vvEPA
   United States
   Environmental Protection
   Agency
   Method 1680: Fecal Coliforms in
   Sewage Sludge (Biosolids) by Multiple-
   Tube Fermentation using Lauryl
   Tryptose Broth (LTB) and EC Medium

   April 2010

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U.S. Environmental Protection Agency
      Office of Water (4303T)
   1200 Pennsylvania Avenue, NW
      Washington, DC 20460
        EPA-821-R-10-003

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                                   Acknowledgments

The contributions of the following persons and organizations to this study are gratefully acknowledged:

Referee Laboratory
•      EPA Office of Research and Development, National Risk Management Research Lab: Mark C.
       Meckes and Karen M. White

Volunteer Participant Laboratories
•      American Interplex: John Overbey and Lizbeth Huggins
•      BioVir Laboratories: Rick Danielson and Jim Truscott
•      City of Los Angeles Bureau of Sanitation Environmental Monitoring Division: Farhana
       Mohamed and Zora Bahariance
•      County Sanitation Districts of Los Angeles County: Shawn Thompson and Julie Millenbach
       Environmental Associates:  Susan Boutros and John Chandler
•      Hampton Roads Sanitation District: Anna Rule and Bob Maunz
       King County Environmental Laboratory: Greg Ma and Bobbie Anderson
•      Hoosier Microbiological Laboratories: Don Hendrickson, Keri Nixon, Katy Bilger, and Lindsey
       Shelton
•      Massachusetts Water Resources Authority: Steve Rhode and Mariya Gofshteyn
•      Milwaukee Metropolitan Sewerage District: Jeff MacDonald and Tim O'Neill
•      University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord
       Utah Department of Health: Sanwat Chaudhuri and Devon Cole

The following facilities provided biosolid matrices for the study:
•      Compost Facility, Columbus, OH: Angela Bianco
       Wastewater Treatment Facility, Sturgeon Bay, WI: Todd Maurina
•      Wastewater Treatment Facility, Fairfield, OH: Drew Young
       Wastewater Treatment Facility, Mason, OH: Ernie Stickler
•      N-Viro Treatment Facility, Toledo, OH: Cindy Drill

Media Photographs
       Mark C. Meckes, NRMRL, US EPA

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                                         Disclaimer

Neither the United States Government nor any of its employees, contractors, or their employees make any
warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use of
or the results of such use of any information, apparatus, product, or process discussed in this report, or
represents that its use by such party would not infringe on privately owned rights.  Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.

Questions concerning this method or its application should be addressed to:

Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov or OSTCWAMethods@epa.gov
                                              IV

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                                   Table of Contents









1.0    Scope and Application 	  1




2.0    Summary of Method	  2




3.0    Definitions	  2




4.0    Interferences  	  3




5.0    Safety  	  3




6.0    Equipment and Supplies	  3




7.0    Reagents and Standards 	  5




8.0    Sample Collection, Handling, and Storage	  8




9.0    Quality Control	  10




10.0   Equipment Calibration and Standardization	  14




11.0   Sample Preparation  	  14




12.0   Lauryl Tryptose Broth (LTB) - EC Broth  Procedure 	  21




13.0   Verification	  23




14.0   Data Analysis and Calculations  	  24




15.0   Sample Spiking Procedure  	  30




16.0   Method Performance	  36




17.0   Pollution Prevention	  36




18.0   Waste Management	  37




19.0   References	  37




20.0   Figures	  37




21.0   Glossary  	  43

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     Method 1680: Fecal Conforms in Sewage Sludge (Biosolids) by
Multiple-Tube Fermentation  using  Lauryl  Tryptose Broth (LIB) and EC
                                        Medium

                                       April 2010
1.0    Scope and Application

1.1     This method describes multiple-tube fermentation procedures [also called the most probable
       number (MPN) procedure] for the detection and enumeration of fecal coliform bacteria in
       biosolids. These methods use culture-specific media and elevated temperature to isolate and
       enumerate fecal coliform organisms. Fecal coliform bacteria, including Escherichia coli (E. coif),
       are commonly found in the feces of humans and other warm-blooded animals, and indicate the
       potential presence of other bacterial and viral pathogens.

1.2     This method is adapted from method 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 U.S. Environmental
       Protection Agency (EPA) in regulating the use and disposal of biosolids under 40 CFR Part 503.
       Subpart D of the 503 regulations protects public health and the environment through requirements
       designed to reduce the potential for contact with disease-bearing microorganisms (pathogens) in
       biosolids applied to land or placed on a surface disposal site.

1.4     Fecal coliform density is expected to correlate with the probability of pathogens present and
       document process performance (vector attraction reduction).

1.5     This method may be used to determine the density of fecal coliform bacteria in Class A and Class
       B biosolids to  satisfy the pathogen reduction requirements of Subpart D of Part 503. A biosolid
       sample is classified as  Class A if it contains a fecal coliform density below 1,000 MPN/g of total
       solids (dry weight basis).  A biosolid sample is classified as Class B if the geometric mean fecal
       coliform density is less than 2 x 106 MPN/g of total solids (dry weight basis).

1.6     To satisfy the pathogen reduction monitoring alternatives for Class B biosolids, seven samples of
       treated biosolids are collected at the time of use or disposal and the geometric mean fecal
       coliform bacterial density of these samples is confirmed not to exceed 2 x 106 MPN/g of total
       solids (dry weight basis).  Although the Part 503 regulation does not specify the total number of
       samples for Class A biosolids, it is recommended that a sampling event extend over two weeks,
       and that at least seven samples be tested to confirm that the mean bacterial density of the samples
       is below 1,000 MPN/g of total solids (dry weight basis). The analysis of seven samples increases
       the method precision by reducing the standard error caused by inherent variations in biosolid
       quality.

1.7     The presence of fecal coliforms may be determined in both Class A and Class B biosolids using
       the MPN procedure.

1.8     Any modification of the method beyond those expressly permitted is subject to the application
       and approval of alternative test procedures under 40 CFR Parts  136.4 and 136.5.
                                                                                   April 2010

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Method 1680
1.9    Method 1680 was submitted to interlaboratory validation in Class A and Class B biosolid
       matrices. A summary of method performance results from this validation study are provided in
       Section 16.0. A comprehensive evaluation of the study results is presented in the validation study
       report (Reference 19.2). For method application please refer to Title 40 Code of Federal
       Regulation Part 136 (40 CFRPart 136).

       Note: Based on the high false positive rates observed for Method  1681 in some matrices, EPA
       recommends that laboratories conduct their own matrix-specific comparisons to determine the
       most appropriate method (1680 or 1681).
2.0   Summary of Method

2.1    Fecal coliform densities of biosolids may be determined by the MPN procedure.

2.2    MPN procedure (Class A and B)

       Method 1680 provides for the enumeration of fecal coliforms in Class A and Class B biosolids
       using the most probable number (MPN) procedure. In Method 1680, LTB is used as a
       presumptive medium followed by EC as confirmation of fecal coliforms.  EC may not be used for
       direct isolation from a biosolid sample because prior enrichment in presumptive medium (LTB) is
       required for optimum recovery of fecal coliforms.

       2.2.1   Summary of the LTB/EC procedure (see Figure 1 in Section 20.0) [see Sections 11.0
               and 12.0 for explanation]

               2.2.1.1    A minimum of four sample dilutions are required, while five or more are
                         preferred. Each sample dilution is inoculated into five test tubes, containing
                         sterile LTB and an inverted vial (gas production).

               2.2.1.2    LTB sample tubes are incubated in a water bath or jacketed incubator at
                         35°C ± 0.5°C. After 24 ± 2 hours, the tubes are examined for presumptive
                         growth and gas production. Gas production is indicated by gas bubble
                         formation within the inverted-vial.  Negative tubes are reincubated for an
                         additional 24  hours and reassessed. Failure to produce gas in LTB medium
                         within 48 ± 3  hours is a negative presumptive test.  EC tubes are incubated in
                         a water bath at 44.5°C ± 0.2°C for 24 ± 2 hours.  Following growth in LTB,
                         gas production in EC broth within 24 ± 2 hours is considered a positive fecal
                         coliform reaction. Failure to produce gas is a negative reaction and indicates
                         fecal coliform bacteria are not present.

               2.2.1.3    A total solids determination is performed on a representative biosolids
                         sample and is used to calculate MPN/g dry weight. Fecal coliform density is
                         reported as MPN/g dry weight.


3.0   Definitions

3.1    Fecal coliform bacteria are gram-negative, non-spore-forming rods that are found in the intestines
       and feces of humans and other warm-blooded animals.  The predominant fecal coliform is E.  coll.
       In this method, fecal coliforms are those bacteria that grow and produce gas in LTB within a total
       of 48 ± 3 hours after incubation at 35°C ± 0.5°C, and that subsequently ferment lactose and

April 2010                                       2

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                                                                                    Method 1680
       produce gas within 24 ± 2 hours in EC broth after incubation at 44.5°C ± 0.2°C.  Since coliforms
       from other sources often cannot produce gas under these conditions, this criterion is used to
       define the fecal component of the coliform group.

3.2    Class A biosolids contain a fecal coliform density below 1,000 MPN/g of total solids (dry weight
       basis).

3.3    Class B biosolids contain a geometric mean fecal coliform density of less than 2  x 106 MPN/g of
       total solids (dry weight basis).

3.4    Definitions for other terms are given in the glossary at the end of the method.
4.0   Interferences

4.1    MPN procedure: Since the MPN tables are based on a Poisson distribution, if the sample is not
       adequately mixed to ensure equal bacterial cell distribution before portions are removed, the
       MPN value will be a misrepresentation of the bacterial density.
5.0   Safety

5.1    The analyst must observe normal safety procedures required in a microbiology laboratory while
       preparing, using, and disposing of media, cultures, reagents, and materials, and while operating
       sterilization equipment.

5.2    Field and laboratory staff collecting and analyzing environmental samples are under some risk of
       exposure to pathogenic microorganisms.  Staff should apply safety procedures used for pathogens
       to handle all samples.

5.3    Mouth-pipetting is prohibited.
6.0   Equipment and  Supplies

6.1    Sterile plastic bags, 1-gallon
6.2    Sterile plastic or glass jars with lids, 1-L
6.3    Sterile auger
6.4    Sterile scoops (do not use curved scoops)
6.5    Ice chest
6.6    Wet ice
6.7    Ice packs, blue ice
6.8    Bubble wrap
6.9    Sterile trowels
6.10   Sterile aluminum foil or kraft paper
6.11   Sterile container, such as a stainless steel or plastic bucket suitable for sample collection
6.12   Flat shovel
6.13   Dilution bottles, borosilicate glass, screw cap, marked at 99 mL or screw cap, borosilicate glass or
       plastic tubes marked at  9 mL

                                               3                                       April 2010

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Method 1680
6.14   Tubes, 16 x 150 mm, borosilicate glass, with loose-fitting aluminum, stainless steel or
       autoclavable caps
6.15   Durham tubes or vials, 10 x 75 mm, borosilicate glass
6.16   Tubes, 16 x 100 mm, screw cap, borosilicate glass, with autoclavable plastic caps
6.17   Test tube racks to hold sterile culture tubes
6.18   Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets
6.19   Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, wide-tip of appropriate volume
6.20   Pipet bulbs, or automatic pipettor
6.21   Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic
       loops
6.22   Sterile disposable applicator sticks
6.23   Bunsen burner or alcohol burner
6.24   Cornwall syringe, sterile, to deliver at least 5 mL
6.25   Media dispensing pump
6.26   Incubator, water- or air-jacketed, humidity-controlled, microbiological type to hold temperature
       at35.0°C±0.5°C
6.27   Gable covered water bath, with circulating system to maintain temperature of 44.5°C ± 0.2°C
6.28   Plastic sterile petri dishes, microbiological grade, 15 mm x  100 mm
6.29   Erlenmeyer flasks, 1-L and 2-L
6.30   Stir bar
6.31   Stir plate
6.32   Sterile blender jars and base
6.33   Water bath maintained at 50°C for tempering agar
6.34   Balance, analytical balance capable of weighing 0.1 mg
6.35   Thermometer, checked against a National Institute of Standards and Technology (NIST) certified
       thermometer, or one that meets the requirements of NIST Monograph SP 250-23
6.36   Latex gloves for handling samples
6.37   pH meter
6.38   Vortex mixer
6.39   Flasks, borosilicate glass, screw-cap, 250-2000 mL volume
6.40   Graduated cylinders, 100- to 1000-mL, covered with aluminum foil or kraft paper and sterilized
6.41   Beakers, glass or plastic, assorted sizes
6.42   Steel pan of water, 30" x 26" x 10"
6.43   Autoclave or steam sterilizer capable of achieving 121°C [15 Ib pressure per square inch (PSI)]
       for 15 minutes
6.44   Crucible or  aluminum evaporating dish
6.45   Drying oven maintained at 103°C - 105°C for tempering agar
April 2010

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                                                                                    Method 1680
7.0   Reagents and Standards

7.1    Reagent-grade chemicals shall be used in all tests.  Unless otherwise indicated, reagents shall
       conform to the specifications of the Committee on Analytical Reagents of the American Chemical
       Society (Reference 19.3).  The agar used in preparation of culture media must be of
       microbiological grade.

