* Approved by Standard Methods Committee, 1997.


9222 D. Fecal Coliform Membrane Filter Procedure


    Fecal coliform bacterial densities may be determined either by the multiple-tube procedure or by the MF technique. See Section 9225 for differentiation of Escherichia coli, the predominant fecal coliform. If the MF procedure is used for chlorinated effluents, demonstrate that it gives comparable information to that obtainable by the multiple-tube test before accepting it as an alternative. The fecal coliform MF procedure uses an enriched lactose medium and incubation temperature of 44.5 0.2C for selectivity. 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 solid heat sink incubator or other incubator that is documented to hold the 44.5C temperature within 0.2C throughout the chamber, over a 24-h period. Areas of application for the fecal coliform method in general are stated in the introduction to the multiple-tube fecal coliform procedures, Section 9221E.


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. See Section 9020 for quality control specifications.

    M-FC medium:


Typtose or biosate...............................  10.0 g
Proteose peptone No. 3 or polypeptone..    5.0 g
Yeast extract.......................................    3.0 g
Sodium chloride, NaCl..........................    5.0 g
Lactose..............................................  12.5 g
Bile salts No. 3 or bile salts mixture......    1.5 g
Aniline blue.........................................    0.1 g
Agar (optional).....................................  15.0 g
Reagent-grade water............................    1   L


    Rehydrate product in 1 L water containing 10 mL 1% rosolic acid in 0.2N NaOH.* Heat to near boiling, promptly remove from heat, and cool to below 50C. Do not sterilize by autoclaving. If agar is used, dispense 5- to 7-mL quantities to 50- X 12-mm petri plates and let solidify. Final pH should be 7.4 0.2. Refrigerate finished medium, preferably in sealed plastic bags or other containers to reduce moisture loss, and discard unused broth after 96 h or unused agar after 2 weeks.

    Test each medium lot against a previously acceptable lot for satisfactory performance as described in Section 9020B, by making dilutions of a culture of E. coli (Section 9020) and filtering appropriate volumes to give 20 to 60 colonies per filter. With each new lot of medium verify 10 or more colonies obtained from several natural samples, to establish the absence of false positives. For most samples M-FC medium may be used without the 1% rosolic acid addition, provided there is no interference with background growth. Such interference may be expected in stormwater samples collected during the first runoff (initial flushing) after a long dry period.

    Before use, test each batch of laboratory-prepared MF medium for performance with positive and negative culture controls. Check for coliform contamination at the beginning and end of each filtration series by filtering 20 to 30 mL of dilution or rinse water through filter. If controls indicate contamination, reject all data from affected samples and request resample.

    b. Culture dishes: tight-fitting plastic dishes are preferred because the membrane filter cultures are submerged in a water bath during incubation. Place fecal coliform cultures in plastic bags or seal individual dishes with waterproof (freezer) tape to prevent leakage during submersion. Specifications for plastic culture dishes are given in Section 9222B.1e.

    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, slower heat transfer from air to the medium, 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 shown to give equivalent results. A temperature tolerance of 44.5 0.2C can be obtained with most types of water baths that also are equipped with a gable top for the reduction of water and heat losses.


*Rosolic acid reagent will decompose if sterilized by autoclaving. Refrigerate stock solution in the dark and discard after 2 weeks or sooner if its color changes from dark red to muddy brown.



2. Procedure


    a. Selection of sample size: Select volume of water sample to be examined in accordance with the information in Table 9222:III. 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 volumes or dilutions to achieve a countable density. Estimate volume and/or dilution 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 prescribed under Section 9222B.5b above.

    c. Preparation of culture dish: Place a sterile absorbent pad in each culture dish and pipet at least 2.0 mL M-FC medium, prepared as directed above, to saturate pad. Carefully remove any excess liquid from culture dish by decanting the plate. Aseptically, place prepared filter on medium-impregnated pad as described in Section 9222B above.

    As a substrate substitution for the nutrient-saturated absorbent pad, add 1.5% agar to M-FC broth as described in Section 9222B above.

    d. Incubation: Place prepared dishes in waterproof plastic bags or seal, invert, and submerge petri dishes in water bath, and incubate for 24 2 h at 44.5 0.2C. Anchor dishes below 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. 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. Count colonies with a low-power (10 to 15 magnifications) binocular wide-field dissecting microscope or other optical device.

    f. Verification: Verify typical blue colonies and any atypical grey to green colonies as described in Section 9020 for fecal coliform analysis. Simultaneous inoculation at both temperatures is acceptable.






3. Calculation of Fecal Coliform Density


    a. General: 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. Calculate fecal coliform density as directed in Section 9222B.6 above. Record densities as fecal coliforms per 100 mL.

    b. Sediment and biosolid samples: For total solid (dry weight basis) see Section 2540G.

    Calculate fecal coliforms per gram dry weight for biosolid analysis as follows:


                                                                           colonies counted

Fecal coliforms per gram dry weight        =                                                      

                                                                    (dilution chosen) X (% dry solids)


where dilution and % dry solids are expressed in decimal form.


    Example 1: There were 22 colonies observed on the 1:10 000 dilution plate of a biosolid with 4% dry solids.



                                      = 5.5 X 106 fecal coliform/g dry weight



    If no filter has a coliform count falling in the ideal range (20 to 60), total the coliform counts on all countable filters and report as fecal coliforms per gram dry weight.



Table 9222:III. Suggested Samples Volumes for Membrane Filter Fecal Coliform Test

   Volume (X) To Be Filtered
Water Source 100 50 10 1 0.1 0.01 0.001 0.0001
Lakes, reservoirs X X
Wells, springs X X
Water supply intake X X X
Natural bathing waters X X X
Sewage treatment plant X X X
Farm ponds, rivers X X X
Stormwater runoff  X X X
Raw municipal sewage X X X
Feedlot runoff      X X X
Sewage sludge X X X



    Example 2: There were 18 colonies observed on the 1:10 000 dilution plate and 2 colonies observed on the 1:100 000 dilution plate of a biosolid sample with 4% dry solids.


                                                   (18 + 2)              

                                                                                 = 4.5 X 106

                                        (0.0001 + 0.00001)(0.04)

    To compute a geometric mean of samples, convert coliform densities of each sample to log10 values. Determine the geometric mean for the given number of samples (usually seven) by averaging the log10 values of the coliform densities and taking the antilog of that value.






4. Bibliography


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


Rose, R.E., E.E. Geldreich & W. Litsky. 1975. Improved membrane filter method for fecal coliform analysis. Appl. Microbiol. 29:532.


Lin, S.D. 1976. Membrane filter method for recovery of fecal coliforms in chlorinated sewage effluents. Appl. Environ. Microbiol. 32:547.


Presswood, W.G. & D.K. Strong. 1978. Modification of M-FC medium by eliminating rosolic acid. Appl. Environ. Microbiol. 36:90.


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


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


Grabow, W.O.K., C.A. Hilner & P. Coubrough. 1981. Evaluation of standard and modified M-FC, MacConkey, and Teepol media for membrane filter counting of fecal coliform in water. Appl. Environ. Microbiol. 42:192.


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


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


U.S. Environmental Protection Agency. 1992. Environmental Regulations and Technology. Control of Pathogens and Vector Attraction in Sewage Sludge. EPA-626/R-92-013, Washington, D.C.


U.S. Environmental Protection Agency. 1993. Standards for the Use or Disposal of Sewage Sludge: Final Rule. 40 CFR Part 257; Federal Register 58:9248, Feb. 19, 1993.



Standard Methods for the Examination of Water and Wastewater. 20th Ed. American Public Health Association, American Water Works Association, Water Environment Federation.