United States

Environmental Protection Agency

Office of Water

(4303-T)

EPA-821-R-14-012

September 2014

Method 1682: Salmonella in Sewage 

Sludge (Biosolids) by Modified 

Semisolid Rappaport-Vassiliadis 

(MSRV) Medium
U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460
Method 1682

Acknowledgments

This method was prepared under the direction of Mark C. Meckes of the National Risk Management 

Research Laboratory within the U.S. Environmental Protection Agency’s (EPA’s) Office of Research and 

Development, and Robin K. Oshiro of the Office of Science and Technology within EPA’s Office of Water.

The contributions of the following persons and organizations to this study are gratefully acknowledged: 

Method Development 

• Richard Danielson and Robert Cooper, BioVir Laboratory, 685 Stone Road Unit # 6, Benicia, CA 

94510 

• William Yanko, Private Consultant, 1111 El Sur Ave, Arcadia, CA 91006 

Referee Laboratory

• EPA Office of Research and Development, National Risk Management Research Laboratory: Mark C. 

Meckes and Karen M. White

Volunteer Participant Laboratories

• American Interplex: John Overbey and Steve Bradford 

• BioVir Laboratories: Rick Danielson and Jim Truscott

• City of Los Angeles Bureau of Sanitation Environmental Monitoring Division: Farhana Mohamed and 

Genevieve Espineda

• County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant (JWPCP): 

Kathy Walker and Debbie Leachman

• County Sanitation Districts of Los Angeles County, San Jose Creek Water Quality Laboratory (SJC): 

Shawn Thompson and Julie Millenbach

• Environmental Associates: Susan Boutros and John Chandler

• Hoosier Microbiological Laboratories: Don Hendrickson, Keri Nixon, Katy Bilger, and Lindsey 

Shelton 

• King County Environmental Laboratory: Greg Ma and Bobbie Anderson 

• Texas A&M University: Suresh Pillai and Jessica Cardenas

• University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord 

• Utah Department of Health: Sanwat Chaudhuri and Devon Cole 

• Wisconsin State Laboratory of Hygiene: Jon Standridge and Linda Peterson 

The following facilities provided biosolid matrices for the study 

• Compost Facility, Columbus, OH: Angela Bianco 

• Hyperion Treatment Plant, Playa del Rey, CA: Steve Fan

• N-Viro Treatment Facility, Toledo, OH: Cindy Drill

• Wastewater Treatment Facility, Sturgeon Bay, WI: Todd Maurina

• Wastewater Treatment Facility, Fairfield, OH: Drew Young 

• Wastewater Treatment Facility, Mason, OH: Ernie Stickler

Media Photographs 

• Mark C. Meckes, NRMRL, US EPA

i September 2014
Method 1682

Disclaimer

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and 

approved this report for publication. Neither the United States Government nor any of its employees, 

contractors, or their employees make any warranty, expressed or implied, or assumes any legal liability or 

responsibility for any third party’s use of or the results of such use of any information, apparatus, product, 

or process discussed in this report, or represents that its use by such party would not infringe on privately 

owned rights. Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use.

Questions concerning this method or its application should be addressed to: 

Robin K. Oshiro

Engineering and Analysis Division (4303T)

U.S. EPA Office of Water, Office of Science and Technology

1200 Pennsylvania Avenue, NW 

Washington, DC 20460 

oshiro.robin@epa.gov 

202-566-1075 

202-566-1054 (facsimile) 

ii September 2014
Method 1682

Introduction

Application of treated biosolids to land can be helpful as a crop nutrient and soil conditioner but may pose 

the risk of releasing pathogens into the environment if proper disinfection and use criteria are not met. 

Among these organisms are Salmonella, which are pathogenic enteric bacteria that can cause salmonellosis 

in animals and humans, if concentrations able to give rise to infections are present. 

The density of Salmonella in Class A biosolids for unrestricted use is to be less than three most probable 

number (MPN) per four grams of total solids (dry weight basis) at the time the biosolids are used or 

disposed. 

Method 1682 is a performance-based method for detecting Salmonella in biosolids. Method 1682 requires 

calculation of the MPN via enrichment, with selection and biochemical confirmation for determination of 

Salmonella. The enrichment step utilizes tryptic soy broth (TSB). After incubation, TSB is spotted onto 

selective modified semisolid Rappaport-Vassiliadis (MSRV) medium. Presumptively identified colonies 

are isolated on xylose-lysine desoxycholate agar (XLD). Biochemical confirmation includes lysine-iron 

agar (LIA), triple sugar iron agar (TSI), and urea broth, followed by serological typing using polyvalent O 

antisera. Calculations for concentration are based on dry weight.

iii September 2014
Method 1682

Table of Contents

Acknowledgments .......................................................................................................................................... i

Disclaimer ..................................................................................................................................................... ii

Introduction .................................................................................................................................................. iii

1.0 Scope and Application ...................................................................................................................... 1

2.0 Summary of Method ......................................................................................................................... 2

3.0 Definitions ......................................................................................................................................... 2

4.0 Interferences ...................................................................................................................................... 2

5.0 Safety ................................................................................................................................................ 2

6.0 Equipment and Supplies.................................................................................................................... 3

7.0 Reagents and Standards .................................................................................................................... 5

8.0 Sample Collection, Handling, and Storage ..................................................................................... 10

9.0 Quality Control ............................................................................................................................... 12

10.0 Equipment Calibration and Standardization ................................................................................... 17

11.0 Sample Preparation ......................................................................................................................... 17

12.0 Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium Procedure ...................................... 19

13.0 Data Analysis and Calculations ...................................................................................................... 23

14.0 Sample Spiking Procedure .............................................................................................................. 26

15.0 Method Performance ....................................................................................................................... 33

16.0 Pollution Prevention ........................................................................................................................ 34

17.0 Waste Management ......................................................................................................................... 34

18.0 References ....................................................................................................................................... 35

19.0 Figures............................................................................................................................................. 36

20.0 Glossary of Definitions and Purposes ............................................................................................. 38

iv September 2014
Method 1682

Method 1682: Salmonella in Sewage Sludge (Biosolids) by 

Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium

September 2014

1.0 Scope and Application

1.1 This method is for the detection and enumeration of Salmonella (CAS registry number 68583-35-7) 

in treated biosolids by enrichment, selection, and characterization. It is intended to enumerate 

Salmonella to help determine the suitability of biosolids for land application in compliance with 40 

Code of Federal Regulations (CFR) Part 503. Although Method 1682 is similar to existing 

recognized procedures using separate media for enrichment, selection, and confirmation of the 

organism, it is intended to be more specific and have greater recovery.

1.2 This method is designed to meet monitoring requirements for Salmonella under 40 CFR Part 503 

Subpart D. Subpart D of the Part 503 regulation defines the requirements for biosolids to be 

classified as either Class A or B with respect to pathogens. Classification of biosolids prior to land 

application provides a means to protect public health and the environment. Following appropriate 

treatment, a biosolid sample is classified as Class A if Salmonella densities are below 3 MPN / 4 

grams of total solids (dry weight basis).

1.3 Although the Part 503 regulation does not specify the total number of samples for Class A 

biosolids, it suggests that a sampling event extend over two weeks, and that at least seven samples 

be tested to confirm that the mean bacterial density of the samples is below 3 MPN / 4 g of total 

solids (dry weight basis). The analysis of seven samples increases the method precision by 

reducing the standard error caused by inherent variations in biosolid quality.

1.4 Although Method 1682 is selective for Salmonella bacteria, it does not differentiate among 

Salmonella species.

Note: H2S negative Salmonella will be missed if translucent pink to red colonies are not 

submitted to biochemical and serological confirmation.

1.5 Method 1682 was submitted to interlaboratory validation in Class A biosolid matrices. A 

comprehensive evaluation of the study results is presented in the validation study report (Reference 

18.2). For method application please refer to Title 40 Code of Federal Regulations Part 136 (40 

CFR Part 136).

1.6 This method is not intended for use in water samples or as a test for microorganisms other than 

Salmonella. Use of this method and appropriate validation for matrices other than Class A 

biosolids is the responsibility of the user.

1.7 Any modification of the method beyond those expressly permitted is subject to the application and 

approval of alternative test procedures under 40 CFR Parts 136.4 and 136.5.

1 September 2014
Method 1682

2.0 Summary of Method

2.1 The modified semisolid Rappaport-Vassiliadis (MSRV) medium protocol presented in Method 

1682 provides enumeration of Salmonella in biosolids based on the most probable number (MPN) 

technique. The determination of Salmonella involves inoculating the enrichment medium, tryptic 

soy broth (TSB), with a measured amount of sample and incubating for 24 hours. After 

incubation, TSB is spotted onto the selective MSRV medium. The MSRV medium uses 

novobiocin and malachite green to inhibit non-Salmonella species, while allowing most 

Salmonella species to grow. Presumptively identified colonies are isolated on xylose-lysine 

desoxycholate agar (XLD), and confirmed using lysine-iron agar (LIA), triple sugar iron agar 

(TSI), and urea broth, followed by positive serological typing using polyvalent O antisera. A total 

solids (% dry weight) determination is performed on a representative biosolids sample and is used 

to calculate MPN / g dry weight. Salmonella density is reported as MPN / 4 g dry weight.

3.0 Definitions

3.1 Salmonella are gram-negative, predominately motile, facultatively-anaerobic, rod-shaped bacteria 

that comprise about 2,000 serovars.

3.2 Class A biosolids are biosolids that meet bacteriological and treatment requirements stipulated in 

the 40 CFR 503 Subpart D.

3.3 Definitions for other terms are provided in the glossary at the end of the method (Section 20.0).

4.0 Interferences

4.1 Low estimates of Salmonella may be caused by the presence of high numbers of competing or 

inhibitory organisms, or toxic substances such as metals or organic compounds.

