Membrane Filtration Method: Fecal Streptococcus and Enterococcus Groups

Soil Microbiology

CSES 4644


1. Fecal Streptococcus Group
The fecal streptococcus group consists of a number of species of the genus Streptococcus, such as S.faecalis, S.faecium, S.avium, S.bovis, S.equinus, and S.gallinarum. They all give a positive reaction with Lancefield's Group D antisera and have been isolated from the feces of warm-blooded animals. In addition, S.avium sometimes reacts with Lancefield's Group Q antisera. S.faecalis subsp. liquefaciens and S.faecalis subsp. zymogenes are differentiated based on the ability of these strains to liquefy gelatin and hemolyze red cells. However, the validity of these subspecies is questionable.

The normal habitat of fecal streptococci is the gastrointestinal tract of warm-blooded animals. S. faecalis and S.faecium once were thought to be more human-specific than other Streptococcus species. Other species have been observed in human feces but less frequently. Similarly, S.bovis, S.equinus, and S.avium are not exclusive to animals, although they usually occur at higher densities in animal feces. Certain streptococcal species predominate in some animal species and not in others, but it is not possible to differentiate the source of fecal contamination based on the speciation of fecal streptococci.

The fecal streptococci have been used with fecal coliforms to differentiate human fecal contamination from that of other warm-blooded animals. Editions of Standard Methods previous to the 17th suggested that the ratio of fecal coliforms (FC) to fecal streptococci (FS) could provide information about the source of contamination. A ratio greater than four was considered indicative of human fecal contamination, whereas a ratio of less than 0.7 was suggestive of contamination by nonhuman sources. The value of this ratio has been questioned because of variable survival rates of fecal streptococcus group species. S.bovis and S.equinus die off rapidly, once exposed to aquatic environments, whereas S.faecalis and S.faecium tend to survive longer. Furthermore, disinfection of wastewaters appears to have a significant effect on the ratio of these indicators, which may result in misleading conclusions regarding the source of contaminants. The ratio is affected also by the methods for enumerating fecal streptococci. The KF membrane filter procedure has a false-positive rate ranging from 10 to 90% in marine and fresh waters. For these reasons, the FC/FS ratio cannot be recommended, and should not be used as a means of differentiating human and animal sources of pollution.

2. Enterococcus
The enterococcus group is a subgroup of the fecal streptococci that includes S.faecalis,S.faecium,S.gallinarum, and S.avium. The enterococci are differentiated from other streptococci by their ability to grow in 6.5% sodium chloride, at pH 9.6, and at 10oC and 45oC.

The enterococci portion of the fecal streptococcus group is a valuable bacterial indicator for determining the extent of fecal contamination of recreational surface waters. Studies at marine and fresh water bathing beaches indicated that swimming-associated gastroenteritis is related directly to the quality of the bathing water and that enterococci are the most efficient bacterial indicator of water quality. Water quality guidelines based on enterococcal density have been proposed for recreational waters. For recreational fresh waters the guideline is 33 enterococci/100mL while for marine waters it is 35/100mL. Each guideline is based on the geometric mean of at least five samples per 30-d period during the swimming season.

3. Selection of Method
The multiple-tube technique is applicable primarily to raw and chlorinated wastewater and sediments, and can be used for fresh and marine waters. The membrane filter technique also may be used for fresh and saline water samples, but it is unsuitable for highly turbid waters.

4. Materials and Culture Media
m Enterococcus agar for fecal streptococci: Heat to dissolve ingredients. Do not autoclave. Dispense into 9- x 50-mm petri plates to a depth of 4 to 5 mm (approximately 4 to 6 mL), and let solidify. Prepare fresh medium for each set of samples. (NOTE: This medium is recommended for Group D streptococci in fresh and marine waters.)
Brain-heart infusion broth: The pH should be 7.4 after sterilization.
Brain-heart infusion agar: Add 15.0 g agar to the ingredients for brain-heart infusion broth. The pH should be 7.4 after sterilization. Tube for slants.
Bile esculin agar: Heat to dissolve ingredients. Dispense 8 to 10 mL into tubes for slants or an appropriate volume into a flask for subsequent pouring into plates. Autoclave at 121oC for 15 min. Do not overheat because this may cause darkening of the medium. Cool to 44 to 46oC and slant the tubes or dispense 15 mL into 15- x 100-mm petri dishes. The final pH should be 6.6 +/- 0.2 after sterilization. Store at 4 to 10oC.

