United States
Environmental Protection
Agency
Method 1680: Fecal Coliforms in
Biosolids by Multiple-Tube Fermentation
Procedures
October 2002 Draft
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-02-026
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Disclaimer
This method is in draft form. It has not been released by the U.S. Environmental Protection Agency and
should not be construed as an Agency-endorsed method. The Office of Science and Technology directed,
managed, and reviewed the work of DynCorp in preparing this report under EPA Contract No. 68-C-98-
139. Neither the United States Government nor any of its employees, contractors, or their employees
make any warranty, expressed or implied, or assumes any legal liability or responsibility for any third
party's use of or the results of such use of any information, apparatus, product, or process discussed in
this report, or represents that its use by such party would not infringe on privately owned rights.
Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov
202-566-1075
202-566-1054 (facsimile)
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Introduction
Application of treated biosolids to land is helpful as a crop nutrient and soil conditioner, but may pose the
risk of releasing pathogens into the environment if disinfection and use criteria are not met. The density
of fecal coliforms in biosolids is used as an indicator of the average density of bacterial and viral
pathogens. Under 40 CFR Part 503, a biosolid sample is classified as Class A if it contains a fecal
coliform density below 1,000 most probable number (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).
Method 1680 is adapted from Standard Methods 9221E (Reference 19.1). Although these methods
currently are approved for the detection of fecal coliform bacteria in biosolids, they were designed for use
in water matrices. Method 1680 was developed to determine the presence of fecal coliforms reliably in
biosolid matrices.
The multiple tube fermentation procedures in Method 1680 are used to detect fecal coliform bacteria.
Although Method 1680 is a performance-based method, fecal coliforms are considered "method-defined
analytes," and requests to modify the procedures are limited, and handled on a case-by-case basis.
IV
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 2
3.0 Definitions 3
4.0 Interferences 3
5.0 Safety 3
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 4
8.0 Sample Collection, Preservation, and Storage 7
9.0 Quality Control 9
10.0 Equipment Calibration and Standardization 10
11.0 Sample Preparation 11
12.0 Multiple-Tube Fermentation Procedures 17
13.0 Data Analysis and Calculations 19
14.0 Method Performance 24
15.0 Reporting Results 24
16.0 Verification Procedures 24
17.0 Pollution Prevention 24
18.0 Waste Management 24
19.0 References 24
20.0 Flowcharts and Validation Data 25
21.0 Glossary 31
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Method 1680: Fecal Conforms in Biosolids by
Multiple-Tube Fermentation Procedures
October 2002 Draft
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, 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 methods 9221E 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 the 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 biosolids. This
method also may be applied specifically 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 Part 136.4 and 136.5.
Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
2.0 Summary of Method
2.1 Fecal coliform densities of biosolids may be determined by the MPN procedure using two media
options.
2.2 MPN procedure (Class A and B)
Two method options are provided in Method 1680 for the MPN procedure: (1) A presumptive
step using lauryl tryptose broth (LTB) plus a confirmation step using EC broth. (EC broth must
not be used for direct fecal coliform isolation from a biosolid sample because prior enrichment is
required in LTB medium for optimum recovery of fecal coliforms). (2) A direct, single step test
using A-l medium. The precision of both tests increases with increasing numbers of replicates per
sample tested.
2.2.1 Summary of the LTB/EC procedure
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° ± 0.2°C for 24 ± 2 hours. 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 Results of the MPN procedure using LTB/EC media are reported in terms of
MPN / g calculated from the number of positive EC tubes and percent total
solids (dry weight basis, see Draft Method 1684, Section 11 for the
determination of total solids).
2.2.2 Summary of the A-l procedure
2.2.2.1 A minimum of four sample dilutions are required, while five or more are
preferred. Each sample dilution is inoculated into five test tubes containing
A-l medium and inverted vials.
2.2.2.2 Sample tubes are incubated in a water bath or jacketed incubator at
35°C ± 0.5 °C for 3 hours, then transferred to a water bath at 44.5°C ± 0.2°C.
After 21 ± 2 hours, tubes are examined for growth and gas production. Gas
production in 24 ± 2 hours or less is a positive reaction indicating the presence
of fecal coliforms.
2.2.2.3 Results of the MPN procedure using A-l medium are reported in terms of the
most probable number (MPN)/g calculated from the number of positive A-l
culture tubes and percent total solids (dry weight basis, see Draft Method 1684
for determination of total solids).
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
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 is E. coll.
In this method, fecal coliforms are those bacteria that ferment lactose and produce gas within 24 ±
2 hours in EC or A-l 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.
4.2 Percent total solids interferences: see Draft Method 1684.
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
Brand names, suppliers, and part numbers are for illustrative purposes only. No endorsement is implied.
Equivalent performance may be achieved using apparatus and materials other than those specified here,
but demonstration of equivalent performance that meets the requirements of this method is the
responsibility of the laboratory.
