Dermatophyte Test Medium (DTM): Composition, Preparation, and the Pathogens It Can Miss

The negative result that wasn't actually negative

A patient with a stubborn, discolored, thickened toenail has a nail clipping sent for fungal culture. The lab plates the sample onto Dermatophyte Test Medium, a convenient single-tube screen: red means dermatophyte, unchanged means probably not. Two weeks later, the tube is still yellow. No color change, no growth reported. Dermatophyte testing negative.

But the true cause of this infection was never a dermatophyte at all. It was a non-dermatophyte mold, one that happens to be sensitive to cycloheximide, the same antifungal DTM relies on to keep saprophytic contaminants from overgrowing the plate. DTM didn't fail here. It worked exactly as designed, suppressing an unwanted organism before it could interfere with the reading. The problem is that "unwanted contaminant" and "genuine pathogen sensitive to the same drug" can look identical to this medium: both simply don't grow.

This is exactly why DTM is a screening tool, not a final answer, and why understanding what it can silently suppress matters as much as understanding what it's designed to detect.

Figure: Dermatophyte Test Medium (DTM) Image source:labm.com

Dermatophyte Test Medium (DTM) is a specialized selective and differential medium used in medical mycology to differentiate dermatophytes (ringworm) from other fungi. Taplin and colleagues developed it by modifying Sabouraud Dextrose Agar (SDA).

DTM uses phenol red as a pH indicator. The medium is yellow at pH below 6.8, turning pink at pH ≥ 8.2 in the presence of alkaline metabolites produced by dermatophytes (Epidermophyton, Microsporum, and Trichophyton spp.). Contaminating saprophytes, if they grow, don't produce these alkaline byproducts within the testing window, typical saprotrophic fungi ferment carbohydrates into acidic byproducts instead, so the medium stays yellow. Culture results should be reported within two weeks of incubation; prolonged incubation increases the risk of false positives, since even slow-growing saprophytes can eventually shift the medium alkaline given enough time.

Composition

Functions of each ingredient:

• Soy peptone provides nitrogenous and carbonaceous compounds essential for microbial growth.
• Dextrose serves as the energy source for metabolism.
• Chloramphenicol acts as a broad-spectrum antibacterial, inhibiting a wide range of Gram-positive and Gram-negative bacteria.
• Cycloheximide, an antifungal agent, inhibits saprophytic fungi, but see the safety note below, this same inhibition can extend to some pathogenic fungi.
• Phenol red is the pH indicator.

The medium's own baseline pH, around 5.6, favors fungal growth generally. This is a separate fact from the phenol red indicator's own color-change thresholds (yellow below pH 6.8, pink at pH ≥ 8.2); the first describes the medium's starting condition, the second describes what the indicator dye does as dermatophytes shift that pH upward through their metabolism.

Preparation of Media

Depending on the requirements, individual laboratories either prepare DTM from dehydrated powder supplied by the commercial manufacturer, or prepared plates or tubes can also be purchased. Theoretically, it can be made in the laboratory using individual ingredients, which is rarely done in most laboratories.

Methods of Inoculation and Incubation

Suitable samples for DTM include plucked hair, skin scrapings, or nail clippings.

• Allow DTM to equilibrate to room temperature before sample inoculation. Make sure the agar surface is dry.
• Place the sample centrally on the surface of the medium and press it gently to ensure firm contact.
• Allow the cap on the tube to remain loose to ensure gaseous exchange during incubation.
• Incubate at 25°C for up to 2 weeks in ambient air.

Culture Results

Dermatophytes produce white or buff-colored growth within 5–10 days along with a red-colored medium; definitive identification requires additional testing; color change appearing only after two weeks is likely a contaminating saprophyte rather than a true positive.

Figure: Dermatophyte test medium (Left- uninoculated vial, middle and right vials showing growth compatible with Trichophyton mentagrophytes (Image source: Jorge Oliveira)

HOW TO REMEMBER

Anchor for the color logic: "red flags alkaline." Dermatophytes shift the medium alkaline as they metabolize, turning it pink/red. Saprophytes, still fermenting sugars into acid, leave it yellow. Red is the signal to take seriously; unchanged doesn't automatically mean "nothing's there."

Why DTM Can Miss the Real Pathogen

Cycloheximide is a double-edged design choice. It's specifically there to suppress fast-growing saprophytic contaminants that would otherwise overrun the plate before a slower-growing dermatophyte gets a chance to be detected. But cycloheximide sensitivity isn't limited to saprophytes: several clinically relevant fungi are also inhibited by it. A sample containing a genuine, cycloheximide-sensitive pathogen will simply fail to grow on DTM, indistinguishable, from the tube alone, from a clean, truly negative result.

This is exactly why DTM should never be the only medium used when a broader fungal differential is clinically relevant. Pairing it with a cycloheximide-free medium (such as plain Sabouraud dextrose agar without added antifungals) protects against exactly the failure mode illustrated in the hook above.

HOW TO REMEMBER

Anchor for cycloheximide's double edge: "the bouncer that turns away a few honest guests too." Cycloheximide is there to keep saprophytic gatecrashers off the plate, but it can't tell the difference between an unwanted contaminant and a legitimate, cycloheximide-sensitive pathogen. Both get turned away at the door, and both look identical from the outside: no growth at all.

Limitations

DTM is a screening test only; saprophytic fungi and yeast may also grow and produce alkaline metabolites, especially from soil-contaminated samples like foot or nail specimens; DTM should not be used as the sole means of dermatophyte identification.

Key exam facts in one table

Where Students Get Confused

• Assuming an unchanged (yellow) tube after two weeks always means no fungal infection. A cycloheximide-sensitive true pathogen can be suppressed just as effectively as an unwanted saprophyte, producing an identical "no growth" result.
• Treating a color change alone as a confirmed diagnosis. DTM is presumptive and screening-level only; definitive dermatophyte identification requires additional testing.
• Confusing the medium's baseline pH (~5.6, favoring growth) with the indicator's color-change threshold (≥8.2, signaling a positive read). These are two different pH facts serving two different purposes.
• Assuming longer incubation is always safer. Past the two-week window, the risk shifts toward false positives, since slow-growing saprophytes can eventually produce alkaline byproducts too.

References and further readings

1. Acharya T., Hare J. (2022) Sabouraud Agar and Other Fungal Growth Media. In: Gupta V.K., Tuohy M. (eds) Laboratory Protocols in Fungal Biology. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-030-83749-5_2
2. Taplin, D., Zaias, N., Rebell, G., & Blank, H. (1969). Isolation and recognition of dermatophytes on a new medium (DTM). Archives of Dermatology.