The laboratory shall follow the additional laboratory practices:
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
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
(C) LES Endo agar
shall be used for the completed test. Refrigerate medium and use within two
(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.
EC Medium (for fecal coliforms):
(A) Autoclave for 12 minutes at
(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
(D) Store prepared medium
in screw cap tubes.
(E) Final pH
shall be 6.9 0.2.
+ 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.
prepared medium in screw cap tubes no longer than three months.
MMO-MUG Test Medium (for Total
Coliform and E. coli):
(A) The laboratory
shall not prepare this medium from basic ingredients.
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.
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.
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.
Heterotrophic Plate Count (HPC) Medium:
(A) Autoclave HPC agar at 121°C for 15
(B) Final pH shall be 7.0
(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.