6.1 Sample bottles—Autoclavable, ground-glass, or plastic wide-mouthed (stoppered or screw cap),
minimum of 125 mL capacity
6.2 Sterile waterproof plastic bags—Whirl-Pak® or equivalent (may be used for collection of solid
samples)
6.3 Dilution containers
6.3.1 Sterile, borosilicate glass, screw cap, marked at 99 mL
6.3.2 Sterile, screw cap, borosilicate glass or plastic tubes marked at 9 mL
6.4 Pipette container—Stainless steel, aluminum or borosilicate glass, for glass pipettes
6.5 Pipettes
6.5.1 Sterile, to deliver (TD) bacteriological or Mohr, glass or plastic, of appropriate volume
3 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
6.5.2 Sterile, wide-mouth
6.6 Volumetric flasks—Borosilicate glass, screw-cap, 250- to 2000-mL volume
6.7 Graduated cylinders—100- to 1000-mL, covered with aluminum foil or kraft paper and sterilized
6.8 Thermometers—0°C to 50°C with 0.2°C graduations checked against a National Institute of
Standards and Technology (NIST) certified thermometer, or one that meets the requirements of
NIST Monograph SP 250-23
6.9 Burner—Bunsen or Fisher type, or electric incinerator unit for sterilizing loops
6.10 pH meter
6.11 Blender and sterile blender jar
6.12 Equipment for MPN procedure
6.12.1 Covered water bath—With circulating system to maintain temperature of 44.5°C ±
0.2°C. Water level should be maintained above the media in immersed tubes.
6.12.2 Autoclave capable of 121°C at 15 psi
6.12.3 Covered water bath or water- or air-jacketed incubator at 35°C ± 0.5°C
6.12.4 Inoculation loops—Nichrome or platinum wire, disposable sterile plastic loops or
sterile wooden applicator, at least 3 mm in diameter
6.12.5 Sterile culture tubes—16 x 150 mm, borosilicate glass
6.12.6 Inverted tubes or vials—10 x 75 mm
6.12.7 Balance—Analytical balance capable of weighing 0.1 mg
6.12.8 Caps—Loose-fitting aluminum, stainless steel, or autoclavable plastic, for 16 mm
diameter test tubes
6.12.9 Test tube racks
6.13 Equipment for percent total solids—see Draft Method 1684
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 19.2). 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 water—Reagent water conforming to Specification Dl 193, Annual Book of ASTM
Standards (Reference 19.3).
7.4 Phosphate buffered dilution water
7.4.1 Prepare stock phosphate buffer solution by dissolving 34.0 g potassium dihydrogen
phosphate (KH2PO4), in 500 mL of reagent-grade water, adjust to pH 7.2 ± 0.5 with 1
N sodium hydroxide (NaOH), and dilute to 1 L with reagent-grade water.
7.4.2 Prepare stock magnesium chloride solution by dissolving 81.1 g of magnesium chloride
hexahydrate (MgCl2-6H2O) to 1 L of reagent-grade water.
7.4.3 To prepare the buffered dilution water, add 1.25 mL stock phosphate buffer solution
and 5.0 mL of the magnesium chloride solution to 1 L reagent-grade water. Dispense in
appropriate amounts for dilutions in bottles or culture tubes. After preparation,
autoclave at 121°C (15 psi) for 15 minutes. 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
Draft October 2002 4
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
placed into an autoclavable pan with a small amount of water to contain breakage and
minimize evaporation from the tubes.
7.5 Heart infusion (HI) broth (DIFCO# 0038-15, BBL# 238300, or equivalent) or agar (DIFCO#
0044-15, BBL# 244300, or equivalent)—For preparation follow procedure as specified on bottle
of media. If dehydrated media is not available, see below for directions
7.5.1 50 g Beef heart, infusion from
7.5.2 10 g Bacto tryptose
7.5.3 5.0 g sodium chloride (NaCl)
7.5.4 15 g Bacto agar
7.5.5 For HI agar, add reagents in Sections 7.5.1 through 7.5.4 to 1-L of reagent-grade water,
mix thoroughly, and heat to dissolve. For HI broth, add reagents in Sections 7.5.1
through 7.5.3 to 1-L of reagent-grade water, mix thoroughly, and heat to dissolve. Stir
well and autoclave at 121°C for 15 minutes. Other general growth media may be used
for QA (Section 9.0) purposes.
7.6 Media for the MPN procedure:
7.6.1 LTB medium (DIFCO# 0241-17, BBL# 224150, or equivalent)—For preparation
follow procedure as specified on bottle of media. If dehydrated media is not available,
see below for directions
7.6.1.1 20.0 g tryptose
7.6.1.2 5.Og lactose
7.6.1.3 2.75 g dipotassium hydrogen phosphate (K2HPO4)
7.6.1.4 2.75 g potassium dihydrogen phosphate (KH2PO4)
7.6.1.5 5.0 g sodium chloride (NaCl)
7.6.1.6 0.1 g sodium lauryl sulfate
7.6.1.7 For single strength LTB, add reagents in Sections 7.6.1.1 through 7.6.1.6
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
hydroxide, if necessary. Prior to sterilization, dispense 10 mL into
16 x 150 mm test tubes with inverted vials. Close tubes with metal or
autoclavable plastic caps and autoclave at 121°C at 15 psi for 15
minutes. After cooling, the medium should fill the inverted vials
completely, leaving no air space.
7.6.1.8 For double strength (2X) LTB, prepare as in Section 7.6.1.7 but use 500
mL of reagent-grade water instead of 1 L. Note: 2XLTB is necessary for
10-mL inoculations, to ensure that the 10-mL inoculation volume does
not excessively dilute the media.
7.6.2 EC medium (DIFCO# 0314-17, BBL# 231430, or equivalent)—Follow procedure as
specified on bottle of media for preparation. If dehydrated media is not available, see
below for directions
7.6.2.1 20.0 g tryptose or trypticase
7.6.2.2 5.Og lactose
7.6.2.3 1.5 g bile salts mixture or bile salts No.3
7.6.2.4 4.0 g dipotassium hydrogen phosphate (K2HPO4)
7.6.2.5 1.5 g potassium dihydrogen phosphate (KH2PO4)
7.6.2.6 5.Og sodium chloride (NaCl)
5 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
7.6.2.7 Add reagents in Sections 7.6.2.1 through 7.6.2.6 to 1-L of reagent-grade
water, mix thoroughly, and heat to dissolve. Adjust pH to 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 at 15 psi for 15 minutes. Medium should fill inverted
tubes leaving no air spaces.
