Dermatophyte Test Medium (DTM): Introduction, Composition, Principle, Test Requirements, Procedure, Result-Interpretation, Colony Characteristics, Modifications,Uses, and Keynotes

Introduction of Dermatophyte Test Medium (DTM)

Dermatophyte Test Medium (DTM) is a selective and differential medium recommended for the cultivation and isolation of pathogenic dermatophytes. The Dermatophytes are a distinct group of fungi that infect the hair, skin, and nails in humans producing a variety of cutaneous infections known as ringworm. Dermatophytes are a group of three genera like Trichophyton, Microsporum, and Epidermatophyton are responsible for most cutaneous fungal infections.

Principle of Dermatophyte Test Medium (DTM)

Soya peptone provides nitrogenous and carbonaceous substances essential for growth. Dextrose( glucose) serves as the energy source for metabolism. The pH indicator, phenol red, is used to detect amine production. Cycloheximide inhibits most of the saprophytic fungi. Chloramphenicol acts as a broad-spectrum antimicrobic that inhibits a wide range of gram-positive and gram-negative bacteria.  The presence of growth on the medium provides presumptive identification of dermatophytes. DTM helps in the isolation and early recognition of members of the Microsporum, Trichophyton by means of the distinct color change from yellow to red. Rapidly growing species may effect a complete color change within 3 days while slow growers will change color proportionately for a longer time. Non-Dermatophytes can be recognized by the absence of color change. A few saprophytes, yeasts, and bacteria change the medium from yellow to red, but can be easily distinguished by colonial morphology.

Composition of Dermatophyte Test Medium (DTM)

           (Hardy Diagnostics)

Dermatophyte Test Medium (DTM) ingredients and their amounts are as follows-

Ingredients	Gms / Litre
Papaic Digest of Soybean Meal	10.0
Dextrose	10.0
Cycloheximide	0.5
Phenol Red	0.2
Chloramphenicol	0.05
Agar	20.0
Distilled water (D/W)	1000 ml
Final pH at 25ºC	5.6 +/- 0.2

Preparation of Dermatophyte Test Medium (DTM)

1. Suspend 40.75 grams of Dermatophyte Test Medium in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 10 minutes. Note:  Avoid overheating at any time.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Mix well before dispensing.
6. Pour Dermatophyte Test Medium into each plate or tube and leave plates or tubes on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Dermatophyte Test Medium (DTM)

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for DTM

• Test specimens ( samples or fungal growth about Specimen Collection: Submit Infectious material 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.)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (For positive control: Trichophyton mentagrophytes
  ATCC 9533, Candida albicans ATCC 10231 and for negative control: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923)
• Biological Safety Cabinet (BSC)

Test procedure for DTM

1. Allow the plates to warm at room temperature, and the agar surface to dry before inoculating.
2. Take plates or tubes.
3. Inoculate the specimen directly onto the medium by pressing the specimen lightly into the surface of the agar.
4. Alternatively, place a small amount of fungus on the agar surface if sub-culturing from another culture medium.
5. A control medium was inoculated in Sabouraud Dextrose Agar parallelly.
6. Incubate media at room temperature (15-30ºC), aerobically, for up to 14 days.
7. Examine media daily and observe for the development of a red color change in the medium.
8. Most pathogenic dermatophytes will produce a color change in three to six days.
9. Examined media daily for up to 14 days.

Result- Interpretation of DTM

• Positive: The appearance of white aerial hyphae and red color around the fungal growth is positive for the presence of dermatophytes fungi.
• Negative: Growth, without a color change to red, indicates that the organism is probably not a dermatophyte. Further biochemical and/or serological testing needs 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 are not typical of dermatophytes even though the media may turn red.
• Control strains: Positive control- Shows growth of Trichophyton mentagrophytes (color change in the medium) and Candida albicans (no color change in the medium) while Negative control- shows no growth of Escherichia coli and Staphylococcus aureus (growth inhibited)

Colony Characteristics of various organisms in Dermatophyte Test Medium (DTM)

