United States
Environmental Protection
Agency
Office of Water
Washington, DC 20460
EPA-821-R-98-004
August 1998
DRAFT
Method 1682: Salmonella spp. in Biosolids by
Enrichment, Selection and Biochemical
Characterization
August 1998
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Acknowledgments
Dr. Richard Danielson, BioVir Laboratories, 685 Stone Rd., Unit G, Benicia, CA 94510, USA
Dr. Robert Cooper, BioVir Laboratories, 685 Stone Rd., Unit G, Benicia, CA 94510, USA
William Yanko, County Sanitation Districts of Los Angeles County, San Jose Water Quality Laboratory,
1965 Workman Mill Road, Whittier, CA 90601, USA
Dr. Mark Meckes, USEPA, Cincinnati, OH 45268
Disclaimer
This method is in draft form. It has not been approved by the U.S. Environmental Protection Agency and
should not be construed as an Agency-endorsed method. It is being circulated for comments on its technical
merit. Mention of trade names or commercial products does not constitute endorsement or recommendation
for use.
Caution: This method is a preliminary draft written from three procedures that have not been
rigorously tested. Because the method has not been validated, problems may occur if
it is applied as written. EPA encourages constructive suggestions for improvement of
the method but cautions reviewers that the method should not be reviewed critically
until the method is validated and revised based on that validation.
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Introduction
Application of treated biosolids to land is helpful as a crop nutrient and soil conditioner but may pose the
risk of release of disease-causing microorganisms into the environment if proper disinfection and use
criteria are not met. Among these organisms are Salmonella spp., pathogenic members of enteric bacteria
that can cause salmonellosis in animals and humans if concentrations able to give rise to infections are
present. Density of Salmonella spp. 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 spp. in biosolids. Method 1682
requires calculation of the MPN via enrichment, with selection and biochemical confirmation for
determination of Salmonella. All calculations are done on a dry weight basis.
Questions concerning this method or its application should be addressed to:
William A. Telliard
Engineering and Analysis Division (4303)
U.S. Environmental Protection Agency
401 M Street, SW
Washington, D.C. 20460
(202)260-7120
Requests for additional copies of this publication should be directed to:
Water Resource Center
Mail Code RC-4100
401 M Street, SW
Washington, D.C. 20460
(202) 260-7786 or (202) 260-2814
Note: This method is performance-based. The laboratory is permitted to modify or omit any step or
procedure, provided that all performance requirements set forth in this method are met. The laboratory
may not omit any quality control analyses. The terms "shall" "must," and "may not" indicate steps and
procedures required for producing reliable results. The terms "should" and "may" indicate optional
steps that may be modified or omitted if the laboratory can demonstrate that the modified method
produces results equivalent or superior to results produced by this method.
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 2
4.0 Interferences 2
5.0 Safety 2
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 4
9.0 Quality Control 8
10.0 Calibration and Standardization 9
11.0 Procedure 10
12.0 Data Analysis and Calculations 15
13.0 Method Performance 18
14.0 Pollution Prevention 18
15.0 Waste Management 18
16.0 References 18
17.0 Tables, Diagrams, Flowcharts, and Validation Data 19
18.0 Glossary of Definitions and Purposes 19
Appendix A: Total Solids in Solid and Semisolid Matrices A-l
IV
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Method 1682: Salmonella spp. in Biosolids by Enrichment,
Selection, and Biochemical Characterization
August 1998 Draft
1.0 Scope and Application
1.1 This method is for the detection and enumeration of Salmonella spp. (CAS registry number 68583-
35-7) in treated sewage sludge (biosolids) by enrichment, selection, and characterization. It is
intended to enumerate Salmonella spp. 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 in using separate media for enrichment, selection,
and confirmation of the organism, it is intended to be more specific with greater recovery. This
method is for use in the Environmental Protection Agency's (EPA's) data gathering and monitoring
programs under the Clean Water Act, the Resource Conservation and Recovery Act, the
Comprehensive Environmental Response, Compensation and Liability Act, and the Safe Drinking
Water Act.
1.2 This method is designed to meet EPA's survey and monitoring requirements of the U.S.
Environmental Protection Agency (EPA) in regulating the use and disposal of biosolids under 40
CFR Part 503 Subpart D and can be specifically applied to determining the density of Salmonella
spp. Subpart D of the Part 503 regulation protects public health and the environment through
requirements designed to reduce the potential for contact with disease-bearing microorganisms
(pathogens) in biosolids applied to land or placed on a surface disposal site. A biosolid is Class A
if the density of Salmonella spp. are less than 3 MPN per 4 grams total solids. This method is
based on existing procedures and techniques for the determination of Salmonella spp. in biosolids
for Class A materials.
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/4g of total
biosolid solids on a 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 Method 1682 can be used for detection of Salmonella spp. bacteria. However, this method cannot
differentiate between serovars of Salmonella spp.
1.5 EPA plans to validate this method for use on digested, dewatered, composted, and heat-dried,
pelletized biosolids.
1.6 The MPN is a statistical estimating technique that states the range of the bacterial population with
95% confidence. MPN is used for samples which require selection from a heterogeneous
population of organisms such as is found in biosolids. The MPN technique will quantify
microorganisms to below the compliance limit of < 3 MPN/4 grams dry weight.
1.7 This method has not been evaluated for use in water samples and is not intended as a test for
organisms other than Salmonella spp.
1.8 Each laboratory that uses this method must demonstrate the ability to generate acceptable results
using the procedure in Section 9.3.1.
1.9 Any modification of the method beyond those expressly permitted is subject to the application and
approval of alternative test procedures under 40 CFR Part 136.4 and 136.5.
2.0 Summary of Method
2.1 The selenite brilliant green sulfa enrichment broth (SBG), Rappaport-Vasilliadis agar medium-
semisolid modification (MSRV), and Rappaport-Vasilliadis (RV) broth protocols presented in
Method 1682 provide enumeration of Salmonella spp. in biosolids based on the most probable
number (MPN) technique. The determination of Salmonella spp. involves initial seeding into either
the selective medium SBG (for the SBG protocol), the enrichment medium tryptic soy broth (TSB)
1 August 1998 Draft
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Method 1682 - Draft
(for the MSRV protocol) or the pre-enrichment medium phosphate buffered peptone water (BPW)
plus enrichment in RV broth (for the RV protocol). Positives from SBG are streaked onto the
selective and differential plating medium xylose-lysine desoxycholate agar (XLD) and modified
lysine iron agar (MLIA). Those organisms that exhibit growth in TSB are spotted onto the
selective MSRV medium. Positive RV tubes are isolated onto lysine-mannitol-glycerol (LMG) agar
and purified onto MacConkey agar. Confirmatory tests for all three protocols are performed with
lysine-iron agar (LIA), triple sugar iron agar (TSI), and urease test medium, followed by positive
serological typing using polyvalent antisera. Percent total solids determination is performed on a
representative biosolids sample for MPN/g dry weight calculations.
3.0 Definitions
3.1 Salmonella spp. are gram-negative, predominately motile, facultatively anaerobic rod-shaped
bacteria that comprise about 2,000 serovars. In this method, Salmonella spp. are those bacteria
that prove positive in the biochemical and serological confirmation methods described.
3.2 Class A biosolids contain Salmonella sp. densities below 3 MPN/4g of total solids on a dry weight
basis.
4.0 Interferences
4.1 Since the most probable number (MPN) tables are based on a Poisson distribution, the MPN value
will be an underestimate of the bacterial density if the sample is not adequately shaken to ensure
equal bacterial cell distribution before portions are removed.
4.2 Low estimates of Salmonella spp. may be caused by the presence of high numbers of competing or
inhibitory organisms, or toxic substances such as metals or organic compounds.
4.3 Bacteria that compete with Salmonella spp., such as Proteus, may overgrow the medium and mask
or prevent detection.
4.4 Percent total solids interferences: See Appendix A.
5.0 Safety
5.1 The biohazard associated with, and the risk of infection from handling Salmonella spp. is high in
this method. Method 1682 does not purport to address all of the safety problems associated with its
use. It is the responsibility of the laboratory to establish appropriate safety and health practices
prior to use of this method. In particular, the analyst/technician must know and observe the safety
procedures required in a microbiology laboratory that handles frank pathogens while collecting,
preparing, using, and disposing of sample reagents and materials. Equipment and supplies that
have come into contact with biohazardous material or suspected of containing biohazardous
material must be sterilized prior to disposal or re-use. 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 handling pathogens to all samples.
5.2 The laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration regulations regarding the safe handling of the chemicals specified in this
method. A reference file of material safety data sheets should be made available to all personnel
involved in these analyses. Additional information on laboratory safety can be found in Reference
16.1.