5.0 Safety

5.1 The analyst must know and observe normal safety procedures required in a microbiology 

laboratory while preparing, using, and disposing of media, cultures, reagents, and materials, 

including operation of sterilization equipment.

5.2 Field and laboratory staff collecting and analyzing environmental samples are under some risk of 

exposure to pathogenic microorganisms. Staff should apply safety procedures used for pathogens 

to handle all samples.

5.3 This method does not address all safety issues associated with its use. It is the responsibility of the 

laboratory to establish appropriate safety and health practices prior to use of this method. A 

reference file of material safety data sheets (MSDSs) should be available to all personnel involved 

in Method 1682 analyses.

5.4 Mouth-pipetting is prohibited.

2 September 2014
Method 1682

6.0 Equipment and Supplies

6.1 Sterile plastic bags, 1-gallon

6.2 Sterile plastic or glass jars with lids, 1-L

6.3 Sterile auger

6.4 Sterile scoops (do not use curved scoops)

6.5 Ice chest

6.6 Wet ice

6.7 Ice packs, blue ice

6.8 Bubble wrap

6.9 Sterile trowels

6.10 Sterile aluminum foil or kraft paper

6.11 Sterile container, such as a stainless steel or plastic bucket suitable for sample collection

6.12 Flat shovel 

6.13 Tubes, 25 × 150 mm, borosilicate glass, with loose-fitting aluminum, stainless steel or autoclavable 

caps

6.14 Tubes, 16 × 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

3 September 2014
Method 1682

6.24 Incubators, water- or air-jacketed, humidity-controlled, microbiological type to hold temperatures 

at 36°C ± 1.5°C and 42°C ± 0.5°C

6.25 Plastic sterile petri dishes, microbiological grade, 15 mm × 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 Water bath maintained at 50°C for tempering agar

6.32 Media filtration equipment, sterile, 0.22-μm pore size syringe filters

6.33 Magnifying glass or dissection scope

6.34 Latex gloves for handling samples and extraction equipment

6.35 pH meter

6.36 Vortex mixer

6.37 Micro pipettor

6.38 Pipet tips to deliver 30 μL

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 lb pressure per square inch [PSI]) for 

15 minutes

6.45 Crucible or aluminum evaporating dish

4 September 2014
Method 1682

7.0 Reagents and Standards

7.1 Purity of reagents: Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, 

reagents shall conform to the specifications of the Committee on Analytical Reagents of the 

American Chemical Society (Reference 18.4). The agar used for preparation of culture media 

must be microbiological grade. 

7.2 Whenever possible, use commercial culture media as a means of quality control. Storage 

temperatures and times for prepared media and reagents are provided in Table 1 in Section 7.16 

below. 

7.3 Purity of reagent water: Reagent-grade water conforming to specifications in Standard Methods for 

the Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or 

141, as applicable), Section 9020 (Reference 18.1). 

7.4 Phosphate Buffered Dilution Water

7.4.1 Composition of stock phosphate buffer solution:

Monopotassium phosphate (KH2PO4) 34.0 g 

Reagent-grade water 500.0 mL 

Preparation: Dissolve KH2PO4 in 500 mL reagent-grade water. Adjust the pH of the 

solution to 7.2 with 1 N NaOH, and bring the volume to 1 L with reagent-grade water. 

Sterilize by filtration or autoclave at 121°C (15 PSI) for 15 minutes. 

7.4.2 Preparation of stock magnesium chloride (MgCl2) solution: Add 38 g anhydrous MgCl2 or 

81.1 g magnesium chloride hexahydrate (MgCl2 • 6H2O) to 1 L reagent-grade water. 

Sterilize by filtration or autoclave at 121°C (15 PSI) for 15 minutes. 

7.4.3 After sterilization, store the stock solutions in the refrigerator until used. If evidence of 

mold or other contamination appears, the affected stock solution should be discarded and a 

fresh solution should be prepared. 

7.4.4 Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer 

and 5 mL of the MgCl2 stock per liter of reagent-grade water. Dispense in appropriate 

amounts for dilutions and/or for use as rinse buffer. Autoclave at 121°C (15 PSI) for 15 

minutes. Final pH should be 7.0 ± 0.2. The amount of time in the autoclave must be 

adjusted for the volume of buffer in the containers and the size of the load.

Note: When test tube racks containing 9.0 mL sterile dilution water are prepared, they 

are placed into an autoclavable pan with a small amount of water to contain 

breakage and minimize evaporation from the tubes.

7.5 Sterile physiological saline (0.85% w/v)

7.5.1 Dissolve 8.5 g NaCl in 1 L reagent-grade water. Dispense 5-10 mL into 16 × 100 mm 

screw cap test tubes, cap and autoclave for 15 minutes at 121°C (15 PSI). Store at room 

temperature.

5 September 2014
Method 1682

7.6 Tryptic soy broth (TSB)

7.6.1 Composition: 

Pancreatic digest of casein 17.0 g 

Enzymatic digest of soybean meal 3.0 g 

Sodium chloride 5.0 g 

Dipotassium phosphate (K2HPO4) 2.5 g 

Dextrose 2.5 g 

Reagent-grade water 1.0 L 

7.6.2 For single strength (1X) 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 × 150 mm culture tubes. The 1X TSB will 

be used for inoculation volumes ≤1 mL. Autoclave for 15 minutes at 121°C (15 PSI).

7.6.3 For triple strength (3X) TSB, prepare as in Section 7.6.2 but use 333 mL of reagent-grade 

water instead of 1 L. Dispense 10 mL and 5 mL volumes into 25 × 150 mm culture tubes. 

The 3X TSB tubes containing 10 mL of media will be inoculated with 20 mL of 

homogenized sample. The 3X TSB tubes containing 5 mL of media will be inoculated 

with 10 mL of homogenized sample. Autoclave for 15 minutes at 121°C (15 PSI). Let 

the media warm to room temperature prior to analysis. 

Note: 3X TSB is necessary for 20- and 10-mL inoculations to ensure that the inoculation 

volume does not excessively dilute the media. 

7.7 Modified Semisolid Rappaport-Vassiliadis (MSRV) medium

7.7.1 Basal medium composition: 

Tryptose 4.59 g 

Casein hydrolysate (acid) 4.59 g 

Sodium chloride 7.34 g 

Monopotassium phosphate (KH2PO4) 1.47 g 

Magnesium chloride, anhydrous (MgCl2) 10.93 g 

Malachite green oxalate 0.037 g (37 mg) 

Agar 2.7 g 

Reagent-grade water 1.0 L 

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, 0.22μm 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. 

6 September 2014
Method 1682

7.7.3 Add reagents for basal medium to 1 L of reagent-grade water, mix thoroughly, and heat to 

boiling to dissolve completely (do not autoclave). Adjust pH to 5.2 ± 0.2 with 1.0 N 

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 × 100 mm petri plates. Do not invert 

plates to store.

Note: If using a commercially prepared novobiocin antimicrobic supplement add 

sufficient volume to achieve a concentration of 0.002% per liter. 

7.8 Xylose-lysine desoxycholate agar (XLD)

7.8.1 Composition: 

Yeast extract 3.0 g 

L-lysine 5.0 g 

Xylose 3.75 g 

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 5.0 g 

Agar 15.0 g 

Phenol red 0.08 g 

Reagent-grade water 1.0 L 

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 × 100 mm sterile petri plates. Let the media warm to room 

temperature prior to inoculation. 

Note: Heating media to boiling sterilizes the media, overheating or autoclaving may 

cause precipitation.

7 September 2014
Method 1682

7.9 Triple sugar iron agar (TSI)

7.9.1 Composition: 

 

Beef extract 3.0 g 

Yeast extract 3.0 g 

Pancreatic digest of casein 15.0 g 

Proteose peptone No. 3 5.0 g 

Dextrose 1.0 g 

Lactose 10.0 g 

Sucrose 10.0 g 

Ferrous sulfate 0.2 g 

Sodium chloride 5.0 g 

Sodium thiosulfate 0.3 g 

Agar 12.0 g 

Phenol red 0.024 g 

Reagent-grade water 1.0 L 

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 × 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 5.0 g 

Yeast extract 3.0 g 

Dextrose 1.0 g 

L-lysine hydrochloride 10.0 g 

Ferric ammonium citrate 0.5 g 

Sodium thiosulfate 0.04 g 

Bromcresol purple 0.02 g 

Agar 15.0 g 

Reagent-grade water 1.0 L 

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 × 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. 

8 September 2014
Method 1682

7.11 Urea broth

7.11.1 Composition: 

Yeast extract 0.1 g 

Monopotassium phosphate (KH2PO4) 9.1 g 

Dipotassium phosphate (K2HPO4) 9.5 g 

Urea 20.0 g 

Phenol red 0.01 g 

Reagent-grade water 1.0 L 

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 μm filter into a 

sterile flask. Aseptically dispense 3 mL into sterile 16 × 100 mm screw cap test tubes 

using a sterile pipet or sterile dispensing syringe. Let the media warm to room 

temperature prior to inoculation.

7.12 Heart infusion agar (HIA)

7.12.1 Composition: 

Beef heart, infusion from 500 g 10.0 g 

Bacto tryptose 10.0 g 

Sodium chloride 5.0 g 

Bacto agar 15.0 g 

Reagent-grade water 1.0 L 

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 × 100 mm sterile petri plates. Let the 

media warm to room temperature prior to inoculation. Other general growth media may 

be used for quality assurance (QA) (Section 9.0) purposes. 

7.13 Salmonella O antiserum Polyvalent Groups A-I and Vi 

7.14 Positive controls

7.14.1 Obtain a stock culture of Salmonella typhimurium ATCC 14028 as a positive control for 

MSRV, XLD, TSI, LIA, and polyvalent O antiserum. 