7.6.3 A-l medium (DIFCO# 1823-17, BBL# 218231, or equivalent)—Follow procedure as
specified on bottle of media for preparation. If dehydrated media is not available, see
below for directions.
7.6.3.1 5.0 g lactose
7.6.3.2 20.0 g tryptone
7.6.3.3 5.Og sodium chloride (NaCl)
7.6.3.4 0.5 g salicin
7.6.3.5 For single strength A-l, add reagents in Sections 7.6.3.1 through 7.6.3.4
to 1-L of reagent-grade water, mix thoroughly, heat to dissolve, and add
1.0 mL polyethylene glycol p-isooctylphenyl ether. Adjust pH to 6.9 ±
0.1 by addition of 1.0 N hydrochloric acid or 1.0 N sodium hydroxide, if
necessary. Prior to sterilization, dispense 10 mL into 16 x 150 mm test
tubes with inverted vials. Make sure there is enough medium to cover the
inverted vial at least halfway after sterilization. Close with metal or
autoclavable plastic caps. Sterilize by autoclaving at 121°C at 15 psi for
10 minutes. Ignore formation of precipitate. Media should fill inverted
tubes leaving no air spaces.
7.6.3.6 For double strength (2X) A-l, prepare as in Section 7.6.3.5 but use 500
mL of reagent-grade water instead of 1 L. Note: 2XA-1 is necessary for
10-mL inoculations, to ensure that the 10-mL inoculation volume does
not excessively dilute the media.
7.7 Positive control—Obtain a stock culture of E. coll (e.g. ATCC # 25922) as a positive control for
LTB, EC, and A-l.
7.8 Negative controls
7.8.1 Obtain a stock culture of Enterobacter aerogenes (e.g. ATCC # 13048) as a negative
control for EC and A-l.
7.8.2 Obtain a stock culture ofPseudomonas (e.g. ATCC # 27853) as a negative control for
LTB.
7.9 The storage times for prepared media used in this method are provided in Table 1 below:
Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
TABLE 1. STORAGE TIMES FOR PREPARED MEDIA (Note: If media is refrigerated, remove from refrigerator 1 -1.5
hours prior to inoculation, so that it reaches room temperature prior to use.)
Media
Agar or broth (EC, LIB, and HI) in loose-cap tubes
Agar or broth (EC, LIB, and HI) in tightly closed screw-cap tubes
Broth (A-1 )
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
7 days
2 weeks
3 months
8.0 Sample Collection, Preservation, 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 outlet pipe used to fill the truck.
8.4.2 Purge the pipe of old biosolids and warm to the digester temperature by allowing
biosolids to flow through the pipe into a bucket.
8.4.3 Position a 1-gal. 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 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 divide 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 stainless steel or
plastic bucket.
8.6.4 After all the samples have been taken, pour the bucket out onto a sterile plastic sheet
7 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
and mix by folding the sample back onto itself several times.
8.6.5 Reduce the sample size by "coning and quartering." Divide the bucket contents into
four even piles. If sample size is still too large, divide each quarter into quarters and
discard half. 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 bucket that has been dumped onto a sterile plastic sheet 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
8.8.2 Date and time
8.8.3 Sample name
8.8.4 Sample location
8.8.5 Parameters
8.8.6 Volume
8.8.7 Observations
8.9 Ensure that the chain of custody form is filled out
8.10 Sample preservation and handling—Ice or refrigerate bacteriological samples at a temperature of
1°C to 4°C during transit to the laboratory. Do not freeze the sample. 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 Chlorinated samples—Add a reducing agent to containers intended for the collection of biosolids
containing residual chlorine or other halogen. Sodium thiosulfate (Na2S2O3) is a satisfactory
dechlorinating agent that neutralizes any residual halogen and prevents continuation of
bactericidal action during sample transport. [If Na2S2O3 is used, add a sufficient volume of
Na2S2O3 to a clean sample bottle, to give a concentration of 100 mg/L in the sample.] In a 120-
mL sample bottle, a volume of 0.1 mL of a 10% solution of Na2S2O3 will neutralize a sample
containing about 15 mg/L residual chlorine.
8.12 Holding time and temperature limitations—Analyses should begin immediately, preferably,
within 2 hours of collection. If it is impossible to examine samples within 2 hours, samples must
be maintained at a temperature of 1°C to 10°C until analysis. Samples must not be frozen. Sample
analysis must begin within 24 hours. Note: Adherence to sample preservation procedures and
holding time limits is critical to the production of valid data. Sample results will be considered
invalid if these conditions are not met.
Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
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
(Section 9.3). Laboratory performance is compared to the performance criteria specified in
Section 14 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 the USEPA
Microbiology Methods Manual, Part IV, C (Reference 19.4).
9.2 General analytical QC procedures
9.2.1 Dilution water sterility check. Check each batch of dilution water for sterility by
adding 20 mL water to 100 mL of a non-selective broth such as HI broth (Section 7.5).
Incubate at 35°C ± 0.5°C for 48 to 72 hours and observe for growth. If any
contamination is indicated, reject analytical data from samples tested with these
materials.
9.2.2 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 (A-l and 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.
9.2.3 Perform duplicate analyses on 10% of samples or one sample per test run, whichever is
greater.
9.2.4 In laboratories with more than one analyst, have each analyst conduct parallel tests on
at least one positive sample monthly.
