&EPA
   United States
   Environmental Protection
   Agency
   Method 1603: Escherichia coli (E. coli) in
   Water by Membrane Filtration Using
   Modified membrane-Thermotolerant
   Escherichia coli Agar (Modified mTEC)
   July 2006

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U.S. Environmental Protection Agency
      Office of Water (4303T)
   1200 Pennsylvania Avenue, NW
      Washington, DC 20460
        EPA-821-R-06-011

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                                 Acknowledgments

The following laboratories are gratefully acknowledged for their participation in the validation of this
method in wastewater effluents:

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

Volunteer Verification Laboratory
•  City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve
   Espineda, and Zora Bahariance

Volunteer Participant Laboratories
•  City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, loannice Lee, Genevieve
   Espineda, and Zora Bahariance

   County Sanitation Districts of Los Angeles County (JWPCP): Kathy Walker, Michele Padilla, and
   Albert Soof

•  County Sanitation Districts of Los Angeles County (SJC): Shawn Thompson and Julie Millenbach

•  Environmental Associates (EA): Susan Boutros and John Chandler

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

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

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

•  North Shore Sanitation District (NSSD): Robert Flood

•  Texas A&M University: Suresh Pillai and Reema Singh

•  University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord

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

   Utah Department of Health: Sanwat Chaudhuri and Devon Cole

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                                        Disclaimer

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and
approved this report for publication. The Office of Science and Technology directed, managed, and
reviewed the work of CSC Biology Studies Group in preparing this report. Neither the United States
Government nor any of its employees, contractors, or their employees make any warranty, expressed or
implied, or assumes any legal liability or responsibility for any third party's use of or the results of such
use of any information, apparatus, product, or process discussed in this report, or represents that its use by
such party would not infringe on privately owned rights. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.

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

Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S.  EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov
202-566-1075
202-566-1053 (facsimile)

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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 	  9




9.0    Quality Control	  9




10.0   Calibration and Standardization  	  14




11.0   Procedure  	  15




12.0   Verification Procedure  	  16




13.0   Data Analysis and Calculations  	  17




14.0   Sample Spiking Procedure  	  17




15.0   Method Performance	  22




16.0   Pollution Prevention	  24




17.0   Waste Management	  24




18.0   References	  25
                                              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 1603: Escherichia coll (E. coll) in Water by Membrane
 Filtration Using Modified membrane- Thermotolerant Escherichia coll
                              Agar (modified mTEC)

                                    August 2006
1.0    Scope and Application

1.1     Method 1603 describes a membrane filter (MF) procedure for the detection and enumeration of
       Escherichia coli bacteria in ambient waters and disinfected wastewaters. This method is a single-
       step modification of EPA Method 1103.1 (mTEC). Unlike the mTEC media method, it does not
       require the transfer of the membrane filter to another substrate.  The modified medium contains a
       chromogen (5-bromo-6-chloro-3-indolyl-p-D-glucuronide), which is catabolized to glucuronic
       acid and a red- or magenta-colored compound by E. coli that produces the enzyme P-D-
       glucuronidase. The apparatus and equipment, and sampling, filtration, and verification
       procedures for the modified mTEC method are identical to those of the original mTEC method.

1.2     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.

1.3     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).


1.4     For method application please refer to Title 40 Code of Federal Regulations Part 136
       (40 CFR Part 13 6).
2.0    Summary of Method

2.1     Method 1603 provides a direct count of E. coli in ambient water or wastewater based on the
       development of colonies that grow on the surface of a membrane filter. A sample is filtered
       through the membrane, which retains the bacteria. After filtration, the membrane is placed on a
       selective and differential medium, modified mTEC agar, 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. The target colonies on modified mTEC agar are red or magenta in color after the
       incubation period.
                                                                                July 2006

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Method 1603
3.0   Definitions

3.1    In Method 1603, E. coll are those bacteria which produce red or magenta colonies on the
       modified mTEC agar.
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 safety issues associated with its use. The laboratory is
       responsible for maintaining a safe work environment and a current awareness file of OSHA
       regulations regarding the safe handling of the chemicals specified in this method. A reference file
       containing material safety data sheets (MSDSs) should be available to all personnel involved in
       these 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   Filter flask, vacuum, usually 1 L, with appropriate tubing