• Trichophyton mentagrophytes: Growth; white colonies and a red color change develop in the medium surrounding the colonies.
• Candida albicans: Growth; small white colonies and no color change in the medium
• Microsporum audouinii: pink-red
• Aspergillus brasiliensis: No growth
• Escherichia coli: No growth
• Staphylococcus aureus: No growth
• Pseudomonas aeruginosa: No growth or poor growth

Trichophyton mentagrophytes colony characteristics on Dermatophyte Test Medium

Trichophyton mentagrophytes on DTM

Trichophyton mentagrophytes in LPCB Tease Mount

Trichophyton rubrum growth on DTM

Trichophyton rubrum in LPCB tease mount

Microsporum ferrugineum growth on DTM

Microsporum ferrugineum in LPCB tease

Fungal infected nail

Fungal infected nail in KOH mount microscopy showing chlamydospores and fungal hyphae

Young culture of Epidermophyton floccosum on DTM

Thin-walled macroconidia in cluster and chlamydospores but lacking microconidia of Epidermophyton floccosum in LPCB tease mount microscopy

Uses of Dermatophyte Test Medium (DTM)

1. Dermatophyte Test Medium (DTM)  is used for the primary isolation and identification of dermatophytes fungi like Epidermophyton, Microsporum, and Trichophyton species from hair, nails, or skin scrapings and scaling scalp lesions.
2. It is also applicable for the selective isolation of dermatophytes in veterinary specimens.

Keynotes on DTM

1. The infection caused by these organisms( dermatophytes) is commonly referred to as a ringworm.
2. The pH indicator is useful in distinguishing dermatophytes, which utilize nitrogenous material for preferred metabolism, producing alkaline by-products, imparting a red color change to the medium while typical saprotrophic fungi utilize carbohydrates in the medium producing acidic by-products and no red color change.
3. The lack of availability of chlortetracycline in late 1992 resulted in the substitution of chloramphenicol for chlortetracycline.
4. Some manufacturers provide antimicrobial agents (i.e. cycloheximide,
   chlortetracycline chloramphenicol, and gentamicin) from outside to add on Dermatophyte Test Agar Base like Oxoid (Cycloheximide and
   Chloramphenicol on Dermasel agar base), Himedia (cycloheximide,
   chlortetracycline and gentamicin on DTM agar base) whereas Hardy Diagnostics, Remel, and BBL  incorporate directly on Dermatophyte Test Medium.
5. Gentamicin inhibits gram-negative bacteria including Pseudomonas species while chlortetracycline inhibits a wide range of gram-positive and gram-negative bacteria.
6. Sterilize DTM by having antimicrobial agents by autoclaving at 121°C for only  10 minutes and also avoid overheating at any time.
7. Himedia suggests aseptically addition of Dermato Supplement ( antimicrobial agents rehydrated vial)  in the cooling step (45-50°C) before pouring into sterile Petri plates whereas Oxoid ( Dermasel selective supplement -vials ) prior to autoclaving.

Dermatophytes in KOH Mount of Skin Scrapping

Limitations of Dermatophyte Test Medium (DTM)

1. Colonies from pure culture for complete identification need other tests like biochemical, immunological, molecular, or mass spectrometry.
2. This medium is more useful as a general screening test, as opposed to an identification medium.
3. Don does not do result interpretations beyond 6 days of incubation otherwise false-positive reactions may result. An alkaline reaction will eventually be produced by most non-dermatophytic fungi that are capable of growing on this medium.
4. Don’t culture the dormant area of an infection otherwise false-negative reactions may arise.
5. Keep loose the caps or lids of inoculated media to assure optimal recovery of dermatophytes.
6. Certain strains of yeast may produce a color change in the medium. These organisms will produce a characteristic white, creamy, bacteria-like colony will and thus allow differentiation from dermatophytic fungi.
7. In heavily contaminated specimens, saprophytic fungi may result in a color change on the medium.  Recognize some of these organisms by their dark green to black hyphae; white aerial hyphae are unique by dermatophytes.

Further Readings

1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
6. https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1365.pd
7. https://catalog.hardydiagnostics.com/cp_prod/content/hugo/DTM.htm
8. https://legacy.bd.com/ds/technicalCenter/inserts/8814091(03).pdf
9. https://himedialabs.com/TD/M188.pdf
10. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp