to each mL of the homogenized biosolid
sample is 1.67 x 10"9 mL per mL [(0.5 mL x 10"6 mL) / 3 00 mL of biosolid],
which is referred to as Vsplked per unit bbsoiids • Proceed to Section 11.2 (inoculation).
14.3.2.2 Solid Samples: Homogenize the Class A biosolid sample (Section 11.1). To
spike a solid sample, add 0.5 mL of spiking suspension dilution "D" to 300 mL
of pH adjusted unspiked homogenized sample (30 g of sample + 270 mL of
sterile phosphate buffered dilution water), cover, and blend on high speed for 1 -
2 minutes. This is the "spiked" sample. The volume (mL) of undiluted
spiking suspension added to each g (wet weight) of the homogenized biosolid
sample is 1.67 x 10"8 mL per g [(0.5 mL x 10"6 mL) / 30 g of biosolid], which is
referred to as Vsplkedper wn biOSoiidS- Proceed to Section 11.2 (inoculation).
27 September 2014
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Method 1682
14.4 Enumeration of undiluted spiking suspension
14.4.1 Prepare heart infusion agar (HIA) according to Section 7.12, add 10 -15 mL of HIA per
100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry.
Note: To ensure that the agar surface is dry prior to use, plates should be made several
days in advance and stored inverted at room temperature or dried using a
laminar-flow hood.
14.4.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine
spread plates:
• Pipet 0.1 mL of dilution "B" onto surface of pre-dried HIA plate [10"5 mL (0.00001
mL) of the original spiking suspension].
• Pipet 0.1 mL of dilution "C" onto surface of pre-dried HIA plate [10"6 mL (0.000001
mL) of the original spiking suspension].
• Pipet 0.1 mL of dilution "D" onto surface of pre-dried HIA plate [10"7 mL (0.0000001
mL) of the original spiking suspension].
14.4.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum over
surface of medium by rotating the dish by hand or on a turntable.
14.4.4 Allow inoculum to absorb into the medium completely.
14.4.5 Invert plates and incubate at 36°C ± 1.5°C for 24 ± 4 hours.
14.4.6 Count and record number of colonies per plate.
14.5 Calculation of Laboratory-Prepared Spike Percent Recovery
Spiked Salmonella typhimurium percent recovery will be calculated in four steps as indicated
below.
Note: The example calculated numbers provided in the tables below have been rounded at the
end of each step. If your laboratory recalculates the examples using a spreadsheet and
rounds only after the final calculation (Step 4), the percent recoveries may be slightly
different.
14.5.1 Step 1: Calculate Concentration of Salmonella typhimurium (CFU / mL) in Undiluted
Spiking Suspension
14.5.1.1 The number of Salmonella typhimurium CFU /mL in the undiluted spiking
suspension will be calculated using all HIA plates yielding counts within the
ideal range of 30 to 300 CFU per plate.
14.5.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if the colonies
are not discrete, results should be recorded as "too numerous to count" (TNTC).
28 September 2014
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Method 1682
14.5.1.3 Calculate the concentration of Salmonella typhimurium (CFU / mL) in the
undiluted spiking suspension according to the following equation. (Example
calculations are provided in Table 7, below.)
Salmonella undilutedspike = (CFUt + CFU2 + ... + CFUn) / (Vt + V2 + ... + Vn)
Where,
Salmonella undiluted spike = Salmonella typhimurium CFU / mL in undiluted
spiking suspension
CFU = Number of colony forming units from HIA plates
yielding counts within the ideal range of 30 to 300
CFU per plate
V = Volume of undiluted sample on each HIA plate
yielding counts within the ideal range of 30 to 300
CFU per plate
n = Number of plates with counts within the ideal range
Table 7. Example Calculations of Salmonella typhimurium Spiking Suspension Concentration
Examples
Example 1
Example 2
CFU / plate (triplicate analyses) from HIA plates
10'5 mL plates
275, 250, 301
TNTC, TNTC,
TNTC
10'6 mL plates
30, 10, 5
TNTC, 299,
TNTC
10~7 mL plates
0, 0, 0
12, 109, 32
Salmonella CFU / mL in undiluted spiking
suspension
(Salmonella undiluted spike)
(275+250+30) /(10'5+ 10'5+10-6) =
5557 (2.1 x10'5) = 26,428,571 =
2.6x107CFU/mL
(299+109+32) /(10'6+ 10'7+10'7) =
440 / (1 .2 x 1 0'6) =366,666,667 =
3.7x108CFU/mL
Salmonella undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per
plate
14.5.2 Step 2: Calculate the Concentration of Spiked Salmonella CFU / mL or CFU / g (wet
weight)
14.5.2.1 The volume of undiluted spiking suspension per unit (mL or g) of spiked
biosolid samples (Vsplkedperunitbiosoiids) is provided in Table 8, below.