9.2.5 Obtain reference cultures from qualified outside sources and use them to establish pure
cultures that are maintained for the laboratory. Use these in routine analytical runs,
quarterly. Review results to correct causes of improper responses and document
actions.
9.2.6 Participate in available interlaboratory performance studies conducted by local, state,
and federal agencies or commercial organizations. Review results to correct
unsatisfactory performance and record corrective actions.
9.3 Analytical QC procedures for MPN procedure—These procedures are required with each new lot
of media, and in conjunction with each batch of samples.
9.3.1 LTB, EC, and A-l positive controls—Inoculate LTB, EC, or A-l (correlating with
procedure being used by the lab) with a known positive (e.g. E. coll ATCC # 25922).
Examine for appropriate responses, record results, and pursue causes of irregularities.
9.3.2 EC and A-l negative controls—Inoculate EC or A-l (correlating with procedure
being used by the lab) with a known negative fecal coliform species (e.g. Enterobacter
aerogenes ATCC # 13048). Examine for appropriate responses, record results, and
pursue causes of irregularities.
9.3.3 LTB negative control—Inoculate LTB (if LTB/EC procedure is being used by the lab)
with a known negative total coliform species (e.g. Pseudomonas ATCC# 27853).
Examine for appropriate responses, record results and pursue causes of irregularities.
9.3.4 Method Blank—Test a 20 mL sterile dilution water sample in the analytical scheme to
verify the sterility of equipment, materials, and supplies.
Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
10.0 Equipment Calibration and Standardization
10.1 Check temperatures in incubators twice daily, with the readings separated by at least 4 hours, to
ensure operation is within stated limits of the method and record daily measurements in the
incubator log book.
10.2 Calibrate thermometers and incubators at least annually against an NIST certified thermometer or
one that meets requirements of NIST Monograph SP 250-23. Check mercury columns for breaks.
10.3 Calibrate top-loading balances monthly with reference weights of ASTM Class 2.
10.4 Calibrate the pH meter prior to each use period with the two standards (pH 4.0, 7.0, and 10.0)
closest to the range being tested.
Draft October 2002 10
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
11.0 Sample Preparation
11.1 Homogenization
Liquid samples are generally defined as samples containing <10% 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.
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 of the "homogenized" sample
contains 10"1 g of the original sample. Adjust the pH to 7.0-7.5 by adding 1.0 N
hydrochloric acid orl.O N sodium hydroxide, if necessary.
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 the number of fecal coliform bacteria in undiluted samples could easily exceed
the detection limits of these procedures, the laboratory must follow the dilution and inoculation
scheme in Section 11.2 or a scheme that results in analysis of the same sample volumes for each
dilution series, at a minimum (additional dilutions may be analyzed as necessary). Please note:
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 B liquid samples—For unspiked samples, four series of five tubes each will be
used for the analysis with 10"3, 10"4, 10"5, and 10"6 mL of the original sample (additional
dilutions may be analyzed as necessary). See Figure 2 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.)
11.2.1.1 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." A 1.0-mL volume 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."
A 1.0-mL volume of dilution "B" is 10"2 mL of the original
sample.
11 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
(C) Use a sterile pipette to transfer 11.0 mL of well-mixed dilution
"B" to 99 mL of sterile dilution water, and mix as before. This is
dilution "C." A 1.0-mL volume of dilution "C" is 10~3 mL of the
original sample.
(D) Use a sterile pipette to transfer 11.0 mL of well mixed dilution
"C" to 99 mL of sterile dilution water, and mix as before. This is
dilution "D." A 1.0-mL volume of dilution "D" is 10'4 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."
A 1.0-mL volume 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."
A 1.0-mL volume 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." A 1.0-mL volume 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." A 1.0-mL volume of dilution "H" is 10'8 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 dilution "C" (unspiked samples only). This
is 10"3 mL of the original sample.
(B) Use a sterile pipette to inoculate each of the second series of
tubes with 1.0 mL of dilution "D" (unspiked samples only). This
is 10"4 mL of the original sample.
(C) Use a sterile pipette to inoculate each of the third series of tubes
with 1.0 mL of "E" (unspiked or spiked samples). This is 10"5
mL of the original sample.
(D) Use a sterile pipette to inoculate each of the fourth series of five
tubes each 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 the five tubes each 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 the five tubes each with
1.0 mL of dilution "H"(spiked samples). This is 10"8 mL of the
original sample.
11.2.1.3 Repeat Section 11.2.1.1 and 11.2.1.2 for each remaining Class B sample.
When inoculations are complete, proceed to Section 12.3.1.4 to continue
the LTB/EC method or to Section 12.4.4 to continue the A-l method.
Draft October 2002 12
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
11.2.2 Class B solid samples—For unspiked samples, four series of five tubes each will
contain 10~3, 10~4, 10~5, and 10~6 g of the original sample (additional dilutions may be
analyzed as necessary). See Figure 3 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 ID'5, ID'6, ID'7, ID'8, and 10'9g of the original sample.)
11.2.2.1 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 and shake vigorously a
minimum of 25 times. This is dilution "A." A volume of 1.0-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."
A volume of 1.0-mL of dilution "B"contains 10"3 g of the
original sample.
(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."
A volume of 1.0-mL of dilution "C" contains 10"4 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."
A volume of 1.0-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."
A volume of 1.0-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." A 1.0-mL volume of dilution "F" is 10"7 mL 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." A 1.0-mL volume of dilution "G" is 10'8 mL of
the original sample.