Note: ATCC recommends that no more than 5 transfers be made before returning to the 

original culture. This will minimize the chance of contamination during transfers 

and genetic shift of the culture. One suggestion is to make your own frozen seed 

stock upon receipt of the organism that can be used for future work. For 

additional information go to http://www.atcc.org. 

7.14.2 Obtain a stock culture of Proteus vulgaris ATCC 13315 as a positive control for urease. 

9 September 2014
Method 1682

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.

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 Reagents 1

Media Storage Temperature Storage Time

Sterile physiological saline (0.85% w/v) room temperature ≤3 months

TSB: loose-capped tubes room temperature ≤2 weeks

MSRV: poured-plates (do not store inverted) room temperature ≤48 hours

XLD: poured plates (store inverted) 1°C to 5°C ≤2 weeks

TSI, LIA, Urea broth: tight-capped tubes 1°C to 5°C ≤3 months

HIA: poured plates (store inverted) 1°C to 5°C ≤2 weeks

2% novobiocin -20°C to -10°C ≤1 year

Polyvalent O antiserum 2° to 8°C lyophilized ≤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% HCl acid wash

10 September 2014
Method 1682

8.3.4 Rinse with distilled water

8.3.5 Allow to air dry

8.3.6 Cover with foil and autoclave for 15 minutes at 121°C (15 PSI)

8.4 Digester biosolids sampling procedure

8.4.1 Collect digester biosolids sample from the discharge pipe.

8.4.2 Purge the discharge pipe of old biosolids and warm to the digester temperature by allowing 

biosolids to flow through the pipe into a container or waste collection device.

8.4.3 Position a 1-gallon sterile bag under the flow so that only the sample touches the inside of 

the bag. Fill the bag, leaving 0.5 inches of head space in the bag for gas production. 

Leaving head room is extremely important when taking samples of biosolids that have 

been anaerobically digested.

8.5 Procedure for sampling conveyor belt biosolid output

8.5.1 Using a sterile scoop, transfer the pressed biosolids directly from the conveyer into a sterile 

container, without mixing or transferring to another area. 

8.5.2 Pack sample into sterile container. Leaving additional head space is not as important as in 

Section 8.4 because there is less gas formation.

8.6 Procedure for sampling from a bin, drying bed, truck bed, or similar container

8.6.1 Remove surface material (upper six inches) and set it aside. Divide the underlying 

material to be sampled into four quadrants.

8.6.2 Use a scoop or core the sample with the auger if material is deep.

8.6.3 Take a sample from each of the quadrants and combine in a sterile container.

8.6.4 After all the samples have been taken, pour the contents of the container out onto a sterile 

surface and mix by folding the sample back onto itself several times.

8.6.5 Reduce the sample size by “coning and quartering.” Divide the container contents into 

four even piles. If sample size is still too large, divide each quarter into quarters and 

discard half. Put into a glass or plastic sampling container.

8.6.6 An alternate method to “coning and quartering” is to randomly take a flat shovel full of 

biosolids from the contents of the container that has been placed on a sterile surface and put 

samples into a sampling container. (Curved scoops have been shown to favor a certain 

size particle and should not be used.)

8.7 Record the following in your log book:

8.7.1 Facility name and location

11 September 2014
Method 1682

8.7.2 Date

8.7.3 Arrival time

8.7.4 Name of facility and contact

8.8 Record the following onto sample container and in log book when known:

8.8.1 Sample number

8.8.2 Date and time

8.8.3 Sampler name

8.8.4 Sample location

8.8.5 Parameters (e.g., type of analysis, field measurements- pH and temperature) 

8.8.6 Volume

8.8.7 Observations

8.9 Ensure that the chain-of-custody form is filled out.

8.10 Sample handling: Maintain bacteriological samples at <10°C during transit to the laboratory. Do 

not allow the sample to freeze. Use insulated containers to ensure proper maintenance of storage 

temperature. Sample bottles should be placed inside waterproof bags, excess air purged, and bags 

sealed to ensure that bottles remain dry during transit or storage. Refrigerate samples upon arrival 

in the laboratory and analyze as soon as possible after collection. Bring samples to room 

temperature before analysis.

8.11 Holding time and temperature limitations: Analyses should begin immediately, preferably, within 

2 hours of collection. If it is impossible to examine samples within 2 hours, samples must be 

maintained at <10°C until analysis. Samples must not be frozen. Sample analysis must begin 

within 6 hours unless otherwise specified in the Code of Federal Regulations Part 503. 

Note: Adherence to sample handling procedures and holding time limits is critical to the 

production of valid data. Sample results will be considered invalid if these conditions are 

not met.

9.0 Quality Control

9.1 Each laboratory that uses Method 1682 is required to operate a formal quality assurance (QA) 

program that addresses and documents instrument and equipment maintenance and performance, 

reagent quality and performance, analyst training and certification, and records storage and 

retrieval. General requirements and recommendations for QA and quality control (QC) 

procedures for microbiological laboratories are provided in Reference 18.3.

12 September 2014
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 BioBallTM 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 BioBallTM

acceptance criteria

Lab-prepared spike 

acceptance criteria

Initial precision and recovery (IPR)

• Mean percent recovery 22% - 126% Detect - 254%

• Precision (as maximum relative standard deviation) 69% 92%

Ongoing precision and recovery (OPR) as percent recovery 1% - 147% Detect - 287%

9.4 Ongoing precision and recovery (OPR): To demonstrate ongoing control of the analytical 

system, the laboratory should routinely process and analyze spiked Milorganite® samples. The 

laboratory should analyze one OPR sample after every 20 field and matrix spike samples or one per 

week that samples are analyzed, whichever occurs more frequently. OPR samples must be 

13 September 2014
Method 1682

accompanied by an acceptable method blank (Section 9.7) and appropriate media sterility checks 

(Section 9.8). The OPR analysis is performed as follows:

9.4.1 Spike a 30-g sample of Milorganite® with Salmonella typhimurium ATCC 14028 

according to the spiking procedure in Section 14.0. Spiking with laboratory-prepared 

suspensions is described in Section 14.3 and spiking with BioBalls is described in Section 

14.6. Process and analyze each OPR sample according to the procedures in Sections 11.0 

and 12.0 and calculate the number of Salmonella MPN / 4 g dry weight according to 

Section 13.0.

9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in 

Section 14.5 or 14.8 for samples spiked with laboratory-prepared spiking suspensions or 

BioBalls, respectively.

9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria 

in Table 2, above. If the OPR result meets the acceptance criteria for recovery, method 

performance is acceptable and analysis of field samples may continue. If the OPR result 

falls outside of the acceptance criteria, system performance is unacceptable. In this event, 

identify the problem by evaluating each step of the analytical process (media, reagents, and 

controls), correct the problem and repeat the OPR analysis.

9.4.4 As part of the laboratory QA program, results for OPR and IPR samples should be charted 

and updated records maintained in order to monitor ongoing method performance. The 

laboratory should also develop a statement of accuracy for Method 1682 by calculating the 

average percent recovery (R) and the standard deviation of the percent recovery (sr). 

Express the accuracy as a recovery interval from R - 2sr to R + 2sr.

9.5 Matrix spikes (MS): MS analysis are performed to determine the effect of a particular matrix on 

Salmonella recoveries. The laboratory should analyze one MS sample when biosolid samples are 

first received from a source from which the laboratory has not previously analyzed samples. 

Subsequently, 5% of field samples (1 per 20) from a given biosolids source should include a MS 

sample. MS samples must be accompanied by the analysis of an unspiked field sample 

sequentially collected from the same sampling site, an acceptable method blank (Section 9.7), and 

appropriate media sterility checks (Section 9.8). When possible, MS analyses should also be 

accompanied by an OPR sample (Section 9.4), using the same spiking procedure 

(laboratory-prepared spiking suspension or BioBalls). The MS analysis is performed as follows:

9.5.1 Prepare two, 30-g field samples that were sequentially collected from the same site. One 

sample will remain unspiked and will be analyzed to determine the background or ambient 

concentration of Salmonella for calculating MS recoveries. The other sample will serve 

as the MS sample and will be spiked with Salmonella typhimurium ATCC 14028 according 

to the spiking procedure in Section 14.0.

9.5.2 Select dilutions based on previous analytical results or anticipated levels of Salmonella in 

the field sample in order to accurately estimate Salmonella density. Neither above or 

below the detection limit of the method.

9.5.3 Spike the MS sample with a laboratory-prepared suspension as described in Section 14.3 or 

with BioBalls as described in Section 14.6. Process and analyze the unspiked and spiked 

field samples according to the procedures in Sections 11.0 and 12.0.

14 September 2014
Method 1682

9.5.4 For the MS sample, calculate the Salmonella MPN / 4 g dry weight according to Section 

13.0 and adjust the density (MPN / 4 g dry weight) based on the ambient concentration of 

Salmonella observed in the unspiked matrix sample.

9.5.5 Calculate the percent recovery (R) for the MS sample (adjust based on ambient Salmonella

in the unspiked sample) using the appropriate equations Section 14.5 or 14.8 for samples 

spiked with laboratory-prepared spiking suspensions or BioBalls, respectively.

9.5.6 Compare the MS result (percent recovery) with the appropriate method performance 

criteria in Table 3. If the MS recovery meets the acceptance criteria, system performance 

is acceptable and analysis of field samples from this biosolid source may continue. If the 

MS recovery is unacceptable and the OPR sample result associated with this batch of 

samples is acceptable, a matrix interference may be causing the poor results. If the MS 

recovery is unacceptable, all associated field data should be flagged.

Table 3. Matrix Spike Precision and Recovery Acceptance Criteria

Performance test BioBallTM acceptance 

criteria

Lab-prepared 

acceptance criteria

Percent recovery for MS or MS/MSD Detect - 158% Detect - 246%

Precision (as maximum relative percent difference of MS/MSD) 177% 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. coli ATCC

25922) and analyzing as described in Section 12.0.