Table 8. Volume of Undiluted Spiking Suspension per Unit (mL or g) of Spiked Biosolid Samples
(V spiked per unit biosolids)
Description of spiked sample
Class A liquid
Class A solid
V spiked per unit biosolids
1.67x10"9 mL per mL of biosolids
1 .67 x 1 0"8 mL per g of biosolids (wet weight)
14.5.2.2 Calculate concentration of Spiked Salmonella wet weight (CFU / mL or CFU /
g) according to the following equation. Example calculations are provided in
Table 9, below.
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Method 1682
Spiked Salmonella wet weight = (Salmonella undiluted spike) x (Vspiked per unit bio
solids/
Spiked;
org
Where,
piked Salmonella wet weight = Number of spiked Salmonella CFU per mL
of biosolid (wet weight)
Salmonella undiluted spike = Salmonella CFU / mL in undiluted spiking
suspension
VSpiked per unit biosoiids = mL of undiluted spiking suspension per mL or
g of spiked biosolid
Table 9.
Salmonella undiluted spike
(Table 7 above)
Example 1:
2.6x107CFU/mL
Example 2:
3.7x108CFU/mL
Vspiked (Table 8 above)
Liquid: 1.67 x 10"9 mL per mL of
biosoiids
Solid: 1 .67 x 1 0"8 mL per g of biosoiids
(wet weight)
Liquid: 1.67 x 10"9 mL per mL of
biosoiids
Solid: 1 .67 x 1 0"8 mL per g of biosoiids
(wet weight)
Spiked Salmonella Wet weight
[CPU / mL or CPU / g (wet weight)]
(2.6 x 107 CPU / mL) x (1.67 x 10'9 mL / mL)
= 0.043 CPU /mL
(2.6 x 1 07 CPU / mL) x (1 .67 x 1 0'8 mL / g)
= 0.43 CPU /g (wet weight)
(3.7 x 108 CPU / mL) x (1.67 x 10'9 mL / mL)
= 0.62 CPU /mL
(3.7 x 108 CPU / mL) x (1.67 x 10'8 mL / g)
= 6.2CFU/g (wet weight)
14.5.3 Step 3: Convert to "True" Spiked Salmonella CFU / 4 g Total Solids (dry weight)
14.5.3.1 Convert to "true" spiked CFU / 4 g total solids dry weight (T) using the spiked
Salmonella mL or g (wet weight) as the numerator in the equation. Examples
are provided in Table 10, below.
Table 10. Examples of Conversion to "True" Spiked Salmonella CFU / 4 g Total Solids (Dry Weight)
Example Total Solids
(Method 1684)
Example 1
Example 2
Class A liquid: 5%
Class A solid: 82%
Class A liquid: 7%
Class A solid: 88%
[(Spiked Salmonella wet weight from Table 9 above) / percent total solids]
= True spiked Salmonella CFU / 4 g dry weight
x4
(0.043 / 0.05) x 4 = 3.5 CFU / 4 g dry weight
(0.43 / 0.82) x 4 = 2.1 CFU / 4 g dry weight
(0.62 / 0.07) x 4 = 35 CFU / 4 g dry weight
(6.2 / 0.88 ) x 4 = 28 CFU / 4 g dry weight
14.5.4 Step 4: Calculate Percent Recovery
14.5.4.1 Calculate percent recovery (R) using the following equation.
R = 100x
(NS-NU)
Where,
R
Ns =
Nu =
T
Percent recovery
Salmonella MPN / 4 g (dry weight) in the spiked sample
Salmonella MPN / 4 g (dry weight) in the unspiked sample
True spiked Salmonella CFU / 4 g (dry weight) in spiked sample
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Method 1682
14.5.4.2 Example percent recovery calculations are provided in Table 11.
Table 11. Example Percent Recovery Calculations
Ns
2.6
2.4
46
16
Nu
0.268
T
Example 1: 3.5
Example 1: 2.1
Example 2: 35
Example 2: 28
Percent recovery (R)
1 00 x (2.6 -0.268) 73.5 = 67%
100 x (2.4 -0.268)72.1 =101%
100 x (46 - 0.268) 7 35 = 130%
100 x (16 -0.268) 7 28 = 56%
14.6 BioBall™ Sample Spiking (Class A Biosolids) and Enumeration
14.6.1 Sample spiking
Since sample homogenization procedures in Method 1682 are specific to either liquid or
solid samples, this spiking procedure is also liquid/solid-specific.
