Coliform bacteria are rod-shaped Gram-negative non-spore forming and motile or non-motile bacteria. Coliforms can ferment lactose with the production of acid and gas when incubated at 35–37°C. Most types of Coliform bacteria do not cause disease, but some can cause mild illness.Typical coliform groups include Citrobacter, Enterobacter, Hafnia, Klebsiella, and Escherichia.
Coliforms are found in water, plants, and soil and are also found in the feces of warm-blooded animals. The presence of coliform bacteria in drinking water may indicate a possible presence of pathogenic organisms such as viruses, parasites, protozoa or bacteria. These waterborne pathogens cause diseases such as hepatitis, giardiasis, and dysentery.
The concentration of different pathogenic organisms in water is large and to test the water sample for the presence of pathogenic organism would be expensive and time-consuming. So the presence of disease-causing bacteria is determined by testing for an “indicator” organism such as coliforms. It is relatively easy to identify coliforms, as they are present in large number than more dangerous pathogens, and respond to the environment similar to many pathogens
The presence of very few fecal coliform bacteria leads to the assumption that the water probably contains no disease‑causing organisms, while the presence of large numbers of fecal coliform bacteria would indicate that the water could contain disease‑producing organisms making it unsuitable for consumption. Therefore, the drinking water standard requires that coliform bacteria be absent from drinking water. The most common method to detect coliform bacteria in a water sample is MPN (most probable number) method. MPN is a relatively simple, reliable, economical and efficient way to know if your water is safe. MPN is the number of organisms that are most likely to be present in a water sample based on the degree of lactose fermentation.
The MPN test is performed in three steps:
1. Presumptive test
2. Confirmed test
3. Completed test
1. PRESUMPTIVE TEST
In the presumptive test, a series of MacConkey broth tubes that contains the indicator bromocresol purple, are inoculated with serially diluted water samples. To increase the sensitivity of the test more test tubes are utilized. Coliforms if present in water utilizes the lactose present in the medium to produce acid and gas. The color of the medium changes into yellow and the presence of gas are detected as gas bubbles collected in the inverted Durham tube present in the medium. The number of total coliforms is estimated by counting the number of tubes giving positive reaction and comparing the pattern of positive results with standard charts to find MPN of total 100 ml water sample.
2. CONFIRMED TEST
Some spore-forming bacteria also produce acid and gas from lactose fermentation giving false positives in the presumptive test. In order to confirm the presence of coliforms, EMB (eosin methylene blue) agar plates are inoculated from positive presumptive tubes and incubated at 44.5°Cand 37°C.The methylene blue in EMB agar allows the growth of Gram-negative coliforms and inhibits the growth of gram-negative bacteria. Presence of greenish metallic sheen on EMB plates at 37°C confirms positive coliform test and those at 44.5°C confirms the presence of thermos-tolerant E.coli.
3. COMPLETED TEST
The completed test is made using colonies from each of the solid-medium plates used for the confirmed test. These organisms are used to inoculate a nutrient agar slant, a tube of lactose broth. After 24 hours at 37°C, the formation of gas in lactose broth and the presence of Gram-negative, non-spore-forming bacilli in the corresponding agar confirm the presence of a member of the coliform group in the sample.
Tube 1: Presence of gas in the Durham tube and the change in color from red to yellow as acid end-products react with the pH indicator confirms the presence of fecal coliforms
Tube 2: The absence of gas in the Durham tube indicates the absence of coliforms Even though the color has changed from red to yellow, gas must be produced for a positive result.
Tube 3: Negative for fecal coliforms.
Advantages of the MPN technique include:
• Easy to interpret the results, either by gas production or color change.
• Coliforms are non -pathogenic
• Effective and inexpensive method of analyzing several samples such as sediments, sludge, mud, etc.
• Requires more tubes and media to increase the sensitivity of the test
• Presence of false positives.
The MPN method is used in the pharmaceutical industry and water testing its applications are:
• Detection of bacteria in food and water to prevent infections and diseases
• Analyzing the thiosulfate-reducing bacteria that contribute to bio-corrosion in oil pipelines
• Enumerating the microbial bacterial populations in environments where steel may suffer from microbiologically influenced corrosion