v>EPA
United States
Environmental Protection
Agency
Assessment of the Effects of Holding
Time on Fecal Coliform and Salmonella
Concentrations in Biosolids
August 2006
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-07-003
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Acknowledgments
This report was prepared by the CSC Microbiology and Biochemistry Studies Group under the direction
of Robin K. Oshiro, of the Office of Science and Technology's Engineering and Analysis Division within
the U.S. Environmental Protection Agency (EPA's) Office of Water.
The contributions of the following volunteer participants and organizations to this study are gratefully
acknowledged:
• A & L Great Lakes Laboratories: Julie Speelman
• Albuquerque Water Utility Authority - Water Quality Laboratory: Steve Glass, Bill Lindberg,
Lauren Tapps
• Alexandria Sanitation Authority: John Benard, Leulu M. Gebremedhin, Lisa Racey, Paul
Carbary, George Floyd
• Amtest Laboratories: Kathy Fugiel, Neila Glidden, Melinda Woomer
• Analytica International: Patryce McKinney, Danielle Carville
• Analytical Laboratories, Incorporated: Sandy Koch, Robert Voermans, Lynn Murray
• Barton Laboratory, Jefferson County Commission: J. Lynn King, Henry Word, Ronstead
Claughton, Don Lovell, Bob Spigner
• Bay County Laboratory Services Division: Carol Monti, Anna Wright
• Central Valley Water Reclamation Facility: Anthony Daw, Lynn Cecil, Dwaine Funk
• City of Everett Water Pollution Control Facility: Jeff Wright, Tim Rickman
• City of Los Angeles, Bureau of Sanitation, Environmental Monitoring Division: Gerald
McGowen, Stan Asato, loannice Lee, Hung Pham, Pauline Nguyen, Marieta Ravelo
• County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant - Water
Quality Laboratory (JWPCP-WQL): Kathy Walker, Debra Leachman, Mark Patterson
• Edge Analytical: Larry Henderson, Kent Oostra, Shannon Kizer
• Energy Laboratories, Incorporated - Casper Branch: Sheryl Garling, N. Lou Miller, Sherri L.
Boatman, Randy Ogden
• Environmental Protection Agency, National Risk Management Research Laboratory: Mark
Meckes, Laura Boczek, Cliff Johnson
• Environmental Science Corporation: Rodney Shinbaum, Kim Johnson, Rachel Freeman
• The Industrial Laboratories Company, Incorporated: Tania Vogel, Geoff Henderson, Lenka
Teodorovic
• Katahdin Analytical Services, Incorporated: Deborah Nadeau, Shelly Brown, Greg Lull, Amy
Broadbent, Jane April, Mandi Greenleaf
• King County Environmental Laboratory: Kate Leone, Despina Strong, Colin Elliot, Joe Calk,
Tami Alley, Bobbie Anderson, Karl Bruun, Eyob Mazengia, Robin Revelle, Debbie Turner,
Jodeen Wieser
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Louisville and Jefferson County Metropolitan Sewer District: Zonetta E. English, Robin R.
Burch, Cliff Wilson
Madison Metropolitan Sewerage District: Kurt Knuth, Montgomery Baker, Kris Farrar, Carol
Mielke
Monroe County Environmental Laboratory at the Frank E. VanLare Waste Treatment Plant:
Drew Smith, Mary Merner, Dave Spanganberg, Stephen Bland
Nova Biologicals, Incorporated: Paul Pearce, Brenda Bates, Donna Reioux, Amber Sutton
Orange County Utilities Central Laboratory: Terri Slifko, Shelley Patterson, Vanessa Perez, Scott
Rampenthal, Theresa Slifko
St. George Regional Water Reclamation Facility Laboratory: Leslie Wentland, Amy Howe
SVL Analytical, Incorporated - Microbiology Laboratory: Linda Johann
Universal Laboratories: Carol Kleemeier, Stacie Splinter, Linda McFarland
Wichita Water and Sewer Wastewater Laboratory: Becky Gagnon, Karen Roberts
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Disclaimer
This document has been reviewed and approved by the EPA/EAD. Mention of company names, trade
names, or commercial products does not constitute endorsement or recommendation for use.
Questions concerning this report 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
202.566.1053 (facsimile)
in
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Table of Contents
Section 1.0 Introduction 1
1.1 Summary of EPA Method 1680 for Fecal Coliforms 1
1.2 Summary of EPA Method 1681 for Fecal Coliforms 1
1.3 Summary of EPA Method 1682 for Salmonella 1
Section 2.0 Study Objectives 2
Section 3.0 Technical Approach 3
3.1 Identification of Laboratories 3
3.2 Sample Collection, Storage Conditions, and Holding Times 3
3.3 Methods 3
3.4 Study Design and Analyses 3
3.5 Quality Control (QC) Analyses 4
Section 4.0 Study Implementation 6
4.1 Study Management 6
4.2 Study Schedule 6
4.3 Participant Laboratories 6
Section 5.0 Data Reporting and Validation 8
5.1 Data Reporting 8
5.2 Data Validation 8
5.3 Censored Data 9
Section 6.0 Results, Data Analysis, and Discussion 10
6.1 Fecal Coliform (Methods 1680/1681) Holding Time Study Results for Class A and Class
B Biosolid Samples 10
6.2 Salmonella (Method 1682) Holding Time Study Results for Biosolid Samples 14
Section 7.0 Conclusions 16
Section 8.0 References 17
Section 9.0 Acronyms 18
IV
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List of Tables
Table 1. Study Analysis Summary 3
Table 2. Positive and Negative Control Cultures 5
Table 3. Laboratories Participating in the Biosolids Holding Time Study 7
Table 4. Fecal Coliform Mean Results (MPN/g dry weight) for Alkaline-Stabilized Samples 12
Table 5. Fecal Coliform Mean Results (MPN/g dry weight) for Composted Samples 12
Table 6. Fecal Coliform Mean Results (MPN/g dry weight) for Heat Dried Samples 13
Table 7. Fecal Coliform Mean Results (MPN/g dry weight) for Thermophilically Digested Samples.. 13
Table 8. Fecal Coliform Mean Results (MPN/g dry weight) for Aerobically Digested Samples 13
Table 9. Fecal Coliform Mean Results (MPN/g dry weight) for Anaerobically Digested Samples 14
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List of Appendices
Appendix A: Laboratory Capabilities Checklist
Appendix B: Instructions for the Biosolids Holding Time Study for Methods 1680/1681 (Class A)
Appendix C: Instructions for the Biosolids Holding Time Study for Methods 1680/1681 (Class B)
Appendix D: Instructions for the Biosolids Holding Time Study for Method 1682
Appendix E: Fecal Coliform Spiking Protocol (Class A)
Appendix F: Fecal Coliform Spiking Protocol (Class B)
Appendix G: Salmonella Spiking Protocol
Appendix H: Alkaline-Stabilized Matrix Instructions
VI
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Executive Summary
This report presents the results of the U.S. Environmental Protection Agency's (EPA's) biosolids holding
time study (the "Study") of most probable number (MPN) procedures for the analysis of fecal coliform
(using EPA Methods 1680 and 1681) concentrations in Class A and Class B matrices and Salmonella
(using EPA Method 1682) concentrations in Class A matrices.
The purpose of this study was to determine whether biosolid samples can be held for 24 hours without
significant change in bacterial densities for these analytes.
Since historical data indicated that ambient concentrations of fecal coliforms and Salmonella were not
present in most of the Class A matrices (alkaline-stabilized, heat dried, and thermophilically digested)
evaluated, fecal coliform and Salmonella samples were spiked. However, it was not necessary to spike
most of the compost matrices analyzed for fecal coliforms because there were a sufficient number of
ambient fecal coliforms to assess the effect of holding time. Spiking was not necessary for Class B
matrices (aerobically digested, anaerobically digested) evaluated during the Study. Samples were
analyzed at 6 and 24 hours after sample collection or spiking.
Four hundred laboratories were contacted for potential study participation in an effort to obtain six or
more useable, valid datasets for every matrix/method combination. A number of reasons, including, but
not limited to, limited production of some matrices, laboratory proximity to biosolids facilities,
scheduling conflicts etc., resulted in limited data for some matrix/method combinations.
Fecal Coliforms
Based on the results of this study, Class A composted, Class B aerobically digested, and Class B
anaerobically digested matrices analyzed for fecal coliforms by Methods 1680 and 1681 may be analyzed
24 hours after sample collection and still generate data comparable to those generated at 6 hours after
sample collection.
All other matrices analyzed for fecal coliforms using Methods 1680 and 1681 should not exceed a 6-hour
maximum sample transport holding time, and must be processed within 2 hours of receipt at the
laboratory. The data for these matrix/method combinations generally showed a tendency to decrease with
extended sample holding times.
Salmonella
Based on the results of this study and the limited data generated, Class A matrices analyzed for
Salmonella using Method 1682 may not be analyzed beyond 6 hours after sample collection and still
generate data comparable to those generated at 6 hours after sample collection. In general, Salmonella
concentrations had a tendency to decrease with extended sample holding times.
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
SECTION 1.0 INTRODUCTION
Land application of biosolids is a critical component of solid waste management. Under Subpart D of
Title 40 Code of Federal Regulations (CFR) Part 503, sewage sludges (biosolids) are required to be
processed prior to land application in order to minimize pathogen levels and the potential public health
risks associated with contact or exposure. Subpart D further defines and classifies sewage sludge
(biosolids) for land application purposes based on pathogen concentrations. EPA has validated Methods
1680 (Reference 8.1) and 1681 (Reference 8.2) for fecal coliforms in Class A and B biosolids and Method
1682 (Reference 8.3) for Salmonella in Class A biosolids to support monitoring requirements. The
objective of this study was to determine whether biosolid samples being analyzed for fecal coliforms and
Salmonella could be held for 24 hours prior to sample analysis without a significant change in bacterial
concentrations.
Holding time. Bacterial analysis of non-potable waters (i.e., wastewater) for compliance monitoring
requires that sample analysis begin within 6 hours of sample collection (40 CFR part 136.3, Table II).
The present study addresses the effect of holding time on fecal coliform and Salmonella concentrations in
biosolids. Samples were analyzed at 6 and 24 hours after sample collection or sample spiking.
1.1 Summary of EPA Method 1680 for Fecal Coliforms
Fecal coliforms were evaluated in Class A and Class B biosolids using Method 1680 (Reference 8.1).