6.11   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

July 2006                                     2

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                                                                                    Method 1603
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
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, 9x50 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   Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic
       loops
6.22   Sterile disposable applicator sticks
6.23   Incubator maintained at 35°C ± 0.5°C, with approximately 90% humidity if loose-lidded petri
       dishes are used
6.24   Waterbath maintained at 44.5°C ± 0.2°C
6.25   Waterbath maintained at 50°C for tempering agar
6.26   Test tubes, 20 x 150 mm, borosilicate glass or plastic
6.27   Test tubes, 10 x 75 mm, borosilicate glass (durham tubes)
6.28   Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes
6.29   Test tubes screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size
6.30   Whirl-Pak® bags or equivalent
6.31   Autoclave or steam sterilizer capable of achieving 121°C [15 Ib pressure per square inch (PSI)]
       for 15 minutes
6.32   Filter paper
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 on Analytical Reagents
       of the American Chemical Society (Reference 18.3). The agar used in preparation of 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 reagent water: Reagent-grade water conforming to specifications in: Standard Methods
       for the Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part
       136 or 141, as applicable), Section 9020 (Reference 18.4).
                                                                                       July 2006

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Method 1603
7.4    Phosphate buffered saline (PBS)

       7.4.1   Composition:

                 Monosodium phosphate (NaH2PO4)                        0.58 g
                 Disodium phosphate (Na^PC^)                           2.5 g
                 Sodium chloride                                         8.5 g
                 Reagent-grade water                                     l.OL


       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 1603 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 7.5) may be substituted for PBS as
               a sample diluent and filtration rinse buffer.

7.5    Phosphate buffered dilution water (Reference 18.5)

       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 (MgCl2) solution: Add 38 g anhydrous MgCl2
               or 81.1 g magnesium chloride hexahydrate (MgCl2oc6H2O) 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 and/or for use as rinse buffer.  Autoclave at 121 °C (15 PSI) for 15
               minutes. Final pH should be 7.0 ± 0.2.
July 2006

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                                                                                   Method 1603
7.6    Modified mTEC agar
    7.6.1   Composition:

             Protease peptone #3                                         5.0g
             Yeast extract                                               3.0g
             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
             Chromogen (5-bromo-6-chloro-3-indolyl-p-D-glucuronide)      0.5 g
             Agar                                                      15.0g
             Reagent-grade water                                         l.OL

       7.6.2  Add dry ingredients to 1 L of reagent-grade water, mix thoroughly, heat to dissolve
              completely. Autoclave at 121°C (15 PSI) for 15 minutes, and cool in a 50°C waterbath;
              adjust pH to 7.3 ± 0.2. with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide. Pour the
              medium into each 9x50 mm culture dish to a 4-5 mm depth (approximately 4-6 mL),
              and allow to solidify. Store in a refrigerator.

7.7    Nutrient agar

       7.7.1  Composition:

              Peptone                                                  5.0g
              Beef extract                                              3.0g
              Agar                                                    15.Og
              Reagent-grade water                                       l.OL
       7.7.2  Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
              completely. Dispense into 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.
                                                                                      July 2006

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Method 1603
7.8    Tryptic/trypticase soy broth
       7.8.1   Composition:

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

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

7.9    Simmons citrate agar

       7.9.1   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.Og
                Reagent-grade water                                      l.OL

       7.9.2   Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve
               completely. Dispense into screw-cap tubes, and autoclave at 121°C (15 PSI) for 15
               minutes. Cool the tubes and slant. Final pH should be 6.9 ± 0.2.
7.10   Tryptone water

       7.10.1  Composition:

                Tryptone                                               lO.Og
                Sodium chloride                                          5.0g
                Reagent-grade water                                      l.OL
       7.10.2 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.
July 2006

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                                                                                   Method 1603
7.11   EC broth

       7.11.1  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                                      1.0 L
       7.11.2 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.

