Yeast Morphology Agar

Intended Use

Yeast Morphology Agar is used for classifying yeasts based on colonial characteristics and cell morphology.

Summary and Explanation

Yeasts are unicellular, eukaryotic, budding cells that are generally round-to-oval or elongate in shape.¹ They multiply principally by the production of blastoconidia (buds).¹ Yeast colonies are moist and creamy or glabrous to membranous in texture.¹ Yeasts are considered opportunistic pathogens.¹
The yeast media cited are prepared according to the formulas of Wickerham.^2-6
These media are included in many applications for the study of yeasts in molecular genetics.^10,11

Principles of the Procedure

Yeast Morphology Agar contains all essential nutrients and vitamins necessary for the cultivation of yeasts, including a source of carbohydrate.

User Quality Control

Identity Specifications
Yeast Morphology Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:                       3.5% solution, soluble in purified water upon boiling. Solution is very light amber, slightly opalescent.
Prepared Appearance:     Very light amber, slightly opalescent without significant precipitate.
Reaction of 3.5%
Solution at 25°C:            pH 5.6 ± 0.2
Cultural Response

Yeast Morphology Agar
Prepare the medium per label directions. Inoculate using the pour plate technique and incubate at 25-30°C for 18-48 hours. Also, inoculate by the Dolman technique (streak and point) and add coverslips. Incubate at 25-30°C for 6-7 days and examine microscopically for hyphae.

Formula

Yeast Morphology Agar
Approximate Formula* Per Liter
Nitrogen Sources
Ammonium Sulfate........................................................ 3.5 g
Asparagine................................................................... 1.5 g
Carbon Source
Dextrose.................................................................... 10.0 g
Amino Acids
L-Histidine Monohydrochloride..................................... 10.0 mg
LD-Methionine........................................................... 20.0 mg
LD-Tryptophan.......................................................... 20.0 mg
Vitamins
Biotin........................................................................... 2.0 μg
Calcium Pantothenate................................................ 400.0 μg
Folic Acid...................................................................... 2.0 μg
Inositol.................................................................. 2,000.0 μg
Niacin....................................................................... 400.0 μg
p-Aminobenzoic Acid.................................................. 200.0 μg
Pyridoxine Hydrochloride............................................. 400.0 μg
Riboflavin................................................................. 200.0 μg
Thiamine Hydrochloride.............................................. 400.0 μg
Compounds Supplying Trace Elements
Boric Acid................................................................. 500.0 μg
Copper Sulfate........................................................... 40.0 μg
Potassium Iodide...................................................... 100.0 μg
Ferric Chloride........................................................... 200.0 μg
Manganese Sulfate.................................................... 400.0 μg
Sodium Molybdate..................................................... 200.0 μg
Zinc Sulfate.............................................................. 400.0 μg
Salts
Monopotassium Phosphate ........................................... 1.0 g
Magnesium Sulfate....................................................... 0.5 g
Sodium Chloride........................................................... 0.1 g
Calcium Chloride.......................................................... 0.1 g
Agar ......................................................................... 18.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

Yeast Morphology Agar
1. Suspend 35 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Yeast Morphology Agar
Inoculate plates using the Dolman technique. This is an excellent method for studying the hyphae of filamentous yeasts.
1. Near one side of the plate (from the relative positions of 10 o’clock to 2 o’clock), lightly inoculate a single streak taken from a slant culture.
2. In addition to the single streak, inoculate two points near the other side of the plate (at the 4 o’clock and 8 o’clock positions).
3. Cover a central section of the streak inoculation and one point inoculation with cover glasses, as follows:
a. With forceps, remove a cover glass from absolute alcohol, drain momentarily, and burn off excess alcohol by passing over a low flame.
b. When the cover glass has cooled, place one edge on the agar and allow it to fall across the central portion of the inoculated streak. Place a second cover glass over one point inoculation.
4. Incubate at 25-30°C for 6-7 days.
5. After incubation, observe with a high dry objective.

Expected Results

Yeast Morphology Agar
Using the high-dry objective, observe for hyphae of filamentous yeasts.

Limitations of the Procedure

Yeasts grown on a rich medium may carry a reserve of nitrogen in the form of protein. Possible errors due to this reserve are eliminated by making two serial transfers in the complete medium. When the first transfer is seven days old, the culture is shaken and one loopful is transferred to a second tube of the complete medium containing the same source of nitrogen. If a positive test is obtained when the second culture is seven days old, the organism being tested assimilates this particular nitrogen source.

*Store at 2-8° C.

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