Biosolid samples are homogenized and inoculated into lauryl tryptose broth (LTB) a presumptive
medium. Following incubation at 35.0°C ± 0.5°C for 24 ± 2 and 48 ± 3 hours, growth from positive tubes
is transferred to EC broth (confirmatory medium) and incubated at 44.5°C ± 0.2°C for 24 ± 2 hours. All
tubes exhibiting turbidity and gas production are considered positive for fecal coliforms.
1.2 Summary of EPA Method 1681 for Fecal Coliforms
Fecal coliforms were evaluated in Class A and Class B biosolids using Method 1681 (Reference 8.2).
Biosolid samples are homogenized and inoculated into A-l medium and incubated at 44.5°C ± 0.2°C for
24 ± 2 hours. All tubes exhibiting turbidity and gas production are considered positive for fecal
coliforms.
1.3 Summary of EPA Method 1682 for Salmonella
Salmonella were evaluated in Class A matrices using Method 1682 (Reference 8.3). The MPN method
requires enrichment, followed by selection and biochemical/serological confirmation. Biosolid samples
are homogenized and inoculated into tryptic soy broth (TSB) an enrichment medium. After incubation,
TSB is spotted onto selective modified semisolid Rappaport-Vassiliadis (MSRV) medium.
Presumptively identified colonies from MSRV are streaked onto xylose-lysine desoxycholate agar (XLD)
for isolation. Biochemical confirmation includes lysine-iron agar (LIA), triple sugar iron
agar (TSI), and urease test medium, followed by serological confirmation using polyvalent O antisera.
August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
SECTION 2.0 STUDY OBJECTIVES
The following study objective was established for the holding time study:
• Evaluate the effect of holding time across multiple laboratories and biosolid matrices on fecal
coliform concentrations using EPA Methods 1680 and 1681 and on Salmonella concentrations
using EPA Method 1682.
To accomplish these objectives, qualified volunteer laboratories analyzed unspiked and/or spiked biosolid
samples for fecal coliforms and Salmonella at 6 and 24 hours from either sample collection or sample
spiking.
The following data quality objective was established for this study:
• Data produced under this study must be generated according to the analytical and QA/QC
procedures as described in each of the analytical methods or approved changes to these
procedures in order to ensure that data will be of known and reliable quality.
August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
SECTION 3.0 TECHNICAL APPROACH
3.1
Identification of Laboratories
Participant laboratories were representative of the general user community, with experience analyzing
biosolid samples for bacteria and had access to representative biosolid matrices. Because the study
assessed holding times, laboratories were located within driving distance (2 hours) of the facility
providing the biosolid sample.
To reduce cost, volunteer laboratories were recruited, from a pool of 400 laboratories contacted for
potential participation in the study. To reduce the burden on the participant laboratories and encourage
volunteer participants, EPA provided the media, reagents, and disposable supplies required for the study.
3.2 Sample Collection, Storage Conditions, and Holding Times
A single bulk sample of at least 1000 g was collected and transported to the laboratory on ice and
maintained at <10°C and above freezing. At the laboratory, the bulk sample was split into replicates, and
spiked (if necessary), and stored in the refrigerator at <10°C and above freezing. After 6 and 24 hours
from sample collection or spiking, replicates were analyzed by the appropriate procedure (Table 1).
3.3 Methods
The holding time for fecal coliforms was evaluated in both Class A and Class B matrices using Methods
1680 and 1681 and Class A matrices for Salmonella using Method 1682.
3.4 Study Design and Analyses
During the holding time study, all three methods (1680, 1681, and 1682) were used to analyze unspiked
and spiked samples at multiple laboratories. Table 1 summarizes the number and type of samples that
were evaluated during the holding time study to meet the objectives listed in Section 2.0.
Table 1. Study Analysis Summary
Matrix
Alkaline- stabilized
Compost
Aerobically digested
Anaerobically digested
Heat Dried
Thermophilically digested
Analyte
fecal coliforms
Salmonella
fecal coliforms
Salmonella
fecal coliforms
fecal coliforms
fecal coliforms
fecal coliforms
Method
1680/1681
1682
1680/1681
1682
1680/1681
1680/1681
1680/1681
1680/1681
No. of Replicates per Holding Time1
6 hours
10
10
10
10
10
10
24 hours
10
10
10
10
10
10
Background levels of fecal coliform bacteria were analyzed during preliminary analyses to determine if spiking was
necessary. All matrices analyzed for Salmonella were spiked.
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
3.4.1 Preliminary Analyses
Preliminary analyses were conducted on unspiked and spiked Class A and Class B biosolid matrices, as
appropriate, prior to the start of the holding time study. The time between preliminary and holding time
study analyses was used to resolve issues that were observed during preliminary analyses. In addition,
preliminary analyses results were used to determine whether spiking was necessary. Holding time
samples were spiked if the ambient fecal coliform concentrations were < 300 MPN/ g dry weight.
3.4.2 Fecal Coliform Sample Holding Time Analyses
Aerobically digested, alkaline-stabilized, anaerobically digested, compost, heat-dried, and
thermophilically digested matrices were analyzed according to Methods 1680 and 1681 and holding time
study instructions (Appendices B and C), as appropriate.
Unspiked Holding Time Samples and QC Samples. A single bulk sample was collected and transported
to the laboratory on ice. The bulk sample was split into 20, 30 g replicates at the laboratory. Sixteen
replicates remained unspiked (8 per holding time) and four replicates were spiked (2 per holding time)
with laboratory-prepared Escherichia coli (ATCC #25922) spiking suspension according to the fecal
coliform spiking procedure (Appendix E). Samples were immediately refrigerated after spiking at <10°C
and above freezing. After 6 and 24 hours from sample collection, 10 replicates were removed from the
refrigerator and analyzed according to Method(s) 1680 and/or 1681.
Spiked Holding Time Samples and QC Samples. A single bulk sample was collected and transported to
the laboratory on ice. The bulk sample was split into 20, 30 g replicates at the laboratory. Four replicates
remained unspiked (2 per holding time) and sixteen replicates were spiked (8 per holding time) with
laboratory-prepared Escherichia coli (ATCC #25922) spiking suspension according to the fecal coliform
spiking procedure (Appendix E). Samples were immediately refrigerated after spiking at <10°C and
above freezing. After 6 and 24 hours from sample collection, 10 replicates were removed from the
refrigerator and analyzed according to Method(s) 1680 and/or 1681.
3.4.3 Salmonella Sample Holding Time Analyses
Compost and alkaline-stabilized matrices were analyzed according to Method 1682 and Salmonella
holding time study instructions (Appendix D). For each matrix, a single bulk sample was collected and
transported to the laboratory on ice. The bulk sample was split into 20, 30 g replicates at the laboratory.
Four replicates remained unspiked (2 per holding time) and sixteen replicates (8 per holding time) were
spiked with laboratory-prepared Salmonella typhimurium (ATCC #14028) spiking suspension according
to the Salmonella spiking procedure (Appendix G). Samples were immediately refrigerated after spiking
at <10°C and above freezing. After 6 and 24 hours from sample spiking, 10 replicates were removed
from the refrigerator and analyzed according to Method 1682.
3.5 Quality Control (QC) Analyses
Participating laboratories completed the following QC requirements: media sterility checks, dilution water
sterility checks, blender jar sterility check, method blanks, positive controls, and negative controls.
Table 2 summarizes the positive and negative control cultures used during the study.
August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
Table 2. Positive and Negative Control Cultures
Method
1680/1681
1680
1680/1681
1682
1682
Medium or Test
EC, A-1
LIB
mEndo
MSRV, XLD, TSI, LIA, Polyvalent O
Urease
Positive Control
Escherichia coli
Escherichia coli
Escherichia coli
Salmonella typhimuriurm
Proteus vulgaris
Negative Control
Enterobacter aerogenes
Pseudomonas aeruginosa
Enterobacter aerogenes
Escherichia coli
Salmonella typhimurium
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
SECTION 4.0 STUDY IMPLEMENTATION
4.1 Study Management
This study was designed under the direction of the Office of Science and Technology, Engineering and
Analysis Division within the U.S. Environmental Protection Agency's (EPA's) Office of Water (OW).
The EPA technical lead was Robin K. Oshiro. Coordination of activities for the Study was performed by
the CSC Microbiology and Biochemistry Studies Group.
4.2 Study Schedule
Fecal coliform analyses were conducted between February 2006 and August 2006. Salmonella analyses
were conducted between May 2006 and August 2006. All laboratories that analyzed biosolid matrices for
Salmonella elected to complete fecal coliform analyses prior to moving on to the Salmonella analyses.
Prior to analyzing holding time study samples each laboratory was required to analyze four initial
precision and recovery (IPRs) samples, one unspiked reference matrix sample, and one matrix spike (MS)
sample to demonstrate method proficiency. Some laboratories repeated IPR analyses due to problems
enumerating the spiking suspension using the spread plate technique. In addition, some of the
laboratories had to repeat the holding time analyses due to fluctuating ambient fecal coliform
concentrations to obtain uncensored data. Discussion of censored data is provided in Section 5.3.
4.3 Participant Laboratories
The 28 participating laboratories involved in the Study are shown in Table 3, below. No endorsement of
these laboratories is implied, nor should any be inferred. Participant laboratories were randomly assigned
numbers for purposes of presenting data in this report.
