Ascospore Agar: Introduction, Principle, Procedure, Colony Morphology, Uses and Keynotes

Ascospore Agar: Introduction, Principle, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Ascospore Agar

Ascospore Agar is recommended medium for the expression of ascospores and therefore it is useful for enrichment and detection of ascosporogenous yeasts. It is the modification of the McClary medium with the addition of potassium acetate in place of sodium acetate. Saccharomyces cerevisiae is the most common yeast used to show expression ascopores.

Principle of Ascospore Agar

Yeast extract and dextrose supply the nutrients required for the growth and also stimulate ascospore formation in yeasts while acetate salt of potassium enhances sporulation in Saccharomyces. Slightly acidic pH of the ascospore agar favors the growth of S. cerevisiae. Agar is the solidifying agent and water is the source of hydrogen and oxygen.

Composition of Ascospore Agar

Ingredients  Gms / Litre
Yeast extract: 2.5
Dextrose:  1.0
Potassium acetate:  10.0
Agar : 30.0
Final pH ( at 25°C) 6.4±0.2

Preparation of Ascospore Agar

  1. Suspend 43.5 grams Ascospore Agar in 1 liter purified/distilled or deionized water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. After autoclaving,  leave for cooling to 45-50°C.
  5. Mix well before dispensing.
  6. Pour Ascospore Agar into each plate and leave plates on the sterile surface until the agar has solidified.
  7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life 

  • 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.
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

  • Test specimens ( samples or fungal growth)
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Control strains (Candida albicans ATCC  10231 and Saccharomyces cerevisiae ATCC 9763)

Procedure of Ascospore Agar,

  1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Inoculate and streak the specimen as soon as possible after collection.
  3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
  4. Streak for isolation with a sterile loop.
  5. Incubate one plate aerobically at room temperature ( 25-30°C) for up to 3-6 days.
  6. Examine the growth and perform LPCB tease mount Microscopy.

Result Interpretation 

In general, ascospores microscopy have no attachment points and are sometimes enclosed in gelatinous sheaths or within a sac.

Presence of ascospores: Positive

Absence of ascospores: Negative

Candida albicans: Luxuriant growth but no ascospores

Saccharomyces cerevisiae: Luxuriant growth with ascospores

Control strains

Candida albicans ATCC  10231: Negative

and Saccharomyces cerevisiae ATCC 9763: Positive

 

Use of Ascospore Agar

It is the medium used for the formation of ascopores of ascosporogenous yeasts.

 

Keynotes on Ascospore Agar

  • Ascospore Agar is the McClary modification medium in which sodium acetate has been replaced by potassium acetate to better enhance the rate of ascospores formation.
  • In general, ascospores have no attachment points and are sometimes enclosed in gelatinous sheaths or within a sac.

Further Readings 

  • https://assets.fishersci.com/TFSAssets/LSG/manuals/IFU9046.pdf
  • https://www.cdhfinechemical.com/images/product/specs/DM%201804.pdf
  • https://himedialabs.com/TD/M804.pdf
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