Prior to receipt in the mycology laboratory, a portion of all tissue samples submitted for culture should also be placed in formalin for submission to the histology laboratory. Histopathologic examination together with appropriate stains are usually necessary to document fungal invasion. These may include the routine hematoxylin and eosin (H&E) stain, Gomori methenamine silver (GMS) stain, periodic acid–Schiff (PAS) stain, and others. Discussion of the use of histopathology and of mycological stains is found in Chap. 4 of this book. See also Appendix C of the CLSI M54-A document [5]. As a part of routine processing, the mycology laboratory should also examine a portion of the specimen directly by microscopy, typically with the use of a potassium hydroxide (KOH) preparation, gram stain, calcofluor white fluorescent stain, India ink stain (limited to cerebrospinal fluid examination for Cryptococcus neoformans), or some other method (Table 2.2). Observation of fungal structures by direct microscopy and/or histopathology is essential to corroborate organism recovery in culture (rule out contamination). Table 9.​4 in Sect. 9.1.1 of the CLSI M54-A document [5] provides a useful listing/description of yeasts and yeast-like fungi seen in clinical specimens.

The media used for primary isolation may vary according to personal preferences; however, certain basic tenets apply to all media used for primary recovery. Material from nonsterile sites should be cultured on media that will support fungal growth but also inhibit bacteria. Antibacterial agents, alone or in combination, are added for this purpose. Common choices include chloramphenicol (< 16 µg/ml), gentamicin (5–100 µg/ml), penicillin (20 µg/ml), streptomycin (40 µg/ml), and ciprofloxacin (5 µg/ml). These agents should not be included, however, when actinomycetes are suspected. Media may also be made selective by the addition of the eukaryotic protein synthesis inhibitor cycloheximide at 0.5 µg/ml. This may be useful in the detection of dimorphic fungi and dermatophytes; however, many clinically significant saprobic fungi may be suppressed, leading to failure in recovering opportunistic etiologic agents in compromised hosts. Therefore, media with and without this agent should routinely be employed. Enriched media with 5–10 %, sheep erythrocytes may be incorporated into the battery for fastidious thermally dimorphic fungi such as H. capsulatum and Blastomyces dermatitidis. Peptone-based versus plant-based media may also be a consideration. Many of the opportunistic filamentous fungi prefer plant-based media, producing more typical colony morphologies and more diagnostic structures, thus increasing the potential to make identification possible from primary plates. Plant-based media may also be made selective with antibacterial agents or cycloheximide. Table 2.3 lists several commercially available media that may be used for both primary isolation and identification. Please consult Sects. 10.2 and 10.3 in the CLSI M54-A document for additional information [5]. The choice of tubed versus plated media is made based upon space constraints, personal preference, and safety. The greater surface area provided by plates is preferred by many laboratorians (and always preferred by the fungi!), as manipulation of cultures, isolation procedures, etc. are more easily performed on plates. When used, plate lids should be firmly attached with an air-permeable material or plates sealed in air-permeable bags to avoid cross-contamination or laboratory worker exposure.

Optimally, cultures should be incubated at 30 °C (± 1 °C). If this temperature is not available, room temperature near 25 °C should be used. Seven-day incubation is generally adequate when screening for yeasts from oropharyngeal or vaginal sites. Although 4-week incubation times have been traditionally recommended, studies suggest that 3 weeks are adequate to detect fungal growth from most other specimens, excluding those from skin, hair, and nails, and in cultures requested specifically to attempt to recover dimorphic pathogens [27]. The time required for development of diagnostic structures, particularly for some coelomycetes and ascomycetes, may be considerably longer, up to several weeks [28, 29].