vvEPA
   United States
   Environmental Protection
   Agency
   Method 1103.1: Escherichia coli (E. coli)
   in Water by Membrane Filtration Using
   membrane-Thermotolerant Escherichia
   co//Agar(mTEC)

   March 2010

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U.S. Environmental Protection Agency
      Office of Water (4303T)
   1200 Pennsylvania Avenue, NW
      Washington, DC 20460
        EPA-821-R-10-002

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                                  Acknowledgments

The following laboratories are gratefully acknowledged for their participation in the validation study for
this method in disinfected wastewater effluents which was conducted in 2004:

Volunteer Research Laboratories
•      EPA Office of Research and Development, National Risk Management Research Lab: Mark C.
       Meckes

       U.S. Army Corps of Engineers, Washington Aqueduct: Elizabeth A. Turner, Michael L.
       Chicoine, and Lisa Neal

Volunteer Verification Laboratory
       Orange County Sanitation District, Environmental Sciences Laboratory: Charles McGee, Michael
       von Winckelmann, Kim Patton, Linda Kirchner, James Campbell, Arturo Diaz, and Lisa McMath

Volunteer Participant Laboratories
       American Interplex: John Overbey, Steve Bradford, and Jessica Young

       County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant
       (JWPCP): Kathy Walker, Michele Padilla, and Albert Soof

•      East Bay Municipal Utility District: Bill Ellgas and Daniel Mills

•      Environmental Associates (EA): Susan Boutros and Madelyn Glase

•      Hampton Roads Sanitation District (HRSD): Anna Rule, Paula Hogg, and Bob Maunz

•      Hoosier Microbiological Laboratories (HML): Don Hendrickson, Katy Bilger, Keri Nixon, and
       Lindsey Shelton

       Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofshteyn

       San Francisco Water: Phil Caskey, Paul McGregor, and Bonnie Bompart

       University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord

       Wisconsin State Laboratory of Hygiene (WSLH): Sharon Kluender, Linda Peterson, and Jeremy
       Olstadt

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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

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                                   Table of Contents









1.0    Scope and Application  	  1




2.0    Summary of Method	  1




3.0    Definitions	  2




4.0    Interferences and Contamination 	  2




5.0    Safety  	  2




6.0    Equipment and Supplies	  2




7.0    Reagents and Standards  	  3




8.0    Sample Collection, Preservation, and Storage  	  10




9.0    Quality Control	  10




10.0   Calibration and Standardization 	  14




11.0   Procedure   	  14




12.0   Verification Procedure  	  15




13.0   Data Analysis and Calculations 	  16




14.0   Sample Spiking Procedure  	  17




15.0   Method Performance	  22




16.0   Pollution Prevention	  26




17.0   Waste Management	  26




18.0   References	  27
                                              IV

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                                  List of Appendices
Appendices A and B are taken from Microbiological Methods for Monitoring the Environment: Water
and Wastes (Reference 18.5).

Appendix A:   Part II (General Operations), Section A (Sample Collection, Preservation, and Storage)

Appendix B:   Part II (General Operations), Sections C.3.5 (Counting Colonies) and C.3.6
              (Calculation of Results)

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              Method  1103.1 Escherichia coli (E. coli) in Water
                          by Membrane Filtration Using
         membrane-Thermotolerant Escherichia coli Agar (mTEC)
                                      March 2010
1.0   Scope and Application
1.1   Method 1103.1 describes a membrane filter (MF) procedure for the detection and enumeration of
      Escherichia coli bacteria in ambient water. E. coli is a common inhabitant of the intestinal tract of
      warm-blooded animals, and its presence in water samples is an indication of fecal pollution and
      the possible presence of enteric pathogens.

1.2   The E. coli test is recommended as a measure of ambient recreational fresh water quality.
      Epidemiological studies have led to the development of criteria which can be used to promulgate
      recreational water standards based on established relationships between health effects and water
      quality. The significance of finding E. coli in recreational fresh water samples is the direct
      relationship between the density of E. coli and the risk of gastrointestinal illness associated with
      swimming in the water (Reference 18.1).

1.3   For method application please refer to Title 40 Code of Federal Regulations Part 136 (40 CFR Part
      136).

1.4   Method 1103.1 and 1603 were submitted to interlaboratory validation in wastewater matrices and
      study results compared.  The estimated percent of total colonies that would have resulted in false
      positives for disinfected, secondary, and combined disinfected/secondary wastewater were
      significantly lower for Method 1603 (0.6%, 1.4%, and 1.0%, respectively) compared to Method
      1103.1  (6.2%, 11.2%, and 10.3%, respectively). The estimated percent of total colonies that
      would have resulted in false negatives for disinfected and combined disinfected/secondary
      wastewater were also significantly lower for Method 1603 (2.5%, and 2.2%, respectively)
      compared to Method 1103.1 (12.3% and 6.0%, respectively).  The estimated percent of total
      colonies that would have resulted in false negatives were not significantly different for secondary
      wastewater samples for Methods 1603  and 1103.1 (2.6% and 2.0%, respectively).

      Based on the high Method  1103.1 false positive and false negative levels when compared to
      Method 1603, Method 1103.1 is not approved for the analysis of disinfected wastewater.
      Laboratories wishing to test for E. coli in wastewater using a membrane filtration method are
      referred to EPA Method 1603. A summary of Method 1103.1 false positive and negative results is
      provided in Section 15.3.2. Detailed Method 1103.1 and 1603 study results are provided in the
      validation study reports (References 18.2 and 18.3,  respectively).
2.0   Summary of Method

2.1    The MF method provides a direct count of bacteria in water based on the development of colonies
       on the surface of the membrane filter (Reference 18.4). A water sample is filtered through the
       membrane which retains the bacteria. After filtration, the membrane is placed on a selective and
       differential medium, mTEC, incubated at 35°C ± 0.5°C for 2 ± 0.5 hours to resuscitate injured or
       stressed bacteria, and then incubated at 44.5°C ± 0.2°C for 22 ± 2 hours. Following incubation,
       the filter is transferred to a filter pad saturated with urea substrate. After 15 minutes, yellow,
       yellow-green, or yellow-brown colonies are counted with the aid of a fluorescent lamp and a
       magnifying lens.
                                                                                 March 2010

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Method 1103.1
3.0   Definitions
3.1    In Method 1103.1, E. coli are those bacteria which produce colonies that remain yellow, yellow-
       green, or yellow-brown on a filter pad saturated with urea substrate broth after primary culturing
       on mTEC medium.
4.0   Interferences and Contamination
4.1    Water samples containing colloidal or suspended particulate material can clog the membrane
       filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with
       enumeration and identification of target colonies.
5.0   Safety
5.1    The analyst must know and observe the normal safety procedures required in a microbiology
       laboratory while preparing, using, and disposing of cultures, reagents, and materials and while
       operating sterilization equipment.
5.2    Mouth-pipetting is prohibited.
5.3    This method does not address all of the safety issues associated with its use. It is the
       responsibility of the laboratory to establish appropriate safety and health practices prior to use of
       this method. A reference file of material safety data sheets (MSDSs) should be available to all
       personnel involved in Method 1103.1 analyses.

6.0   Equipment and  Supplies
6.1    Glass lens with magnification of 2-5X, or stereoscopic microscope
6.2    Lamp, with a cool, white fluorescent tube
6.3    Hand tally or electronic counting device
6.4    Pipet  container, stainless steel, aluminum or borosilicate glass, for glass pipets
6.5    Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume
6.6    Sterile graduated cylinders, 100-1000 mL, covered with aluminum foil  or kraft paper
6.7    Sterile membrane filtration units (filter base and  runnel), glass, plastic, or stainless steel, wrapped
       with aluminum foil or kraft paper
6.8    Ultraviolet unit for sanitization of the filter funnel between filtrations (optional)
6.9    Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source (In an emergency or
       in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of
       air, can be used)
6.10   Flask, filter, vacuum, usually 1 L, with appropriate tubing
6.11   A filter manifold to hold a number of filter bases (optional)
6.12   Flask for safety trap placed between the filter flask and the vacuum source
6.13   Forceps, straight or curved, with smooth tips to handle filters without damage
6.14   Ethanol, methanol, or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps
6.15   Burner, Bunsen, or Fisher type, or electric incinerator unit for sterilizing loops and needles