7.2    Whenever possible, use commercial dehydrated culture media.

7.3    Purity of reagent water: Reagent-grade water conforming to specifications in Standard Methods
       for the Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part
       136 or 141, as applicable), Section 9020 (Reference 19.1).

7.4    Phosphate buffered dilution water

       7.4.1   Composition of stock phosphate buffer solution:

               Potassium dihydrogen phosphate (KH2PO4)        34.0 g
               Reagent-grade water                          500.0 mL

               Preparation: Dissolve KH2PO4 in 500 mL reagent-grade water. Adjust the pH of the
               solution to 7.2 with  1 N NaOH, and bring the volume to 1 L with reagent-grade water.
               Sterilize by filtration or autoclave at  121°C (15 PSI) for 15 minutes.

       7.4.2   Preparation of stock magnesium chloride (MgCl2) solution:  Add 38 g anhydrous MgCl2
               or 81.1 g magnesium chloride hexahydrate (MgCl2 • 6H2O)  to 1 L reagent-grade water.
               Sterilize by filtration or autoclave at  121°C (15 PSI) for 15 minutes.

       7.4.3   After sterilization, store the stock solutions in the refrigerator until used.  If evidence of
               mold or other contamination appears, the affected stock solution should be discarded and
               a fresh solution should be prepared.

       7.4.4   Working phosphate  buffered dilution water: Mix 1.25 mL of the stock phosphate buffer
               and 5 mL of the MgCl2 stock per liter of reagent-grade water. Dispense in appropriate
               amounts for dilutions and/or for use as rinse buffer. Autoclave at 121 °C (15 PSI) for 15
               minutes. Final pH should be 7.0 ± 0.2. The amount of time in the autoclave must be
               adjusted for the volume of buffer in the containers and the size of the load.

               Note: When test tube racks containing 9.0 mL sterile dilution water are prepared, they are
               placed into an autoclavable pan with  a small amount of water to contain breakage and
               minimize evaporation from the tubes.
                                                                                      April 2010

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Method 1680
7.5    Heart infusion agar (HIA)

       7.5.1   Composition:

               Beef heart, infusion from 500 g                   10. Og
               Bacto tryptose                                  10.0 g
               Sodium chloride (NaCl)                           5.0 g
               Bacto agar                                      15.0g
               Reagent-grade water                              l.OL

       7.5.2   Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve.  Adjust
               pH to 7.4 ± 0.2 with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide. Stir well and
               autoclave at 121°C (15 PSI) for 15 minutes.  Pour into 15 x 100 mm sterile petri plates.
               Let the media warm to room temperature prior to inoculation. Other general growth
               media may be  used for quality assurance (QA) (Section 9.0) purposes.

7.6    LTB medium

       7.6.1   Composition:

               Tryptose                                      20.0 g
               Lactose                                        5.0 g
               Dipotassium hydrogen phosphate (K2HPO4)        2.75 g
               Potassium dihydrogen phosphate (KH2PO4)         2.75 g
               Sodium chloride (NaCl)                          5.0 g
               Sodium lauryl sulfate                            0.1 g
               Reagent-grade water                             1.0 L

       7.6.2   For single strength (IX) LTB, add reagents to 1 L of reagent-grade water, mix
               thoroughly, and heat to dissolve.  Adjust pH to 6.8 ± 0.2 with 1.0 N hydrochloric acid or
               1.0 N sodium hydroxide.  Dispense 10-mL volumes into 25 x 150 mm culture tubes. IX
               TSB will be used for inoculation volumes < 1 mL. Autoclave at 121°C (15  PSI) for 15
               minutes.

       7.6.3   For double strength (2X) LTB, prepare as in Section 7.6.2 but use 500 mL of reagent-
               grade water instead of 1 L.

               Note: 2X LTB is necessary for 10-mL inoculations, to ensure that the 10-mL inoculation
               volume does not excessively dilute the media.
April 2010

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                                                                                     Method 1680
7.7    EC medium

       7.7.1   Composition:

               Tryptose or trypticase                           20.0 g
               Lactose                                         5.0g
               Bile salts mixture or bile salts No.3                1.5 g
               Dipotassium hydrogen phosphate (K2HPO4)        4.0 g
               Potassium dihydrogen phosphate (KH2PO4)        1.5 g
               Sodium chloride (NaCl)                          5.0 g
               Reagent-grade water                             l.OL

       7.7.2   Add reagents 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 (15 PSI) for 15
               minutes. Medium should fill inverted tubes leaving no air spaces.

7.8    Positive control

       7.8.1   Obtain a stock culture of E. coll (e.g., ATCC # 25922) as a positive control for LTB and
               EC medium. Note: ATCC recommends that no more than 5 transfers be made before
               returning to the original culture.  This will minimize the chance of contamination during
               transfers and genetic shift of the culture. One suggestion is to make your own frozen
               seed stock upon receipt of the organism that can be used for future work. For additional
               information go to http: //www. atcc. org.

7.9    Negative controls

       7.9.1   Obtain a stock culture of Enterobacter aerogenes (e.g., ATCC # 13048) as a negative
               control for EC medium.

       7.9.2   Obtain a stock culture of Pseudomonas (e.g., ATCC # 27853) as a negative control for
               LTB.

7.10   The storage times for prepared media used in this method are provided in Table 1.


Table 1.  Storage Times for Prepared Media
Media
Agar or broth (EC, LTB, and HIA) in loose-cap tubes
Agar or broth (EC, LTB, and HIA) in tightly closed screw-cap tubes
Poured agar plates (should be stored inverted)
Large volume of agar in tightly closed screw-cap flask or bottle
Storage Time
2 weeks
3 months
2 weeks
3 months
Note'. If media is refrigerated, remove from refrigerator 1-1.5 hours prior to inoculation, so that it reaches room
temperature prior to use.
                                                                                        April 2010

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Method 1680
7.11   Milorganite® (CAS 8049-99-8) or equivalent

       Milorganite® (heat-dried Class A biosolid) is produced by Milwaukee Metropolitan Sewerage
       District.  It is available in many home gardening centers.

       Obtain Milorganite® as the reference matrix for initial precision and recovery (IPR) and ongoing
       precision and recovery (OPR) analyses.  Milorganite® is used as the reference matrix because it
       is easily accessible, inexpensive, generally does not contain the analyte of interest, and is of
       consistent quality.
8.0   Sample Collection,  Handling, and Storage

8.1    The most appropriate location for biosolid sample collection is the point prior to leaving the
       wastewater treatment plant. Samples may be taken from pipes, conveyor belts, bins, compost
       heaps, drying beds, and stockpiles.

8.2    Collect samples in sterile, non-toxic glass, or plastic containers with leak-proof lids. All
       sampling containers and equipment must be clean and sterile.

8.3    Equipment and container cleaning procedure

       8.3.1  Wash apparatus with laboratory-grade detergent and water

       8.3.2  Rinse with tap water

       8.3.3  Rinse with 10% HC1 acid wash

       8.3.4  Rinse with distilled water

       8.3.5  Allow to air dry

       8.3.6  Cover with foil and autoclave for 15 minutes at 121°C (15 PSI)

8.4    Digester biosolids sampling procedure

       8.4.1  Collect digester biosolids sample from the discharge pipe.

       8.4.2  Purge the discharge pipe of old biosolids and warm to the digester temperature by
              allowing biosolids to flow through the pipe into a container or waste collection device

       8.4.3  Position a 1-gallon sterile bag under the flow so that only the  sample touches the inside
              of the bag. Fill the bag, leaving 0.5 inches of head space in the bag for gas production.
              Leaving head room is extremely important when taking samples of biosolids that have
              been anaerobically digested.

8.5    Procedure for sampling conveyor belt biosolid output

       8.5.1  Using a sterile scoop, transfer the pressed biosolids directly from the conveyer into a
              sterile container, without mixing or transferring to another area.
April 2010

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                                                                                     Method 1680
       8.5.2   Pack sample into sterile container.  Leaving additional head space is not as important as
               in Section 8.4 because there is less gas formation.

8.6    Procedure for sampling from a bin, drying bed, truck bed, or similar container

       8.6.1   Remove surface material (upper six inches) and set it aside. Divide the underlying
               material to be sampled into four quadrants.

       8.6.2   Use a scoop or core the sample if material is deep.

       8.6.3   Take a sample from each of the quadrants and combine in a sterile container.

       8.6.4   After all the samples have  been taken, pour the contents of the container out onto a sterile
               surface and mix by folding the sample back onto itself several times.

       8.6.5   Reduce the sample size by "coning and quartering." Divide the container contents into
               four even piles.  If sample  size is still too large, divide each quarter  into quarters and
               discard half.  Put into a glass or plastic sampling container.

       8.6.6   An alternate method to "coning and quartering" is to randomly take a flat shovel full of
               biosolids from the  contents of the container that has been placed on a sterile surface and
               put samples into a  sampling container. (Curved scoops have been shown to favor a
               certain size particle and should not be used.)

8.7    Record the following in your log book:

       8.7.1   Facility name and  location

       8.7.2   Date

       8.7.3   Arrival time

       8.7.4   Name of facility and contact

8.8    Record the following onto  sample container and in log book when known:

       8.8.1   Sample number

       8.8.2   Date and time

       8.8.3   Sampler name

       8.8.4   Sample location

       8.8.5   Parameters (e.g., type of analysis, field measurements- pH and temperature)

       8.8.6   Volume

       8.8.7   Observations

8.9    Ensure that the chain-of-custody form is filled out.


                                               9                                       April 2010

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Method 1680
8.10   Sample handling: Maintain bacteriological samples at <10°C during transit to the laboratory. Do
       not allow the sample to freeze. Use insulated containers to ensure proper maintenance of storage
       temperature.  Sample bottles should be placed inside waterproof bags, excess air purged, and bags
       sealed to ensure that bottles remain dry during transit or storage. Refrigerate samples upon
       arrival in the  laboratory and analyze as soon as possible after collection. Bring samples to room
       temperature before analysis.

8.11   Holding time and temperature limitations: For fecal coliform samples for sewage sludge
       (biosolids) only, the holding time is extended to 24 hours for the following sample types using
       either EPA Method 1680 (LTB-EC) or 1681 (A-l): Class A composted, Class B aerobically
       digested, and Class B anaerobically digested. All other matrices should be analyzed within 8
       hours of sample collection, 6 hour maximum transport and 2 hours for sample processing.

       Note: Adherence to sample handling procedures and holding time limits is critical to the
       production of valid data. Sample results will be considered invalid if these conditions are not
       met.
9.0   Quality Control

9.1    Each laboratory that uses this method is required to operate a formal quality assurance (QA)
       program.  The minimum requirements of this program consist of an initial demonstration of
       laboratory capability through the analysis of positive and negative control samples and blanks
       (Sections  9.6 and 9.7).  Laboratory performance is compared to the performance criteria specified
       in Section 16.0 to determine whether the results of the analyses meet the performance
       characteristics of the method. Specific quality control (QC) requirements for Method 1680 are
       provided below.  General recommendations on QA and QC for facilities, personnel, and
       laboratory equipment, instrumentation, and supplies used in microbiological analyses are
       provided in Reference 19.4.

9.2    The minimum analytical QC requirements for the analysis of samples using Method  1680 include
       an initial demonstration of laboratory capability through performance of the initial precision and
       recovery (IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through
       performance of the ongoing precision and recovery (OPR) analysis (Section 9.4) and matrix spike
       (MS) analysis (Section 9.5, disinfected wastewater only), and the routine analysis of positive and
       negative controls (Section 9.6), method blanks (Section 9.7), and media sterility checks (Section
       9.8). For the IPR, OPR and MS analyses, it is necessary to spike samples with
       laboratory-prepared spiking suspensions as described in Section 15.0.