9.6.1.2 Negative controls are conducted by inoculating urea broth with a known 

negative control (e.g., Salmonella typhimurium ATCC 14028) and analyzing as 

described in Section 12.0.

9.6.1.3 If a negative control fails to exhibit the appropriate response, check and/or 

replace the associated media or reagents, and/or the negative control, and 

reanalyze the appropriate negative control.

9.6.2 Positive controls: The laboratory should analyze positive controls to ensure that the 

MSRV, XLD, TSI, LIA, urea broth, and polyvalent O antiserum are performing properly. 

Positive controls should be analyzed whenever a new batch of media or reagent is used. 

On an ongoing basis, the laboratory should perform a positive control every day that 

15 September 2014
Method 1682

samples are analyzed. An OPR sample (Section 9.4) may take the place of a positive 

control. Positive and negative control results are provided in Table 4.

9.6.2.1 Positive controls are conducted by inoculating MSRV, XLD, TSI, LIA, and 

polyvalent O antiserum with a known positive culture (e.g., Salmonella 

typhimurium ATCC 14028) and analyzing as described in Section 12.0.

9.6.2.2 Positive controls are conducted by inoculating urea broth with a known positive 

culture (e.g., Proteus vulgaris ATCC 13315) and analyzing as described in 

Section 12.0.

9.6.2.3 If the positive control fails to exhibit the appropriate response, check and/or 

replace the associated media or reagents, and/or the positive control, and 

reanalyze the appropriate positive control.

9.7 Method blank. Test a 20-mL sterile dilution water sample in the analytical scheme to verify the 

sterility of equipment, materials, and supplies. Absence of growth indicates freedom of 

contamination from the target organism. On an ongoing basis, the laboratory should perform a 

method blank every day that samples are analyzed.

9.8 Media sterility check. To test sterility of media, incubate a representative portion of each batch 

at 36°C ± 1.5°C (TSB, XLD, TSI, LIA, HIA, and urea broth) or 42°C ± 0.5°C (MSRV) for 24 ± 2 

hours and observe for growth. A batch is defined as one tube/plate out of 50 in each lot, or one 

tube/plate if the lot contains less than 50 tubes/plates.

Table 4. Positive and Negative Control Results

Medium Salmonella

result Positive Method 1682 reaction Negative Method 1682 reaction

Tryptic Soy Broth (TSB) Positive Turbidity No turbidity

Modified Semisolid 

Rappaport-Vassiliadis 

(MSRV) medium

Positive

Migrated cells visible as a 

gray-white turbid zone (halo) 

extending out from inoculations

Medium remains blue-green 

around inoculations with no 

gray-white turbid zone (halo) (E. 

coli has marked inhibition)

Xylose lysine 

desoxycholate agar 

(XLD) Positive Pink to red colonies with black centers

Other colors with or without black 

centers (e.g. E. coli is yellow 

without black center)

Triple sugar iron agar 

(TSI) Positive

Good growth with alkaline slant 

(red) with acid butt (yellow) with or 

without H2S production (which may 

result in a black butt)

Other color combinations (e.g., E. 

coli is yellow slant and butt)

Lysine iron agar (LIA) Positive

Alkaline slant (purple) with alkaline 

butt (purple) with or without H2S 

production (which may result in a 

black butt)

Other color combinations (e.g., E. 

coli is red to red/purple slant and 

butt without H2S production) 

Urea broth Negative Pink No color change (Salmonella is 

urease negative)

Polyvalent O Positive Agglutination No agglutination

16 September 2014
Method 1682

10.0 Equipment Calibration and Standardization

10.1 Check temperatures in incubators/waterbaths twice daily, a minimum of four hours apart, to ensure 

operation is within stated limits of the method and record daily measurements in incubator log 

book.

10.2 Check temperatures in refrigerators/freezers at least once daily to ensure operation is within stated 

limits of the method. Record daily measurements in refrigerator/freezer log book.

10.3 Calibrate thermometers and incubators semiannually against a NIST certified thermometer or one 

that meets the requirements of NIST Monograph SP 250-23 (Reference 18.1). Check mercury 

columns for breaks.

10.4 Calibrate pH meter prior to each use with two standards (pH 4.0, 7.0, and 10.0) closest to the range 

being tested.

10.5 Calibrate top-loading balances once per month with reference weights of ASTM Class 2.

11.0 Sample Preparation

11.1 Homogenization

Sample homogenization procedures are based on whether the sample is a liquid or a solid. If 

sample is alkaline-stabilized (liquid or solid), adjust the pH as described in Section 11.1.3. Liquid 

samples are generally defined as samples containing ≤7% total solids (dry weight).

11.1.1 Liquid samples: Homogenize 300 mL of sample in a sterile blender on high speed for one 

to two minutes. Adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid or 1.0 N 

sodium hydroxide, if necessary. This is the “homogenized” sample. When adjusting pH 

do not exceed the homogenized sample volume by greater than 5% (15 mL).

11.1.2 Solid samples: Weigh out 30.0 g ± 0.1 g of well-mixed sample in a sterile dish. Whenever 

possible, the sample tested should contain all materials that will be included in the biosolid. 

For example, if wood chips are part of the biosolid compost, some mixing or grinding may 

be needed to achieve homogeneity before testing. Large pieces of wood that are not easily 

ground may be discarded before homogenizing. Transfer the sample to a sterile blender. 

Use 270 mL of sterile buffered dilution water (Section 7.4) to rinse any remaining sample 

into the blender. Alternatively, the sample may be directly weighed in the sterile blender 

jar. Cover and blend on high speed for one to two minutes. This is the “homogenized” 

sample. A volume of 10-mL of the “homogenized” sample contains 1.0 g of the original 

sample. Adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid or 1.0 N sodium 

hydroxide, if necessary. When adjusting pH do not exceed the homogenized sample 

volume by greater than 5% (15 mL). 

Note: Do not suspend bacteria in dilution water for more than 30 minutes at room 

temperature. Chill on wet ice or at 4°C ± 1°C to slow replication between spiking 

samples.

17 September 2014
Method 1682

11.1.3 Alkaline-stabilized: The alkaline-stabilized biosolid samples generally have a pH of 

approximately 12. Prior to analysis, the alkaline-stabilized biosolid sample must be 

neutralized to a pH of approximately 7.5. Do not add lab-prepared spikes or BioBalls to 

the samples prior to pH adjustment. 

11.1.3.1 Adjustment of pH should be done in a fume hood. Prior to adjusting the pH of 

the sample, calibrate/standardize the pH meter with pH buffers 7.0 and 10.0. 

Weigh out 30 g of sample into a sterile 600 mL beaker, add 250 mL of sterile 

buffered dilution water and a sterile magnetic stir bar. Place beaker on a mixing 

plate, insert pH probe into mixture, begin stirring, and take an initial pH reading. 

To minimize the amount of volume added to each sample, pH should be adjusted 

using 10 N HCl. 

Note: The addition of the 10 N HCl will produce fumes, do not be alarmed. 

The addition of the acid should be done incrementally to ensure that the 

pH does not drop instantaneously below 5.0. It is recommended that 

the pH adjustment be completed within 10-15 minutes and monitored 

for an additional 15 minutes to ensure that the sample is able to maintain 

a constant pH of around 7.5. Pour pH adjusted sample into blender jar, 

use the remaining sterile buffered dilution water (15 mL) to rinse the 

beaker twice and pour rinse water into the blender jar. 

11.2 Inoculation

Media inoculation procedures are based on whether the original sample was a liquid or a solid. 

For some transfers, it may be convenient to use a sterile, wide-mouth pipette, capable of 

transferring particulate matter. If samples are being spiked, a maximum of 1 hour may elapse 

between initial unspiked sample homogenization and analysis of spiked samples. 

11.2.1 Liquid samples: For unspiked and spiked samples, three series of five tubes will be used for 

the analysis with 20-, 10-, and 1.0-mL of the original sample. See Figure 1 in Section 

19.0 for an overview of the inoculation scheme. 

11.2.1.1 Inoculation 

(A) Use a sterile pipette to inoculate each of the first series of five tubes 

(containing 10 mL of 3X TSB) with 20.0 mL of the original “homogenized” 

sample per tube.

(B) Use a sterile pipette to inoculate each of the second series of tubes 

(containing 5 mL of 3X TSB) with 10.0 mL of the original “homogenized” 

sample per tube.

(C) Use a sterile pipette to inoculate each of the third series of tubes (containing 

10 mL of 1X TSB) with 1.0 mL of the original “homogenized” sample per 

tube. 

11.2.1.2 Repeat Section 11.2.1.1 for the remaining liquid samples. When inoculations 

are complete, go to Section 12.0 to continue the analyses. 

18 September 2014
Method 1682

11.2.2 Solid samples: For unspiked and spiked samples, three series of five tubes will be used for 

the analysis with 2.0-, 1.0-, and 0.1-g of the original sample (20.0, 10.0, and 1.0 mL of the 

homogenized sample). The first two series of tubes must contain 3X TSB. See Figure 2 

in Section 19.0 for an overview of the inoculation scheme. 

11.2.2.1 Inoculation 

(A) Use a sterile pipette to inoculate each of the first series of tubes (containing 

10 mL of 3X TSB) with 20.0 mL of the “homogenized” sample per tube. 

This is 2.0 g of the original sample. Solids that will not separate easily 

and/or may float should be submerged into the broth with a sterile loop. 

(B) Use a sterile pipette to inoculate each of the second series of tubes 

(containing 5 mL of 3X TSB) with 10.0 mL of the “homogenized” sample 

per tube. This is 1.0 g of the original sample. Solids that will not separate 

easily and/or may float should be submerged into the broth with a sterile 

loop. 

