yeast-7.pdf

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Microbiology
Yeast–7
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YEAST SPORULATION
Yeast sporulation is useful for identification of some
species of wild yeast, because lager yeast generally
sporulates poorly, if at all. Sporulation can also be used
to help characterize yeast and to produce spores for ge-
netic studies.
Use of a presporulation medium (4–6) initiates
aerobic growth and enhances yeast sporulation after
transfer to the potassium acetate medium (1–4). A heavy
loopful of culture from the presporulation medium
should be used to facilitate recovery of yeast cells from
the sporulation medium. To help eliminate confusion
when identifying spores from a poorly sporulating
culture, familiarization with the analysis is recom-
mended.
Medium
(a)
MYGP presporulation medium
Malt extract
Yeast extract
Peptone
Glucose
Agar
Distilled water to
(b)
Acetate sporulation medium
Potassium acetate
Agar
Distilled water to
Apparatus
(a) Autoclave.
(b) Water bath, 45°C.
(c) Petri dishes or test tubes.
(d) Pipettes.
(e) Platinum loop.
(f) Incubator, 28°C.
(g) Staining apparatus, as in
Yeast-3B.
Method
Streak a loopful of a pure culture of the yeast to be
tested onto MYGP presporulation agar and incubate 24
h at 28°C. Transfer a heavy loopful of yeast from the
presporulation medium onto the acetate sporulation agar
and incubate the culture for a minimum of 3 days at
28°C. Transfer a film of the yeast to a glass slide, air
dry, heat fix, and stain as in
Yeast-3B.
Ascospores are
stained green to blue-green and vegetative cells pink to
red.
Count 1,000–1,500 cells with the oil immersion
objective. Report the percentage of sporulated cells and
the typical number of spores per ascus.
References
0.5 g
1.5 g
100 mL
1. American Society of Brewing Chemists. Report of Subcom-
mittee on Microbiology.
Journal
43:16, 1985.
2. European Brewery Convention. Analytica Microbiologica, Part
II. Method 2.3.2.3. Sporulation.
J. Inst. Brew.
87:308, 1981.
3. Fowell, R. R. Pages 305–385 in:
The Yeasts.
A. H. Rose and J.
S. Harrison, Eds. Academic Press, London, 1969.
4. Ingledew, W. M., and Casey, G. P.
Brew. Dig.
57:18, 1982.
5. Ingledew, W. M., and Casey, G. P.
Brew. Dig.
57:22, 1982.
6. Wickerham, L. J.
Taxonomy of Yeasts.
Pages 1–56 in: U.S.
Department of Agriculture Tech. Bull. No. 1029, 1951.
0.3 g
0.3 g
0.5 g
1.0 g
2.0 g
100 mL
Sterilize media for 20 min at 121°C. After cooling,
pour into sterile petri dishes or prepare slants.
Reagents
(a)
Malachite green,
as in
Yeast-3B.
(b)
Safranin-O,
in
Yeast-3B.
(c)
Ethyl alcohol,
95%.
(d)
Immersion oil,
for microscope.
1985, Rev 2010
doi: 10.1094/ASBCMOA-Yeast-7

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