LAB 19




   The purpose of the bacteriological examination of water is to determine if there is a possibility of pathogens being present. Infectious diseases such as salmonellosis, typhoid fever, shigellosis, cholera, hepatitis A, amoebic dysentery, Campylobacter gastroenteritis, giardiasis, and other fecal-oral route diseases may be transmitted by fecally-contaminated water. The identification of pathogens, however, is quite difficult. Pathogens may not survive long in water and are usually present only in small quantity. Therefore, one usually tests for the presence of coliforms in water.

   Coliforms are gram‑negative, lactose-fermenting rods of the family Enterobacteriaceae. Escherichia coli, a fecal coliform, is normal flora of the intestines in humans and animals and is, therefore, a direct indicator of fecal contamination of the water. The presence of coliforms would then indicate the possibility of fecal pathogens being present.

   Two tests are frequently performed to monitor water: the fecal coliform count and the total coliform count.

            1. The fecal coliform count tests specifically for the fecal coliform E. coli. M‑FC medium is used in this test and the plates are incubated at 45.5°C. This temperature is selective for fecal coliforms (nonfecal coliforms will not grow at this temperature) that produce blue colonies. This test, however, requires a special water bath incubator to assure a temperature of 45.5°C.

            2. The total coliform count will detect any coliforms (fecal and nonfecal) present in the water. It is not as specific an indicator of fecal contamination, but is a useful screening test. M-coliform medium is used in this test and the plates are incubated at 37°C. Both fecal and nonfecal coliforms will grow and produce metallic green colonies. Coliforms would indicate the possibility of fecal contamination of the water.

   Both of these tests use the micropore membrane filter method. Different amounts of the water sample being tested are passed through a membrane filter (Lab 19). The water passes through and the bacteria are trapped on the surface of the filter. The filter is then placed in a petri plate on pads containing either M‑FC or M‑coliform medium. Colonies then form on the filter. By counting the number of colonies and knowing the volume of water sample used, the number of fecal coliforms or total coliforms per ml of water can be determined.



            1. Take three 50mm petri plates and aseptically place a sterile pad in the bottom of each. Label the plates 0.1ml, 1.0ml, and 10.0ml.

            2. Using a 10ml pipette, add 2.0ml of M‑coliform broth to each pad.

            3. Using alcohol-flamed forceps, remove a 0.45µm pore-diameter gridded membrane filter and place it grid‑side‑up in the filter set‑up.

            4. Secure the funnel to the filter set-up.

            5. Pour about one inch of sterile saline into the filter set‑up.

            6. Using a 1.0ml pipette, add 0.1ml of the water sample being tested to the sterile water and mix.

            7. Vacuum the water through the filter.

            8. Add another inch of sterile saline to the funnel and swirl. This washes the bacteria off the sides of the funnel onto the filter.

            9. Vacuum.

            10. Using alcohol-flamed forceps, remove the filter and place it grid-side-up in the plate labeled 0.1ml. Make sure the entire filter makes contact with the M-coliform-containing pad.

            11. Repeat using 1.0ml of the water sample.

            12. Repeat using 10.0ml of the water sample.

            13. Incubate the 3 plates at 37°C until the next lab period.

            14. Observe the M-coliform plates. Both fecal and non-fecal coliforms will produce metallic green colonies. If there are a feasible number of coliform colonies for counting, determine the number of total coliforms per ml of water.


Creative Commons License
Microbiology Laboratory Manual by Gary E. Kaiser, PhD, Professor of Microbiology
is licensed under a Creative Commons Attribution 4.0 International License.
Last updated: September, 2017