9.3    Initial precision and recovery (IPR):  The IPR analyses are used to demonstrate acceptable
       method performance (recovery and precision) and should be performed by each laboratory before
       the method is used for monitoring field samples. EPA recommends but does not require that an
       IPR be performed by each analyst. IPR samples should be accompanied by an acceptable method
       blank (Section 9.7) and appropriate media sterility checks (Section 9.8).  The IPR analyses are
       performed as follows:

       9.3.1   Prepare four, 30-g samples of Milorganite® and spike each sample with E. coli ATCC
               #  25922  according to the spiking procedure in Section 15.0. Process and analyze each
               IPR sample according to the procedures in Sections 11.0 and 12.0 and calculate the fecal
               coliform MPN/g dry weight according to Section 14.0.
April 2010                                       10

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                                                                                    Method 1680
       9.3.2   Calculate the percent recovery (R) for each IPR sample using the appropriate equation in
               Section 15.7.

       9.3.3   Using the percent recoveries of the four analyses, calculate the mean percent recovery
               and the relative standard deviation (RSD) of the recoveries.  The RSD is the standard
               deviation divided by the mean, multiplied by 100.

       9.3.4   Compare the mean recovery and RSD with the corresponding IPR criteria in Table 2,
               below. If the mean and RSD for recovery of fecal coliforms meet acceptance criteria,
               system performance is acceptable and analysis of field samples may begin. If the mean
               recovery or the RSD fall outside of the required range for recovery, system performance
               is unacceptable. In this event, identify the problem by evaluating each step of the
               analytical process, media, reagents, and controls, correct the problem and repeat the IPR
               analyses.
Table 2.  Initial and Ongoing Precision and Recovery (IPR and OPR
Performance test
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
LTB/EC acceptance criteria
65% -221%
84%
37% -391%
Acceptance Criteria
9.4    Ongoing precision and recovery (OPR): To demonstrate ongoing control of the analytical
       system, the laboratory should routinely process and analyze spiked Milorganite® samples.  The
       laboratory should analyze one OPR sample after every 20 field and matrix spike samples or one
       per week that samples are analyzed, whichever occurs more frequently.  OPR samples must be
       accompanied by an acceptable method blank (Section 9.7) and appropriate media sterility checks
       (Section 9.8). The OPR analysis is performed as follows:

       9.4.1  Spike a 30-g sample of Milorganite® with E. coll ATCC # 25922 according to the
              spiking procedure in Section 15.0. Process and analyze each OPR sample according to
              the procedures in Sections 11.0 and 12.0 and calculate the number of fecal coliform
              MPN/g dry weight according to Section 14.0.

       9.4.2  Calculate the percent recovery (R) for the OPR sample using the appropriate equations
              in Section 15.7.

       9.4.3  Compare the  OPR result (percent recovery) with the corresponding OPR recovery
              criteria in Table 2, above. If the OPR result meets the acceptance criteria for recovery,
              method performance is acceptable and analysis of field samples may continue. If the
              OPR result falls outside of the acceptance criteria, system performance is unacceptable.
              In this event, identify the problem by evaluating each step of the analytical process
              (media, reagents, and controls), correct the problem and repeat the OPR analysis.

       9.4.4  As part of the laboratory QA program, results for OPR and IPR samples should be
              charted and updated records maintained in order to monitor ongoing method
              performance.  The laboratory should also develop a statement of accuracy for Method
              1680 by calculating the average percent recovery (R)  and the standard deviation of the
              percent recovery (sr). Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
                                              11
                      April 2010

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Method 1680
9.5    Matrix spikes (MS): MS analysis are performed to determine the effect of a particular matrix on
       fecal coliform recoveries.  The laboratory should analyze one MS sample when biosolid samples
       are first received from a source from which the laboratory has not previously analyzed samples.
       Subsequently, 5% of field samples (1 per 20) from a given biosolids source should include a MS
       sample. MS samples must be accompanied by the analysis of an unspiked field sample
       sequentially collected from the same sampling site, an acceptable method blank (Section 9.7), and
       appropriate media sterility checks (Section 9.8). When possible, MS analyses should also be
       accompanied by an OPR sample (Section 9.4). The MS analysis is performed as follows:

       9.5.1   Prepare two, 30-g field samples that were sequentially collected from the same site.
               One sample will remain unspiked and will be analyzed to determine the background or
               ambient concentration of fecal coliforms for calculating MS recoveries (Section 9.5.3).
               The other sample will serve as the MS sample and will be spiked with E. coll ATCC #
               25922 according to the spiking procedure in Section 15.0.

       9.5.2   Select dilutions based on previous analytical results or anticipated levels of fecal
               coliforms in the field sample in order to accurately estimate fecal coliform density.
               Neither above or below the detection limit of the method. Section 11.0 includes possible
               dilution schemes for both Class A and Class B biosolids.

       9.5.3   Spike the MS sample with a laboratory-prepared suspension as described in Section 15.0.
               Process and analyze  the unspiked and spiked field samples according to the procedures in
               Sections 11.0 and 12.0.

       9.5.4   For the MS sample, calculate the fecal coliform MPN/g dry weight according to Section
               14.0 and adjust the density (MPN/g dry weight) based on the ambient concentration of
               fecal coliforms observed in the unspiked matrix sample.

       9.5.5   Calculate the percent recovery (R) for the MS sample  (adjusted based on ambient fecal
               coliform in the unspiked sample) using the appropriate equations in Section 15.7.

       9.5.6   Compare  the MS result (percent recovery) with the appropriate method performance
               criteria in Table 3. If the MS recovery meets the acceptance criteria, system performance
               is acceptable and analysis of field samples from this biosolid source may continue. If the
               MS recovery is unacceptable and the OPR sample result associated with this batch of
               samples is acceptable, a matrix interference may be causing the poor results.  If the MS
               recovery is unacceptable, all associated field data should be flagged.

Table 3.  Matrix Spike Precision and Recovery Acceptance Criteria
Performance test
Class A Biosolids: Matrix spike (MS)
MS percent recovery
Class B Biosolids: Matrix spike (MS)
MS percent recovery
Class A Biosolids: Matrix spike, matrix spike duplicate (MS/MSD)
Percent recovery for MS/MSD
Precision (as maximum relative percent difference of MS/MSD)
Class B Biosolids: Matrix spike, matrix spike duplicate (MS/MSD)
Percent recovery for MS/MSD
Precision (as maximum relative percent difference of MS/MSD)
LTB/EC acceptance criteria
30 - 424%
8 - 709%
30 - 424%
150%
8 - 709%
125%
April 2010                                       12

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                                                                                     Method 1680
       9.5.7   Laboratories should record and maintain a control chart comparing MS recoveries for all
               matrices to batch-specific and cumulative OPR sample results analyzed using Method
               1680. These comparisons should help laboratories recognize matrix effects on method
               recovery and may also help to recognize inconsistent or sporadic matrix effects from a
               particular source.

9.6    Culture Controls

       9.6.1   Negative controls: The laboratory should analyze negative controls to ensure that the
               LTB and EC are performing properly. Negative controls should be analyzed whenever a
               new batch of media or reagents is used. On an ongoing basis, the laboratory should
               perform a negative  control  every day that samples are analyzed.

               9.6.1.1     Negative controls are conducted by inoculating LTB with a known negative
                          total coliform species (e.g., Pseudomonas ATCC # 27853) and EC with a
                          known negative fecal coliform species (e.g., Enterobacter aerogenes ATCC
                          # 13048) and analyzing as described in Section 12.0. Viability of the
                          negative controls should be demonstrated using anon-selective media (e.g.,
                          nutrient agar or tryptic soy agar).

               9.6.1.2     If a negative control fails to exhibit the  appropriate response, check and/or
                          replace the associated media or reagents, and/or the negative control, and re-
                          analyze the appropriate negative control.

       9.6.2   Positive controls: The laboratory should analyze positive controls to ensure that the
               LTB and EC are performing properly. Positive controls should be analyzed whenever a
               new batch of media or reagents is used. On an ongoing basis, the laboratory should
               perform a positive control every day that samples are analyzed.  An OPR sample (Section
               9.4) may take the place of a positive control.

               9.6.2.1     Positive controls are conducted by inoculating LTB and EC with a known
                          positive fecal coliform species (e.g., E.  coli ATCC # 25922) and analyzing as
                          described in Section 12.0.

               9.6.2.2     If the positive control fails to exhibit the appropriate response, check and/or
                          replace the associated media or reagents, and/or the positive  control, and
                          reanalyze the appropriate positive control.

9.7    Method blank.  Test a 20-mL sterile dilution water sample  in the analytical scheme to verify the
       sterility of equipment, materials, and supplies. Absence of growth indicates freedom of
       contamination from the target organism.  On an ongoing basis, the laboratory should perform a
       method blank every day that samples are analyzed.

9.8    Media sterility check. To test sterility of media, subject a representative portion of each
       batch to incubation at 35°C ± 0.5°C (LTB)  or 44.5°C ± 0.2°C (EC) for 48 ± 3 or 24 ± 2 hours
       respectively and observe for growth.  With  respect to media, a batch is defined as one tube/plate
       out of 50 in each lot or one tube/plate, if the lot contains less than 50 tubes/plates. Absence of
       growth indicates media sterility.  On an ongoing basis, the laboratory should perform a media
       sterility check every day that samples are analyzed.
                                               13                                       April 2010

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Method 1680
10.0  Equipment Calibration  and Standardization

10.1   Check temperatures in incubators/water baths twice daily, a minimum of four hours apart, to
       ensure operation is within stated limits of the method and record daily measurements in incubator
       log book.

10.2   Check temperatures in refrigerators/freezers at least once daily to ensure operation is within stated
       limits of the method.  Record daily measurements in refrigerator/freezer log book.

10.3   Calibrate thermometers and incubators at least annually against an NIST certified thermometer or
       one that meets requirements of NIST Monograph SP 250-23 (Reference 19.1).  Check mercury
       columns for breaks.

10.4   Calibrate the pH meter prior to each use period with the two standards (pH 4.0, 7.0, and 10.0)
       closest to the range being tested.

10.5   Calibrate top-loading balances monthly with reference weights of ASTM Class 2.
11.0  Sample Preparation

11.1   Homogenization

       Sample homogenization procedures are based on whether the sample is a liquid or a solid.  If
       sample is alkaline-stabilized (liquid or solid), adjust the pH as described in Section 11.1.3.
       Liquid samples are generally defined as samples containing <7% total solids (dry weight).

       11.1.1 Liquid samples: Homogenize 300 mL of sample in a sterile blender on high speed for one
              to two minutes. Adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid or 1.0 N
              sodium hydroxide, if necessary. This is the "homogenized" sample. When adjusting the
              pH do not exceed the homogenized sample volume by greater than 5% (15 mL).

       11.1.2 Solid samples: Weigh out 30.0 ± 0.1 g of well-mixed sample in a sterile dish.  Whenever
              possible, the sample tested should contain  all materials that will be included in the
              biosolid. For example, if wood chips  are part of the biosolid compost, some mixing or
              grinding may be needed to achieve homogeneity before testing.  Large pieces of wood
              that are not easily ground may be discarded before homogenizing.  Transfer the sample to
              a sterile blender.  Alternatively, the sample may be weighed directly into the sterile
              blender jar. Use 270 mL of sterile dilution water (Section 7.4) to rinse any remaining
              sample into the blender.  Cover and blend  on high speed for one minute. This is the
              "homogenized" sample.  A volume of 1.0-mL of the "homogenized" sample contains 10"1
              g of the original sample.  Adjust the pHto 7.0-7.5 by adding 1.0 N hydrochloric acid or
              1.0 N sodium hydroxide, if necessary.

              Note: Do not suspend bacteria in dilution water for more than 30 minutes at room
              temperature.  Chill on wet ice or at 4°C ± 1°C to slow replication between spiking
              samples.

       11.1.3 Alkaline -stabilized: The alkaline-stabilized biosolid samples generally have a pH
              of approximately 12.  Prior to analysis, the alkaline-stabilized biosolid samples must be
              neutralized to a pH of approximately 7.5.  Do not add lab-prepared spikes to the samples
              prior to pH adjustment.

