Isolation and Identification of Yeasts from Natural ... - Library Science

6 Spencer and Spencer
3. Saboraud’s glucose broth: glucose (40 g), mycological peptone (10 g),
agar (15 g).
4. Yeast extract-peptone-dextrose agar: This medium is used extensively for
maintaining and culture of yeast strains for genetic investigations. It con-
tains: Glucose (dextrose) (20 g), yeast extract (10 g), peptone (10 g), and
agar (15 g). The agar is omitted if the yeasts are to be grown in liquid
culture. For some purposes, such as in fermentation tests, the peptone is
omitted whether the yeasts are to be grown in liquid or on solid medium.
5. Wickerham’s chemically defined medium (I): This medium was intro-
duced by L. J. Wickerham about 1950, and has become the standard chemi-
cally defined medium for investigations of yeasts of most species. It is
available from Difco (Detroit, MI) in the dehydrated form, but can be made
up in large batches in the laboratory if desired. The reagents must be of
very high quality, as some grades and some brands contain toxic impuri-
ties that inhibit yeast growth.
a. Carbon source (for yeast-carbon base): glucose, 10 g.
b. Nitrogen source (for yeast-nitrogen base): ammonium sulfate, 3.5 g.
c. Amino acids: L-Histidine (10 mg), DL-methionine (20 mg), oL-tryp-
tophan (20 mg).
d. Salts mixture: KH2P04 (1 g), MgS04.7Hz0 (0.5 g), NaCl (0.5 g),
CaC12*6Hz0 (0.5 g).
e. Trace elements mixture: H3B03 (500 pg), MnS04*4H,0 (400 pg)
ZnS04.7H,0 (400 pg), FeC13*6H20 (200 pg), Na2Mo04*2H20 (200 pg),
KI (100 pg), CuS04*5Hz0 (40 pg).
f. Growth factor mixture: Myo-Inositol (10 mg), calcium pantothen-
ate (2 mg), niacin or nicotinic acid (400 pg), pyridoxine HCl(400 pg),
thiamine-HCl (400 pg), p-aminobenzoic acid (200 pg), riboflavin
(200 pg), biotin (20 pg), folic acid (2 pg).
The trace elements and growth factor mixtures can be made up as
1 OX or more concentrated mixtures and added in appropriate amounts
if the medium is to be filter-sterilized, which is desirable for the most
accurate determinations of growth and utilization of carbon and nitro-
gen sources. If it is to be autoclaved, then the vitamin mixture should
be filter-sterilized and added aseptically after the rest of the medium
has cooled.
For determination of assimilation of sugars and related compounds,
the sugar solution should be sterilized separately, preferably by filtration.
Separate sterilization of the yeast-carbon base is less important for
determination of assimilation of nitrite and nitrate, though it may be
desirable.