(C) Use a sterile pipette to inoculate each of the third series of tubes (containing 

10 mL of 1X TSB) with 1.0 mL of the “homogenized” sample per tube. 

This is 0.1 g of the original sample. 

11.2.2.2 Repeat Section 11.2.2.1 for remaining solid samples. When inoculations are 

complete, go to Section 12.0 to continue the analysis. 

Note: For regulatory purposes, Salmonella monitoring is required only for 

Class A biosolids in 40 CFR Part 503. If this procedure is being used to 

enumerate Salmonella in samples other than Class A biosolid samples, 

additional dilutions may be required prior to analysis. When 

attempting to quantify samples containing higher Salmonella

concentrations than can be evaluated using the 20-10-1 scheme, it may 

be necessary to evaluate additional dilution volumes of 0.1-, 0.01-, and 

0.001-mL. The MPN table in Standard Methods would then be used, 

instead of Table 5 in Section 13.0. 

Although other dilution and inoculation schemes may be used for the analysis of 

samples with higher Salmonella concentrations, the first transfer from the 

“homogenized” sample should always be 11 mL of homogenized sample to 99 

mL dilution water or 10 mL of homogenized sample to 90 mL dilution water. 

This will ensure that a sufficient amount of the original biosolid sample is 

transferred at the beginning of the dilution scheme. 

12.0 Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium 

Procedure

12.1 In this protocol, the modified semisolid Rappaport-Vassiliadis (MSRV) medium MPN technique is 

used to determine Salmonella densities in Class A biosolid samples. Although the Part 503 

regulation does not specify the total number of samples for Class A biosolids, it suggests that a 

sampling event extend over two weeks, and that at least seven samples be tested to confirm that the 

19 September 2014
Method 1682

mean bacterial density of the samples is below 3 MPN / 4 g of total solids (dry weight basis). The 

analysis of seven samples increases the method precision by reducing the standard error caused by 

inherent variations in biosolid quality. The precision of the test increases with increasing numbers 

of replicates per sample tested. For an overview of the MPN procedure refer to Figure 3 in 

Section 19.0.

12.2 Enrichment phase

12.2.1 Prepare TSB media and dispense into tubes as directed in Section 7.6. 

Note: If media is refrigerated, remove from refrigerator 1-1.5 hours 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 1X 

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-μL drops from each TSB tube onto a corresponding MSRV plate 

that has been labeled with sample ID, date, and original inoculation volume (e.g., 20.0, 

10.0, or 1.0 mL). Space the drops evenly over the entire plate. In addition, inoculate an 

MSRV plate with positive and negative controls. Do not invert the plates. Allow the 

drops to absorb into the agar for approximately 1 hour at room temperature and incubate 

plates at 42°C ± 0.5°C for 16 to 18 hours in a humidity-controlled hot air incubator. If a 

humidity-controlled hot air incubator is not available, an open pan of water placed in the 

bottom of the incubator will suffice.

12.3.2 Examine plates for the appearance of motility surrounding the inoculations, as evidenced 

by a “whitish halo” of growth approximately 2 cm from the center of the spot.

12.3.3 Using a sterile inoculating loop, stab into a halo from the outer edge of a target colony on 

the MSRV plate and streak onto an XLD plate. Since Salmonella are predominately 

located within the MSRV media, the loop should penetrate the MSRV at least half-way. 

20 September 2014
Method 1682

Repeat this step using another target colony from the MSRV plate. In addition, inoculate 

XLD positive and negative controls. (See Photo 1) 

Photo 1. Salmonella spp. produce halos indicating motility on MSRV plates.

12.3.4 Incubate XLD plates for 18 to 24 hours at 36°C ± 1.5°C. After incubation, refrigerate one 

of the XLD plates at 1°C to 5°C and submit the other plate to biochemical confirmation. 

(If issues arise in subsequent steps of the method, the laboratory may wish to return to the 

refrigerated XLD plate.) 

Note: XLD plates may be refrigerated over the weekend prior to submitting to 

biochemical confirmation.

12.3.5 Black and pink to red colonies with black centers are considered Salmonella. (See Photo 2) 

Photo 2. Salmonella spp. produce pink to red colonies with black centers on XLD plates. 

12.4 Biochemical confirmation phase

12.4.1 Label all tubes with inoculation date, sample identification, and original inoculation 

volume (e.g., 20.0, 10.0, or 1.0 mL). Pick isolated colonies exhibiting Salmonella

morphology (pink to red colonies with black centers) and inoculate triple sugar iron agar 

(TSI) slants, lysine iron agar (LIA) slants, and urea broth. Inoculate slants by stabbing the 

butt and streaking the slant. Use the same XLD colony to inoculate all three media. This 

will require going back to the same XLD colony multiple times to ensure sufficient 

21 September 2014
Method 1682

inoculum for each medium. In addition, inoculate each medium with the appropriate 

positive and negative controls. Incubate for 24 ± 2 hours at 36°C ± 1.5°C. If only 

atypical colony morphology is observed on XLD plates, pick from an atypical colony and 

inoculate TSI slants, LIA slants, and urea broth and incubate as described above.

12.4.1.1 TSI: A positive TSI reaction is an acid butt (yellow in color) and an alkaline slant 

(red in color) with or without H2S gas production. When H2S gas production is 

present, the butts of both the LIA and TSI may be black, which would be 

considered a positive reaction for Salmonella. H2S is more likely but acid butt 

is also possible (but rare).

12.4.1.2 LIA: A positive LIA reaction is an alkaline butt (purple in color) and an alkaline 

slant (purple in color) with or without H2S gas production. When H2S gas 

production is present, the butts of both the LIA and TSI may be black, which 

would be considered a positive reaction for Salmonella. 

12.4.1.3 Urea broth: Urea is an orange medium and will change to pink or deep 

purplish-red if positive. A negative urease test is one that exhibits no color 

change after inoculation. Salmonella are negative for urease.

Note: H2S negative Salmonella will be missed if translucent pink to red 

colonies are not submitted to biochemical and serological confirmation.

12.4.2 To confirm cultures via polyvalent O antiserum: 

Emulsify growth on the slant portion of TSI (regardless of whether TSI is positive or 

negative) using sterile physiological saline (Section 7.5). Place two discrete drops of 

emulsified growth onto slide. To the first drop of emulsified growth, add one drop of 

polyvalent O antiserum. To the second drop of emulsified growth, add one drop of sterile 

saline (as a visual comparison). Observe under magnification for an agglutination 

reaction which indicates a positive result. Appropriate positive and negative controls 

from TSI must be analyzed for each batch of samples.

12.4.3 In order for the original TSB tube to be considered positive for Salmonella, the associated 

inoculations must be MSRV positive, XLD positive, either TSI or LIA positive, urease 

negative, and polyvalent-O positive (Table 4). Correlate all positive plates and tubes to 

original TSB tube and record results. Determine the MPN from this information (see 

Section 13.0). Record all data clearly into a laboratory notebook. 

12.5 Total solids determination

12.5.1 Determination of percent dry weight - When sample results are to be calculated on a dry 

weight basis, a second portion of sample should be weighed at the same time as the portion 

used for analytical determination. 

WARNING: The drying oven should be contained in a hood or be vented. Significant 

laboratory contamination may result from drying a heavily contaminated 

sample.

12.5.2 Immediately after weighing the sample for microbiological examination, weigh 10-30 g of 

sample into a tarred crucible or aluminum evaporating dish. Dry this aliquot overnight at 

22 September 2014
Method 1682

103°C to 105°C Allow to cool in a desiccator before weighing. Calculate the % dry 

weight as follows: 

% dry weight = 

g dry sample 

g sample x 100 

13.0 Data Analysis and Calculations

The estimated density of Salmonella bacteria is calculated as the most probable number (MPN). Salmonella

results from biosolid samples are reported as MPN / 4 g total solids (dry weight basis), which is calculated 

according to the following steps:

1. Selection of MPN / mL (wet weight)

2. Conversion to MPN / g (dry weight) and calculation of MPN / 4 g total solids (dry weight) 

13.1 Step 1: Obtain MPN / mL (wet weight)

Obtain the MPN index value from Table 5 using the number of positive tubes in the three 

significant dilutions series. Since Table 5 assumes that 20.0, 10.0, and 1.0 mL of homogenized 

sample were inoculated into TSB and because liquid samples are not diluted in the homogenization 

step (Section 11.0), the MPN index = MPN / mL for liquid samples. 

Since solid samples were diluted in the homogenization step (Section 11.1), the dilution must be 

taken into account when calculating MPN / mL (wet weight). As a result the MPN index value 

from Table 5 is divided by 0.1 to account for diluting the sample during the homogenization step. 

