United States Office of Water EPA-821-R-14-009
Environmental Protection Agency (4303-T) September 2014
&EPA
United States
Environmental Protection
Agency
Method 1680: Fecal Conforms in
Sewage Sludge (Biosolids) by
Multiple-Tube Fermentation using
Lauryl Tryptose Broth (LTB) and EC
Medium
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
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Method 1680
Acknowledgments
The contributions of the following persons and organizations to this study are gratefully acknowledged:
Referee Laboratory
• EPA Office of Research and Development, National Risk Management Research Lab: Mark C. Meckes
and Karen M. White
Volunteer Participant Laboratories
• American Interplex: John Overbey and Lizbeth Huggins
• BioVir Laboratories: Rick Danielson and Jim Truscott
• City of Los Angeles Bureau of Sanitation Environmental Monitoring Division: Farhana Mohamed and
Zora Bahariance
• County Sanitation Districts of Los Angeles County: Shawn Thompson and Julie Millenbach
Environmental Associates: Susan Boutros and John Chandler
• Hampton Roads Sanitation District: Anna Rule and Bob Maunz
King County Environmental Laboratory: Greg Ma and Bobbie Anderson
• Hoosier Microbiological Laboratories: Don Hendrickson, Keri Nixon, Katy Bilger, and Lindsey Shelton
• Massachusetts Water Resources Authority: Steve Rhode and Mariya Gofshteyn
• Milwaukee Metropolitan Sewerage District: Jeff MacDonald and Tim O'Neill
University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord
• Utah Department of Health: Sanwat Chaudhuri and Devon Cole
The following facilities provided biosolid matrices for the study:
• Compost Facility, Columbus, OH: Angela Bianco
Wastewater Treatment Facility, Sturgeon Bay, WI: Todd Maurina
• Wastewater Treatment Facility, Fairfield, OH: Drew Young
• Wastewater Treatment Facility, Mason, OH: Ernie Stickler
N-Viro Treatment Facility, Toledo, OH: Cindy Drill
Media Photographs
Mark C. Meckes, NRMRL, US EPA
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Method 1680
Disclaimer
Neither the United States Government nor any of its employees, contractors, or their employees make any
warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use of or the
results of such use of any information, apparatus, product, or process discussed in this report, or represents that its
use by such party would not infringe on privately owned rights. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov or OSTCWAMethods@epa.gov
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Method 1680
Table of Contents
Acknowledgments i
Disclaimer ii
1.0 Scope and Application 1
2.0 Summary of Method 2
3.0 Definitions 2
4.0 Interferences 3
5.0 Safety 3
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 5
8.0 Sample Collection, Handling, and Storage 8
9.0 Quality Control 10
10.0 Equipment Calibration and Standardization 14
11.0 Sample Preparation 14
12.0 Lauryl Tryptose Broth (LTB) - EC Broth Procedure 21
13.0 Verification 23
14.0 Data Analysis and Calculations 23
15.0 Sample Spiking Procedure 28
16.0 Method Performance 34
17.0 Pollution Prevention 35
18.0 Waste Management 35
19.0 References 35
20.0 Figures 36
21.0 Glossary 41
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Method 1680
Method 1680: Fecal Conforms in Sewage Sludge (Biosolids) by
Multiple-Tube Fermentation using Lauryl Tryptose Broth (LIB)
and EC Medium
September 2014
1.0 Scope and Application
1.1 This method describes multiple-tube fermentation procedures [also called the most probable number
(MPN) procedure] for the detection and enumeration of fecal coliform bacteria in biosolids. These
methods use culture-specific media and elevated temperature to isolate and enumerate fecal coliform
organisms. Fecal coliform bacteria, including Escherichia coli (E. coif), are commonly found in the
feces of humans and other warm-blooded animals, and indicate the potential presence of other bacterial
and viral pathogens.
1.2 This method is adapted from method 922 IE in Standard Methods for the Examination of Water and
Wastewater, 20th Edition, for the determination of fecal coliform bacteria in a variety of matrices
(Reference 19.1).
1.3 This method is designed to meet the survey and monitoring requirements of U.S. Environmental
Protection Agency (EPA) in regulating the use and disposal of biosolids under 40 CFR Part 503.
Subpart D of the 503 regulations protects public health and the environment through requirements
designed to reduce the potential for contact with disease-bearing microorganisms (pathogens) in
biosolids applied to land or placed on a surface disposal site.
1.4 Fecal coliform density is expected to correlate with the probability of pathogens present and document
process performance (vector attraction reduction).
1.5 This method may be used to determine the density of fecal coliform bacteria in Class A and Class B
biosolids to satisfy the pathogen reduction requirements of Subpart D of Part 503. A biosolid sample is
classified as Class A if it contains a fecal coliform density below 1,000 MPN/g of total solids (dry weight
basis). A biosolid sample is classified as Class B if the geometric mean fecal coliform density is less
than 2 x 106 MPN/g of total solids (dry weight basis).
1.6 To satisfy the pathogen reduction monitoring alternatives for Class B biosolids, seven samples of treated
biosolids are collected at the time of use or disposal and the geometric mean fecal coliform bacterial
density of these samples is confirmed not to exceed 2 x 106 MPN/g of total solids (dry weight basis).
Although the Part 503 regulation does not specify the total number of samples for Class A biosolids, it is
recommended that a sampling event extend over two weeks, and that at least seven samples be tested to
confirm that the mean bacterial density of the samples is below 1,000 MPN/g of total solids (dry weight
basis). The analysis of seven samples increases the method precision by reducing the standard error
caused by inherent variations in biosolid quality.
1.7 The presence of fecal coliforms may be determined in both Class A and Class B biosolids using the
MPN procedure.
1.8 Any modification of the method beyond those expressly permitted is subject to the application and
approval of alternative test procedures under 40 CFR Parts 136.4 and 136.5.
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Method 1680
1.9 Method 1680 was submitted to interlaboratory validation in Class A and Class B biosolid matrices. A
summary of method performance results from this validation study are provided in Section 16.0. A
comprehensive evaluation of the study results is presented in the validation study report (Reference
19.2). For method application please refer to Title 40 Code of Federal Regulation Part 136 (40 CFR
Part 136).
Note: Based on the high false positive rates observed for Method 1681 in some matrices, EPA
recommends that laboratories conduct their own matrix-specific comparisons to determine the
most appropriate method (1680 or 1681).
2.0 Summary of Method
2.1 Fecal coliform densities of biosolids may be determined by the MPN procedure.
2.2 MPN procedure (Class A and B)
Method 1680 provides for the enumeration of fecal coliforms in Class A and Class B biosolids using the
most probable number (MPN) procedure. In Method 1680, LTB is used as a presumptive medium
followed by EC as confirmation of fecal coliforms. EC may not be used for direct isolation from a
biosolid sample because prior enrichment in presumptive medium (LTB) is required for optimum
recovery of fecal coliforms.
2.2.1 Summary of the LTB/EC procedure (see Figure 1 in Section 20.0) [see Sections 11.0 and
12.0 for explanation]
2.2.1.1 A minimum of four sample dilutions are required, while five or more are preferred.
Each sample dilution is inoculated into five test tubes, containing sterile LTB and an
inverted vial (gas production).
2.2.1.2 LTB sample tubes are incubated in a water bath or jacketed incubator at 35°C ±
0.5°C. After 24 ± 2 hours, the tubes are examined for presumptive growth and gas
production. Gas production is indicated by gas bubble formation within the
inverted-vial. Negative tubes are reincubated for an additional 24 hours and
reassessed. Failure to produce gas in LTB medium within 48 ± 3 hours is a negative
presumptive test. EC tubes are incubated in a water bath at 44.5°C ± 0.2°C for 24 ±
2 hours. Following growth in LTB, gas production in EC broth within 24 ± 2 hours
is considered a positive fecal coliform reaction. Failure to produce gas is a negative
reaction and indicates fecal coliform bacteria are not present.
2.2.1.3 A total solids determination is performed on a representative biosolids sample and is
used to calculate MPN/g dry weight. Fecal coliform density is reported as MPN/g
dry weight.
3.0 Definitions
3.1 Fecal coliform bacteria are gram-negative, non-spore-forming rods that are found in the intestines and
feces of humans and other warm-blooded animals. The predominant fecal coliform can be E. coll. In
this method, fecal coliforms are those bacteria that grow and produce gas in LTB within a total of
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Method 1680
48 ± 3 hours after incubation at 35°C ± 0.5°C, and that subsequently ferment lactose and produce
gas within 24 ± 2 hours in EC broth after incubation at 44.5°C ± 0.2°C. Since coliforms from
other sources often cannot produce gas under these conditions, this criterion is used to define the
fecal component of the coliform group.
3.2 Class A biosolids contain a fecal coliform density below 1,000 MPN/g of total solids (dry weight basis).
3.3 Class B biosolids contain a geometric mean fecal coliform density of less than 2 x 106 MPN/g of total
solids (dry weight basis).
3.4 Definitions for other terms are given in the glossary at the end of the method.
4.0 Interferences
4.1 MPN procedure: Since the MPN tables are based on a Poisson distribution, if the sample is not
adequately mixed to ensure equal bacterial cell distribution before portions are removed, the MPN value
will be a misrepresentation of the bacterial density.
5.0 Safety
5.1 The analyst must observe normal safety procedures required in a microbiology laboratory while
preparing, using, and disposing of media, cultures, reagents, and materials, and while operating
sterilization equipment.
5.2 Field and laboratory staff collecting and analyzing environmental samples are under some risk of
exposure to pathogenic microorganisms. Staff should apply safety procedures used for pathogens to
handle all samples.
5.3 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Sterile plastic bags, 1-gallon
6.2 Sterile plastic or glass jars with lids, 1-L
6.3 Sterile auger
6.4 Sterile scoops (do not use curved scoops)
6.5 Ice chest
6.6 Wet ice
6.7 Ice packs, blue ice
6.8 Bubble wrap
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Method 1680
6.9 Sterile trowels
6.10 Sterile aluminum foil or kraft paper
6.11 Sterile container, such as a stainless steel or plastic bucket suitable for sample collection
6.12 Flat shovel
6.13 Dilution bottles, borosilicate glass, screw cap, marked at 99 mL or screw cap, borosilicate glass or plastic
tubes marked at 9 mL
6.14 Tubes, 16 x 150 mm, borosilicate glass, with loose-fitting aluminum, stainless steel or autoclavable caps
6.15 Durham tubes or vials, 10 x 75 mm, borosilicate glass
6.16 Tubes, 16 x 100 mm, screw cap, borosilicate glass, with autoclavable plastic caps
6.17 Test tube racks to hold sterile culture tubes
6.18 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets
6.19 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, wide-tip of appropriate volume
6.20 Pipet bulbs, or automatic pipettor
6.21 Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic loops
6.22 Sterile disposable applicator sticks
6.23 Bunsen burner or alcohol burner
6.24 Cornwall syringe, sterile, to deliver at least 5 mL
6.25 Media dispensing pump
6.26 Incubator, water- or air-jacketed, humidity-controlled, microbiological type to hold temperature at
35.0°C±0.5°C
6.27 Gable covered water bath, with circulating system to maintain temperature of 44.5°C ± 0.2°C
6.28 Plastic sterile petri dishes, microbiological grade, 15 mm x 100 mm
6.29 Erlenmeyer flasks, 1-Land 2-L
6.30 Stir bar
6.31 Stir plate
6.32 Sterile blender jars and base
6.33 Water bath maintained at 50°C for tempering agar
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Method 1680
6.34 Balance, analytical balance capable of weighing 0.1 mg
6.35 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified
thermometer, or one that meets the requirements of NIST Monograph SP 250-23
6.36 Latex gloves for handling samples
6.37 pH meter
6.38 Vortex mixer
6.39 Flasks, borosilicate glass, screw-cap, 250-2000 mL volume
6.40 Graduated cylinders, 100- to 1000-mL, covered with aluminum foil or kraft paper and sterilized
6.41 Beakers, glass or plastic, assorted sizes
6.42 Steel pan of water, 30" x 26" x 10"
6.43 Autoclave or steam sterilizer capable of achieving 121°C [15 Ib pressure per square inch (PSI)] for 15
minutes
6.44 Crucible or aluminum evaporating dish
6.45 Drying oven maintained at 103°C - 105°C for tempering agar
7.0 Reagents and Standards
7.1 Reagent-grade chemicals shall be used in all tests. Unless otherwise indicated, reagents shall conform
to the specifications of the Committee on Analytical Reagents of the American Chemical Society
(Reference 19.3). The agar used in preparation of culture media must be of microbiological grade.