11.2.2.2 Inoculation
(A) 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) Inoculate each of the second series of tubes with 1.0 mL of
dilution "C"(unspiked samples only). This is 10"4 g of the
original sample.
(C) Inoculate each of the third series of tubes with 1.0 mL of "D"
(unspiked or spiked samples) This is 10"5 g of the original
sample.
13 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
(D) Inoculate each of the fourth series of five tubes each 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 the five tubes each with
1.0 mL of dilution "F" (spiked samples). This is 10"7 mL of the
original sample.
(F) Use a sterile pipette to inoculate each of the five tubes each with
1.0 mL of dilution "G" (spiked samples). This is 10"8 mL of the
original sample.
11.2.2.3 When inoculations are complete, go to Section 12.3.1.4 to continue the
LTB/EC method or to Section 12 A A to continue the A-l method.
11.2.3 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 4 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"4,
and 10"5 mL of the original sample.)
11.2.3.1 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." A volume of 1.0-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."
A 1.0-mL volume of dilution "B" is 10"2 mL of the original
sample.
(C) Use a sterile pipette to transfer 11.0 mL of well mixed dilution
"B" to 99 mL of sterile dilution water, and mix as before. This is
dilution "C." A volume of 1.0-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 well mixed
dilution "C" to 99 mL of sterile dilution water, and mix as
before. This is dilution "D." A 1.0-mL volume of dilution
"D" is 10"4 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." A 1.0-mL volume of dilution "E" is 10"5 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 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 the third series of tubes
with 1.0 mL of dilution "B" (unspiked or spiked samples). This
is 10"2 mL of the original sample.
Draft October 2002 14
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
(D) Use a sterile pipette to inoculate each of the fourth series of 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 the five tubes with 1.0
mL of dilution "D" (spiked samples). This is 10"4 mL of the
original sample.
(F) Use a sterile pipette to inoculate each of the five tubes with 1.0
mL of "E" (spiked samples). This is 10"5 mL of the original
sample.
11.2.3.3 Repeat steps 11.2.3.1 and 11.2.3.2 for the remaining Class A samples.
When inoculations are complete, go to Section 12.3.1.4 to continue the
LTB/EC method or to Section 12 A A to continue the A-l method.
11.2.4 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 contain 2X media. See Figure 5 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"4, and 10"5 g of the original sample.)
11.2.4.1 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." A
volume of 1.0-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."
A volume of 1.0-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." A volume of 1.0-mL of dilution "C" contains
10"4 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." A volume of 1.0-mL of dilution "D" contains
10"5 g of the original sample.
11.2.4.2 Inoculation
(A) Use a sterile pipette to inoculate each of the first series of 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.
15 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
(B) Use a sterile pipette to inoculate each of the second series of
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 the third series of 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 the fourth series of tubes
with 1.0 mL of dilution "B" (unspiked or spiked samples). This
is 10"3 g of the original sample.
(E) Inoculate each of the five tubes with 1.0 mL of dilution "C"
(spiked samples). This is 10"4 g of the original sample.
(F) Inoculate each of the five tubes with 1.0 mL of "D" (spiked
samples). This is 10"5 g of the original sample.
11.2.4.3 Repeat Section 11.2.4.1 and 11.2.4.2 for remaining Class A solid
samples. When inoculations are complete, go to Section 12.3.1.4 to
continue the LTB/EC method or to Section 12.4.4 to continue the A-l
method.
Draft October 2002 16
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
12.0 Multiple-Tube Fermentation Procedures
12.1 The MPN procedure may be used to determine fecal coliform densities in unknown, Class A, and
Class 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. There are two options included in Method 1680.
For the first option, 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.
The second option is a direct test using A-1 medium in a single step procedure (not requiring a
presumptive phase). The precision of both tests increases with increasing numbers of replicates
per sample tested. For an overview of the MPN procedure, refer to Figure 1.
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 7.6.1.
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: 2XLTB is needed
for 10-mL inoculations, to ensure that the 10-mL inoculation volume
does not excessively dilute the LTB.
12.3.1.3 Based on the matrix (i.e. Class A solid, Class B liquid),
dilute and inoculate samples according to 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. 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 (Section 12.3.2).
12.3.2 Confirmation phase for fecal coliforms using EC medium
12.3.2.1 Prepare EC broth tubes as described in Section 7.6.2. 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
17 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
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.
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 13.1.
12.4 A-l procedure
12.4.1 Prepare A-l broth tubes as directed in Section 7.6.3. Note: If media is
refrigerated, remove from refrigerator 1-1.5 hours prior to inoculation, so that it reaches
room temperature prior to use.
12.4.2 For each sample, arrange test tubes in four rows of five tubes each (Section 11.2). Use
10 mL of 2X A-l broth for 10-mL inoculations. Clearly label each row of tubes to
identify the sample and dilution volume to be inoculated. Note: 2XA-1 is needed for
10-mL inoculations, to ensure that the 10-mL inoculation volume does not excessively
dilute the A-l.
12.4.3 Dilute and inoculate samples depending on the matrix (i.e. Class A solid, Class
B liquid), as described in Section 11.2.
12.4.4 Incubate inoculated A-l tubes at 35°C ± 0.5°C for 3 hours ± 15 minutes.
12.4.5 Transfer A-1 tubes to a water bath at 44.5°C ± 0.2°C and incubate for an additional 21
± 2 hours. Maintain water level above the media in immersed tubes. Total
incubation time should not exceed 24 ± 2 hours.