Table 5. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five 

Tubes are used per 20.0, 10.0, and 1.0 mL Homogenized Sample Inoculation Volumes a

Combination of 

Positives MPN Index

95% Confidence Limits Combination of 

Positives MPN Index

95% Confidence Limits

Lower Upper Lower Upper

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

0-0-1 0.0065 0.0012 0.0223 1-3-1 0.0393 0.0092 0.0821

0-0-2 0.0130 0.0012 0.0352 1-3-2 0.0475 0.0132 0.0967

0-0-3 0.0195 0.0012 0.0472 1-3-3 0.0559 0.0173 0.1119

0-0-4 0.0262 0.0033 0.0589 1-3-4 0.0644 0.0216 0.1277

0-0-5 0.0328 0.0062 0.0706 1-3-5 0.0730 0.0260 0.1444

0-1-0 0.0067 0.0012 0.0228 1-4-0 0.0409 0.0099 0.0849

0-1-1 0.0134 0.0012 0.0360 1-4-1 0.0495 0.0141 0.1002

0-1-2 0.0202 0.0012 0.0483 1-4-2 0.0583 0.0185 0.1163

0-1-3 0.0270 0.0037 0.0604 1-4-3 0.0672 0.0231 0.1331

0-1-4 0.0339 0.0067 0.0725 1-4-4 0.0763 0.0277 0.1509

0-1-5 0.0408 0.0099 0.0847 1-4-5 0.0855 0.0324 0.1700

0-2-0 0.0138 0.0012 0.0367 1-5-0 0.0517 0.0152 0.1042

0-2-1 0.0208 0.0012 0.0495 1-5-1 0.0609 0.0199 0.1212

0-2-2 0.0279 0.0040 0.0619 1-5-2 0.0703 0.0247 0.1391

0-2-3 0.0350 0.0072 0.0745 1-5-3 0.0799 0.0296 0.1583

0-2-4 0.0422 0.0106 0.0871 1-5-4 0.0897 0.0346 0.1790

0-2-5 0.0494 0.0141 0.1001 1-5-5 0.0998 0.0397 0.2015

0-3-0 0.0215 0.0012 0.0507 2-0-0 0.0155 0.0012 0.0404

0-3-1 0.0288 0.0044 0.0636 2-0-1 0.0226 0.0018 0.0526

0-3-2 0.0362 0.0077 0.0766 2-0-2 0.0303 0.0051 0.0662

0-3-3 0.0437 0.0113 0.0898 2-0-3 0.0382 0.0087 0.0801

0-3-4 0.0512 0.0051 0.1243 2-0-4 0.0462 0.0125 0.0943

0-3-5 0.0588 0.0095 0.1428 2-0-5 0.0543 0.0165 0.1090

23 September 2014
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 MPN Index

95% Confidence Limits Combination of 

Positives MPN Index

95% Confidence Limits

Lower Upper Lower Upper

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

0-4-1 0.0375 0.0084 0.0789 2-1-1 0.0315 0.0056 0.0683

0-4-2 0.0453 0.0121 0.0927 2-1-2 0.0397 0.0094 0.0827

0-4-3 0.0531 0.0160 0.1069 2-1-3 0.0480 0.0134 0.0976

0-4-4 0.0611 0.0200 0.1216 2-1-4 0.0565 0.0177 0.1131

0-4-5 0.0691 0.0241 0.1369 2-1-5 0.0652 0.0221 0.1293

0-5-0 0.0390 0.0090 0.0814 2-2-0 0.0327 0.0062 0.0705

0-5-1 0.0470 0.0129 0.0958 2-2-1 0.0413 0.0101 0.0856

0-5-2 0.0553 0.0170 0.1107 2-2-2 0.0501 0.0144 0.1013

0-5-3 0.0636 0.0212 0.1262 2-2-3 0.0590 0.0189 0.1176

0-5-4 0.0720 0.0255 0.1425 2-2-4 0.0681 0.0236 0.1349

0-5-5 0.0806 0.0299 0.1596 2-2-5 0.0774 0.0283 0.1533

1-0-0 0.0072 0.0012 0.0241 2-3-0 0.0431 0.0110 0.0887

1-0-1 0.0139 0.0012 0.0369 2-3-1 0.0523 0.0155 0.1053

1-0-2 0.0209 0.0012 0.0497 2-3-2 0.0617 0.0203 0.1227

1-0-3 0.0281 0.0041 0.0623 2-3-3 0.0714 0.0252 0.1412

1-0-4 0.0353 0.0073 0.0749 2-3-4 0.0813 0.0303 0.1611

1-0-5 0.0425 0.0107 0.0878 2-3-5 0.0914 0.0354 0.1826

1-1-0 0.0144 0.0012 0.0377 2-4-0 0.0547 0.0168 0.1098

1-1-1 0.0217 0.0013 0.0509 2-4-1 0.0647 0.0218 0.1284

1-1-2 0.0290 0.0045 0.0640 2-4-2 0.0750 0.0271 0.1484

1-1-3 0.0365 0.0079 0.0771 2-4-3 0.0855 0.0325 0.1700

1-1-4 0.0441 0.0115 0.0905 2-4-4 0.0964 0.0380 0.1937

1-1-5 0.0517 0.0153 0.1043 2-4-5 0.1076 0.0436 0.2201

1-2-0 0.0224 0.0017 0.0523 2-5-0 0.0681 0.0235 0.1349

1-2-1 0.0301 0.0050 0.0658 2-5-1 0.0791 0.0292 0.1566

1-2-2 0.0379 0.0085 0.0795 2-5-2 0.0904 0.0349 0.1805

1-2-3 0.0457 0.0123 0.0935 2-5-3 0.1021 0.0409 0.2070

1-2-4 0.0537 0.0162 0.1079 2-5-4 0.1143 0.0469 0.2372

1-2-5 0.0618 0.0203 0.1229 2-5-5 0.1268 0.0531 0.2725

3-0-0 0.0255 0.0028 0.0585 4-3-0 0.0797 0.0295 0.1579

3-0-1 0.0330 0.0063 0.0710 4-3-1 0.0937 0.0366 0.1877

3-0-2 0.0417 0.0103 0.0863 4-3-2 0.1086 0.0441 0.2228

3-0-3 0.0506 0.0147 0.1023 4-3-3 0.1245 0.0520 0.2656

3-0-4 0.0598 0.0193 0.1191 4-3-4 0.1414 0.0602 0.3218

3-0-5 0.0691 0.0241 0.1368 4-3-5 0.1595 0.0686 0.4067

3-1-0 0.0344 0.0069 0.0734 4-4-0 0.1012 0.0404 0.2049

3-1-1 0.0435 0.0112 0.0896 4-4-1 0.1181 0.0489 0.2476

3-1-2 0.0529 0.0159 0.1065 4-4-2 0.1364 0.0578 0.3038

3-1-3 0.0626 0.0207 0.1244 4-4-3 0.1563 0.0672 0.3890

3-1-4 0.0725 0.0258 0.1434 4-4-4 0.1780 0.0770 0.5273

3-1-5 0.0827 0.0310 0.1640 4-4-5 0.2015 0.0873 0.6411

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

3-2-1 0.0555 0.0171 0.1112 4-5-1 0.1524 0.0653 0.3687

3-2-2 0.0657 0.0223 0.1303 4-5-2 0.1769 0.0766 0.5210

3-2-3 0.0763 0.0277 0.1510 4-5-3 0.2046 0.0886 0.6528

3-2-4 0.0872 0.0333 0.1735 4-5-4 0.2357 0.1015 0.7516

3-2-5 0.0984 0.0390 0.1984 4-5-5 0.2708 0.1150 0.8426

3-3-0 0.0583 0.0186 0.1164 5-0-0 0.0549 0.0162 0.1116

3-3-1 0.0693 0.0241 0.1371 5-0-1 0.0637 0.0213 0.1265

3-3-2 0.0806 0.0299 0.1597 5-0-2 0.0763 0.0277 0.1510

3-3-3 0.0924 0.0359 0.1847 5-0-3 0.0896 0.0345 0.1787

3-3-4 0.1046 0.0421 0.2128 5-0-4 0.1037 0.0417 0.2107

3-3-5 0.1173 0.0484 0.2452 5-0-5 0.0953 0.0165 0.2234

3-4-0 0.0733 0.0262 0.1450 5-1-0 0.0678 0.0234 0.1344

3-4-1 0.0856 0.0325 0.1700 5-1-1 0.0816 0.0304 0.1618

3-4-2 0.0984 0.0390 0.1982 5-1-2 0.0963 0.0379 0.1936

3-4-3 0.1118 0.0457 0.2307 5-1-3 0.1121 0.0459 0.2316

3-4-4 0.1258 0.0526 0.2695 5-1-4 0.1291 0.0542 0.2796

3-4-5 0.1405 0.0597 0.3184 5-1-5 0.1293 0.0304 0.3090

24 September 2014
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 MPN Index

95% Confidence Limits Combination of 

Positives MPN Index

95% Confidence Limits

Lower Upper Lower Upper

3-5-0 0.0913 0.0354 0.1825 5-2-0 0.0879 0.0337 0.1751

3-5-1 0.1055 0.0426 0.2150 5-2-1 0.1046 0.0421 0.2128

3-5-2 0.1204 0.0500 0.2538 5-2-2 0.1227 0.0511 0.2605

3-5-3 0.1362 0.0577 0.3029 5-2-3 0.1427 0.0608 0.3267

3-5-4 0.1529 0.0656 0.3715 5-2-4 0.1646 0.0710 0.4385

3-5-5 0.1707 0.0738 0.4795 5-2-5 0.1767 0.0503 0.5230

4-0-0 0.0381 0.0082 0.0809 5-3-0 0.1151 0.0474 0.2394

4-0-1 0.0461 0.0125 0.0942 5-3-1 0.1368 0.0580 0.3050

4-0-2 0.0563 0.0175 0.1126 5-3-2 0.1614 0.0695 0.4183

4-0-3 0.0668 0.0229 0.1323 5-3-3 0.1895 0.0821 0.5899

4-0-4 0.0777 0.0284 0.1537 5-3-4 0.2216 0.0957 0.7101

4-0-5 0.0890 0.0342 0.1773 5-3-5 0.2527 0.0814 0.7971

4-1-0 0.0484 0.0136 0.0983 5-4-0 0.1571 0.0676 0.3935

4-1-1 0.0592 0.0190 0.1181 5-4-1 0.1907 0.0826 0.5954

4-1-2 0.0705 0.0248 0.1395 5-4-2 0.2319 0.0999 0.7409

4-1-3 0.0822 0.0308 0.1631 5-4-3 0.2834 0.1196 0.8726

4-1-4 0.0945 0.0370 0.1894 5-4-4 0.3475 0.1417 1.0160

4-1-5 0.1072 0.0434 0.2193 5-4-5 0.4256 0.1437 1.1800

4-2-0 0.0626 0.0207 0.1244 5-5-0 0.2398 0.0762 0.7629

4-2-1 0.0748 0.0269 0.1479 5-5-1 0.3477 0.1172 1.0160

4-2-2 0.0875 0.0335 0.1742 5-5-2 0.5422 0.1791 1.4190

4-2-3 0.1009 0.0403 0.2041 5-5-3 0.9178 0.2672 2.2010

4-2-4 0.1150 0.0473 0.2392 5-5-4 1.6090 0.3837 4.1030

4-2-5 0.1299 0.0546 0.2820 5-5-5 >1.609000 0.3837 ---- a Table was developed using the MPN calculator developed by Albert Klee (Reference 18.5)

13.2 Step 2: Convert to MPN / g (dry weight) and calculate MPN / 4 g total solids (dry weight): 

For analysis and calculation of percent total solids, refer to Section 12.5. 