5. Procedures
a. mE Method
1. Selection of sample size and filtration--Filter appropriate sample volumes through a 0.45-micrometer, gridded, sterile membrane to give 20 to 60 colonies on the membrane surface. Transfer filter to agar medium in petri dish, avoiding air bubbles beneath the membrane.
2. Incubation--Invert culture plates and incubate at 41oC +/- 0.5oC for 48h.
3. Substrate test--After 48 h incubation, carefully transfer filter to EIA medium. Incubate at 41oC +/- 0.5oC for 20 min.
4. Counting--Pink to red enterococci colonies develop a black or reddish-brown precipitate on the underside of the filter. Count colonies using a fluorescent lamp and a magnifying lens.

b. m Enterococcus method
1. Selection of sample size and filtration--See fecal coliforms
2. Incubation--Let plates stand for 30 min, then invert and incubate at 35 +/- 0.5oC for 48 h.
3. Counting--Count all light and dark red colonies as enterococci. Count colonies using a fluorescent lamp and a magnifying lens.

6. Calculation of Fecal Streptococci or Enterococci Density
Compute density from sample quantities producing membrane filter counts within the desired 20- to 60-fecal streptococcus or enterococcus colony range. Calculate as done with fecal coliforms. Record densities as fecal streptococci or enterococci per 100mL.

Membrane filtration on mEnterococcus agar. The plate at the bottom is uninoculated. The red colonies typical of the Enterococci are clearly visible on the white membrane filters.

7. Verification Tests
Pick selected typical colonies from a membrane and streak for isolation onto the surface of a brain-heart infusion agar plate. Incubate at 35oC +/- 0.5oC for 24 to 48 h.
Transfer a loopful of growth from a well-isolated colony on brain-heart infusion agar into a brain-heart infusion broth tube and to each of two clean glass slides. Incubate the brain-heart infusion broth at 35oC +/- 0.5oC for 24 h. Add a few drops of freshly prepared 3% hydrogen peroxide to the smear on a slide. The appearance of bubbles constitutes a positive catalase test and indicates that the colony is not a member of the fecal streptococcus group. If the catalase test is negative, i.e., no bubbles, make a Gram stain of the second slide. Fecal streptococci and enterococci are gram-positive, ovoid cells, 0.5 to 1.0 micrometers in diameter, mostly in pairs or short chains.
Transfer a loopful of growth from the brain-heart infusion broth to each of the following media: bile esculin agar (incubate at 35oC +/- 0.5oC for 48 h); brain-heart infusion broth (incubate at 45oC +/- 0.5oC for 48 h); brain-heart infusion broth with 6.5% NaCl (incubate at 35oC +/- 0.5oC for 48 h).
Growth of catalase-negative, gram-positive cocci on bile esculin agar and at 45oC in brain-heart infusion broth verifies that the colony is of the fecal streptococcus group. Growth at 45o and in 6.5% NaCl broth indicates that the colony belongs to the enterococcus group.

8. References

Ericksen, T.H., C.Thomas and A.Dufour. 1983. Comparison of two selective membrane filter methods for enumerating fecal streptococci in freshwater samples. Abs.Annual Meeting, American Soc. Microbiology, p. 279.

Cabelli, V.J. 1983. Health Effects Criteria for Marine Waters. EPA-600/1-80-031, U.S. Environmental Protection Agency, Cincinnati, Ohio.

Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters. EPA-600/1-84-004, U.S.Environmental Protection Agency, Cincinnati, Ohio.

U.S.Environmental Protection Agency. 1986. Ambient Water Quality Criteria for Bacteria--1986. EPA-440/5-84-002, U.S.Environmental Protection Agency, Washington, D.C.

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