14.6.1.1 Liquid Samples: Homogenize an unspiked Class A biosolid sample. Open
BioBall™ vial by removing the crimp and cap. To spike a liquid sample,
aseptically add 1 BioBall™ to 300 mL of pH adjusted unspiked homogenized
sample, cover, and blend on high speed for 1 - 2 minutes. This is the "spiked"
sample. Proceed to Section 11.2 (inoculation).
14.6.1.2 Solid Samples: Homogenize the Class A biosolid sample (Section 11.1). Open
BioBall™ vial by removing the crimp and cap. To spike a solid sample,
aseptically add 1 BioBall™ to 300 mL of pH adjusted unspiked homogenized
sample (30 g of sample + 270 mL of sterile phosphate buffered dilution water),
cover, and blend on high speed for 1-2 minutes. This is the "spiked" sample.
Proceed to Section 11.2 (inoculation).
14.7 Enumeration of BioBall™
14.7.1 Prepare heart infusion agar (HIA) according to Section 7.12, add 10-15 mL of HIA per
100 x 15 mm petri dish, and allow to solidify. For larger plates, adjust volume
appropriately. Ensure that agar surface is dry.
Note: To ensure that the agar surface is dry prior to use, plates should be made several
days in advance and stored inverted at room temperature or dried using a
laminar-flow hood.
14.7.2 Each of the following will be conducted in triplicate, resulting in the evaluation of three
spread plates:
• Open BioBall™ vial by removing the crimp and cap. Aseptically place one
BioBall™ onto the center of each HIA plate by tipping the vial over the medium.
• Immediately pipette 200 (iL of sterile physiological saline solution (0.85%) directly
onto the BioBall™.
Allow the BioBall™ to dissolve.
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Method 1682
14.7.3 For each spread plate, using a sterile bent glass rod or spreader, distribute the BioBall™
inoculum over surface of medium by rotating the dish by hand or on a turntable and cover
the plate.
14.7.4 Allow inoculum to absorb into the medium completely.
14.7.5 Invert plates and incubate at 36°C ± 1.5°C for 24 ± 4 hours.
14.7.6 Count and record number of colonies per plate.
14.8 Calculation of BioBall™ Spike Percent Recovery
Spiked BioBall™ percent recovery will be calculated in 4 steps as indicated.
Note: The example calculated numbers provided in the tables below have been rounded at the
end of each step. If your laboratory recalculates the examples using a spreadsheet and
rounds only after the final calculation (Step 4), the percent recoveries may be slightly
different.
14.8.1 Step 1: Calculate Concentration of Salmonella typhimurium (CFU) per BioBall™
14.8.1.1 The number of Salmonella typhimurium (CFU) in the BioBalls will be
calculated using all HIA plates. Count the number of CPUs on all three plates
and calculate the mean. (An example is provided in Table 12.)
Table 12 Example calculation for mean Salmonella CFU per BioBall™
CFU / plate (triplicate analyses) from HIA plates
HIA plate count #1
40
HIA plate count #2
38
HIA plate count #3
48
Mean Salmonella CFU per BioBall™
(40 + 38 + 48) / 3 = 42
14.8.2 Step 2: Calculate the Concentration of Spiked Salmonella in the Homogenized
Sample [CFU / mL or CFU / g (wet weight)]
14.8.2.1 Since sample homogenization procedures in Method 1682 are specific to either
liquid or solid samples, the concentration of spiked Salmonella in the
homogenized sample will be reported as CFU / mL for liquid samples or CFU / g
for solid samples. Calculate the concentration of spiked Salmonella in the
homogenized sample using the equation below. Examples are provided in
Table 13, below.