7.2 Whenever possible, use commercial dehydrated culture media.
7.3 Purity of reagent water: Reagent-grade water conforming to specifications in Standard Methods for the
Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or 141, as
applicable), Section 9020 (Reference 19.1).
7.4 Phosphate buffered dilution water
7.4.1 Composition of stock phosphate buffer solution:
Potassium dihydrogen phosphate (KH2PO4) 34.0 g
Reagent-grade water 500.0 mL
Preparation: Dissolve KH2PO4 in 500 mL reagent-grade water. Adjust the pH of the solution
to 7.2 with 1 N NaOH, and bring the volume to 1 L with reagent-grade water. Sterilize by
filtration or autoclave at 121°C (15 PSI) for 15 minutes.
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Method 1680
7.4.2 Preparation of stock magnesium chloride (MgCl2) solution: Add 38 g anhydrous MgCl2 or 81.1
g magnesium chloride hexahydrate (MgCl2 • 6H2O) to 1 L reagent-grade water. Sterilize by
filtration or autoclave at 121°C (15 PSI) for 15 minutes.
7.4.3 After sterilization, store the stock solutions in the refrigerator until used. If evidence of mold or
other contamination appears, the affected stock solution should be discarded and a fresh
solution should be prepared.
7.4.4 Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer and 5
mL of the MgCl2 stock per liter of reagent-grade water. Dispense in appropriate amounts for
dilutions and/or foruse as rinse buffer. Autoclave at 121°C (15 PSI) for 15 minutes. Final pH
should be 7.0 ± 0.2. The amount of time in the autoclave must be adjusted for the volume of
buffer in the containers and the size of the load.
Note: When test tube racks containing 9.0 mL sterile dilution water are prepared, they are
placed into an autoclavable pan with a small amount of water to contain breakage and
minimize evaporation from the tubes.
7.5 Heart infusion agar (HIA)
7.5.1 Composition:
Beef heart, infusion from 500 g 10. Og
Bacto tryptose 10.0 g
Sodium chloride (NaCl) 5.0 g
Bacto agar 15.Og
Reagent-grade water 1.0 L
7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve. AdjustpHto
7.4 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide. Stir well and autoclave at
121°C (15 PSI) for 15 minutes. Pour into 15 x 100 mm sterile petri plates. Letthe media
warm to room temperature prior to inoculation. Other general growth media may be used for
quality assurance (QA) (Section 9.0) purposes.
7.6 LTB medium
7.6.1 Composition:
Tryptose 20.0 g
Lactose 5.0g
Dipotassium hydrogen phosphate (K2HPO4) 2.75 g
Potassium dihydrogen phosphate (KH2PO4) 2.75 g
Sodium chloride (NaCl) 5.0 g
Sodium lauryl sulfate 0.1 g
Reagent-grade water 1.0 L
7.6.2 For single strength (IX) LTB, add reagents to 1 L of reagent-grade water, mix thoroughly, and
heat to dissolve. Adjust pH to 6.8 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium
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Method 1680
hydroxide. Dispense 10-mL volumes into 25 x 150 mm culture tubes. IX TSB will be used
for inoculation volumes <1 mL. Autoclave at 121°C (15 PSI) for 15 minutes.
7.6.3 For double strength (2X) LTB, prepare as in Section 7.6.2 but use 500 mL of reagent-grade
water instead of 1 L.
Note: 2X LTB is necessary for 10-mL inoculations, to ensure that the 10-mL inoculation
volume does not excessively dilute the media.
7.7 EC medium
7.7.1 Composition:
Tryptose or trypticase 20.0 g
Lactose 5.0g
Bile salts mixture or bile salts No.3 1.5 g
Dipotassium hydrogen phosphate (K2HPO4) 4.0 g
Potassium dihydrogen phosphate (KH2PO4) 1.5 g
Sodium chloride (NaCl) 5.0 g
Reagent-grade water 1.0 L
7.7.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve. AdjustpHto
6.9 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide, if necessary. Prior to
sterilization, dispense 10 mL per 16 x 150 mm test tubes, each with an inverted vial, and
sufficient medium to cover the inverted vial halfway after sterilization. Close tubes with metal
or heat-resistant plastic caps. Autoclave at 121°C (15 PSI) for 15 minutes. Medium should
fill inverted tubes leaving no air spaces.
7.8 Positive control
7.8.1 Obtain a stock culture of E. coli (e.g., ATCC 25922) as a positive control for LTB and EC
medium.
Note: ATCC recommends that no more than 5 transfers be made before returning to the
original culture. This will minimize the chance of contamination during transfers and
genetic shift of the culture. One suggestion is to make your own frozen seed stock
upon receipt of the organism that can be used for future work. For additional
information go to http://www.atcc.org.
7.9 Negative controls
7.9.1 Obtain a stock culture of Enterobacter aerogenes (e.g., ATCC 13048) as a negative control for
EC medium.
7.9.2 Obtain a stock culture of Pseudomonas (e.g., ATCC 27853) as a negative control for LTB.
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Method 1680
7.10 The storage times for prepared media used in this method are provided in Table 1.
Table 1. Storage Times for Prepared Media
Media
Agar or broth (EC, LIB, and HIA) in loose-cap tubes
Agar or broth (EC, LIB, and HIA) in tightly closed screw-cap tubes
Poured agar plates (should be stored inverted)
Large volume of agar in tightly closed screw-cap flask or bottle
Storage Time
2 weeks
3 months
2 weeks
3 months
Note'. If media is refrigerated, remove from refrigerator 1-1.5 hours prior to inoculation, so that it reaches room temperature
prior to use.
7.11 Milorganite® (CAS 8049-99-8) or equivalent
Milorganite® (heat-dried Class A biosolid) is produced by Milwaukee Metropolitan Sewerage District.
It is available in many home gardening centers.
Obtain Milorganite® as the reference matrix for initial precision and recovery (IPR) and ongoing
precision and recovery (OPR) analyses. Milorganite® is used as the reference matrix because it is
easily accessible, inexpensive, generally does not contain the analyte of interest, and is of consistent
quality.
8.0 Sample Collection, Handling, and Storage
8.1 The most appropriate location for biosolid sample collection is the point prior to leaving the wastewater
treatment plant. Samples may be taken from pipes, conveyor belts, bins, compost heaps, drying beds,
and stockpiles.
8.2 Collect samples in sterile, non-toxic glass, or plastic containers with leak-proof lids. All sampling
containers and equipment must be clean and sterile.
8.3 Equipment and container cleaning procedure
8.3.1 Wash apparatus with laboratory-grade detergent and water
8.3.2 Rinse with tap water
8.3.3 Rinse with 10% HC1 acid wash
8.3.4 Rinse with distilled water
8.3.5 Allow to air dry
8.3.6 Cover with foil and autoclave for 15 minutes at 121°C (15 PSI)
8.4 Digester biosolids sampling procedure
8.4.1 Collect digester biosolids sample from the discharge pipe.
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Method 1680
8.4.2 Purge the discharge pipe of old biosolids and warm to the digester temperature by allowing
biosolids to flow through the pipe into a container or waste collection device
8.4.3 Position a 1-gallon sterile bag under the flow so that only the sample touches the inside of the
bag. Fill the bag, leaving 0.5 inches of head space in the bag for gas production. Leaving head
room is extremely important when taking samples of biosolids that have been anaerobically
digested.
8.5 Procedure for sampling conveyor belt biosolid output
8.5.1 Using a sterile scoop, transfer the pressed biosolids directly from the conveyer into a sterile
container, without mixing or transferring to another area.
8.5.2 Pack sample into sterile container. Leaving additional head space is not as important as in
Section 8.4 because there is less gas formation.
8.6 Procedure for sampling from a bin, drying bed, truck bed, or similar container
8.6.1 Remove surface material (upper six inches) and set it aside. Divide the underlying material to
be sampled into four quadrants.
8.6.2 Use a scoop or core the sample if material is deep.
8.6.3 Take a sample from each of the quadrants and combine in a sterile container.
8.6.4 After all the samples have been taken, pour the contents of the container out onto a sterile
surface and mix by folding the sample back onto itself several times.
8.6.5 Reduce the sample size by "coning and quartering." Divide the container contents into four
even piles. If sample size is still too large, divide each quarter into quarters and discard half
Put into a glass or plastic sampling container.
8.6.6 An alternate method to "coning and quartering" is to randomly take a flat shovel full of
biosolids from the contents of the container that has been placed on a sterile surface and put
samples into a sampling container. (Curved scoops have been shown to favor a certain size
particle and should not be used.)
8.7 Record the following in your log book:
8.7.1 Facility name and location
8.7.2 Date
8.7.3 Arrival time
8.7.4 Name of facility and contact
8.8 Record the following onto sample container and in log book when known:
8.8.1 Sample number
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Method 1680
8.8.2 Date and time
8.8.3 Sampler name
8.8.4 Sample location
8.8.5 Parameters (e.g., type of analysis, field measurements- pH and temperature)
8.8.6 Volume
8.8.7 Observations
8.9 Ensure that the chain-of-custody form is filled out.
8.10 Sample handling: Maintain bacteriological samples at <10°C during transit to the laboratory. Do not
allow the sample to freeze. Use insulated containers to ensure proper maintenance of storage
temperature. Sample bottles should be placed inside waterproof bags, excess air purged, and bags
sealed to ensure that bottles remain dry during transit or storage. Refrigerate samples upon arrival in the
laboratory and analyze as soon as possible after collection. Bring samples to room temperature before
analysis.
8.11 Holding time and temperature limitations: For fecal coliform samples for sewage sludge (biosolids)
only, the holding time is extended to 24 hours for the following sample types using either EPA Method
1680 (LTB-EC) or 1681 (A-l): Class A composted, Class B aerobically digested, and Class B
anaerobically digested. All other matrices should be analyzed within 8 hours of sample collection, 6
hour maximum transport and 2 hours for sample processing.
Note: Adherence to sample handling procedures and holding time limits is critical to the production of
valid data. Sample results will be considered invalid if these conditions are not met.
9.0 Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance (QA) program.
The minimum requirements of this program consist of an initial demonstration of laboratory capability
through the analysis of positive and negative control samples and blanks (Sections 9.6 and 9.7).
Laboratory performance is compared to the performance criteria specified in Section 16.0 to determine
whether the results of the analyses meet the performance characteristics of the method. Specific quality
control (QC) requirements for Method 1680 are provided below. General recommendations on QA and
QC for facilities, personnel, and laboratory equipment, instrumentation, and supplies used in
microbiological analyses are provided in Reference 19.4.
9.2 The minimum analytical QC requirements for the analysis of samples using Method 1680 include an
initial demonstration of laboratory capability through performance of the initial precision and recovery
(IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through performance of the
ongoing precision and recovery (OPR) analysis (Section 9.4) and matrix spike (MS) analysis (Section
9.5, disinfected wastewater only), and the routine analysis of positive and negative controls (Section
9.6), method blanks (Section 9.7), and media sterility checks (Section 9.8). For the IPR, OPR and MS
analyses, it is necessary to spike samples with laboratory-prepared spiking suspensions as described in
Section 15.0.
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Method 1680
9.3 Initial precision and recovery (IPR): The IPR analyses are used to demonstrate acceptable method
performance (recovery and precision) and should be performed by each laboratory before the method is
used for monitoring field samples. EPA recommends but does not require that an IPR be performed by
each analyst. IPR samples should be accompanied by an acceptable method blank (Section 9.7) and
appropriate media sterility checks (Section 9.8). The IPR analyses are performed as follows:
9.3.1 Prepare four, 30-g samples of Milorganite® and spike each sample with E. coli ATCC 25922
according to the spiking procedure in Section 15.0. Process and analyze each IPR sample
according to the procedures in Sections 11.0 and 12.0 and calculate the fecal coliform MPN/g
dry weight according to Section 14.0.
