Laboratory Diagnostic in Medical Mycology

INTRODUCTION
Daily responsibilities of the clinical microbiology laboratory Opportunistic fungi is increasing at an impressive rate Laboratory technologists must recognize large group of potential fungal pathogen

Detection and recovery of fungi from clinical specimens

Dermatophytosis and Agents of Superficial Mycosis

Specimen and direct microscopic examination Skin, nail scraping and hair shaft Placed in one or two drops of 10-20% KOH A cover slip is placed on top and the preparation is heated gently Nails may require a strong alkali solution (25%KOH) and long clearing time N. B. combination of KOH plus Dimethyl sulfaoxide (DMSO) may be used for nail specimens

OBSERVATIONS
Hair

Endothrix hair invasion

Ectothrix hair invasion

Skin or nail

KOH microscopic examination of skin or nail

Isolation media
1. SDA plus antibiotics and cycloheximide (SDACGC) 2. SDACG) SDA Plus Antibiotics

Identification of etiologic agents Colony characteristics Color of the surface and reverse of colony texture of the surface (powdery, granules, woolly, cottony) rate of growth

Observation
Trichophyton violaceum Color Pink Texture Waxy Microscopic canis Color White with yellow reverse color Texture Woolly

Observation
Trichophyton rubrum Color White with brown to red reverse color Texture downy to fluffy

Microscopic morphology
Arrangement and type of the conidia macroconidia or macroconidia other structure determine by teased mounts or slide culture preparations mounted in lactophenol cotton blue special media used such as Corn Meal Agar or Potato Dextrose Agar to stimulate sporluation.

Microscopic morphology

Macroconidia of microsporum canis

chlamydospore of T. violaceum

Microconidia of T. rubrum

Pityriasis versicolor (Tinea versicolor)

A. Direct microscopic examination Skin scrapings are mounted in 10% KOH plus India ink or in lactophenol Cotton blue. Observation Short hyphae spaghetti like appearance Curved hyphae and yeast cells
A. Isolation media SDACG plus 2% Olive oil or Tween 80

Identification of yeast of medical importance with reference to Candida spp using the following scheme
Germ tube test Negative Corn meal agar Hyphae absent Unease test +Chlamydospores hyphae present arthroconidia (+) Trichosporon species or geotrichum spp (+) (-) Small cells or C. glabrate Mucoid with Capsule or red with capsule Cryptococcus spp Rhodoturula spps large cell S. cervisiae blastoconidia (-) Candida spp Biochemical tests positive mostly C. albicans

Identification of C. albicans and other Candida species

a. Gram stain Gram positive oval or round shape b. Germ tube test A small portion of an isolate colony suspended in a test tube containing 0.5 ml of rabbit or human serum or plasma The test tube is inoculated at 30C for 23hr examined microscopically for the presence of germ tubes N.B: Other Candida species produce pseudohyphae and true hyphae Observations: C. glabrate produces yeast form only C. albicans produce germ tube

Identification of C. albicans and other Candida species

c. Chlamydospore production For the differentiation of C. albicans from other Candida species using Corn Meal Agar plus 0.02 % Tween 80 Observation C. albicans produce chlamydospore after 24hr incubation at room temperature, while other Candida species produce only pseudohyphae and true hyphae. N. B. C. glabrate produces yeast form only

Aspergillus

Colony appearance Microscopic appearance Microscopic appearance

Structure of Aspergillus

Zygomycosis
Colony appearance

Microscopic appearance

structures