Membrane Filtration Method: Fecal Coliforms


Virginia Polytechnic Institute and State University


The membrane filter (MF) technique is highly reproductible, can be used to test relatively large volumes of sample, and yields numerical results more rapidly than the multiple-tube procedure. The membrane filter technique is extremely useful in monitoring drinking water and a variety of natural waters. However, the MF technique has limitations, particularly when testing waters with high turbidity or noncoliform (background) bacteria. For such waters or when the membrane filter technique has not been used previously, it is desirable to conduct parallel tests with the multiple-tube fermentation technique to demonstrate applicability and comparability.

As related to the membrane filter technique, the coliform group may be defined as comprising all aerobic and many facultative anaerobic, gram-negative, nonspore-forming, rod-shaped bacteria that develop a red colony with a metallic sheen within 24h at 35oC on an Endo-type medium containing lactose. Some members of the total coliform group may produce a dark red or nucleated colony without a metallic sheen. When verified these are classified as atypical coliform colonies. When purified cultures of coliform bacteria are tested they produce a negative cytochrome oxidase (CO) and positive Beta-galactosidase (ONPG) reaction. Generally, all red, pink, blue, white, or colorless colonies lacking sheen are considered non-coliforms by this technique.

Fecal Coliform Membrane Filter Procedure

Fecal coliform bacterial densities may be determined either by the multiple-tube procedure or by a membrane filter (MF) technique. If the MF procedure is used for chlorinated effluents, demonstrate that it gives comparable information to that obtained by the multiple-tube test before accepting it as an alternative. The MF procedure uses an enriched lactose medium and incubation temperature of 44.5 +/- 0.2oC for selectivity and gives 93% accuracy in differentiating between coliforms found in the feces of warm-blooded animals and those from other environmental sources. Because incubation temperature is critical, submerge waterproofed (plastic bag enclosures) MF cultures in a water bath for incubation at the elevated temperature or use an appropriate, accurate solid heat sink incubator. Alternatively, use an equivalent incubator that will hold the 44.5oC temperature within 0.2oC (throughout the chamber), over a 24-h period, while located in an environment of ambient air temperatures ranging from 5oC to 35oC.

1. Materials and Culture Medium

a. M-FC medium: The need for uniformity dictates the use of dehydrated media. Never prepare media from basic ingredients when suitable dehydrated media are available. Follow manufacturer's directions for rehydration. Commercially prepared media in liquid form (sterile ampule or other) also may be used if known to give equivalent results.

b. Culture Dishes: Use tight-fitting plastic dishes because the MF cultures are submerged in a water bath during incubation. Enclose groups of fecal coliform cultures is plastic bags or seal individual dishes with waterproof (freezer) tape to prevent leakage during submersion.

c. Incubator: The specificity of the fecal coliform test is related directly to the incubation temperature. Static air incubation may be a problem in some types of incubators because of potential heat layering within the chamber and the slow recovery of temperature each time the incubator is opened during daily operations. To meet the need for greater temperature control use a water bath, a heat-sink incubator, or a properly designed and constructed incubator giving equivalent results. A temperature tolerance of 44.5 +/- 0.2oC can be obtained with most types of water baths that also are equipped with a gable top for the reduction of heat and water losses. A circulating water bath is excellent but may not be essential to this test if the maximum permissible variation of 0.2oC in temperature can be maintained with other equipment.

2. Procedure

a. Selection of sample size: Use sample volumes that will yield counts between 20 and 60 fecal coliform colonies per membrane. When the bacterial density of the sample is unknown, filter several decimal volumes to establish fecal coliform density. Estimate volume expected to yield a countable membrane and select two additional quantities representing one-tenth and ten times this volume, respectively.

b. Filtration of sample: Follow the same procedure and precautions as described above.

c. Preparation of culture dish: Place a sterile absorbent pad in each culture dish and pipet approximately 2 mL M-FC medium, prepared as directed above, to saturate pad. Carefully remove any excess liquid from the culture dish. Place prepared filter on medium-impregnated pad.

As a substrate substitution for the nutrient-saturated absorbent pad, add 1.5% agar to M-FC broth.

d. Incubation: Place prepared cultures in waterproof plastic bags or seal petri dishes, submerge in water bath, and incubate for 24 +/- 2h at 44.5 +/- 0.2oC. Anchor dishes below the water surface to maintain critical temperature requirements. Place all prepared cultures in the water bath within 30 min after filtration. Alternatively, use an appropriate, accurate solid heat sink or equivalent incubator.

e. Counting: Colonies produced by fecal coliform bacteria on M-FC medium are various shades of blue. Pale yellow colonies may be atypical E.coli; verify for gas production in mannitol at 44.5oC. Nonfecal coliform colonies are gray to cream-colored. Normally, few nonfecal coliform colonies will be observed on M-FC medium because of selective action of the elevated temperature and addition of rosolic acid salt reagent. Elevating the temperature to 45.0 +/- 0.2oC may be useful in eliminating environmental Klebsiella from the fecal coliform population. Count colonies with a low power (10-15 magnifications) binocular wide-field dissecting microscope or other optical device.

3. Calculation of Fecal Coliform Density

Compute the density from the sample quantities that produced MF counts within the desired range of 20 to 60 fecal coliform colonies. This colony density range is more restrictive than the 20 to 80 total coliform range because of larger colony size on M-FC medium. Record densities as fecal coliforms per 100 mL.

Compute the count by the following equation:

(total) Coliform colonies/100mL = (coliform colonies counted x 100) / (mL sample filtered)

For verified coliform counts, adjust the initial count based upon the positive verificationpercentage and report as "verified coliform count per 100 mL."
Percentage verified coliforms =
(number of verified colonies) / (total number of coliform colonies subjected to verification) x 100.

The plate in the upper left shows typical dark blue fecal coliform colonies on a membrane filter after incubation on M-FC agar. The plate in the lower center (purple) is M-FC agar before use, and the plate in the upper right (blue) is a control plate streaked with an E.coli culture.

4. Bibliography

Geldreich, E.E., H.F.Clark, C.B.Huff and L.C.Best. 1965. Fecal-coliform-organism medium for the membrane filter technique.J.Amer. Water Works Assoc. 57:208.

Green, B.L., W. Litsky and K.J.Sladek. 1980. Evalutation of membrane filter methods for enumeration of faecal coliforms from marine waters. Mar. Environ. Res. 67:267

Greenberg, A.E., L.S. Clesceri, and A.D.Eaton(eds). 1992. Standard Methods for the Examination of Water and Wastewater. Amer.Public Health Assoc. Washington, D.C.

Sartory, D.P. 1980. Membrane filtration faecal coliform determinations with unmodified and modified M-FC medium. Water SA 6:113.

Rychert, R.C. and G.R.Stephenson. 1981. Atypical Escherichia coli in streams. Appl. Environ. Microbiol. 41:1276.

Pagel, J.E., A.A.Qureshi, D.M.Young and L.T.Vlassoff. 1982. Comparison of four membrane filter methods for fecal coliform enumeration. Appl. Environ. Microbiol. 43:787.

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