9.3.2 Calculate the percent recovery (R) for each IPR sample using the appropriate equation in
Section 15.7.
9.3.3 Using the percent recoveries of the four analyses, calculate the mean percent recovery and the
relative standard deviation (RSD) of the recoveries. The RSD is the standard deviation divided
by the mean, multiplied by 100.
9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 2, below.
If the mean and RSD for recovery of fecal coliforms meet acceptance criteria, system
performance is acceptable and analysis of field samples may begin. If the mean recovery or the
RSD fall outside of the required range for recovery, system performance is unacceptable. In
this event, identify the problem by evaluating each step of the analytical process, media,
reagents, and controls, correct the problem and repeat the IPR analyses.
Table 2. Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria
Performance test
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
LTB/EC acceptance criteria
65% -221%
84%
37% -391%
9.4 Ongoing precision and recovery (OPR): To demonstrate ongoing control of the analytical system, the
laboratory should routinely process and analyze spiked Milorganite® samples. The laboratory should
analyze one OPR sample after every 20 field and matrix spike samples or one per week that samples are
analyzed, whichever occurs more frequently. OPR samples must be accompanied by an acceptable
method blank (Section 9.7) and appropriate media sterility checks (Section 9.8). The OPR analysis is
performed as follows:
9.4.1 Spike a 30-g sample of Milorganite® withE! coli ATCC 25922 according to the spiking
procedure in Section 15.0. Process and analyze each OPR sample according to the procedures
in Sections 11.0 and 12.0 and calculate the number of fecal coliform MPN/g dry weight
according to Section 14.0.
9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equations in
Section 15.7.
9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria in
Table 2, above. If the OPR result meets the acceptance criteria for recovery, method
performance is acceptable and analysis of field samples may continue. If the OPR result falls
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Method 1680
outside of the acceptance criteria, system performance is unacceptable. In this event, identify
the problem by evaluating each step of the analytical process (media, reagents, and controls),
correct the problem and repeat the OPR analysis.
9.4.4 As part of the laboratory QA program, results for OPR and IPR samples should be charted and
updated records maintained in order to monitor ongoing method performance. The laboratory
should also develop a statement of accuracy for Method 1680 by calculating the average percent
recovery (R) and the standard deviation of the percent recovery (sr). Express the accuracy as a
recovery interval from R - 2sr to R + 2sr.
9.5 Matrix spikes (MS): MS analysis are performed to determine the effect of a particular matrix on fecal
coliform recoveries. The laboratory should analyze one MS sample when biosolid samples are first
received from a source from which the laboratory has not previously analyzed samples. Subsequently,
5% of field samples (1 per 20) from a given biosolids source should include a MS sample. MS samples
must be accompanied by the analysis of an unspiked field sample sequentially collected from the same
sampling site, an acceptable method blank (Section 9.7), and appropriate media sterility checks (Section
9.8). When possible, MS analyses should also be accompanied by an OPR sample (Section 9.4). The
MS analysis is performed as follows:
9.5.1 Prepare two, 30-g field samples that were sequentially collected from the same site. One
sample will remain unspiked and will be analyzed to determine the background or ambient
concentration of fecal coliforms for calculating MS recoveries (Section 9.5.3). The other
sample will serve as the MS sample and will be spiked with E. coll ATCC 25922 according to
the spiking procedure in Section 15.0.
9.5.2 Select dilutions based on previous analytical results or anticipated levels of fecal coliforms in
the field sample in order to accurately estimate fecal coliform density. Neither above or below
the detection limit of the method. Section 11.0 includes possible dilution schemes for both
Class A and Class B biosolids.
9.5.3 Spike the MS sample with a laboratory-prepared suspension as described in Section 15.0.
Process and analyze the unspiked and spiked field samples according to the procedures in
Sections 11.0 and 12.0.
9.5.4 For the MS sample, calculate the fecal coliform MPN/g dry weight according to Section 14.0
and adjust the density (MPN/g dry weight) based on the ambient concentration of fecal
coliforms observed in the unspiked matrix sample.
9.5.5 Calculate the percent recovery (R) for the MS sample (adjusted based on ambient fecal coliform
in the unspiked sample) using the appropriate equations in Section 15.7.
9.5.6 Compare the MS result (percent recovery) with the appropriate method performance criteria in
Table 3. If the MS recovery meets the acceptance criteria, system performance is acceptable
and analysis of field samples from this biosolid source may continue. If the MS recovery is
unacceptable and the OPR sample result associated with this batch of samples is acceptable, a
matrix interference may be causing the poor results. If the MS recovery is unacceptable, all
associated field data should be flagged.
12 September 2014
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Method 1680
Table 3. Matrix Spike Precision and Recovery Acceptance Criteria
Performance test
Class A Biosolids: Matrix spike (MS)
MS percent recovery
Class B Biosolids: Matrix spike (MS)
MS percent recovery
Class A Biosolids: Matrix spike, matrix spike duplicate (MS/MSD)
Percent recovery for MS/MSD
Precision (as maximum relative percent difference of
MS/MSD)
Class B Biosolids: Matrix spike, matrix spike duplicate (MS/MSD)
Percent recovery for MS/MSD
Precision (as maximum relative percent difference of
MS/MSD)
LTB/EC acceptance criteria
30 - 424%
8 - 709%
30 - 424%
150%
8 - 709%
125%
9.5.7 Laboratories should record and maintain a control chart comparing MS recoveries for all
matrices to batch-specific and cumulative OPR sample results analyzed using Method 1680.
These comparisons should help laboratories recognize matrix effects on method recovery and
may also help to recognize inconsistent or sporadic matrix effects from a particular source.
9.6 Culture Controls
9.6.1 Negative controls: The laboratory should analyze negative controls to ensure that the LTB and
EC are performing properly. Negative controls should be analyzed whenever a new batch of
media or reagents is used. On an ongoing basis, the laboratory should perform a negative
control every day that samples are analyzed.
9.6.1.1 Negative controls are conducted by inoculating LTB with a known negative total
coliform species (e.g., Pseudomonas ATCC 27853) and EC with a known negative
fecal coliform species (e.g.,Enterobacter aerogenes ATCC 13048) and analyzing as
described in Section 12.0. Viability of the negative controls should be demonstrated
using a non-selective media (e.g., nutrient agar or tryptic soy agar).
9.6.1.2 If a negative control fails to exhibit the appropriate response, check and/or replace
the associated media or reagents, and/or the negative control, and re-analyze the
appropriate negative control.
9.6.2 Positive controls: The laboratory should analyze positive controls to ensure that the LTB and
EC are performing properly. Positive controls should be analyzed whenever a new batch of
media or reagents is used. On an ongoing basis, the laboratory should perform a positive
control every day that samples are analyzed. An OPR sample (Section 9.4) may take the place
of a positive control.
9.6.2.1 Positive controls are conducted by inoculating LTB and EC with a known positive
fecal coliform species (e.g.,E. coll ATCC 25922) and analyzing as described in
Section 12.0.
9.6.2.2 If the positive control fails to exhibit the appropriate response, check and/or replace
the associated media or reagents, and/or the positive control, and reanalyze the
appropriate positive control.
9.7 Method blank. Test a 20-mL sterile dilution water sample in the analytical scheme to verify the
sterility of equipment, materials, and supplies. Absence of growth indicates freedom of contamination
13
September 2014
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Method 1680
from the target organism. On an ongoing basis, the laboratory should perform a method blank every
day that samples are analyzed.
9.8 Media sterility check. To test sterility of media, subject a representative portion of each batch to
incubation at 35°C ± 0.5°C (LTB) or 44.5°C ± 0.2°C (EC) for 48 ± 3 or 24 ± 2 hours respectively and
observe for growth. With respect to media, a batch is defined as one tube/plate out of 50 in each lot or
one tube/plate, if the lot contains less than 50 tubes/plates. Absence of growth indicates media sterility.
On an ongoing basis, the laboratory should perform a media sterility check every day that samples are
analyzed.
10.0 Equipment Calibration and Standardization
10.1 Check temperatures in incubators/water baths twice daily, a minimum of four hours apart, to ensure
operation is within stated limits of the method and record daily measurements in incubator log book.
10.2 Check temperatures in refrigerators/freezers at least once daily to ensure operation is within stated limits
of the method. Record daily measurements in refrigerator/freezer log book.
10.3 Calibrate thermometers and incubators at least annually against an NIST certified thermometer or one
that meets requirements of NIST Monograph SP 250-23 (Reference 19.1). Check mercury columns for
breaks.
10.4 Calibrate the pH meter prior to each use period with the two standards (pH4.0, 7.0, and 10.0) closest to
the range being tested.
10.5 Calibrate top-loading balances monthly with reference weights of ASTM Class 2.
11.0 Sample Preparation
11.1 Horn ogenization
Sample homogenization procedures are based on whether the sample is a liquid or a solid. If sample is
alkaline-stabilized (liquid or solid), adjust the pH as described in Section 11.1.3. Liquid samples are
generally defined as samples containing <7% total solids (dry weight).
11.1.1 Liquid samples: Homogenize 300 mL of sample in a sterile blender on high speed for one to
two minutes. Adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid or 1.0 N sodium
hydroxide, if necessary. This is the "homogenized" sample. When adjusting the pH do not
exceed the homogenized sample volume by greater than 5% (15 mL).
11.1.2 Solid samples: Weigh out 30.0 ± 0.1 g of well-mixed sample in a sterile dish. Whenever
possible, the sample tested should contain all materials that will be included in the biosolid.
For example, if wood chips are part of the biosolid compost, some mixing or grinding may be
needed to achieve homogeneity before testing. Large pieces of wood that are not easily ground
may be discarded before homogenizing. Transfer the sample to a sterile blender.
Alternatively, the sample may be weighed directly into the sterile blender jar. Use 270 mL of
sterile dilution water (Section 7.4) to rinse any remaining sample into the blender. Cover and
blend on high speed for one minute. This is the "homogenized" sample. A volume of 1.0-mL
14 September 2014
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Method 1680
of the "homogenized" sample contains 10"1 g of the original sample. Adjust the pHto 7.0-7.5
by adding 1.0 N hydrochloric acid or 1.0 N sodium hydroxide, if necessary.
Note: Do not suspend bacteria in dilution water for more than 30 minutes at room
temperature. Chill on wet ice or at 4°C ± 1°C to slow replication between spiking
samples.
11.1.3 Alkaline-stabilized: The alkaline-stabilized biosolid samples generally have a pH of
approximately 12. Prior to analysis, the alkaline-stabilized biosolid samples must be
neutralized to apH of approximately 7.5. Do not add lab-prepared spikes to the samples prior
to pH adjustment.
11.1.3.1 Adjustment of pH should be done in a fume hood. Prior to adjusting the pH of the
sample, calibrate/standardize the pH meter with pH buffers 7.0 and 10.0. Weigh
out 30 g of sample into a sterile 600 mL beaker, add 250 mL of sterile buffered
dilution water and a sterile magnetic stir bar. Place beaker on a mixing plate,
insert pH probe into mixture, begin stirring, and take an initial pH reading. To
minimize the amount of volume added to each sample, pH should be adjusted
using 10 NHC1.
Note: The addition of the ION HC1 will produce fumes, do not be alarmed.
The addition of the acid should be done incrementally to ensure that the
pH does not drop instantaneously below 5.0. It is recommended that the
pH adjustment be completed within 10-15 minutes and monitored for an
additional 15 minutes to ensure that the sample is able to maintain a
constant pH of around 7.5. Pour pH adjusted sample into blender jar, use
the remaining sterile buffered dilution water (15 mL) to rinse the beaker
twice and pour rinse water into the blender jar.
11.2 Dilution and Inoculation
Biosolid samples analyzed for fecal coliforms using this method may require dilution prior to analysis.
