Chlamydospore formation: Introduction, Test Requirements, Test Procedure and Result Interpretation

Chlamydospore

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.

Test Requirements

  • Test specimens ( samples or fungal growth)
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains (Candida albicans ATCC 10231 and Candida krusei ATCC 6258)
  • Biological Safety Cabinet (BSC)

Test procedure

  1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Take plates.
  3. 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”.
  4. 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.
  5. 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.
  6. 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.
  7.  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.
  8. Examine microscopically for characteristic structures.
  9. 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)

  1. Corn Meal Agar (CMA) is a well-established fungal medium for chlamydospore production by Candida species.
  2. It is also useful for the cultivation of fungi.
  3. 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.
  4. 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.

Gram stain: single or budding yeast cells with or without pseudohyphae  and gram-positive

Germ tube test: Positive

The test is carried out using 0.5 ml rabbit or human serum in which test yeast cells are inoculated and incubated at 37°C for 2-3 hours.

Put a drop of this after 2-3 hours incubation on the slide and cover with the coverslip. Focus at 10X objective and finally observe at high power objective (40X) of a compound microscope.

Result Interpretation

Germ tube test (GTT) positive:  Presence of sprouting yeast cells

Germ tube test negative: Absence of sprouting yeast cells

Chlamydospore formation

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-

Media used-

Sabouraud Dextrose Agar (SDA)

Potato Dextrose Agar (PDA)

Corn Meal Agar (CMA) Candida CHROMAgar

Antifungal Sensitivity Test Medium (AFST)

Colonial morphology -Colour of colonies on CHROMAgar

Gram staining under microscopy

Germ Tube Test (GTT)

Sugar Assimilation

#Candida growth on Cystine electrolyte Deficient agar ( CLED) agar-

#Sugar assimilation test medium preparation in the laboratory -manually as shown in video-

Further Readings

  1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3rd ed 1988 Publisher WB Saunder co, Philadelphia.
  3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. A Textbook of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
  5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  6. http://himedialabs.com/TD/M146.pdf
  7. http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp
  8. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CornMealAgarTween.htm
  9. https://www.sigmaaldrich.com/catalog/product/sial/42347
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