5.3 Samples may contain high concentrations of biohazardous compounds and must be handled with
gloves. If there is evidence of pressure build-up in the sample container from gasses, these sample
containers must be opened in a biological safety cabinet. Any positive reference materials must
also be handled with gloves and the analyst/technician must never place gloves in or near the face
after exposure to media known or suspected to contain salmonella organisms. Laboratory
August 1998 Draft
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Method 1682 - Draft
personnel must change gloves after handling sewage sludge (biosolids), media tubes or any other
items which carry pathogenic organisms. Mouth pipetting is prohibited.
6.0 Equipment and Supplies
Note: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and materials
other than those specified here, but demonstration of equivalent performance that meets the
requirements of this method is the responsibility of the laboratory.
6.1 Equipment for collection and transport of samples
6.1.1 Sterile plastic bags—1-gal
6.1.2 Plastic or glass jars—1-L
6.1.3 Sterile auger
6.1.4 Sterile scoops— Scienceware, Bel Art No. 436904, or equivalent
6.1.5 Ice chest—Igloo, Coleman, or equivalent
6.1.6 Ice
6.1.6.1 Wet ice—purchased locally, or
6.1.6.2 Ice packs—Blue Ice, UTek cat. no. 429, or equivalent, frozen for use
Note: Blue Ice and similar products may be used, provided the sample is protected from
freezing. Do not allow sample to be in direct contact with ice or blue ice because of freezing
potential.
6.1.7 Bubble wrap
6.2 Equipment for mixing biosolids
6.2.1 Sterile trowels
6.2.2 Sterile plastic sheet
6.3 Equipment for growth of microorganisms
6.3.1 Sterile culture tubes—18 x 150 mm and 16 x 150 mm, autoclavable slip caps and culture
tube racks
6.3.2 5- and 10-mL sterile serological pipets and 10-mL plastic wide-mouthed sterile serological
pipets—Falcon, Kimble, or equivalent
6.3.3 Pipet bulbs, or automatic pipettor—Pipet-Aid, or equivalent
6.3.4 Nichrome wire inoculating loop and needle
6.3.5 Bunsen burner or alcohol burner
6.3.6 Cornwall syringe—sterile, to deliver at least 5 mL
6.3.7 Media dispensing pump—Unispense II, or equivalent
6.3.8 Incubator, water-jacketed, humidity-controlled, microbiological type to hold temperatures
at either 37°C and 42°C ± 0.5°C—Precision, VWR, or equivalent
6.3.9 Plastic sterile petri dishes—microbiological grade, 25 mm x 100 mm
6.3.10 Glass templates or slides for agglutination test
6.3.11 Erlenmeyer flasks—1-L and 2-L Corning, Nalgene, Kimble, or equivalent
6.3.12 Stir bar—Fisher cat. no. 14-511-93, or equivalent
6.3.13 Stir plate—Fisher cat. no. 14-493-120S, or equivalent
August 1998 Draft
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Method 1682 - Draft
6.3.14 Homogenization equipment
6.3.14.1 Sterile blender jars and motor base—Oster, Corning or equivalent
6.3.14.2 Stomacher and stomacher bags—Oster or equivalent
6.3.15 Water bath to temper agar media with a range from room temperature to
100°C—Precision, VWR Scientific, or equivalent
6.3.16 Media filtration equipment—Sterile, 0.22-/^m pore size Millex syringe filters or larger
diameter Millipore 0.22-^m pore size
6.3.17 Sterile cotton-tipped applicators
6.3.18 Magnifying glass or dissection scope—Zeiss, or equivalent
6.3.19 Latex gloves for handling samples and extraction equipment—Microflex, San Francisco,
CA, stock no. UL-315-L, or equivalent
6.3.20 pH meter—Beckman, Corning, or equivalent
6.3.21 Vortex mixer—Vortex Genie, or equivalent
6.4 Equipment for percent total solids determination—see Appendix A
6.5 Miscellaneous labware and supplies
6.5.1 Flasks—Erlenmeyer, various sizes
6.5.2 Beakers—glass or plastic, assorted sizes
6.5.3 Lint-free tissues—KimWipes or equivalent
6.5.4 Steel pan of water—30" x 26" x 10"
7.0 Reagents and Standards
Note: 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. The agar used in preparation of culture media must be of microbiological
grade.
7.1 Deionized water—conforming to Specification D 1193, Annual Book of ASTM Standards.
7.2 Phosphate buffered water—Prepare phosphate buffer stock by adding 34.0 g potassium dihydrogen
phosphate (KH2PO4) into 500 mL of deionized water. Dissolve completely , then using a pH meter,
adjust pH to 7.2 ± 0.5 with approximately 175 mL 1 N NaOH. Dilute to 1 L with deionized water.
Prepare magnesium chloride stock by adding 81.1 g MgCl2.*6H2O into 1 L of deionized water and
mixing thoroughly to dissolve. Add 1.25 mL stock phosphate buffer solution and 5.0 mL stock
magnesium chloride solution to 1 L of deionized water. Autoclave for 15 min at 121°C. If buffer is
to be used for making sample suspensions, add 0.1% Tween-80, mix well, dispense into erlenmeyer
flasks, and autoclave at 121°C for 15 min. Store at room temperature.
7.3 Buffered peptone water (BPW)—(Oxoid cat. No. CM509, or equivalent) Add 20 g of BPW to 1 L
of deionized water and mix well. Dispense 10 mL volumes per 16 mm x 150 mm culture tube;
sterilize in autoclave at 121°C for 15 min. Store at 4°C. To make 2X BPW, add 40 g of BPW
medium to 1 L of deionized water and mix well. Dispense 10 mL volumes per 18 x 150 mm
culture tubes and sterilize in autoclave at 121°C for 15 min. Store at 4°C.
Note: Tween lowers surface tension of water. Buffer with 0.1% Tween-80 may "bump" or flash
boil when removed from the autoclave. Do not fill flask more than half-full for autoclaving.
Remove from autoclave carefully, pointing flask top away from face.
7.4 Tween-80—Sigma Chemical Co. cat. no. P1754, or equivalent.
August 1998 Draft 4
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Method 1682 - Draft
7.5 Tryptic soy broth (TSB)—(DIFCO cat. no. 0370-15, or equivalent) Add 30 g of TSB in 1 L of
deionized water and mix well. Adjust pH with enough 1.0 N NaOH or HC1, if necessary, to
achieve a pH of 7.3 ± 0.1 at 25°C. Warm to approximately 80°C and mix to dissolve completely.
Dispense 10 mL volumes per 16 mm x 150 mm culture tube; sterilize in autoclave for 15 min at
121°C. 2X TSB is prepared by adding 30 g TSB to 500 mL deionized water, adjusting pH as
above. Dispense 10 mL volumes into 18 mm x 150 mm culture tubes and autoclave. Store at room
temperature in the dark.
7.6 Lysine-mannitol-glycerol agar (LMG) (Commercially unavailable)—Prepare LMG agar by adding
3.0 g proteose peptone, 5.0 g yeast extract, 5.0 g lysine, 5.0 g mannitol, 5.0 g glycerol, 5.0 g NaCl,
1.0 g sodium desoxycholate, 4.0 g sodium thiosulfate, 1.0 g ferric ammonium citrate, 0.1 g phenol
red and 15.0 g agar into 1 L of deionized water. Boil for 2 -3 min while mixing to dissolve
thoroughly and cool to 45 - 50°C. Dispense immediately into 25 x 100 mm sterile petri plates at 15
- 20 mL per plate.
7.7 Rappaport-Vasilliadis agar medium-semisolid modification (MSRV) (DIFCO cat. no. 1868-
17)—Add 31.6 g of MSRV to 1 L deionized water and mix well. Boil the medium, but do not
autoclave. Cool to 50°C. Add 1.0 mL of a 2% stock solution of Novobiocin (Section 7.11) per liter
of medium. Swirl the medium to mix. Immediately pour thick plates with approximately 25 mL
each. Since the medium will not be firm, do not invert plates to store. Store at room temperature
and use within 48 hours.
7.8 Rappaport-Vassilliadis broth (RV) (Oxoid cat. No. CM669)—Add 30.0 g of RV to 1 L deionized
water and mix well. Heat gently until dissolved completely. Dispense 10 mL per 16 x 150 mm
screw cap or slip cap tube. Autoclave for 15 min at 115°C. Store at 4°C. These tubes will be used
for the most probable number analysis in the RV broth method. Prepare at least 15 tubes per
sample, plus enough to carry the positive controls through the test.