April 2010                                      14

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                                                                                     Method 1680
               11.1.3.1    Adjustment of pH should be done in a fume hood.  Prior to adjusting the pH
                          of the sample, calibrate/standardize the pH meter with pH buffers 7.0 and
                          10.0. Weigh out 30 g of sample into a sterile 600 mL beaker, add 250 mL of
                          sterile buffered dilution water and a sterile magnetic stir bar. Place beaker on
                          a mixing plate, insert pH probe into mixture, begin stirring, and take an
                          initial pH reading. To minimize the amount of volume added to each sample,
                          pH should be adjusted using ION HC1.

                          Note: The addition of the 10 N HC1 will produce fumes, do not be  alarmed.
                          The addition of the acid should be done incrementally to ensure that the pH
                          does not drop instantaneously below 5.0.  It is recommended that the pH
                          adjustment be completed within 10-15 minutes and monitored for an
                          additional 15 minutes to ensure that the sample is able to maintain  a constant
                          pH of around 7.5. Pour pH adjusted sample into blender jar, use the
                          remaining sterile buffered dilution water (15 mL) to rinse the beaker twice
                          and pour rinse water into the blender jar.

11.2   Dilution and Inoculation

       Biosolid samples analyzed for fecal coliforms using this method may require dilution prior to
       analysis. An ideal sample volume will yield results that accurately estimate fecal coliform
       density. Because fecal coliform concentrations in undiluted samples could easily exceed the
       analytical range of this procedure, the laboratory must follow the dilution and inoculation
       schemes in Section  11.2.1 (liquid) or 11.2.2 (solid), if necessary additional dilutions may be
       analyzed to ensure results obtained are not censored (less-than or greater-than) values.  Although
       other dilution and inoculation schemes may be used, the first transfer from the "homogenized"
       sample should always be 11 mL of homogenized sample to 99 mL dilution water or 10 mL of
       homogenized sample to 90 mL dilution water. This will ensure that a sufficient amount of the
       original biosolid sample is transferred at the beginning of the  dilution scheme.
       Note: Do not suspend bacteria in dilution water for more than 30 minutes at room temperature.
       For some transfers, it may be convenient to use a sterile, wide-mouth pipette, capable of
       transferring particulate matter. If samples are being spiked, a maximum of 1 hour may elapse
       between initial unspiked sample homogenization and analysis of spiked samples.

       11.2.1  Class A liquid samples:  Four series of five tubes each will contain 1.0, 10"1, 10"2, and
               10"3 mL of the original sample. See Figure 2 in Section 20.0 for an overview of this
               dilution and inoculation scheme.  (For spiked samples, four series of five tubes each will
               be used for the analysis with 10"2,  10"3, 10"4, and 10"5 mL of the  original sample.)

               11.2.1.1  Dilution

                        (A)   Use a sterile pipette to transfer 11.0 mL of "homogenized" sample
                              (Section 11.1.1) to 99 mL of sterile dilution water (Section 7.4), cap, and
                              mix by vigorously shaking the bottle a minimum of 25 times.  This is
                              dilution "A." One mL of dilution "A" contains 10"1 mL of the  original
                              sample.

                        (B)   Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of
                              sterile dilution water, and mix as before.  This is dilution "B."  One mL
                              of dilution "B" is 10"2 mL of the original sample.
                                               15                                      April 2010

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Method 1680
                        (C)   Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of
                              sterile dilution water, and mix as before. This is dilution "C."  One mL
                              of dilution "C" is 10~3 mL of the original sample.

                        (D)   Additional dilutions for analysis of spiked samples.

                              •   Use a sterile pipette to transfer 11.0 mL of dilution "C" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "D." One
                                 mL 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." One
                                 mL of dilution "E" is 10~5 mL of the original sample.

               11.2.1.2 Inoculation

                        (A)   Use a sterile pipette to inoculate each of the first series of five tubes with
                              1.0 mL of the original "homogenized" sample per tube (unspiked
                              samples only).

                        (B)   Use a sterile pipette to inoculate each of the second series of tubes with
                              1.0 mL of dilution "A" (unspiked samples only).  This is 10"1 mL of the
                              original sample.

                        (C)   Use a sterile pipette to inoculate each of five tubes with  1.0 mL of
                              dilution "B" (unspiked or spiked samples).  This is 10"2 mL of the
                              original sample.

                        (D)   Use a sterile pipette to inoculate each of five tubes with  1.0 mL of
                              dilution "C" (unspiked or spiked samples).  This is 10"3 mL of the
                              original sample.

                        (E)   Use a sterile pipette to inoculate each of five tubes with  1.0 mL of
                              dilution "D" (spiked samples). This is 10"4 mL of the  original sample.

                        (F)   Use a sterile pipette to inoculate each of five tubes with  1.0 mL of
                              dilution "E" (spiked samples). This is 10"5 mL of the original sample.

               11.2.1.3 Repeat steps 11.2.1.1 and 11.2.1.2 for the remaining Class A samples. When
                        inoculations are complete, go to Section 12.3.1.4 to continue the LTB/EC
                        method.

        11.2.2 Class A solid samples: For unspiked samples, four series of five tubes will be used for the
               analysis with 1.0,  10"1, 10"2 and 10"3 g of the original sample. The first series of tubes
               must contain 2X media. See Figure 3 in  Section 20.0 for a summary of this dilution and
               inoculation scheme. (For spiked samples, four series of five tubes each will be used for
               the analysis with 10"2, 10"3, 10"4, and 10"5 g of the original sample.)

               11.2.2.1 Dilution

                        (A)   A 1.0-mL volume of the "homogenized" sample (Section 11.1.2)
                              contains 10"1 g of the original sample.


April 2010                                       16

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                                                                       Method 1680
         (B)   Use a sterile pipette to transfer 11.0 mL of the blender contents to 99 mL
               of sterile dilution water (Section 7.4) and shake vigorously a minimum of
               25 times.  This is dilution "A."  One mL of dilution "A" contains 10~2 g
               of the original sample.

         (C)   Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of
               sterile dilution water and mix as before. This is dilution "B."  One mL of
               dilution "B" contains 10"3 g of the original sample.

         (D)   Additional dilutions for analysis of spiked samples:

               •   Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of
                  sterile dilution water, and mix as before. This is dilution "C."  One
                  mL of dilution "C" contains 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." One
                  mL of dilution "D" contains 10"5 g of the original sample.
11.2.2.2 Inoculation

         (A)   Use a sterile pipette to inoculate each of the first series of five tubes with
               10.0 mL of the "homogenized" sample  (unspiked samples only). This
               series of tubes must contain 2X media.  This is 1.0 g of the original
               sample. Since test tubes with inverted vials are being used, shaking is
               not practical.  Solids that will not separate easily and/or may float should
               be submerged into the broth with a sterile loop.

         (B)   Use a sterile pipette to inoculate each of five tubes with 1 mL of the
               "homogenized" mixture (unspiked samples only).  This is 10"1 g of the
               original sample.

         (C)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
               dilution "A" (unspiked or spiked samples).  This is 10"2 g of the original
               sample.

         (D)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
               dilution "B" (unspiked or spiked samples). This is 10"3 g of the original
               sample.

         (E)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
               dilution "C" (spiked samples). This is 10"4 g of the original sample.

         (F)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
               dilution "D" (spiked samples).  This is  10"5 g of the original sample.

11.2.2.3 Repeat Sections 11.2.2.1 and 11.2.2.2 for remaining Class A solid samples.
         When inoculations are complete, go to Section 12.3.1.4 to continue the
         LTB/EC method.
                                17                                       April 2010

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Method 1680
        11.2.3 Class B liquid samples: For unspiked samples, four series of five tubes each will be used
               for the analysis with 10~3, 10~4, 10~5, and 10~6 mL of the original sample (additional
               dilutions may be analyzed as necessary). See Figure 4 in Section 20.0 for a summary of
               this dilution and  inoculation scheme.  (For spiked samples, five series of five tubes each
               will be used for the analysis with 10"5, 10"6, 10"7, 10"8, and 10"9 mL of the original sample.)

               11.2.3.1 Dilution

                        (A)   Use a sterile pipette to transfer 11.0 mL of homogenized sample (from
                              Section 11.1.1) to 99 mL of sterile dilution water (Section 7.4), cap, and
                              mix by vigorously shaking the bottle a minimum of 25 times. This is
                              dilution "A." One mL of dilution "A" is 10"1 mL of the original sample.

                        (B)   Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of
                              sterile dilution water, and mix as before. This is dilution "B." One mL
                              of dilution "B" is 10"2 mL of the original sample.

                        (C)   Use a sterile pipette to transfer 11.0 mL of dilution "B" to 99 mL of
                              sterile dilution water, and mix as before. This is dilution "C." One mL
                              of dilution "C" is 10"3 mL of the original sample.

                        (D)   Use a sterile pipette to transfer 11.0 mL of dilution "C" to 99 mL of
                              sterile dilution water, and mix as before. This is dilution "D." One mL
                              of dilution "D" is 10"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." One mL
                              of dilution "E" is 10"5 mL of the original sample.

                        (F)   Use a sterile pipette to transfer 11.0 mL of dilution "E" to 99 mL of
                              sterile dilution water, and mix as before. This is dilution "F." One mL
                              of dilution "F" is 10"6 mL of the original sample.

                        (G)   Additional dilutions for analysis of spiked samples:

                                 Use a sterile pipette to transfer 11.0 mL of dilution "F" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "G." One
                                 mL of dilution "G" is 10"7 mL of the original sample.

                                 Use a sterile pipette to transfer 11.0 mL of dilution "G" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "H." One
                                 mL of dilution "H" is 10"8 mL of the original sample.

                                 Use a sterile pipette to transfer 11.0 mL of dilution "H" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "I." One
                                 mL of dilution "I" is 10"9 mL of the original sample.

               11.2.3.2 Inoculation

                        (A)   Use a sterile pipette to inoculate each of the first series of five tubes with
                              1.0 mL of dilution "C" (unspiked samples only). This is 10"3 mL of the
                              original sample.
April 2010                                        18

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                                                                             Method 1680
                (B)   Use a sterile pipette to inoculate each of five 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 five tubes with 1.0 mL of
                      dilution "E" (unspiked or spiked samples). This is 10"5 mL of the
                      original sample.

                (D)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                      dilution "F" (unspiked or spiked samples). This is 10"6 mL of the
                      original sample.

                (E)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                      dilution "G" (spiked samples).  This is 10"7 mL of the original sample.

                (F)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                      dilution "H" (spiked samples).  This is 10"8 mL of the original sample.

                (G)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                      dilution "I" (spiked samples). This is 10"9 mL of the original sample.

       11.2.3.3 Repeat Sections 11.2.3.1  and 11.2.3.2 for each remaining Class B sample.
                When inoculations are complete, proceed to Section 12.3.1.4 to continue the
                LTB/EC method.

11.2.4 Class B solid samples: For unspiked samples, four series of five tubes each will
       contain 10"3, 10"4, 10"5, and 10"6 g of the original sample (additional dilutions may be
       analyzed as necessary). See Figure 5 in Section 20.0 for a summary of this dilution and
       inoculation scheme. (For spiked samples, five series of five tubes each will be  used for
       the analysis with 10'5, 10'6, 10'7, 10'8, and 10'9 g of the original sample.)

       11.2.4.1 Dilution

                (A)   A volume of 1.0-mL of the "homogenized" sample (Section 11.1.2)
                      contains 10"1 g of the original sample.

                (B)   Use a sterile pipette to transfer 11.0 mL of the blender contents to 99 mL
                      of sterile dilution water (Section 7.4) and shake vigorously a minimum of
                      25 times. This is dilution "A."  One mL of dilution "A" contains 10"2 g
                      of the original sample.

                (C)   Use a sterile pipette to transfer 11.0 mL of dilution "A" to 99 mL of
                      sterile dilution water, and mix as before.  This is dilution "B."  One 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."  One 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."  One mL
                      of dilution "D" contains  10"5 g of the original sample.


                                        19                                       April 2010

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Method 1680
                        (F)   Use a sterile pipette to transfer 11.0 mL of dilution "D" to 99 mL of
                              sterile dilution water and mix as before.  This is dilution "E." One mL of
                              dilution "E" contains  10~6 g of the original sample.

                        (G)   Additional dilutions for analysis of spiked samples:

                              •   Use a sterile pipette to transfer 11.0 mL of dilution "E" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "F." One
                                 mL of dilution "F" is 10"7 g of the original sample.

                              •   Use a sterile pipette to transfer 11.0 mL of dilution "F" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "G." One
                                 mL of dilution "G" is 10"8 g of the original sample.

