Fungal isolation protocol

Protocol for processing of biopsy, tissue and lymph node specimens for fungal isolation

Prepared by Dr Evelina N Lagamayo

Edited by the AFWG committee

1. Why do I need this protocol:

To increase culture yield rate 

2. General recommendations

Collection and container	Sterile container with 1 mL of sterile NSS or BHI broth.
Processing	Cut tissue into small pieces (1 mm2) or mince on sterile petri dish with sterile scalpel blade and homogenize in sterile tube with sterile NSS/BHI broth. – If non-septate hyphae are observed, avoid grinding the tissue. – If septate hyphae are observed, cut/mince tissue into smaller pieces to increase the chance of isolation of the fungi. Inoculate the homogenate as well as the small pieces of minced tissue on appropriate agar plates (1x BHIA plate, 2x SDA plates and 1x SDA plate containing cycloheximide and chloramphenicol). Label plates accordingly and incubate until 30 days.
Media and incubation temperatures	Incubate the BHIA and 1x SDA (no antibiotics) plates at 35°C. Incubate 1x SDA plate (no antibiotics) and 1x SDA plate (with antibiotics) at room temperature (25-27°C).
NSS, normal saline solution; BHI, brain heart infusion; BHIA, brain heart infusion agar; SDA, Sabouraud dextrose agar

3. Special considerations

Corneal scrapings	Inoculate directly on culture media
If the tissue is hard	In addition to mincing, a tissue grinder or sterile glass beads can be used with sterile NSS or distilled water; vortex for a few seconds before inoculating on appropriate culture media.
If mucormycosis-causing species is suspected	Inoculate tissue directly on SDA plate after mincing with a sterile scalpel blade. Avoid grinding the tissue.
NSS, normal saline solution; SDA, Sabouraud dextrose agar

Note: This protocol is a guideline for increasing the yield of fungal isolation from specimens. Any laboratory can apply or use any other media as options, such as blood agar.