7.9 Selenite brilliant green sulfa enrichment broth (SBG) (DIFCO cat. no. 0715-17)—Dissolve 24.2 g
SBG in 1 L deionized water for single-strength medium. Use 24.2 g in 500 mL for double strength
medium. Prepare fresh on the day of use by heating in a 70°C water bath for 1 hr instead of boiling
for 10 min as indicated on the manufacturer's instructions. Cool to room temperature prior to use.
7.10 Xylose-lysine desoxycholate agar (XLD) (DIFCO cat. no. 0788)—Add 57.0 g of XLD agar to 1 L
deionized water. Heat to boiling to dissolve completely but do not autoclave. Cool to 47°C and
pour into 25 x 100 mm sterile petri plates. Plated medium may be stored for up to 2 weeks at 2 -
8°C.
7.11 Novobiocin (2%) stock solution (DIFCO cat. no. 3197)—Dissolve 50 mg into 25 mL of deionized
water and filter sterilize using a 0.22 ^m 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. Can be stored for up to 1 year at -
20°C.
7.12 Modified lysine iron agar (MLIA) (DIFCO cat. no. 0849)—Dissolve 34.5 g LIA into 1 L of
deionized water. Add 10 g sucrose, 10 g lactose and 1.5 g bile salts No. 3 (DIFCO # 0130). Heat
to boiling and cool to 47°C. Add 3.7 mL stock Novobiocin (Section 7.11) and stir before pouring
into 25 x 100 mm sterile petri dishes. Store at 4°C.
7.13 Triple sugar iron agar (TSI) (DIFCO cat. no. 0265)—Weigh out 65 g TSI into 1 L of deionized
water. Heat until boiling to dissolve completely. Dispense 5 mL amounts into 16 x 100 mm test
tubes in slant racks, cap and sterilize for 15 min at 121°C and 15 psi. Allow the autoclaved
medium to solidify in the slant rack such that half the surface area is on a slant and half is in the
butt of the tube. Store at 4°C.
7.14 Lysine iron agar (LIA) (DIFCO cat. no. 0849)—Add 34.5 g into 1 L of reagent-grade water and
mix. Heat to boiling to dissolve completely. Dispense 5 mL amounts into 16 x 100 mm test tubes
and sterilize at 121°C for 12 min. Allow the autoclaved medium to solidify in the slant rack such
that half the surface area is on a slant and half is in the butt of the tube. Store at 4°C.
5 August 1998 Draft
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Method 1682 - Draft
7.15 Urease test broth (DIFCO# 0283-15)—Dissolve 38.7 g urease test broth in 1 L deionized water.
Filter sterilize using a 0.22 ^m filter into a sterile flask. Aseptically dispense 3 mL into sterile 16 x
100 mm test tubes using a sterile pipet or sterile Cornwall syringe. Store at 4°C.
7.16 Sterile physiological saline (0.85% w/v)— Dissolve 8.5 g NaCl in 1 L deionized water. Autoclave
for 20 minutes at 121°C. Store at room temperature.
7.17 MacConkey agar without salt (DIFCO cat.no.0331-17)— Dissolve 47 g MacConkey agar in 1 L
deionized water. Sterilize by autoclaving for 15 min at 121°C but avoid prolonged heating. Cool to
45 - 50°C and dispense 25 mL per 25 x 100 mm sterile petri plates. Plated medium may be stored
for up to 2 weeks at 2 - 8°C in the dark.
8.0 Sample Collection, Preservation, and Storage
8.1 The most appropriate location of biosolids 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.
Note: If samples are not to be tested within 1 hour after collection, they should be cooled to
below 10°C during a maximum transport time of 6 hours. If samples are brought to 4°C by
prompt chilling, 24 hours between sampling and analysis should not adversely affect the results.
Samples should not be frozen.
8.2 All sampling containers and equipment must be clean and sterile to prevent contamination or
overgrowth by other bacteria or mold.
8.3 Equipment and container cleaning procedure
8.3.1 Wash apparatus with laboratory grade detergent and water.
8.3.2 Rinse with tap water.
8.3.3 Rinse with 10% HC1 acid wash.
8.3.4 Rinse with distilled water.
8.3.5 Allow to air dry or rinse with an organic solvent such as hexane to promote drying
process.
8.3.6. Cover with foil and autoclave for 15 minutes at 121°C.
8.4 Procedure for sampling digester biosolids
8.4.1. Collect the digester biosolids sample from the outlet pipe used to fill the truck.
8.4.2 Purge the pipe of old biosolids and warm to the digester temperature by allowing biosolid
to flow through the pipe into a bucket.
8.4.3 Position a 1-gal. sterile bag under the flow so that nothing but sample touches the inside of
the bag. Fill the bag, but leave 0.5 in. 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
8.5.1 Collect the biosolids sample directly into the sample container without mixing or
transferring to another area.
8.5.2 Using a sterile scoop, transfer the pressed biosolids directly off of the conveyer and into a
sterile container.
8.5.3 Pack the sample into the container. Head space is not as important as with a liquid sample
because there is less gas formation.
August 1998 Draft
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Method 1682 - Draft
8.6 Procedure for sampling from a bin, drying bed, truck bed or similar container
8.6.1 Divide the material to be sampled into four quadrants.
8.6.2 Use a scoop or core the sample if material is deep.
8.6.3 Take a sample from each of the quadrants and combine in a stainless steel or plastic
bucket.
8.6.4 After all the samples have been taken, pour the bucket contents out onto a plastic sheet and
mix by folding the sample back onto itself several times.
8.6.5 Reduce the sample size by "coning and quartering." Divide the bucket contents into four
even piles. If sample size is still too large, divide each quarter into quarters and discard
half of each quarter. Put into glass or plastic sampling container.
8.6.6 An alternate method to "coning and quartering" is to randomly take flat shovel-fulls of
biosolid from the bucket that has been dumped onto a plastic sheet and put those samples
into a sampling container. (Curved scoops have been shown to favor a certain size particle
and therefore should not be used.)
8.7 Record the following into your log book:
8.7.1 Facility name and location
8.7.2 Date
8.7.3 Arrival time
8.7.4 Name of facility and contact
8.8 Record the following onto sample container when known:
8.8.1 Sample number
8.8.2 Date and time
8.8.3 Sample name
8.8.4 Sample location
8.8.5 Parameters
8.8.6 Volume
8.8.7 Observations
8.9 Ensure that the chain of custody form is filled out
8.10 Sample preservation and handling—Ice or refrigerate bacteriological samples at a temperature of
1 °C to 4°C during transit to the laboratory. Do not freeze sample. Use insulated containers to
assure proper maintenance of storage temperature. Sample bottles should be placed inside scalable
ziplock-type 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 Chlorinated samples—Add a reducing agent to containers intended for the collection of biosolids
containing residual chlorine or other halogen. Sodium thiosulfate (Na2S2O3) is a satisfactory
dechlorinating agent that neutralizes any residual halogen and prevents continuation of bactericidal
action during sample transport. If Na2S2O3 is used, add a sufficient volume of Na2S2O3 to a clean
sample bottle, to give a concentration of 100 mg/L in the sample. In a 120-mL sample bottle, a
volume of 0.1 mL of a 10% solution of Na2S2O3 will neutralize a sample containing about 15 mg/L
residual chlorine.
8.12 Holding time limitations—Analyses should begin as soon as possible, preferably, within 8 hours of
collection. If it is impossible to examine samples within 8 hours, refrigerate samples at 4°C and
analyze within 24 hours.
August 1998 Draft
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Method 1682 - Draft
Note: Adherence to sample preservation procedures and holding time limits is critical to the
production of valid data. Sample results will be considered invalid if those conditions are not
met.
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance (QA)
program. The minimum requirements of this program consist of an initial demonstration of
laboratory capability through analysis of positive and negative control samples (Section 9.4) and
blanks (Section 9.5), and analysis of positive and negative control samples and blanks as tests of
continued performance. Laboratory performance is compared to the performance criteria specified
in Sections 9.4 and 9.5 to determine if the results of analyses meet the performance characteristics
of the method. Specific quality control (QC) requirements for Method 1682 are provided below.
General recommendations on QA and QC for facilities, personnel, and laboratory equipment,
instrumentation, and supplies used in microbiological analyses are provided in the USEPA
microbiology methods manual, Part IV, C (4) (Reference 16.2).
9.2 If an analytical technique other than the techniques specified in this method is used, that technique
must have a specificity and recovery equal to or belter than the specificity and recovery of the
techniques in this method for Salmonella in the sample of interest. Specificity is defined as
producing results equivalent to the results produced by this method for Salmonella, and that meet
all of the QC acceptance criteria stated in this method. Recovery is defined as the ability of the test
to enumerate viable Salmonella spp. without losses as a consequence of environmental parameters
or the test method.