                              •   Use a sterile pipette to transfer 11.0 mL of dilution "G" to 99 mL of
                                 sterile dilution water, and mix as before. This is dilution "H." One
                                 mL of dilution "H" is 10"9 g of the original sample.

               11.2.4.2 Inoculation

                        (A)   Use a sterile pipette to inoculate each of the first series of five tubes with
                              1.0 mL of dilution "B" (unspiked  samples only).  This is  10"3 g of the
                              original sample.

                        (B)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                              dilution "C" (unspiked samples only). This is 10"4 g of the original
                              sample.

                        (C)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                              dilution "D" (unspiked or spiked samples).  This is 10"5 g of the original
                              sample.

                        (D)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                              dilution "E" (unspiked or spiked samples). This is 10"6 g of the original
                              sample.

                        (E)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                              dilution "F" (spiked samples). This is 10"7 g of the original sample.

                        (F)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                              dilution "G" (spiked samples). This is 10"8 g of the original sample.

                        (G)   Use a sterile pipette to inoculate each of five tubes with 1.0 mL of
                              dilution "H" (spiked samples). This is 10"9 g of the original sample.

               11.2.4.3 When inoculations are complete, go to Section 12.3.1.4 to continue the
                        LTB/EC method.
April 2010                                       20

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                                                                                   Method 1680
12.0  Lauryl Tryptose Broth (LIB) - EC Broth Procedure

12.1   In this protocol, the Lauryl-Tryptose Broth - EC medium is used to determine fecal coliform
       densities in Class A and B biosolid samples. Analysis of seven samples collected at the time of
       disposal using this procedure will satisfy the requirements of the monitoring alternative for
       demonstrating pathogen reduction in both Class A and Class B biosolids.  In Method 1680, LTB
       is used as a presumptive medium followed by EC as confirmation of fecal coliforms. EC may not
       be used for direct isolation from a biosolid sample because prior enrichment in presumptive
       medium (LTB) is required for optimum recovery of fecal coliforms. Precision of the test
       increases with increasing numbers of replicates per sample tested. For an overview of the MPN
       procedure, refer to Figure 1 in Section 20.0.

12.2   Since sample fecal coliform densities are expected to be variable, it is recommended that at least
       seven biosolid samples be analyzed using this method. The geometric mean fecal coliform
       density of the seven biosolids samples should not exceed 2 x 106 MPN/g of total solids (dry
       weight basis) to qualify as Class B biosolids.  Although there is not a specific number of samples
       required for Class A biosolids, it is recommended that a sampling event extend over two weeks
       and that at least seven samples be collected and determined to be below 1,000 MPN/g of total
       solids (dry weight basis) to qualify as Class A biosolids.

12.3   LTB/EC Procedure

       12.3.1 Presumptive phase with LTB medium

              12.3.1.1 Prepare LTB media and dispense into tubes as directed in Section 7.6. Note: If
                       media is refrigerated, remove from refrigerator  1-1.5 hours prior to inoculation,
                       so that it reaches room temperature prior to use.

              12.3.1.2 For each sample, arrange test tubes in four rows of five tubes each (Section
                       11.2).  When 10 mL of sample or dilution is used, tubes should contain 10 mL
                       of 2X LTB media.  Clearly label each row of tubes to identify the sample and
                       dilution to be inoculated. Note: 2X LTB is needed for 10 mL inoculations, to
                       ensure that the 10-mL inoculation volume does not excessively dilute the LTB.

              12.3.1.3 Dilute  and inoculate samples depending on the matrix (i.e. Class A solid, Class
                       B liquid), as described in Section 11.2.

              12.3.1.4 Incubate inoculated tubes at 35°C ± 0.5°C. After 24 ± 2 hours, swirl each tube
                       gently  and examine it for growth and gas production. If no gas has formed,
                       reincubate for an additional 24 ± 2 hours and reassess. Final assessment should
                       be within a total of 48 ± 3 hours.

              12.3.1.5 For tubes with growth, the presence of gas in inverted vials within 48 ± 3 hours
                       signifies a positive presumptive reaction.

                       Note: The presence of gas in the absence of growth is usually due to
                       mishandling or improper shaking of the tubes after inoculation.

              12.3.1.6 For tubes with a positive presumptive reaction, proceed to the confirmation
                       phase (See Photo 1).
                                              21                                      April 2010

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Method 1680
        12.3.2  Confirmation phase for fecal coliforms using EC medium

               12.3.2.1  Prepare EC broth tubes as described in Section 7.7. For each positive LTB
                        tube, one EC tube will be inoculated. Note: If media is refrigerated, remove
                        from refrigerator 1-1.5 hours prior to inoculation, so that it reaches room
                        temperature prior to use.

               12.3.2.2  Gently shake tubes from  presumptive test showing positive reaction.

               12.3.2.3  Using a sterile 3- to 3.5-mm-diameter loop or sterile wooden applicator stick,
                        transfer growth from each positively presumptive LTB tube to corresponding
                        tubes containing EC broth.

               12.3.2.4  Place all EC tubes in a 44.5°C ± 0.2°C water bath within 30 minutes of
                        inoculation and incubate  for 24 ± 2 hours.  Maintain water level above the
                        media in immersed tubes.

               12.3.2.5  After incubation, examine each tube for growth and gas production. Gas
                        production with growth in EC broth at 24 ± 2 hours is considered a positive
                        fecal coliform reaction (See Photo 1). Failure to produce gas constitutes a
                        negative reaction.  Note:  The presence of gas in the absence of growth is
                        usually due to mishandling or improper shaking of the tubes after inoculation.

               12.3.2.6  Record positive and negative reactions for the EC tubes. Calculate MPN / g of
                        total solids (dry weight) from the number of positive EC tubes as described in
                        Section 14.0.
                                                            tubes on left with silver caps) and EC
                                                            with red caps), fecal coliforms produce
                                                            (tubes 1 and 3, when counting from the
               Photo 1.  In LTB (two
               (two tubes on right
               turbidity and gas
               left).

12.4   Total solids determination
        12.4.1  Determination of                            I percent dry weight - When sample
        results are to be calculated on a  I                     I dry
               weight basis, a second                         portion of sample should be weighed at
               the same time as the portion used for analytical determination.

               WARNING: The drying  oven should be contained in a hood or be vented.  Significant
               laboratory contamination may result from drying a heavily contaminated sample.

        12.4.2  Immediately after weighing the sample for microbiological examination, weigh 10-30 g
               of the sample  into a tarred crucible or aluminum evaporating dish. Dry this aliquot
               overnight at 103°C to 105°C. Allow to cool in a desiccator before weighing. Calculate
               the % dry weight as follows:
                           % dry weight =
                                           g dry sample

                                            g sample
x100
April 2010
                                               22

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                                                                                      Method 1680
13.0  Verification

13.1   Individual biochemical tests including oxidase, citrate, lactose fermentation, indole, ONPG
       hydroloysis, methyl red, and Voges-Proskauer may be used to verify positive and negative
       results.

13.2   Alternately, commercially available multi-test identification systems may be used to verify
       positive and negative results.  Such identification systems for Enterobacteriaceae must include
       lactose fermentation, o-nitrophenyl-p-D-galactopyranoside (ONPG), and cytochrome oxidase test
       reactions.

       Note: Due to the high false positive and negative rates, it is recommended that analysts be
       required to submit all positive and negative tubes with growth to verification for at least one
       biosolid sample for each type of biosolid that the analyst normally evaluates on a monthly basis.
       This should increase analyst proficiency in using these procedures.
14.0   Data Analysis and Calculations

The estimated density of fecal coliform bacteria, based on the confirmation test using EC, is calculated in
terms of most probable number (MPN). Due to the extreme variability in the solid content of biosolids,
fecal coliform results from biosolid samples are reported as MPN/g total solids (dry weight basis). MPN /
g total solids (dry weight) is calculated in three steps (Sections  14.1,  14.2, and  14.3):

            Selection of significant dilutions
        •    Calculation of MPN/mL (wet weight)
            Conversion to MPN/g total solids (dry weight)

The calculation of geometric means is provided in Section 14.4.

14.1    Step 1: Select Significant Dilutions

        A dilution refers to the mL (liquid samples) or g (solid  samples) of original sample that was
        inoculated into each series of tubes. For example, with Class B solid samples (Section 11.2.4),
        four, five-tube dilutions are used, with 10~3,  10~4, 10~5, and 10~6 g 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 5, below. For these examples,
        the numerator represents the number of positive tubes per sample dilution series and the
        denominator represents the total number of tubes inoculated per dilution series.

        14.1.1 Choose the highest dilution (the most dilute, with the least amount of sample) giving
              positive results in all five tubes inoculated and  the two succeeding higher (more dilute)
              dilutions (For Table 5, Example A,  10~4 is higher/more dilute than 10~3.)

        14.1.2 If the lowest dilution (least dilute) tested has less than five tubes with positive results,
               select it and the two next succeeding higher dilutions (Table 5, Examples B and C).

        14.1.3 When a positive result occurs in a dilution higher (more dilute) than the three significant
              dilutions selected according to the rules above, change the selection to the lowest dilution
               (least dilute) that has less than five positive results and the next two higher dilutions
               (more dilute) (Table 5, Example D).

                                               23                                       April 2010

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Method 1680
        14.1.4 When the selection rules above have left unselected any higher dilutions (more dilute)
               with positive results, add those higher-dilution positive results to the results for the
               highest selected dilution (Table 5, Example E).

        14.1.5 If there were not enough higher dilutions tested to select three dilutions, then select the
               next lower dilution (Table 5, Example F).

14.2    Step 2: Calculate MPN/mL (wet weight)

        14.2.1  Obtain the MPN index value from Table 4 using the number of positive tubes in the three
               significant dilutions series and calculate MPN/mL using the following equation.  The
               95% confidence limits may also be obtained from Table 4.  Table 4 has been adjusted by
               a factor of 10 to eliminate having to multiply the MPN index by 10 in the equation
               provided below.

        Note: The example calculated numbers provided in the tables below have been rounded at the end
        of each step. If your laboratory recalculates the examples using a spreadsheet and rounds only
        after the final calculation, values may be slightly different.

                                                  MPN Index from Table 4
      MPN/mL     =    	
                               Largest volume tested in the dilution series used for MPN determination

        14.2.2  When using MPN tables other than those provided in this method (e.g. Table 9221:IV,
               Standard Methods for Water and Wastewater, Reference 19.1), additional
               steps/calculations are required to determine final reporting value of MPN/g dry weight.
               For example, Table 9221:IV MPN index is per 100 mL (MPN/100 mL); which will need
               to be converted to MPN/mL . In addition, the MPN index must be multiplied by a factor
               of 10 when using the largest volume tested in the dilution series used for MPN
               determination.

