Introduction

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

Presumptive stage, confirmed stage and completed test.

Presumptive Stage: A series of lauryl tryptose broth primary fermentation tubes are inoculated

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.

Formationofgasinanyamountwithin48+3hr is a positive presumptive test.

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

the tube indicates a positive confirmed test.

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

gram-negative, non-spore-forming, rod-shaped bacteria in the agar culture may be considered a

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

methodology is the ability to estimate a microbial population size based on a process-related

attribute.