„..,„, „ Salmonella mean CFU
Spiked Salmonella weiweighi = meanC*u
» homogenized sample
Where,
Spiked Salmonella wet weigi,t = Number of spiked Salmonella CFU per mL or g
of biosolid (wet weight)
Salmonella mean CFU = Mean CFU of Salmonella in BioBalls
Vhomogenized sample = mL or g of spiked homogenized biosolid
sample
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Method 1682
Table 13. Example Calculation of Spiked Salmonella in the Homogenized Sample
(Salmonella wet weight)
Salmonella mean CPU
(Table 12 above)
42 CPU
V homogenized sample
Liquid: 300 ml_
Solid: 30 g
Spiked Salmonella wet weight
[CPU / mL or CPU / g (wet weight)]
42 CPU / 300 mL = 0.14 CPU / mL (wet weight)
42 CPU / 30 g = 1 .4 CPU / g (wet weight)
14.8.3 Step 3: Convert to "True" Spiked Salmonella CFU / 4 g Total Solids (dry weight)
14.8.3.1 Convert to "true" spiked CFU / 4 g total solids dry weight (T) using the spiked
Salmonella per mL or g (wet weight) as the numerator in the equation.
Examples are provided in Table 14, below.
Table 14. Examples of Conversion to "True" Spiked Salmonella CFU / 4 g Total Solids (Dry Weight)
Example Total Solids
Class A liquid: 5%
Class A solid: 82%
[(Spiked Salmonella wet weight from Table 13 above) / percent total solids]
= True spiked Salmonella CFU / 4 g (dry weight)
x4
(0.14 / 0.05) x 4 = 1 1 .2 CFU / 4 g dry weight
(1.4/0.82)x4 = 6.8 CFU /4g dry weight
14.8.4 Step 4: Calculate Percent Recovery
14.8.4.1 Calculate percent recovery (R) using the following equation.
R = 100x
(N. - Nu)
Where,
R = Percent recovery
Ns = Salmonella MPN / 4 g (dry weight) in the spiked sample
Nu = Salmonella MPN / 4 g (dry weight) in the unspiked sample
T = True spiked Salmonella CFU / 4 g (dry weight) in spiked sample
14.8.4.2 Example percent recovery calculations are provided in Table 15.
Table 15. Example Percent Recovery Calculations
Ns
12
4.8
Nu
0.268
T
11.2
6.8
Percent recovery (R)
100x(12-0.268)/11. 2 = 105%
1 00 x (4.8 -0.268) 76.8 = 67%
15.0 Method Performance
15.1 Interlaboratory Validation of Method 1682
15.1.1 Twelve volunteer laboratories and a referee laboratory participated in the U.S.
Environmental Protection Agency's (EPA's) interlaboratory validation study of EPA
Method 1682. The purposes of the study were to characterize method performance across
multiple laboratories and multiple biosolid matrices and to develop quantitative quality
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September 2014
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Method 1682
control (QC) acceptance criteria. A detailed description of the study and results are
provided in the validation study report (Reference 18.2). 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 March 2003 draft
version of EPA Method 1682.
15.1.2 Recovery - Method 1682 mean recoveries of Salmonella from compost, thermophilically
digested liquid, thermophilically digested solid, and alkaline-stabilized biosolids, spiked
with BioBall™ spikes were 42%, 13%, 91%, and 19%, respectively. Mean recoveries of
Salmonella from compost, thermophilically digested liquid, thermophilically digested
solid, and alkaline-stabilized biosolids, spiked with laboratory-spiked spikes were 100%,
5.6%, 87%, and 55%, respectively. Mean Salmonella recoveries for Milorganite®
(reference matrix) samples spiked with BioBalls and laboratory-prepared spikes were 81%
and 120%, respectively.
15.1.3 Precision - Method 1682 overall relative standard deviations (RSDs) from biosolids,
spiked with BioBalls ranged from 62% to 150%. For biosolid samples spiked with
laboratory-prepared spiking suspensions, RSDs ranged from 61% to 180%.
16.0 Pollution Prevention
16.1 The solutions and reagents used in this method pose little threat to the environment when recycled
and managed properly.
16.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
17.0 Waste Management
17.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly hazardous waste identification rules and land disposal restrictions,
and for protecting 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. An overview of requirements can be found in Environmental Management Guide
for Small Laboratories (EPA 233-B-98-001).
17.2 Samples, reference materials, and equipment known or suspected to have viable bacteria or viral
contamination must be sterilized prior to disposal.
