vvEPA
United States
Environmental Protection
Agency
Method 1682: Salmonella in Sewage
Sludge (Biosolids) by Modified
Semisolid Rappaport-Vassiliadis
(MSRV) Medium
July 2006
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-06-14
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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
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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)
IV
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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.
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Table of Contents
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 4
8.0 Sample Collection, Handling, and Storage 10
9.0 Quality Control 12
10.0 Equipment Calibration and Standardization 17
11.0 Sample Preparation 18
12.0 Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium Procedure 20
13.0 Data Analysis and Calculations 24
14.0 Sample Spiking Procedure 27
15.0 Method Performance 36
16.0 Pollution Prevention 37
17.0 Waste Management 37
18.0 References 38
19.0 Figures 39
20.0 Glossary of Definitions and Purposes 41
VI
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Method 1682: Salmonella in Sewage Sludge (Biosolids) by
Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium
July 2006
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 referto 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.
July 2006
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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.
July 2006 2
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Method 1682
5.4 Mouth-pipetting is prohibited.
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
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 jars and base
6.31 Waterbath maintained at 50°C for tempering agar
6.32 Media filtration equipment, sterile, 0.22-(im pore size syringe filters
3 July 2006
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Method 1682
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
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.
July 2006
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Method 1682
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.
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).
July 2006
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Method 1682
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 (MgQ2) 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.
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.
July 2006
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Method 1682
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 0.8 g
Sodium thiosulfate 6.8 g
Sodium chloride 5.0g
Agar 15.0g
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
1.0 N 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 mediate boiling sterilizes the media, overheating or autoclaving may
cause precipitation.
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
July 2006
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Method 1682
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 3.0 g
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.
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.
July 2006 8
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Method 1682
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 apositive control for urease.
7.15 Negative controls
7.15.1 Obtain a stock culture of Escherichia coli 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.
July 2006
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Method 1682
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
1 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
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)
July 2006
10
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Method 1682
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.)
11 July 2006
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Method 1682
8.7 Record the following in your log book:
8.7.1 Facility name and location
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.
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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
0% - 254%
92%
0% - 287%
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Method 1682
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
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.
July 2006 14
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Method 1682
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.
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
0%-158%
177%
Lab-prepared
acceptance criteria
0% - 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.
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Method 1682
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
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.
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Method 1682
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 H2S production (which
may result in a black butt)
Alkaline slant (purple) with
alkaline butt (purple) with or
without H2S 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) (£. coli
has marked inhibition)
Other colors with or without black
centers (e.g. £ coli is yellow
without black center)
Other color combinations (e.g., £
coli is yellow slant and butt)
Other color combinatiions (e.g., £.
coli is red to red/purple slant and
butt without H2S production)
No color change (Salmonella is
urease negative)
No agglutination
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.
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July 2006
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Method 1682
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 pHto 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.
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 ION HC1.
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
July 2006 18
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Method 1682
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.
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.
19 July 2006
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Method 1682
(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 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.
July 2006 20
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Method 1682
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 prior to 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.
Repeat this step using another target colony from the MSRV plate. In addition, inoculate
XLD positive and negative controls. (See Photo 1)
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Method 1682
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.
July 2006
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Method 1682
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
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.
23 July 2006
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Method 1682
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 103°C to 105°C Allow to cool in a desiccator before weighing. Calculate the % dry
weight as follows:
g dry sample
% dry weight = x 100
g sample
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.
July 2006 24
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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
0-0-0
0-0-1
0-0-2
0-0-3
0-0-4
0-0-5
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
0-2-0
0-2-1
0-2-2
0-2-3
0-2-4
0-2-5
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
0-4-0
0-4-1
0-4-2
0-4-3
0-4-4
0-4-5
0-5-0
0-5-1
0-5-2
0-5-3
0-5-4
0-5-5
1-0-0
1-0-1
1-0-2
1-0-3
1-0-4
1-0-5
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
1-2-0
1-2-1
1-2-2
1-2-3
1-2-4
1-2-5
MPN Index
<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
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
95% Confidence Limits
Lower I 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
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 0.0241
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
Combination of
Positives
1-3-0
1-3-1
1-3-2
1-3-3
1-3-4
1-3-5
1-4-0
1-4-1
1-4-2
1-4-3
1-4-4
1-4-5
1-5-0
1-5-1
1-5-2
1-5-3
1-5-4
1-5-5
2-0-0
2-0-1
2-0-2
2-0-3
2-0-4
2-0-5
2-1-0
2-1-1
2-1-2
2-1-3
2-1-4
2-1-5
2-2-0
2-2-1
2-2-2
2-2-3
2-2-4
2-2-5
2-3-0
2-3-1
2-3-2
2-3-3
2-3-4
2-3-5
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
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
0.0234
0.0315
0.0397
0.0480
0.0565
0.0652
0.0327
0.0413
0.0501
0.0590
0.0681
0.0774
0.0431
0.0523
0.0617
0.0714
0.0813
0.0914
0.0547
0.0647
0.0750
0.0855
0.0964
0.1076
0.0681
0.0791
0.0904
0.1021
0.1143
0.1268
95% Confidence Limits
Lower I 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
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
0.0110 0.0887
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
! Table was developed using the MPN calculator developed by Albert Klee (Reference 18.5)
25
July 2006
-------
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 (cont.)