7.12   Oxidase reagent

       7.12.1 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 and are recommended for
              colony verification (Section 12.0).

7.13   Kovacs indole reagent

       7.13.1 Composition:

                p-dimethylaminobenzaldehyde                           10.0 g
                Amy 1 or isoamyl alcohol                               150.0mL
                Concentrated (12 M) hydrochloric acid                   50.0 mL
       7.13.2  Preparation: Dissolve p-dimethylaminobenzaldehyde in alcohol, slowly add hydrochloric
               acid, and mix.
                                                                                      July 2006

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Method 1603
7.14   Tryptic soy agar (TSA)

       7.14.1 Composition:

                Pancreatic digest of casein                                15.0 g
                Enzymatic digest of soybean meal                          5.0 g
                Sodium chloride                                          5.0g
                Agar                                                   15. Og
                Reagent- grade water                                     1.0 L

       7.14.2 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.

7.15   Lauryl tryptose broth (LTB)

       7.15.1 Composition:

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

       7.15.1 Preparation: Add reagents (Section 7.15.1) to 1 L of reagent-grade water, heat with
              frequent mixing, and boil for one minute to dissolve completely.  Autoclave at 121°C (15
              PSI) for 15 minutes.  Final pH should be 6.8 ± 0.2.

7.16   Control cultures

       7.16.1 Positive control and/or spiking organism (either of the following are acceptable):
              •   Stock cultures ofEscherichia coli (E. coli} ATCC #11775
              •   E. coli ATCC #11775 BioBalls (BTF Pty, Sydney, Australia)

       7.16.2 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
July 2006

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                                                                                  Method 1603
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 in 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 1603 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. Additional recommendations for QA and quality control (QC) procedures for
       microbiological laboratories are provided in Reference 18.5.

9.2    The minimum analytical QC requirements for the analysis of samples using Method 1603 include
       an initial demonstration of laboratory capability through performance of the initial precision and
       recovery (IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through
       performance of the ongoing precision and recovery (OPR) analysis (Section 9.4) and matrix spike
       (MS) analysis (Section 9.5, disinfected wastewater only), and the routine analysis of positive and
       negative controls (Section 9.6), filter sterility checks (Section 9.8), method blanks (Section 9.9),
       and media sterility checks (Section 9.11). For the IPR, OPR and MS analyses, it is necessary to
       spike samples with either laboratory-prepared spiking suspensions or BioBalls as described in
       Section 14.
                                                                                     July 2006

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Method 1603
       Note: Performance criteria for Method 1603 are based on the results of the interlaboratory
       validation of Method 1603 in PBS and disinfected wastewater matrices. Although the matrix
       spike recovery criteria (Section 9.5, Table 2) pertain only to disinfected wastewaters, the IPR
       (Section 9.3) and OPR (Section 9.4) recovery criteria (Table 1) are valid method performance
       criteria that should be met, regardless of the matrix being evaluated.

9.3    Initial precision and recovery (IPR)—The IPR analyses are used to demonstrate acceptable
       method performance (recovery and precision) and should be performed by each laboratory before
       the method is used for monitoring field samples. EPA recommends but does not require that an
       IPR be performed by each analyst. IPR samples should be accompanied by an acceptable method
       blank (Section 9.9) and appropriate media sterility checks (Section 9.11). The IPR analyses are
       performed as follows:

       9.3.1   Prepare four, 100-mL samples of PBS and spike each sample with E.  coli 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. coli 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 14.2.2 or  14.3.4 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. coli meet acceptance criteria, system
               performance is  acceptable and analysis of field samples may begin. If the mean recovery
               or the RSD fall outside of the required range for recovery, system performance  is
               unacceptable.  In this event, identify the problem by evaluating each step of the analytical
               process, media, reagents, and controls, correct the problem and repeat the IPR analyses.
Table 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
46% -119%
36%
38% -127%
BioBall™
acceptance criteria
detect -144%
61%
detect -144%
July 2006
10

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                                                                                   Method 1603
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 must be
       accompanied by an acceptable method blank (Section 9.9) and appropriate media sterility checks
       (Section 9.11). 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
              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 laboratory-prepared spiking suspensions
              or BioBalls, respectively.

