The mycetoma causative organisms can be identified by their textural description, morphological and biological activities in pure culture. The biological activity may include acid fastness, optimal temperature, proteolytic activity, utilization of sugars and nitrogenous compounds.
To culture the causative agents of mycetoma, sizeable amount of grains are needed. They should be soaked and stored in saline for culture, washed several times by normal saline and inoculated onto suitable culture media on sterilized condition using either safety cabinet or flame sterilized area. Modified Sabouraud agar supplemented with 0.5% yeast extract, blood agar, brain-heart infusion agar, and Löwenstein agar are the commonly recommended media.
For the isolation of actinomycetes, culture media should not contain antibiotics while the eumycetes culture should contain antibiotics. The commonly used antibiotics are penicillin G (20 U/ml), gentamicin sulphate (400 µg/ml), streptomycin (40 µg/ml), or chloramphenicol (50 µg/ml).
Nocardiae typically produce substrate and aerial hyphae that look like rods- and coccoids. Streptomyces form a yellowish substrate mycelium and lack aerial hyphae, while identification of Madurellais mainly based on the morphology of the fruiting bodies and morphology of the colonies.
Photograph showing M. mycetomatis growth
The phenotypic criteria of the causative organisms are important for their identification. These include the production of β-glucuronidase, degradation of adenine, casein and hypoxanthine, growth on adonitol, aesculin hydrolysis, glycerol, glycogen, meso-inositol, D-raffinose, L-rhamnose, D-turanose, D-xylose and L-aspartic acid. The later, as sole carbon sources play role in identification of pathogenic Streptomyces spp, A. maduraewas found to be positive for α-glucosidase and negative for N-acetyl-β-glucosaminidase.
Because of limited information available on phenotypic properties and assimilation patterns for identification of eumycetoma agents; new system, the API 20C AUX kits was recently used and was able to identify M. fahalii, M. pseudomycetomatis and M. Tropicana.
Differentiation of the various dematiaceous fungi, based on morphology is sometimes difficult, and time consuming, culture usually takes about three weeks to give accurate result and it need good experience.
In general, the culture technique is time consuming and accidental contamination may give a false positive result. It also requires experience to identify the causative organisms.