North Carolina Administrative Code › Title 10A - HEALTH AND HUMAN SERVICES › Chapter 42 - LABORATORY SERVICES › Subchapter D - CERTIFICATION AND IMPROVEMENT › Section .0200 - LABORATORY CERTIFICATION › Section 42D .0248 - MICROBIOLOGY GENERAL LABORATORY PRACTICES

Current through Register Vol. 39, No. 2, July 15, 2024
(a) The
				  general laboratory practices for microbiological analyses shall be in
				  accordance with those listed in the EPA "Manual for the Certification of
				  Laboratories Analyzing Drinking Water", Chapter 5, Section 4, General
				  Laboratory Practices, which is hereby incorporated by reference including any
				  subsequent amendments and editions, except that Sections 4.6.2-4.6.11 are not
				  incorporated by reference. A copy is available for inspection at the Division
				  of Public Health, 1915 Mail Service Center, Raleigh, North Carolina 27699-1915.
				  Nonprofit organizations or government agencies may obtain a copy by contacting
				  the EPA Drinking Water Hotline at 800-426-4791. Other organizations may obtain
				  a copy from the National Technical Information Service at 800-336-4700 for
				  thirty five dollars ($35.00).
(b)
				  The laboratory shall follow the additional laboratory practices:
(1) Membrane Filter Media:
Use m-Endo broth or agar or LES Endo broth or agar in the
					 single step or enrichment techniques. Ensure that ethanol used in rehydration
					 procedure is not denatured. Prepare medium in a sterile flask and use a boiling
					 water bath or, if constantly attended, a hot plate with a stir bar to bring
					 medium to the boiling point. Do not boil medium. Final pH shall be 7.2
					 0.2.
(2) Multiple Tube
				  Fermentation (MTF) Media:
(A) Use double
				  strength lauryl sulfate broth or lactose broth in the presumptive test and
				  single strength brilliant green lactose bile (BGLB) broth in the confirmed
				  test. Autoclave media at 121°C for 12 minutes. Final pH shall be6.8 0.2 or
				  7.2 0.2 for BGLB broth.
(B) If MTF
				  media are refrigerated after sterilization, incubate overnight at 35°C
				  0.5°C before use. Discard tubes showing growth or bubbles. Use MTF media
				  prepared in tubes with loose fitting closures within one week. Store broth
				  media in screw cap tubes or bottles no longer than three months, provided media
				  are stored in the dark. Discard media if evaporation exceeds 10 percent of
				  original volume.
(C) LES Endo agar
				  shall be used for the completed test. Refrigerate medium and use within two
				  weeks.
(3) Clark's Total
				  Coliform Medium:
(A) Autoclave for 12 minutes
				  at 121°C. Allow space between bottles.
(B) Final pH shall be 6.8 0.2.
(C) Store prepared medium in screw capped
				  culture bottle no longer than three months; discard if evaporation exceeds 10
				  percent of original volume.
(4) EC Medium (for fecal coliforms):
(A) Autoclave for 12 minutes at
				  121°C.
(B) Examine tubes after
				  sterilization to insure that inverted inner tubes are free of air bubbles and
				  that the vials are at least partially covered with medium.
(C) Incubate refrigerated sterilized medium
				  overnight at 35°C 0.5°C; discard tubes that show growth or
				  bubbles.
(D) Store prepared medium
				  in screw cap tubes.
(E) Final pH
				  shall be 6.9 0.2.
(5) EC
				  + MUG Medium (for detection of fecal coliforms-E. coli):
(A) Autoclave medium at 121°C (gas tubes
				  shall not be used).
(B) Final pH
				  shall be 6.9 0.2.
(C) Store
				  prepared medium in screw cap tubes no longer than three months.
(6) MMO-MUG Test Medium (for Total
				  Coliform and E. coli):
(A) The laboratory
				  shall not prepare this medium from basic ingredients.
(B) Each lot purchased shall be tested for
				  performance by inoculation with three control bacteria: Escherichia coli, a
				  total coliform other than E. coli (e.g., Klebsiella pneumoniae) and a
				  non-coliform (e.g. Pseudomonas aeruginosa).
These control organisms shall be stock cultures or
					 commercially available discs impregnated with the organism. Incubate these
					 controls at 35°C 0.5°C for 24 hours, and read and record result.
(C) Do not autoclave.
(7) Fecal Coliform Membrane Filter
				  Medium (for enumeration of fecal coliform in source water) .
(A) Rehydrate medium in reagent water
				  containing 10 ml of 1 percent rosolic acid in 2N NaOH. Bring it to the boiling
				  point; do not autoclave.
(B)
				  Autoclave for 12 minutes at 121°C.
(C) Final pH shall be 7.4 0.2.
(D) Refrigerate unused prepared medium;
				  discard after 96 hours.
(8) Heterotrophic Plate Count (HPC) Medium:
(A) Autoclave HPC agar at 121°C for 15
				  minutes.
(B) Final pH shall be 7.0
				  0.2.
(C) Temper melted agar at
				  44°-46°C before pouring.
(D) Hold melted agar no longer than four
				  hours. Do not melt sterile agar medium more than once.