       9.4.3  Compare the  OPR result (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, 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
              1603 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 - 2 sr to R + 2sr.

9.5    Matrix spikes (MS)—MS analysis  are performed to determine the effect of a particular matrix
       on E. coll recoveries.  The laboratory should analyze one MS sample when disinfected
       wastewater samples are first received from a source from which the laboratory has not previously
       analyzed samples. Subsequently, 5% of field samples (1 per 20) from a given disinfected
       wastewater source should include a  MS sample. MS samples must be accompanied  by the
       analysis of an unspiked field sample sequentially collected from the same sampling site, an
       acceptable method blank (Section 9.9), and appropriate media sterility checks (Section 9.11).
       When possible, MS analyses should also be accompanied by an OPR sample (Section 9.4), using
       the same spiking procedure  (laboratory-prepared spiking suspension or BioBalls). The MS
       analysis is performed as follows:

       9.5.1  Prepare two,  100-mL field samples that were  sequentially collected from the same site.
              One sample will remain unspiked and will be analyzed to determine the background or
              ambient concentration of E.  coll for calculating MS recoveries (Section 9.5.3). The other
              sample will serve as the MS sample and will be spiked with E. coll ATCC #11775
              according to the spiking procedure in Section 14.
                                              11                                      July 2006

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Method 1603
       9.5.2   Select sample volumes based on previous analytical results or anticipated levels of
               E. coli in the field sample in order to achieve the recommended target range of E. coli
               (20-80 CPU, including spike) per filter. If the laboratory is not familiar with the matrix
               being analyzed, it is recommended that a minimum of three dilutions be analyzed to
               ensure that a countable plate is obtained for the MS and associated unspiked sample. If
               possible, 100-mL of sample should be analyzed.

       9.5.3   Spike the MS sample volume(s) with a laboratory-prepared suspension as described in
               Section 14.2 or with BioBalls as described in Section 14.3. Immediately filter and
               process the unspiked and spiked field samples according to the procedures in Section 11.

               Note: When analyzing smaller sample volumes (e.g, <20 mL), 20-30 mL of PBS should
               be added to the funnel or an aliquot of sample should be dispensed into a 20-30  mL
               dilution blank prior to filtration. This will allow even distribution of the sample on the
               membrane.

       9.5.4   For the MS  sample, calculate the number of E. coli (CFU/100 mL) according to Section
               13 and adjust the colony counts based on any background E.  coli observed in the
               unspiked matrix sample.

       9.5.5   Calculate the percent recovery  (R) for the MS sample (adjusted based on ambient E. coli
               in the unspiked sample) using the appropriate equation in Section 14.2.2 or 14.3.4 for
               samples  spiked with laboratory-prepared spiking suspensions or BioBalls, respectively.

       9.5.6   Compare the MS result (percent recovery) with the appropriate method performance
               criteria in Table 2, below. If the MS recovery meets the acceptance criteria, system
               performance is  acceptable and  analysis of field samples from this disinfected wastewater
               source may continue.  If the MS recovery is unacceptable and the OPR sample result
               associated with this batch of samples  is acceptable, a matrix interference may be causing
               the poor results. If the MS  recovery is unacceptable, all associated field data should be
               flagged.

       9.5.7   Acceptance criteria for MS recovery (Table 2) are based on data from spiked disinfected
               wastewater matrices and are not appropriate for use with other matrices (e.g., ambient
               recreational waters).
Table 2.    Matrix Spike Precision and Recovery Acceptance Criteria
Performance test
Percent recovery for MS
Lab-prepared acceptance
criteria
12% -149%
BioBall™ acceptance
criteria
17% -117%
       9.5.8   Laboratories should record and maintain a control chart comparing MS recoveries for all
               matrices to batch-specific and cumulative OPR sample results analyzed using Method
               1603.  These comparisons should help laboratories recognize matrix effects on method
               recovery and may also help to recognize inconsistent or sporadic matrix effects from a
               particular source.
July 2006                                      12

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                                                                                     Method 1603
9.6    Culture Controls

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

               9.6.1.1     Negative controls are conducted by 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).

               9.6.1.2     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.

