How to perform and interpret dermatophyte cultures

How to perform and interpret dermatophyte cultures

Use this guide—put together by a veterinary dermatologist—to maximize your success with this indispensable in-house test.
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Jul 01, 2010

Dermatophyte cultures can be challenging to perform and interpret correctly. However, knowing how to best collect samples for culture, select and incubate culture media, and identify media culture changes and fungal colony morphology will help you avoid a misdiagnosis.

COLLECTING SAMPLES FOR CULTURE

Hair pluck

To obtain samples for dermatophyte culture, use a sterile hemostat to pluck hairs from around the periphery of a newly formed or expanding skin lesion, avoiding areas that may have been recently medicated. Ideal hairs to select are those in areas of active crusting and hairs that appear damaged or misshapen.1

Toothbrush technique


Figure 1. The Mackenzie brush technique is used to collect samples for dermatophyte culture.
Hair plucks can potentially miss infected hairs and may not sample infected epithelium adequately, so it is ideal to also obtain samples using the Mackenzie brush technique. With this technique, a new toothbrush is removed from its packaging and is rubbed gently over the suspect area, including the skin and haired margins of alopecic or scaly lesions (Figure 1).1 Brush the unaffected area first, and then brush the lesions to avoid spreading spores to unaffected areas and to avoid losing spores from affected areas. Then gently embed the toothbrush bristles into the fungal culture media (Figure 2), taking care not to embed the bristles too deeply, which risks displacing the culture media when the bristles are removed. Use a sterile hemostat to remove hair and debris caught among the bristles, and place the material on the culture medium surface.


Figure 2. The toothbrush bristles have been gently pressed onto the fungal culture media.
The Mackenzie brush technique is helpful to screen for asymptomatic carriers and to obtain samples from animals undergoing antifungal treatment in which skin lesions have clinically resolved. In these cases, stroke the toothbrush over the entire body, concentrating especially on areas with prior lesions and, in cats, on the face, ears, and paws. It is recommended to brush for one minute or to brush the length of the animal 10 times.2 In animals undergoing antifungal therapy, repeat cultures every two or three weeks, and continue treatment until two negative culture results are obtained.2

In cases of suspected onychomycosis, the toothbrush can be used on the affected claw fold. Additionally, samples of claw fold fur can be obtained with a sterile hemostat, and the proximal affected nail can be sampled by using a scalpel blade to shave off small pieces of keratin. (Precleaning the nail with alcohol is recommend to help reduce accumulated saprophytic or environmental fungal organisms.) If an avulsed claw is considered for fungal culture, discard the distal part of the nail, and obtain samples by scraping the proximal concave surface of the claw.1

Toothbrushes can be obtained inexpensively in bulk from dollar stores or from online distributors. They can be used once and discarded or gas sterilized for repeated use.

SELECTING AND INCUBATING CULTURE MEDIA

Dermatophyte test medium contains Sabouraud's dextrose agar with cycloheximide, gentamicin, and chlortetracycline as antifungal and antibacterial agents to retard the growth of contaminant organisms. The pH indicator phenol red is also added.

Dermatophytes preferentially metabolize protein in the culture medium, releasing alkaline metabolites that turn the yellow fungal culture medium to red at the same time the dermatophyte colony appears. Most other fungi initially use carbohydrates and produce acidic metabolites; these saprophytic fungi can eventually consume protein and cause media color change, but it occurs several days after fungal growth appears.1,3 Daily observation and logging of fungal growth correlated with media color change is, thus, important in correctly interpreting dermatophyte test medium culture results.

Culture plates are recommended over vials, as the vial openings are usually too narrow to pass toothbrush heads for inoculation or to easily sample fungal colonies for microscopic analysis.4 To facilitate fungal sporulation and identification, it may be helpful to use a dermatophyte test medium plate that has a separate area of plain Sabouraud's agar or rapid sporulation medium, which does not contain inhibiting agents. For example, the Dermatoplate-Duo (Vetlab Supply) culture plate has dermatophyte test medium on one side and enhanced sporulation agar on the other side.

According to recommendations from a fungal culture media manufacturer, culture media should be stored at 36 to 77 F (2 to 25 C) and protected from light before inoculation.5 The plates should be warmed to room temperature (77 to 86 [25 to 30 C]) before inoculation. Before and during the inoculation procedure, the plates should be handled in a manner that minimizes exposure of the media to the environment. Do not use expired plates or plates that exhibit drying, cracking, discoloration, microbial contamination, or other signs of deterioration. Excessive condensation may appear in plates that have been damaged by exposure to temperature extremes.5

Fungal cultures should be incubated at room temperature (77 to 86 F [25 to 30 C]) with 30% humidity.1,5 If room temperature is not maintained, use an incubator, or send the samples to a reference laboratory for culture.4

Most organisms will appear within seven to 10 days; however, plates should be kept for 21 days, especially when no growth is seen initially or when the sample has been obtained from a pet receiving antifungal therapy. According to a fungal culture media manufacturer, dermatophyte culture plates may be incubated in full light, although some authors recommend incubation in the dark to avoid UV light-induced inhibition of fungal growth.1,5 In dry climates, it is suggested that plates be placed in plastic bags or containers to prevent dehydration of the media, which can inhibit organism growth.5 After 48 to 72 hours, begin examining the plates daily for characteristic media color changes and fungal growth.