9.2.1 Each time a modification is made to this method, the analyst is required to repeat the
positive and negative control samples (Section 9.4) to demonstrate that the modification
produces results equivalent or superior to results produced by this method.
9.2.2 The laboratory is required to maintain records of modifications made to this method. These
records include the following, at a minimum:
9.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s) who
performed the analyses and modification, and of the quality control officer who
witnessed and will verify the analyses and modification.
9.2.2.2 A listing of the analyte measured (Salmonella spp.).
9.2.2.3 A narrative stating reason(s) for the modification.
9.2.2.4 Results from all QC tests comparing the modified method to this method,
including initial precision and recovery (analysis of positive and negative
control samples (Section 9.4), and analysis of blanks (Section 9.6).
9.2.2.5 Data that will allow an independent reviewer to validate each determination by
tracing the processing and analysis steps leading to the final result.
9.3 Analytical QC procedures required per batch of field samples (batch is defined as 20 samples or
one 12-hour laboratory shift, whichever comes sooner).
9.3.1 Perform one positive control sample and one negative control sample (Section 9.4) per
batch of field samples
9.3.2 Perform one blank sample (Section 9.5) per batch of field samples
9.3.3 Perform duplicate analyses on at least one sample per batch. In laboratories with more
than one analyst, have each make parallel analyses on at least one positive sample
monthly.
August 1998 Draft
-------
Method 1682 - Draft
9.3.4 Check dilution water for sterility by adding 20 mL water to 100 mL of a nonselective broth
such as tryptic soy broth. Incubate at 35 ± 0.5° C for 24 hours and observe growth. If any
contamination is indicated, reject analytical data from samples tested with these materials.
9.3.5 Test medium sterility by subjecting a representative portion of each batch to incubation at
37 ±0.5°C for 24 to 48 hours and observe for indications of growth.
9.4 Positive and negative control samples. Obtain reference cultures from qualified outside sources and
use these to establish pure stock cultures that are maintained for the laboratory. Analyze positive
and negative control samples with each batch of field samples and with each new lot of media. For
positive control, inoculate the medium with a known positive Salmonella sp.(e.g. S. typhimurium);
for negative control, inoculate the medium with a known negative Salmonella sp. (e.g.
Escherichia. coll). Test each batch of each individual medium, reagent, or antisera with both
positive and negative controls to verify correct response.
9.5 Process a positive sample through the entire analytical procedure and examine for appropriate
responses. Process a negative control sample until it is verified that culture is negative.
Table 1. Positive and Negative Control Results
Medium
Selenite brilliant green
Rappaport-Vasilliadis broth
Tryptic soy broth
Lysine mannitol glycerol agar
Rappaport-Vasilliadis agar medium-
semisolid modification
Xylose lysine desoxycholate agar
MacConkey Agar
Modified lysine iron agar
Lysine iron agar
Triple sugar iron agar
Urease test broth
Reaction for
Salmonella spp.
"Brick" red color
Negative Reaction for
Salmonella spp.
Medium remains clear green color
Turbidity indicates growth of multiple types of microorganisms
General purpose growth medium. Growth of any organism results in turbid solution.
Colonies have black centers surrounded
by a yellow-orange to pink zone.
S. typhi is yellow with a yellow zone and a
black center after 48 h.
Salmonella spp. which are H2S negative
are pink or colorless with pink zones.
Migrated cells visible as a gray-white
turbid zone extending out from the
inoculated drop
Pink to red colonies with black centers.
(Shigella spp. are red)
Transparent or slightly opaque colonies
Purple medium with colonies having "fried
egg" appearance and smooth edges.
Centers are black and nucleated.
Alkaline slant (purple) with alkaline butt
(purple) with or without H2S
Good growth with alkaline slant (red) with
acid butt (yellow) and H2S production
(Black butt)
No color change (negative urease means
it's positive for Salmonella spp.)
Yellow colonies with yellow zone.
Proteus spp. are small, with dark
greenish-grey or black centers
surrounded by a pink zone.
Medium remains blue-green around
the drop with no gray-white turbid
zone
Enterococcus spp. and E. coll are
yellow with bile ppt
Other color indicators are organisms
but not Salmonella spp.
Atypical growth and color change to
yellow if Enterobacteriaceae are
present
Other color indicators are organisms
but not Salmonella spp.
Other color indicators are organisms
but not Salmonella spp.
Pink (positive urease, but negative
for Salmonella spp.)
August 1998 Draft
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Method 1682 - Draft
10.0 Calibration and Standardization
10.1 Check temperatures in incubators daily to ensure operation is within stated limits of the method
and record daily measurements in incubator log book.
10.2 Check/calibrate thermometers twice per year against a NIST certified thermometer or one that
meets the requirements of NIST Monograph SP 250-23. Check mercury columns for breaks.
10.3 Calibrate pH meter prior to each use using standards of pH 4.0 and 7.0.
10.4 Calibrate top-loading balances once per month with reference weights of ASTM Class 2.
10.5 All equipment must be calibrated according to manufacturer's instructions and maintained on a
schedule as dictated in their maintenance manual.
11.0 Procedure
11.1 Three types of procedures are given in the following sections. All procedures entail identical
homogenization, biochemical and serological confirmation steps, however, the beginning steps of
each method are different. The first method is the selenite brilliant green sulfa enrichment broth
(SBG) procedure which involves enrichment in SBG followed with selection in xylose lysine agar
(XLD) and modified lysine iron agar (MLIA). The second procedure is the Rappaport-Vasilliadis
agar medium-semisolid modification (MSRV) procedure that enriches with tryptic soy broth (TSB)
then selects in MSRV. The third procedure is the Rappaport-Vasilliadis broth (RV) method which
pre-enriches with buffered peptone water, enriches with RV broth, isolates onto LMG agar with
purification of isolates onto MacConkey's agar. All procedures are followed by identical
biochemical and serological confirmation steps.
11.2 Homogenization
11.2.1 Prepare sample suspensions of biosolids by weighing out a sufficient amount of biosolids
so that at least 4 g dry weight will be present. If the sample contains more than 10% solids
in the sample then use 50 g wet weight. If less solids are present, then use a sample known
to contain at least 4 g dry weight. Add Tween-80 directly to liquid samples to a 0.1% v/v
concentration.
11.2.2 Place the material in a stomacher bag or blender and add 100 mL of phosphate buffered
water plus 0.1% Tween-80 if 4g dry weight is used or 500 mL phosphate buffered water
plus 0.1% Tween-80 if 50 g is weighed out.
Note: This procedure may be altered as a result of testing to standardize biosolids preparation.
11.2.2.1 Homogenization using a using a stomacher
11.2.2.1.1 Seal the stomacher bag and stomach for approximately 5 min. at
normal speed.
11.2.2.1.2 Cut almost through the top corner seal of the stomacher bag with
scissors. This will avoid contamination of the scissors and the
contents of the bag.
11.2.2.1.3 Tear off the bag corner and pour the contents into a 250 mL
sterile disposable centrifuge tube.
11.2.2.2 Homogenization using a mechanical mixer:
11.2.2.2.1 Place a cover on the blender and mix at medium speed for 2 to not
more than 5 min.
August 1998 Draft 10
-------
Method 1682 - Draft
11.3 Enrichment and selection
11.3.1 Selenite brilliant green sulfa enrichment broth (SBG) procedure
11.3.1.1 Preparation
11.3.1.1.1 Prepare phosphate buffered water (Section 7.2) and Novobiocin
stock solution (Section 7.11) according to their recipes. Follow
instructions for storage.
11.3.1.1.2 Prepare selenite brilliant green sulfa enrichment broth (SBG) (IX
and 2X) (Section 7.9), and cool to room temperature. Prepare
most probable number (MPN) tubes by aseptically dispensing 10
mL of 2X SBG into five sterile 18 mm x 150 mm tubes per each
biosolids sample. Aseptically dispense 10 mL of IX SBG into
twelve sterile 16 mm x 150 mm tubes per each biosolid sample.
Note: SBG must be used on the same day it is made.
11.3.1.1.3 Prepare 400 mL of each of the primary isolation media: XLD
(Section 7.10) and MLIA (Section 7.12 ) per biosolids sample.
Pour 15 plates (25 mL per plate) of each media per sample.
Allow plates to solidify and excess moisture to evaporate prior to
use. Follow instructions for storage.
11.3.1.1.4 Prepare 30 tubes of the biochemical medium TSI (Section 7.13),
LIA (Section 7.14) and urease test broth (Section 7.15) for each
sample.