For the conversion to MPN / g total solids (dry weight), we assume that,

MPN / mL wet weight = MPN / g wet weight.

Therefore, we may calculate MPN / 4 g total solids (dry weight) for liquid samples using the 

following equation: 

MPN / 4 g (dry 

weight) = [MPN / mL wet weight from Step 1] × 4

Percent total solids expressed as a decimal

Example calculations are provided in Table 6, below. 

25 September 2014
Method 1682

Table 6. Example Calculation of Salmonella Density

Example

Volume of homogenized 

sample used to inoculate TSB Step 1:

MPN / mL 

(wet weight)

Percent total 

solids

(dry weight)

Step 2:

MPN / 4 g

(dry weight) 20.0 mL 10.0 mL 1.0 mL

A (liquid) 0/5 1/5 0/5 0.0067 1% (0.0067) (4) / (0.01) = 

2.68 MPN / 4 g

B (liquid) 3/5 1/5 1/5 0.0435 2% (0.0435) (4) / (0.02) = 

8.7 MPN / 4 g

C (liquid) 5/5 5/5 2/5 0.5422 3% (0.5422) (4) / (0.03) = 72 

MPN / 4 g

D (solid) 0/5 1/5 0/5 0.0067 / 0.11 = .067 96% (0.067) (4) / (0.96) = 

0.28 MPN / 4 g

E (solid) 4/5 4/5 4/5 0.1181 / 0.11 = 1.181 18% (1.181) (4) / (0.18) = 26 

MPN / 4 g

F (solid) 5/5 5/5 2/5 0.5422 / 0.11 = 5.422 43% (5.422) (4) / (0.43) = 50 

MPN / 4 g

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.

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 108 Salmonella typhimurium colony forming 

units (CFU) per mL. This is referred to as the “undiluted spiking suspension”.

14.3 Laboratory-Prepared Sample Spiking (Class A Biosolids) 

Since the objective of spiking the biosolid sample is to establish percent recovery, it is necessary to 

determine the number of Salmonella typhimurium in the undiluted spiking suspension. 

26 September 2014
Method 1682

14.3.1 Sample spiking

14.3.1.1 Dilute spiking suspension

Mix the undiluted spiking suspension by vigorously shaking the bottle a 

minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the undiluted 

spiking suspension to 99 mL of sterile phosphate buffered dilution water 

(Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 

times. This is spiking suspension dilution “A”. A 1.0-mL volume of dilution 

“A” is 10-2 mL of the original undiluted spiking suspension. 

14.3.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspension dilution “A” to 99 

mL of sterile phosphate buffered dilution water, cap, and mix by vigorously 

shaking the bottle a minimum of 25 times. This is spiking suspension dilution 

“B”. A 1.0-mL volume of dilution “B” is 10-4 mL of the original undiluted 

spiking suspension. 

14.3.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “B” to 99 

mL of sterile phosphate buffered dilution water, cap, and mix by vigorously 

shaking the bottle a minimum of 25 times. This is spiking suspension dilution 

“C”. A 1.0-mL volume of dilution “C” is 10-5 mL of the original undiluted 

spiking suspension. 

14.3.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “C” to 99 

mL of sterile phosphate buffered dilution water, cap, and mix by vigorously 

shaking the bottle a minimum of 25 times. This is spiking suspension dilution 

“D”. A 1.0-mL volume of dilution “D” is 10-6 mL of the original undiluted 

spiking suspension. 

14.3.2 Spike sample(s)

Since sample homogenization procedures in Method 1682 are specific to either liquid or 

solid samples, this spiking procedure is also liquid/solid-specific. 

14.3.2.1 Liquid Samples: Homogenize an unspiked Class A biosolid sample (Section 

11.1). To spike a liquid sample, add 0.5 mL of spiking suspension dilution “D” 

to 300 mL of pH adjusted unspiked homogenized sample, cover, and blend on 

high speed for 1 - 2 minutes. This is the “spiked” sample. The volume (mL) of 

undiluted spiking suspension added to each mL of the homogenized biosolid 

sample is 1.67 x 10-9 mL per mL [(0.5 mL x 10-6 mL) / 300 mL of biosolid], 

which is referred to as Vspiked per unit biosolids. 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 × 10-8 mL per g [(0.5 mL × 10-6 mL) / 30 g of biosolid], which is 

referred to as Vspiked per unit biosolids. Proceed to Section 11.2 (inoculation). 

27 September 2014
Method 1682

14.4 Enumeration of undiluted spiking suspension

14.4.1 Prepare heart infusion agar (HIA) according to Section 7.12, add 10 - 15 mL of HIA per 

100 × 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. 

Note: To ensure that the agar surface is dry prior to use, plates should be made several 

days in advance and stored inverted at room temperature or dried using a 

laminar-flow hood. 

14.4.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine 

spread plates:

• Pipet 0.1 mL of dilution “B” onto surface of pre-dried HIA plate [10-5 mL (0.00001 

mL) of the original spiking suspension].

• Pipet 0.1 mL of dilution “C” onto surface of pre-dried HIA plate [10-6 mL (0.000001 

mL) of the original spiking suspension].

• Pipet 0.1 mL of dilution “D” onto surface of pre-dried HIA plate [10-7 mL (0.0000001 

mL) of the original spiking suspension].

14.4.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum over 

surface of medium by rotating the dish by hand or on a turntable. 

14.4.4 Allow inoculum to absorb into the medium completely. 

14.4.5 Invert plates and incubate at 36°C ± 1.5°C for 24 ± 4 hours. 

14.4.6 Count and record number of colonies per plate. 

14.5 Calculation of Laboratory-Prepared Spike Percent Recovery

Spiked Salmonella typhimurium percent recovery will be calculated in four steps as indicated 

below. 

Note: The example calculated numbers provided in the tables below have been rounded at the 

end of each step. If your laboratory recalculates the examples using a spreadsheet and 

rounds only after the final calculation (Step 4), the percent recoveries may be slightly 

different.

14.5.1 Step 1: Calculate Concentration of Salmonella typhimurium (CFU / mL) in Undiluted 

Spiking Suspension

14.5.1.1 The number of Salmonella typhimurium CFU / mL in the undiluted spiking 

suspension will be calculated using all HIA plates yielding counts within the 

ideal range of 30 to 300 CFU per plate. 

14.5.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if the colonies 

are not discrete, results should be recorded as “too numerous to count” (TNTC).

28 September 2014
Method 1682

14.5.1.3 Calculate the concentration of Salmonella typhimurium (CFU / mL) in the 

undiluted spiking suspension according to the following equation. (Example 

calculations are provided in Table 7, below.)

 Salmonella undiluted spike = (CFU1 + CFU2 + ... + CFUn) / (V1 + V2 + ... + Vn) 

Where,

Salmonella undiluted spike = Salmonella typhimurium CFU / mL in undiluted 

spiking suspension 

CFU = Number of colony forming units from HIA plates 

yielding counts within the ideal range of 30 to 300 

CFU per plate

V = Volume of undiluted sample on each HIA plate 

yielding counts within the ideal range of 30 to 300 

CFU per plate

n = Number of plates with counts within the ideal range

Table 7. Example Calculations of Salmonella typhimurium Spiking Suspension Concentration

Examples

CFU / plate (triplicate analyses) from HIA plates Salmonella CFU / mL in undiluted spiking 

suspension

(Salmonella undiluted spike) a 10-5 mL plates 10-6 mL plates 10-7 mL plates 

Example 1 275, 250, 301 30, 10, 5 0, 0, 0

(275+250+30) / (10-5+ 10-5+10-6) =

555 / (2.1 x 10-5) = 26,428,571 =

2.6 x 107 CFU / mL

Example 2 TNTC, TNTC, 

TNTC

TNTC, 299, 

TNTC 12, 109, 32

(299+109+32) / (10-6+ 10-7+10-7) =

440 / (1.2 x 10-6) =366,666,667 =

3.7 x 108 CFU / mL

a Salmonella undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per 

plate

14.5.2 Step 2: Calculate the Concentration of Spiked Salmonella CFU / mL or CFU / g (wet 

weight)

14.5.2.1 The volume of undiluted spiking suspension per unit (mL or g) of spiked 

biosolid samples (Vspiked per unit biosolids) is provided in Table 8, below. 

Table 8. Volume of Undiluted Spiking Suspension per Unit (mL or g) of Spiked Biosolid Samples 

(V spiked per unit biosolids)

Description of spiked sample V spiked per unit biosolids

Class A liquid 1.67 x 10-9 mL per mL of biosolids

Class A solid 1.67 x 10-8 mL per g of biosolids (wet weight)

14.5.2.2 Calculate concentration of Spiked Salmonella wet weight (CFU / mL or CFU / 

g) according to the following equation. Example calculations are provided in 

Table 9, below. 