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

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

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

       9.6.3   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 3.
Table 3.    Verification Controls
Medium
Cytochrome oxidase reagent
Kovac's indole reagent
Simmons citrate agar
EC broth (44.5°C ± 0.2°C)
Positive Control
E. faecalis
E. coli
E. aerogenes
E. coli
Negative Control
E. coli
E. aerogenes
E. coli
E. aerogenes
9.7    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 12.0.
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Method 1603
9.8    Filter sterility check—Place at least one membrane filter per lot of filters 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.

9.9    Method blank—Filter a 50-mL volume of sterile PBS or phosphate-buffered dilution water,
       place the filter on a modified 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.

9.10   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 PBS buffer and filtration assembly.
9.11   Media sterility check—The laboratory should test media sterility by incubating one unit (tube
       or plate) from each batch of medium (TSA, modified mTEC, 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.

9.12   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.


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 the modified mTEC agar as directed in Section 7.6.

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.



July 2006                                      14

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                                                                                   Method 1603
11.5   Select sample volumes based on previous knowledge of the pollution level, 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 (20-60 enterococci colonies) is obtained.

11.6   Smaller sample sizes 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 may 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 buffered
       rinse water.  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 modified 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.

       Note: 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 plates from the waterbath, count and record the number of red or
       magenta colonies with the aid of an illuminated lens with a 2-5X magnification or a  stereoscopic
       microscope (See Photo  1).
                Photo 1. E. coli colonies on modified mTEC agar are red to magenta.
                                              15
July 2006

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Method 1603
12.0  Verification Procedure

12.1   Red or magenta colonies 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.

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.

       12.4.1  Incubate the Simmons citrate agar for 4 days at 35°C ± 2°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 broth for 18-24 hours at 35°C ± 2°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.
July 2006                                      16

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                                                                                 Method 1603
13.0  Data Analysis and Calculations

       Use the following general rules to calculate the E. coll count (CPU) per 100 mL of sample:

13.1   If possible, select a membrane filter with 20-80 magenta or red colonies, 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 Microbiological Methods for Monitoring the Environment: Water
       and Wastes, Part II, Sections C.3.5 and C.3.6 (see Appendix B).

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


14.0  Sample Spiking Procedure

14.1   Method 1603 QC requirements (Section 9.0) include the preparation and analysis of spiked
       reference (PBS) and matrix samples in order to monitor initial and ongoing method performance.
       For the IPR (Section 9.3), OPR (Section 9.4), and MS (Section 9.5) 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 of laboratory-prepared spikes

              14.2.1.1   Stock Culture.  Prepare a stock culture by inoculating a TSA slant (or other
                         non-selective media) with Escherichia coli 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 (Section 14.2.1.2) 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.

                                              17                                    July 2006

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Method 1603
                          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.

                          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 using laboratory-prepared suspensions

               14.2.2.1    Add 0.3 mL of the spiking suspension dilution "D" to 100 mL of PBS or
                          appropriate volume of sample 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.  Filter the spiked sample and analyze
                          the filter according to the procedures in Section 11.

       14.2.3  Enumeration of laboratory-prepared 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.


July 2006                                      18

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                                                                                   Method 1603
               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. coll (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.

       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 4, below.
                . coli undilutedspike = (CFU1 + CFU2 + ...+ CFUn) / (VI + V2 + ... + Vn)
d spik
  ike = E. coll (CPU / mL) in undiluted spiking suspension
              E. coli u

              Where,

              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 of 30 to 300 CPU
                         per plate

              Note: The example calculated numbers provided in the tables below have been rounded at
              the end of each step for simplification purposes.  Generally, rounding should only occur
              after the  final calculation.