                                               10  x MPN Index from Table 9221 :VI
       MPN/100 mL    =   	
                                Largest volume tested in the dilution series used for MPN determination
                                                        MPN/100mL
        MPN/mL       =   	
                                                            100
April 2010                                       24

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                                                                                                          Method 1680
Table 4.  MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
	When Five Tubes are Used per Dilutiona	
  Combination of
     Positives
MPN Index
    ml
95% Confidence Limits
Lower	Upper
Combination of
   Positives
MPN Index
    mL
                                                                                               95% Confidence Limits
                                                                                               Lower
                                                                                                             Upper
      0-0-0
      0-0-1
      0-0-2
      0-0-3
      0-0-4
                 0.03
                 0.03
                 0.03
                 0.08
                 0.15
                 0.03
                 0.03
                 0.03
                 0.08
                 0.15
                 0.23
                 0.03
                 0.03
                 0.08
                 0.15
                 0.23
                 0.31
                 0.03
                 0.09
                 0.16
                 0.23
                 0.31
                 0.39
                 0.09
                 0.16
                 0.24
                 0.32
                 0.40
                 0.48
                 0.16
                 0.24
                 0.32
                 0.40
                 0.48
                 0.56
                 0.03
                 0.03
                 0.03
                 0.11
                 0.19
                 0.28
                 0.03
                 0.03
                 0.11
                 0.19
                 0.28
                 0.37
                 0.03
                 0.12
                 0.20
                 0.29
                 0.38
                 047
               0.63
               1.01
               1.37
               1.74
               2.12
               0.63
               1.01
               1.38
               1.75
               2.14
               2.56
               1.02
               1.39
               1.76
               2.15
               2.58
               3.07
               1.40
               1.77
               2.17
               2.60
               3.10
               3.72
               1.79
               2.19
               2.63
               3.13
               3.77
               4.62
               2.21
               2.65
               3.17
               3.82
               4.70
               5.63
               0.68
               1.08
               1.49
               1.91
               2.36
               2.87
               1.09
               1.50
               1.92
               2.38
               2.90
               3.54
               1.51
               1.94
               2.40
               2.93
               3.59
               451
    1-3-0
    1-3-1
    1-3-2
    1-3-3
    1-3-4
    1-3-5
    1-4-0
    1-4-1
    1-4-2
    1-4-3
    1-4-4
    1-4-5
    1-5-0
    1-5-1
    1-5-2
    1-5-3
    1-5-4
    1-5-5
    2-0-0
    2-0-1
    2-0-2
    2-0-3
    2-0-4
    2-0-5
    2-1-0
    2-1-1
    2-1-2
    2-1-3
    2-1-4
    2-1-5
    2-2-0
    2-2-1
    2-2-2
    2-2-3
    2-2-4
    2-2-5
    2-3-0
    2-3-1
    2-3-2
    2-3-3
    2-3-4
    2-3-5
    2-4-0
    2-4-1
    2-4-2
    2-4-3
    2-4-4
    2-4-5
    2-5-0
    2-5-1
    2-5-2
    2-5-3
    2-5-4
    7-5-5
   0.83
   1.04
   1.25
   1.47
   1.69
   1.91
   1.05
   1.27
   1.48
   1.70
   1.93
   2.15
   1.28
   1.50
   1.72
   1.95
   2.17
   2.40
   0.45
   0.68
   0.91
   1.15
   1.39
   1.64
   0.68
   0.92
   1.16
   1.41
   1.66
   1.92
   0.93
   1.18
   1.43
   1.68
   1.94
   2.21
   1.19
   1.44
   1.70
   1.97
   2.23
   2.51
   1.46
   1.72
   1.99
   2.26
   2.54
   2.82
   1.74
   2.01
   2.29
   2.57
   2.86
   3 15
0.12
0.20
0.29
0.38
0.48
0.57
0.21
0.30
0.39
0.48
0.58
0.67
0.30
0.40
0.49
0.58
0.68
0.77
0.03
0.06
0.15
0.25
0.35
0.46
0.06
0.15
0.25
0.36
0.46
0.57
0.16
0.26
0.36
0.47
0.58
0.69
0.26
0.37
0.48
0.59
0.70
0.82
0.38
0.49
0.60
0.72
0.83
0.94
0.50
0.61
0.73
0.84
0.95
1 07
1.96
2.43
2.96
3.64
4.60
5.66
2.45
3.00
3.70
4.68
5.75
6.57
3.03
3.75
4.77
5.83
6.64
7.31
1.19
1.64
2.13
2.69
3.38
4.37
1.66
2.16
2.72
3.43
4.47
5.71
2.18
2.76
3.49
4.56
5.81
6.75
2.79
3.55
4.67
5.91
6.83
7.59
3.61
4.77
6.00
6.92
7.68
8.36
4.88
6.10
7.00
7.76
8.45
9 10
1 Table was developed using the MPN calculator developed by Albert Klee
                                                          25
                                                                                         April 2010

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Method 1680
Table 4.  MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
          When Five Tubes are Used per Dilution (cont.)a
Combination of
Positives
3-0-0
3-0-1
3-0-2
3-0-3
3-0-4
3-0-5
3-1-0
3-1-1
3-1-2
3-1-3
3-1-4
3-1-5
3-2-1
3-2-2
3-2-3
3-2-4
3-2-5
3-3-0
3-3-1
3-3-2
3-3-3
3-3-4
3-3-5
3-4-1
3-4-2
3-4-3
3-4-4
3-4-5
3-5-0
3-5-1
3-5-2
3-5-3
3-5-4
3-5-5
4-0-0
4-0-1
4-0-2
4-0-3
4-0-4
405
4-1-0
4-1-1
4-1-2
4-1-3
4-1-4
4-1-5
4-2-0
4-2-1
4-2-2
4-2-3
4-2-4
4-7-5
MPN Index
ml
0.79
1.06
1.35
1.65
1.96
2.29
1.07
1.37
1.67
1.99
2.32
2.67
1.70
2.02
2.36
2.71
3.08
1.72
2.05
2.40
2.76
3.13
3.52
2.44
2.81
3.19
3.58
3 99
2.48
2.86
3.25
3.65
4.07
4.50
1 .30
1.66
2.07
2.53
3.02
3 55
1.69
2.12
2.58
3.10
3.65
4.25
2.16
2.64
3.17
3.75
4.38
504
95% Confidence
Lower
0.10
0.21
0.33
0.46
0.59
0.73
0.22
0.34
0.47
0.60
0.74
0.88
0.48
0.62
0.76
0.90
1.04
0.49
0.63
0.77
0.92
1.06
1.20
0.79
0.93
1.08
1.23
1 37
0.80
0.95
1.10
1.25
1.40
1.54
0.31
0.46
0.64
0.82
1.02
1 21
0.48
0.66
0.85
1.05
1.25
1.45
0.67
0.87
1.08
1.29
1.50
1 71
Limits
Upper
1.88
2.46
3.23
4.40
5.89
6.99
2.50
3.29
4.52
6.01
7.10
8.00
4.64
6.13
7.20
8.10
8.94
4.77
6.24
7.31
8.21
9.06
9.89
6.36
7.42
8.33
9.18
10.02
10 86
7.53
8.44
9.31
10.17
11.03
11.89
3.11
4.45
6.31
7.64
8.81
9.96
4.60
6.46
7.79
8.98
10.16
11.38
6.61
7.94
9.15
10.37
11.64
1797
Combination of
Positives
4-3-0
4-3-1
4-3-2
4-3-3
4-3-4
4-3-5
4-4-0
4-4-1
4-4-2
4-4-3
4-4-4
4-4-5
4-5-1
4-5-2
4-5-3
4-5-4
4-5-5
5-0-0
5-0-1
5-0-2
5-0-3
5-0-4
5-0-5
5-1-0
5-1-1
5-1-2
5-1-3
5-1-4
5-1-5
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-2-5
5-3-1
5-3-2
5-3-3
5-3-4
5-3-5
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
mL
2.71
3.26
3.86
4.51
5.21
5.93
3.35
3.98
4.66
5.39
6.15
6.93
4.83
5.59
6.39
7.22
8.06
2.40
3.14
4.27
5.78
7.59
9.53
3.29
4.56
6.31
8.39
10.62
12 93
4.93
7.00
9.44
12.05
14.79
17.67
7.92
10.86
14.06
17.50
21.22
25.27
12.99
17.24
22.12
27.81
34.54
42.56
23.98
34.77
54.22
91.78
160.90
>16090
95% Confidence Limits
Lower
0.90
1.11
1.32
1.54
1.76
1.96
1.14
1.37
1.59
1.81
2.02
2.23
1.64
1.87
2.09
2.30
2.50
0.76
1.06
1.46
1.92
2.39
1.65
_
1.56
2.07
2.57
3.04
3 04
1.67
2.24
2.80
3.31
3.81
5.03
2.47
3.08
3.68
4.34
5.29
8.14
3.48
4.29
5.63
8.82
11.59
14.37

11.72
17.91
26.72
38.37

Upper
8.09
9.34
10.60
11.92
13.31
14.77
9.53
10.84
12.23
13.68
15.21
16.81
12.56
14.09
15.70
17.39
19.16
7.63
9.08
11.42
14.46
18.16
22.34
9.40
12.02
15.53
19.85
24.85
30.90
12.76
16.94
22.13
28.43
37.14
52.30
1 o.OD
25.44
34.45
51.31
67.98
79.71
31.08
49.75
70.87
86.00
101.10
118.00
76.29
101.60
1 41 .90
220.10
410.30

1 Table was developed using the MPN calculator developed by Albert Klee
April 2010
26

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                                                                                       Method 1680
Examples of MPN/mL calculations are provided in Table 5.




Table 5. Examples of Significant Dilution Selection and Calculation of MPN/mL;
Example
(liquid
or solid)
A
B
C
D
E
F
1Q-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
(7.92 / ID'4) = 79,200 MPN / mL
79,000 MPN/mL
(4.8 3/10-3) = 4830 MPN/mL
4800 MPN / mL
(0.18/10-3)= 180 MPN/mL
(1.37/10-4)= 13,700 MPN/mL
14, 000 MPN/mL
(3.98 /10-3) = 3980 MPN/mL
4000 MPN / mL
(54.22 / ID'4) = 542,200 MPN / mL
540,000 MPN/mL
a Significant dilutions are underlined and largest significant dilutions highlighted







14.3    Step 3: Convert to MPN/g Total Solids (dry weight)




        For analysis and calculation of percent total solids see Section 12.4.




        For the conversion to MPN/g total solids (dry weight), we assume that,




        MPN/mL wet weight = MPN/g wet weight.




        Therefore, we may convert to MPN/g total solids (dry weight) using the following equation:
       MPN /g (dry weight)
                                                    MPN/mL (wet weight) from step 2
                                               Percent total solids (expressed as a decimal)
                                                27
April 2010

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Method 1680
Examples of the conversion to MPN/g (dry weight) are provided in Table 6.

Table 6.  Examples of Conversion to MPN/g Total Solids (Dry Weight), Continuing From Step 2 in Table 5
Example
(liquid or solid)
A
B
C
D
E
F
Total
Solids
4%
60%
56%
22%
18%
43%
Step 3:
(MPN / ml_ wet weight from step 2) / percent total solids = MPN / g dry weight
79,000 / 0.04 = 1,975,000 = 2.0 x 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
14.4   Calculation of Geometric Mean

       To satisfy pathogen reduction requirements for Class B biosolids in Subpart D of Part 503, seven
       biosolid samples are collected and the geometric mean density of fecal coliforms is calculated.
       The geometric mean is calculated by:

       • converting each sample's MPN fecal coliforms/g (dry weight) to the Iog10 value,
       • averaging the Iog10 values, and
       • taking the antilog of the mean Iog10 value.

       An example is provided in Table 7.

Table 7. Calculation of Geometric Mean Fecal Coliform Density for Biosolid Samples
Sample No.
1
2
3
4
5
6
7
MPN Fecal coliforms / g (dry weight)
600,000 = 6.0 X105
4,200,000 = 4.2 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)
April 2010
28

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                                                                                     Method 1680
15.0  Sample Spiking Procedure

15.1   Method 1680 QC requirements (Section 9.0) include the preparation and analysis of spiked
       reference (Milorganite®) samples in order to monitor initial and ongoing method performance.
       For the IPR (Section 9.3) and OPR (Section 9.4) analyses it is necessary to spike samples with
       laboratory-prepared spiking suspensions. Section 15.0 is arranged in the following order:
       preparation of the E. coll  spiking suspension (Section 15.2), spiking suspension dilution (Section
       15.3), spiking suspension enumeration (Section 15.4), Class A sample spiking (Section 15.5),
       Class B sample spiking (Section 15.6), and calculation of spiked E. coll percent recovery (Section
       15.7).

15.2   Preparation  of E. coli Spiking Suspension (Class A or B)

       15.2.1   Stock Culture.  Prepare a stock culture by inoculating a heart infusion agar (HIA) slant
                [or other non-selective media (e.g., Tryptic Soy Agar)] with Escherichia coli ATCC #
                25922 and incubating at 35°C ± 3°C for 20 ± 4 hours. This stock culture may be stored
                in the dark at room temperature for up to 30 days.

       15.2.2   1% Laurvl Tryptose Broth (LTB). Prepare a  1% solution of LTB by combining 99 mL
                of sterile phosphate buffered dilution water and 1 mL of sterile single strength lauryl
                tryptose broth in a sterile screw cap bottle or re-sealable dilution water container.
                Shake to mix.

       15.2.3   Spiking Suspension (Undiluted). From the stock culture of E. coli ATCC # 25922,
                transfer a small loopful of growth to the 1% LTB solution and vigorously shake a
                minimum of 25 times.  Incubate at 35°C ± 3°C for 20 ± 4 hours. The resulting spiking
                suspension contains approximately 1.0 x 107 to  1.0 x 10s E. coli colony forming units
                (CPU) per mL. This is referred to as the "undiluted spiking suspension."