March 2010                                    2

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                                                                                 Method 1103.1
6.16   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
6.17   Petri dishes, sterile, plastic, 9 x 50 mm with tight-fitting lids; and 15 x 100 mm with loose fitting
       lids
6.18   Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, 125 mL volume
6.19   Flasks, borosilicate glass, screw-cap, 250-2000 mL volume
6.20   Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 (im pore size
6.21   Absorbent pads, sterile, 47 mm diameter
6.22   Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic
       loops
6.23   Sterile disposable applicator sticks
6.24   Incubator maintained at 35°C ± 0.5°C
6.25   Waterbath maintained at 44.5°C ± 0.2°C
6.26   Waterbath maintained at 50°C for tempering agar
6.27   Test tubes, 20 x 150 mm, borosilicate glass or plastic
6.28   Test tubes, 10 x 75 mm, borosilicate glass (durham tubes)
6.29   Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes
6.30   Test tubes screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size
6.31   Filter Paper
6.32   Whirl-Pak® bags
6.33   Autoclave or steam sterilizer capable of achieving 121°C [15 Ib pressure per square inch (PSI)]
       for 15 minutes
7.0   Reagents and Standards
7.1    Purity of Reagents: Reagent-grade chemicals shall be used in all tests. Unless otherwise
       indicated, reagents shall conform to the specifications of the Committee of Analytical Reagents of
       the American Chemical Society (Reference 18.5). The agar used in preparation of the culture
       media must be of microbiological grade.
7.2    Whenever possible,  use commercial culture media as a means of quality control.
7.3    Purity of 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 18.6).
                                                                                    March 2010

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Method 1103.1
7.4    Phosphate buffered saline

       7.4.1   Composition:

               Monosodium phosphate (NaH2PO4)                    0.58 g
               Disodium phosphate (N^HPC^)                       2.5  g
               Sodium chloride                                     8.5  g
               Reagent-grade water                                 1.0  L

       7.4.2   Dissolve the ingredients in 1 L of reagent-grade water, and dispense in appropriate
               amounts for dilutions in screw-cap bottles or culture tubes, and/or into containers for use
               as rinse water. Autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ±
               0.2.

               Note: The initial and ongoing precision and recovery (IPR and OPR) performance criteria
               established for Method 1103.1  were determined using spiked PBS samples (Section 9.3,
               Table 1). Laboratories must use  PBS when performing IPR and OPR sample analyses.
               However, phosphate-buffered dilution water (Section 7.6) may be substituted for PBS as
               a sample diluent and filtration rinse buffer.
7.5    Phosphate buffered dilution water (Reference 18.7)

       7.5.1   Composition of stock phosphate buffer solution:

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

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

       7.5.4   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 in screw-cap bottles or culture tubes, and/or into containers for use
               as rinse water.  Autoclave at 121 °C (15 PSI) for 15 minutes. Final pH should be 7.0 ±
               0.2.
March 2010

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                                                                                Method 1103.1
7.6    mTEC agar

       7.6.1   Composition:

               Protease peptone #3                                 5.0  g
               Yeast extract                                       3.0  g
               Lactose                                           10.0  g
               Sodium chloride                                    7.5  g
               Dipotassium phosphate (K2HPO4)                     3.3  g
               Monopotassium phosphate (KH2PO4)                 1.0  g
               Sodium lauryl sulfate                                0.2  g
               Sodium desoxycholate                              0.1  g
               Bromcresol purple                                  0.08 g
               Bromphenol red                                    0.08 g
               Agar                                             15.0  g
               Reagent-grade water                                1.0  L

       7.6.2   Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve.
               Autoclave at 121°C (15 PSI) for 15 minutes, and cool in a 50°C waterbath. Pour the
               medium into each 9x50 mm culture dish to a 4-5 mm depth (approximately 4-6 mL),
               and allow to solidify.  Final pH should be 7.3 ± 0.2.  Store in a refrigerator.
7.7    Urea substrate medium

       7.7.1   Composition:

               Urea                                       2.0 g
               Phenol red                                  0.01 g
               Reagent-grade water                       100.0 mL

       7.7.2   Add reagents to 100 mL reagent-grade water and mix thoroughly to dissolve. Adjust to
               pH 5.0 ± 0.2 with 1 N HC1. The substrate solution should be a straw-yellow color at this
               pH (See Photo 1). The substrate solution should be stored at 6-8°C for no more than one
               week.
                                                                                    March 2010

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Method 1103.1
Photo 1.   Urea substrate medium. After adjusting the pH of the medium to 5 ± 0.2, the urea substrate
           medium should be straw-yellow in color.
7.8    Tryptic soy agar (TSA)

       7.8.1  Composition:
              Pancreatic digest of casein
              Enzymatic digest of soybean meal
              Sodium chloride
              Agar
              Reagent-grade water
15.0g
 5.0 g
 5.0g
15.0 g
 l.OL
       7.8.2  Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
              completely. Autoclave at 121°C (15 PSI) for 15 minutes and cool in a 50°C waterbath.
              Pour the medium into each 15 x 100 mm culture dish to a 4-5 mm depth, and allow to
              solidify. Final pH should be 7.3 ± 0.2.
March 2010

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                                                                                 Method 1103.1
7.9    Lauryl Tryptose Broth (LTB)

       7.9.1   Composition
               Tryptose                                    20.0  g
               Lactose                                      5.0  g
               Dipotassium phosphate (K2HPO4)              2.75 g
               Monopotassium phosphate (KH2PO4)           2.75 g
               Sodium chloride                              5.0  g
               Sodium lauryl sulfate                          0.1  g

       7.8.2   Add reagents to 1 L of reagent-grade water, mix thoroughly, and warm to dissolve
               completely. Dispense into fermentation tubes (20 x  150 mm tubes containing inverted 10
               x 75 mm tubes). Autoclave at 121°C (15 PSI) for 15 minutes. Final pH should 6.8 ± 0.2.
7.10   Nutrient agar (NA)

       7.10.1  Composition:

               Peptone                                     5.0g
               Beef extract                                  3.0g
               Agar                                       15. Og
               Reagent-grade water                          l.OL

       7.10.2  Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to boiling to
               dissolve completely. Dispense in screw-cap tubes, and autoclave at 121°C (15 PSI) for
               15 minutes. Remove the tubes and slant. Final pH should be 6.8 ± 0.2.
7.11   Tryptic/trypticase soy broth (TSB)

       7.11.1  Composition:

               Pancreatic digest of casein                    17.Og
               Enzymatic/papaic digest of soybean meal        3.0 g
               Sodium chloride                              5.0g
               Dextrose                                     2.5 g
               Dipotassium phosphate (K2HPO4)              2.5 g
               Reagent-grade water                          l.OL

       7.11.2  Add reagents to  1 L of reagent-grade water, mix thoroughly, and heat to dissolve
               completely. Dispense in screw-cap tubes, and autoclave at 121°C (15 PSI) for 15
               minutes. Final pH should be 7.3 ± 0.2.
                                                                                    March 2010

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Method 1103.1
7.12   Simmons citrate agar slants

       7.12.1  Composition:

               Magnesium sulfate (MgSO4)                         0.2  g
               Monoammonium phosphate (NH4H2PO4)              1.0  g
               Dipotassium phosphate (K2HPO4)                    1.0  g
               Sodium citrate (Citric acid)                          2.0  g
               Sodium chloride                                    5.0  g
               Bromthymol blue                                   0.08 g
               Agar                                              15.0  g
               Reagent-grade water                                1.0  L

       7.12.2  Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to boiling to
               dissolve completely. Dispense into screw-cap tubes, and autoclave at 121°C (15 PSI) for
               15 minutes. After sterilization, slant until solid. Final pH should be 6.9 ± 0.2.

7.13   Tryptone water

       7.13.1  Composition:

               Tryptone                                    lO.Og
               Sodium chloride                              5.0 g
               Reagent-grade water                          l.OL

       7.13.2  Add reagents to 1 L of reagent grade water and mix thoroughly to dissolve.  Dispense in
               5-mL volumes into tubes, and autoclave at 121°C (15 PSI) for 15 minutes.  Final pH
               should be 7.3 ± 0.2.

7.14   EC broth

       7.14.1  Composition:

               Tryptose or trypticase peptone                       20.0 g
               Lactose                                             5.0g
               Bile salts No. 3                                       1.5g
               Dipotassium phosphate (K2HPO4)                      4.0 g
               Monopotassium phosphate (KH2PO4)                   1.5 g
               Sodium chloride                                      5.0 g
               Reagent-grade water                                  l.OL

       7.14.2  Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
               completely. Dispense into fermentation tubes (20 x 150 mm tubes containing inverted 10
               x 75 mm tubes). Autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 6.9 ±
               0.2.

               Note: Do not use tubes if the inverted tubes (durham tubes) are not completely filled with
               medium after sterilization.

7.15   Oxidase reagent

       7.15.1  Composition:

               N, N, N',N'-tetramethyl-p-phenylenediamine dihydrochloride, 1% aqueous solution (1 g
               per 100 mL sterile reagent-grade water)

               Note: Prepared oxidase test slides are commercially available.

