Tissue Biopsies from Visceral Organs
Ideally, tissue specimens should include both normal tissue and the center and edge of the lesion.
Tissue samples should be kept moist with saline or BHI broth and be transported to the laboratory as soon as possible. It is essential that histopathology also receive a portion of the tissue in formalin for rapid frozen sectioning with staining by H&E, GMS & PAS.
Tissue specimens received for culture should be aseptically teased apart in a sterile petri dish. If areas of pus and necrosis are present, inoculate these directly onto the isolation media listed below. Also perform a smear for direct microscopic examination. If there are no areas of pus or necrosis then process the specimen by mincing it into pieces as small as possible with a sterile scalpel blade, or for soft tissues by grinding in a sterile glass tissue grinder. Inoculate the minced or homogenized material onto the same media listed below.
Zygomycetous fungi will not survive the chopping up or tissue grinding process. If on clinical and/or radiological evidence zygomycosis is suspected then gently tease the tissue apart and inoculate it directly onto the isolation media. If you are not sure maintain the specimen in saline or BHI broth until the results of the frozen sections are known. If zygomycetous hyphae are present proceed as above, otherwise homogenize the specimen and plate out.
For direct microscopy examination of tissue sections stained with H&E, GMS & PAS is essential. Direct smears may also be made by smearing a small amount of tissue onto slides for Gram stain, and if necessary, Ziehl Neelsen stain and modified Ziehl Neelsen stain.
(a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and 35C; and
(b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26C for 4 weeks to exclude the presence of a slow growing fungus.