17.3 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less Is Better: Laboratory Chemical Management for Waste Reduction,
both available from the American Chemical Society's Department of Government Relations and
Science Policy, 1155 16th Street NW, Washington, DC 20036.
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Method 1682
18.0 References
18.1 American Public Health Association, American Water Works Association, and Water Environment
Federation. 1998. Standard Methods for Water and Wastewater. 20th Edition. Sections 9020, 9030,
9040, 9050, and 9221.
18.2 USEPA. Results of the Interlab oratory Validation of EPA Method 1682 (MSRV)for Salmonella in
Biosolids. EPA-821-B-04-008. September 2004.
18.3 Bordner, R., J.A. Winter and P.V. Scarpino (eds.), Microbiological Methods for Monitoring the
Environment, Water and Wastes, EPA-600/8-78-017. Office of Research and Development,
USEPA
18.4 American Chemical Society (ACS). 2000. Reagent Chemicals, American Chemical Society
Specifications. American Chemical Society, New York. For suggestions of the testing of reagents
not listed by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals,
BDH, Poole, Dorset, UK and the United States Pharmacopeia.
18.5 Klee, A. J. 1993. A computer program for the determination of the most probable number and its
confidence limits. Journal of Microbiological Methods. 18:91-98.
35 September 2014
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Method 1682
19.0 Figures
Method1682Class A Sample Homogenization and Inoculation
Figurel. Liquid Sample (Sections 11.1.1 and11.2.1)
Liquid sample (SOOmq
(homogenized sample from section 11.1.1)
2Oml_ of original sample
in 10ml_ ofSXTSB
1Oml_ of original sample
inSmL of 3X TSB
1 .OmL of original sample
in 10ml_ oflXTSB
Figure2. Solid Sample (Sections 11.1.2 and 11.2.2)
Rinse sample into blender with27Oml_ of
sterile buffered dilution water
(homogenized sample from section 1 1.1.2)
2.Og of original sample
in 1 OmL of3X TSB
1 .Og of original sample
in 5mL of3X TSB
0.1 g of original sample
in 1OmL of 1X TSB
Figures 1 and 2. Liquid and Solid Samples
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Method 1682
Figure3. Method 1682MSRV Procedure
(Repeat the following for each TSB tube)
CEnrichment Phase
Section 12.2
Selection Phase
Section 12.3
Biochemical Confirmation Phase
Section 12.4
Use the same XLD colony to inoculate all three media.
If typical colonies are not present submit atypical colony.
MSRV
Incubate for24±2 h
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Method 1682
20.0 Glossary of Definitions and Purposes
These definitions and purposes are specific to this method but have been conformed to common usage as
much as possible.
20.1 Units of weight and measure and their abbreviations
20.1.1 Symbols
°C degrees Celsius
< less than
> greater than
% percent
± plus or minus
(iL microliter
20.1.2 Alphabetical characters
ASTM American Society for Testing and Materials
ATCC formerly known as American Type Culture Collection
CFR Code of Federal Regulations
EPA United States Environmental Protection Agency
g gram
L liter
mg milligram
mL milliliter
mm millimeter
MPN most probable number
NIST National Institute of Standards and Technology
QA quality assurance
QC quality control
TD to deliver
20.2 Definitions, acronyms, and abbreviations (in alphabetical order).
Analyte—The microorganism tested for by this method. The analyte in this method is
Salmonella.
Enrichment—A non-selective culture media for enhanced growth.
Liquid samples—Generally, samples containing <7 % total solids (dry weight).
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Method blank—An aliquot of sterile reagent water that is treated exactly as a sample including
exposure to all glassware, equipment, media, procedures that are used with samples. The method
blank is used to verify the sterility of equipment, materials, and supplies.
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Method 1682
Most probable number method (MPN)—A statistical determination of the number of bacteria per
weight or volume of sample. It is based on the fact that the greater the number of bacteria in a
sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to
grow in a dilution series.
Must—This action, activity, or procedural step is required.
Negative control—A control culture that, when analyzed exactly like a field sample, will produce a
known negative result for a given type of media.
Positive control—A control culture that, when analyzed exactly like a field sample, will produce a
known positive result for a given type of media.
Preparation blank—See Method blank.
Selective media—A culture media designed to suppress the growth of unwanted microorganisms
and encourage the growth of desired ones.
Should—This action, activity, or procedural step is suggested but not required.
Solid samples—Generally, samples containing >7 % total solids (dry weight).
39 September 2014
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