March 2010                                     8

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                                                                                Method 1103.1
7.16   Kovacs indole reagent

       7.16.1 Composition:

              p-dimethylaminobenzaldehyde                 10.0 g
              Amy 1 or Isoamyl alcohol                      ISO.OmL
              Concentrated (12 M) hydrochloric acid          50.0 mL

       7.16.2 Dissolve P-dimethylaminobenzaldehyde in alcohol, slowly add hydrochloric acid, and
              mix.

7.17   Control cultures

       7.17.1 Positive control and/or spiking organism (either of the following are acceptable)

              •   Stock cultures of Escherichia coll (E. coli) ATCC #11775

              •   E. coli ATCC #11775 BioBalls (BTF Pty, Sydney, Australia)

       7.17.2 Negative control organism (either of the following are acceptable)

              •   Stock cultures of Enterococcus faecalis (E. faecalis} ATCC #19433

              •   E. faecalis ATCC # 1943 3 BioBalls (BTF Pty, Sydney, Australia)

                                    OR

              •   Stock cultures of Enterobacter aerogenes (E. aerogenes} ATCC #13048
                                                                                   March 2010

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Method 1103.1
8.0   Sample Collection, Handling, and Storage

8.1    Sampling procedures are briefly described below. Detailed sampling methods can be found in
       Microbiological Methods for Monitoring the Environment: Water and Wastes, Part II, Section A
       (see Appendix A). Adherence to sample handling procedures and holding time limits is critical to
       the production of valid data. Samples should not be analyzed if these conditions are not met.

       8.1.1  Sampling Techniques

              Samples are collected by hand or with a sampling device if the sampling site has difficult
              access such as a dock, bridge, or bank adjacent to a surface water.  Composite samples
              should not be collected, since such samples do not display the range of values found in
              individual samples.  The sampling depth for surface water samples should be 6-12 inches
              below the water surface. Sample containers should be positioned such that the mouth of
              the container is  pointed away from the sampler or sample point.  After removal of the
              container from the water, a small portion of the sample should be discarded to allow for
              proper mixing before analyses.

       8.1.2  Storage Temperature and Handling Conditions

              Ice or refrigerate water samples at a temperature of <10°C during transit to the
              laboratory. Do  not freeze the samples. Use insulated containers to assure proper
              maintenance of storage temperature.  Take care that sample bottles are not totally
              immersed in water during transit or storage.

       8.1.3  Holding Time Limitations

              Sample  analysis should begin immediately, preferably within 2 hours of collection.  The
              maximum transport time to the laboratory is 6 hours, and samples should be processed
              within 2 hours of receipt at the laboratory.
9.0   Quality  Control

9.1    Each laboratory that uses Method 1103.1 is required to operate a formal quality assurance (QA)
       program that addresses and documents instrument and equipment maintenance and performance,
       reagent quality and performance, analyst training and certification, and records storage and
       retrieval. General requirements and recommendations for QA and quality control (QC)
       procedures  for microbiological laboratories are provided in Reference 18.7.

9.2    The minimum analytical QC requirements for the analysis of samples using Method 1103.1
       include routine analysis of positive and negative controls (Section 9.5), filter sterility checks
       (Section 9.7), method blanks (Section 9.8), and media sterility checks (Section 9.10). Additional
       analytical QC for the analysis of samples using Method 1103.1 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).  For the IPR and OPR analyses, it
       is necessary to spike PBS samples with either laboratory-prepared spiking suspensions or
       BioBalls as described in Section 14.
March 2010                                    10

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                                                                                Method 1103.1
9.3    Initial precision and recovery (IPR)—The IPR analyses are used to demonstrate acceptable
       method performance (recovery and precision) by each laboratory before the method is used for
       monitoring field samples. EPA recommends but does not require that IPR analyses be performed
       by each analyst.  IPR samples should be accompanied by an acceptable method blank (Section
       9.8) and appropriate media sterility checks (Section 9.10).  The IPR analyses are performed as
       follows:

       9.3.1  Prepare four, 100-mL samples of PBS and spike each sample with E. coll ATCC #11775
              according to the spiking procedure in Section 14. Spiking with laboratory-prepared
              suspensions is described in Section 14.2 and  spiking with BioBalls is described in
              Section 14.3. Filter and process each IPR sample according to the procedures in Section
              11 and calculate the number of E. coll per 100 mL according to Section 13.

       9.3.2  Calculate the percent recovery (R) for each IPR sample using the appropriate equation in
              Section 14.2.4.3 or 14.3.2 for samples spiked with laboratory-prepared spiking
              suspensions or BioBalls, respectively.

       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 1,
              below.  If the mean and RSD for recovery of E. coll meet acceptance criteria, system
              performance is acceptable and analysis of field samples may begin. If the mean 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 IPR analyses.
Table 1.    Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria
Performance test
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
Lab-prepared spike
acceptance criteria
76% -124%
41%
54o/0. 146o/0
BioBall™ spike
acceptance criteria
68% - 96%
25%
58% -106%
9.4    Ongoing precision and recovery (OPR)—To demonstrate ongoing control of the analytical
       system, the laboratory should routinely process and analyze spiked PBS 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 should be
       accompanied by an acceptable method blank (Section 9.8) and appropriate media sterility checks
       (Section 9.10). The OPR analysis is performed as follows:

       9.4.1  Spike a 100-mL PBS sample with E. coll ATCC #11775 according to the spiking
              procedure in Section 14. Spiking with laboratory-prepared suspensions  is described in
              Section 14.2 and spiking with BioBalls is described in Section 14.3. Filter and process


                                              11                                    March 2010

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Method 1103.1
               each OPR sample according to the procedures in Section 11 and calculate the number of
               E. coll per 100 mL according to Section 13.

       9.4.2   Calculate the percent recovery (R) for the OPR sample using the appropriate equation in
               Section 14.2.2 or 14.3.4 for samples spiked with BioBalls or laboratory-prepared spiking
               suspensions, respectively.

       9.4.3   Compare the OPR results (percent recovery) with the corresponding OPR recovery
               criteria in Table 1, 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, method recovery 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
               1103.1 by calculating the average percent recovery (R) and the standard deviation of the
               percent recovery (sr). Express the accuracy as a recovery interval from R - 2sr to R + 2sr.

9.5    Culture Controls

       9.5.1   Negative controls—The laboratory should analyze negative controls to ensure that the
               mTEC agar and urea substrate 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.

               9.5.1.1     Negative controls are conducted by filtering a dilute suspension of viable E.
                         faecalis (e.g., ATCC  #19433) and analyzing as described in Section  11.
                          Viability of the negative controls should be demonstrated using a
                          non-selective media (e.g., nutrient agar or tryptic soy agar).

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

       9.5.2   Positive controls—The laboratory should analyze positive controls to ensure that the
               mTEC agar and urea substrate 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.

               9.5.2.1     Positive controls are conducted by filtering a dilute suspension of viable E.
                          coll (e.g., ATCC #11775) and analyzing as described in Section 11.

               9.5.2.2     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.
March 2010                                    12

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                                                                                 Method 1103.1
       9.5.3   Controls for verification media—All verification media should be tested with
               appropriate positive and negative controls whenever a new batch of media and/or
               reagents are used.  On an ongoing basis, the laboratory should perform positive and
               negative controls on the verification media with each batch of samples submitted to
               verification.  Examples of appropriate controls for verification media are provided in
               Table 2.

Table 2.    Verification Controls
Medium
Cytochrome oxidase reagent
Kovacs indole reagent
Simmons citrate agar
EC broth (44.5°C ± 0.2°C)
Positive Control
P. aeruginosa
E. co//
E. aerogenes
E. co//
Negative Control
£. co//
E. aerogenes
S. flexneri
E. aerogenes
9.6    Colony verification—The laboratory should verify 10 typical colonies (positive) and 10 atypical
       colonies (negative) per month or 1 typical colony and 1 atypical colony from 10% of all positive
       samples, whichever is greater. Verification procedures are provided in Section 12.

9.7    Filter sterility check—Place at least one membrane filter on a TSA plate, and incubate for 24 ± 2
       hours at 35°C ± 0.5°C. Absence of growth indicates sterility of the filter. On an ongoing basis,
       the laboratory should perform a filter sterility check every day that samples are analyzed.

9.8    Method blank—Filter a 50-mL volume of sterile PBS or phosphate-buffered dilution water,
       place the filter on an mTEC agar plate and process according to Section  11. 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.

9.9    Filtration blank—Filter a 50-mL volume of sterile PBS or phosphate-buffered dilution water
       before beginning sample filtrations. Place the filter on a TSA plate, and incubate for 24 ± 2 hours
       at 35°C ± 0.5°C. Absence of growth indicates  sterility of the buffer and filtration assembly.