Table 4.    Example Calculations of E. coli Spiking Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
TSA plates in Section 2.2.5
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 undj|Uted 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|Utedspike is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
                                               19
                                                             July 2006

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Method 1603
               14.2.4.2   Calculate true concentration (CPU / 100 mL) of spiked E. coll (T splked£ coU)
                          according to the following equation. Example calculations are provided in
                          Table 5, below.
                            spiked £. coli
                                     = (E. co//
                      undiluted spike
ike) X (" spil
spiked per 100 mL sample/
               Where,

               T
                 spiked E. coli

               E. coli
                     undiluted spike
               * spiked per 100 mL sample
               Number of spiked E. coli (CPU / 100 mL)

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

               mL of undiluted spiking suspension per 100 mL sample
Table 5.    Example Calculations of Spiked E. coli
FP
^w undiluted spike
9.6x107CFU/mL
2.8x107CFU/mL
" spiked per 100 mL sample
3.0 X10-7mL per 100mL of
sample
3.0 X10-7mL per 100mL of
sample
Tspiked E. coli
(9.6 x 1 07 CPU / mL) x (3.0 x 1 0'7 mL / 1 00 mL) =
28.8 CPU /1 00 mL
(2.8 x 107 CPU / mL) x (3.0 x 10'7 mL / 100 mL) =
8.4 CPU/ 100 mL
               14.2.4.3   Calculate percent recovery (R) of spiked E. coli (CPU /100 mL) according
                          to the following equation. Example calculations are provided in Table 6,
                          below.
                                R =  100 x
                                                   (Ns - 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 (Section 14.2.4.2)
July 2006
                     20

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                                                                                Method 1603
Table 6.    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 7, below.
                              R  =  100 x
                                                (Ns - Nu)
              Where,

              R  =

              Ns =

              Nu =

              T  =
Percent  recovery

E. coll (CPU / 100 mL) in the spiked sample (Section 13)

E. coll (CPU / 100 mL) in the unspiked sample (Section 13)

True spiked E. coll (CPU / 100 mL) in spiked sample based on the lot mean
value provided by manufacturer
Table 7.    Example Percent Recovery Calculations
Ns(CFU/100mL)
24
36
Nu(CFU/100ml_)
<1
10
T(CFU/100ml_)
32
32
Percent recovery (R)
100 x (24- 1)732 = 72%
100 x (36-10)732 = 81%
                                             21
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Method 1603
15.0  Method Performance

15.1   Performance Characteristics (Reference 18.6)

       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 modified
              mTEC method precision was found to be fairly representative of what would be expected
              from counts with a Poisson distribution.

       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 modified mTEC method has
              been reported to be -2% of the true value.

       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 modified mTEC medium averaged 6% for marine and fresh
              water samples.  Five percent of the E. coll colonies observed gave a false negative
              reaction.

       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. coll
              on modified mTEC medium has been reported as 80 colonies per filter.

15.2   Interlaboratory validation of Method 1603 in disinfected wastewater (Reference 18.2)

       15.2.1 Eight volunteer laboratories, an E. coll verification laboratory, and a research laboratory
              participated in the U.S. Environmental Protection Agency's (EPA's) interlaboratory
              validation study of EPA Method 1603. 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. A detailed description of
              the of the study and results are provided in the validation study report (Reference 18.2).
              Results submitted by laboratories were validated using a standardized data review process
              to confirm that results were generated in accordance with study-specific instructions and
              the September 2002 version of EPA Method 1603.

       15.2.2 Recovery - Method 1603 was characterized by mean laboratory-specific recoveries of E.
              coll from disinfected wastewater samples spiked with  laboratory-prepared spikes ranging
              from 47.8% to 106%, with an overall mean recovery of 80.7%. For PBS samples spiked
              with laboratory spiking suspensions,  mean laboratory-specific recoveries ranged from
              70.7% to 109.7%, with an overall mean recovery of 82.9%.

