Section 465.350  General Laboratory Practices

a)         The following requirements 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 UV sterilizer or boiling water may be used on membrane filter assemblies for at least two minutes to prevent bacterial carryover 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 or 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." The pH of stock phosphate buffer solution is 7.2 ± 0.5.

2)         The phosphate buffer solution and magnesium chloride solution shall be autoclaved or filter sterilized, labeled, dated, and stored at 1º to 5ºº 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)         Alternatively, commercially prepared phosphate buffer and magnesium chloride solution may be used when preparing rinse and dilution water.  The date received, expiration date, proof of sterility, and pH of phosphate buffer shall be recorded.

d)         The following minimum requirements 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)         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 two weeks after refrigeration or discarded.

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

8)          The laboratory using commercially manufactured prepared 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.  Media shall be discarded by the manufacturer's expiration date.

9)          Each new lot of prepared commercial medium and each batch of laboratory prepared medium shall be checked before use with positive and negative culture controls.  Additionally, each batch of prepared media (whether commercially prepared or laboratory prepared) shall be checked for sterility.  Control organisms (total coliform, fecal coliform, and/or E. coli, as appropriate) shall be either known stock cultures (periodically checked for purity) or commercially available cultures impregnated with the organism. Results shall be recorded. The following table identifies a few positive and negative culture controls that laboratories might consider.

10)       Examples of appropriate American Type Culture Collection strains include the following:

Enterococcus faecalis ATCC 11700

Enterococcus faecium ATCC 6057

Enterobacter aerogenes ATCC 13048

Escherichia coli ATCC 8739 or 25922

Klebsiella pneumoniae (thermotolerant) ATCC 13883

Proteus vulgaris ATCC 13315

Pseudomonas aeruginosa ATCC 27853

Serratia marcesenes ATCC 14756

Staphylococcus aureus ATCC 6538

(Source:  Amended at 35 Ill. Reg. 14494, effective August 12, 2011)