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
Table 3. Laboratories Participating in the Biosolids Holding Time Study
A&L Great Lakes Laboratories
Julie Speelman
3505 Conestoga Drive, Fort Wayne, IN 46808
Albuquerque Water Utility Authority
Water Quality Laboratory
Lauren Tapps
4201 2nd Street, S.W., Albuquerque, NM 87105-0511
Alexandria Sanitation Authority
John Benard, Leulu M. Gebremedhin, Lisa Racey,
Paul Carbary, George Floyd
1500 Eisenhower Avenue, Alexandria, VA22314
Amtest Laboratories
KathyFugiel, Neila Glidden, Melinda Woomer
14603 NE 87th Street, Redmond, WA 98052
Analytica International
Patryce McKinney, Danielle Carville
3330 Industrial Avenue, Fairbanks, AK 99701
Analytical Laboratories, Incorporated
Sandy Koch, Robert Voermans, Lynn Murray
1804 North 33rd Street, Boise, ID 83703-5814
Barton Laboratory, Jefferson County Commission
J. Lynn King, Henry Word, Ronstead Claughton, Don
Lovell, Bob Spigner
1290 Oak Grove Road, Homewood, AL 35209-6961
Bay County Laboratory Services Division
Carol Monti, Anna Wright
3420 Transmitter Road, Panama City, FL 32404
Central Valley Water Reclamation Facility
Anthony Daw, Lynn Cecil, Dwaine Funk
800 West Central Valley Road
Salt Lake City, UT 84119-3379
City of Everett Water Pollution Control Facility
Jeff Wright, Tim Rickman
4027 4th Street S.E., Everett, WA 98205
City of Los Angeles, Bureau of Sanitation,
Environmental Monitoring Division
Gerald McGowen, Stan Asato, loannice Lee, Hung Pham,
Pauline Nguyen, Marieta Ravelo
12000 Vista del Mar, 452 Playa del Ray, CA 90293
County Sanitation Districts of Los Angeles County
(JWPCP)
Kathy Walker, Debra Leachman, Mark Patterson
24501 S. Figueroa Street, Carson, CA 90745
Edge Analytical
Larry Henderson, Kent Oostra, Shannon Kizer
805 West Orchard Drive, Suite 4, Bellingham, WA 98225
Energy Laboratories, Incorporated - Casper Branch
Sheryl Garling, N. Lou Miller, Sherri Boatman, Randy
Ogden
2393 Salt Creek Highway, Casper, WY 82601
Environmental Protection Agency, National Risk
Management Research Laboratory
MarkMeckes, Laura Boczek, Cliff Johnson
26 West Martin Luther King Drive, Cincinnati, OH 45268
Environmental Science Corporation
Rodney Shinbaum, Kim Johnson, Rachael Freeman
12065 Lebanon Road, Mt. Juliet, TN 37122
The Industrial Laboratories Company, Incorporated
Tania Vogel, Geoff Henderson, Lenka Teodorovic
4046 Youngfield Street, Wheat Ridge, CO 80033
Katahdin Analytical Services, Incorporated
Deborah Nadeau, Shelly Brown, Greg Lull, Amy Broadbent,
Jane April, Mandi Greenleaf
340 County Road #5, Westbrook, ME 04092
King County Environmental Laboratory
Kate Leone, Despina Strong, Colin Elliot, Joe Calk, Tami Alley,
Bobbie Anderson, Karl Bruun, Eyob Mazengia, Robin Revelle,
Debbie Turner, Jodeen Wieser
322 West Ewing Street, Seattle, WA 981 19-1507
Louisville and Jefferson County Metropolitan Sewer
District
Zonetta E. English, Robin R. Burch, Cliff Wilson
4522 Algonquin Parkway, Louisville, KY 40211
Madison Metropolitan Sewerage District
Kurt Knuth, Montgomery Baker, Kris Farrar, Carol Mielke
1610 Moorland Road, Madison, Wl 53713
Monroe County Environmental Laboratory at the Frank E.
VanLare Waste Treatment Plant
Drew Smith, Mary Merner, Dave Spanganberg, Stephen Bland
1574 Lakeshore Blvd, Admin Bldg #7, Rochester, NY 14617
Nova Biologicals, Incorporated
Paul Pearce, Brenda Bates, Donna Reioux, Amber Sutton
1775 East Loop 336, Suite 4, Conroe, TX 77301
Orange County Utilities Central Laboratory
Terri Slifko, Shelley Patterson, Vanessa Perez, Scott
Rampenthal, Theresa Slifko
9124 Curry Ford Road, Orlando, FL 32825
St. George Regional Water Reclamation Facility Laboratory
Leslie Wentland, Amy Howe
3780 South 1550 West, St. George, UT 84790
SVL Analytical, Incorporated - Microbiology Laboratory
Linda Johann
2195 Ironwood Court, Suite C, Coeur d'Alene, ID 83814
Universal Laboratories
Carol Kleemeier, Stacie Splinter, Linda McFarland
20 Research Drive, Hampton, VA 23666
Wichita Water and Sewer Wastewater Laboratory
Becky Gagnon, Karen Roberts
2305 East 57th Street South, Wichita, KS 67216
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
SECTION 5.0 DATA REPORTING AND VALIDATION
5.1 Data Reporting
Laboratories submitted the following data to CSC for review and validation:
• Completed cover sheet with sample collection and QC information
• Completed sample-specific reporting forms
• Documentation of any additional information that would assist in evaluating the data
5.2 Data Validation
Data review checklists were used to ensure that each data package was complete and to ensure that each
sample result met the study-specific and method-specific requirements. Items reviewed for each sample
included the following:
• Confirmation that original forms were submitted
• Confirmation that all QC checks were performed and exhibited the appropriate response
• Confirmation that all method-specific incubation times and temperatures were met
• Confirmation that all media and reagents were used within expiration dates
• Confirmation that samples were spiked with appropriate dilution
• Confirmation that all calculations were correct
This process was performed independently by two data reviewers, each of whom entered the results into
separate spreadsheets designed for data review and validation for this study. The results were compared
to verify consistency and identify potential data entry errors.
The following issues were encountered during laboratory analyses:
Laboratory 12 (Method 1680, Class B, aerobically digested): Did not meet the QC acceptance criteria for
OPR and MS samples during the holding time study, which may have been due to TSA enumeration
difficulties, which likely resulted in underestimating the spike concentration. TSA counts were
considerably lower compared to other laboratories, and Laboratory 12 results obtained during preliminary
analyses. As a result, data was considered invalid and not included in subsequent data analyses.
Laboratory 16 (Method 1680, Class A, compost): Did not meet OPR and matrix spike (MS) criteria. As
a result, data from Laboratory 16 was considered invalid and not included in subsequent data analyses.
Laboratory 17 (Method 1680, Class B, anaerobic): During the week of Class B sample analyses, the
laboratory spiked OPR and MS samples according to the Class A spiking instructions, resulting in
censored values for the OPRs (less-than values) and 0 percent recoveries for matrix spike samples. Since
the laboratory failed to meet the Class B OPR and MS criteria as a result of spiking according to Class A
spiking instructions, data was considered invalid and not included in subsequent data analyses.
Laboratory 23 (Method 1681, Class A, compost): Did not meet OPR and matrix spike (MS) criteria;
recoveries for both OPR and MS samples were less than 1 percent. In addition, most of the analytical
results observed during the holding time study were censored (less-than) values. As a result, data from
Laboratory 23 was considered invalid and not included in subsequent data analyses.
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
Most laboratories were not familiar with the reference matrix (Milorganite®) which resulted in some
laboratories not meeting IPR criteria for Methods 1680 and 1681 during preliminary analyses. These
laboratories generally experienced difficulties homogenizing the Milorganite® samples. For samples
being analyzed by Method 1681, excessive foaming during homogenization made it difficult to interpret
A-l results. As a result of the expedited study schedule, some laboratories moved forward with the
holding time study, despite having failed to meet IPR criteria as specified by the method. However,
unless specifically noted in this section all of the laboratories that failed the IPR criteria did meet the OPR
criteria during the holding time study. While it would have been preferable for the laboratories to repeat
the IPR analyses prior to moving forward with the holding time study, it was not possible due to
scheduling constraints, and since the laboratories met OPR criteria, data from these laboratories was
considered valid and included in subsequent data analyses.
Laboratory 6 (Methods 1680 and 1681, Class A alkaline-stabilized): As expected, fecal coliform results
for all of the replicates analyzed at 6 and 24 hours were censored (less-than) values, because samples
were not neutralized prior to spiking. Although results were censored, this data was included in
subsequent data analyses.
Laboratory 1: A-l (Method 1681) and MSRV (Method 1682) media was expired (less-than 5 days
beyond expiration date); all controls exhibited the appropriate response so data was included in
subsequent data analyses.
Laboratory 2: EC tubes (Method 1680) were read 48 hours after incubation (instead of being read at 24
and 48 hours), results were considered valid and included in subsequent data analyses.
Laboratory 13 (Method 1681, compost): Did not analyze MS samples during the holding time study.
However, the laboratory did analyze MS samples during the week of preliminary analyses, and because
the laboratory analyzed less than 20 matrix samples during the study, the laboratory met the method-
specific MS criteria. Although the laboratory did not meet the study-specific requirements for
performance of MS samples during the week of the holding time study, data from this laboratory was
considered valid and included in subsequent data analyses because method-specific QC criteria were met.
Laboratory 19 (Method 1681, Class B, anaerobically digested): Both OPR and MS samples resulted in
censored (less-than) values during the holding time study. Evaluation of the TSA spike enumeration
indicated that the E. coll spiking suspension was not viable. The laboratory did meet IPR and MS criteria
during the preliminary analyses. Since the spiking suspension used during the holding time study was not
viable and because the laboratory was clearly in-control and performing acceptably throughout the study,
data was considered valid and included in subsequent data analyses.
Laboratories 1 and 5 (Method 1682, compost): Analyzed samples that were shipped from facilities not
within driving distance during the holding time study. Because all matrices were spiked with S.
typhimurium, results from these samples were included in subsequent data analyses.
5.3 Censored Data
During the evaluation of holding time study samples, results below the analytical range of the method
(less-than results) were observed for all of the spiked alkaline-stabilized (Class A) replicates evaluated by
Methods 1680 and 1681 at Laboratory 6 and unspiked compost replicates evaluated by Method 1680 at
Laboratory 15. Censored (less-than) values were observed for all of the unspiked samples analyzed for
Salmonella, regardless of matrix. In addition, all of the spiked alkaline-stabilized replicates analyzed for
Salmonella resulted in censored (less-than) values. All censored values were set to one-half the detection
limit (less-than value) prior to data analyses.
August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
SECTION 6.0 RESULTS, DATA ANALYSIS, AND DISCUSSION
This section includes results of unspiked and spiked biosolid samples which were analyzed for fecal
coliforms or Salmonella. Only valid results are included in this section; a detailed description of data
invalidation information is included in Section 5.0.
6.1 Fecal Coliform (Methods 1680/1681) Holding Time Study Results for Class A and
Class B Biosolid Samples
All fecal coliform results were natural log-transformed prior to performing any statistical analyses as a
result of the skewed distribution of the results, as recommended in Statistical Methods for Environmental
Pollution Monitoring (Reference 8.4). Results were stratified by EPA Methods (1680 and 1681), matrix,
matrix class, and spike type (i.e., spiked or unspiked). For each stratification, a two-way Analysis of
Variance (ANOVA) model was fit to assess the effect of holding time and laboratory on the log-
transformed concentrations, and to assess whether there was a significant interaction between holding
time and laboratory. For 14 of the 17 method/matrix/matrix class/spike types, there was no significant
interaction between holding time and laboratory, and, as a result, a single comparison was performed in a
single ANOVA model using data from all laboratories for these 14 method/matrix/matrix class/spike type
combinations. Pairwise comparisons were not necessary for this analysis because samples were analyzed
at only two holding times. For the other three method/matrix/matrix class/spike type combinations, there
was a significant interaction between holding time and laboratory, and therefore a single two-sample t-test
was performed for each laboratory separately.