9.10   Media sterility check—The laboratory should test media sterility by incubating one unit (tube or
       plate) from each batch of medium  (TSA, mTEC, urea substrate, and verification media) as
       appropriate and observing for growth. Absence of growth indicates media sterility. On an
       ongoing basis, the laboratory should perform a media sterility check every day that samples are
       analyzed.

9.11   Analyst colony counting variability—Laboratories with two or more analysts should compare
       each analyst's colony counts from one positive field sample per month.  Colony counts should be
       within 10% between analysts. Laboratories with a single analyst should have that analyst
       perform duplicate colony counts of a single membrane filter each month. Duplicate colony
       counts should be within 5% for a single analyst.  If no positive field samples are available, a OPR
       sample may be substituted for these determinations.
                                               13
March 2010

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Method 1103.1
10.0  Calibration and Standardization

10.1   Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to
       ensure operation within stated limits.

10.2   Check thermometers at least annually against a NIST certified thermometer or one that meets the
       requirements of NIST monograph SP 250-23.  Check mercury columns for breaks.

10.3   Refrigerators used to store media and reagents should be monitored daily to ensure proper
       temperature control.
11.0  Procedure

11.1   Prepare mTEC agar and urea substrate as directed in Sections 7.6 and 7.7, respectively.

11.2   Mark the petri dish and report form with the sample identification and volume.

11.3   Place a sterile membrane filter on the filter base, grid side up, and attach the funnel to the base so
       that the membrane filter is held between the funnel and the base.

11.4   Shake the sample bottle vigorously at least 25 times to distribute the bacteria uniformly, and
       measure the desired volume of sample or dilution into the funnel.

11.5   Select sample volumes based on previous knowledge of E. coll concentration, to produce 20-80
       E. coli colonies on the membranes. It is recommended that a minimum of three dilutions be
       analyzed to ensure that a countable plate (20-80 E.  coli colonies) is obtained.

11.6   Smaller sample volumes or sample dilutions can be used to minimize the interference of turbidity
       or for high bacterial densities.  Multiple volumes of the same sample or sample dilutions should
       be filtered.
       Note: When analyzing smaller sample volumes (e.g., < 20 mL), 20-30 mL of PBS or phosphate-
       buffered dilution water should be added to the funnel or an aliquot of sample should be dispensed
       into a dilution blank prior to filtration. This will allow even distribution of the sample on the
       membrane.

11.7   Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffer.
       Turn off the vacuum,  and remove the funnel from the filter base.

11.8   Use sterile forceps to  aseptically remove the membrane filter from the filter base, and roll it onto
       the mTEC agar to avoid the formation of bubbles between the membrane and the agar surface.
       Reseat the membrane if bubbles occur.  Run the forceps around the edge of the filter outside the
       area of filtration, close to the edge of the dish, to be sure that the filter is properly seated on the
       agar. Close the dish, invert, and incubate 35°C ± 0.5°C for 2 ± 0.5 hours.

11.9   After a 2 ±  0.5 hour incubation at 35°C ± 0.5°C, transfer the plates to a Whirl-Pak® bag, seal the
       bag, and submerge in a 44.5°C ± 0.2°C waterbath for 22 ± 2 hours.

       Do not overfill the Whirl-Pak® bag because this will prevent proper sealing allowing liquid to
       enter the bag and possibly contaminating the plates.

11.10  After 22 ± 2 hours, remove the plate from the waterbath.  Place an absorbent pad in the lid  of the
       same petri dish, and saturate the pad with urea substrate medium. Aseptically transfer the
       membrane from mTEC agar to the absorbent pad saturated with urea substrate medium, and allow
       to sit at room temperature for 15-20 minutes. (See Photo 2)
March 2010                                    14

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                                                                                Method 1103.1
Photo 2.   Escherichia coll produces yellow, yellow-green, or yellow-brown colonies on mTEC agar.
11.11  After incubation on the urea substrate at room temperature, count and record colonies on those
       membrane filters containing 20-80 yellow, yellow-green, or yellow-brown colonies. Use
       magnification for counting and a small fluorescent lamp to give maximum visibility of colonies.
       (See Photo 3)
Photo 3.   E. coll colonies remain yellow, yellow-green, or yellow-brown when the filter is placed on
           the urea substrate medium, while non-target colonies turn pink or purple.
12.0  Verification Procedure

12.1   Yellow, yellow-green, or yellow-brown colonies from the urease test are considered "typical" E.
       coll. Verification of typical and atypical colonies may be required in evidence gathering and is
       also recommended as a means of quality control. The verification procedure follows.
12.2   Using a sterile inoculating loop or needle, transfer growth from the centers of at least 10 well-
       isolated typical and 10 well-isolated atypical colonies to nutrient agar plates or slants and to
       tryptic/trypticase soy broth. Incubate the agar and broth cultures for 24 ± 2 hours at 35°C ±
       0.5°C.
                                              15
March 2010

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Method 1103.1
12.3   After incubation, transfer growth from the nutrient agar slant and perform cytochrome oxidase
       test. If the area where the bacteria were applied turns deep purple within 15 seconds, the test is
       positive.
       Note: Use only platinum, plastic or wooden applicators to perform the oxidase test. Do not use
       iron or other reactive wire because it may cause false positive reactions.


12.4   Transfer growth from the tryptic/trypticase soy broth tube to Simmons citrate agar, tryptone
       water, and an EC broth fermentation tube.

       12.4.1  Incubate the Simmons citrate agar for 4 days at 35°C ± 2.0°C in an aerobic atmosphere.
               A positive reaction is indicated by growth with an intense blue color on the slant. E. coll
               is citrate negative, and thus for this organism on this medium there should be either no
               growth or trace growth with no change in agar color (i.e., medium remains dark green).

       12.4.2  Incubate the EC broth at 44.5°C ± 0.2°C in a waterbath for 24 ± 2 hours. The water level
               must be  above the level of the EC broth in the tube. A positive test is indicated by
               turbidity and production of gas as seen in the inner durham tube.

       12.4.3  Incubate the tryptone water for 18-24 hours at 35°C ± 2.0°C with loosened caps. After
               the incubation period, add 0.5 mL of Kovacs indole reagent and shake the tube gently.
               Allow the tubes to stand for 5-10 minutes at room temperature.  A positive test for indole
               is indicated by a deep red color which develops in the alcohol layer on top of the broth.

12.5   E. coll are oxidase-negative,  citrate-negative, EC growth and gas-positive, and indole-positive.

12.6   Alternately, commercially available multi-test identification systems may be used to verify
       colonies.  Inoculate the colonies into an identification system for Enterobacteriaceae that
       includes lactose  fermentation, o-nitrophenyl-p-D-galactopyranoside  (ONPG), and cytochrome
       oxidase test reactions.
13.0  Data Analysis and Calculations

Use the following general rules to calculate the E. coll count per 100 ml of sample:
13.1   If possible, select a membrane filter with 20-80 yellow, yellow-green, or yellow-brown colonies
       on the urea substrate, and calculate the number of E. coll per 100 mL according to the following
       general formula:

                                             Number of E. coli colonies
                  E. coli/ 100mL =       	  x   100
                                            Volume of sample filtered (mL)


13.2   See general counting rules in Reference 18.7 (see Appendix B).

13.3   Report results as E. coli CPU per 100 mL of sample.
March 2010                                    16

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                                                                                Method 1103.1
14.0  Sample Spiking Procedure

14.1   Method 1103.1 QC requirements (Section 9) include the preparation and analysis of spiked
       reference (PBS) samples in order to monitor initial and ongoing method performance. For the
       IPR (Section 9.3) and OPR (Section 9.4) tests it is necessary to spike samples with either
       laboratory-prepared spiking suspensions (Section 14.2) or BioBalls (Section 14.3) as described
       below.

14.2   Laboratory-Prepared Spiking Suspensions

       14.2.1 Preparation

              14.2.1.1   Stock Culture. Prepare a stock culture by inoculating a TSA slant (or other
                         non-selective media) with Escherichia coll ATCC #11775 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.

              14.2.1.2   Undiluted Spiking Suspension. Prepare a 1% solution of lauryl tryptose
                         broth (LTB) by combining 99 mL of sterile PBS and 1 mL of sterile single-
                         strength LTB in a sterile screw-cap bottle or re-sealable dilution water
                         container. Inoculate the 1% LTB using a small amount of growth from the
                         stock culture. Disperse the inoculum by vigorously  shaking the broth culture
                         and incubate at 35°C ± 3°C for 20 ± 4 hours. This culture is referred to as
                         the undiluted spiking  suspension and should contain approximately
                         1.0 x 107 - 1.0 x 10s E. coli colony forming  units (CPU) per mL of culture.

