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.
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
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.
||To illustrate procedures for bacteriologic examination of water
||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
: 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.
- Presumptive Test
- Inoculate 1.0 ml of the sample of tap water into a tube of lactose broth. Label the tube “Tap, Presumptive.”
- Repeat procedure 1 with the spring water sample. Label the tube “Spring, Presumptive.”
- Incubate both tubes at 35°C for 24 to 48 hours.
- Confirmed and Completed Tests
- Examine the inoculated EMB plate streaked from a positive presumptive test, noting the color of colonies.
- Pick a coliform type of colony and inoculate it into a tube of lactose broth and onto a nutrient agar slant. Label these tubes
- 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.”
- Incubate these cultures at 35°C for 24 to 48 hours.
- Read all lactose broths for gas formation.
- Prepare a Gram stain from each agar slant.
Record results of presumptive, confirmed, and completed tests in the following tables.