DERMATOPHYTE MILK AGAR

Cat. no. L75 Dermatophyte Milk Agar, 16x100mm Tube, 6.0ml 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Dermatophyte Milk Agar is recommended for use in the cultivation and differentiation of certain dermatophytes, particularly Trichophyton rubrum , Trichophyton mentagrophytes , and Microsporum persicolor .

SUMMARY

Hardy Diagnostics Dermatophyte Milk Agar is prepared according the the formulation of Bromcresol purple-milk solids-glucose medium. Fischer and Kane, in 1971, proposed Bromcresol purple-milk solids-glucose medium as an aid in the identification and purification of dermatophyte isolates. (10) The indicator, bromcresol purple, was incorporated into the medium to allow detection of bacterial contaminants which are usually noted by the production of an acidic (yellow) medium. Through the course of their study, Fischer and Kane found that pure cultures of T. mentagrophytes resulted in diffuse, spreading growth and a marked alkaline pH change at 7 days, while T. rubrum produced restricted growth patterns and no pH changes within a 7 day incubation period.

Alkalinization of Dermatophyte Milk Agar is noted by a color change in the medium from its original blue-gray appearance to a violet purple color. Alkali is produced when casein, present in the milk solids, is utilized by certain dermatophytes. Acidification of the medium, noted by a color change from blue-gray to yellow, usually indicates bacterial or yeast contamination. (7,8) Filamentous fungi other than dermatophytes may also result in an acidic (yellow) medium. (8)

FORMULA

Ingredients per liter of deionized water:*

Skim Milk Powder 40.0gm
Glucose 20.0gm
Bromcresol Purple 16.0mg
Ethanol 1.0ml
Agar 15.0gm

Final pH 6.6 +/- 0.3 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

Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Consult listed references for information on specimen collection. (1-5)

Method of Use: The medium should be brought to room temperature, and the agar surface should be dry prior to inoculation. A small fragment of the suspect colony should be inoculated to the Dermatophyte Milk Agar. Incubate at 25ºC. for up to seven days. Examine media daily and observe for development of a violet-purple color change in the medium. Note any change in pH and the colony growth rate. Do not hold negative cultures longer than seven days.

INTERPRETATION OF RESULTS

Development of a violet-purple color around the fungal growth is indicative of an alkaline reaction.

Trichophyton mentagrophytes produces diffuse growth and an alkaline reaction within seven days of incubation.

Trichophyton rubrum produces restrictive growth and produces no color change in the medium within seven days of incubation. Typical T. rubrum isolates produce the typical red colony reverse pigment of the species within four to six days on Dermatophyte Milk Agar. (7) This pigment can be easily distinguished from a positive alkaline reaction. Further biochemical and/or serological testing is recommended for complete identification.

Microsporum persicolor isolates grow rapidly and diffusely on Dermatophyte Milk Agar but do not produce alkalinity after seven days, or more extended periods, of incubation. (7)

LIMITATIONS

Christensen Urea Agar and Vitamin-free Casamino Acids Agar may be inoculated in parallel to Dermatophyte Milk Agar to aid in the differentiation and identification of dermatophytes.

False-alkaline-positive reactions may result, if interpretations are made beyond 7 days of incubation. An alkaline reaction will eventually be produced by T. rubrum . Thus, results must be read within one week or immediately after the first growth and color reaction is observed.

It has been reported that, in rare instances, isolates contaminated with strongly caseolytic bacteria resulted in stunted growth and partially or completely suppressed alkaline reactions of T. mentagrophytes on Dermatophyte Milk Agar. (7)

Slow growing nodular isolates of T. mentagrophytes may produce slightly alkaline reactions after seven days of incubation. It may be necessary to extend incubation for an additional three days for development of a full positive alkaline reaction. Since T. rubrum will produce an alkaline result with extended incubation, it is recommended that further biochemical tests be performed for complete identification.

The caps of inoculated media must be kept loose to assure optimal recovery of dermatophytes.

Isolates contaminated with bacteria or yeast occasionally produce an acidic (yellow) reaction which seldomly hinders the separation of T. rubrum and T. mentagrophytes . (7)

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Trichophyton mentagrophytes
ATCC ® 9533
G 3-7 days 15-30°C Aerobic Profuse growth; violet-purple color in medium around colonies
Trichophyton rubrum
ATCC ® 28188
G 7 days 15-30°C Aerobic Restricted growth; no color change in medium

User Quality Control

PHYSICAL APPEARANCE

Dermatophyte Milk Agar should appear opaque, and blue-gray in color.

T. mentagrophytes growing on Dermatophyte Milk Agar

Trichophyton mentagrophytes (ATCC ® 9533) growing on Dermatophyte Milk Agar (Cat. no. L75). Incubated aerobically for 5 days at 30ºC.

T. rubrum growing on Dermatophyte Milk Agar

Trichophyton rubrum (ATCC ® 28188) growing on Dermatophyte Milk Agar (Cat. no. L75). Incubated aerobically for 5 days at 30ºC.

Dermatophyte Milk Agar

Uninoculated tube of Dermatophyte Milk Agar
(Cat. no. L75).

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Rebell, E. and Taplin. 1970. Dermatophytes, 2nd ed. University of Miami Press, Miami.

7. Summerbell, R.C., et al. 1988. J. Clin. Micro., Vol. 26, No. 11. American Society for Microbiology.

8. St. Germain, Guy, et al. 1996. Identifying Filamentous Fungi. Star Publishing Company, Belmont, CA.

9. Campbell, M.C. and Stewart, J.L. 1980. The Medical Mycology Handbook, John Wiley and Sons, New York.

10. Fischer, J.B. and Kane. 1971. J. Mycopathol. Mycol. Appl.; 43:169-180.

11. Fischer, J.B. and Kane. 1974. J. Can. J. Microbiol.; 20:167-182.


ATCC is a registered trademark of the American Type Culture Collection.

022316gr