Combination of
Positives
3-0-0
3-0-1
3-0-2
3-0-3
3-0-4
3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
3-1-5
3-2-0
3-2-1
3-2-2
3-2-3
3-2-4
3-2-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-0
3-4-1
3-4-2
3-4-3
3-4-4
3-4-5
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
4-0-1
4-0-2
4-0-3
4-0-4
4-0-5
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0
4-2-1
4-2-2
4-2-3
4-2-4
4-2-5
MPN Index
0.0255
0.0330
0.0417
0.0506
0.0598
0.0691
0.0344
0.0435
0.0529
0.0626
0.0725
0.0827
0.0456
0.0555
0.0657
0.0763
0.0872
0.0984
0.0583
0.0693
0.0806
0.0924
0.1046
0.1173
0.0733
0.0856
0.0984
0.1118
0.1258
0.1405
0.0913
0.1055
0.1204
0.1362
0.1529
0.1707
0.0381
0.0461
0.0563
0.0668
0.0777
0.0890
0.0484
0.0592
0.0705
0.0822
0.0945
0.1072
0.0626
0.0748
0.0875
0.1009
0.1150
0.1299
95% Confidence Limits
Lower I Upper
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
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
0.0342 0.1773
0.0136 0.0983
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
Combination of
Positives
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
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0
5-3-1
5-3-2
5-3-3
5-3-4
5-3-5
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
0.0797
0.0937
0.1086
0.1245
0.1414
0.1595
0.1012
0.1181
0.1364
0.1563
0.1780
0.2015
0.1304
0.1524
0.1769
0.2046
0.2357
0.2708
0.0549
0.0637
0.0763
0.0896
0.1037
0.0953
0.0678
0.0816
0.0963
0.1121
0.1291
0.1293
0.0879
0.1046
0.1227
0.1427
0.1646
0.1767
0.1151
0.1368
0.1614
0.1895
0.2216
0.2527
0.1571
0.1907
0.2319
0.2834
0.3475
0.4256
0.2398
0.3477
0.5422
0.9178
1 .6090
>1 .609000
95% Confidence Limits
Lower I Upper
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
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
1 Table was developed using the MPN calculator developed by Albert Klee (Reference 18.5)
July 2006
26
-------
Method 1682
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] x 4
Percent total solids expressed as a decimal
Example calculations are provided in Table 6, below.
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 / 0.1 1=. 067
0.1181 /0.11 = 1.181
0.5422 / 0.1 1 = 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 /4g
(0.0435) (4) / (0.02) =
8.7 MPN /4g
(0.5422) (4) / (0.03) =
72MPN/4g
(0.067) (4) / (0.96) =
0.28 MPN /4g
(1.181)(4)/(0.18) = 26
MPN/4g
(5.422) (4) / (0.43) = 50
MPN/4g
1 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.
27
July 2006
-------
Method 1682
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.
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.
July 2006 28
-------
Method 1682
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 samplers')
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) / 300 mL of biosolid], which is
referred to as Vsplkedperumtblosollds. 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 Vsplked
perumtbiosoiids- Proceed to Section 11.2 (inoculation).
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.
29 July 2006
-------
Method 1682
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).
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.)
July 2006 30
-------
Method 1682
Salmonella
undiiuted spike
Where,
Salmonella
CFU
V
undiluted spike
+ CFU2+... + CFUn)/(V1+V2 + ...+Vn)
Salmonella typhimurium CFU / mL in undiluted spiking
suspension
Number of colony forming units from HIA plates
yielding counts within the ideal range of 30 to 300 CPU
per plate
Volume of undiluted sample on each HIA plate yielding
counts within the ideal range of 30 to 300 CPU per plate
Number of plates with counts within the ideal range
Table 7. Example Calculations of Salmonella typhimurium Spiking Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from HIA plates
lO^mL plates
275, 250, 301
TNTC, TNTC,
TNTC
10'6mL 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 SDike) a
(275+250+30) /(10-5+ 10-5+1Q-6) =
5557 (2.1 x10-5) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10-6+ 10-7+1Q-7) =
440 / (1 .2 x ID'6) =366,666,667 =
3.7x108CFU/mL
^Salmonella undi|Uted spikeis calculated using all plates yielding counts within the ideal range of 30 to 300 CPU 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 (Vsplkedperunitblosollds) is provided in Table 8, below.
Table 8. Volume of Undiluted Spiking Suspension per Unit (mL or g) of Spiked Biosolid Samples
" snikpri npr unit hinsnliris/
Description of spiked sample
Class A liquid
Class A solid
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)
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Method 1682
14.5.2.2 Calculate concentration of Spiked Salmonella wet weight (CPU / mL or
CPU / g) according to the following equation. Example calculations are
provided in Table 9, below.