12.4.6 After incubation, remove tubes from the water bath, swirl each tube gently, and
examine for growth and gas production. Gas production with growth
is considered a positive fecal coliform reaction. Please note that for the A-l
procedure, any evolution of gas is considered a positive result. Collection of gas in the
durham tube is not necessary. The presence of gas in the absence of growth is usually
due to mishandling or improper shaking of the tubes after inoculation.
12.4.7 Record positive and negative A-l results and calculate MPN / g total solids (dry
weight) from the number of positive A-l broth tubes as described in 14.1.
Draft October 2002 18
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
13.0 Data Analysis and Calculations
The estimated density of fecal coliform bacteria, based on the confirmation test
using EC or the direct test with A-l, 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
13.1, 13.2, and 13.3):
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 13.4.
13.1 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 liquid samples (Section 11.2.1), four, five-tube dilutions are used, with 10"
3, 10~4, 10~5, and 10~6 mL of the original sample in each tube. Only three of the
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 3, 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.
13.1.1 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 Example A
from Table 3, 10~4 is higher/more dilute than 10~3.)
13.1.2 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 3, Examples B and C).
13.1.3 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 3, Example D).
13.1.4 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 3, Example E).
13.1.5 If there were not enough higher dilutions tested to select three
dilutions, then select the next lower dilution (Table 3, Example F).
19 Draft October 2002
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
13.2 Step 2: Calculate MPN / mL (wet weight)
Obtain the MPN index value from Table 2 using the number of positive tubes in the
three significant dilutions series and calculate MPN / mL using the following equation.
(95% confidence limits may also be obtained from Table 4.)
MPN index value from Table 2
MPN / ml_ =
largest volume tested in dilution series used for MPN determination
MPN / mL for significant dilution combinations not appearing in Table 4 may be estimated by the
following derivation of Thomas' formula (Equation 2, Reference 19.6). If such unlikely tube
combinations occur in more than 1% of the samples, it is an indication that the procedure is faulty
or that the statistical assumptions underlying the MPN estimate are not being fulfilled.
number of positive test tubes
MPN / ml =
sample in negative tubes X ml sample in all tubes)
Draft October 2002 20
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Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
Table 2. MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
When Five Tubes are Used per Dilution
Combination
of Positives
v '/M\i-' y
0-1-1
0-2-0
0-2-1
;"i-£M3 ; ' ',
' ;, J^-l' ) y ,
1-0-2
1-1-0
1-1-1
V * ]l'iv!'"V ? ' t
„ g. *| v*£ k 1 % /
1-3-0
1-3-1
1-4-0
2-1-0
2-1-1
2-1-2
•'"•:;'fcS'.iy
2-3-0
2-3-1
2-4-0
v . /3-M,';.,,
^*''"3VcK2:'{,\."'.
3-1-0
3-1-1
3-1-2
/•> 3-5-4 ''V
3-3-0
3-3-1
3-3-2
v yi4-f>
4-0-0
4-0-1
4-0-2
MPN Index
- v f ' , 1 ( „ ,*
0.36
0.37
0.55
r- &2p"'y
:. ';.'0,40;, ,";
0.60
0.40
0.61
, ;^2S/v ;
0.83
1.0
1.0
-• !< exit _ \;
0.68
0.92
1.2
\y!ly'r
1.2
1.4
1.5
''.'•'tf.7$v; •<;',
..^ii.t'y.
''• .'• t'3 [••':
1.1
1.4
1.7
^'%;r'<
1.7
2.1
2.4
•i.'vS'v^
1.3
1.7
2.1
95% Confidence Limits
Lower
, ;,- ^.009- .; •
0.07
0.07
0.18
; - ; 0,t8 , , ^~
v\- "A$$ ,",;;;;
'. ''"o.O?. • ^-
0.18
0.071
0.18
;''!'lteVv
0.34
0.35
0.35
''• ' 'C.34 V
0.18
0.34
0.41
V'-'JJj1'-;
0.41
0.59
0.59
; '^kmf^
-'• '-'O-^S,''.-^':
0.35
0.56
0.60
y>-;y
0.68
0.68
0.98
'•••'{St
0.41
0.59
0.68
Upper
-;>!-•:
1.0
1.0
1.5
y'- i,o '" . .;
\^, yto- ;"-•'.;'
1.5
1.2
1.5
\ > - f ,, ' t \
2.2
2.2
2.2
1.7
2.2
2.6
^::£r:..;->
2.6
3.6
3.6
'y -","2,3 .' .',
'•"' ''",'^S ';'" .'
2.6
3.6
3.6
-y'tty
4.0
4.0
7.0
";.';,:;J°: :'•
3.5
3.6
4.0
Combination
of Positives
''! , n ft * \
4-1-2
4-1-3
4-2-0
' '\$-2-1' _',-''
- •; 4-2-2 ' •" - ,
'•"•'4&£'-: ', ;
4-3-0
4-3-1
4-3-2
1 s( f«.»fjk,isLf" f« f
tv Ai4»^' * k
4-5-0
4-5-1
5-0-0
v .,'§-0';2"'V, '
5-1-0
5-1-1
5-1-2
' W*\! '•
;-' "iJl:"-:\'"
5-2-2
5-2-3
5-2-4
*•'•', £&£''>'' ^
5-3-3
5-3-4
5-4-0
:-;.J|iy;
5-4-4
5-4-5
5-5-0
v i/ 5-4-2 , y
5-5-4
5-5-5
MPN Index
y-^r;
2.6
3.1
2.2
' > , >B ^ , \f
l,';',:;^.,"-'"' :
•" ",3!,8 '.,;
2.7
3.3
3.9
y:fy:%:
4.1
4.8
2.3
3.3
4.6
6.3
y w y.