An ideal sample volume will yield results that accurately estimate fecal coliform density. Because fecal
coliform concentrations in undiluted samples could easily exceed the analytical range of this procedure,
the laboratory must follow the dilution and inoculation schemes in Section 11.2.1 (liquid) or 11.2.2
(solid), if necessary additional dilutions may be analyzed to ensure results obtained are not censored
(less-than or greater-than) values. Although other dilution and inoculation schemes may be used, the
first transfer from the "homogenized" sample should always be 11 mL of homogenized sample to 99 mL
dilution water or 10 mL of homogenized sample to 90 mL dilution water. This will ensure that a
sufficient amount of the original biosolid sample is transferred at the beginning of the dilution scheme.
Note: Do not suspend bacteria in dilution water for more than 30 minutes at room temperature. For
some transfers, it may be convenient to use a sterile, wide-mouth pipette, capable of transferring
particulate matter. If samples are being spiked, a maximum of 1 hour may elapse between
initial unspiked sample homogenization and analysis of spiked samples.
11.2.1 Class A liquid samples: Four series of five tubes each will contain 1.0, 10"1, 10"2,and
10"3 mL of the original sample. See Figure 2 in Section 20.0 for an overview of this dilution
and inoculation scheme. (For spiked samples, four series of five tubes each will be used for the
analysis with 10"2, 10"3, 10^, and 10"5 mL of the original sample.)
15 September 2014
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Method 1680
11.2.1.1 Dilution
(A) Use a sterile pipette to transfer 11.0 mL of "homogenized" sample (Section
11.1.1) to 99 mL of sterile dilution water (Section 7.4), cap, and mix by
vigorously shaking the bottle a minimum of 25 times. This is dilution "A."
One mL of dilution "A" contains 10"1 mL of the original sample.
(B) Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of sterile
dilution water, and mix as before. This is dilution "B." One mL of dilution
"B" is 10~2 mL of the original sample.
(C) Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of sterile
dilution water, and mix as before. This is dilution "C." One mL of dilution
"C" is 10~3 mL of the original sample.
(D) Additional dilutions for analysis of spiked samples.
• Use a sterile pipette to transfer 11.0 mL of dilution "C" to 99 mL of sterile
dilution water, and mix as before. This is dilution "D." OnemLof
dilution "D" is 10^ mL of the original sample.
• Use a sterile pipette to transfer 11.0 mL of dilution "D" to 99 mL of sterile
dilution water, and mix as before. This is dilution "E." OnemLof
dilution "E" is 10"5 mL of the original sample.
11.2.1.2 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of five tubes with 1.0
mL of the original "homogenized" sample per tube (unspiked samples only).
(B) Use a sterile pipette to inoculate each of the second series of tubes with 1.0 mL
of dilution "A" (unspiked samples only). This is 10"1 mL of the original
sample.
(C) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "B"
(unspiked or spiked samples). This is 10"2 mL of the original sample.
(D) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "C"
(unspiked or spiked samples). This is 10"3 mL of the original sample.
(E) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "D"
(spiked samples). This is 10^ mL of the original sample.
(F) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "E"
(spiked samples). This is 10"5 mL of the original sample.
11.2.1.3 Repeat steps 11.2.1.1 and 11.2.1.2 for the remaining Class A samples. When
inoculations are complete, go to Section 12.3.1.4 to continue the LTB/EC method.
11.2.2 Class A solid samples: For unspiked samples, four series of five tubes will be used for the
analysis with 1.0, 10"1,10"2 and 10"3 g of the original sample. The first series of tubes must
16 September 2014
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Method 1680
contain 2X media. See Figure 3 in Section 20.0 for a summary of this dilution and inoculation
scheme. (For spiked samples, four series of five tubes each will be used for the analysis with
10"2, 10"3, 10^, and 10"5 g of the original sample.)
11.2.2.1 Dilution
(A) A 1.0-mL volume of the "homogenized" sample (Section 11.1.2) contains 10"1
g of the original sample.
(B) Use a sterile pipette to transfer 11.0 mL of the blender contents to 99 mL of
sterile dilution water (Section 7.4) and shake vigorously a minimum of 25
times. This is dilution "A." One mL of dilution "A" contains 10"2 g of the
original sample.
(C) Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of sterile
dilution water and mix as before. This is dilution "B." One mL of dilution
"B" contains 10"3 g of the original sample.
(D) Additional dilutions for analysis of spiked samples:
• Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of sterile
dilution water, and mix as before. This is dilution "C." One mL of
dilution "C" contains \0A g of the original sample.
• Use a sterile pipette to transfer 11.0 mL of dilution "C" to 99 mL of sterile
dilution water and mix as before. This is dilution "D." One mL of
dilution "D" contains 10"5 g of the original sample.
11.2.2.2 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of five tubes with 10.0
mL of the "homogenized" sample (unspiked samples only). This series of
tubes must contain 2X media. This is 1.0 g of the original sample. Since
test tubes with inverted vials are being used, shaking is not practical. Solids
that will not separate easily and/or may float should be submerged into the
broth with a sterile loop.
(B) Use a sterile pipette to inoculate each of five tubes with 1 mL of the
"homogenized" mixture (unspiked samples only). This is 10"1 g of the
original sample.
(C) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "A"
(unspiked or spiked samples). This is 10"2 g of the original sample.
(D) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "B"
(unspiked or spiked samples). This is 10"3 g of the original sample.
(E) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "C"
(spiked samples). This is 10^ g of the original sample.
17 September 2014
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Method 1680
(F) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "D"
(spiked samples). This is 10~5 g of the original sample.
11.2.2.3 Repeat Sections 11.2.2.1 and 11.2.2.2 for remaining Class A solid samples. When
inoculations are complete, go to Section 12.3.1.4 to continue the LTB/EC method.
11.2.3 Class B liquid samples: For unspiked samples, four series of five tubes each will be used for the
analysis with 10~3, 10^, 10~5, and 10~6 mL of the original sample (additional dilutions may be
analyzed as necessary). See Figure 4 in Section 20.0 for a summary of this dilution and
inoculation scheme. (For spiked samples, five series of five tubes each will be used for the
analysis with 10"5, 10"6, 10"7, 10"8, and 10"9 mL of the original sample.)
11.2.3.1 Dilution
(A) Use a sterile pipette to transfer 11.0 mL of homogenized sample (from Section
11.1.1) to 99 mL of sterile dilution water (Section 7.4), cap, and mix by
vigorously shaking the bottle a minimum of 25 times. This is dilution "A."
One mL of dilution "A" is 10"1 mL of the original sample.
(B) Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of sterile
dilution water, and mix as before. This is dilution "B." One mL of dilution
"B" is 10"2 mL of the original sample.
(C) Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of sterile
dilution water, and mix as before. This is dilution "C." One mL of dilution
"C" is 10"3 mL of the original sample.
(D) Use a sterile pipette to transfer 11.0 mL of dilution "C" to 99 mL of sterile
dilution water, and mix as before. This is dilution "D." One mL of dilution
"D" is 10^ mL of the original sample.
(E) Use a sterile pipette to transfer 11.0 mL of dilution "D" to 99 mL of sterile
dilution water, and mix as before. This is dilution "E." One mL of dilution
"E" is 10"5 mL of the original sample.
(F) Use a sterile pipette to transfer 11.0 mL of dilution "E" to 99 mL of sterile
dilution water, and mix as before. This is dilution "F." One mL of dilution
"F" is 10"6 mL of the original sample.
(G) Additional dilutions for analysis of spiked samples:
• Use a sterile pipette to transfer 11.0 mL of dilution "F" to 99 mL of sterile
dilution water, and mix as before. This is dilution "G." One mL of
dilution "G" is 10"7 mL of the original sample.
• Use a sterile pipette to transfer 11.0 mL of dilution "G" to 99 mL of sterile
dilution water, and mix as before. This is dilution "H." OnemLof
dilution "H" is 10"8 mL of the original sample.
18 September 2014
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Method 1680
• Use a sterile pipette to transfer 11.0 mL of dilution "H" to 99 mL of sterile
dilution water, and mix as before. This is dilution "I." OnemLof
dilution "I" is 10~9 mL of the original sample.
11.2.3.2 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of five tubes with 1.0
mL of dilution "C" (unspiked samples only). This is 10~3 mL of the original
sample.
(B) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "D"
(unspiked samples only). This is 10^ mL of the original sample.
(C) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "E"
(unspiked or spiked samples). This is 10~5 mL of the original sample.
(D) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "F"
(unspiked or spiked samples). This is 10~6 mL of the original sample.
(E) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "G"
(spiked samples). This is 10~7 mL of the original sample.
(F) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "H"
(spiked samples). This is 10~8 mL of the original sample.
(G) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "I"
(spiked samples). This is 10~9 mL of the original sample.
11.2.3.3 Repeat Sections 11.2.3.1 and 11.2.3.2 for each remaining Class B sample. When
inoculations are complete, proceed to Section 12.3.1.4 to continue the LTB/EC
method.
11.2.4 Class B solid samples: For unspiked samples, four series of five tubes each will
contain 10~3,10^, 10~5, and 10~6 g of the original sample (additional dilutions may be analyzed as
necessary). See Figure 5 in Section 20.0 for a summary of this dilution and inoculation
scheme. (For spiked samples, five series of five tubes each will be used for the analysis with
10-5, 10-6, 10-7, 10-8, and 10'9 g of the original sample.)
11.2.4.1 Dilution
(A) A volume of 1.0-mL of the "homogenized" sample (Section 11.1.2) contains
10"1 g of the original sample.
(B) Use a sterile pipette to transfer 11.0 mL of the blender contents to 99 mL of
sterile dilution water (Section 7.4) and shake vigorously a minimum of 25
times. This is dilution "A." One mL of dilution "A" contains 10"2 g of the
original sample.
(C) Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of sterile
dilution water, and mix as before. This is dilution "B." OnemLof dilution
"B" contains 10"3 g of the original sample.
19 September 2014
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Method 1680
(D) Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of sterile
dilution water, and mix as before. This is dilution "C." One mL of dilution
"C" contains 10^ g of the original sample.
(E) Use a sterile pipette to transfer 11.0 mL of dilution "C" to 99 mL of sterile
dilution water and mix as before. This is dilution "D." One mL of dilution
"D" contains 10~5 g of the original sample.
(F) Use a sterile pipette to transfer 11.0 mL of dilution "D" to 99 mL of sterile
dilution water and mix as before. This is dilution "E." One mL of dilution
"E" contains 10~6 g of the original sample.
(G) Additional dilutions for analysis of spiked samples:
• Use a sterile pipette to transfer 11.0 mL of dilution "E" to 99 mL of sterile
dilution water, and mix as before. This is dilution "F." One mL of
dilution "F" is 10~7 g of the original sample.
• Use a sterile pipette to transfer 11.0 mL of dilution "F" to 99 mL of sterile
dilution water, and mix as before. This is dilution "G." One mL of
dilution "G" is 10~8 g of the original sample.
• Use a sterile pipette to transfer 11.0 mL of dilution "G" to 99 mL of sterile
dilution water, and mix as before. This is dilution "H." One mL of
dilution "H" is 10~9 g of the original sample.
11.2.4.2 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of five tubes with 1.0
mL of dilution "B" (unspiked samples only). This is 10~3 g of the original
sample.
(B) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "C"
(unspiked samples only). This is 10^ g of the original sample.
(C) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "D"
(unspiked or spiked samples). This is 10~5 g of the original sample.
(D) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "E"
(unspiked or spiked samples). This is 10~6 g of the original sample.
(E) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "F"
(spiked samples). This is 10~7 g of the original sample.
(F) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "G"
(spiked samples). This is 10~8 g of the original sample.
(G) Use a sterile pipette to inoculate each of five tubes with 1.0 mL of dilution "FT
(spiked samples). This is 10~9 g of the original sample.
20 September 2014
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Method 1680
11.2.4.3 When inoculations are complete, go to Section 12.3.1.4 to continue the LTB/EC
method.
