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Specific equipment and supplies are needed for collection and analysis of indicator bacteria by membrane filtration procedures. The equipment listed in table 7.1-2 should be sufficient to begin analysis of fecal indicator bacteria using these procedures.

Table 7.1-2

Equipment for collection and analysis of bacterial samples must be clean and sterile (table 7.1-3). Wrap equipment in kraft paper, autoclavable bags, or aluminum foil. Sterilize and store the equipment in a clean area. Resterilize equipment if foil, bag, or kraft paper is torn.

Add sodium thiosulfate (Na2S2O3) to sample bottles before sterilization if the water to be collected contains residual chlorine or other halogens added for disinfection. Residual chlorine can be found in samples collected from sources such as treated potable-water taps, in effluents, and surface-water samples collected from the mixing zones of wastewater-treatment plants. A 10-percent solution of Na2S2O3 is prepared in the following manner. In a volumetric flask, dissolve 100 g Na2S2O3 into 500 mL of deionized or distilled water; stir until dissolved, and fill flask to 1,000 mL (Bordner and Winter, 1978, p. 6; American Public Health Association and others, 1992, p. 9-18). Add 0.1 mL of 10-percent Na2S2O3 solution for every 100 mL of sample. Keep Na2S2O3 refrigerated and in a dark bottle; after 6 months prepare a fresh solution.

Add ethylenediaminetetraacetic acid (EDTA) to sample bottles when water to be collected contains trace elements such as copper, nickel, and zinc at concentrations greater than 10 mg/L (Britton and Greeson, 1989, p. 5-6; Bordner and Winter, 1978, p. 6; American Public Health Association and others, 1992, p. 9-18). A 15-percent solution of EDTA is prepared by dissolving 372 mg in 1,000 mL of distilled or deionized water. Before sterilization, add 0.3 mL of the EDTA solution per 100 mL of sample to sample bottles. EDTA can be combined with the Na2S2O3 solution in the sample bottle before sterilization.

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Sterilize the filtration apparatus between sites or for each sample collected at the same site at different times. Autoclaving is the preferred method of sterilization. Use only autoclaves that have temperature, pressure, and liquid- and dry-utensil-cycle controls. Steam sterilizers and vertical autoclaves are not recommended because the temperature cannot be held constant.

Table 7.1-3

Take care to ensure that materials to be autoclaved, such as tubing and containers, are thermally stable. Polymers (such as polycarbonate, polypropylene, polyallomer, and polymethylpentene) and Teflons and Tefzel (such as perfluoroalkyoxy-polymers or PFA, ethylenetetrafluoro-ethylene or ETFE, fluorinated ethylene propylene or FEP, and polytetrafluoroethylene polymers or PTFE) can be autoclaved. Each has different thermal characteristics and tolerances to repeated autoclaving.

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Only Millipore Hydrosol field filtration units are designed to be flame sterilized with methanol. Formaldehyde gas, a by-product of methanol combustion, kills all microorganisms in the unit. The following sterilization procedure is acceptable for the Hydrosol unit in field situations where other sterilization techniques are not practicable (Millipore, 1973, p. 48-49).


  1. Remove the stainless steel flask from the base of the filter-holder assembly.

  2. Saturate the asbestos ring (wick) around the base assembly with methanol.

  3. Ignite the methanol on the asbestos wick and allow to burn for 30 seconds.

  4. Invert the stainless steel flask over the funnel and the burning asbestos ring, and seat the flask on the base of the filter-holder assembly. Leave the flask in place for 15 minutes. Before filtering the next sample, rinse flask and funnel thoroughly with sterile buffered water to remove all residues of formaldehyde.

  5. Repeat sterilization procedure before processing the next sample.

Quality control. Use a sterilization indicator, such as autoclave tape or StreamClox, to help determine whether adequate temperature and pressure have been attained during autoclaving. Keep a log book of equipment and include quality-control procedures with the date, the results, and the name of the analyst. The 18th edition of Standard Methods for the Examination of Water and Wastewater (American Public Health Association and others, 1992, p. 9-9, Table 9020:III) contains specifications for the length of time and temperature for autoclave sterilization of various media, apparatus, and cultures to be discarded. Overloading the autoclave with equipment or materials will result in incomplete sterilization. Make sure that steam can circulate around all equipment, utensils, and bottles. Cultures of bacteria to be discarded must be autoclaved for at least one-half hour.

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Maintainer: Office of Water Quality
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Last Modified: 12Dec03 imc