              14.2.1.3   Mix the undiluted spiking suspension thoroughly by shaking the bottle a
                         minimum of 25 times and prepare a series of dilutions (4 total) in the
                         following manner:

                         14.2.1.3.1     Dilution  "A"—Aseptically transfer 1.0 mL of the undiluted
                                       spiking suspension to 99 mL of sterile PBS and mix
                                       thoroughly by shaking the bottle a minimum of 25 times.
                                       This is spiking suspension dilution "A" and 1 mL contains
                                        10"2 mL of the original undiluted spiking suspension.

                         14.2.1.3.2    Dilution  "B"—Aseptically transfer 1.0 mL of dilution "A" to
                                       99 mL of sterile PBS and mix thoroughly by shaking the
                                       bottle a minimum of 25 times. This is spiking suspension
                                       dilution "B" and 1 mL contains 10"4 mL of the original
                                       undiluted spiking suspension.

                         14.2.1.3.3    Dilution  "C"—Aseptically transfer 11.0 mL of dilution "B"
                                       to 99 mL of sterile PBS and mix thoroughly by shaking the
                                       bottle a minimum of 25 times. This is spiking suspension
                                       dilution "C" and 1 mL contains 10"5 mL of the original
                                       undiluted spiking suspension.
                                              17                                   March 2010

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Method 1103.1
                          14.2.1.3.4    Dilution "D"—Aseptically transfer 11.0 mL of dilution "C"
                                        to 99 mL of sterile PBS and mix thoroughly by shaking the
                                        bottle a minimum of 25 times.  This is spiking suspension
                                        dilution "D" and 1 mL contains 10~6 mL of the original
                                        undiluted spiking suspension.

       14.2.2  Sample Spiking

               14.2.2.1    Add 0.3 mL of the spiking suspension dilution "D" to 100 mL of PBS and
                          mix thoroughly by shaking the bottle a minimum of 25 times. The volume of
                          undiluted spiking suspension added to each 100 mL sample is 3.0 x 10"7 mL
                          [(0.3 mL x 10"6 mL) per 100 mL of sample] which is referred to as Vsplkedper 100
                          mL sample ^n Section 14.2.4.2 below.  Filter the spiked sample and analyze the
                          filter according to the procedures in Section 11.

       14.2.3  Enumeration of Spiking Suspension

               14.2.3.1    Prepare trypticase soy agar (TSA) spread plates, in triplicate, for spiking
                          suspension dilutions "B", "C", and "D".

                          Note: Agar plates must be dry prior to  use. To ensure that the agar surface is
                          dry, plates should be made several days in advance and stored inverted at
                          room temperature or dried using a laminar-flow hood.

               14.2.3.2    Mix dilution "B"  by shaking the bottle a minimum of 25 times. Pipet 0.1 mL
                          of dilution "B" onto the surface of each TSA  plate in triplicate.

               14.2.3.3    Mix dilution "C"  by shaking the bottle a minimum of 25 times. Pipet 0.1 mL
                          of dilution "C" onto the surface of each TSA  plate in triplicate.

               14.2.3.4    Mix dilution "D"  by shaking the bottle a minimum of 25 times. Pipet 0.1 mL
                          of dilution "D" onto the surface of each TSA plate in triplicate.

               14.2.3.5    Use a  sterile bent glass rod or spreader to distribute the inoculum over the
                          surface of plates by rotating the dish by hand or on a turntable. Note: Ensure
                          that the inoculum is evenly distributed over the entire  surface of the plate.

               14.2.3.6    Allow the inoculum to absorb into the  medium of each plate completely.
                          Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours.

               14.2.3.7    Count and record number of colonies per plate. Refer to Section 14.2.4 for
                          calculation of E. coll concentration in the undiluted spiking suspension. The
                          number of E. coli (CPU / mL) in the undiluted spiking suspension will be
                          calculated using all TSA plates yielding counts within the countable range of
                          30 to 300 CPU per plate.
March 2010                                    18

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                                                                                 Method 1103.1
       14.2.4  Recovery calculations for samples spiked with laboratory-prepared spiking
               suspensions

               14.2.4.1    Calculate the concentration of E. coll (CPU / mL) in the undiluted spiking
                          suspension (Section 14.2.1.2) according to the following equation. Example
                          calculations are provided in Table 3, below.
              E. coli undilutedspike = (CFUt + CFU2 + ...+ CFUn) / (Vt + V2 + ... + Vn)

              Where,

              E. coli undilutedspike = E. coli (CPU / mL) in undiluted spiking suspension

              CPU =    Number of colony forming units from TSA plates yielding counts within the
                         countable range of 30 to 300 CPU per plate

              V    =    Volume  of undiluted sample on each TSA plate yielding counts within the
                         countable range of 30 to 300 CPU per plate

              n    =    Number of plates with counts within the countable range (30 to 300 CPU/
                         plate)

              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, the percent recoveries may be slightly
              different.

Table 3.    Example Calculations of E. coli Spiking Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
TSA plates in Section 14.2.3.7
10 5 ml. plates
TNTC, TNTC,
TNTC
269, 289, 304
10s ml_ plates
94, 106,89
24, 30, 28
10 7 ml_ plates
10, 0,4
0,2, 0
E. coli CPU / mL in undiluted
spiking suspension
(EC undiluted spike)
(94+106+89) /(10-6+10-6+10-6) =
289 / (3.0 x ID'6) = 96,333,333 =
9.6x107CFU/mL
(269+289+30) / ( 1 0~5+ 1 0~5+ 1 0'6) =
588 / (2.1 x 10-5) = 28,000,000 =
2.8x107CFU/mL
*EC undi|Utedspikeis calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
                                               19
March 2010

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Method 1103.1
               14.2.4.2   Calculate true concentration (CPU / 100 mL) of spiked E. coll (T splked£ coh)
                          according to the following equation.  Example calculations are provided in
                          Table 4, below.
               -*- spiked E. co,

               Where,

               T

  • -------
                                                                                  Method 1103.1
    Table 5.    Example Percent Recovery Calculations
    Ns(CFU/100mL)
    42
    34
    16
    10
    Nu(CFU/100mL)
    <1
    10
    <1
    <1
    T(CFU/100mL)
    28.8
    28.8
    8.4
    8.4
    Percent recovery (R)
    100x(42-1)/28.8
    = 142%
    100 x (34 -10) 728.8
    = 83%
    100x(16-1)/8.4
    = 179%
    100x(10-1)/8.4
    = 107%
    14.3   BioBall™ spiking procedure
           14.3.1 Aseptically add 1 BioBall™ to 100 mL (or appropriate volume) of sample and mix by
                  vigorously shaking the sample bottle a minimum of 25 times.  Analyze the spiked sample
                  according to the procedures in Section 11.
           14.3.2 Recovery calculations for samples spiked with BioBalls—Calculate percent recovery (R)
                  of spiked E. coll (CPU / 100 mL) according to the following equation. Example
                  calculations are provided in Table 6, below.
                                  R  =  100x
                            (N, - Nu)
                  Where,
                  R  =
                  Ns =
                  Nu =
                  T  =
    Percent recovery
    E. coli (CPU / 100 mL) in the spiked sample (Section 13)
    E. coli (CPU / 100 mL) in the unspiked sample (Section 13)
    True spiked E. coli (CPU / 100 mL) in spiked sample based on the
    lot mean value provided by manufacturer
                                                21
                                                             March 2010
    

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    Method 1103.1
    Table 6.   Example Percent Recovery Calculations
    Ns (CPU/ 100
    ml_)
    24
    36
    Nu (CPU/ 100
    ml_)
    <1
    10
    T(CFU/100ml_)
    32
    32
    Percent recovery (R)
    100 x (24-1)732 = 72%
    100 x (36 -10) 732 = 81%
    15.0  Method Performance
    
    15.1   Performance Characteristics
    
           15.1.1 Precision - The degree of agreement of repeated measurements of the same parameter
                  expressed quantitatively as the standard deviation or as the 95% confidence limits of the
                  mean computed from the results of a series of controlled determinations. The mTEC
                  method precision was found to be fairly representative of what would be expected from
                  counts with a Poisson distribution (Reference 18.3).
    
           15.1.2 Bias - The  persistent positive or negative deviation of the  average value of the method
                  from the assumed or accepted true value. The bias of the mTEC method has been
                  reported to  be -2% of the true value (Reference  18.3).
    
           15.1.3 Specificity  - The ability of a method to select and or distinguish the target bacteria under
                  test from other bacteria in the same water sample. The specificity characteristic of a
                  method is usually reported as the percent of false positive  and false negative results. The
                  false positive rate reported for mTEC medium averaged 9% for marine and fresh water
                  samples.  Less than 1% of the E. coli colonies observed gave a false negative reaction
                  (Reference  18.3).
    