12.0 Lauryl Tryptose Broth (LIB) - EC Broth Procedure
12.1 In this protocol, the Lauryl-Tryptose Broth - EC medium is used to determine fecal coliform densities in
Class A and B biosolid samples. Analysis of seven samples collected at the time of disposal using this
procedure will satisfy the requirements of the monitoring alternative for demonstrating pathogen
reduction in both Class A and Class B biosolids. In Method 1680, LTB is used as a presumptive
medium followed by EC as confirmation of fecal coliforms. EC may not be used for direct isolation
from a biosolid sample because prior enrichment in presumptive medium (LTB) is required for optimum
recovery of fecal coliforms. Precision of the test increases with increasing numbers of replicates per
sample tested. For an overview of the MPN procedure, refer to Figure 1 in Section 20.0.
12.2 Since sample fecal coliform densities are expected to be variable, it is recommended that at least seven
biosolid samples be analyzed using this method. The geometric mean fecal coliform density of the
seven biosolids samples should not exceed 2 x 106 MPN/g of total solids (dry weight basis) to qualify as
Class B biosolids. Although there is not a specific number of samples required for Class A biosolids, it
is recommended that a sampling event extend over two weeks and that at least seven samples be
collected and determined to be below 1,000 MPN/g of total solids (dry weight basis) to qualify as Class
A biosolids.
12.3 LTB/EC Procedure
12.3.1 Presumptive phase with LTB medium
12.3.1.1 Prepare LTB media and dispense into tubes as directed in Section 7.6.
Note: If media is refrigerated, remove from refrigerator 1-1.5 hours prior to
inoculation, so that it reaches room temperature prior to use.
12.3.1.2 For each sample, arrange test tubes in four rows of five tubes each (Section 11.2).
When 10 mL of sample or dilution is used, tubes should contain 10 mL of 2X LTB
media. Clearly label each row of tubes to identify the sample and dilution to be
inoculated.
Note: 2X LTB is needed for 10 mL inoculations, to ensure that the 10-mL
inoculation volume does not excessively dilute the LTB.
12.3.1.3 Dilute and inoculate samples depending on the matrix (/'.e., Class A solid, Class B
liquid), as described in Section 11.2.
12.3.1.4 Incubate inoculated tubes at 35°C ± 0.5°C. After 24 ± 2 hours, swirl each tube
gently and examine it for growth and gas production. If no gas has formed,
reincubate for an additional 24 ± 2 hours and reassess. Final assessment should be
within a total of 48 ± 3 hours.
12.3.1.5 For tubes with growth, the presence of gas in inverted vials within 48 ± 3 hours
signifies a positive presumptive reaction.
21 September 2014
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Method 1680
Note: The presence of gas in the absence of growth is usually due to mishandling
or improper shaking of the tubes after inoculation.
12.3.1.6 For tubes with a positive presumptive reaction, proceed to the confirmation phase
(See Photo 1).
Photo 1. In LTB (two tubes on left with silver caps) and EC (two tubes on right with
red caps), fecal coliforms produce turbidity and gas (tubes 1 and 3, when
counting from the left).
12.3.2 Confirmation phase for fecal coliforms using EC medium
12.3.2.1 Prepare EC broth tubes as described in Section 7.7. For each positive LTB tube, one
EC tube will be inoculated.
Note: If media is refrigerated, remove from refrigerator 1-1.5 hours prior to
inoculation, so that it reaches room temperature prior to use.
12.3.2.2 Gently shake tubes from presumptive test showing positive reaction.
12.3.2.3 Using a sterile 3- to 3.5-mm-diameter loop or sterile wooden applicator stick, transfer
growth from each positively presumptive LTB tube to corresponding tubes
containing EC broth.
12.3.2.4 Place all EC tubes in a 44.5°C ± 0.2°C water bath within 30 minutes of inoculation
and incubate for 24 ± 2 hours. Maintain water level above the media in immersed
tubes.
12.3.2.5 After incubation, examine each tube for growth and gas production. Gas production
with growth in EC broth at 24 ± 2 hours is considered a positive fecal coliform
reaction (See Photo 1). Failure to produce gas constitutes a negative reaction.
Note: The presence of gas in the absence of growth is usually due to mishandling
or improper shaking of the tubes after inoculation.
12.3.2.6 Record positive and negative reactions for the EC tubes. Calculate MPN / g of total
solids (dry weight) from the number of positive EC tubes as described in Section
14.0.
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Method 1680
12.4 Total solids determination
1 2.4. 1 Determination of percent dry weight - When sample results are to be calculated on a dry weight
basis, a second portion of sample should be weighed at the same time as the portion used for
analytical determination.
WARNING: The drying oven should be contained in a hood or be vented. Significant
laboratory contamination may result from drying a heavily contaminated sample.
1 2.4.2 Immediately after weighing the sample for microbiological examination, weigh 10-30 g of the
sample into a tarred crucible or aluminum evaporating dish. Dry this aliquot overnight at
103°C to 105°C. Allow to cool in a desiccator before weighing. Calculate the % dry weight
as follows:
g dry sample
% dry weight = g sample x 100
13.0 Verification
1 3.1 Individual biochemical tests including oxidase, citrate, lactose fermentation, indole, ONPG hydroloysis,
methyl red, and Voges-Proskauer may be used to verify positive and negative results.
1 3.2 Alternately, commercially available multi-test identification systems may be used to verify positive and
negative results. Such identification systems for Enterobacteriaceae must include lactose fermentation,
o-nitrophenyl-(3-D-galactopyranoside (ONPG), and cytochrome oxidase test reactions.
Note: Due to the high false positive and negative rates, it is recommended that analysts be required to
submit all positive and negative tubes with growth to verification for at least one biosolid
sample for each type of biosolid that the analyst normally evaluates on a monthly basis. This
should increase analyst proficiency in using these procedures.
14.0 Data Analysis and Calculations
The estimated density of fecal coliform bacteria, based on the confirmation test using EC, is calculated in terms of
most probable number (MPN). Due to the extreme variability in the solid content of biosolids, fecal coliform
results from biosolid samples are reported as MPN/g total solids (dry weight basis). MPN / g total solids (dry
weight) is calculated in three steps (Sections 14.1, 14.2, and 14.3):
Selection of significant dilutions
• Calculation of MPN/mL (wet weight)
• Conversion to MPN/g total solids (dry weight)
The calculation of geometric means is provided in Section 14.4.
14.1 Step 1: Select Significant Dilutions
A dilution refers to the mL (liquid samples) or g (solid samples) of original sample that was inoculated
into each series of tubes. For example, with Class B solid samples (Section 11.2.4), four, five-tube
dilutions are used, with 10"3, 10^, 10"5, and 10"6 g of the original sample in each tube. Only three of the
23 September 2014
-------
Method 1680
four dilution series will be used to estimate the MPN. The three selected dilutions are called significant
dilutions and are selected according to the following criteria. Examples of significant dilution selections
are provided in Table 5, below. For these examples, the numerator represents the number of positive
tubes per sample dilution series and the denominator represents the total number of tubes inoculated per
dilution series.
14.1.1 Choose the highest dilution (the most dilute, with the least amount of sample) giving positive
results in all five tubes inoculated and the two succeeding higher (more dilute) dilutions (For
Table 5, Example A, 10^ is higher/more dilute than 10~3.)
14.1.2 If the lowest dilution (least dilute) tested has less than five tubes with positive results, select it
and the two next succeeding higher dilutions (Table 5, Examples B and C).
14.1.3 When a positive result occurs in a dilution higher (more dilute) than the three significant
dilutions selected according to the rules above, change the selection to the lowest dilution (least
dilute) that has less than five positive results and the next two higher dilutions (more dilute)
(Table 5, Example D).
14.1.4 When the selection rules above have left unselected any higher dilutions (more dilute) with
positive results, add those higher-dilution positive results to the results for the highest selected
dilution (Table 5, Example E).
14.1.5 If there were not enough higher dilutions tested to select three dilutions, then select the next
lower dilution (Table 5, Example F).
14.2 Step 2: Calculate MPN/mL (wet weight)
14.2.1 Obtain the MPN index value from Table 4 using the number of positive tubes in the three
significant dilutions series and calculate MPN/mL using the following equation. The 95%
confidence limits may also be obtained from Table 4. Table 4 has been adjusted by a factor of
10 to eliminate having to multiply the MPN index by 10 in the equation provided below.
Note: The example calculated numbers provided in the tables below have been rounded at the
end of each step. If your laboratory recalculates the examples using a spreadsheet and
rounds only after the final calculation, values may be slightly different.
MPN Index from Table 4
Largest volume tested in the dilution series used for MPN determination
14.2.2 When using MPN tables other than those provided in this method (e.g. Table 9221 :FV, Standard
Methods for Water and Wastewater, Reference 19.1), additional steps/calculations are required
to determine final reporting value of MPN/g dry weight. For example, Table 9221:FV MPN
index is per 100 mL (MPN/100 mL); which will need to be converted to MPN/mL . In addition,
the MPN index must be multiplied by a factor of 10 when using the largest volume tested in the
dilution series used for MPN determination.