11.3.1.1.5 Assemble five 2X SBG tubes and 12, IX SBG tubes per biosolid
sample for MPN determination. (One IX SBG tube will be used
for positive control and one IX SBG tube will be used for
negative control.)
11.3.1.2 Enrichment procedure required for calculation of most probable
number (MPN)
11.3.1.2.1 Pipet 10 mL of the homogenized biosolids from Section 11.2 into
each of the 5 tubes containing 10 mL 2X SBG.
11.3.1.2.2 Pipet 1.0 mL of the homogenized biosolids into each of the 5
tubes of IX SBG, and 0.1 mL into each of the remaining 5 tubes
of IX SBG.
11.3.1.2.3 Inoculate one tube IX SBG with a Salmonella culture and one
tube SBG with an E. coll culture for positive and negative
controls, respectively.
11.3.1.2.4 The controls will be carried through to the plating step as a QC
measure to detect media or incubator failure.
11.3.1.2.5 Incubate SBG tubes for 20 to 24 hours at 37°C.
11.3.1.2.6 Record all "brick red" tubes as presumptive positive along with
sample number in lab notebook.
11.3.1.3 Selection
11.3.1.3.1 Streak the positive SBG tubes, including positive control tubes, to
plates containing the selective medium xylose-lysine
desoxycholate agar (XLD) and modified lysine iron agar (MLIA)
11 August 1998 Draft
-------
Method 1682 - Draft
for primary isolation of Salmonella spp. Incubate plates for 18 to
24 hr at 37°C. Inoculate plates with fresh negative control.
11.3.1.3.2 Do not discard the SBG broth tubes. Instead, retain the SBG
broth tubes for an additional 24 hours at 37°C until after the
isolation plates have been incubated and examined.
11.3.1.3.3 Examine the primary isolation plates (XLD and MLIA) and
determine whether the pattern of Salmonella spp. isolation
follows a logical dilution distribution pattern. The pattern should
exhibit more total positives in the least dilute and fewer positives
in the most dilute. If this pattern is not established then inoculate
1 mL from the least dilute/negative 48 hour SBG tubes into fresh
SBG tubes, and incubate 18 to 24 hours at 37°C to find the
Salmonella.
11.3.1.3.4 Proceed to Section 11.4 for biochemical confirmation of
Salmonella and biochemical characterization using TSI, LIA ,
urease test broth and polyvalent O antisera reaction.
11.3.2 Rappaport-Vasilliadis agar medium-semisolid modification (MSRV) procedure
11.3.2.1 Preparation
11.3.2.1.1 Prepare phosphate buffered water (Section 7.2) and Novobiocin
stock solution (Section 7.11) according to their recipes. Follow
instructions for storage.
11.3.2.1.2 Prepare tryptic soy broth (TSB) (IX and 2X) (Section 7.5)
according to directions. Prepare MPN tubes by aseptically
dispensing 10 mL of the 2X TSB into 5 sterile 18 mm x 150 mm
tubes for each biosolid sample. Aseptically dispense 10 mL of the
IX TSB into 10 sterile 16 mm x 150 mm tubes per each biosolid
sample. Dispense 2 more tubes of IX TSB for controls per set of
samples.
11.3.2.1.3 Prepare 400 mL of the MSRV primary isolation medium (Section
7.6) per biosolids sample. Pour 15 plates (25 mL per plate) of the
medium per biosolid sample. Allow to cool and solidify before
use. Store up right as media will not be firm.
11.3.2.1.4 Prepare 400 mL of the isolation medium xylose-lysine
desoxycholate agar (XLD) (Section 7.10) per biosolids sample.
Pour 15 plates (25 mL per plate) of the media per biosolid
sample. Allow plates to solidify and dry slightly prior to use.
Follow instructions for storage.
11.3.2.1.5 Prepare 30 slant tubes of each of the biochemical confirmation
media: TSI (Section 7.13), LIA (Section 7.14) and urease test
broth (Section 7.15).
11.3.2.1.6 Assemble five 2X TSB tubes and twelve IX TSB tubes per
biosolid sample for MPN determination. (One IX TSB tube will
be used for positive control and one IX TSB tube will be used for
negative control.)
11.3.2.2 Enrichment procedure required for calculation of most probable
number (MPN)
11.3.2.2.1 Add 10 mL aliquots of homogenized biosolid sample from Section
11.2 to five tubes containing 10 mL of 2X TSB.
August 1998 Draft 12
-------
Method 1682 - Draft
11.3.2.2.2 Pipet 1.0 mL of homogenized sample into each of five tubes of
IX TSB, and 0.1 mL to the remaining 5 tubes of IX TSB. The
MPN enrichment is carried out using 10 mL of inoculum per 10
mL 2X broth. The IX broth is inoculated with 1.0 and 0.1 mL
quantities of sample.
11.3.2..2.3 For positive and negative controls, inoculate one tube of IX TSB
with a loopful of Salmonella culture and one tube TSB with a
loopful of an E. coll culture, respectively. The controls will be
carried through to the plating step as a QC measure to detect
media or incubator failure.
11.3.2.2.4 Incubate the TSB MPN tubes and controls for 24 hours at 35°C.
11.3.2.2.5 Record all turbid tubes as positive along with sample number in
lab notebook. Because of the non-inhibitory nature of the
enrichment medium, all tubes will be positive in most instances. If
the tubes do not appear positive, then this may indicate the
presence of atoxic substance.
11.3.2.3 Selection
11.3.2.3.1 Apply six, 30-^L drops from each positive tube onto a
corresponding MSRV plate. Allow the drops to absorb into the
agar for 1 hour at room temperature, and incubate each plate,
inoculated side up, at 42 ±0.5°C for 16 to 18 hours in a humidity-
controlled hot air incubator. An open pan of water placed in the
bottom of the incubator will serve the same purpose.
11.3.2.3.2 Examine plates for the appearance of motility surrounding the
spots, as evidenced by a "whitish halo" of growth approximately
2 cm from the center of the spot.
11.3.2.3.3 Material from the outer edge of the halo is taken up with a sterile
inoculating needle and streaked onto XLD agar for isolation.
11.3.2.3.4 Incubate XLD plates for 18 to 24 hours at 35°C.
11.3.2.3.5 Proceed to Section 11.4 for confirmation of Salmonella spp. and
biochemical characterization using TSI, LIA , urease test broth
and polyvalent O antisera reaction.
11.3.3 Rappaport-Vasilliadis broth (RV) method procedure
11.3.3.1 Preparation
11.3.3.1.1 Prepare buffered peptone water (BPW) (Section 7.3). Follow
instructions for storage.
11.3.3.1.2 Prepare Rappaport-Vasilliadis (RV) broth (Section 7.8), and cool
to room temperature prior to use.
11.3.3.1.3 Prepare lysine-mannitol-glycerol (LMG) agar according to its
recipe. (Section 7.6). Pour 15 plates (25 mL per plate) of the
medium per biosolid sample. Allow plates to solidify and dry
slightly prior to use. Follow instructions for storage.
11.3.3.1.4 Prepare MacConkey agar (Section 7.17) and follow instructions
for storage. Pour 15 plates (25 mL per plate) of the media per
biosolid sample. Allow plates to solidify and dry slightly prior to
use. Follow instructions for storage.
11.3.3.2 Pre-enrichment procedure for calculation of most probable
number (MPN)
13 August 1998 Draft
-------
Method 1682 - Draft
11.3.3.2.1 Inoculate 10 mL of the homogenized biosolid sample from
Section 11.2 into each of the 5 tubes containing 10 mL of 2X
BPW.
11.3.3.2.2 Pipet 1.0 mL of the homogenized biosolids into each of 5 tubes of
IX BPW, and 0.1 mL into each of the remaining 5 tubes of IX
BPW.
11.3.3.2.3 Inoculate one tube IX BPW with a lab stock Salmonella culture
and one tube BPW with an E. coll culture for positive and
negative controls, respectively.
11.3.3.2.4 The controls will be carried through to the plating step as a QC
measure to detect media or incubator failure.
11.3.3.2.5 Incubate the pre-enrichment PBW MPN tubes and controls at
37°C for 24 hours.
11.3.3.2.6 Record all turbid tubes as positive along with sample number in
lab notebook. Because of the non-inhibitory nature of the
enrichment medium, all tubes will be positive in most instances. If
the tubes do not appear positive, then this may indicate the
presence of atoxic substance.
11.3.3.3 Enrichment step for Salmonella spp. in RV broth
11.3.3.3.1 Transfer 0.1 mL from each turbid buffered peptone water pre-
enrichment tube to a corresponding tube of RV broth.
11.3.3.3.2 Incubate the enrichment RV tubes and controls at 43°C for 48
hours.