Fecal coliform results are summarized in Tables 4 through 9. Significant decreases in bacterial
concentrations at 24-hour compared to 6-hour results are indicated by a "D".
Based on statistical analysis of the fecal coliform results, the following observations were made:
Alkaline-Stabilized Class A Matrices Analyzed Using Methods 1680 and 1681
Fecal coliform concentrations were significantly decreased by the 24-hour holding time compared to
the 6-hour holding time at Laboratory 7. Only two laboratories analyzed alkaline-stabilized Class A
samples during the holding time study resulting in two data sets for both methods.
Alkaline-Stabilized Class B Matrices Analyzed Using Methods 1680 and 1681
• Fecal coliform concentrations were significantly decreased by the 24-hour holding time compared to
the 6-hour holding time at Laboratory 22. A wide variation of fecal coliform concentrations was
observed in alkaline-stabilized Class B matrices during the holding time study. This might be
attributed to variability of pH across replicates, as a result of the treatment process. In general,
replicates with a consistent pH of 12 would significantly reduce bacterial levels, while replicates with
a lower pH or uneven distribution of pH could result in replicates with much higher bacterial levels or
delayed die-off
During preliminary analyses, fecal coliform concentrations were considerably lower than Class A
regulatory limits (<1000 MPN/g dry weight), even though the matrix evaluated by Laboratory 22
generally falls into the Class B category. Alkaline-stabilized matrices were not neutralized prior to
spiking to ensure that the matrices evaluated were representative of real-world alkaline-stabilized
matrices. Spiked replicates analyzed at 6 hours resulted in fecal coliform concentrations that ranged
from <65 MPN/g dry weight to 6.0 x 106 MPN/g dry weight, despite having been spiked at
approximately 3.0 x 106 MPN/g dry weight. These censored values (<65 MPN/g dry weight) for
samples spiked at such high-levels would only be expected if the pH of the replicate was very high
August 2006 10
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
(e.g., pH 12). Conversely, the 6.0 x 106 MPN/g dry weight would only have been observed if there
were high ambient concentrations; which would not be possible in samples with an extremely high
pH. Additionally, laboratories reported observing chunks of lime in some (but not all) of the
replicates. The preliminary results, the 6-hour results, the laboratory's observations, and the
expectation that this was a Class B matrix supports the supposition that the treatment process resulted
in a highly variable pH. The significant decrease in fecal coliform density at 24 hours may have
simply been a result of pH variability in the replicates spiked and analyzed, with replicates with a
wide range of pH being analyzed at the 6-hour holding time and replicates with high pH being
analyzed at the 24-hour holding time.
Composted Class A Matrices Analyzed Using Methods 1680 and 1681
• Fecal coliform concentrations were not significantly different at the 24-hour holding time compared
to the 6-hour holding time at all five laboratories (3, 13, 15, 17, and 29) that analyzed a composted
matrix during the holding time study.
Heat Dried Class A Matrices Analyzed Using Methods 1680 and 1681
• Fecal coliform concentrations were significantly decreased by the 24-hour holding time compared to
the 6-hour holding time at Laboratory 7. Only two laboratories analyzed heat dried samples during
the holding time study resulting in two data sets for Method 1680 and one data set for 1681.
Thermophilically Digested Class A and Class B Matrices Analyzed Using Method 1680
• Only two thermophilically digested matrices were evaluated during the study, both by Method 1680.
Fecal coliform concentrations were not significantly different at the 24-hour holding time compared
to the 6-hour holding time regardless of matrix class.
Aerobically Digested Class B Matrices Analyzed Using Methods 1680 and 1681
• Fecal coliform concentrations were not significantly different at the 24-hour holding time compared
to the 6-hour holding time at all five laboratories (3, 8, 24, and 27).
Anaerobically Digested Class B Matrices Analyzed Using Methods 1680 and 1681
• Fecal coliform concentrations were not significantly different at the 24-hour holding time compared
to the 6-hour holding time at all twelve laboratories (1, 5, 9, 11, 14, 18, 19, 20, 25, 26, and 28).
11 August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
Table 4. Fecal Coliform Mean Results (MPN/g dry weight) for Alkaline-Stabilized Samples
Laboratory
6
7
2
22
Mean
RSDb
Mean
RSDb
Class
A
A
B
B
A
A
B
B
Method 1680 (LTB/EC)
6 hours
24
24,342
128,508
1,028,871
12,183
58
578,689
192
24 hours
24
3,875 Da
19,205
5,925 Da
1,949
56
12,565
230
Method 1681 (A-1)
6 hours
24
27,242
22,320
13,633
25
22,320
155
24 hours
24
3,516 Da
1,954
1,770
40
1,954
114
Shading indicates laboratories did not conduct sample analyses with corresponding methods
3 Fecal coliform concentrations were significantly lower at 24-hours compared to 6-hour results as indicated by "D"
b Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of
the squared lab RSDs
Table 5. Fecal Coliform Mean Results (MPN/g dry weight) for Composted Samples
Laboratory
3
13
15
17a
29a
Mean (unspiked)
RSDb(unspiked)
Mean (spiked)
RSDb (spiked)
Method 1680 (LTB/EC)
6 hours
1
0
46,153
0
56
46,153
76
24 hours
3
0
28,858
2
150
28,858
59
Method 1681 (A-1)
6 hours
0
1
744
1
29
744
43
24 hours
1
1
1,168
1
130
1,168
44
Shading indicates laboratories did not conduct sample analyses with corresponding methods
a Laboratory analyzed spiked replicates; preliminary analyses indicated spiking was necessary
b Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of
the squared lab RSDs
August 2006
12
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
Table 6. Fecal Coliform Mean Results (MPN/g dry weight) for Heat Dried Samples
Laboratory
7
21
Mean
RSDb
Method 1680 (LTB/EC)
6 hours
8,128
149
4,139
123
24 hours
5,664
89
2,876
84
Method 1681 (A-1)
6 hours
7,228
7,228
55
24 hours
1,463Da
1,463
47
Shading indicates laboratories did not conduct sample analyses with corresponding methods
a Fecal coliform concentrations were significantly lower at 24-hours compared to 6-hour results as indicated by "D"
b Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of
the squared lab RSDs
Table 7. Fecal Coliform Mean Results (MPN/g dry weight) for Thermophilically Digested
Samples
Laboratory
10
22
Mean
RSDa
Mean
RSDa
Class
A
B
A
A
B
B
Method 1680 (LTB/EC)
6 hours
2,228
226,951
2,228
71
226,951
93
24 hours
9,305
111,353
9,305
75
111,353
78
None of the participant laboratories evaluated thermophilically digested samples using method 1681
a Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean
of the squared lab RSDs
Table 8. Fecal Coliform Mean Results (MPN/g dry weight) for Aerobically Digested Samples
Laboratory
3
8
24
27
Mean
RSDa
Method 1680 (LTB/EC)
6 hours
128,138
4,242
8,509,642
2,880,674
50
24 hours
105,693
4,079
7,932,045
2,680,606
41
Method 1681 (A-1)
6 hours
122,108
5,368
63,738
70
24 hours
81,852
6,051
43,951
72
Shading indicates laboratories did not conduct sample analyses with corresponding methods
a Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean
of the squared lab RSDs
13
August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
Table 9. Fecal Coliform Mean Results (MPN/g dry weight) for Anaerobically Digested Samples
Laboratory
1
5
9
11
14
18
19
20
25a
26
28
Mean
RSDb'
Mean
RSDb
Class
B
B
B
B
B
B
B
B
A
B
B
A
A
B
B
Method 1680 (LTB/EC)
6 hours
221,480
79,316
141
3,936
818,406
59,211
319,430
214,560
81
24 hours
353,130
103,986
125
3,685
532,167
31,417
295,914
188632
115
Method 1681 (A-1)
6 hours
132,072
276,318
69
23,446
937
101,974
937
85
106,776
109
24 hours
179,295
209,209
211
11,532
3,253
146,687
3,253
198
109,387
143
Shading indicates laboratories did not conduct sample analyses with corresponding methods
a Laboratory analyzed a spiked Class A anaerobically digested sample
b Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of
the squared lab RSDs
6.2 Salmonella (Method 1682) Holding Time Study Results for Biosolid Samples
All Salmonella results were natural log-transformed prior to performing any statistical analyses as a result
of the skewed distribution of the results, as recommended in Statistical Methods for Environmental
Pollution Monitoring (Reference 8.4). Results were stratified by matrix. For the alkaline-stabilized
matrix, a single comparison was performed using a two-sample t-test based on the data from the one
laboratory that performed analyses for the given matrix. For the composted matrix, a two-way Analysis
of Variance (ANOVA) model was fit to assess the effect of holding time and laboratory on the log-
transformed concentrations, and to assess whether there was a significant interaction between holding
time and laboratory. There was no significant interaction between holding time and laboratory for
compost samples, and, as a result, a single comparison was performed in a single ANOVA model using
data from all laboratories for that matrix. Pairwise comparisons were not necessary for this analysis
because samples were analyzed at only two holding times.
Salmonella results are summarized in Table 10. Significantly different holding times are indicated by a
"D," indicating a significant decrease in concentration compared to 6-hour results.
Based on statistical analysis of the Salmonella results, the following observations were made:
August 2006
14
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
Alkaline-Stabilized Class A Matrix
• Salmonella concentrations were not significantly different at the 24-hour holding time compared to
the 6-hour holding time.
Composted Class A Matrices
• Salmonella concentrations were significantly decreased by the 24-hour holding time compared to the
6-hour holding time at Laboratories 1 and 5.
Table 10. Salmonella Mean Results (CPU/ 4g dry weight) from Biosolid Samples
Laboratory
6
1
5
Mean
RSDb
Mean
RSDb
Matrix
Alkaline-stabilized
Compost
Compost
Alkaline-stabilized
Alkaline-stabilized
Compost
Compost
Method 1682
6 hours
0.29
9.95
7.78
0.29
0
8.87
48.50
24 hours
0.29
5.34 Da
5.22 Da
0.29
0
5.28
73.80
a Salmonella concentrations were significantly lower compared to "6" hour results
b Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of
the squared lab RSDs
15
August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
SECTION 7.0 CONCLUSIONS
Land application of biosolids is a critical component of solid waste management. Under Subpart D of the
40 Code of Federal Regulations (CFR) Part 503, sewage sludges (biosolids) are required to be processed
prior to land application in order to minimize pathogen levels and the potential public health risks
associated with contact or exposure. Subpart D further defines and classifies biosolids for land
application purposes based on pathogen concentrations. EPA has validated Methods 1680 and 1681 for
fecal coliforms in Class A and B biosolids and Method 1682 to support the monitoring requirements for
Salmonella in Class A biosolids. The main objective of this study was to determine whether biosolid
samples being analyzed for fecal coliforms and Salmonella can be held for 24 hours prior to sample
analysis without a significant change in bacterial concentrations.