,.nM/,nn , 10 x MPN Index from Table 9221 :VI
MPN/100 mL =
MPN / mL
Largest volume tested in the dilution series used for MPN determination
MPN/100 mL
100
24 September 2014
-------
Method 1680
Table 4. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five Tubes
are Used per Dilutiona
Combination of
Positives
0-0-0
0-0-1
0-0-2
U 0-0-3
0-0-4
0-0-5
0-1-0
0-1-1
0-1-2
0-1-3
0-1-4
0-1-5
0-2-0
0-2-1
0-2-2
0-2-3
0-2-4
0-2-5
0-3-0
0-3-1
0-3-2
0-3-3
0-3-4
0-3-5
0-4-0
0-4-1
0-4-2
0-4-3
0-4-4
r 0-4-5
0-5-0
0-5-1
0-5-2
0-5-3
0-5-4
0-5-5
1-0-0
1-0-1
1-0-2
1-0-3
1-0-4
105
1-1-0
1-1-1
1-1-2
1-1-3
1-1-4
1-1-5
1-2-0
1-2-1
1-2-2
1-2-3
1-2-4
1-2-5
MPN Index
ml
<0.1803
0.18
0.36
0.54
0.72
0.91
0.18
0.36
0.55
0.73
0.91
1.10
0.37
0.55
0.74
0.92
1.11
1.29
0.56
0.74
0.93
1.12
1.30
1.49
0.75
0.94
1.12
1.31
1.50
1.69
0.94
1.13
1.33
1.52
1.71
1.90
0.20
0.40
0.60
0.81
1.01
1 22
0.40
0.61
0.81
1.02
1.23
1.44
0.61
0.82
1.03
1.24
1.46
1 67
95% Confidence Limits
Lower
0.03
0.03
0.03
0.08
0.15
0.03
0.03
0.03
0.08
0.15
0.23
0.03
0.03
0.08
0.15
0.23
0.31
0.03
0.09
0.16
0.23
0.31
0.39
0.09
0.16
0.24
0.32
0.40
0.48
0.16
0.24
0.32
0.40
0.48
0.56
0.03
0.03
0.03
0.11
0.19
0.03
0.03
0.11
0.19
0.28
0.37
0.03
0.12
0.20
0.29
0.38
047
Upper
0.63
1.01
1.37
1.74
2.12
0.63
1.01
1.38
1.75
2.14
2.56
1.02
1.39
1.76
2.15
2.58
3.07
1.40
1.77
2.17
2.60
3.10
3.72
1.79
2.19
2.63
3.13
3.77
4.62
2.21
2.65
3.17
3.82
4.70
5.63
0.68
1.08
1.49
1.91
2.36
2.87
1.09
1.50
1.92
2.38
2.90
3.54
1.94
2.40
2.93
3.59
451
Combination of
Positives
1-3-0
1-3-1
1-3-2
1-3-3
1-3-4
1-3-5
1-4-0
1-4-1
1-4-2
1-4-3
1-4-4
1-4-5
1-5-0
1-5-1
1-5-2
1-5-3
1-5-4
1-5-5
2-0-0
2-0-1
2-0-2
2-0-3
2-0-4
2-0-5
2-1-0
2-1-1
2-1-2
2-1-3
2-1-4
2-1-5
2-2-0
2-2-1
2-2-2
2-2-3
2-2-4
2-2-5
2-3-0
2-3-1
2-3-2
2-3-3
2-3-4
2-3-5
2-4-0
2-4-1
2-4-2
2-4-3
2-4-4
2-4-5
2-5-0
2-5-1
2-5-2
2-5-3
2-5-4
2-5-5
MPN Index
mL
0.83
1.04
1.25
1.47
1.69
1.91
1.05
1.27
1.48
1.70
1.93
2.15
1.28
1.50
1.72
1.95
2.17
2.40
0.45
0.68
0.91
1.15
1.39
1.64
0.68
0.92
1.16
1.41
1.66
1.92
0.93
1.18
1.43
1.68
1.94
2.21
1.44
1.70
1.97
2.23
2.51
1.46
1.72
1.99
2.26
2.54
2.82
1.74
2.01
2.29
2.57
2.86
315
95% Confidence Limits
Lower
0.12
0.20
0.29
0.38
0.48
0.57
0.21
0.30
0.39
0.48
0.58
0.67
0.30
0.40
0.49
0.58
0.68
0.77
0.03
0.06
0.15
0.25
0.35
0.46
0.06
0.15
0.25
0.36
0.46
0.57
0.16
0.26
0.36
0.47
0.58
0.69
0.26
0.37
0.48
0.59
0.70
0.82
0.38
0.49
0.60
0.72
0.83
0.94
0.50
0.61
0.73
0.84
0.95
1 07
Upper
1.96
2.43
2.96
3.64
4.60
5.66
2.45
3.00
3.70
4.68
5.75
6.57
3.03
3.75
4.77
5.83
6.64
7.31
1.19
1.64
2.13
2.69
3.38
4.37
1.66
2.16
2.72
3.43
4.47
5.71
2.18
2.76
3.49
4.56
5.81
6.75
2.79
3.55
4.67
5.91
6.83
7.59
3.61
4.77
6.00
6.92
7.68
8.36
4.88
6.10
7.00
7.76
8.45
910
25
September 2014
-------
Method 1680
Table 4. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results When Five Tubes
are Used per Dilutiona
Combination of
Positives
3-0-0
3-0-1
3-0-2
3-0-3
3-0-4
3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
r3-1-5
3-2-0
3-2-1
3-2-2
3-2-3
3-2-4
3-2-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-0
3-4-1
3-4-2
3-4-3
3-4-4
3-4-5
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
4-0-1
4-0-2
4-0-3
4-0-4
4-0-5
r 4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0
4-2-1
4-2-2
4-2-3
4-2-4
4-2-5
MPN Index
ml
0.79
1.06
1.35
1.65
1.96
2.29
1.07
1.37
1.67
1.99
2.32
2.67
1.38
1.70
2.02
2.36
2.71
3.08
1.72
2.05
2.40
2.76
3.13
3.52
2.09
2.44
2.81
3.19
3.58
3.99
2.48
2.86
3.25
3.65
4.07
450
1.30
1.66
2.07
2.53
3.02
3.55
1.69
2.12
2.58
3.10
3.65
4.25
2.16
2.64
3.17
3.75
4.38
5.04
95% Confidence Limits
Lower
0.10
0.21
0.33
0.46
0.59
0.73
0.22
0.34
0.47
0.60
0.74
0.88
0.35
0.48
0.62
0.76
0.90
1.04
0.49
0.63
0.77
0.92
1.06
1.20
0.64
0.79
0.93
1.08
1.23
1.37
0.80
0.95
1.10
1.25
1.40
1 54
0.31
0.46
0.64
0.82
1.02
1.21
0.48
0.66
0.85
1.05
1.25
1.45
0.67
0.87
1.08
1.29
1.50
1.71
Upper
1.88
2.46
3.23
4.40
5.89
6.99
2.50
3.29
4.52
6.01
7.10
8.00
3.35
4.64
6.13
7.20
8.10
8.94
4.77
6.24
7.31
8.21
9.06
9.89
6.35
7.42
8.33
9.18
10.02
10.86
7.53
8.44
9.31
10.17
11.03
11 89
3.11
4.45
6.31
7.64
8.81
9.96
4.60
6.46
7.79
8.98
10.16
11.38
6.61
7.94
9.15
10.37
11.64
12.97
Combination of
Positives
4-3-0
4-3-1
4-3-2
4-3-3
4-3-4
4-3-5
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-0
4-5-1
4-5-2
4-5-3
4-5-4
4-5-5
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-1-0
5-1-1
5-1-2
5-1-3
5-1-4
5-1-5
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-0
5-3-1
5-3-2
5-3-3
5-3-4
5-3-5
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
mL
2.71
3.26
3.86
4.51
5.21
5.93
3.35
3.98
4.66
5.39
6.15
6.93
4.11
4.83
5.59
6.39
7.22
8.06
2.40
3.14
4.27
5.78
7.59
9.53
3.29
4.56
6.31
8.39
10.62
12.93
4.93
7.00
9.44
12.05
14.79
17.67
7.92
10.86
14.06
17.50
21.22
25.27
12.99
17.24
22.12
27.81
34.54
42.56
23.98
34.77
54.22
91.78
160.90
>1 60.90
95% Confidence Limits
Lower
0.90
1.11
1.32
1.54
1.76
1.96
1.14
1.37
1.59
1.81
2.02
2.23
1.41
1.64
1.87
2.09
2.30
2.50
0.76
1.06
1.46
1.92
2.39
1.65
1.12
1.56
2.07
2.57
3.04
3.04
2.24
2.80
3.31
3.81
503
2.47
3.08
3.68
4.34
5.29
8.14
3.48
4.29
5.63
8.82
11.59
14.37
7.62
11.72
17.91
26.72
38.37
Upper
8.09
9.34
10.60
11.92
13.31
14.77
9.53
10.84
12.23
13.68
15.21
16.81
11.11
12.56
14.09
15.70
17.39
19.16
7.63
9.08
11.42
14.46
18.16
22.34
9.40
12.02
15.53
19.85
24.85
30.90
12.76
16.94
22.13
28.43
37.14
52.30
18.86
25.44
34.45
51.31
67.98
79.71
31.08
49.75
70.87
86.00
101.10
118.00
76.29
101.60
141.90
220.10
410.30
Table was developed using the MPN calculator developed by Albert Klee
26
September 2014
-------
Method 1680
Examples of MPN/mL calculations are provided in Table 5.
Table 5. Examples of Significant Dilution Selection and Calculation of MPN/mL'
Example
(liquid
or solid)
A
B
C
D
E
F
io-3
mLorg
5/5
4/5
0/5
5/5
4/5
5/5
1C'4
mLorg
5/5
5/5
1/5
3/5
4/5
5/5
io-5
mLorg
3/5
1/5
0/5
1/5
0/5
5/5
io-6
mLorg
0/5
0/5
0/5
1/5
1/5
215
Stepl:
Significant
Dilutions
5-3-0
4-5-1
0-1-0
3-1-1
4-4-1
5-5-2
Step 2:
(MPN from Table 2 / largest sig. dilution)
= MPN / mL wet weight
(7.92 / 1 0'4) = 79,200 MPN / mL
79,000 MPN/mL
(4.8 3/10'3) = 4830 MPN/mL
4800 MPN / mL
(0.18 /10'3) = 180 MPN/mL
(1. 37 /10'4) = 13,700 MPN/mL
14, 000 MPN/mL
(3.98 /10'3) = 3980 MPN/mL
4000 MPN / mL
(54.22 / 10'4) = 542,200 MPN / mL
540,000 MPN / mL
a Significant dilutions are underlined and largest significant dilutions highlighted
14.3 Step 3: Convert to MPN/g Total Solids (dry weight)
• For analysis and calculation of percent total solids see Section 12.4.
For the conversion to MPN/g total solids (dry weight), we assume that,
• MPN/mL wet weight = MPN/g wet weight.
Therefore, we may convert to MPN/g total solids (dry weight) using the following equation:
MPN/mL (wet weight) from step 2
MPN/g (dry weight)
Percent total solids (expressed as a decimal)
Examples of the conversion to MPN/g (dry weight) are provided in Table 6.
Table 6. Examples of Conversion to MPN/g Total Solids (Dry Weight), Continuing From Step 2 in Table 5
Example
(liquid or solid)
A
B
C
D
E
F
Total
Solids
4%
60%
56%
22%
18%
43%
Step 3:
(MPN / mL wet weight from step 2) / percent total solids = MPN / g dry weight
79,000 / 0.04 = 1 ,975,000 = 2.0 x 1 0s MPN / g dry weight
4800 / 0.6 = 8000 = 8.0 x 1 03 MPN / g dry weight
180 / 0.56 = 321 = 3.2 x 1 02 MPN / g dry weight
14,000 / 0.22 = 63,636 = 6.4 x 104 MPN / g dry weight
4,000 / 0.18 = 22,222 = 2.2 x 104 MPN / g dry weight
540,000/0.43 = 1,255,81 4 = 1.3x1 0s MPN/g dry weight
27
September 2014
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Method 1680
14.4 Calculation of Geometric Mean
To satisfy pathogen reduction requirements for Class B biosolids in Subpart D of Part 503, seven
biosolid samples are collected and the geometric mean density of fecal coliforms is calculated. The
geometric mean is calculated by:
converting each sample's MPN fecal coliforms/g (dry weight) to the logio value,
averaging the logio values, and
• taking the antilog of the mean logio value.
An example is provided in Table 7.
Table 7. Calculation of Geometric Mean Fecal Coliform Density for Biosolid Samples
Sample No.
1
2
3
4
5
6
7
MPN Fecal coliforms/g (dry weight)
600,000 = 6.0 X105
4,200,000 = 4.2X1 0s
1,700,000 =1.7X1 0s
1,400,000 =1.4X1 0s
400,000 = 4.0 X105
1, 100,000 =1.1X 10s
510,000 = 5.1 X105
logio
5.78
6.62
6.23
6.15
5.60
6.04
5.71
Mean of logio values = (5.78 + 6.62 + 6.23 + 6.1 5 + 5.60 + 6.04 + 5.71 ) / 7 = 6.02
Antilog of 6.02 = 1,047,128 = 1.0 x 10s geometric mean MPN of fecal coliforms/g (dry weight)
15.0 Sample Spiking Procedure
15.1 Method 1680 QC requirements (Section 9.0) include the preparation and analysis of spiked
reference (Milorganite®) samples in order to monitor initial and ongoing method performance. For the
IPR (Section 9.3) and OPR (Section 9.4) analyses it is necessary to spike samples with
laboratory-prepared spiking suspensions. Section 15.0 is arranged in the following order: preparation
of the E. coli spiking suspension (Section 15.2), spiking suspension dilution (Section 15.3), spiking
suspension enumeration (Section 15.4), Class A sample spiking (Section 15.5), Class B sample spiking
(Section 15.6), and calculation of spiked E. coli percent recovery (Section 15.7).
15.2 Preparation of El coli Spiking Suspension (Class A or B)
15.2.1 Stock Culture. Prepare a stock culture by inoculating a heart infusion agar (FflA) slant [or
other non-selective media (e.g., Tryptic Soy Agar)] with Escherichia coli ATCC 25922 and
incubating at35°C±3°Cfor20±4 hours. This stock culture may be stored in the dark at room
temperature for up to 30 days.
15.2.2 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 lauryl tryptose broth
in a sterile screw cap bottle or re-sealable dilution water container. Shake to mix.
15.2.3 Spiking Suspension (Undiluted). From the stock culture ofE. coli ATCC 25922, 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
28
September 2014
-------
Method 1680
approximately 1.0 x 107 to 1.0 x 108 E. coli colony forming units (CPU) per mL. This is
referred to as the "undiluted spiking suspension."
15.3 Spiking Suspension Dilution
15.3.1 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 to 99 mL of sterile dilution
water (Section 7.4), cap, and mix by vigorously shaking the bottle a minimum of 25 times.