11.3.3.4 Isolation
11.3.3.4.1 After 48 hours, subculture each of the positive RV broth tubes
onto an individual plate of lysine-mannitol-glycerol (LMG) agar.
Subculturing may be performed by transferring the organisms
using a sterile cotton swab or flamed loop. The plate should be
streaked for single cell colony formation.
11.3.3.4.2 Incubate the LMG plates at 37°C for 24 hours.
11.3.3.5 Purification
11.3.3.5.1 After the 24 hour incubation, colonies suspected of being
Salmonella spp. are purified onto MacConkey agar. The suspect
colony is picked up via sterile loop from the LMG plate and is
streaked for single cell colony formation onto MacConkey agar.
11.3.3.5.2 Incubate MacConkey agar plates at 37°C for 24 hours.
Note: Experienced analysts In the Isolation of single cell colonies may be able to culture
isolated Salmonella, colonies on the LMG plate such that purification is redundant. If this is the
case, It Is allowable that biochemical confirmation (Section 11.4) follows the Isolation step
(Section 11.3.3.4).
11.3.3.5.3 Proceed to Section 11.4 for biochemical confirmation of
Salmonella and biochemical characterization using TSI, LIA ,
urease test broth and polyvalent O antisera reaction.
August 1998 Draft 14
-------
Method 1682 - Draft
11.4 Biochemical confirmation
11.4.1 Label all tubes with date, sample, and experiment. Pick those isolated colonies exhibiting
Salmonella sp. morphology as defined in Table 1 for the medium being used, from the
plate to slants of triple sugar iron agar (TSI), lysine iron agar (LIA), and urease test broth.
Use the same colony to inoculate all three media. Inoculate positive and negative control
organisms into each medium. Inoculate agar medium with a needle into the butt of the tube
and streak along the slant. Incubate for 24 hours at 35°C. A positive TSI reaction is an
acid butt (yellow in color) and an alkaline slant (purple in color) with or without H2S gas
production. A positive LIA reaction is an alkaline butt, alkaline slant with or without the
production of H2S. Urease is an orange medium and will change to pink or cerise if
positive. A negative urease test is one that exhibits no color change after inoculation.
Salmonella spp. are negative for urease.
11.4.2 To confirm cultures via polyvalent O antiserum: Emulsify isolates on the slant portion of
either LIA or TSI using sterile physiological saline (0.85%). Place one drop of suspension
into each of 2 slide wells. Add one drop of sterile saline to one well as a control and one
drop of polyvalent O antiserum (DIFCO) to the second well. Observe the template under
magnification for an agglutination reaction.
11.4.3 Correlate all positive plates and tubes to original positive tube from which sample was
plated. Record all positive samples. Determine the MPN from this information (see Section
12.0). Record all data clearly into a laboratory notebook. When percent total solids has
been determined, record the information with results.
11.5 Percent total solids determination: See Appendix A
12.0 Data Analysis and Calculations
Note: See the USEPA microbiology manual, Part II, Section C, 3.5, for general counting rules
(2). The Most Probable Number (MPN) procedure is used to estimate bacterial density following
the multiple-tube fermentation technique. Conversion is made from MPN/100 mL to MPN/g.
Bacterial density is recorded as MPN/g dry weight.
12.1 Estimation of Salmonella spp. density
12.1.1 The estimated density of Salmonella spp. bacteria, based on the confirmation test using the
polyvalent O antiserum reaction, is computed in terms of the most probable number
(MPN) procedure. The MPN value may be obtained from the MPN table (Table 3) below
using the number of positive tubes in three significant dilutions.
12.1.2 Only three of the four series of five selection tubes will be used for estimating the MPN or
"MPN code." The MPN code represents the total number of positive tubes for Salmonella
spp. per dilution. The three dilutions are called significant dilutions.
12.1.3 Scenarios for selection of significant dilutions
12.1.3.1 When more than three dilutions are positive, use results from only
three dilution series to compute MPN. Choose the highest dilution
(the most dilute, with least sample volume, e.g., 0.01 is higher
than 0.1) giving positive results in all five tubes inoculated and
the two succeeding higher dilutions.
12.1.3.2 If there are positive tubes in higher dilutions than the dilutions
selected, positive results are moved up from these dilutions to
increase the positive tubes in the next highest dilution selected.
15 August 1998 Draft
-------
Method 1682 - Draft
12.1.3.3 If all tubes are positive, choose the three highest dilution series. If
all tubes are negative, choose the three lowest dilution series.
12.1.3.4 In the significant dilution example table (Table 2), the numerator
represents the number of positive tubes per sample dilution and
the denominator represents total number of tubes inoculated per
sample dilution.
12.1.4 Use Table 3 to estimate the MPN index/100 mL from the significant dilutions chosen.
12.1.5 The MPN for combinations not appearing in the Table 3 may be estimated by Thomas'
formula (Equation 2). If such unlikely tube combinations occur in more than 1% of the
samples, it is an indication that the procedure is faulty or that the statistical assumptions
underlying the MPN estimate are not being fulfilled.
Equation 2 (Thomas' formula)
MPN / 100 mL =
number of positive test tubes X 100
i](mL sample in negative tubes X mL sample in all tubes)
12.1.6 Due to the extreme variability of the solid content of biosolids, results from biosolid
samples are converted to MPN/g using the Equation 3.
Equation 3
MPN / g =
10 (MPN index from Table 3)
(largest significant dilution volume)(% total solids)
12.1.7 After the MPN/g has been calculated, the biosolid sample can be evaluated according to
Class A biosolid pathogen requirements.
TABLE 2. SIGNIFICANT DILUTIONS IN DETERMINING MPN INDEX (SIGNIFICANT
DILUTIONS ARE UNDERLINED)
Sample
A
B
C
10.0 mL
5/5
5/5
0/5
1.0 mL
5/5
3/5
1/5
0.1 mL
3/5
1/5
0/5
0.01 mL
0/5
1/5
0/5
Significant
Dilution Results
5-3-0
5-3-2
0-1-0
August 1998 Draft
16
-------
Method 1682 - Draft
Table 3. MPN Table
Combination of
Positives
0-0-0
0-0-1
0-1-0
0-2-0
MPN Index/
100mL
<2
2
2
4
95% Confidence Limits
Lower
...
1.0
1.0
1.0
Upper
...
10
10
13
1-0-0
1-0-1
1-1-0
1-1-1
1-2-0
2
4
4
6
6
1.0
1.0
1.0
2.0
2.0
11
15
15
18
18
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
4
7
7
9
9
12
1.0
2.0
2.0
3.0
3.0
5.0
17
20
21
24
25
29
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
8
11
11
14
14
17
3.0
4.0
4.0
6.0
6.0
7.0
24
29
29
35
35
40
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
13
17
17
21
26
5.0
7.0
7.0
9.0
12
38
45
46
55
63
Combination of
Positives
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
MPN
Index/
lOOmL
22
26
27
33
34
95% Confidence Limits
Lower
9.0
12
12
15
16
Upper
56
65
67
77
80
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1
5-3-2
5-3-3
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
23
30
40
30
50
60
50
70
90
80
110
140
170
130
170
220
280
350
240
300
500
900
1600
>1600
9.0
10
20
10
20
30
20
30
40
30
40
60
80
50
70
100
120
160
100
100
200
300
600
—
86
110
140
120
150
180
170
210
250
250
300
360
410
390
480
580
690
820
940
1300
2000
2900
5300
—
17
August 1998 Draft
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Method 1682 - Draft
13.0 Method Performance
13.1 Expected performance data will be included into this method when the validation studies have been
completed.
14.0 Pollution Prevention
14.1 Selenite is a carcinogen and should be disposed of as indicated on the media package. The other
solutions and reagents used in this method pose little threat to the environment when recycled and
managed properly.
14.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
15.0 Waste Management
15.1 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).
15.2 Samples, reference materials, and equipment known or suspected to have viable Salmonella spp.
attached or contained must be sterilized prior to disposal.
15.3 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 N.W., Washington D.C. 20036.
16.0 References
16.1 American Public Health Association, American Water Works Association, and Water Environment
Federation. 1995. Standard Methods for Water and Wastewater. 19th Edition. Sections 9020,
9030, 9040, 9050, and 9221.
16.2 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
16.3 Yanko, William A.,Walker, Alan S., Jackson, James L., Leticia L. Libao, and April L. Garcia.
1995. Enumerating Salmonella in Biosolids for Compliance with Pathogen Regulations. Water
Environment Research. 67: 364 - 370.
