Introduction of Corn Meal Agar (CMA)
Corn Meal Agar (CMA) is a well established fungal medium which 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. Corn meal 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.
Principle of Corn Meal Agar (CMA)
The medium consists of only corn meal infusion and agar which is a very simple formulation.However this infusion has enough nutrients to
enhance the growth of fungi. Agar acts as solidifying agent that provides solid physical form. Corn meal agar with tween 80, tween 80, a mixture of oleic esters, stimulates the production of chlamydospores by C. albicans, C. stellatoidea and occasionally C. tropicalis and also useful for the maintenance of fungal stock cultures.
Composition of Corn Meal Agar (CMA)
Corn Meal Agar (CMA) ingredients and their amounts are as follows-
Ingredients Gms / Litre
- Corn Meal Infusion: 2.0gm
- Tween 80: 7.0ml
- Agar: 15.0
- Distilled water(D/W): 1000 ml
Final pH 6.2 +/- 0.3 at 25ºC.
Preparation of Corn Meal Agar (CMA)
- Suspend 17.0 grams Corn Meal Agar (CMA) in 1 liter purified / distilled or deionized water.
- Heat to boiling to dissolve the medium completely.
- Add 7 ml Tween 80.
- Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- After autoclaving, leave for cooling to 45-50°C.
- Mix well before dispensing.
- Pour Corn Meal Agar into each plate and leave plates on the sterile surface until the agar has solidified.
- Store the plates in a refrigerator at 2-8°C.
Storage and Shelf life of Corn Meal Agar (CMA)
- Store at 2-8ºC and away from direct light.
- Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
- Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.
- Test specimens ( samples or fungal growth)
- Inoculating loop
- Bunsen burner
- Control strains (Candida albicans ATCC® 10231 and Candida krusei ATCC® 6258)
- Biological Safety cabinet (BSC)
- Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
- Take plates.
- 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 coverslip has cooled, place 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 cover slip for detection of chlamydospore formation.
- Follow steps one through six of the above procedure for the examination of sporulation of molds. Incubate until mold is visible and mount cover slip in glycine.
- Examine microscopically for characteristic structures.
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 reverse side of 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.
- Corn meal 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.
Key Notes on CMA
- The addition of `Tween 80’ (e.g. 1%) to Corn Meal Agar greatly enhances the development of chlamydospores on the medium
- Mackenzie found that all 163 isolates of Candida albicans obtained from laboratories in the United Kingdom produced chlamydospores on Oxoid Corn Meal Agar but Dawson using only 27 isolates of Candida albicans, found that Oxoid Czapek Dox Agar and rice infusion agar were slightly superior for chlamydospore production
- A single Petri dish containing Corn Meal Agar may be used to identify four or five different colonies of Candida grown on Sabouraud Dextrose Agar. Using a straight wire, pick a colony off the surface of the latter medium and make a deep cut in the Corn Meal Agar (i.e. a horizontal furrow). Repeat for each colony. Place a flamed sterile coverslip over the line of inoculum. After incubation for 24 to 48 hours at 22°C, the streaks are examined microscopically, through the cover slip, using a low power objective. Along such streaks, Candida albicans produces mycelium-bearing ball-like clusters of budding cells and the characteristic thick-walled round chlamydospores.
- Glucose supplemented Corn Meal Agar should not be used for chlamydospore production.
- Corn Meal Agar with `Tween 80’ (or other wetting agents) will allow Candida stellatoides and Candida tropicalis to produce chlamydospores.
- Some Candida strains lose their ability to produce chlamydospores after repeated subculturing.
Limitations of Corn Meal Agar (CMA)
- Recommended temperature is 25ºC for the best results of Chlamydospore formation because of inhibition of Chlamydospore At 30-37ºC.
- Further tests need colonies from pure culture for complete identification like biochemical, immunological, molecular, or mass spectrometry
- A non-selective and selective medium should be inoculated for isolation of fungi from potentially contaminated specimens.
- Repeated subculturing of some Candida strains result in a loss of their ability to produce chlamydospores.
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