Four hundred laboratories were contacted for potential study participation in an effort to obtain six or
more useable, valid datasets for every matrix/method combination. A number of reasons, including, but
not limited to, limited production of some matrices, laboratory proximity to biosolids facilities,
scheduling conflicts etc., resulted in limited data for some matrix/method combinations.
Fecal Coliforms
Based on the results of this study, Class A composted, Class B aerobically digested, and Class B
anaerobically digested matrices analyzed for fecal coliforms by Methods 1680 and 1681 may be analyzed
24 hours after sample collection and still generate data comparable to those generated at 6 hours after
sample collection.
All other matrices analyzed for fecal coliforms using Methods 1680 and 1681 should not exceed a 6-hour
maximum sample transport holding time, and must be processed within 2 hours of receipt at the
laboratory. The data for these matrix/method combinations generally showed a tendency to decrease with
extended sample holding times.
Salmonella
Based on the results of this study and the limited data generated, Class A matrices analyzed for
Salmonella using Method 1682 may not be analyzed beyond 6 hours after sample collection and still
generate data comparable to those generated at 6 hours after sample collection. In general, Salmonella
concentrations had a tendency to decrease with extended sample holding times.
August 2006 16
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Holding Time Effects on Fecal Coliforms and Salmonella in Biosolids
SECTION 8.0 REFERENCES
8.1 US EPA. 2005. EPA Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-
Tube Fermentation using Lauryl Tryptose Broth (LTB) and EC Medium
8.2 US EPA. 2005. EPA Method 1681: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple
Tube Fermentation using A-1 Medium
8.3 US EPA. 2005. EPA Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified
Semisolid Rappaport-Vassiliadis (MSRV) Medium
8.4 Gilbert, R.O. 1987. Statistical Methods for Environmental Pollution Monitoring. VanNostrand
Reinhold, New York.
17 August 2006
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Holding Time Effects on Fecal Coliforms and Salmonella Concentrations in Biosolids
SECTION 9.0 ACRONYMS
LTB Lauryl tryptose broth
LIA Lysine iron agar
MPN Most probable number
MSRV Modified semisolid Rappaport-Vassiliadis medium
QC Quality control
RSD Relative standard deviation
SAS Statistical analysis software
SD Standard deviation
TSA Tryptic soy agar
TSB Tryptic soy broth
TSI Triple sugar iron agar
XLD Xylose-lysine desoxycholate agar
August 2006 18
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Appendix A:
Laboratory Capabilities Checklist
-------
Laboratory Capabilities Checklist
Biosolids Holding Time Study
(March 13, 2006)
EPA plans to invite utility and commercial laboratories to participate in a study to evaluate the effect of holding
time on fecal coliforms in biosolids using EPA Methods 1680 and 1681 and Salmonella concentrations in
biosolids using EPA Method 1682. Samples will be analyzed at 6 and 24 hours following collection and/or
spiking. In addition, the study will include an assessment of false positive and false negative confirmation rates
for EPA Methods 1680 and 1681. Since Method 1682 includes serological confirmation, assessment of false
positive and false negative confirmation rates will not be done during this study. EPA will provide the media,
reagents, and disposable supplies necessary for this study. Volunteer laboratories and participants will be
acknowledged in the study reports.
If your laboratory is interested in participating in this study, please provide the requested information below and
fax the signed, completed checklist to Darcy Gibbons at 703.461.8056. Darcy will confirm receipt of the
checklist. If you have any questions pertaining to the information requested below or the holding time study,
please do not hesitate to contact Yildiz Chambers at 703.461.2165 or ychambers@csc.com.
Section 1. Laboratory Capabilities and Experience
A. Please complete the requested laboratory capabilities and experience information below, if this
information has not been previously provided to CSC. The information requested in Table 1 pertains to
experience with a given method for biosolids analyzed.
Table 1.
Analyst Experience
Analyst
Years of experience or estimated number of samples analyzed
1680(LTB/EC)
1681 (A-1)
1682(MSRV)
B. Name of primary analyst participating in study:
C. Primary analyst's years of experience performing biosolids analyses:
D. What certifications does your laboratory have for microbial analyses?
E.
Additional comments:
-------
Section 2. Background Information
A.
B.
C.
D.
Does your laboratory have access to biosolid samples?
If yes, please indicate in Table 2, below.
Is your laboratory located within two hours driving distance of
the biosolid facility (sample collection site)?
Has your laboratory ever participated in a biosolids study?
Yes No
Yes No
Yes No
If your laboratory has experience analyzing biosolid samples for fecal coliforms and/or Salmonella,
please place a check "•/" next to the biosolid type which you have analyzed and indicate both: the
method(s) used for analysis, and typical concentrations of each analyte (Table 2, below).
Table 2.
Fecal coliforms and Salmonella
Access?
Example
i/'
Biosolids Type
Composted
Heat-dried
Composted
Thermophilically
digested
Alkaline-
stabilized
Aerobically
digested
Anaerobically
digested
Class A
or B
A
Monitoring
Frequency
2
Fecal coliforms
Methods
I §80
Typical
Range
30 x 1 05
Salmonella
Methods
Typical
Range
12 x 103
E. CSC recognizes that your laboratory may have access to multiple facilities with biosolid matrices. For
each facility, please describe the treatment process (e.g., composted, anaerobically digested). Please be as
descriptive as possible.
Facility 1:
Facility 2:
Facility 3:
Will your laboratory be prepared to collect and analyze biosolid samples within the 6-hour holding time?
Yes No
-------
I certify that the information provided above is accurate and complete:
Primary Analyst:
Phone Number:
Email Address:
Lab Manager:
Phone Number:
Email Address:
Laboratory Name:
Laboratory FedEx Shipping Address:
Signature (lab manager):
Date:
-------
Appendix B:
Instructions for the Biosolids Holding Time Study
for Methods 1680/1681 (Class A)
-------
Instructions for the Biosolids Holding Time Study
Method 1680 and 1681: Class A Fecal Conforms
Holding Time Study Schedule
The same analytical procedures described for preliminary analyses will be used during the Biosolids Holding Time Study.
The following schedule may be used as a time-line for sample analyses. Note: During the holding time study, unspiked
samples with growth will be submitted to verification as described in the verification instructions.
Sample Analyses: LTB/EC and A-l
Option A: If ambient fecal coliform concentration is < 300 MPN / g dry weight
2 unspiked matrix samples per holding time
8 spiked matrix samples per holding time
1 unspiked OPR sample per holding time (Milorganite® [reference matrix])
2 spiked OPR samples per holding time (Milorganite® spiked with E. coll ATCC # 25922 spiking suspension)
Total =13 samples per holding time
Option B: If ambient fecal coliform concentration is > 300 MPN / g dry weight
8 unspiked matrix samples per holding time
2 spiked matrix samples per holding time
1 unspiked OPR sample per holding time (Milorganite® [reference matrix])
2 spiked OPR samples per holding time (Milorganite® spiked with E. coli ATCC # 25922 spiking suspension)
Total =13 samples per holding time
Example Schedule
Monday (0800)
Inoculate spiking suspension (see spiking protocol)
Tuesday (0800)
• Dilute spiking suspension and plate onto TSA (see spiking protocol)
• Collect a 1000 g bulk sample
• Follow Option A instructions if ambient fecal coliform concentration is < 300 MPN / g dry weight
• Follow Option B instructions if ambient fecal coliform concentration is > 300 MPN / g dry weight
• Weigh out 20, 30 g samples
Weigh out 6, 30 g samples of Milorganite® for OPR analysis (1 unspiked and 2 spiked per holding time)
• Option A: Spike 16 of the 20 samples
• Liquid samples: 3 mL of spiking suspension "B" per 300 mL of sample
• Solid samples: 3 mL of spiking suspension "B" per 30 g of sample
• Option B: Spike 4 of the 20 samples
• Liquid samples: 3 mL of spiking suspension "B" per 300 mL of sample
• Solid samples: 3 mL of spiking suspension "B" per 30 g of sample
Holding Time Instructions - Fecal Coliforms: Class A
-------
• Refrigerate all samples at < 10°C and above freezing
Option A: Unspiked matrix: 2 (6 hour) and 2 (24 hour)
Spiked matrix: 8 (6 hour) and 8 (24 hour)
Unspiked OPR: 1 (6 hour) and 1 (24 hour)
Spiked OPR: 2 (6 hour) and 2 (24 hour)
Option B: Unspiked matrix: 8 (6 hour) and 8 (24 hour)
Spiked matrix: 2 (6 hour) and 2 (24 hour)
Unspiked OPR: 1 (6 hour) and 1 (24 hour)
Spiked OPR: 2 (6 hour) and 2 (24 hour)
Tuesday - 6 Hour Hold Time (1400)
Spike 2, 30 g Milorganite® samples (OPRs)
Option A: Remove 2 unspiked and 8 spiked samples from refrigerator
Option B: Remove 8 unspiked and 2 spiked samples from refrigerator
• Homogenize by adding 30 g samples to 270 mL of sterile buffer water
Inoculate LTB and/or A-l tubes with the following undiluted and diluted homogenate
Unspiked liquid: 1.0 mL, 10'1, 102, 103
Unspiked solid: 10 mL, 1.0 mL, 10'1, 10'2
Spiked liquid: 102, 103, 104, 105
Spiked solid: 10'1, 10'2, 10'3, 10'4
Unspiked OPR: Analyze 1 unspiked Milorganite® sample by inoculating 5 tubes of 2X media with 10 mL undiluted
homogenate
Incubate LTB tubes at 35.0°C ± 0.5°C for 24 ± 2 hours
Incubate A-l tubes at 35.0°C ± 0.5°C for 3 hours ± 15 minutes
Transfer A-l tubes to a water bath at 44.5°C ± 0.2°C for 21 ± 2 hours
Wednesday - 24 Hour Hold Time (0800)
• Spike 2, 30 g Milorganite® samples (OPRs)
• Option A: Remove 2 unspiked and 8 spiked samples from refrigerator
• Option B: Remove 8 unspiked and 2 spiked samples from refrigerator
Homogenize by adding 30 g samples to 270 mL of sterile buffer water
• Inoculate LTB and/or A-l tubes with the following undiluted and diluted homogenate
Unspiked liquid: 1.0 mL, 10 \ 102, 103
Unspiked solid: 10 mL, 1.0 mL, 10'1, 10'2
Spiked liquid: 10'2, 10'3, 10'4, 10'5
Spiked solid: 10'1, 10'2, 10'3, 10'4
• Unspiked OPR: Analyze 1 unspiked Milorganite® sample by inoculating 5 tubes of 2X media with 10 mL undiluted
homogenate
Incubate LTB tubes at 35.0°C ± 0.5°C for 24 ± 2 hours
Incubate A-l tubes at 35.0°C ± 0.5°C for 3 hours ± 15 minutes
Transfer A-l tubes to a water bath at 44.5°C ± 0.2°C for 21 ± 2 hours
Read TSA plates and record results (see spiking protocol)
Holding Time Instructions - Fecal Coliforms: Class A
-------
Wednesday Cont. (1200 -1400)
Read A-l (6 hour) tubes at 24 ±2 hours and record results; refrigerate A-l tubes from unspiked samples (with
growth) for verification
Read LTB (6 hour) tubes at 24 ±2 hours and record results
• Transfer growth from LTB tubes with growth and gas production to EC tubes
Reincubate LTB for an additional 24 ±2 hours at 35°C ± 0.5°C
Incubate EC broth in the waterbath at 44.5°C ± 0.2°C for 24 ±2 hours
Thursday (0600-1000)
Read A-l (24 hour) tubes at 24 ±2 hours and record results; refrigerate A-l tubes from unspiked samples (with
growth) for verification
Read LTB (24 hour) tubes at 24 ±2 hours and record results
• Transfer growth from LTB tubes with growth and gas production to EC tubes
Reincubate LTB for an additional 24 ± 2 hours at 35°C ± 0.5°C
Incubate EC broth in the waterbath at 44.5°C ± 0.2°C for 24 ± 2 hours
Thursday (1200-1600)
Read LTB (6 hour) tubes at 48 ± 3 hours and record results
• Transfer growth to EC tubes and incubate at 44.5°C ± 0.2°C for 24 ± 2 hours
Read EC (24 hour) tubes at 24 ±2 hours and record results; refrigerate EC tubes from unspiked samples (with
growth) for verification
Friday (0600-1000)
Read LTB (24 hour) tubes at 48 ± 3 hours and record results
• Transfer growth from presumptive tubes to EC
Incubate EC tubes at 44.5°C ± 0.2°C for 24 ± 2 hours
Saturday (0600-1000)
• Read EC tubes at 24 ±2 hours and record results; refrigerate EC tubes from unspiked samples (with growth) for
verification
Monday
Remove EC and A-l tubes from refrigerator and begin verifications (see verification instructions)
Note: Please fax and send a hard copy of results and any accompanying documentation from holding time study to Darcy
Gibbons at 703-461-8056.