16.4 Yanko, William A., and Dr. Constantine Skanavis. 1994. Evaluation of Composted Sewage
Sludge Based Soil Amendments for Potential Risks ofSalmonellosis. Journal of Environmental
Health 56 (7): 19-23.
16.5 Walker, Alan S., and William A. Yanko. 1987. Analytical Techniques and Residuals
Management in Water Pollution Control, Water Pollution Control Federation, Specialty
Conference Series: 331 - 337.
16.6 Dr. Robert C. Cooper: Paper in Progress: Detection of Salmonella in Biosolids Using Rappaport-
Vassiliadis Semisolid Agar. BioVir Laboratories, Benicia, CA.
16.7 De Smedt, Jozef M., Bolderdijk, Robert F., Helmut Rappold, and Dieter Lautenschlaeger. 1986.
Rapid Salmonella Detection in Foods by Motility Enrichment on a Modified Semi-Solid
Rappaport-Vassiliadis Medium. Journal of Food Protection. 49 (7): 510 - 514.
August 1998 Draft 18
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Method 1682 - Draft
16.8 Rappold, Helmut, and Robert F. Bolderdijk. Modified Lysine Iron Agarfor Isolation of
Salmonella from Food. Applied and Environmental Microbiology. 1979. 38 (1): 162 -163.
16.9 Hu, Catherine J., Gibbs, R.A., and Goen E. Ho. 1995. Detection of Salmonella in Composted
Wastewater Sludge. Environmental Science Report No.95/5. Murdoch University. ISBN No. 0-
86905-429-5.
17.0 Tables, Diagrams, Flowcharts, and Validation Data
To be included in future draft, as required
18.0 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.
18.1 Units of weight and measure and their abbreviations
18.1.1 Symbols
°C degrees Celsius
^L microliter
< less than
> greater than
% percent
18.1.2 Alphabetical characters
cm centimeter
gal gallon
g gram
L liter
M molar
mg milligram
min minute
mL milliliter
mm millimeter
MPN most probable number
OD outside diameter
s second
v/v volume per unit volume
w/v weight per unit volume
18.2 Definitions, acronyms, and abbreviations (in alphabetical order).
Analyte—The microorganism tested for by this method. The analytes in this method are
Salmonella spp.
Differential medium—A solid culture medium that makes it easier to distinguish colonies of the
desired organism.
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Deionized water—water that has been through mixed bed resin columns and is demonstrated to be
free from the analytes of interest and potentially interfering substances.
Enrichment—Using a culture medium to increase growth of the target organism prior to isolation
of that organism.
Field blank—An aliquot of sterile biosolids or other reference matrix that is placed in a sample
container in the laboratory or the field, and treated as a sample in all respects, including exposure
to sampling site conditions, storage, and all analytical procedures. The purpose of the field blank is
to determine if the field or sample transporting procedures and environments have contaminated the
sample.
Laboratory blank—See Method blank.
Laboratory reagent blank—See Method blank.
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 biosolids or designated matrix 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 determine if analytes or interferences are present in the
laboratory environment.
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 the tubes in a dilution series.
Must—This action, activity, or procedural step is required.
Negative control—A suspension of bacteria that will not give a positive reaction during any
portion of this method such as E. coll.
Positive control—See Ongoing precision and recovery standard.
Preferred—Optional
Preparation blank—See Method blank.
Selective medium—A culture medium 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.
Stock suspension—A suspension containing an analyte that is prepared using a reference material
traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will
attest to the purity and authenticity of the reference material.
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Method 1682 - Draft
Appendix A: Total Solids in Solid and Semisolid Matrices
1.0 Scope and Application
1.1 This procedure is applicable to the determination of total solids in such solid and semisolid samples
as soils, sediments, biosolids separated from water and wastewater treatment processes, and
biosolid cakes from vacuum filtration, centrifugation, or other biosolid dewatering processes.
1.2 This procedure is taken from EPA Method 1684: Total, Fixed, and Volatile Solids in Solid and
Semi-Solid Matrices.
1.3 Method detection limits (MDLs) and minimum levels (MLs) have not been formally established for
this draft procedure. These values will be determined during the validation of Method 1684.
1.4 This procedure is performance based. The laboratory is permitted to omit any step or modify any
procedure (e.g. to overcome interferences, to lower the cost of measurement), provided that all
performance requirements in this procedure are met. Requirements for establishing equivalency are
given in Section 9.1.2 of Method 1682.
1.5 Each laboratory that uses this procedure must demonstrate the ability to generate acceptable results
using the procedure in Section 9.2 of this appendix.
2.0 Summary of Method
2.1 Sample aliquots of 25-50 g are dried at 103°C to 105°C to drive off water in the sample.
2.2 The mass of total solids in the sample is determined by comparing the mass of the sample before
and after each drying step.
3.0 Definitions
3.1 Total Solids-The residue left in the vessel after evaporation of liquid from a sample and
subsequent drying in an oven at 103°C to 105°C.
3.2 Additional definitions are given in Sections 3.0 and 18.0 of Method 1682.
4.0 Interferences
4.1 Sampling, subsampling, and pipeting multi-phase samples may introduce serious errors (Reference
13.1). Make and keep such samples homogeneous during transfer. Use special handling to ensure
sample integrity when subsampling. Mix small samples with a magnetic stirrer. If visible
suspended solids are present, pipet with wide-bore pipets. If part of a sample adheres to the sample
container, intensive homogenization is required to ensure accurate results. When dried, some
samples form a crust that prevents evaporation; special handling such as extended drying times are
required to deal with this. Avoid using a magnetic stirrer with samples containing magnetic
particles.
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4.2 The temperature and time of residue drying has an important bearing on results (Reference 1).
Problems such as weight losses due to volatilization of organic matter, and evolution of gases from
heat-induced chemical decomposition, weight gains due to oxidation, and confounding factors like
mechanical occlusion of water and water of crystallization depend on temperature and time of
heating. It is therefore essential that samples be dried at a uniform temperature, and for no longer
than specified. Each sample requires close attention to desiccation after drying. Minimize the time
the desiccator is open because moist air may enter and be absorbed by the samples. Some samples
may be stronger desiccants than those used in the desiccator and may take on water.
4.3 Residues dried at 103° C to 105° C may retain some bound water as water of crystallization or as
water occluded in the interstices of crystals. They lose CO2 in the conversion of bicarbonate to
carbonate. The residues usually lose only slight amounts of organic matter by volatilization at this
temperature. Because removal of occluded water is marginal at this temperature, attainment of
constant weight may be very slow.
4.4 Results for residues high in oil or grease may be questionable because of the difficulty of drying to
constant weight in a reasonable time.
4.5 The determination of total solids is subject to negative error due to loss of ammonium carbonate
and volatile organic matter during the drying step at 103°C to 105°C. Carefully observe specified
ignition time and temperature to control losses of volatile inorganic salts if these are a problem.
5.0 Safety
5.1 Refer to Section 5.0 of Method 1682 for safety precautions.
6.0 Equipment and Supplies
Note: Brand names, suppliers, and part numbers are cited for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using equipment and materials
other than those specified here, but demonstration of equivalent performance that meets the
requirements of this method is the responsibility of the laboratory.
6.1 Evaporating Dishes-Dishes of 100-mL capacity. The dishes may be made of porcelain (90-mm
diameter), platinum, or high-silica glass.
6.2 Watch glass-Capable of covering the evaporating dishes (Section 6.1).
6.3 Steam bath.
6.4 Desiccator-Moisture concentration in the desiccator should be monitored by an instrumental
indicator or with a color-indicator desiccant.
6.5 Drying oven-Thermostatically-controlled, capable of maintaining a uniform temperature of 103 °C
to 105°C throughout the drying chamber.
6.6 Analytical balance-Capable of weighing to 0.1 mg for samples having a mass up to 200 g.
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6.7 Container handling apparatus-Gloves, tongs, or a suitable holder for moving and handling hot
containers after drying.
6.8 Bottles-Glass or plastic bottles of a suitable size for sample collection
6.9 Rubber gloves (Optional)
6.10 No. 7 Cork borer (Optional)
7.0 Reagents and Standards
7.1 Reagent water-Deionized, distilled, or otherwise purified water.
7.2 Sodium chloride-potassium hydrogen phthalate standard (NaCl-KHP).
7.2.1 Dissolve 0.10 g sodium chloride (NaCl) in 500 mL reagent water. Mix to dissolve.
7.2.2 Add 0.10 g potassium hydrogen phthalate (KHP) to the NaCl solution (Section 7.2.1) and
mix. If the KHP does not dissolve readily, warm the solution while mixing. Dilute to 1 L
with reagent water. Store at 4°C. Assuming 100% volatility of the acid phthalate ion, this
solution contains 200 mg/L total solids, 81.0 mg/L volatile solids, and 119 mg/L fixed
solids.
