Laboratory Diagnosis of Fungal Infections
Fungi are in the airTypes of specimens
Common fungal primary recovery culture mediaSAB agar (sabouraud dextrose agar ) *Brain-heart infusion agar *Potato flake agar These media used for primary recovery of saprophytic & dimorphic fungi.
Dermatophyte test medium *Mycosel Thesemedia used for identification of dermatophytes .
Direct examination Potassium hydroxide (10 % to 20 %) The most rapid method for direct examination of infected material . Gently heating the slide will increase the rate of clearing & the fungus should be more readily observed.Cultivation of fungi*Sabouraud glucose agar with antibiotics (penicillin &chloramphenicol )andcycloheximide will support most of the pathogenic fungi.*Place a small amount of hyphae or spores or both on the center of the agar medium in a petri dish using a stiff 22 –gauge nichrome wire in an inoculating needle holder
Temperature requirement Majority of fungi – 37°CSuperficial mycosis – 30°CDimorphic fungi – 25°C & 37°C Incubation timeAt least 4 weeksUsually positive cultures are obtained in 7-10 daysCandida & Aspergillus - 24 to 72 hrs
Identification of fungal cultures
Colony morphology – colour, texture, pigment production
Specimens should be cultured on agar slants:
Safe Require less space More resistant to drying during prolonged incubation Blood cultures should be inoculated in to biphasic blood culture bottlesDirect mounts
*Using sterile technique ,remove a small portion of the colony from near the center. *Place material in a drop of lactophenol with or without cotton blue mounting medium . The cotton blue dye is desirable for light- colored or colorless fungi.If a material is dense ,tease a part with two sterile needles & add a cover slip. *Examine under low magnification of the microscope , then higher magnification for smaller organisms.
Slide culture
In order to avoid disturbing the arrangement of the fungus structures ,slide culture techniques can be utilized& permanently stained mounts made from the slide and cover glassSlide culture technique
MycologySaprophytes: Hyaline Members - Aspergillus spp. Growth rate varies, colors vary, surface velvety to cottony. Mycelium - septate and hyaline with unbranched condiophores A. fumigatus is considered a potential pathogen, especially if from a pulmonary source.
Saprophytes: Hyaline Members - Penicillium spp. Commonly rapid growing; white to bluish-green. Conidiophores characteristically form a brush-shaped structure. Sterigmata are flask shaped.
Saprophytes: Dematiaceous Members - Cladosporium spp. Rapid growth. Green colonies, reverse is black. Septate, dematiaceous mycelium. Conidia are borne in chains.
Saprophytes: Aseptate Members - All are susceptible to cycloheximide. Rapid growers. Some have root-like structures termed rhizoids. Spore bearing structures are called sporangiophores.