Spiked Salmonella wetweight = (Salmonella
Where,
Spiked Salmonella wetweight
Salmonella
undiluted spike
like) X
spiked per unit biosolidsJ
undiluted spike
V,
spiked per unit biosolids
Number of spiked Salmonella CPU per mL or g
of biosolid (wet weight)
Salmonella CPU / mL in undiluted spiking
suspension
mL of undiluted spiking suspension per mL or g
of spiked biosolid
Table 9. Example Calculations of Spiked Salmonella,
Salmonella undMuted spike
(Table 7 above)
Example 1:
2.6x107CFU/mL
Example 2:
3.7x108CFU/mL
V spiked (Table 8 above)
Liquid: 1.67 x 10~9 mL per mL of
biosolids
Solid: 1 .67 x 1 0~8 mL per g of biosolids
(wetweight)
Liquid: 1.67 x 10~9 mL per mL of
biosolids
Solid: 1 .67 x 1 0'8 mL per g of biosolids
(wetweight)
Spiked Sa/mone//awetweight
[CPU / mL or CPU / g (wet weight)]
(2.6 x 1 07 CPU / mL) x (1 .67 x 10'9 mL / mL)
= 0.043 CPU /mL
(2.6x107CFU/mL)x(1.67x10-8 mL/g)
= 0.43 CPU /g (wet weight)
(3.7x1 08 CPU / mL) x (1 .67 x 1 O'9 mL / mL)
= 0.62 CPU /mL
(3.7x108CFU/mL)x(1.67x10-8 mL/g)
= 6. 2 CPU /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 we, 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
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Method 1682
14.5.4 Step 4: Calculate Percent Recovery
14.5.4.1
Calculate percent recovery (R) using the following equation.
R = 100 x
(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 CPU / 4 g (dry weight) in spiked sample
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%
1 00 x (2.4-0.268)72.1 =101%
1 00 x (46 -0.268) 7 35 = 130%
1 00 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).
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Method 1682
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.
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.)
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Method 1682
Table 12.
Example calculation for mean Salmonella CFU per BioBall™
CPU / 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.
Spiked Salmonella
wet weight
Salmonella
mean CFU
V,
homogenized sample
Where,
Spiked Salmonella wetweight
Salmonella
mean CFU
V,
homogenized sample
Number of spiked Salmonella CFU per mL or g
of biosolid (wet weight)
Mean CFU of Salmonella in BioBalls
mL or g of spiked homogenized biosolid sample
Table 13. Example Calculation of Spiked Salmonella in the Homogenized Sample (Salmonella^
weight)
Salmonella meanCFU
(Table 12 above)
42 CFU
" homogenized sample
Liquid: 300 mL
Solid: 30 g
Spiked Salmonella wetweigh,
[CFU / mL or CFU / g (wet weight)]
42 CFU / 300 mL = 0. 1 4 CFU / mL (wet weight)
42 CFU / 30 g = 1 .4 CFU / 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.
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July 2006
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Method 1682
Table 14. Examples of Conversion to "True" Spiked Salmonella CPU / 4 g Total Solids (Dry Weight)
Example Total Solids
Class A liquid: 5%
Class A solid: 82%
[(Spiked Salmonella wetweightfrom Table 13 above) / percent total solids]
= True spiked Salmonella CPU / 4 g (dry weight)
x4
(0.14 / 0.05) x 4 = 11. 2 CPU /4g dry weight
(1.4 / 0.82) x 4 = 6.8 CPU /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
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 CPU / 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 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.
July 2006
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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 Interlaboratory Validation of EPA Method 1682 (MSRV) for Salmonella
inBiosolids. 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.
July 2006 38
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Method 1682
19.0 Figures
Figures 1 and 2. Liquid and Solid Samples
Method 1682 Class A Sample H om o g e n izatio n and Inoculation
Figure 1. Liquid Sample (Sections 11.1.1 and 11.2.1)
Liquid sample (300 m L)
in 10 mL of 3X TSB
in 10 mL of 1X TSB
Figure 2. Solid Sample (Sections 11.1.2 and 11.2.2)
Rinse sample into blenderwith 270 ml of
sterile buffered dilution water
2.0 g of origin al sample
in 10 mL of 3X TSB
1.0 g of origin al sample
in 5 mL of 3X TSB
0.1 g of origin al sample
in 10 mL of 1X TSB
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July 2006
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Figure 3. Method 1682 MSRV Procedure
(Repeat the following for each TSB tube)
C Enrichment
Section 12.2 J
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.
XLD
Incubate for21 ±3 h
@36°C±1.5°C
Incubate for 17 ± 1 h
@ 42°C ± 0.5°C
) Indicates a "halo"
Incubate for 24 ± 2 h
@36°C±1.5°C
Store one XLD @
1°C - 5°C for backup
K Emulsify growth on the slant portion of TSI (regardless of whether TSI is positive or negative)
K Place two separate drops of emulsified growth onto slide
K To the first drop of emulsified growth, add one drop of polyvalent O antiserum
K To the second drop of emulsified growth, add one drop of sterile saline as a control
Polyvalent O
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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.
41 July 2006
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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).
July 2006 42
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