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.
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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.
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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.
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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.
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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.
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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.
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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

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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.
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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

-------
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

-------
                                   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

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                                   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

-------