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Laboratory Diagnosis
CLINICAL MATERIAL: Skin or nail scrapings, urine, sputum and bronchial washings, pleural fluid and blood, tissue biopsies from various visceral organs. The specimen from a patient with suspected isolates of Candida has to be collected with meticulous care aseptically
Culture
Sample are immediately transferred to Sabourauds Dextrose media (agar or broth) with chloramphenicol Incubation period range from 8 to15 days @ (37 & RT ) The colonies can be apparent in 3-8 days On Sabouraud's dextrose agar colonies are white to cream colored, smooth, glabrous, opaque and yeast-like in appearance The colonies are composed of blastoconidia that reproduce by simple budding.
Culture contd
Should
be made with freshly collected samples. Isolates also grows on bacteriological media ( i.e. blood agar)
Macro morphology
At
37 C as long pseudohyphae, hyphae and blastoconidia (3-8 subspherical gemantes cells x 2-7 um)
to develop into a rich substrate proteins (serum albumin) projections (germ tubes). The ability to produce chlamydospores (forms of resistance) media poor in carbohydrates.
a. Candida albicans do not have constrictions at the point of origin, this is an Important diagnostic hallmark b. Candida dubliniensis has a germ tube with restriction. germ tubenegative Candida albicans strains tested further, by perfoming a chlamydospore test
Laboratory diagnosis
DIRECT EXAMINATION. Microscopic observation hyphae fresh preparation of some fluid with 10% KOH, or Gram staining. (Gems blastoconidias). The slide is observed for Candida morphology under oil immersion objective lens (100 X) of a Bright Field microscope.
Laboratory diagnosis
India
Ink Preparation: Negative - no capsules present. Dalmau Plate Culture on Cornmeal and Tween 80 Agar: Pseudohyphae with blastoconidia and terminal vesicles (chlamydoconidia).
The suspected Candida cultures are inoculated into 0.5 ml of human/horse serum in a small tube and incubated at 37 C for 2-2.5 hr. After desired period of incubation, a loop-full of culture is placed on a glass slide and overlaid with a coverslip. The preparation is examined for germinating blastospores i.e. Germ Tube
After human serum has been inoculated and incubated in 90 minutes at 37 C, the isolate begins to form hyphae called germ tube Pseudohyphae = yeast cell that continues to get longer, almost looking like hyphae Candida sp. can all produce pseudohyphae If see pseudohyphae,most likely a Candida sp. Growing evidence supports the view that hyphal growth is a response to nutrient deprivation, especially low nitrogen The extension of germ tubes into elongated hyphae by Candida albicans is essential for damage of host cells
Other tests
Methods of isolation characterization and identification of yeasts are very different from those of the mycelial fungi. Morphology is much less important, although C. albicans will form chlamydospores, Most yeasts, however, look pretty much the same. Physiology is far more important, therefore physiological tests used in the lab include: carbohydrate assimilation and fermentation Germ Tube test is Positive within 3 hours (discussed) Hydrolysis of Urea is Negative Growth on Cycloheximide medium is Positive Growth at 37C is Positive
Fermentation Reactions
Where