15.3   Spiking Suspension Dilution

       15.3.1   Mix the spiking suspension by vigorously shaking the bottle a minimum of 25 times.
                Use a sterile pipette to transfer 1.0 mL of the undiluted spiking suspension to 99 mL of
                sterile dilution water (Section 7.4), cap, and mix by vigorously shaking the bottle a
                minimum of 25 times.  This is spiking suspension dilution "A." A 1.0-mL dilution "A"
                is 10"2 mL of the original undiluted spiking suspension.

       15.3.2   Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "A" to 99 mL of
                sterile dilution water, cap, and mix by vigorously shaking the bottle a minimum of 25
                times. This is spiking suspension dilution "B."  A 1.0-mL dilution "B" is 10"3 mL of
                the original  undiluted spiking suspension.

       15.3.3   Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "B" to 99 mL
                of sterile dilution water, cap, and mix by vigorously shaking the bottle a minimum of
                25 times.  This  is spiking suspension dilution "C." A 1.0-mL dilution "C" is 10"4 mL of
                the original  undiluted spiking suspension.

       15.3.4   Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "C" to 99 mL
                of sterile dilution water, cap, and mix by vigorously shaking the bottle a minimum of
                25 times.  This  is spiking suspension dilution "D." A 1.0-mL dilution "D" is 10"5 mL
                of the original undiluted spiking suspension.
                                               29                                      April 2010

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Method 1680
        15.3.5    Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution "D" to 99 mL
                 of sterile dilution water, cap, and mix by vigorously shaking the bottle a minimum of
                 25 times.  This is spiking suspension dilution "E." A 1.0-mL dilution "E" is 10~6 mL of
                 the original undiluted spiking suspension.

15.4    Spiking Suspension Enumeration

        15.4.1    Prepare heart infusion agar (HIA) (Section 7.5), add 10 - 15 mL of HIA per 100 x 15
                 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note: To ensure
                 that the agar surface is dry prior to use, plates should be made several days in advance
                 and stored inverted at room temperature or dried using a laminar-flow hood.

        15.4.2    Each of the following will be conducted in triplicate, resulting in the evaluation of
                 nine spread plates:

               Pipet 0.1 mL of dilution "C" onto surface of pre-dried HIA plate [10~5 mL (0.00001) of
               the original spiking suspension].

               Pipet 0.1 mL of dilution "D" onto surface of pre-dried HIA plate [10'6 mL (0.000001) of
               the original spiking suspension].

               Pipet 0.1 mL of dilution "E" onto surface of pre-dried HIA plate [10'7 mL (0.0000001) of
               the original spiking suspension].

        15.4.3    For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum
                 over surface of medium by rotating the dish by hand or on a turntable.

        15.4.4    Allow inoculum to absorb into the medium completely.

        15.4.5    Invert plates and incubate at 35°C ± 0.5°C for 24 ± 4 hours.

        15.4.6    Count and record number of colonies perplate. Referto Section 15.7 for calculation of
                 spiking suspension concentration.

15.5    Class A Biosolid Sample Spiking

        Homogenize the unspiked Class A biosolid sample (Section 11.1). For the enumeration of fecal
        coliforms in the unspiked sample,  dilute and inoculate according to Section 11.2. After the
        unspiked sample has been diluted and media inoculated, spike the biosolid sample as indicated
        below.

        Since the objective of spiking the biosolid sample is to establish percent recovery, it is necessary
        to determine the number of E. coli in the undiluted spiking suspension. Instructions for spiking
        suspension enumeration are provided below.

        15.5.1    Liquid Samples: Since the unspiked, homogenized sample was analyzed by the
                 LTB/EC procedure and a dilution series was prepared (Section 11.2), 284 mL of the
                 original 300 mL of unspiked, homogenized sample remains. To spike the sample, add
                 1.0 mL of spiking suspension dilution "B" (from Section 15.3.2) for every 100-mL of
                 unspiked homogenized sample remaining, cover, and blend on high speed for 1 - 2
                 minutes. This is the "spiked, homogenized" sample. The volume  (mL) of undiluted
                 spiking suspension added to each mL of the spiked biosolid sample is 1.0 x 10"5 mL per


April 2010                                       30

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                                                                                     Method 1680
                mL [(2.8 mL x 10"3 mL) / 284 mL of biosolid], which is referred to as Vsplkedperumtblosollds
                below.  Proceed to Section 11.2.1 (dilution and inoculation).

       15.5.2   Solid Samples: Since the unspiked, homogenized sample was analyzed by the LTB/EC
                procedure and a dilution series was prepared (Section 11.2), 234 mL of the original 300
                mL of unspiked, homogenized sample remains. To spike the sample, add 1.0 mL of
                spiking suspension dilution "B" (from Section 15.3.2) for every 100-mL of unspiked
                homogenized sample remaining, cover, and blend on high speed for 1-2 minutes.  This
                is the "spiked, homogenized" sample.  The volume (mL) of undiluted spiking
                suspension added to each g (wet weight) of the spiked biosolid sample is 1.0 x 10"4 mL
                per g [(2.3 mL x 10'3 mL) / 23.4 g of biosolid], which is referred to as Vsplkedperunitblosollds
                below.  Proceed to Section 11.2.2 (dilution and inoculation).

15.6   Class B Biosolid  Sample Spiking

       Homogenize the unspiked Class B biosolid sample (Section 11.1). For the enumeration of fecal
       coliforms in the unspiked sample, dilute and inoculate according to Section 11.2. After the
       unspiked sample has been diluted and media inoculated, spike the biosolid sample as indicated
       below.

       Since the objective of spiking the biosolid sample is to establish percent recovery, it is necessary
       to determine the number of E. coli in the undiluted spiking suspension. Instructions for spiking
       suspension enumeration are provided below.
       Since a dilution series was prepared from the unspiked, homogenized sample (Section 11.2),  289
       mL of the original 300 mL of unspiked, homogenized sample remains. To spike the sample,  add
       1.0 mL of well-mixed undiluted spiking suspension, for every 100-mL of unspiked homogenized
       sample remaining after unspiked sample evaluation and dilution preparation, cover, and blend on
       high speed  for 1 - 2 minutes. This is the "spiked, homogenized" sample.

       Note: The volumes of undiluted spiking suspensions added per mL or g (wet weight) of the
       spiked Class B biosolids are different from those for the Class A biosolids, since different
       volumes of the unspiked, homogenized sample remains.

       15.6.1   Liquid Samples: The volume (mL) of undiluted spiking suspension added to each Ml of
                the spiked biosolid sample is 1.0 x 10~2 mL [(2.9 mL spiking suspension) / 289 mL of
                biosolid], which is referred to as Vsplkedperumtblosollds below.  Proceed to Section  11.2.3
                (dilution and inoculation).

       15.6.2   Solid Samples: The volume (mL) of undiluted spiking suspension added to each g  (wet
                weight) of the spiked biosolid sample is  1.0 x 10"1 mL per g [(2.9 mL spiking
                suspension) / 28.9 g of biosolids], which is referred to as Vsplkedperumtblosollds below.
                Proceed to Section 11.2.4 (dilution and inoculation).

15.7   Calculation of Spiked E. coli Percent Recovery

       Spiked E. coli percent recovery will be conducted in four steps as indicated below. Note: The
       example calculated numbers provided in the tables below have been rounded at the end of each
       step.  If your laboratory recalculates the examples using a spreadsheet and rounds only after the
       final calculation (Step 4), the percent recoveries may be slightly different.
                                               31                                       April 2010

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Method 1680
        15.7.1  Step 1: Calculate Concentration of E. coli (CFU / mL) in Undiluted Spiking
               Suspension

               15.7.1.1  The number of E. coli CFU / mL in the spiking suspension will be calculated
                        using all plates yielding counts within the ideal range of 30 to 300 CFU per
                        plate.

               15.7.1.2 If the number of colonies exceeds the upper range (i.e. >300) or if the colonies
                        are not discrete, results should be recorded as "too numerous to count"
                        (TNTC).

               15.7.1.3 Calculate the concentration of E. coli (CFU / mL) in the undiluted spiking
                        suspension according to the following equation. Example calculations are
                        provided in Table 8, below.
                        EC
                                                  CFU
                           undiluted spike
               Where:
               J-'*-' undiluted spike
               CFU
               V
=   E. coli CFU / mL in undiluted spiking suspension
=   number of colony forming units from HIA plates yielding counts
    within the ideal range of 30 to 300 CFU per plate
=   volume of undiluted sample in each HIA plate yielding counts within
    the ideal range of 30 to 300 CFU per plate
=   number of plates with counts within the ideal range of 30 to 300
    CFU per plate
Table 8.  Example Calculations of E. coli Spiking Suspension Concentration
Examples
Example 1
Example 2
CFU / plate (triplicate analyses) from
HIA plates
1Q-5 mL plates
275, 250, 301
TNTC, TNTC,
TNTC
10"6 mL plates
30, 10, 5
TNTC, 299, TNTC
10'7 mL plates
0, 0, 0
12, 109, 32
E. coli CFU / mL in undiluted
spiking suspension
(EC undiluted spike)
(275+250+30) /(10-5+10-5+10-6) =
5557 (2.1 x10-5) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10-6+10-7+10-7) =
440 / (1 .2 x ID'6) =366,666,667 =
3.7x108CFU/mL
' EC undNuted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate
April 2010
                 32

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                                                                                      Method 1680
        15.7.2 Step 2: Calculate Spiked E. coli [CFU / mL or g (wet weight)]

               15.7.2.1  The volume of undiluted spiking suspension per unit (mL or g) of spiked
                        biosolid samples (Vsplkedperumtblosollds) is provided in Table 9.
Table 9.        Volume of Undiluted Spiking Suspension per Unit (mL or g) of Spiked Biosolid
               Sam pies (VSDikedD6runitbio    *
Description of spiked sample
Class A liquid
Class A solid
Class B liquid
Class B solid
spiked per unit biosoiids
1 .0 X 1 0"5 mL per mL of biosoiids
1 .0 x 1 0"4 mL per g of biosoiids (wet weight)
1 .0 X 1 0"2 mL per mL of biosoiids
1 .0 x 1 0~1 mL per g of biosoiids (wet weight)
               15.7.2.2 Calculate concentration of spiked E. coli (wet weight) in biosolid sample
                        according to the following equation.
                      Spiked EC
                                'wet weight
                        = (ECUI
                               undiluted spike/
              'spiked per unit biosoiids.
               Where:

               Spiked ECwetweight

               EC,,
               V,
  -'undiluted spike

  spiked per unit biosoiids
Number of spiked E. coli CFU per mL or g of biosolid
(wet weight)
E. coli CFU / mL in undiluted spiking suspension
mL of undiluted spiking suspension per mL or g of
spiked biosolid
Examples are provided in Table 10.
Table 10.
Example Calculations of Spiked EC
                                                 'wet weight
FP
cv> undiluted spike
2.6x107CFU/mL
3.7x108CFU/mL
" spiked
Class A liquid: 1.0 X 10~5 mL per mL of
biosoiids
Class A solid: 1 .0 x 1 0"4 mL per g of
biosoiids (wet weight)
Class B liquid: 1.0 x 10~2 mL per mL of
biosoiids
Class B solid: 1 .0 x 1 0~1 mL per g of
biosoiids (wet weight)
Class A liquid: 1.0 X 10~5 mL per mL of
biosoiids
Class A solid: 1 .0 x 1 0"4 mL per g of
biosoiids (wet weight)
Class B liquid: 1 .0 x 1 0"2 mL per mL of
biosoiids
Class B solid: 1 .0 x 1 0"1 mL per g of
biosoiids (wet weight)
Spiked ECwe, weigh,
(2.6 x 107 CFU / mL) x (1.0 x 10'5 mL / mL)
= 2.6x102CFU/mL
(2.6 x 107 CFU / mL) x (1.0 x 10'4 mL / g)
= 2.6 x 1 03 CFU / g (wet weight)
(2.6 x 1 07 CFU / mL) x (1 .0 x 1 0-2 mL / mL)
= 2.6x105CFU/mL
(2.6x107CFU/mL)x(1.0x10-1 mL/g)
= 2.6 x 1 06 CFU / g (wet weight)
(3.7x1 08 CFU / mL) x (1 .0 x 1 0-5 mL / mL)
= 3.7x103CFU/mL
(3.7 x 108 CFU / mL) x (1 .0 x 10'4 mL / g)
= 3.7 x 1 04 CFU / g (wet weight)
(3.7x1 08 CFU / mL) x (1 .0 x 1 0-2 mL / mL)
= 3.7x106CFU/mL
(3.7x108CFU/mL)x(1.0x10-1 mL/g)
= 3.7x107CFU/g (wet weight)
        15.7.3 Step 3: Convert to "True" Spiked E. coli CFU / g Total Solids (dry weight)
                                               33
                                                                         April 2010

