KF STREPTOCOCCUS AGAR

Cat. no. G276 KF Streptococcus Agar, 15x60mm Plate, 15ml 10 plates/bag
Cat. no. G376 KF Streptococcus Agar, 15x100mm Plate, 19ml 10 plates/bag

INTENDED USE

Hardy Diagnostics KF Streptococcus Agar is intended for the selective isolation and enumeration of fecal streptococci (including Enterococcus ) from water and food samples by direct culture or membrane filtration. (1,2)

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

The natural habitat of fecal streptococci is the gastrointestinal tract of warm-blooded animals. The enterococcus subgroup of fecal streptococcus is a valuable bacteriological indicator for determining the extent of fecal contamination of recreational surface waters.

KF (Kenner Fecal) Streptococcus Agar is a selective medium developed by Kenner, Clark and Kabler for the isolation and enumeration of fecal streptococci in surface waters, food and other materials by direct culture or membrane filtration. (1,2,5,6) In the early 1960s, Kenner et al. compared the performance of this medium to other streptococcal media and showed that KF Streptococcus Agar yielded higher cell densities using both MPN tests and membrane filter counts; they recommended use of this medium for membrane filtration over the multiple tube technique. (5,7) As a result, current isolation and enumeration techniques for detecting fecal streptococci as an indicator of pollution using KF Streptococcus Agar are made according to the APHA recommended guidelines for the examination of surface waters and foods. (1,2)

Hardy Diagnostics KF Streptococcus Agar is based on Kenner, Clark and Kabler's formulation and includes peptone as a source of nitrogen, amino acids and carbon. Yeast extract provides a source of vitamins, amino acids and essential trace elements. Maltose and lactose provide carbon as an energy source. Sodium azide is a selective agent added to inhibit the growth of gram-negative microorganisms. Bromcresol purple dye acts as an indicator. Triphenyl Tetrazolium Chloride (TTC) is a colorless redox agent added to differentiate colonies on agar or membrane filters. When irreversibly reduced to formazan by actively growing cells, TTC forms a cellular insoluble red pigment that can be exploited for more efficient enumeration. (3,4)

FORMULA

Ingredients per liter of deionized water:*

Maltose 20.0gm
Proteose Peptone No. 3 10.0gm
Yeast Extract 10.0gm
Sodium Glycerophosphate 10.0gm
Sodium Chloride 5.0gm
Lactose 1.0gm
Sodium Azide 0.4gm
Bromcresol Purple 15.0mg
1% Triphenyl Tetrazolium Chloride 10.0ml
Agar 20.0gm

Final pH 7.2 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Direct Plating Procedure:

1. Inoculate plates using a 10 -3 or greater dilution using the spread plate technique to obtain isolated colonies.

2. Incubate plates inverted at 35 +/- 2ºC. for 46 to 48 hours.

3. Count all red to pink colonies and report as the number of fecal enterococci calculated per sample.

Membrane Filter Procedure:

1. Filter a suitable volume of sample through a sterile membrane filter.

2. Place the inoculated membrane on the agar surface, inoculum side up, using a rolling motion to ensure proper contact with the surface and to avoid entrapment of air bubbles.

3. Incubate plates inverted at 35 +/- 2ºC. for 46 to 48 hours.

4. Count all red to pink colonies and report as the number of fecal enterococci calculated per volume of sample.

INTERPRETATION OF RESULTS

Fecal enterococci will appear as red to pink centered colonies. Using a dissecting microscope with 15X magnification or a colony counter can aid in determining colony counts.

LIMITATIONS

Due to varying nutritional requirements, some strains may grow poorly or fail to grow at all on this medium.

Some strains of Streptococcus bovis and Streptococcus equinus may be inhibited by azide.

The growth of orange, yellow, white or other colored colonies should not be counted.

Tropical marine water samples should be incubated anaerobically to avoid excessive numbers of false-positive presumptive counts for enterococci.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, membrane filters, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Enterococcus faecalis
ATCC ® 29212
A 46-48hr 35°C Aerobic Growth; colonies with red to pink centers
Escherichia coli
ATCC ® 25922
B 48hr 35°C Aerobic Partial to complete inhibition

USER QUALITY CONTROL

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Gershman, M., D.C. O'Meara and H.L. Chute. 1959. Use of Tetrazolium Salt for an Easily Discernible Sulfide-Motility Reaction. J. Bact.; 78(5):739-740.

4. Kelly, A.T. and M. Fulton. 1953. Use of Triphenyl Tetrazolium Chloride in Motility Test Medium. Am. J. Clin. Pathol.; 23:512.

5. Kenner, B.A., H.F. Clark and P.W. Kabler. 1960. Quantification of Streptococci in Faeces. Am. J. Publ. Health; 50:1553-1559.

6. Kenner, B.A., H.F. Clark and P.W. Kabler. 1961. Cultivation and Enumeration of Streptococci in Surface Waters. Appl. Microbiol.; 9:15-20.

7. Slanetz, L.W. and C.H. Bartley. 1964. Detection and Sanitary Significance of Fecal Streptococci in Water. Am. J. Pub. Health Nat. Health; 54(4):609-614.


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