           15.1.4 Upper Counting Limit (UCL) - That colony count above which there is an unacceptable
                  counting error. The error may be due to overcrowding or  antibiosis. The UCL for
                  E. coli on mTEC medium has been reported as 80 colonies per filter (Reference 18.3).
    
    15.2   Collaborative Study Data for Ambient Waters
    
           15.2.1 A collaborative study was conducted among eleven volunteer laboratories, each with two
                  analysts who  independently tested local fresh and marine recreational waters and sewage
                  treatment plant effluent samples, in duplicate. The data were reported to the
                  Environmental Monitoring and Support Laboratory - Cincinnati, U.S. Environmental
                  Protection Agency, for statistical calculations.
    
           15.2.2 The  results  of the study are shown in Figure 1 where S0 equals the pooled standard
                  deviation among replicate counts from a single analyst for three groupings (counts less
                  than 30, counts from 30 to 50, and counts greater than 50) and SB equals the pooled
                  standard deviation between means of duplicates from analysts in the same laboratory for
                  the same groupings. The precision estimates from this study did not show any difference
                  among the water types analyzed.
    March 2010
    22
    

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                                                                               Method 1103.1
    15.2.3 By linear regression, the precision of the method can be generalized as:
    
           S0 = 0.028 count/100 mL + 6.11 (dilution factor) and
           SB = 0.233 count/100 mL + 0.82 (dilution factor)
                                                             100
                         Where dilution factor =
                                                Volume of Original Sample Filtered
    
    15.2.4 Because of the instability of microbial populations in water samples, each laboratory
           analyzed its own sample series and no full measure of recovery or bias was possible.
           However, all laboratories analyzed a single surrogate sample prepared from a freeze-
           dried culture of E. coll. The mean count (x) and the overall standard deviation of the
           counts (ST) (which includes the variability among laboratories for this standardized E.
           coll sample) were 31.6 colonies/membrane and 7.61 colonies/membrane, respectively.
                                            23                                    March 2010
    

    -------
    I
    O
    ~v
    O
    CD_
    
    
    O
    Q.
    to
                   FIGURE  1.   Precision Estimates for I. coli in Water by the Membrane  Filter/mTEC Procedure.
    

    -------
                                                                                       Method 1103.1
    15.3   Interlaboratory Validation Study Results for Wastewater
    
           15.3.1  Ten volunteer participant laboratories, an E. coll verification laboratory, and two research
                   laboratories participated in the U.S. Environmental Protection Agency's (EPA's)
                   interlaboratory validation study of EPA Method 1103.1 for the analysis of E. coll in
                   disinfected wastewater.  The purposes of the Study were to characterize method
                   performance across multiple laboratories and disinfected wastewater matrices and to
                   develop quantitative quality control (QC) acceptance criteria. The false positive and
                   negative assessments are provided in Tables 7 and 8, respectively. Detailed results of the
                   study are provided in the validation study report (Reference 18.2).
    
           15.3.2 Method 1103.1 is not approved  for the analysis of E. coll in disinfected wastewater
                   because of the high false positive and false negative levels observed during the validation
                   study compared to Method  1603.  Laboratories wishing to test for E. coll in wastewater
                   using a membrane filtration method are referred to EPA Method 1603.
    Table 7.   False Positive Assessment of Unspiked Disinfected and Unspiked Secondary
               Wastewater Effluents
    Matrix
    Disinfected
    Secondary
    Disinfected + Secondary
    Total colonies
    Typical
    272
    1190
    1462
    Atypical
    725
    347
    1072
    False positive (FP) assessment
    Typical
    colonies
    submitted
    70
    104
    174
    No. FP
    colonies
    16
    15
    31
    FP confirmation
    rate (%) a
    22.9%
    14.4%
    17.8%
    Estimated % of total
    colonies that would have
    been a FPb
    6.2%
    1 1 .2%
    10.3%
    a False positive confirmation rate = number of false positive colonies / number of typical colonies submitted
    b Percent of total colonies estimated to be false positives = [(total typical colonies * pp confirmation rate) / (total
    number of typical and atypical colonies observed)] x 100; e.g., [(1190 x(i5/i04))/(1190+347)] x 100 = 11.2%
                                                    25
    March 2010
    

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    Method 1103.1
    Table 8.        False Negative Assessment of Unspiked Disinfected and Unspiked Secondary
                   Wastewater Effluents
    Matrix
    Disinfected
    Secondary
    Disinfected + Secondary
    Total colonies
    Typical
    272
    1190
    1462
    Atypical
    725
    347
    1072
    False negative (FN) assessment
    Atypical
    colonies
    submitted
    89
    45
    134
    No. FN
    colonies
    15
    4
    19
    FN confirmation
    rate (%) a
    16.9%
    8.9%
    1 4.2%
    Estimated % of total
    colonies that would have
    been a FN b
    12.3%
    2.0%
    6.0%
    a False negative confirmation rate = number of false negative colonies / number of atypical colonies submitted
    b Percent of total colonies estimated to be false negatives = [(total atypical colonies * FN confirmation rate) / (total
      number of typical and atypical colonies observed)] x 100; e.g., [(347x(4/45))/(1190+347)] x 100 = 2.0%
    16.0  Pollution Prevention
    
    16.1   The solutions and reagents used in this method pose little threat to the environment when
           recycled and managed properly.
    16.2   Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
           the volume of expired materials to be disposed.
    17.0  Waste Management
    
    17.1   It is the laboratory's responsibility to comply with all federal, state, and local regulations
           governing waste management, particularly the biohazard and hazardous waste identification rules
           and land disposal restrictions, and to protect 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.
    
    17.2   Samples, reference materials, and equipment known or suspected to have viable E. coli attached
           or contained must be  sterilized prior to disposal.
    
    17.3   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.
    March 2010
    26
    

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                                                                                  Method 1103.1
    18.0  References
    
    
    18.1   Dufour, A.P. Health Effects Criteria for Fresh Recreational Waters, EPA-600/1-84-004.
           Research Triangle Park, NC: U.S. Environmental Protection Agency, 1984.
    18.2   USEPA. 2004. Results of the Interlab oratory Validation of EPA Method 1103.1 (mTEC) for E.
           coli in Wastewater Effluent. EPA-821-R-04-020. December 2004.
    
    18.3   USEPA. 2004. Results of the Interlab oratory Validation of EPA Method 1603 (modified mTEC)
           forE. coli in Wastewater Effluent. EPA-821-R-04-020. December 2004.
    
    18.4   Dufour, A.P., E.R. Strickland, V.J. Cabelli. 1981. Membrane filter method for enumerating
           Escherichia coli. Appl. Environ. Microbiol. 41:1152-1158.
    
    18.5   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.
    
    18.6   APHA. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition.
           American Public Health Association, Washington D.C.
    
    18.7   Bordner, R., J.A. Winter, and P.V. Scarpino (eds.).  Microbiological Methods for Monitoring the
           Environment, Water and Wastes, EPA-600/8-78-017. Cincinnati, OH: U.S. Environmental
           Protection Agency,  1978.
                                                 27                                   March 2010
    

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                        Appendix A:
    Part II (General Operations), Section A (Sample Collection,
                  Preservation, and Storage)
    

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                                        Sample Collection1
    1.0   Sample Containers
          1.1      Sample Bottles: bottles must be resistant to sterilizing conditions and the solvent action
                   of water. Wide-mouth borosilicate glass bottles with screw-cap or ground-glass stopper
                   or heat-resistant plastic bottles may be used if they can be sterilized without producing
                   toxic materials (see examples A and C in Figure 1).  Screw-caps must not produce
                   bacteriostatic or nutritive compounds upon sterilization.
                   Figure 1.  Suggested sample containers.
    
          1.2      Selection and Cleaning of Bottles: Samples bottles should be at least 125 mL volume
                   for adequate sampling and for good mixing.  Bottles of 250 mL, 500 mL, and 1000 mL
                   volume are often used for multiple analyses. Discard bottles which have chips, cracks,
                   and etched surfaces. Bottle closures must be water-tight. Before use, thoroughly cleanse
                   bottles and closures with detergent and hot water, followed by a hot water rinse to
                   remove all trace of detergent.  Then rinse them three times with laboratory-pure water.
    
          1.3      Dechlorinating Agent: The agent must be placed in the bottle when water and
                   wastewater samples containing residual chlorine are anticipated.  Add sodium thiosulfate
                   to the bottle before sterilization at a concentration of 0.1 mL of a 10% solution for each
                   125 mL sample volume. This concentration will neutralize approximately 15 mg/L of
                   residue chlorine.
    
