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Candida albicans Laboratory diagnosis

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

Morphogenesis of Candida albicans


At

25 C is single budding yeast

At

37 C as long pseudohyphae, hyphae and blastoconidia (3-8 subspherical gemantes cells x 2-7 um)

There are two ways to identify Candida


Ability

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).

How Germ Tube test is done

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

Germ Tube develops


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

fermentation means the production of gas and is independent of pH changes.