Holding Time Instructions - Fecal Coliforms: Class A
-------
Appendix C: Instructions for the Biosolids Holding Time Study
for Methods 1680/1681 (Class B)
-------
Instructions for the Biosolids Holding Time Study
Method 1680 and 1681: Class B Fecal Conforms
Holding Time Study Schedule
During the holding time study, follow the same analytical procedures as described above for the preliminary analyses.
Once preliminary analyses are complete, results may be used to determine appropriate dilutions to be analyzed. The
following schedule may be used as a time-line for sample analyses. Note: During the holding time study, samples with
growth will be submitted to verification as described in the verification instructions.
Sample Analyses: LTB/EC and A-l
8 unspiked matrix samples per holding time
2 spiked matrix samples per holding time
1 unspiked OPR sample per holding time (Milorganite® [reference matrix])
2 spiked OPR samples per holding time (Milorganite® spiked with E. coll ATCC # 25922 spiking suspension)
Total =13 samples per holding time
Example Schedule
Monday (0800)
Inoculate spiking suspension (see spiking protocol)
Tuesday (0800)
• Dilute spiking suspension and plate onto TSA (see spiking protocol)
• Collect a 1000 g bulk sample
Weigh out 20, 30 g samples (8 unspiked matrix and 2 spiked matrix per holding time)
Weigh out 6, 30 g samples of Milorganite® for OPR analysis (1 unspiked and 2 spiked per holding time) as follows
• Unspiked matrix: 8 (6 hour) and 8 (24 hour)
• Spiked matrix: 2 (6 hour) and 2 (24 hour)
Unspiked OPR: 1 (6 hour) and 1 (24 hour)
Spiked OPR: 2 (6 hour) and 2 (24 hour)
• Refrigerate all samples at < 10°C and above freezing
Tuesday - 6 Hour Hold Time (1400)
Spike 2, 30 g Milorganite® samples (OPRs)
• Remove the 8 unspiked and 2 spiked samples from refrigerator
Homogenize by adding 30 g samples to 270 mL of sterile buffer water
• After homogenization for 1 minute, spike matrix and OPR samples with 3 ml of undiluted spiking suspension, then
homogenize for 1 additional minute
Holding Time Instructions - Fecal Coliforms: Class B
-------
• Inoculate LTB and/or A-l tubes with the following serially diluted homogenate
• Unspiked liquid: 103, 104, 105, 106
Unspiked solid: 10'2, 10'3, 10'4, 10'5
Spiked liquid: 10'5, 10'6, 10'7, 10'8
Spiked solid: 10'4, 10'5, 10'6, 10'7
• Unspiked OPR: Analyze 1 unspiked Milorganite® sample by inoculating 5 tubes of 2X media with 10 mL
undiluted homogenate
Incubate LTB tubes at 35.0°C ± 0.5°C for 24 ± 2 hours
Incubate A-l tubes at 35.0°C ± 0.5°C for 3 hours ± 15 minutes
Transfer A-l tubes to a water bath at 44.5°C ± 0.2°C for 21 ± 2 hours
Wednesday - 24 Hour Hold Time (0800)
• Spike 2, 30 g Milorganite® samples (OPRs)
Remove the 8 unspiked and 2 spiked samples from refrigerator
• Homogenize by adding 30 g samples to 270 mL of sterile buffer water
• After homogenization for 1 minute, spike matrix and OPR samples with 3 ml of undiluted spiking suspension, then
homogenize for 1 additional minute
• Inoculate LTB and/or A-l tubes with the following serially diluted homogenate
Unspiked liquid: 103, 104, 105, 106
Unspiked solid: 10'2, 10'3, 10'4, 10'5
Spiked liquid: 10'5, lO'6, 10'7, 10'8
Spiked solid: 10'4, 10'5, 10'6, 10'7
Unspiked OPR: Analyze 1 unspiked Milorganite® sample by inoculating 5 tubes of 2X media with 10 mL
undiluted homogenate
Incubate LTB tubes at 35.0°C ± 0.5°C for 24 ± 2 hours
Incubate A-l tubes at 35.0°C ± 0.5°C for 3 hours ± 15 minutes
Transfer A-l tubes to a water bath at 44.5°C ± 0.2°C for 21 ± 2 hours
Read TSA plates and record results (see spiking protocol)
Wednesday (1200 -1400)
Read A-l (6 hour) tubes at 24 ±2 hours and record results; refrigerate A-l tubes from unspiked samples (with
growth) for verification
Read LTB (6 hour) tubes at 24 ±2 hours and record results
• Transfer growth from LTB tubes with growth and gas production to EC tubes
Reincubate LTB for an additional 24 ± 2 hours at 35°C ± 0.5°C
Incubate EC broth in the waterbath at 44.5°C ± 0.2°C for 24 ± 2 hours
Thursday (0600-1000)
Read A-l (24 hour) tubes at 24 ±2 hours and record results; refrigerate A-l tubes from unspiked samples (with
growth) for verification
Read LTB (24 hour) tubes at 24 ±2 hours and record results
• Transfer growth from LTB tubes with growth and gas production to EC tubes
Reincubate LTB for an additional 24 ± 2 hours at 35°C ± 0.5°C
Incubate EC broth in the waterbath at 44.5°C ± 0.2°C for 24 ± 2 hours
Holding Time Instructions - Fecal Coliforms: Class B
-------
Thursday (1200-1600)
Read LTB (6 hour) tubes at 48 ± 3 hours and record results
• Transfer growth to EC tubes and incubate at 44.5°C ± 0.2°C for 24 ± 2 hours
Read EC (24 hour) tubes at 24 ±2 hours and record results; refrigerate EC tubes from unspiked samples (with
growth) for verification
Friday (0600-1000)
Read LTB (24 hour) tubes at 48 ± 3 hours and record results
• Transfer growth from presumptive tubes to EC
Incubate EC tubes at 44.5°C ± 0.2°C for 24 ± 2 hours
Saturday (0600-1000)
• Read EC tubes at 24 ±2 hours and record results; refrigerate EC tubes (with growth) for verification
Monday
• Remove EC and A-1 tubes from refrigerator and begin verifications (see verification instructions)
Note: Please fax and send a hard copy of results and any accompanying documentation from holding time study to Darcy
Gibbons at 703-461-8056.
Holding Time Instructions - Fecal Coliforms: Class B
-------
Appendix D: Instructions for the Biosolids Holding Time Study
for Method 1682
-------
Instructions for the Biosolids Holding Time Study
Method 1682: Salmonella
Holding Time Study Schedule
The same analytical procedures described for preliminary analyses will be used during the Biosolids Holding Time Study.
The following schedule may be used as a time-line for sample analyses. Note: One positive and negative control
tube/plate should be inoculated with the appropriate positive/negative control organism for each medium used during
sample analysis.