          1.4      Chelating Agent: A chelating agent should be added to sample bottles used to collect
                   samples suspected of containing >0.01 mg/L concentrations of heavy metals such as
                   copper, nickel or zinc, etc. Add 0.3 mL of a 15% solution of ethylenediaminetetraacetic
                   acid (EDTA) tetrasodium salt, for each 125 mL sample volume prior to sterilization.
           lrThe text is taken from Part II, Section A, of the EPA publication "Microbiological Methods for
    Monitoring the Environment" EPA-600/8-78-017, December 1978.
    
                                                   1
    

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           1.5      Wrapping Bottles: Protect the tops and necks of glass stoppered bottles from
                   contamination by covering them before sterilization with aluminum foil or kraft paper.
    
           1.6      Sterilization of Bottles: Autoclave glass or heat-resistant plastic bottles at 121°C for 15
                   minutes. Alternatively, dry glassware may be sterilized in a hot oven at  170°C for not
                   less than two hours.  Ethylene oxide gas sterilization is acceptable for plastic containers
                   that are not heat-resistant. Sample bottles sterilized by gas should be stored overnight
                   before being used to allow the last traces of gas to dissipate.
    
           1.7      Plastic Bags:  The commercially available bags (Whirl-pak) (see example B in Figure 1)
                   are a practical substitute for plastic or glass samples bottles in sampling soil, sediment, or
                   biosolids. The bags are sealed in manufacture and opened only at time of sampling. The
                   manufacturer states that such bags are sterilized.
    
    2.0    Sampling Techniques
    
           Samples are collected by hand or with a sampling device if the sampling site has difficult access
           such as a bridge or bank adjacent to a surface water.
    
           2.1   Chlorinated Samples: When samples such as treated waters, chlorinated wastewaters or
                recreational waters are collected, the sample bottle must contain a dechlorinating agent (see
                section 1.3 above).
    
           2.2   Composite Sampling: In no  case should a composite sample be collected for bacteriologic
                examination.  Data from individual samples show a range of values. A composite sample
                will not display this range.  Individual results will give information about industrial process
                variations in flow and composition.  Also, one or more portions that make up a composite
                sample may contain toxic or nutritive materials and cause erroneous results.
    
           2.3   Surface Sampling by Hand: A grab sample is obtained using a sample bottle prepared as
                described in (1) above.  Identify the sampling site on the bottle label and on a field log sheet.
                Remove the bottle covering and  closure and protect from contamination.  Grasp the bottle at
                the base with one hand and plunge the bottle mouth down into the water to avoid introducing
                surface scum (Figure 2). Position the mouth of the bottle into the current away from the
                hand of the collector and, if applicable, away from the side of the sampling platform.  The
                sampling depth should be 15-30 cm (6-12 inches) below the water surface.  If the water body
                is static, an artificial current can be created, by moving the bottle horizontally in the
                direction it is pointed and away from the sampler. Tip the bottle slightly upwards to allow
                air to exit and the bottle to fill. After removal of the bottle from the stream, pour out a small
                portion of the sample to allow an air space of 2.5-5 cm (1-2 inches) above each sample for
                proper mixing of the sample  before analyses. Tightly stopper the bottle and place on ice (do
                not freeze) for transport to the laboratory.
    

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                              Figure 2. Grab sampling technique for surface waters.
    3.0   Selection of Sampling Sites and Frequency
    
          These will be described for streams, rivers, estuarine, marine, and recreational waters as well as
          domestic and industrial wastewaters.
    
          3.1  Stream Sampling: The objectives of the initial survey dictate the location, frequency and
               number of samples to be collected.
    
               3.1.1 Selection of Sampling Sites: A typical stream sampling program includes sampling
                     locations upstream of the area of concern, upstream and downstream of waste
                     discharges, upstream and downstream from tributary entrances to the river and
                     upstream of the mouth of the tributary. For more complex situations, where several
                     waste discharges are involved, sampling includes sites upstream and downstream from
                     the combined discharge area and samples taken directly from each industrial or
                     municipal waste discharge.  Using available bacteriological, chemical and discharge
                     rate data, the contribution of each pollution source can be determined.
    
               3.1.2 Small Streams: Small streams  should be sampled at background stations upstream of
                     the pollution sources and at stations downstream from pollution sources.  Additional
                     sampling sites should be located downstream to delineate the zones of pollution.
                     Avoid sampling areas where stagnation may occur (e.g., backwater of a tributary) and
                     areas located near the inside bank of a curve in the stream which may not be
                     representative of the main channel.
    

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         3.1.3 Large Streams and Rivers: Large streams are usually not well mixed laterally for long
               distances downstream from the pollution sources. Sampling sites below point source
               pollution should be established to provide desired downstream travel time and
               dispersal as determined by flow rate measurements. Particular care must be taken to
               establish the proper sampling points. Occasionally, depth samples are necessary to
               determine vertical mixing patterns.
    
    3.2  Estuarine and Marine Sampling: Sampling estuarine and marine waters requires the
         consideration of other factors in addition to those usually recognized in fresh water
         sampling. They include tidal cycles, current patterns, bottom currents and counter-currents,
         stratification, seasonal fluctuations, dispersion of discharges and multi-depth samplings.
    
         The frequency of sampling varies with the objectives. When a sampling program is started,
         it may be necessary to sample every hour around the clock to establish pollution loads and
         dispersion patterns. The sewage discharges may occur continuously or intermittently.
    
         When the sampling strategy for a survey is planned, data may be available from previous
         hydrological studies done by the Coast Guard, Corps of Engineers, National Oceanic and
         Atmospheric Administration (NOAA), U.S. Geological Survey, or university and private
         research investigations. In a survey, float studies and dye  studies are often carried out to
         determine surface and undercurrents.  Initially depth samples are taken on the bottom and at
         five feet increments between surface and bottom. A random grid pattern for selecting
         sampling sites is established statistically.
    
         3.2.1 Estuarine Sampling: When a survey is made on an estuary, samples are often taken
               from a boat, usually making an end to end traverse of the  estuary.  Another method
               involves taking samples throughout a tidal cycle, every hour or two hours from a
               bridge or from an anchored boat at a number of fixed points.
    
               In a large bay or estuary where many square miles of area are involved, a grid or
               series of stations may be necessary.  Two sets of samples  are usually taken from an
               area on a given day, one at ebb or flood slack water, and the other three hours earlier,
               or later, at the half tidal interval.  Sampling is scheduled so that the mid-sampling time
               of each run coincides with the calculated occurrence of the tidal condition.
    
               In location sampling sites, one must consider points at which tributary waters enter the
               main stream or estuary, location of shellfish beds and bathing beaches. The sampling
               stations can be adjusted  as data accumulate.  For example, if a series of stations half
               mile apart consistently show similar values,  some of these stations may be dropped
               and other stations added in areas where data shows more variability.
    
               Considerable stratification can occur between the salt water from the sea and the fresh
               water supplied by a river. It is essential when starting a survey of an unknown estuary
               to find out whether there is any marked stratification. This can be done by chloride
               determinations at different locations and depths. It is possible for stratification to
               occur in one part of an estuary and not in another.
    

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               On a flood tide, the more dense salt water pushing up into the less dense fresh river
               water will cause an overlapping with the fresh water flowing on top. A phenomenon
               called a salt water wedge can form.  As a result, stratification occurs.  If the discharge
               of pollution is in the salt water layer, the contamination will be concentrated near the
               bottom at the flood tide. The flow or velocity of the fresh water will influence the
               degree of stratification which occurs. If one is sampling only at the surface, it is
               possible that the data will not show the polluted underflowing water which was
               contaminated at the point below the  fresh water river.  Therefore, where stratification
               is suspected, samples at different depths will be needed to measure vertical
               distribution.
    
         3.2.2 Marine Sampling: In ocean studies, the environmental conditions are most diverse
               along the coast where shore, atmosphere and the surf are strong influences. The
               shallow coastal waters are particularly susceptible to daily fluctuations in temperature
               and seasonal changes.
    
               Sampling during the entire tidal cycle or during a half cycle may be required. Many
               ocean studies such as sampling over the continental shelf involve huge areas and no
               two areas of water are the  same.
    
               Selection of sampling sites and depths are most critical in marine waters.  In winter,
               cooling of coastal waters can result in water layers which approach 0°C. In summer,
               the shallow waters warm much faster than the deeper waters.  Despite the  higher
               temperature, oxygen concentrations  are higher in  shallow than in deeper waters due to
               greater water movement, surf action and photosynthetic activity from macrophytes
               and the plankton.
    
               Moving from the shallow waters to the intermediate depths, one observes  a
               moderation of these shallow water characteristics. In the deeper waters, there is a
               marked stabilization of conditions.  Water temperatures are lower and more stable.
               There is limited turbulence, little  penetration of light, sparse vegetation and the ocean
               floor is covered with a layer of silts and sediments.
    
