The bacteriological examination of water is performed routinely by water utilities and many governmental agencies to ensure a safe supply of water for drinking, bathing, swimming and other domestic and industrial uses. The examination is intended to identify water sources which have been contaminated with potential disease-causing microorganisms. Such contamination generally occurs either directly by human or animal feces, or indirectly through improperly treated sewage or improperly functioning sewage treatment systems. The organisms of prime concern are the intestinal pathogens, particularly those that cause typhoid fever and bacillary dysentery. Since human fecal pathogens vary in kind (viruses, bacteria, protozoa) and in number, it would be impossible to test each water sample for each pathogen. Instead, it is much easier to test for the presence of nonpathogenic intestinal organisms such as E. coli. E. coli is a normal inhabitant of the intestinal tract and is not normally found in fresh water. Therefore, if it is detected in water, it can be assumed that there has been fecal contamination of the water. In order to determine whether water has been contaminated by fecal material, a series of tests are used to demonstrate the presence or absence of coliforms. The coliform group is comprised of Gram-negative, nonspore-forming, aerobic to facultatively anaerobic rods, which ferment lactose to acid and gas. Two organisms in this group include E. coli and Enterobacter aerogenes; however, the only true fecal coliform is E. coli, which is found only in fecal material from warm-blooded animals. The presence of this organism in a water supply is evidence of recent fecal contamination and is sufficient to order the water supply closed until tests no longer detect E. coli. In this exercise, you will be testing water samples for the presence of coliforms. There will be three principal tests: the presumptive, confirmed and completed tests (see flow-chart).
STANDARD WATER ANALYSIS
The Presumptive Test
In the presumptive test, a series of lactose broth tubes are inoculated with measured amounts of the water sample to be tested. The series of tubes may consist of three or four groups of three, five or more tubes. The more tubes utilized, the more sensitive the test. Gas production in any one of the tubes is presumptive evidence of the presence of coliforms. The most probable number (MPN) of coliforms in 100 ml of the water sample can be estimated by the number of positive tubes (see MPN Table).
The Confirmed Test
If any of the tubes inoculated with the water sample produce gas, the water is presumed to be unsafe. However, it is possible that the formation of gas may not be due to the presence of coliforms. In order the confirm the presence of coliforms, it is necessary to inoculate EMB (eosin methylene blue) agar plates from a positive presumptive tube. The methylene blue in EMB agar inhibits Gram-positive organisms and allows the Gram-negative coliforms to grow. Coliforms produce colonies with dark centers. E. coli and E. aerogenes can be distinguished from one another by the size and color of the colonies. E. coli colonies are small and have a green metallic sheen, whereas E. aerogenes forms large pinkish colonies. If only E. coli or if both E. coli and E. aerogenes appear on the EMB plate, the test is considered positive. If only E. aerogenes appears on the EMB plate, the test is considered negative. The reasons for these interpretations are that, as previously stated, E. coli is an indicator of fecal contamination, since it is not normally found in water or soil, whereas E. aerogenes is widely distributed in nature outside of the intestinal tract.
The Completed Test
The completed test is made using the organisms which grew on the confirmed test media. These organisms are used to inoculate a nutrient agar slant and a tube of lactose broth. After 24 hours at 37°C, the lactose broth is checked for the production of gas, and a Gram stain is made from organisms on the nutrient agar slant. If the organism is a Gram-negative, nonspore-forming rod and produces gas in the lactose tube, then it is positive that coliforms are present in the water sample.