Sample Analyses
2 unspiked matrix samples per holding time
8 spiked matrix samples per holding time
1 unspiked reference matrix (Milorganite®) sample per holding time
2 spiked reference matrix (Milorganite® spiked with Salmonella typhimurium ATCC # 14028 spiking suspension)
samples per holding time (OPR)
Total =13 samples per holding time
Example Schedule
Sunday (1200)
• Inoculate spiking suspension (see spiking protocol)
Monday (1200)
• Dilute spiking suspension and plate onto TSA (see spiking protocol)
Collect a single bulk sample of approximately 1000 g and transport to the laboratory on ice; maintain sample at
< 10°C and above freezing
• Weigh out 20, 30 g samples (2 unspiked and 8 spiked per holding time)
Weigh out 6, 30 g samples of Milorganite® for OPR analysis (1 unspiked and 2 spiked per holding time)
• Spike 16 of the 20 matrix samples with 0.5 mL of spiking suspension "D" (10~6 of the original spiking suspension);
see spiking protocol
• Refrigerate all samples at < 10°C and above freezing
• Unspiked matrix: 2 (6 hour) and 2 (24 hour)
Spiked matrix: 8 (6 hour) and 8 (24 hour)
• Unspiked OPR: 1 (6 hour) and 1 (24 hour)
• Spiked OPR: 2 (6 hour) and 2 (24 hour)
Monday - 6 Hour Hold Time (1800)
Spike 2, 30 g Milorganite® samples (OPRs)
Remove 2 unspiked and 8 spiked matrix samples from refrigerator
• Homogenize by adding each 30 g sample to 270 mL sterile buffer
Inoculate TSB tubes with the following volumes of homogenized unspiked or spiked samples
• Add 20 mL homogenized sample to 10 mL 3X TSB
• Add 10 mL homogenized sample to 5 mL 3X TSB
• Add 1 mL homogenized sample to 10 mL IX TSB
Unspiked OPR: Inoculate 5 tubes of 3X media with 20 mL homogenized sample
• Incubate inoculated TSB tubes at 36°C ± 1.5°C for 24 ± 2 hours
Holding Time Instructions - Salmonella 1
-------
Tuesday - 24 Hour Hold Time (1200)
Spike 2, 30 g Milorganite® samples (OPRs)
Remove 2 unspiked and 8 spiked matrix samples from refrigerator
• Homogenize by adding each 30 g sample to 270 mL sterile buffer
• Inoculate TSB tubes with the following volumes of homogenized unspiked or spiked samples
• Add 20 mL homogenized sample to 10 mL 3X TSB
• Add 10 mL homogenized sample to 5 mL 3X TSB
• Add 1 mL homogenized sample to 10 mL IX TSB
Unspiked OPR: Inoculate 5 tubes of 3X media with 20 mL homogenized sample
• Incubate inoculated TSB tubes at 36°C ± 1.5°C for 24 ± 2 hours
• Examine TSA plates (spiking suspension enumeration) and record results (see spiking protocol)
Tuesday (1600-2000)
• Read TSB (6 hour) tubes at 24 ±2 hour incubation and record results
Transfer 6, 30 (il drops from each TSB tube (6 hour) to corresponding MSRV plates; allow drops to absorb for
approximately 1 hour at room temperature
Do not invert MSRV plates
• Incubate MSRV plates (6 hour) at 42°C ± 0.5°C for 16-18 hours in a humidity-controlled incubator
Wednesday (0800 -1200)
• Read TSB (24 hour) tubes at 24 ±2 hour incubation and record results
Transfer 6, 30 (il drops from each TSB tube (24 hour) to corresponding MSRV plates; allow drops to absorb for
approximately 1 hour at room temperature
Do not invert MSRV plates
• Incubate MSRV plates (24 hour) at 42°C ± 0.5°C for 16-18 hours in a humidity-controlled incubator
Wednesday (1200-1400)
Remove MSRV plates (6 hour) from incubator and examine plates for the appearance of a "whitish halo" of growth;
record results
Choose 2 target colonies from MSRV plates (6 hour) and streak growth onto 2 separate XLD plates
• Incubate XLD plates (6 hour) at 36°C ± 1.5°C for 18 to 24 hours
Thursday (0600)
• Remove MSRV plates (24 hour) from incubator and examine for the appearance of a "whitish halo" of growth; record
results
Choose 2 target colonies from MSRV plates (24 hour) and streak growth onto two separate XLD plates
• Incubate XLD plates (24 hour) at 36°C ± 1.5°C for 18 to 24 hours
Thursday (1200)
• Remove XLD plates (6 hour) from incubator; refrigerate one of the XLD plates at < 10°C and above freezing and
submit the other plate to biochemical confirmation. Note: Both XLD plates may be refrigerated at < 10°C and above
freezing over the weekend prior to submitting to biochemical confirmation.
Holding Time Instructions - Salmonella
-------
Friday (0600)
• Remove XLD plates (24 hour) from incubator; refrigerate one of the XLD plates at < 10°C and above freezing and
submit the other plate to biochemical confirmation. Note: XLD plates may be refrigerated at < 10°C and above
freezing over the weekend prior to submitting to biochemical confirmation.
Monday (0800)
Biochemical Confirmation
Remove one XLD plate from refrigerator (6 hour and 24 hour) and examine for growth
• Pick a well isolated colony from each XLD plate (6 hour and 24 hour) exhibiting Salmonella morphology and
inoculate TSI, LIA, and Urease broth by stabbing the butt and streaking the slant; use the same XLD colony to
inoculate all three media
• TSI/LIA/Urease broth: Incubate (6 hour and 24 hour) slants and broth at 36°C ± 1.5°C for 18 to 24 hours
Tuesday (0800)
Remove TSI/LIA/Urease slants (6 hour and 24 hour) from the incubator and examine for positive/negative reactions;
record results
Serology
• Emulsify growth on the slant portion of TSI; place two discrete drops of emulsified growth onto a 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 slide under magnification and record results
Note: 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. Correlate all
positive plates and tubes to original TSB tube and record results.
Note: Please fax and send a hard copy of results and any accompanying documentation from holding time study to Darcy
Gibbons at 703-461-8056.
Holding Time Instructions - Salmonella
-------
Appendix E: Fecal Coliform Spiking Protocol (Class A)
-------
Fecal Coliform Spiking Procedure
Class A Biosolids: Methods 1680 and 1681
The purpose of this protocol is to provide laboratories with a fecal coliform spiking procedure for the Method 1680 and
1681 Holding Time Study.
1.0 Preparation of E. coli Spiking Suspension
1.1 Stock Culture. Prepare a stock culture by inoculating a tryptic soy agar (TSA) slant (or other non-selective
media) with Escherichia coli ATCC # 25922 and incubating at 35°C ± 3°C for 20 ± 4 hours. This stock culture
may be stored in the dark at room temperature for up to 30 days.
1.2 1% Lauryl Tryptose Broth (LTB). Prepare a 1% solution of LTB by combining 99 mL of sterile phosphate
buffered dilution water and 1 mL of sterile single strength LTB in a sterile screw cap bottle or re-sealable dilution
water container (Hardy Diagnostics D699 or equivalent). Shake to mix.
1.3 Spiking Suspension (Undiluted). From the stock culture of E. coli ATCC # 25922 in Section 1.1, transfer a
small loopful of growth to the 1% LTB solution and vigorously shake a minimum of 25 times. Incubate at 35°C ±
3°C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0 x 107 - 1.0 x 10s E. coli
colony forming units (CPU) per mL. This is referred to as the "undiluted spiking suspension." Note: Spiking
suspension should be inoculated the day prior to preliminary and holding time study analyses.
2.0 Class A Biosolid Sample Spiking and Enumeration of Spiking Suspension
Homogenize the unspiked Class A biosolid sample according to study instructions. For preliminary analyses,
four, 30 g Milorganite® samples (IPR) per method and one, 30 g matrix sample per method will be spiked prior to
analyses. During the holding time study, four, 30 g Milorganite® samples (OPR) per method and either sixteen,
30 g matrix samples per method (Option A), or four, 30 g matrix samples per method (Option B) will be spiked.
2.1 Sample spiking: Class A
2.1.1 Dilute spiking suspension
2.1.1.1 Mix the 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 (from Section 1.3) to 99
mL of sterile dilution water, 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.
2.1.1.2 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "A" to 99 mL of
sterile 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"3 mL of the
original undiluted spiking suspension.
2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" to 99 mL of
sterile 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"4 mL of the
original spiking suspension.
2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" to 99 mL of
sterile 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"5 mL of the
original spiking suspension.
Fecal Coliforms: Class A - Spiking Protocol
-------
2.1.1.5 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "D" to 99 mL of
sterile dilution water, cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "E." A 1.0-mL volume of dilution "E" is 10~6 mL of the
original spiking suspension.
2.1.2 Spike sampled
2.1.2.1 Liquid Samples
For every 300 mL of liquid sample, add 3.0 mL of spiking suspension "B". Note:
Laboratories spiking liquid samples will receive additional verbal instructions from CSC.
Following spiking, inoculate appropriate medium according to the study instructions.
2.1.2.2 Solid Samples
For each 30-g sample, add 3.0 mL of spiking suspension "B". Following spiking, inoculate
appropriate medium according to the study instructions.
2.2 Enumeration of undiluted spiking suspension (prepared in Section 1.3)
2.2.1 Prepare TSA by adding 10 - 15 mL of TSA 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.
2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread plates:
• Pipet 0.1 mL of dilution "C" onto surface of pre-dried TSA plate [10~5 mL of the original spiking
suspension].
• Pipet 0.1 mL of dilution "D" onto surface of pre-dried TSA plate [10~6 mL of the original spiking
suspension].
• Pipet 0.1 mL of dilution "E" onto surface of pre-dried TSA plate [10~7 mL of the original spiking
suspension].
2.2.3 Immediately after inoculating 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. Shake spiking
suspension 25 times before inoculating each plate.
2.2.4 After spreading, allow inoculum to absorb into the medium completely for approximately 1-3 minutes.
2.2.5 Invert plates and incubate at 35°C ± 0.5°C for 24 ± 4 hours.
2.2.6 Count and record number of colonies per plate.
Fecal Coliforms: Class A - Spiking Protocol
-------
3.0 Calculate Concentration of E. coli (CPU / ml_) in Undiluted Spiking Suspension
3.1 The number of E. coli CPU / mL in the spiking suspension will be calculated using all plates yielding counts
within the ideal range of 30 - 300 CPU per plate.
3.2 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).
3.3 Calculate the concentration of E. coli (CPU / mL) in the undiluted spiking suspension according to the following
equation. (Example calculations are provided in Table 1.)
