Microorganisms can be found everywhere, whether in air, land, or water. Some of these
microorganisms are harmless to humans, while some can cause disease. One of the
microorganisms that can be found in these environments is the coliform bacteria. Coliform
bacteria are present in the environment and feces of all warm-blooded animals and humans.
Coliform bacteria are unlikely to cause illness. However, their presence in drinking water
indicates that disease-causing organisms (pathogens) could be in the water system. Most
pathogens that can contaminate water supplies come from the feces of humans or animals.
Coliform bacteria are a commonly used indicator of the sanitary quality of food and
water. They are defined as rod-shaped Gram-negative non-spore forming and motile or non-
motile bacteria which can ferment lactose with the production of acid and gas when incubated at
3537C.
Two techniques are commonly used to detect the presence of coliforms in water.
The first of these is called the multiple fermentation tube or most probable number
technique. In this method measured portions of a water sample are placed in test-tubes
containing a culture medium. The tubes are then incubated for a standard time at a standard
temperature.
The multiple-tube fermentation technique is a three stage procedure in which the results
are statistically expressed in terms of the Most Probable Number (MPN). These stages the
with graduated quantities of the sample to be tested. The inoculated tubes are incubated at 35 +
0.5EC for 24 + 2 hr, at which time the tubes are examined for gas formation. For the tubes in
which no gas is formed, continue incubation and examine for gas formation attheendof48+3hr.
Confirmed Stage: The confirmed stage is used on all primary fermentation tubes showing gas
formation during the 24-hr and 48-hr periods. Fermentation tubes containing brilliant green
lactose bile broth are inoculated with medium from the tubes showing a positive result in the
presumptive test. Inoculation should be performed as soon as possible after gas formation occurs.
The inoculated tubes are incubated for 48 + 3 hr at 35 + 0.5EC. Formation of gas at any time in
Completed Test: The completed test is performed on all samples showing a positive result in the
confirmed test. It can also be used as a quality control measure on 20% of all samples analyzed.
One or more plates of eosin methylene blue are streaked with sample to be analyzed. The
streaked plates are incubated for 24 + 2 hr at 35 + 0.5EC. After incubation, transfer one or more
typical colonies (nucleated, with or without metallic sheen) to a lauryl tryptose broth
fermentation tube and a nutrient agar slant. The fermentation tubes and agar slants are incubated
at 35 + 0.5EC for 24 + 2 hr, or for 48 + 3 hr if gas is not produced. From the agar slants
corresponding to the fermentation tubes in which gas formation occurs, gram-stained samples are
examined microscopically. The formation of gas in the fermentation tube and the presence of
satisfactorily completed test, demonstrating the positive presence of coliform bacteria in the
analyzed sample.
The Most Probable Number (MPN) technique is an important technique in estimating
microbial populations in soils, waters, and agricultural products. Many soils are heterogeneous,
therefore exact cell numbers of an individual organism can be impossible to determine. The
MPN technique is used to estimate microbial population sizes in situations like this. The
technique does not rely on quantitative assessment of individual cells; instead it relies on specific
qualitative attributes of the microorganism being counted. The important aspect of MPN
attribute.