EPA Region 9 Laboratory SOP 1102
http://www.epa.gov/region09/lab/sops/sop1102.html
MULTIPLE TUBE FERMENTATION ANALYSIS FOR TOTAL AND FECAL COLIFORMS
Summary
The Multiple Tube Fermentation procedure or Most Probable Number (MPN) is a tube-dilution method using a nutrient
rich medium, which is less sensitive to toxicity and supports the growth of environmentally stressed organisms. The
multiple tube fermentation technique is applicable for the analysis of drinking water, fresh, salt, and brackish surface
waters, influents, chlorinated effluents. The multiple tube procedure is also appropriate for muds, sediments and
sludges because particulates do not interfere visibly with the test.
The coliform group consists of several genera of bacteria belonging to the family Enterobacteriaceae. The historical
definition of the group has been based on the method used for detection (lactose fermentation) rather than on the
tenets of systematic bacteriology. Accordingly when the fermentation technique is used, the group is defined as all
aerobic and facultative anaerobic, gram negative, non-spore forming rod-shaped bacteria that ferment lactose with
gas and acid formation within 48h at 35°C.
The fecal coliforms are part of the total coliform group. They are defined as gram-negative nonspore forming rods
that ferment lactose in 24 ± 2 hours at 44.5 ± 0.2°C with the production of gas in a multiple-tube procedure or
produce acidity with blue colonies in a membrane filter procedure.
This method detects and estimates the total coliforms in water samples by the multiple fermentation tube technique.
The total coliform method has two stages: the Presumptive and the Confirmed Tests. In the Presumptive Test, a
series of lauryl tryptose broth fermentation tubes are inoculated with decimal dilutions of the sample. The formation of
gas at 35 C within 48 h constitutes a positive Presumptive Test for members of the total coliform group. However, the
MPN must be carried through the Confirmed Test for valid results. In this test, inocula from positive Presumptive tubes
are transferred to tubes of Brilliant Green lactose bile (BGLB) broth. The BGLB medium contains selective and
inhibitive agents to suppress the growth of all non coliform organisms. Gas production after incubation at 24 or 48
hours at 35 C constitutes a positive Confirmed test for total coliforms. For fecal coliform determination, gas positive
LTB tubes are also transferred aseptically to EC medium and incubated at 44.5 ± 0.2°C for 24 ± 2 hours. The
production of gas in EC at the elevated temperature after 24 hours constitutes a positive fecal coliform test.
The multiple tube fermentation technique results are reported in terms of the Most Probable Number (MPN) or
organisms present. This number is based on certain probability formulas, and is an estimate of the mean density of
coliforms in the sample. Coliform density, together with other information obtained by engineering or sanitary surveys,
provides the best assessment of water treatment effectiveness and sanitary quality of untreated water.