    3.3  Recreational Waters (Bathing Beaches'): Sampling sites at bathing beaches or other
         recreational areas should include upstream or peripheral areas and locations adjacent to
         natural drains that would discharge storm water, or run-off areas draining septic wastes from
         restaurants, boat marinas, or garbage  collection areas. Samples of bathing beach water
         should be collected at locations and times of heaviest use.  Daily sampling, preferably in the
         afternoon, is the optimum frequency during the season.  Weekends  and holidays which are
         periods of highest use must be included in the sampling  program. Samples of estuarine
         bathing waters should be obtained at high tide, ebb tide and low tide in order to determine
         the cyclic water quality and deterioration that must be monitored during the swimming
         season.
    

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    3.4  Domestic and Industrial Waste Discharges: It is often necessary to sample secondary and
         tertiary wastes from municipal waste treatment plants and various industrial waste treatment
         operations. In situations where the plant treatment efficiency varies considerably, grab
         samples are collected around the clock at selected intervals for a three to five day period.  If
         it is known that the process displays little variation, fewer samples are needed. In no case
         should a composite sample be collected for bacteriological examination. The National
         Pollution Discharge Elimination System (NPDES) has established wastewater treatment
         plant effluent limits for all dischargers.  These are often based on maximum and mean
         values.  A sufficient number of samples must be collected to satisfy the permit and/or to
         provide statistically sound data and give a fair representation of the bacteriological quality of
         the discharge.
    

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                           Appendix B:
    Part II (General Operations), Sections C.3.5 (Counting Colonies)
                 and C.3.6 (Calculation of Results)
    

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                                        Counting Colonies1
    
    1.0   Counting Colonies
    
          Colonies should be counted using a fluorescent lamp with a magnifying lens. The flourescent lamp
          should be nearly perpendicular to the membrane filter. Count colonies individually, even if they
          are in contact with each other.  The technician must learn to recognize the difference between two
          or more  colonies which have grown into contact with each other and single, irregularly shaped
          colonies which sometimes develop on membrane filters.  The latter colonies are usually associated
          with a fiber or particulate material and the colonies conform to the shape and size of the fiber or
          particulates.  Colonies which have grown together almost invariably show a very fine line of
          contact.
    
    2.0   Calculation of Results
    
          2.1    Select the membrane filter with the number of colonies in the acceptable range and calculate
                count per 100 mL according to the general formula:
    
                Count per 100 mL = (No. of colonies counted/Volume  of sample filtered, in mL) x 100
    
          2.2   Counts Within the Acceptable Limits
    
                The acceptable range of colonies that are countable on  a membrane is a function of the
                method.  Different methods may have varying acceptable count ranges. All examples in this
                appemdix assume that the acceptable range of counts is between 20-80 colonies per
                membrane.
    
                For example, assume that filtration of volumes of 50, 15, 5, 1.5, and 0.5 mL produced
                colony counts of 200, 110, 40, 10, and 5, respectively.
    
                An analyst would not actually count the colonies on all filters.  By inspection the analyst
                would select the membrane filter with the acceptable range of target colonies, as defined by
                the method, and then limit the actual counting to such membranes.
    
                After selecting the best membrane filter for counting, the analyst counts colonies and applies
                the general formula as in section 2.1 above to calculate the count/100 mL.
    
          2.3   More Than One Acceptable Count
    
                2.3.1  If there are acceptable counts on replicate plates, carry counts independently to final
                      reporting units, then calculate the arithmetic mean of these counts to obtain the final
                      reporting value.
           lrThe text is largely taken from Part II, Section C, of the EPA publication "Microbiological
    Methods for Monitoring the Environment" EPA-600/8-78-017, December 1978. Some examples were
    kindly provided by Kristen Brenner, US EPA.
    
                                                   1
    

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          Example, if the counts are 24 and 36 for replicate plates of 100 mL each, then the arithmetic
          mean is calculated as follows:
    
                     (24 CFU/100 mL + 36 CFU/100 mL)
                	 = 30 CPU/100 mL
       2.3.2 If there is more than one dilution having an acceptable range of counts, independently
             carry counts to final reporting units, then average for final reported value.
    
             For example, if volumes of 100, 10, 1 and 0.1 mL produced colony counts of Too
             Numerous To Count (TNTC), 75, 30, and 1, respectively, then two volumes, 10 mL and 1
             mL, produced colonies in the acceptable counting range.
    
             Independently carry each MF count to a count per 100 mL:
    
                          75
                                        x100        =750CFU/100mL
                          10
    
                                    and
    
                          30
                                        x100       =3000 CPU/100 mL
             Calculate the arithmetic mean as in section 2.3.1 above:
    
                      (750 CFU/100 mL + 3000 CFU/100 mL)
                                                         = 1875CFU/100mL
             Report this as 1875 CFU/100 mL.
    2.4   If all MF counts are below the lower acceptable count limit, select the most nearly
          acceptable count.
    
          2.4.1 For example, sample volumes of 100, 10 and 1 mL produced colony counts of 17, 1
               and 0, respectively.
    
               Here, no colony count falls within recommended limits.  Calculate on the basis of the
               most nearly acceptable plate count, 17, and report as 17 CFU/100 mL.
    
               Note that in this case, because no calculations were done (i.e. this is the count for 100
               mL), the count is reported as  17 CFU/100 mL rather than an  "estimated count of
               17 CFU/100 mL"
    

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          2.4.2 As a second example, assume a count in which sample volumes of 10 and 1 mL
               produced colony counts of 18 and 0, respectively.
    
               Here, no colony count falls within recommended limits.  Calculate on the basis of the
               most nearly acceptable plate count,  18, and calculate as in section 2.3.2 above.
                          18
                         	     x-ioo        =180 CPU/100mL
                          10
                Report this as an estimated count of 180 CFU/100 mL.
    
    2.5   If counts from all membranes are zero, calculate using count from largest filtration volume.
    
          For example, sample volumes of 25, 10, and 2 mL produced colony counts of 0, 0, and 0,
          respectively, and no actual calculation is possible, even as an estimated report. Calculate the
          number of colonies per 100 mL that would have been reported if there had been one colony
          on the filter representing the largest filtration volume. In this example, the largest volume
          filtered was 25 mL and thus the calculation would be:
                                         x100         =4 CPU 7100 mL
                          25
          Report this as < (less than) 4 CFU/100 mL.
    
    2.6   If all membrane counts are above the upper acceptable limit, calculate count using the
          smallest volume filtered.
    
          For example, assume that the volumes 1, 0.3, and 0.01 mL produced colony counts of
          TNTC, 150, and 110 colonies, respectively. Since all colony counts are above the
          acceptable limit, use the colony count from the smallest sample volume filtered and estimate
          the count as:
    
                          110
                                 	     x-ioo     =1,100,000 CPU/100mL
                         0.01
          Report this as estimated count 1.1 x 106 CFU/100 mL
    
    2.7   If typical colonies are too numerous to count (TNTC), use upper limit count with smallest
          filtration volume.
    

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          For example, assume that the volumes 1, 0.3, and 0.01 mL all produced too many typical
          colonies, and that the laboratory bench record indicated TNTC.
    
          Use the upper acceptable count for the method (80 colonies in this example) as the basis of
          calculation with the smallest filtration volume and estimate the count as:
                          80
                                 	     x 100       = 800,000 CPU /100 mL
                          0.01
          Report this as > (greater than) 8 x 105 CPU/100 mL
    
    2.8   If colonies are both above and below the upper and lower acceptable limits (i.e., no counts
          are within the acceptable limits), select the most nearly acceptable count.
    
          2.8.1  For example, sample volumes of 100, 10 and 1 mL produced colony counts of 84, 8
                and 0, respectively.
    
                Here, no colony count falls within recommended limits. Calculate on the basis of the
                most nearly acceptable plate count,  84, and report as 84 CFU/100 mL.
    
                Note that in this case, because no calculations were done (i.e. this is the count for 100
                mL), the count is reported as 84 CFU/100 mL rather than an "estimated count of
                84 CFU/100 mL"
    
          2.8.2  As a second example, assume a count in which sample volumes of 100, 10 and 1 mL
                produced colony counts of 98, 18, and 0, respectively.
    
                Here, no colony count falls within recommended limits. Calculate on the basis of the
                most nearly acceptable plate count,  18, and calculate as in section 2.3.2 above.
    
                           18
                  	     x-ioo        =180 CPU/100mL
                          10
    
                Report this as estimated count 180 CFU/100 mL.
    
    2.9   If there is no result because of a confluent growth, > 200 atypical colonies (TNTC), lab
          accident, etc., report as No Data and specify the reason.
    

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