Blood and Bone Marrow
The laboratory should be informed by the physician if fungal septicemia is suspected because special media are necessary for the optimum recovery of fungi.
Numerous blood culture systems are available; however all systems must be vented to atmospheric air and incubated at 30C to maximize the rate and time of recovery of fungal organisms.
Aseptically collect 10 ml of blood and prepare several smears for Giemsa, Gram and PAS staining. Culture the remaining specimen by one or more of the following methods. With bone marrow aspirates the initial material is generally used for making smears for Giemsa staining, the remaining 3-5 ml of marrow and blood may be cultured on the media listed below.
1. Direct culture method.
Inoculate 0.5-1.0 ml of buffy coat, prepared by centrifuging 5-10 ml of blood, onto the surface of the media listed below. This inoculum can then be spread over the surface of the agar with a sterile inoculating loop and the plate incubated aerobically at 30C. This method is suitable for small low volume laboratories where there are few requests for fungal blood cultures. Cultures should be maintained for 4 weeks.
2. Biphasic filter technique.
The recovery of fungi from blood may be enhanced by using a biphasic bottle containing a slant of brain heart infusion agar and 60-100 ml of BHI broth. A ratio of 1:10 to 1:20 (blood to broth) is recommended, a minimum of 5.0 ml of blood is required for each culture bottle. The biphasic culture bottle is kept vented and is tilted daily to allow broth to flow over the agar surface. These cultures must be carefully checked daily for growth. Because fungi will not turn the broth very cloudy it is imperative to frequently Gram stain the bottle contents to detect fungal elements. Cultures should be incubated at 30C and maintained for 4 weeks.
3. Membrane filter technique.
This is a superior technique to the vented biphasic blood bottle used to concentrate and culture specimens of blood and CSF. Briefly, specimens are treated sequentially with Triton-X and sodium carbonate solutions to lyse blood cells and then filtered by vacuum through a 0.45 um membrane. This membrane is then placed onto the media listed below.
4. Lysis centrifugation isolator system.
The Wampole Isolator system has been found to significantly improve the recovery of fungi from blood and is strongly recommend by Koneman and Roberts (1985) as the method of choice for processing blood cultures from patients with suspected fungal septicemia. The Isolator utilizes a tube that contains components that lyse leukocytes and erythrocytes and also inactivate plasma complement and certain antibiotics. Once lysed, the cells release the microorganisms contained within them, and the centrifugation step in the procedure serves to concentrate the organisms in the blood sample. This concentrate is then inoculated onto the surface of appropriate culture media listed below. Ten milliliters of blood are required for each tube and cultures should be incubated at 30oC and maintained for 4 weeks.
Bactec have produced a special fungal media (BACTEC Fungal Medium) for enhanced fungal blood culture using their non-radiometric (NR) instruments. Once again, blood cells are lysed by the medium to enhance recovery of fungi. Note antimicrobials have also been added to limit the growth of bacteria.
Primary isolation media for blood and bone marrow culture: (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.
Note: 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.