>:.vvS.''"::-
9.4
12
15
.v'^y'y
"'! 34;''*'-
17
21
13
:y^'2;;!y
35
43
24
;:y'|;;';
160
>160
95% Confidence Limits
Lower
v^Sr-1'
0.98
1.0
0.68
' ;'ft"9$-''; ;'
- ' 1,0,: •'"-••
]*' i,4\ '-. "
0.99
1.0
1.4
V ;,"}', 4 '_ A
1.4
1.5
0.68
1.0
1.4
2.2
"^"4-\!:
• i.^'y''
3.4
3.6
5.8
* ''•' $;2,; ;. •
7.0
7.0
3.6
;":-^ - r.
10
15
7.0
v" '22--«,;v
40
70
Upper
' •' ,'W - ,
7.0
7.0
5.0
;;, -7-.0-. '
r>vl7.e -'•"_;
:. >j'lft; /''
7.0
7.0
10
'XiH'1, v
,' "JQ*,1' ',
10
12
7.0
10
12
15
'•"'Cl*«^'
23
25
40
'''.' ~40' "•"•,'
40
40
40
'^ l-'i" '
71
110
71
.J.'/^Jy'
460
—
Draft October 2002
-------
Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
Examples of MPN / mL calculations are provided in Table 3, below.
TABLE 3. EXAMPLES OF SIGNIFICANT DILUTION SELECTION AND CALCULATION OF MPN / ML
(SIGNIFICANT DILUTIONS ARE UNDERLINED AND LARGEST SIGNIFICANT DILUTIONS HIGHLIGHTED)
Example
(liquid
or solid)
A
B
C
D
E
F
io-3
mL or g
5/5
4/5
0/5
5/5
4/5
5/5
1Q-4
mL or g
5/5
5/5
1/5
3/5' .
4/5
5/5
io-5
mL or g
3/5
1/5
0/5
1/5
0/5
5/5
io-6
mL or g
0/5
0/5
0/5
1/5
1/5
2/5
Step 1 :
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
(7.9 /10-4) = 79,000 MPN / mL
(4.8 / 10-3) = 4800 MPN / mL
(0.18 /10-3) = 180 MPN /mL
(1.4/10-4)= 14,000 MPN /mL
(4.0/10-3) = 4,OOOMPN/mL
(54 / ID'4) = 540,000 MPN / mL
13.3 Step 3: Convert to MPN / g total solids (dry weight)
For analysis and calculation of percent total solids, refer to Draft Method 1684 (Section 11).
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 / g (dry weight) =
MPN / mL (wet weight) from step 2
percent total solids (expressed as a decimal)
Examples of the conversion to MPN / g (dry weight) are provided in Table 4.
Draft October 2002
22
-------
Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
TABLE 4. EXAMPLES OF CONVERSION TO MPN / g TOTAL SOLIDS (DRY WEIGHT), CONTINUING FROM STEP
2 IN TABLE 3.
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 106 MPN / g dry weight
4800 / 0.6 = 8000 = 8.0 x 103 MPN / g dry weight
180 / 0.56 = 321 = 3.2 x 102 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 ,255,814 = 1 .3 x 106 MPN / g dry weight
13.4 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 Iog10 value,
averaging the Iog10 values, and
• taking the antilog of the mean Iog10 value.
An example is provided in Table 5 below.
TABLE 5.
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 = 4.2 X106
1,700,000= 1.7X106
1,400,000= 1.4X106
400,000 = 4.0 X105
1,100,000= 1.1 X106
510,000 = 5.1 X105
'og10
5.78
6.62
6.23
6.15
5.60
6.04
5.71
Mean of Iog10 values = (5.78 + 6.62 + 6.23 + 6.15 + 5.60 + 6.04 + 5.71 ) / 7 = 6.02
Antilog of 6.02 = 1,047,128 = 1.0x 106 geometric mean MPN of fecal coliforms /g (dry weight)
23
Draft October 2002
-------
Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
14.0 Method Performance
[This section will be updated based on validation study results]
15.0 Reporting Results
15.1 Report sample results as MPN fecal coliforms / g of total solids when using the multiple-tube
fermentation procedures. See Draft Method 1684 for determination of total solids.
16.0 Verification Procedures
[Section will be updated after the validation study]
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. 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 (EPA 233-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 American Society for Testing and Materials. Reagent Chemicals, American Chemical Society
Specifications. American Chemical Society. Washington, DC. For suggestions of the testing of
reagents not listed by the American Chemical Society, see Reference 19.7.
19.3 Annual Book of ASTMStandards. Vol. 11.01. American Society for Testing and Materials.
Philadelphia, PA 19103.
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.
19.5 Environmental Regulations and Technology: Control of Pathogens and Vector Attraction in
Draft October 2002 24
-------
Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
Biosolids. 1992. EPA/625/R-92/013. Office of Research and Development. USEPA.
19.6 Thomas, H. A. Jr. 1942. Bacterial densities from fermenation tubes. Journal of the American
Water Works Association. 34:572.
19.7 Analytical Standards for Laboratory Chemicals. BDH Ltd. Poole, Dorset, UK, and the United
States Pharmacopeia.
19.8 Bordner, R. H., C. F. Frith and J. A. Winter (eds.). 1977. Proceedings of the Symposium on the
Recovery of Indicator Organisms Employing Membrane Filters. EPA600/9-77-024. USEPA,
Environmental Monitoring and Support Laboratory, Cincinnati, OH.
19.9 Lin, S. D. 1973. Evaluation of coliform tests for chlorinated secondary effluents. Journal of the
Water and Pollution Control Federation. 45:498.