EC
CFU1 + CFU2+ ... + CFUn
undiluted spike
V2+ ... +Vn
Where
FT
-LjV-x undiluted spike
CPU
V
E. coli CPU / mL in undiluted spiking suspension
number of colony forming units from plates yielding counts within the ideal range of
30-300 CPU per plate
volume of undiluted sample in each TSA plate yielding counts within the ideal range of
30-300 CPU per plate
number of plates with counts within the ideal range
TABLE 1. EXAMPLE CALCULATIONS OF E. COLI SPIKING SUSPENSION CONCENTRATION
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
TSA plates
1Q-5 mL plates
275, 250, 301
TNTC, TNTC, TNTC
10'6 mL plates
30, 10,5
TNTC, 299, TNTC
10'7 mL plates
0, 0, 0
12, 109, 32
E. coli CPU / mL in undiluted
spiking suspension
(EC undiluted spike)
(275+250+30) / ( 1 0~5+ 1 0~5+ 1 0'6) =
5557 (2.1 x10-5) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10-6+10-7+10-7) =
440 / (1.2 x ID'6) =366,666,667 =
3.7x108CFU/mL
*EC undi|Utedspikeis calculated using all plates yielding counts within the ideal range of 30 - 300 CPU per plate
Fecal Coliforms: Class A - Spiking Protocol
-------
Appendix F: Fecal Coliform Spiking Protocol (Class B)
-------
Fecal Coliform Spiking Procedure
Class B Biosolids: Methods 1680 and 1681
The purpose of this protocol is to provide laboratories with a fecal coliform spiking procedure for the Method 1680 and
1681 Holding Time Study.
1.0 Preparation of E. coli Spiking Suspension
1.1 Stock Culture. Prepare a stock culture by inoculating a tryptic soy agar (TSA) slant (or other non-selective
media) with Escherichia coli ATCC # 25922 and incubating at 35°C ± 3°C for 20 ± 4 hours. This stock culture
may be stored in the dark at room temperature for up to 30 days.
1.2 1% Lauryl Tryptose Broth (LTB). Prepare a 1% solution of LTB by combining 99 mL of sterile phosphate
buffered dilution water and 1 mL of sterile single strength LTB in a sterile screw cap bottle or re-sealable dilution
water container (Hardy Diagnostics D699 or equivalent). Shake to mix.
1.3 Spiking Suspension (Undiluted). From the stock culture of E. coli ATCC # 25922 in Section 1.1, transfer a
small loopful of growth to the 1% LTB solution and vigorously shake a minimum of 25 times. Incubate at 35°C ±
3°C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0 x 107 - 1.0 x 10s E. coli
colony forming units (CPU) per mL. This is referred to as the "undiluted spiking suspension." Note: Spiking
suspension should be inoculated the day prior to preliminary and holding time study analyses.
2.0 Class B Biosolid Sample Spiking and Enumeration of Spiking Suspension
Homogenize the unspiked Class B biosolid sample according to study instructions. For preliminary analyses,
four, 30 g Milorganite® samples (IPR) per method and one, 30 g matrix sample per method will be spiked prior to
analyses. During the holding time study, four, 30 g Milorganite® samples (OPR) per method and four, 30 g
matrix samples per method will be spiked.
2.1 Sample spiking: Class B
2.1.1 Dilute spiking suspension
2.1.1.1 Mix the 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 (from Section 1.3) to 99
mL of sterile dilution water, 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.
2.1.1.2 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "A" to 99 mL of
sterile 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"3 mL of the
original undiluted spiking suspension.
2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" to 99 mL of
sterile 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"4 mL of the
original spiking suspension.
2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" to 99 mL of
sterile 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"5 mL of the
original spiking suspension.
Fecal Coliforms: Class B - Spiking protocol
-------
2.1.1.5 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "D" to 99 mL of
sterile dilution water, cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "E." A 1.0-mL volume of dilution "E" is 10~6 mL of the
original spiking suspension.
2.1.2 Spike sample(s)
2.1.2.1 Liquid Samples
For every 300 mL of liquid sample, add 3.0 mL of the undiluted spiking suspension. Note:
Laboratories spiking liquid samples will receive additional verbal instructions from CSC.
Following spiking, inoculate appropriate medium according to the study instructions.
2.1.2.2 Solid Samples
For each 30-g sample, add 3.0 mL of the undiluted spiking suspension. Following spiking,
inoculate appropriate medium according to the study instructions.
2.2 Enumeration of undiluted spiking suspension (prepared in Section 1.3)
2.2.1 Prepare TSA by adding 10 - 15 mL of TSA 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.
2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread plates:
Pipet 0.1 mL of dilution "C" onto surface of pre-dried TSA plate [10~5 mL of the original spiking
suspension].
Pipet 0.1 mL of dilution "D" onto surface of pre-dried TSA plate [10~6 mL of the original spiking
suspension].
Pipet 0.1 mL of dilution "E" onto surface of pre-dried TSA plate [10~7 mL of the original spiking
suspension].
2.2.3 Immediately after inoculating 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. Shake spiking
suspension 25 times before inoculating each plate.
2.2.4 After spreading, allow inoculum to absorb into the medium completely for approximately 1-3 minutes.
2.2.5 Invert plates and incubate at 35°C ± 0.5°C for 24 ± 4 hours.
2.2.6 Count and record number of colonies per plate.
Fecal Coliforms: Class B - Spiking protocol
-------
3.0 Calculate Concentration of E. coli (CPU / ml_) in Undiluted Spiking Suspension
3.1 The number of E. coli CPU / mL in the spiking suspension will be calculated using all plates yielding counts
within the ideal range of 30 - 300 CPU per plate.
3.2 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).
3.3 Calculate the concentration of E. coli (CPU / mL) in the undiluted spiking suspension according to the following
equation. (Example calculations are provided in Table 1.)
EC
CFU1 + CFU2+ ... + CFUn
undiluted spike
V2+ ... +Vn
Where
FT
-LjV-x undiluted spike
CPU
V
E. coli CPU / mL in undiluted spiking suspension
number of colony forming units from plates yielding counts within the ideal range of
30-300 CPU per plate
volume of undiluted sample in each TSA plate yielding counts within the ideal range of
30-300 CPU per plate
number of plates with counts within the ideal range
TABLE 1. EXAMPLE CALCULATIONS OF E. COLI SPIKING SUSPENSION CONCENTRATION
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
TSA plates
1Q-5 mL plates
275, 250, 301
TNTC, TNTC, TNTC
10'6 mL plates
30, 10,5
TNTC, 299, TNTC
10'7 mL plates
0, 0, 0
12, 109, 32
E. coli CPU / mL in undiluted
spiking suspension
(EC undiluted spike)
(275+250+30) / ( 1 0~5+ 1 0~5+ 1 0'6) =
5557 (2.1 x10-5) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10-6+10-7+10-7) =
440 / (1.2 x ID'6) =366,666,667 =
3.7x108CFU/mL
*EC undi|Utedspikeis calculated using all plates yielding counts within the ideal range of 30 - 300 CPU per plate
Fecal Coliforms: Class B - Spiking protocol
-------
Appendix G: Salmonella Spiking Protocol
-------
Salmonella Spiking Procedure
Method 1682
The purpose of this protocol is to provide laboratories with a Salmonella typhimurium spiking procedure for the Method
1682 Holding Time Study.
1.0 Preparation of Laboratory-Prepared Spiking Suspensions
1.1 Stock Culture. Prepare a stock culture by inoculating a tryptic soy agar (TSA) 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.
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.
1.3 Spiking Suspension (Undiluted). From the stock culture of S. typhimurium ATCC # 14028, in section 1.1,
aseptically transfer a small loopful of growth to the 1% TSB solution and vigorously shake a minimum of 25
times. Incubate at 36°C ± 1.5°C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0 x
107 to 1.0 x 10s S. typhimurium colony forming units (CPU) per mL. This is referred to as the "undiluted spiking
suspension."
2.0 Sample Spiking and Enumeration of Spiking Suspension
Homogenize the unspiked Class A biosolid sample according to study instructions. For preliminary analyses,
four, 30 g Milorganite® samples (IPR) and one, 30 g matrix sample will be spiked prior to analyses. During the
holding time study, four, 30 g Milorganite® samples (OPR) and sixteen, 30 g matrix samples will be spiked.
2.1 Sample spiking
2.1.1 Dilute spiking suspension
2.1.1.1 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 1.3), 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.
2.1.1.2 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.
2.1.1.3 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.
2.1.1.4 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.
Spiking Protocol: Salmonella
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2.1.2 Spike sample(s)
2.1.2.2 For each 30 g sample, add 0.5 mL of spiking suspension "D". Following spiking, inoculate
appropriate medium according to the study instructions.
2.2 Enumeration of undiluted spiking suspension (prepared in Section 1.3)
2.2.1 Prepare TSA by adding 10 - 15 mL of TSA 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.
2.2.2 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 TSA plate [10~5 mL of the original spiking
suspension].
• Pipet 0.1 mL of dilution "C" onto surface of pre-dried TSA plate [10~6 mL of the original spiking
suspension].
• Pipet 0.1 mL of dilution "D" onto surface of pre-dried TSA plate [10~7 mL of the original spiking
suspension].
2.2.3 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.
2.2.4 Allow inoculum to absorb into the medium completely for approximately 1-3 minutes.
2.2.5 Invert plates and incubate at 36°C ± 1.5°C for 24 ± 4 hours.
2.2.6 Count and record number of colonies per plate.
3.0 Calculate Concentration of S. typhimurium (CPU / mL) in Undiluted Spiking Suspension
3.1 The number of S. typhimurium CPU / mL in the undiluted spiking suspension will be calculated using all TSA
plates yielding counts within the ideal range of 30 to 300 CPU per plate.
3.2 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).
3.3 Calculate the concentration of S. typhimurium (CPU / mL) in the undiluted spiking suspension according to the
following equation. (Example calculations are provided in Table 1, below.)
Salmonella undilutedspike = (CPU, + CFU2 + ... + CFUn) / (V, + V2 + ... + Vn)
Where,
Salmonella undlluted splke = S. typhimurium CPU / mL in undiluted spiking suspension
CPU = Number of colony forming units from TSA plates yielding
counts within the ideal range of 30 to 300 CPU per plate
V = Volume of undiluted sample on each TSA plate yielding counts
within the ideal range of 30 to 300 CPU per plate
n = Number of plates with counts within the ideal range
Spiking Protocol: Salmonella
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Table 1. Example Calculations of S. typhimuhum Spiking Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from TSA plates
ID'5 mL plates
275, 250, 301
TNTC, TNTC,
TNTC
10'6 mL plates
30, 10, 5
TNTC, 299,
TNTC
10'7 mL plates
0, 0, 0
12, 109, 32
Salmonella CPU / mL in undiluted spiking
suspension
(Salmonella undiluted spike) a
(275+250+30) /(10-5+ 10-5+1Q-6) =
5557 (2.1 x10-5) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10-6+ 10-7+1Q-7) =
440 / (1 .2 x 1 0-6) =366,666,667 =
3.7x108CFU/mL
aSalmonella undNuted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
Spiking Protocol: Salmonella
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