29 September 2014
Method 1682

Spiked Salmonella wet weight = (Salmonella undiluted spike) x (Vspiked per unit biosolids) 

Where,

Spiked Salmonella wet weight = Number of spiked Salmonella CFU per mL or g 

of biosolid (wet weight) 

Salmonella undiluted spike = Salmonella CFU / mL in undiluted spiking 

suspension

Vspiked per unit biosolids = mL of undiluted spiking suspension per mL or 

g of spiked biosolid 

Table 9. Example Calculations of Spiked Salmonella wet weight

Salmonella undiluted spike

(Table 7 above) V spiked (Table 8 above) Spiked Salmonella wet weight 

[CFU / mL or CFU / g (wet weight)]

Example 1:

2.6 x 107 CFU / mL

Liquid: 1.67 x 10-9 mL per mL of 

biosolids

(2.6 x 107 CFU / mL) x (1.67 x 10-9 mL / mL)

= 0.043 CFU / mL

Solid: 1.67 x 10-8 mL per g of biosolids 

(wet weight)

(2.6 x 107 CFU / mL) x (1.67 x 10-8 mL / g)

= 0.43 CFU / g (wet weight)

Example 2:

3.7 x 108 CFU / mL

Liquid: 1.67 x 10-9 mL per mL of 

biosolids

(3.7 x 108 CFU / mL) x (1.67 x 10-9 mL / mL)

= 0.62 CFU / mL

Solid: 1.67 x 10-8 mL per g of biosolids 

(wet weight)

(3.7 x 108 CFU / mL) x (1.67 x 10-8 mL / g)

= 6.2 CFU / 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)

[(Spiked Salmonella wet weight from Table 9 above) / percent total solids] × 4

= True spiked Salmonella CFU / 4 g dry weight

Example 1 Class A liquid: 5% (0.043 / 0.05) × 4 = 3.5 CFU / 4 g dry weight

Class A solid: 82% (0.43 / 0.82) × 4 = 2.1 CFU / 4 g dry weight

Example 2

Class A liquid: 7% (0.62 / 0.07) × 4 = 35 CFU / 4 g dry weight

Class A solid: 88% (6.2 / 0.88 ) × 4 = 28 CFU / 4 g dry weight

14.5.4 Step 4: Calculate Percent Recovery

14.5.4.1 Calculate percent recovery (R) using the following equation. 

R = 100 x (Ns - Nu) 

T 

Where, 

R = Percent recovery

Ns = Salmonella MPN / 4 g (dry weight) in the spiked sample 

Nu = Salmonella MPN / 4 g (dry weight) in the unspiked sample 

T = True spiked Salmonella CFU / 4 g (dry weight) in spiked sample 

30 September 2014
Method 1682

14.5.4.2 Example percent recovery calculations are provided in Table 11. 

Table 11. Example Percent Recovery Calculations

Ns Nu T Percent recovery (R)

2.6

0.268

Example 1: 3.5 100 x (2.6 - 0.268) / 3.5 = 67%

2.4 Example 1: 2.1 100 x (2.4 - 0.268) / 2.1 = 101%

46 Example 2: 35 100 x (46 - 0.268) / 35 = 130%

16 Example 2: 28 100 x (16 - 0.268) / 28 = 56%

14.6 BioBallTM 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 

BioBallTM vial by removing the crimp and cap. To spike a liquid sample, 

aseptically add 1 BioBallTM 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 

BioBallTM vial by removing the crimp and cap. To spike a solid sample, 

aseptically add 1 BioBallTM to 300 mL of pH adjusted unspiked homogenized 

sample (30 g of sample + 270 mL of sterile phosphate buffered dilution water), 

cover, and blend on high speed for 1 - 2 minutes. This is the “spiked” sample. 

Proceed to Section 11.2 (inoculation). 

14.7 Enumeration of BioBallTM 

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 BioBallTM vial by removing the crimp and cap. Aseptically place one 

BioBallTM onto the center of each HIA plate by tipping the vial over the medium. 

• Immediately pipette 200 μL of sterile physiological saline solution (0.85%) directly 

onto the BioBallTM.

• Allow the BioBallTM to dissolve. 

31 September 2014
Method 1682

14.7.3 For each spread plate, using a sterile bent glass rod or spreader, distribute the BioBallTM 

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 BioBallTM Spike Percent Recovery

Spiked BioBallTM 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 BioBallTM 

14.8.1.1 The number of Salmonella typhimurium (CFU) in the BioBalls will be 

calculated using all HIA plates. Count the number of CFUs on all three plates 

and calculate the mean. (An example is provided in Table 12.)

Table 12 Example calculation for mean Salmonella CFU per BioBallTM

CFU / plate (triplicate analyses) from HIA plates

HIA plate count #1 HIA plate count #2 HIA plate count #3 Mean Salmonella CFU per BioBallTM

40 38 48 (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

Vhomogenized sample

Where,

Spiked Salmonella wet weight = Number of spiked Salmonella CFU per mL or g 

of biosolid (wet weight)

Salmonella mean CFU = Mean CFU of Salmonella in BioBalls

Vhomogenized sample = mL or g of spiked homogenized biosolid 

sample

32 September 2014
Method 1682

Table 13. Example Calculation of Spiked Salmonella in the Homogenized Sample 

(Salmonella wet weight)

Salmonella mean CFU

(Table 12 above) V homogenized sample

Spiked Salmonella wet weight 

[CFU / mL or CFU / g (wet weight)]

42 CFU Liquid: 300 mL 42 CFU / 300 mL = 0.14 CFU / mL (wet weight)

Solid: 30 g 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. 

Table 14. Examples of Conversion to “True” Spiked Salmonella CFU / 4 g Total Solids (Dry Weight)

Example Total Solids [(Spiked Salmonella wet weight from Table 13 above) / percent total solids] × 4

= True spiked Salmonella CFU / 4 g (dry weight)

Class A liquid: 5% (0.14 / 0.05) × 4 = 11.2 CFU / 4 g dry weight

Class A solid: 82% (1.4 / 0.82) × 4 = 6.8 CFU / 4 g dry weight

14.8.4 Step 4: Calculate Percent Recovery

14.8.4.1 Calculate percent recovery (R) using the following equation. 

R = 100 x 

(Ns - Nu) 

T 

Where,

R = Percent recovery

Ns = Salmonella MPN / 4 g (dry weight) in the spiked sample 

Nu = Salmonella MPN / 4 g (dry weight) in the unspiked sample 

T = True spiked Salmonella CFU / 4 g (dry weight) in spiked sample 

14.8.4.2 Example percent recovery calculations are provided in Table 15. 

Table 15. Example Percent Recovery Calculations

Ns Nu T Percent recovery (R)

12 0.268 11.2 100 x (12 - 0.268) / 11.2 = 105%

4.8 6.8 100 x (4.8 - 0.268) / 6.8 = 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 

33 September 2014
Method 1682

control (QC) acceptance criteria. A detailed description of the study and results are 

provided in the validation study report (Reference 18.2). Results submitted by 

laboratories were validated using a standardized data review process to confirm that results 

were generated in accordance with study-specific instructions and the March 2003 draft 

version of EPA Method 1682.

15.1.2 Recovery - Method 1682 mean recoveries of Salmonella from compost, thermophilically 

digested liquid, thermophilically digested solid, and alkaline-stabilized biosolids, spiked 

with BioBallTM 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.

34 September 2014
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 in 

Biosolids. EPA-821-B-04-008. September 2004.

18.3 Bordner, R., J.A. Winter and P.V. Scarpino (eds.), Microbiological Methods for Monitoring the 

Environment, Water and Wastes, EPA-600/8-78-017. Office of Research and Development, 

USEPA

18.4 American Chemical Society (ACS). 2000. Reagent Chemicals, American Chemical Society 

Specifications. American Chemical Society, New York. For suggestions of the testing of reagents 

not listed by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals, 

BDH, Poole, Dorset, UK and the United States Pharmacopeia.

18.5 Klee, A. J. 1993. A computer program for the determination of the most probable number and its 

confidence limits. Journal of Microbiological Methods. 18:91-98.

35 September 2014
Method 1682

19.0 Figures

Figures 1 and 2. Liquid and Solid Samples

 Solid sample (30 g)

(unhomogenized)

Rinse sample into blender with 270 mL of 

sterile buffered dilution water

(homogenized sample from section 11.1.2)

10 mL per ut be

20 mL per tube

1 mL per tube

2.0 g of original sample

in 10 mL of 3X TSB

1.0 g of original sample

in 5 mL of 3X TSB

0.1 g of original sample

in 10 mL of 1X TSB

10 mL per tube

20 mL per tube

1 mL per tube

20 mL of original sample

in 10 mL of 3X TSB

10 mL of original sample

in 5 mL of 3X TSB

1.0 mL of original sample

in 10 mL of 1X TSB

Liquid sample (300 mL)

(homogenized sample from section 11.1.1)

Figure 1. Liquid Sample (Sections 11.1.1 and 11.2.1)

Figure 2. Solid Sample (Sections 11.1.2 and 11.2.2)

Method 1682 Class A Sample Homogenization and Inoculation

36 September 2014
Method 1682

37 September 2014
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

μL 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.

38 September 2014
Method 1682

Most probable number method (MPN)—A statistical determination of the number of bacteria per 

weight or volume of sample. It is based on the fact that the greater the number of bacteria in a 

sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to 

grow in a dilution series.

Must—This action, activity, or procedural step is required.

Negative control—A control culture that, when analyzed exactly like a field sample, will produce a 

known negative result for a given type of media.

Positive control—A control culture that, when analyzed exactly like a field sample, will produce a 

known positive result for a given type of media.

Preparation blank—See Method blank.

Selective media—A culture media designed to suppress the growth of unwanted microorganisms 

and encourage the growth of desired ones.

Should—This action, activity, or procedural step is suggested but not required.

Solid samples—Generally, samples containing >7 % total solids (dry weight).

39 September 2014