       15.2.3 Precision - Method 1603 was characterized by laboratory-specific relative standard
              deviations (RSDs) from disinfected wastewater samples spiked with laboratory-prepared
              spikes ranging from 6.1% to  51.4%, with an overall pooled, within-laboratory RSD of
              25.9%. For PBS samples spiked with laboratory-prepared spiking suspensions,
              laboratory-specific RSDs ranged from 7.7% to 29.6%, with an overall pooled, within-
              laboratory RSD of 19.6%.
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                                                                                       Method 1603
        15.2.4 False positive rates - Method 1603 laboratory-specific false positive rates determined from
               all unspiked disinfected and secondary results combined, ranged from 0% - 6.7%.  For
               secondary wastewater (excluding disinfected results), only one of 41 typical colonies
               submitted to verification was non-E1. coli, resulting in a false positive rate of 2.4%. For
               disinfected wastewater (excluding secondary results), only one of 67 typical colonies
               submitted to verification was non-E1. coli, resulting in a false positive rate of 1.5%. Since
               all 785 typical colonies observed during the study could not be submitted to confirmation,
               the percent of total colonies that would have resulted in a false positive result was
               estimated (see Table 6, Reference 18.2).  It is estimated that 0.6% and 1.4% of the total
               colonies would have resulted in a false positive for disinfected wastewater and secondary
               wastewater, respectively.

        15.2.5 False negative rates - Method 1603 laboratory-specific false negative rates determined
               from all unspiked disinfected and secondary results combined, also ranged from 0% -
               6.7%.  For secondary wastewater (excluding disinfected results), two of 33 atypical
               colonies submitted to verification were identified as E. coli, resulting in a false negative
               rate of 6.1%.  For disinfected wastewater (excluding secondary results), three of 75
               atypical colonies submitted to verification were identified as E. coli, resulting in a false
               negative rate of 4.0%. Since all 732 atypical colonies observed during the study could not
               be submitted to confirmation, the percent of total colonies that would have resulted in a
               false negative result was estimated. It is  estimated that 2.5% and 2.6% of the total
               colonies would have resulted in a false negative for disinfected wastewater and  secondary
               wastewater, respectively. The false positive and negative  assessments are provided in
               Table 8.
Table 8.   False Positive and False Negative Assessment for Unspiked Disinfected and Unspiked
           Secondary Wastewater Effluents


Matrix

Disinfected
Secondary
Disinfected &
Secondary
Total colonies

Typical

163
622
785

Atypical

263
469
732
False positive (FP) assessment

Typical
colonies
submitted

67
41
108

No. FP
colonies

1
1
2

FP
confirmation
rate (%) a

1.5%
2.4%
1.9%
Estimated
% of total
colonies
that would
have been
aFPb
0.6%
1.4%
1.0%
False negative (FN) assessment

Atypical
colonies
submitted

75
33
108

No. FN
colonies

3
2
5

FN
confirmation
rate (%) c

4.0%
6.1%
4.6%
Estimated
% of total
colonies
that would
have been a
FNd
2.5%
2.6%
2.2%
    False positive confirmation rate = number of false positive colonies / number of typical colonies submitted
    Percent of total colonies estimated to be false positives = [(total typical colonies * FP confirmation rate) / (total
    number of typical and atypical colonies observed)] x 100; e.g., [(622 x(i/4i))/(622+469)] x 100 = 1.4%
    False negative confirmation rate = number of false negative colonies / number of atypical colonies submitted
    Percent of total colonies estimated to be false negatives = [(total atypical colonies x FN  confirmation rate) / (total
    number of typical and atypical colonies observed)] x 100; e.g., [(469 x(2/33))/(622+469)] x 100 = 2.6%
                                                 23
July 2006

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Method 1603
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. coll attached or
       contained must be sterilized prior to disposal.

17.3   Samples preserved with HC1 to pH <2 are hazardous and must be neutralized before being
       disposed, or must be handled as hazardous waste.

17.4   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.
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 1603 (modified mTEC)
       forE. coli in Wastesater Effluent. EPA-821-R-04-009. December 2004.

18.3   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, seeAnalaR Standards for Laboratory Chemicals, BDH, Poole, Dorset, UK and the United
       States Pharmacopeia.

18.4   APHA. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition.
       American Public Health Association, Washington D.C.

18.5   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.

18.6   Smith, B. G. and A. P. Dufour. 1997. A Modified mTEC Medium for Monitoring Recreational
       Waters. American Society for Microbiology General Meeting, Miami Beach, FL, May 1997.
July 2006                                     24

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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.
                          k;     A
               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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