Bacteriological Analysis of Water

The principal means through which pathogenic microorganisms reach water supplies is fecal contamination. The most important waterborne diseases are typhoid fever and other salmonelloses, cholera, bacillary and amebic dysentery, and giardiasis. The viral agents of infectious hepatitis and poliomyelitis and the parasite Cryptosporidium are fecal organisms and may also be spread in contaminated water.

The method for bacteriologic examination of water is designed to provide an index of fecal contamination. Pathogenic microorganisms do not necessarily multiply in water, and therefore they may be present in small numbers that are difficult to demonstrate in culture. Escherichia coli, other coliform bacteria, and enterococci, however, are not only abundant in feces but also usually multiply in water, so that they are present in large, readily detectable numbers if fecal contamination has occurred. Thus, culture demonstration of E. coli and enterococci in water indicates a fecal source of the organisms. In water from sources subjected to purification processes (such as reservoirs), the presence of E. coli or enterococci may mean that chlorination is inadequate. By bacteriologic standards, water for drinking (i.e., potable water) should be free of coliforms and enterococci and contain not more than 500 organisms per milliliter. The term “coliform,” which refers to lactose-fermenting gramnegative enteric bacilli, is now obsolete except in sanitary bacteriology.

A presumptive test for coliforms is performed by inoculating a sample of water into tubes of lactose broth containing Durham tubes. After 24 hours of incubation, the tubes are examined for the presence of acid and gas as an indication of lactose fermentation. Other than coliforms, few organisms found in water can ferment lactose rapidly with production of gas. Gaseous fermentation of lactose within 24 to 48 hours provides presumptive evidence of the presence of coliforms. The test must be confirmed, however, to exclude the possibility that another type of organism provided the positive lactose result.

The confirmed test is done by plating a sample of the positive lactose broth culture onto a differential agar medium. Eosin methylene blue (EMB) agar is frequently used. Coliform colonies ferment the lactose of EMB and consequently have a deep purple color with a coppery, metallic sheen. This characteristic appearance of the growth provides confirmation of the presumptive test.

A completed test requires inoculation of another lactose broth and an agar slant with isolated colonies from EMB. Gas formation in the lactose broth and microscopic demonstration of gram-negative, nonsporing rods on the agar slant are considered complete evidence of the presence of coliform organisms in the original sample.

Total plate counts are also made of water samples to determine whether they meet the criteria for potability. Instead of performing the broth procedure for the presumptive and confirmed tests, in some public health laboratories a specified volume of water is passed through a cellulose membrane filter that retains bacteria. The filter is placed on an agar medium, such as EMB, the plate is incubated, and then examined for the presence and numbers of coliform colonies growing on the filter. In this way, the presumptive and confirmed tests, as well as the quantitative count are performed simultaneously. The test for the presence of enterococci is performed similarly by filtration.

In this laboratory session you will perform presumptive, confirmed, and completed tests for E. coli in water samples.

Purpose	To illustrate procedures for bacteriologic examination of water
Materials	Sample of spring water Sample of tap water Sterile 1.0-ml pipettes Lactose broth with Durham tubes Nutrient agar slants EMB plates inoculated from a positive presumptive test

Procedures
Note: The entire procedure for the bacteriologic analysis of water requires several days to complete. Therefore, the instructor will provide some material that has been inoculated and incubated in advance to conserve classroom time.

1. Presumptive Test
   1. Inoculate 1.0 ml of the sample of tap water into a tube of lactose broth. Label the tube “Tap, Presumptive.”
   2. Repeat procedure 1 with the spring water sample. Label the tube “Spring, Presumptive.”
   3. Incubate both tubes at 35°C for 24 to 48 hours.


2. Confirmed and Completed Tests
   1. Examine the inoculated EMB plate streaked from a positive presumptive test, noting the color of colonies.
   2. Pick a coliform type of colony and inoculate it into a tube of lactose broth and onto a nutrient agar slant. Label these tubes “Coliform, Completed.”
   3. Pick a colony that is not of coliform type and inoculate it into a tube of lactose broth and onto a nutrient agar slant. Label these tubes “Noncoliform, Completed.”
   4. Incubate these cultures at 35°C for 24 to 48 hours.
   5. Read all lactose broths for gas formation.
   6. Prepare a Gram stain from each agar slant.

Results
Record results of presumptive, confirmed, and completed tests in the following tables.