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Method 1680
               Convert to "true" spiked CPU / g total solids (dry weight) as indicated in Section 14.3
               using the E. coll mL or g (wet weight) from Section 15.7.2 as the numerator in the
               equation.  Examples are provided in Table 11.
Table 1 1 . Examples of Conversion to "True" Spiked E. coli CPU / g Total Solids (Dry Weight)
Example Total Solids
Class A liquid: 9%
Class A solid: 82%
Class B liquid: 4%
Class B solid: 23%
Class A liquid: 7%
Class A solid: 88%
Class B liquid: 3%
Class B solid: 40%
(CPU / mL or g) / percent total solids =True spiked E. coli CPU / g dry weight
2.6 x 102 / 0.09 = 2889 = 2.9 x 103 CPU / g dry weight
2.6 x 1 03 / 0.82 = 31 71 = 3.2 x 1 03 CPU / g dry weight
2.6 x 105 / 0.04 = 6,500,000 = 6.5 x 106 CPU / g dry weight
2.6 x106 70.23= 11,304,348= 1.1 x 107 CPU / g dry weight
3.7 x 103 / 0.07 = 52,857 = 5.3 x 1 04 CPU / g dry weight
3.7 x 104 / 0.88 = 42,045 = 4.2 x 1 04 CPU / g dry weight
3.7x106 70.03= 123,333,333= 1.2 x 108 CPU / g dry weight
3.7 x 107 / 0.40 = 92,500,000 = 9.3 x 107 CPU / g dry weight
       15.7.4  Step 4: Calculate Percent Recovery

               15.7.4.1  Calculate percent recovery (R) using the following equation:

                               R= 100 x
              Where:
              R   =
              Ns  =
              Nu  =
              T   =
                                                T
Percent recovery
Fecal coliform MPN/g (dry weight) in the spiked sample
Fecal coliform MPN/g (dry weight) in the unspiked sample
True spiked E. coli CPU /g (dry weight) in spiked sample
Example percent recovery calculations are provided in Table 12.

Table 12.  Example Percent Recovery Calculations
Matrix
Class A liquid
Class A solid
Class B liquid
Class B solid
Ns
2.5 x103
3.9 x103
1.4x108
8.3 x106
Nu
1.5x101
5.0 x102
1.7x106
8.0 x105
T
2.9 x103
3.2 x103
1.2x108
1.1 x107
Percent recovery (R)
100 x [(2.5 x 103) - (1.5 x 101)] / 2.9 x 103 = 86%
100 x [(3.9 x 103) - (5.0 x 102)] / 3.2 x 103 = 107%
100x[(1.4x108)-(1.7x106)]/1.2x108= 115%
100 x [(8.3 x 106) - (8.0 x 105)] / 1.1 x 107 = 68%
April 2010
                       34

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                                                                                   Method 1680
16.0  Method Performance

16.1   Intel-laboratory Validation of Method 1680

       16.1.1 Twelve volunteer laboratories and a referee laboratory participated in U.S.
              Environmental Protection Agency's (EPA's) interlaboratory validation study of EPA
              Method 1680. The purposes of the study were to characterize method performance
              across multiple laboratories and multiple biosolid matrices and to develop quantitative
              quality control (QC) acceptance criteria. A detailed description of the study and results
              are provided in the validation study report (Reference 19.2). Results submitted by
              laboratories were validated using a standardized data review process to confirm that
              results were generated in accordance with study-specific instructions and the October
              2002 Draft version of EPA Method 1680.

       16.1.2 Recovery - Method 1680 mean recoveries of fecal coliforms from Class A matrices,
              compost and thermophilically digested biosolids, spiked with laboratory-prepared spikes
              were 190% and 140%, respectively.  Median recoveries of fecal coliforms from compost
              and thermophilically digested biosolids, spiked with laboratory-prepared  spikes were
              130% and 120%, respectively. Mean recoveries of fecal coliforms from Class B
              matrices, aerobically and anaerobically digested biosolids, spiked with laboratory-
              prepared spikes were 2000% and 890%, respectively. Median recoveries of fecal
              coliforms from aerobically and anaerobically digested biosolids, spiked with laboratory-
              prepared spikes were 88% and 86%, respectively. For Milorganite® (reference matrix)
              samples spiked with laboratory spiking suspensions, the mean recovery was 130%, with a
              median percent recovery of 120%.

       16.1.3 Precision - Method 1680 was characterized by high variability. For percent recovery, the
              overall relative standard deviations (RSDs) ranged from  81% to 130% for Class A
              matrices and from 170% to 360% for Class B matrices for EPA-prepared spikes.  For
              ambient (unspiked) fecal coliform results, the overall RSDs ranged from 22% to 250%
              for Class A matrices and from 140% to 470% for Class B matrices.

       16.1.4 False positive rates - Method 1680  false positive rates were relatively low (6%) for
              thermophilically digested matrices  but high (32%) for the composted matrices.  False
              positive rates for Milorganite® could not be accurately assessed because  only two
              positive tubes were observed during the study.  False positive rates for aerobically
              digested and anaerobically digested samples were relatively low compared to Class A
              rates and ranged from 5% to 10%, dependent on procedure and matrix.

       16.1.5 False negative rates - Method 1680 false negative rates for Class A matrices were
              relatively low compared to Class B rates and ranged from 0% to 6%. False negative rate
              for aerobically digested matrices was slightly higher (10%) compared to Class A rates.
              In contrast, the false negative rate for anaerobically digested matrices was 22%.

17.0  Pollution Prevention

17.1   The solutions and reagents used in this method pose little threat to the environment when
       recycled and managed properly.

17.2   Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
       the volume of expired materials to be disposed.
                                              35                                      April 2010

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Method 1680
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   USEPA. 2004. Results of the Interlab oratory Validation of EPA Method 1680 (LTB/EC) for
       Fecal Coliforms in Biosolids. EPA-821-R-04-009. December 2004.

19.3   American Chemical Society (ACS). 2000. Reagent Chemicals, American Chemical Society
       Specifications. American Chemical Society, New York. For suggestions of the testing of reagents
       not listed by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals,
       BDH, Poole, Dorset, UK and the United States Pharmacopeia.

19.4   Bordner, R., J.A. Winter, and P.V. Scarpino (eds.).  1978. Microbiological Methods for
       Monitoring the Environment, Water and Wastes. EPA-600/8-78-017. Office of Research and
       Development. USEPA.

19.5   Klee, A. J. 1993. A computer program for the determination of the most probable number and its
       confidence limits. Journal of Microbiological Methods. 18:91-98.
20.0  Figures

       The following pages include flow charts summarizing the dilution and inoculation schemes
       (Section 11.0) and procedures (Section 12.0). Schemes for dilution and inoculation are
       dependent on sample Class (A or B) and matrix (solid) or (liquid).
April 2010                                     36

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                 FIGURE 1.   MULTIPLE TUBE FERMENTATION PROCEDURE
                                           LTB/EC Method
                                      Inoculate LTB and incubate at
                                       35°C ± 0.5°C for 24 ± 2 hr
            Growth and gas production
                                           No gas production
                                                                   Incubate additional 24 hr
                                                                     (total of 48 ± 3 hr)
            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

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                             FIGURE 2.   CLASS A LIQUID SAMPLE DILUTION AND INOCULATION SCHEME
Delivery Volume
Class A
liquid sample
"Homogenized sample"


                                       -11.0ml-
  99 ml of sterile
buffered dilution water
       "A"
      (ID'1)
                                                                        -11.0 ml-
99 ml of sterile
buffered dilution water
"B"
(ID'2)


                                                                                                         -11.0 ml-
99 ml of sterile
buffered dilution water
"C"
(ID'3)


                       1.0mL
    1.0ml
                                                                                          1.0ml
                                                                                                                           1.0ml
                        o
                        3
                        —
      q

      3
      i—
      2, w
      o 5
                                                                                           ,  w
                                                                                           O  1)

                                                                                             t
                                                                                           3
                                                                                          -&_
                                                                                           o>
q
3
r~
o 5|
f is

is
3

-------
                          FIGURE 3.    CLASS A SOLID SAMPLE DILUTION AND INOCULATION SCHEME
        Class A
      solid sample
Delivery Volume
 Rinse sample into blender
  with 270 ml_ of sterile
  buffered dilution water
 "Homogenized Sample"
         (10-1)
                                   10.0mL
  o
_. CQ
  O
  -
                                   =
                                   NJ
                                   X

                                   05'
                                   Q. CD
     CO
     CD
       -11.0mL-
                   I.OmL
CQ
O
O  W
§  CD

<9i CD'
3  cn
9L 10
cn
CD

-&_
CD
   99 ml_ of sterile
buffered dilution water
        "A"
                                                                                 (10
                                                                                   i-2\
-11.0mL-
   99 mL of sterile
buffered dilution water
        "B"
                                                            (10
                                                                                                                      i-3\
                                             1.0 mL
                                                                               1.0 mL
       CQ
       O

       O W
       § CD

       <9i CD'
       3 cn
                                              cn
                                              Q)
                                              3
                   CQ
                   O

                   O W
                   ° CD
                                                                                                                    cn
                                                                                                                    CD

                                                                                                                    -

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                                        FIGURE 4.  CLASS B LIQUID SAMPLE DILUTION AND INOCULATION SCHEME
     Class B
      liquid
     sample
"Homogenized sample"
—11.0 ml_-
                     —H.OmL-
                                           -11.0 ml_-
99.0 mLof
sterile
buffered
dilution
water
"C"
(ID'3)


99.0 mLof
sterile
buffered
dilution
water
"D"
(ID'4)


                                                                                       -H.OmL-
                                         Delivery Volume
99.0 mLof
sterile
buffered
dilution
water
"E"
(ID'5)


                                                                                                             -11.0mL-
99.0 mLof
sterile
buffered
dilution
water
"F"
(10-6)


                                                                        lOmL
                                                                                              lOmL
                                                                                                                    LOmL
                                                                                                                                           LOmL
                                                                         o ro
                                                                                               o ro

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                                           FIGURE 5.   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"
        (ID'1)
                                -11.0 ml_
                                                           11.0 ml_
                                                                                    11.0 ml_
                                                                                                             -11.0 ml_-
                                                                                                                                      -11.0 ml_-
                                          Delivery Volume
                                                                                  1.0 ml_
                                                                                                           1.0 ml_
                                                                                                                                     1.0 mL
                                                                                                                                                              1.0 mL

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Method 1680
21.0  Glossary
       The definitions and purposes are specific to this method but have been conformed to common
       usage as much as possible.

21.1   Units of weight and measure and their abbreviations

       21.1.1  Symbols:

               °C     degrees Celsius
               <      less than
               >      greater than
               %     percent
               ±      plus or minus

       21.1.2  Alphabetical characters:

               EC     Escherichia coll
               EPA   United States Environmental Protection Agency
               g      gram
               L      liter
               LTB   lauryl tryptose broth
               mg     milligram
               mL     milliliter
               mm    millimeter
               MPN   most probable number
               NIST   National Institute of Standards and Technology
               QA     quality assurance
               QC     quality control
               TD     to deliver

21.2   Definitions,  acronyms, and abbreviations (in alphabetical order):

       Analyte—The microorganism tested for by this method.  The analytes in this method are fecal
       coliforms.

       Enrichment—A non-selective culture media for enhanced growth.

       Liquid samples—Generally defined as samples containing <7% total solids (dry weight).

       May—This action, activity, or procedural step is neither required nor prohibited.

       May not—This action, activity, or procedural step is prohibited.

       Method blank—An aliquot of sterile reagent water that is treated exactly as a sample including
       exposure to all glassware, equipment, media, procedures that are used with samples. The method
       blank is used to verify the sterility of equipment, materials, and supplies.
April 2010                                       42

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                                                                              Method 1680
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.

Negative control—A control culture that, when analyzed exactly like a field sample, will produce
a known negative result for a given type of media.

Positive control—A control culture that, when analyzed exactly like a field sample, will produce
a known positive result for a given type of media.

Preparation blank—See Method blank.

Selective media—A culture media designed to suppress the growth  of unwanted microorganisms
and encourage the growth of desired ones.

Should—This action, activity, or procedural step is suggested but not required.

Solid samples—Generally, samples containing >7 % total solids (dry weight).
                                        43                                      April 2010

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