Section 465.350  General Laboratory Practices

a)         The following standards shall apply to sterilization procedures:

1)         Autoclaving of the following items shall be carried out at 121º + 1º C for the durations specified below:

2)         Membrane filters and pads and all media shall be removed from the autoclave immediately after completion of the sterilization cycle.

3)         The maximum elapsed time for exposure of carbohydrate-containing media to any heat (from the time of closing the loaded autoclave to unloading) shall be 45 minutes.

4)         Membrane filter assemblies shall be autoclaved between each sample filtration series.  A filtration series ends when 30 minutes or more have elapsed between sample filtrations.  A UV sterilizer or boiling water may be used on membrane filter assemblies for at least 2 minutes to prevent bacterial carry-over between sample filtrations, but shall not be used as a substitute for autoclaving between sample filtration series.

5)         Dried glassware to be sterilized in a hot-air sterilizing oven shall be kept at 175º + 5º C for at least 2 hours.

6)         Empty sample containers shall be moistened with several drops of distilled water before autoclaving to prevent an "airlock" sterilization failure.

b)         Laboratory pure water, which may be distilled, deionized, or other processed water, shall meet the standards set forth in Section 465.380.  Only water determined to be laboratory pure water shall be used for performing bacteriological analyses.

c)         Rinse and dilution water shall be prepared in the following manner:

1)         A stock phosphate buffer solution of potassium dihydrogen phosphate (KH₂PO₄) and a magnesium chloride solution shall be prepared as specified in "Standard Methods for the Examination of Water and Wastewater."

2)         The phosphate buffer solution and magnesium chloride solution shall be autoclaved or filter sterilized, labeled, dated, and stored at 1º to 4.4º C.

3)         The stored stock phosphate buffer solution and magnesium chloride solution shall be free of turbidity.

4)         Rinse and dilution water shall be prepared by adding 1.25 ml of stock phosphate buffer solution and 5.0 ml of magnesium chloride solution per liter of laboratory pure water.

5)         Check each batch of prepared or each lot of commercial dilution/rinse water for sterility by adding 50 ml of water to 50 ml of double-strength, nonselective broth.  Incubate at 35.0º + 0.5º C for 24 hours and check for growth.

6)         Check each batch of prepared or each lot of commercial dilution water blanks for pH; pH shall be 7.2 + 0.2.

7)         Check 1 of 25 dilution water blanks per batch of prepared or lot of commercial dilution water blanks for volume using a Class A graduated cylinder or a MacCaffrey flask. Volume must be 99 ml + 2 ml.

d)         The following minimum standards shall be met for storing and preparing media:

1)         Laboratories shall use commercial dehydrated media or commercially manufactured prepared media for routine bacteriological procedures.

2)         All media shall be prepared according to the media specifications of "Standard Methods for the Examination of Water and Wastewater."

3)         Dehydrated media containers shall be kept tightly closed and stored in a cool, dry location.  Discolored or caked dehydrated media shall not be used.

4)         All water used shall be laboratory pure water.

5)         Dissolution of the media shall be completed before dispensing to culture tubes or bottles.

6)         Membrane filter broths and agar media shall be heated in a boiling water bath or, if constantly attended, a hot plate with a stir bar, until completely dissolved.  The medium shall not be boiled.  Denatured ethanol shall not be used.

7)         Membrane filter broths shall be stored and refrigerated no longer than 96 hours prior to use. Membrane filter agar media shall be stored in a refrigerator, and used within 2 weeks after preparation.  Prepared plates shall be stored in sealed plastic bags or containers to minimize evaporation.

8)         Multiple Tube Fermentation (MTF) media, when prepared in tubes with loose-fitting caps, shall be used within one week after preparation.  If MTF media are refrigerated after sterilization, they shall be incubated overnight at 35º C to confirm usability.  Tubes of MTF media showing growth or gas bubbles shall be discarded.  Refrigerated M Endo agar LES shall be used within 2 weeks after refrigeration or discarded.

9)         MTF media in screw cap containers may be held up to 3 months, provided the media are stored in the dark and evaporation does not exceed 1.0 ml per 10 ml total volume.

10)        Heterotrophic plate count agar in screw cap containers shall be stored and refrigerated no longer than 3 months.

11)        Commercially prepared media may be used, provided the media has been prepared in a microbiological water laboratory certified by the regulatory agency having responsibility for laboratory certification in the states where media is manufactured.  The laboratory using the media shall record the date received, type of medium, lot number, sample performance when checked against cultures known to give positive and negative results, and pH verification.  Medium shall be discarded by manufacturer's expiration date.

12)        Each new lot of dehydrated medium shall be checked before use against a lot that has previously tested to be acceptable with positive and negative samples or culture controls.  In addition, each batch of laboratory prepared medium shall include positive and negative culture controls. These control organisms shall be either stock cultures (periodically checked for purity) or commercially available disks impregnated with the organism.  Results shall be recorded.

13)        Preparation of ONGP-MUG medium from basic ingredients by the laboratory is not permitted.  Medium shall be protected from light.  Ingredients and containers supplied by manufacturers are sterile and shall not be autoclaved.

14)        Each lot of fluorogenic medium shall be checked before use with a 366 nm ultraviolet light with a 6-watt bulb. If the media exhibit faint fluorescence, the laboratory shall use another lot that does not fluoresce. Records shall be maintained in accordance with Section 465.420.

15)       If the Quanti-Tray or Quanti-Tray 2000 test is used, the sealer shall be checked monthly by adding a dye (e.g., bromcresol purple) to the water.  If dye is observed outside the wells another sealer shall be obtained. Records shall be maintained.