Culture Media Preparation Tips

DERMATOPHYTE TEST MEDIUM (DTM)

Written by MicroDok

Dermatophyte test medium (DTM) is a specialized agar used in medical mycology (Fig. 1). It is based on Sabouraud’s dextrose agar with added cycloheximide to inhibit saprotrophic growth, antibiotic to inhibit bacterial growth, and phenol red a pH indicator. DTM can be used with clinical specimen suspected for fungal infection such as: Scraping of skin, hair,nail lesion, scaling scalp lesions.

Fig 1. Illustration of DTM in a slant

Dermatophyte Test Medium is a modification of a commercial formulation made by Taplin in 1969. Nitrogenous and carbonaceous compounds essential for microbial growth are provided by soy peptone. Dextrose serves as the energy source for metabolism. Chloramphenicol acts as a broad spectrum antimicrobic which inhibits a wide range of gram-positive and gram-negative bacteria. Cycloheximide is added to inhibit saprophytic fungi. Phenol red, the pH indicator, is affected by the presence of dermatophytes (Epidermophyton, Microsporum, and Trichophyton spp.), which all produce alkaline metabolites. Production of alkali results in the medium changing from yellow-orange to red in color.

Other organisms that may grow on the medium can be recognized as non-dermatophytes by their color and colony morphology. Bacteria and certain yeast can grow on this medium showing characteristic white or creamy bacteria like colonies. Contaminating saprophytes can turn Dermatophyte Test Medium from its yellow-orange color to red, but can be ruled out due to the green to black hyphae produced. Dermatophytes typically produce white aerial hyphae.

DTM fungal cultures are used to isolate and identify dermatophyte organisms such as Microsporum species, and Epidermophyton species, Trichophyton species. DTM is made with special ingredients that inhibit bacterial growth and turn red when dermatophytes grow. The pH indicator in DTM is useful in distinguishing a dermatophyte fungus, which utilizes nitrogenous material for preferred metabolism, producing alkaline by-products, imparting a red color change to the medium. Typical saprotrophic fungi utilize carbohydrates in the medium producing acidic by-products and no red color change.

COMPONENTS OF DTM

Ingredients per liter of deionized water (Table 1):*

Final pH 5.6 +/- 0.2 at 25ºC.

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

Table 1. Components of DTM

DIRECTION ON PREPARATION OF DTM

Suspend 20.10 grams in 500 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Aseptically add the rehydrated contents of one vial of Dermato Supplement (FD015). Mix well before pouring into sterile Petri plates.

PROCEDURE ON USAGE OF DTM

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation. Consult listed references for information on specimen collection.

Method of Use: The medium should be brought to room temperature, and the agar surface should be dry prior to inoculation. Once received by the lab, the specimen should be inoculated directly onto the medium by pressing the specimen lightly into the surface of the agar. Alternatively, a small amount of fungus may be placed on the agar surface if subculturing from another culture medium. A control medium, Sabouraud Dextrose Agar (Cat. no. L40), may be inoculated in parallel. Incubate media at room temperature (15-30ºC.), aerobically, for up to fourteen days. Examine media daily and observe for development of a red color change in the medium. Most pathogenic dermatophytes will produce a color change in three to six days.

INTERPRETATION OF RESULTS USING DTM

Media should be examined daily for up to fourteen (14) days.

Positive result: Appearance of white aerial hyphae and red color around the fungal growth is positive for the presence of dermatophytic fungi.

Negative result: Growth, without a color change to red, indicates that the organism is probably not a dermatophyte. Further biochemical and/or serological testing is recommended for complete identification.

If growth appears on the control medium (Sabouraud Dextrose Agar) and no growth appears on DTM, the organism is not a dermatophyte. Colonies with green or black hyphae is not typical of dermatophytes even though the media may turn red.

PRINCIPLE AND INTERPRETATION

The Dermatophytes are a distinct group of fungi that infect the hair, skin and nails of humans and animals producing a variety of cutaneous infections known as ringworm. Dermatophytes like Trichophyton, Microsporum and Epidermatophyton are responsible for most of the cutaneous fungal infections. DTM Agar Base was developed by Taplin as a selective and differential medium for detection and identification of dermatophytes. On this medium identification of Dermatophytes are based on morphology and alkaline metabolites production. A combination of three antimicrobial agents (cycloheximide, chlortetracycline and gentamicin) inhibits bacteria and saprophytic yeasts and moulds. Dermatophytes are presumptively identified based on gross morphology and the production of alkaline metabolites, which raise the pH and cause the phenol red indicator to change the color of the medium from yellow to pink-red. Soya peptone provides nitrogenous and carbonaceous substances essential for growth. Glucose is the energy source. The pH indicator, phenol red, is used to detect amine production. Cycloheximide (as FD) inhibits most of the saprophytic fungi. Gentamicin inhibits gram-negative bacteria including Pseudomonas species while chlortetracycline inhibits a wide range of gram-positive and gram-negative bacteria. The presence of growth on the medium provides presumptive identification of dermatophytes. DTM Agar helps in isolation and early recognition of members of the Microsporum, Trichophyton by means of the distinct colour change from yellow to red. Rapidly growing species may effect a complete colour change within 3 days while slow growers will change colour in proportionately longer time. Non-Dermatophytes can be recognized by the absence of colour change. A few saprophytes, yeasts and bacteria change the medium from yellow to red, but can be easily distinguished by colonial morphology. Complete classification of Dermatophytes depends on microscopic observations along with biochemical and serological tests.

LIMITATIONS OF DTM

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

This medium is more useful as a general screening test, as opposed to an identification medium.

False-positive reactions may result, if interpretations are made beyond 14 days of incubation. An alkaline reaction will eventually be produced by most non-dermatophytic fungi that are capable of growing on this medium.

If the dormant area of an infection is cultured, false-negative reactions may arise.

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

A color change in the medium may be produced by certain strains of yeast. A characteristic white, creamy, bacteria-like colony will be produced by these organisms and thus allow differentiation from dermatophytic fungi.

If the specimen is heavily contaminated, saprophytic fungi may result in a color change on the medium. Some of these organisms may be recognized by their dark green to black hyphae; white aerial hyphae is exhibited by dermatophytes.

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. Taplin, D. 1965. J. Invest. Der.; 45:545.
  8. Taplin, D., et al. 1969. Arch. Derm.; 99:203.
  9. Campbell, M.C., and J.L. Stewart. 1980. The Medical Mycology Handbook, John Wiley & Sons, New York.

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