20.0 Flowcharts and Validation Data
20.1 The following pages contain flow charts of dilution and inoculation schemes (Section 11) and for
the procedures (Section 12). Schemes for dilution and inoculation are dependent on Class (A or
B) and matrix (solid) or (liquid).
25 Draft October 2002
-------
FIGURE 1. MULTIPLE TUBE FERMENTATION PROCEDURE
LTB/EC Method
Choose either LTB/EC
orA-1 method
A-1 Method
Inoculate LTB and incubate at
35°C ± 0.5°C for 24 ± 2 hr
Inoculate A-1 and incubate at
35°C ± 0.5°C for 3 hr
Growth and gas production
No gas production
Transfer to water bath at
44.5°C ± 0.2°C for additional 21 ± 2 hr
Incubate additional 24 hr
(total of 48 ± 3 hr)
Failure to produce gas within
24 ± 2 hr:
NEGATIVE fecal coliform
Growth and gas production
within 24 ± 2 hr:
POSITIVE fecal coliform
Inoculate EC and incubate
at 44.5°C ± 0.2°C
for 24 ± 2 hr
Growth and gas production
Failure to produce gas
within 48 ± 3 hr:
NEGATIVE fecal coliform
Growth and gas
production within
24 ± 2 hr:
POSITIVE fecal coliform
Failure to produce gas
within 24 ± 2 hr:
NEGATIVE fecal coliform
-------
FIGURE 2. CLASS B LIQUID SAMPLE DILUTION AND INOCULATION SCHEME
Class B
liquid
sample
"Homogenized sample"
—11.0ml-
— 11.0ml-
-11.0ml-
Delivery Volume
-11.0ml-
1.0mL
y, w
o 5
-11.0 mL-
1.0 ml
y, 03
o ro
-11.0ml-
1.0mL
3,03
o (^
ft
a co
1.0ml
,03
-------
FIGURE 3. CLASS B SOLID SAMPLE DILUTION AND INOCULATION SCHEME
Class B
solid
sample
30 g-*
Rinse sample into blender
with 270 mL of sterile
buffered dilution water
"Homogenized Sample"
(10-1)
-11.0 mL
11.0mL
Delivery Volume
1.0 mL
CD
O
11.0mL
11.0mL
1.0 mL
CD
O
€i-
3
5L ro
1.0 mL
11.0mL
1.0 mL
CD
o
8
-------
FIGURE 4. CLASS A LIQUID SAMPLE DILUTION AND INOCULATION SCHEME
Class A
liquid sample
"Homogenized sample"
Delivery Volume
-11.0ml-
99 ml of sterile
buffered dilution water
"A"
(10-1)
-11.0ml-
99 ml of sterile
buffered dilution water
"B"
(10-2)
-11.0 ml-
99 ml of sterile
buffered dilution water
"C"
(10-3)
1.0mL
1.0 ml
1.0 ml
1.0mL
o
3
Q
3
I—
2,0)
f 8
T? K 1
03
&_ CO
en
Q)
3
ro
p
w
3
i—
2,0)
-------
FIGURE 5. CLASS A SOLID SAMPLE DILUTION AND INOCULATION SCHEME
Class A
solid sample
-30 g-
Delivery Volume
Rinse sample into blender
with 270 ml of sterile
buffered dilution water
"Homogenized Sample"
(10-1)
10.0mL
-11.0ml-
1.0ml
99 ml of sterile
buffered dilution water
"A"
(10-2)
-11.0ml-
99 ml of sterile
buffered dilution water
"B"
(10-3)
1.0mL
1.0ml
o
(Q
03
(D I!'
Q. Q)_
=' cn
3|
•o_
n>
CQ
o
I*
(Q ;;:•
CQ
o
•o.
n>
-------
Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
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 coli
EPA Environmental Protection Agency
g gram
L liter
LTB lauryl tryptose broth
mg milligram
mL milliliter
mm millimeter
MPN most probable number (in this method, multiple tube fermentation)
NIST National Institute of Standards and Technology
TD to deliver
QC quality control
21.2 Definitions, acronyms, and abbreviations (in alphabetical order).
Analyst—The analyst must have two years of college lecture and laboratory course work in
microbiology or a closely related field. The analyst also must have at least 6 months bench
experience, must have at least 3 months experience with plating procedures, and must have
successfully analyzed at least 50 biosolid samples for fecal coliforms. Six months of additional
experience in the above area may be substituted for two years of college. The analyst must also
demonstrate acceptable performance during an on-site evaluation.
Analyte—The microorganism tested for by this method. The analytes in this method are fecal
coliforms.
Enrichment—Using a culture media for preliminary isolation that favors the growth of a
particular kind of organism.
Liquid samples—Generally defined as samples containing <10% 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.
Most probable number method (MPN)—A statistical determination of the number of bacteria per
weight or volume of sample. It is based on the fact that the greater the number of bacteria in a
sample, the more dilution is needed to reduce the density to the point at which no bacteria are left
to grow in a dilution series.
Must—This action, activity, or procedural step is required.
31 Draft October 2002
-------
Method 1680 (Fecal Conforms in Biosolids by Multiple-tube Fermentation)
Preferred—Optional
Preparation blank—See Method blank.
Quantitative transfer—the process of transferring a solution from one container to another using a
pipette in which as much solution as possible is transferred, followed by rinsing of the walls of
the source container with a small volume of rinsing solution (e.g., PBS), followed by transfer of
the rinsing solution, followed by a second rinse and transfer.
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 defined as samples containing >10% total solids (dry weight).
Technician—See Analyst.
Draft October 2002 32
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