This is spiking suspension dilution "A." A 1.0-mL dilution "A" is 10~2 mL of the original
undiluted spiking suspension.
15.3.2 Use a sterile pipette to transfer ll.OmL 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 dilution "B" is 10~3 mL of the original undiluted
spiking suspension.
15.3.3 Use a sterile pipette to transfer ll.OmL 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 dilution "C" is 10^ mL of the original undiluted
spiking suspension.
15.3.4 Use a sterile pipette to transfer ll.OmL 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 dilution "D" is 10"5 mL of the original undiluted
spiking suspension.
15.3.5 Use a sterile pipette to transfer ll.OmL 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 dilution "E" is 10"6 mL of the original undiluted
spiking suspension.
15.4 Spiking Suspension Enumeration
15.4.1 Prepare heart infusion agar (HIA) (Section 7.5), add 10 -15 mL of FflA per 100 x 15 mm petri
dish, and allow to solidify. Ensure that agar surface is dry.
Note: To ensure that the agar surface is dry prior to use, plates should be made several days in
advance and stored inverted at room temperature or dried using a laminar-flow hood.
15.4.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread
plates:
• Pipet 0.1 mL of dilution "C" onto surface of pre-dried HIA plate [10"5 mL (0.00001) of the
original spiking suspension].
• Pipet 0.1 mL of dilution "D" onto surface of pre-dried HIA plate [10"6 mL (0.000001) of the
original spiking suspension].
• Pipet 0.1 mL of dilution "E" onto surface of pre-dried HIA plate [10"7 mL (0.0000001) of
the original spiking suspension].
29 September 2014
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Method 1680
15.4.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculumb over
surface of medium by rotating the dish by hand or on a turntable.
15.4.4 Allow inoculum to absorb into the medium completely.
15.4.5 Invert plates and incubate at 35°C ± 0.5°C for 24 ± 4 hours.
15.4.6 Count and record number of colonies per plate. Refer to Section 15.7 for calculation of spiking
suspension concentration.
15.5 Class A Biosolid Sample Spiking
Homogenize the unspiked Class A biosolid sample (Section 11.1). Forthe enumeration of fecal
coliforms in the unspiked sample, dilute and inoculate according to Section 11.2. After the unspiked
sample has been diluted and media inoculated, spike the biosolid sample as indicated below.
Since the objective of spiking the biosolid sample is to establish percent recovery, it is necessary to
determine the number ofE. coli in the undiluted spiking suspension. Instructions for spiking suspension
enumeration are provided below.
15.5.1 Liquid Samples: Since the unspiked, homogenized sample was analyzed by the LTB/EC
procedure and a dilution series was prepared (Section 11.2), 284 mL of the original 300 mL of
unspiked, homogenized sample remains. To spike the sample, add 1.0 mL of spiking
suspension dilution "B" (from Section 15.3.2) for every 100-mL of unspiked homogenized
sample remaining, cover, and blend on high speed for 1 - 2 minutes. This is the "spiked,
homogenized" sample. The volume (mL) of undiluted spiking suspension added to each mL
of the spiked biosolid sample is 1.0 x 10'5 mL per mL [(2.8 mL x 10'3 mL) / 284 mL of
biosolid], which is referred to as Vspjkedperumtbiosoiids below. Proceed to Section 11.2.1 (dilution
and inoculation).
15.5.2 Solid Samples: Since the unspiked, homogenized sample was analyzed by the LTB/EC
procedure and a dilution series was prepared (Section 11.2), 234 mL of the original 300 mL of
unspiked, homogenized sample remains. To spike the sample, add 1.0 mL of spiking
suspension dilution "B" (from Section 15.3.2) for every 100-mL of unspiked homogenized
sample remaining, cover, and blend on high speed for 1 - 2 minutes. This is the "spiked,
homogenized" sample. The volume (mL) of undiluted spiking suspension added to each g
(wet weight) of the spiked biosolid sample is 1.0 x 10^ mL per g [(2.3 mL x 10"3 mL) / 23.4 g
of biosolid], which is referred to as Vspjked per unit biosoiMs below. Proceed to Section 11.2.2
(dilution and inoculation).
15.6 Class B Biosolid Sample Spiking
Homogenize the unspiked Class B biosolid sample (Section 11.1). For the enumeration of fecal
coliforms in the unspiked sample, dilute and inoculate according to Section 11.2. After the unspiked
sample has been diluted and media inoculated, spike the biosolid sample as indicated below.
Since the objective of spiking the biosolid sample is to establish percent recovery, it is necessary to
determine the number of E. coli in the undiluted spiking suspension. Instructions for spiking suspension
enumeration are provided below.
30 September 2014
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Method 1680
Since a dilution series was prepared from the unspiked, homogenized sample (Section 11.2), 289 mL of
the original 300 mL of unspiked, homogenized sample remains. To spike the sample, add 1.0 mL of
well-mixed undiluted spiking suspension, for every 100-mL of unspiked homogenized sample
remaining after unspiked sample evaluation and dilution preparation, cover, and blend on high speed for
1-2 minutes. This is the "spiked, homogenized" sample.
Note: The volumes of undiluted spiking suspensions added per mL or g (wet weight) of the spiked
Class B biosolids are different from those for the Class A biosolids, since different volumes of
the unspiked, homogenized sample remains.
15.6.1 Liquid Samples: The volume (mL) of undiluted spiking suspension added to each Ml of the
spiked biosolid sample is 1.0 x 10"2 mL [(2.9 mL spiking suspension) / 289 mL of biosolid],
which is referred to as Vspiedperumtbiosoiids below. Proceed to Section 11.2.3 (dilution and
inoculation).
15.6.2 Solid Samples: The volume (mL) of undiluted spiking suspension added to each g (wet weight)
of the spiked biosolid sample is 1.0 x 10"1 mL per g [(2.9 mL spiking suspension) / 28.9 g of
biosolids], which is referred to as Vs^ pa-unit biosolids below. Proceed to Section 11.2.4 (dilution
and inoculation).
15.7 Calculation of Spiked E. coli Percent Recovery
Spiked E. coli percent recovery will be conducted in four steps as indicated below.
Note: The example calculated numbers provided in the tables below have been rounded at the end of
each step. If your laboratory recalculates the examples using a spreadsheet and rounds only
after the final calculation (Step 4), the percent recoveries may be slightly different.
15.7.1 Step 1: Calculate Concentration of K coli (CFU / mL) in Undiluted Spiking Suspension
15.7.1.1 The number of E. coli CFU / mL in the spiking suspension will be calculated using
all plates yielding counts within the ideal range of 30 to 300 CFU per plate.
15.7.1.2 If the number of colonies exceeds the upper range (i.e., >300) or ifthe colonies are
not discrete, results should be recorded as "too numerous to count" (TNTC).
15.7.1.3 Calculate the concentration of £! coli (CFU / mL) in the undiluted spiking
suspension according to the following equation. Example calculations are provided
in Table 8, below.
CFU, +CFU, + ... + CFU
pr = ! 1 n-
undiluted spike I/ _i_ I/ _i_ _i_ T/
Where:
EC united spike = E. coli CFU / mL in undiluted spiking suspension
CFU = number of colony forming units from HIA plates yielding counts
within the ideal range of 30 to 300 CFU per plate
31 September 2014
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Method 1680
V = volume of undiluted sample in each HIA plate yielding counts
within the ideal range of 30 to 300 CPU per plate
n = number of plates with counts within the ideal range of 30 to 300
CPU per plate
Table 8. Example Calculations of E. coli Spiking Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from HIA plates
10"5mL plates
275, 250, 301
TNTC, TNTC,
TNTC
10"6mL plates
30, 10,5
TNTC, 299,
TNTC
10~7 mL plates
0,0,0
12,109,32
E. coli CFU / mL in undiluted spiking
suspension
(EC undiluted spike)
(275+250+30) / (1 0'5+1 0'5+1 0'6) =
555 / (2.1 x10'5) = 26,428, 571 =
2.6x107CFU/mL
(299+1 09+32) / (1 0'6+1 0'7+1 0'7) =
440 / (1.2 x 10'6) =366,666,667 =
3.7x108CFU/mL
ECundiiuted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
15.7.2 Step 2: Calculate Spiked K coli [CFU / mL or g (wet weight)]
15.7.2.1 The volume of undiluted spiking suspension per unit (mL or g) of spiked biosolid
samples (Vsplkedpermitblosoilds) is provided in Table 9.
Table 9. Volume of Undiluted Spiking Suspension per Unit (mL or g) of Spiked Biosolid Samples
("spiked per unit biosolids)
Description of spiked sample
Class A liquid
Class A solid
Class B liquid
Class B solid
V spiked per unit biosolids
1 .0 X 1 0"5 mL per mL of biosolids
1 .0 x 1 0"4 mL per g of biosolids (wet weight)
1 .0 X 1 0"2 mL per mL of biosolids
1 .0 x 1 0"1 mL per g of biosolids (wet weight)
15.7.2.2 Calculate concentration of spiked E. coli (wet weight) in biosolid sample according
to the following equation.
Spiked ECwet weight" (ECmdilutedspike) X (V,
Where:
Spiked ECwetweight
-'-'^undiluted spike
» spiked per unit biosolids
spiked per unit biosolids/
Number of spiked E. coli CFU per mL or g of biosolid (wet
weight)
E. coli CFU / mL in undiluted spiking suspension
mL of undiluted spiking suspension per mL or g of spiked
biosolid
32
September 2014
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Method 1680
Examples are provided in Table 10.
Table 10. Example Calculations of Spiked ECwet weight
EC undiluted spike
2.6x107CFU/mL
3.7x108CFU/mL
V spiked
Class A liquid: 1.0X10"5mL permLof
biosolids
Class A solid: 1 .0 x 1 0"4 ml_ per g of
biosolids (wet weight)
Class B liquid: 1.0 x 10"2mL per ml_ of
biosolids
Class B solid: 1 .0 x 1 0"1 ml_ per g of
biosolids (wet weight)
Class A liquid: 1.0 X10"5mL permLof
biosolids
Class A solid: 1 .0 x 1 0"4 ml_ per g of
biosolids (wet weight)
Class B liquid: 1 .0 x 1 0"2 ml_ per ml_ of
biosolids
Class B solid: 1 .0 x 1 0"1 ml_ per g of
biosolids (wet weight)
Spiked ECwet weight
(2.6 x 1 07 CPU / ml_) x (1 .0 x 1 0'5 ml_ / ml_)
= 2.6x102CFU/mL
(2.6 x107 CFU /mL)x(1.0x10'4 mL/g)
= 2.6x1 03 CPU / g (wet weight)
(2.6 x 1 07 CPU / ml_) x (1 .0 x 1 0'2 mL / mL)
= 2.6x105CFU/mL
(2.6x107CFU/mL)x(1.0x10'1 mL/g)
= 2.6 x 1 0e CPU / g (wet weight)
(3.7x108CFU/mL)x(1.0x10'5mL/mL)
= 3.7x103CFU/mL
(3.7 x108 CFU /mL)x(1.0x10'4 mL/g)
= 3.7 x 1 04 CFU / g (wet weight)
(3.7 x 1 08 CFU / mL) x (1 .0 x 1 0'2 mL / mL)
= 3. 7x1 0s CFU /mL
(3.7x108CFU/mL)x(1.0x10'1 mL/g)
= 3.7 x 1 07 CFU / g (wet weight)
15.7.3 Step 3: Convert to "True" Spiked E. coli CFU / g Total Solids (dry weight)
Convert to "true" spiked CFU / g total solids (dry weight) as indicated in Section 14.3 using the
E. coli mL or g (wet weight) from Section 15.7.2 as the numerator in the equation. Examples
are provided in Table 11.
