Chlamydospore formation: Introduction, Test Requirements, Test Procedure and Result Interpretation
Chlamydospores of Candida alibicans
Chlamydospores develop in a nutritionally deficient medium e.g. cornmeal agar(CMA) at 20°C. They can see at the end of pseudohyphae as shown above image. Corn Meal Agar (CMA) is a well-established fungal medium that is a suitable substrate for chlamydospore production by Candida albicans and the maintenance of fungal stock cultures. This is a very simple formulation containing only cornmeal infusion and agar. The addition of glucose (0.2 g% w/v) to CMA will enhance the chromogenesis of some species of Trichophyton e.g. Trichophyton rubrum. Cornmeal agar is an enrichment medium developed by Hazen and Reed for use in the cultivation of fungi. Walker and Huppert, in 1960, found that the addition of Tween 80 to CMA resulted in rapid and abundant chlamydospore formation.
Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
Using an inoculating needle, obtain visible paste of the organism. Draw the needle through the agar making two perpendicular lines in the shape of an “x”.
Flame a coverslip and allow it to cool. Once the coverslip has cooled, place it over the central area of the “x” in order to reduce oxygen tension. Reduced oxygen tension stimulates chlamydospore production. Leave some growth uncovered.
Seal the plate with tape or MycoSeals and incubate aerobically at room temperature (25-30ºC) for up to 72 hours in the dark. Examine daily for typical colonial growth and morphology.
Invert the plate and examine microscopically using a low power objective (10 X). View along the edge of the coverslip for detection of chlamydospore formation.
Follow steps one through six of the above procedure for the examination of sporulation of molds. Incubate until the mold is visible and mount coverslip in glycine.
Examine microscopically for characteristic structures.
For better observation of fungal structures, LPCB can be used as follows-
Take a clean and grease-free glass slide.
Put a large drop of LPCB with a Pasteur pipette or dropper.
Transfer a small quantity of the culture to the drop.
Tease the culture (in case of a mold) well with teasing needles so as to get a uniform spread.
Put on a coverslip gently to avoid entrapment of air bubbles.
Examine under low- (10 X) and high-power (40 X) objectives.
Observe the morphological features carefully as shown below.
Result Interpretation of CMA
Using low power magnification, examine for the presence of budding cells, hyphae, blastospores, and chlamydospores. Most strains of C. albicans and C. stellatoidea form typical chlamydospores after 24-48 hours incubation. Examine daily for up to four days. C. dubliniensis will also form chlamydospores, but in clusters, rather than singles as with the C. albicans. Control strains, Candida albicans: Good growth; white colonies and chlamydospores while Candida krusei: Good growth; white / cream colonies, no chlamydospores.
Colony Characteristics of various organisms in CMA
Candida albicans: Growth; smooth white colonies after 24-48 hours
Trichophyton rubrum: Growth seen in 7 days and it may take 3-4 weeks for red color on the reverse side of the colony to be visible.
Uses of Corn Meal Agar (CMA)
Corn Meal Agar (CMA) is a well-established fungal medium for chlamydospore production by Candida species.
It is also useful for the cultivation of fungi.
Cornmeal agar is a nutritionally impoverished medium and thus it is useful for the maintenance of stock cultures of fungi, especially the black-pigmented varieties.
The addition of glucose (0.2 g% w/v) to CMA is applicable for the chromogenesis of some species of Trichophyton e.g. Trichophyton rubrum.
Related Video on Candida
Identification of Candida albicans
Wet mount preparation: Single or budding yeast with or without pseudohyphae.
It forms in Cornmeal tween agar after incubation for 48-72 hours at 22-25°C. Chlamydospores are spherical, thick-walled, and usually produced on supporting cells that occur along pseudohyphae or at the tip of hyphae.
#Methenamine silver borate stained smear of Candida albicans under the microscope as shown below video clip-
#Carbohydrate Fermentation Test of Candida albicans–
#Candida albicans under Fluorescence microscope stained with Acridine Orange-
#Generally budding and rarely blastospore of Candida albicans in Gram’s stain-
# Use of chromagar for detection of various Candida species and they are-
C. albicans -light green color
C. dubliniensis -dark-green
C. tropicalis -blue to blue-gray color with a paler pink edge
C. krusei – a pink colony with a pale-pink to white edge
Trichosporon species- blue-green colony
#Candida glabrata isolation as shown below video-
Candida glabrata isolated by using the following tools and techniques- Growing the organism on SDA also on Candida chromagar Performing GTT and also further biochemical tests
# Candida tropicalis and its Laboratory Diagnosis-