8.0 Sample Collection, Preservation, and Storage
8.1 Use resistant-glass or plastic bottles to collect sample for solids analysis, provided that the material
in suspension does not adhere to container walls. Sampling should be done in accordance with
Reference 13.2. Begin analysis as soon as possible after collection because of the impracticality of
preserving the sample. Refrigerate the sample at 4°C up to the time of analysis to minimize
microbiological decomposition of solids. Preferably do not hold samples more than 24 hours.
Under no circumstances should the sample be held more than seven days. Bring samples to room
temperature before analysis.
9.0 Quality Control
9.1 Quality control requirements and requirements for performance-based methods are given in Section
9.0 of Method 1682.
9.2 Initial demonstration of laboratory capability - The initial demonstration of laboratory capability is
used to characterize laboratory performance and method detection limits.
9.2.1 Method detection limit (MDL) - The method detection limit should be established for total
solids using diluted NaCl-KHP standard (Section 7.2). To determine MDL values, take
seven replicate aliquots of the diluted NaCl-KHP solution and process each aliquot
through each step of the analytical method. Perform all calculations and report the
concentration values in the appropriate units. MDLs should be determined every year or
whenever a modification to the method or analytical system is made that will affect the
method detection limit.
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9.2.2 Initial Precision and Recovery (IPR) - To establish the ability to generate acceptable preci-
sion and accuracy, the analyst shall perform the following operations:
9.2.2.1 Prepare four samples by diluting NaCl-KHP standard (Section 7.2) to 1-5
times the MDL. Using the procedures in Section 11, analyze these samples for
total solids.
9.2.2.2 Using the results of the four analyses, compute the average percent recovery
(x) and the standard deviation (s, Equation 1) of the percent recovery for total
solids.
Equation 1
Is-2-
c. 1
(I-2)
n
\ n-\
Where:
n = number of samples
x = % recovery in each sample
s = standard deviation
9.2.2.3 Compare s and x with the corresponding limits for initial precision and
recovery in Table 2 (to be determined in validation study). If s and x meet the
acceptance criteria, system performance is acceptable and analysis of samples
may begin. If, however, s exceeds the precision limit or x falls outside the
range for recovery, system performance is unacceptable. In this event, correct
the problem, and repeat the test.
9.3 Laboratory blanks
9.3.1 Prepare and analyze a laboratory blank initially (i.e. with the tests in Section 9.2) and with
each analytical batch. The blank must be subjected to the same procedural steps as a
sample, and will consist of approximately 25 g of reagent water.
9.3.2 If material is detected in the blank at a concentration greater than the MDL (Section 1.3),
analysis of samples must be halted until the source of contamination is eliminated and a
new blank shows no evidence of contamination. All samples must be associated with an
uncontaminated laboratory blank before the results may be reported for regulatory compli-
ance purposes.
9.4 Ongoing Precision and Recovery
9.4.1 Prepare an ongoing precision and recovery (OPR) solution identical to the IPR solution
described in Section 9.2.2.1.
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9.4.2 An aliquot of the OPR solution must be analyzed with each sample batch (samples started
through the sample preparation process (Section 11) on the same 12-hour shift, to a
maximum of 20 samples).
9.4.3 Compute the percent recovery of total solids in the OPR sample.
9.4.4 Compare the results to the limits for ongoing recovery in Table 2 (to be determined in
validation study). If the results meet the acceptance criteria, system performance is
acceptable and analysis of blanks and samples may proceed. If, however, the recovery of
total solids falls outside of the range given, the analytical processes are not being
performed properly. Correct the problem, reprepare the sample batch, and repeat the OPR
test. All samples must be associated with an OPR analysis that passes acceptance criteria
before the sample results can be reported for regulatory compliance purposes.
9.4.5 Add results that pass the specifications in Section 9.4.4 to IPR and previous OPR data.
Update QC charts to form a graphic representation of continued laboratory performance.
Develop a statement of laboratory accuracy for each analyte by calculating the average
percent recovery (R) and the standard deviation of percent recovery (SR). Express the
accuracy as a recovery interval from R-2SR to R+2SR. For example, if R=05% and
SR=5%, the accuracy is 85-115%.
9.5 Duplicate analyses
9.5.1 Ten percent of samples must be analyzed in duplicate. The duplicate analyses must be
performed within the same sample batch (samples whose analysis is started within the
same 12-hour period, to a maximum of 20 samples).
9.5.2 The total solids of the duplicate samples must be within 10%.
10.0 Calibration and Standardization
10.1 Calibrate the analytical balance at 2 mg and 1000 mg using class "S" weights.
10.2 Calibration shall be within ± 10% (i.e. ±0.2 mg) at 2 mg and ± 0.5% (i.e. ±5 mg) at 1000 mg. If
values are not within these limits, recalibrate the balance.
11.0 Procedure
11.1 Preparation of evaporating dishes-Heat dishes and watch glasses at 103°C to 105°C for 1 hour in
an oven. Cool and store the dried equipment in a desiccator. Weigh each dish and watch glass prior
to use (record combined weight as "Wdlsh").
11.2 Preparation of samples
11.2.1 Fluid samples-If the sample contains enough moisture to flow readily, stir to homogenize,
place a 25 to 50 g sample aliquot on the prepared evaporating dish. If the sample is to be
analyzed in duplicate, the mass of the two aliquots may not differ by more than 10%.
Spread each sample so that it is evenly distributed over the evaporating dish. Evaporate the
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Method 1682 - Draft
samples to dryness on a steam bath. Cover each sample with a watch glass, and weigh
(record weight as "Wsample").
Note: Weigh wet samples quickly because wet samples tend to lose weight by evaporation.
Samples should be weighed immediately after aliquots are prepared.
11.2.2 Solid samples-If the sample consists of discrete pieces of solid material (dewatered
biosolid, for example), take cores from each piece with a No. 7 cork borer or pulverize the
entire sample coarsely on a clean surface by hand, using rubber gloves. Place a 25 to 50 g
sample aliquot of the pulverized sample on the prepared evaporating dish. If the sample is
to be analyzed in duplicate, the mass of the two aliquots may not differ by more than 10%.
Spread each sample so that it is evenly distributed over the evaporating dish. Cover each
sample with a watch glass, and weigh (record weight as "Wsample").
11.3 Dry the samples at 103 °C to 105 °C for a minimum of 12 hours, cool to balance temperature in an
individual desiccator containing fresh desiccant, and weigh. Heat the residue again for 1 hour, cool
it to balance temperature in a desiccator, and weigh. Repeat this heating, cooling, desiccating, and
weighing procedure until the weight change is less than 5% or 50 mg, whichever is less. Record the
final weight as "Wtotal."
Note: It is imperative that dried samples be weighed quickly since residues often are very
hygroscopic and rapidly absorb moisture from the air. Samples must remain in the dessicator
until the analyst is ready to weigh them.
12.0 Data Analysis and Calculations
12.1 Calculate the % solids or the mg solids/kg biosolid for total solids (Equation 2).
Equation 2
W -W
% total solids = —^—-^- * 100
W — W
''sample ''dish
or
mg total solids Wtotal - Wdish
kg sludge W,-Wd
* 1,000,000
sample dish
Where:
Wdlsh=Weightofdish (mg)
Wsampk=Weight of wet sample and dish (mg)
Wtotal=Weight of dried residue and dish (mg)
12.2 Sample results should be reported as % solids or mg/kg to three significant figures. Report results
below the ML as < the ML, or as required by the permitting authority or in the permit.
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13.0 Method Performance
13.1 Method performance (MDL and quality control acceptance criteria) will be determined during the
multi-lab validation of this method.
13.2 Total solids duplicate determinations must agree within 10% to be reported for permitting
purposes. If duplicate samples do not meet this criteria, the problem must be discovered and the
sample must be run over.
14.0 Pollution Prevention
14.2 Pollution prevention details are given in Section 14 of Method 1682.
15.0 Waste Management
15.1 Waste management details are given in Section 15 of Method 1682.
16.0 References
16.1 "Standard Methods for the Examination of Water and Wastewater," 18th ed. and later revisions,
American Public Health Association, 1015 15th Street NW, Washington, DC 20005. 1-35: Section
1090 (Safety), 1992.
16.2 U.S. Environmental Protection Agency, 1992. Control of Pathogens and Vector Attraction in
Sewage Sludge. Publ 625/R-92/013. Office of Research and Development, Washington, DC.
17.0 Tables, Diagrams, Flowcharts, and Validation Data
17.1 Tables containing method requirements for QA/QC will be added after the validation study has
been performed.
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