Table 1 1 . Examples of Conversion to "True" Spiked E. coli CFU / g Total Solids (Dry Weight)
Example Total Solids
Class A liquid: 9%
Class A solid: 82%
Class B liquid: 4%
Class B solid: 23%
Class A liquid: 7%
Class A solid: 88%
Class B liquid: 3%
Class B solid: 40%
(CFU / mL or g) / percent total solids =True spiked E. coli CFU / g dry weight
2.6 x 1 02 / 0.09 = 2889 = 2.9 x 1 03 CFU / g dry weight
2.6 x 1 03 / 0.82 = 31 71 = 3.2 x 1 03 CFU / g dry weight
2.6 x 105/0.04 = 6,500,000 = 6.5 x 10s CFU /g dry weight
2.6 x 106/0.23 = 1 1,304,348 = 1.1x107 CFU /g dry weight
3.7 x 1 03 / 0.07 = 52,857 = 5.3 x 1 04 CFU / g dry weight
3.7 x 1 04 / 0.88 = 42,045 = 4.2 x 1 04 CFU / g dry weight
3.7x 1 0s / 0.03 = 1 23,333,333 = 1.2 x108 CFU /g dry weight
3.7 x 1 07 / 0.40 = 92,500,000 = 9.3 x 1 07 CFU / g dry weight
15.7.4 Step 4: Calculate Percent Recovery
15.7.4.1 Calculate percent recovery (R) using the following equation:
T
Where:
R = Percent recovery
Ns = Fecal coliform MPN/g (dry weight) in the spiked sample
33
September 2014
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Method 1680
Nu = Fecal coliform MPN/g (dry weight) in the unspiked sample
T = True spiked E. coll CPU /g (dry weight) in spiked sample
Example percent recovery calculations are provided in Table 12.
Table 12. Example Percent Recovery Calculations
Matrix
Class A liquid
Class A solid
Class B liquid
Class B solid
Ns
2.5 x103
3.9 x103
1.4x108
8.3x1 0s
Nu
1.5x101
5.0 x102
1.7x106
8.0 x105
T
2.9 x103
3.2 x103
1.2x108
1.1x107
Percent recovery (R)
100x[(2.5x103)-(1.5x101)]/2.9x103 = 86%
100 x [(3.9 x 103) - (5.0 x 102)] 73.2 x 103 = 107%
100x[(1.4x108)-(1.7x106)]/1.2x108=115%
1 00 x [(8.3 x 1 0s) - (8.0 x 1 05)] / 1 . 1 x 1 07 = 68%
16.0 Method Performance
16.1 Intel-laboratory Validation of Method 1680
16.1.1 Twelve volunteer laboratories and a referee laboratory participated in U.S. Environmental
Protection Agency's (EPA's) interlaboratory validation study of EPA Method 1680. The
purposes of the study were to characterize method performance across multiple laboratories and
multiple biosolid matrices and to develop quantitative quality control (QC) acceptance criteria.
A detailed description of the study and results are provided in the validation study report
(Reference 19.2). Results submitted by laboratories were validated using a standardized data
review process to confirm that results were generated in accordance with study-specific
instructions and the October 2002 Draft version of EPA Method 1680.
16.1.2 Recovery - Method 1680 mean recoveries of fecal coliforms from Class A matrices, compost
and thermophilically digested biosolids, spiked with laboratory-prepared spikes were 190% and
140%, respectively. Median recoveries of fecal coliforms from compost and thermophilically
digested biosolids, spiked with laboratory-prepared spikes were 130% and 120%, respectively.
Mean recoveries of fecal coliforms from Class B matrices, aerobically and anaerobically
digested biosolids, spiked with laboratory-prepared spikes were 2000% and 890%, respectively.
Median recoveries of fecal coliforms from aerobically and anaerobically digested biosolids,
spiked with laboratory-prepared spikes were 88% and 86%, respectively. For Milorganite®
(reference matrix) samples spiked with laboratory spiking suspensions, the mean recovery was
130%, with a median percent recovery of 120%.
16.1.3 Precision - Method 1680 was characterized by high variability. For percent recovery, the
overall relative standard deviations (RSDs) ranged from 81% to 130% for Class A matrices and
from 170% to 360% for Class B matrices for EPA-prepared spikes. For ambient (unspiked)
fecal coliform results, the overall RSDs ranged from 22% to 250% for Class A matrices and
from 140% to 470% for Class B matrices.
16.1.4 False positive rates - Method 1680 false positive rates were relatively low (6%) for
thermophilically digested matrices but high (32%) for the composted matrices. False positive
rates for Milorganite® could not be accurately assessed because only two positive tubes were
observed during the study. False positive rates for aerobically digested and anaerobically
digested samples were relatively low compared to Class A rates and ranged from 5% to 10%,
dependent on procedure and matrix.
34
September 2014
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Method 1680
16.1.5 False negative rates - Method 1680 false negative rates for Class A matrices were relatively low
compared to Class B rates and ranged from 0% to 6%. False negative rate for aerobically
digested matrices was slightly higher (10%) compared to Class A rates. In contrast, the false
negative rate for anaerobically digested matrices was 22%.
17.0 Pollution Prevention
17.1 The solutions and reagents used in this method pose little threat to the environment when recycled and
managed properly.
17.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the
volume of expired materials to be disposed.
18.0 Waste Management
18.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly hazardous waste identification rules and land disposal restrictions, and
for protecting the air, water, and land by minimizing and controlling all releases from fume hoods and
bench operations. Compliance with all sewage discharge permits and regulations is also required. An
overview of requirements can be found in Environmental Management Guide for Small Laboratories
(EPA233-B-98-001).
18.2 Samples, reference materials, and equipment known or suspected to have viable bacteria or viral
contamination must be sterilized prior to disposal.
18.3 For further information on waste management, consult The Waste Management Manual for Laboratory
Personnel and Less Is Better: Laboratory Chemical Management for Waste Reduction, both available
from the American Chemical Society's Department of Government Relations and Science Policy, 1155
16th Street NW, Washington, DC 20036.
19.0 References
19.1 American Public Health Association, American Water Works Association, and Water Environment
Federation. 1995. Standard Methods for Water and Wastewater. 20th Edition. Sections: 9020, 9221,
9222.
19.2 USEPA. 2004. Results of the Interlaboratory Validation of EPA Method 1680 (LTB/EC)for Fecal
Coliforms in Biosolids. EPA-821-R-04-009. December 2004.
19.3 American Chemical Society (ACS). 2000. Reagent Chemicals, American Chemical Society
Specifications. American Chemical Society, New York. For suggestions of the testing of reagents not
listed by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals, BDH,
Poole, Dorset, UK and the United States Pharmacopeia.
19.4 Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). 1978. Microbiological Methods for Monitoring the
Environment, Water and Wastes. EPA-600/8-78-017. Office of Research and Development. USEPA.
35 September 2014
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Method 1680
19.5 Klee, A. J. 1993. A computer program for the determination of the most probable number and its
confidence limits. Journal of Microbiological Methods. 18:91-98.
20.0 Figures
The following pages include flow charts summarizing the dilution and inoculation schemes (Section
11.0) and procedures (Section 12.0). Schemes for dilution and inoculation are dependent on sample
Class (A or B) and matrix (solid) or (liquid).
FIGURE1. MULTIPLE TUBE FERMENTATION PROCEDURE
LTB/EC Method
Inoculate LTB and incubate at
35°C±0.5°Cfor24±2hr
Growth and gas production
No gas production
Incubate additiona!24hr
(total of48±3hi)
Inoculate EC and incubate
at44.5°C±0.2°C
for24±2hr
Growth and gas production
Failure to produce gas
within48±3hr
NEGATIVE fecal coliform
Growth and gas
production within
24±2hr
POSITIVE fecal coliform
Failure to produce gas
within24±2hr
NEGATIVE fecal coliform
36
September 2014
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Method 1680
FIGURE2. CLASS A LIQUID SAMPLE DILUTION AND INOCULATION SCHEME
Class A
liquid sample
"Homogenized sample"
-11.0mL-
Delivery Volume
1.0mL
o
3
i—
S-eo
O CD
Z!. Z!.
(Q CD
5' m
9L -^
en
01
99 ml of sterile
buffered dilution water
"A"
(10-1)
-11.0mL-
1.0mL
o
3
m
3
•g.
(D
99mL of sterile
buffered dilution water
"B"
(10-2)
-11.0mL-
1.0mL
O CD
I'
99mL of sterile
buffered dilution water
"C"
(10-3)
q
3
i—
a
o
37
April 2010
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Method 1680
FIGURE 3. CLASS A SOLID SAMPLE DILUTION AND INOCULATION SCHEME
Class A
solid sample
-30 g-
Rinse sample into blender
with 270 ml of sterile
buffered dilution water
"Homogenized Sample"
(ID'1)
Delivery Volume
10.0mL
1.0 ml
-11.0ml-
1
'
b
_. (Q
»a
t*i
i. m
• ro'
5 ^
Cm
U)
'I
ro"
i
q
(Q
^
0
"9.
su
tn
w
3
nT
»
n>'
M
99 ml of sterile
buffered dilution water
"A"
(ID'2)
-H.OmL-
1.0mL
q
(Q
99 ml of sterile
buffered dilution water
"B"
(ID'3)
1.0ml
q
(Q
38
April 2010
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Method 1680
FIGURE4. CLASS B LIQUID SAMPLE DILUTION AND INOCULATION SCHEME
Class B
liquid
sample
"Homogenized sample"
—1LOmL-
—H.OmL-
H.OmL-
gg.omLof
sterile
buffered
dilution
water
"C"
(ID'3)
-11.0mL-
gg.omLof
sterile
buffered
dilution
water
"D"
(ID'4)
-HOrnL-
gg.omLof
sterile
buffered
dilution
water
"E"
(ID'5)
-11.0mL-
gg.omLof
sterile
buffered
dilution
water
"F"
(ID'6)
Delivery Volume
lOmL
lOmL
LOmL
LOmL
a
o
Q3_ CO
8
39
April 2010
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Method 1680
FIGURES. CLASS B SOLID SAMPLE DILUTION AND INOCULATION SCHEME
Class B
solid
sample
-30g—*
Rinse sample into blender
with 270ml_ of sterile
buffered dilution water
"Homogenized Sample"
(ID'1)
-11.0mL-
11.0mL-
Delivery Volume
11.0mL-
fa
11.0mL-
1.0mL
fa
11.0mL-
CQ
Iff
<° o
=! W
91 co
40
April 2010
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Method 1680
21.0 Glossary
The definitions and purposes are specific to this method but have been conformed to common usage as
much as possible.
21.1 Units of weight and measure and their abbreviations
21.1.1 Symbols:
°C degrees Celsius
< less than
> greater than
% percent
± plus or minus
21.1.2 Alphabetical characters:
EC Escherichia coll
EPA United States Environmental Protection Agency
g gram
L liter
LTB lauryl tryptose broth
mg milligram
mL milliliter
mm millimeter
MPN most probable number
NIST National Institute of Standards and Technology
QA quality assurance
QC quality control
TD to deliver
21.2 Definitions, acronyms, and abbreviations (in alphabetical order):
Analyte—The microorganism tested for by this method. The analytes in this method are fecal
conforms.
Enrichment—A non-selective culture media for enhanced growth.
Liquid samples—Generally defined as samples containing <7% total solids (dry weight).
May—This action, activity, or procedural step is neither required nor prohibited.
May not—This action, activity, or procedural step is prohibited.
Method blank—An aliquot of sterile reagent water that is treated exactly as a sample including exposure
to all glassware, equipment, media, procedures that are used with samples. The method blank is used to
verify the sterility of equipment, materials, and supplies.
Most probable number method (MPN)—A statistical determination of the number of bacteria per weight
or volume of sample. It is based on the fact that the greater the number of bacteria in a sample, the more
41 April 2010
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Method 1680
dilution is needed to reduce the density to the point at which no bacteria are left to grow in a dilution
series.
Must—This action, activity, or procedural step is required.
Negative control—A control culture that, when analyzed exactly like a field sample, will produce a
known negative result for a given type of media.
Positive control—A control culture that, when analyzed exactly like a field sample, will produce a
known positive result for a given type of media.
Preparation blank—See Method blank.
Selective media—A culture media designed to suppress the growth of unwanted microorganisms and
encourage the growth of desired ones.
Should—This action, activity, or procedural step is suggested but not required.
Solid samples—Generally, samples containing >7 % total solids (dry weight).
42 April 2010
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