Table Of ContentCHAPTER 1
Isolation and Identification
of Yeasts from Natural Habitats
John E l! Spencer and Dorothy M. Spencer
1. Introduction
Yeasts are relatively easy to isolate from natural habitats, the technique
being to weigh out a sample of the substrate, suspend it in sterile water,
and streak or spread a suitable dilution on either a nonselective medium
(usually acidified malt extract agar to suppressb acterial growth) to obtain
specimens of the total population, or on a selective medium to obtain cul-
tures of particular species (1,2). After the plates have been incubated at
an appropriate temperature and colonies have developed, these are exam-
ined under a dissecting microscope to select at least one of each colony
type. Unfortunately, many yeasts cannot be distinguished immediately
by visual examination of either the vegetative cells or the colonial mor-
phology, so that it is better to isolate as many colonies as can be handled,
for identification.
In samples containing a low density of the yeast population, such as
natural water, it may be necessary to concentrate the samples rather than
dilute them, by passing the sample (usually l&50 mL) through a sterile
membrane filter, and then placing the filter on a plate of the isolation
medium. Colonies are usually small and discrete and can be picked easily.
2. Materials
1. Acidified malt extract medium: Autoclave the malt extract agar, and let
cool to about 60°C.A dd approx 0.7 mLJ100 mL. of medium, of lNHC1, to
a final pH of 3.7-3.8, mix thoroughly, and pour plates (I).
From* Methods m Molecular Biology, Vol 53 Yeast Protocols
Edited by. I. Evans Humana Press Inc., Totowa, NJ
1
2 Spencer and Spencer
2. Yeast-nitrogen base, 0.67% (all percentages are w/v, unless otherwise
stated),p lus various selective carbon sources, 1% unless otherwise stated( 2).
a. For Cryptococc~s species,e specially Cryptococc~s cereanu.s from rot-
ting cactus, meso-mositol.
b. For Candida sonorensis, 0.5% methanol.
c. For Pichia cactophila, Pichiapseudocactophila, and Pichla norvegensis,
glucosamme.
d. For Pichia mexicana, a species of Clavispora, Debaryomyces hansenii,
and some species of Cryptococcus, N-acetylglucosamine.
3. Yeast-carbon base, plus different nitrogen sources (2). Nitrate, to select for
specieso f Hansenula (now, nitrate-positive specieso f Achia, Citeromyces,
and some species of Candida). This method can be used to detect the pres-
ence of Candida utzlis in commercial baker’s yeast. Other selective nitro-
gen sources include lysine, creatinme, ethylamine, and nitrite.
4. Millipore or other membrane filter apparatus, sterile filters, and flat twee-
zers for handling filters.
5. Dissecting microscope for examining colonies.
6. Other standard equipment for mlcroblological investigation.
7. Collecting vessels and tools. These can be sterile jars, flasks, or tubes for
liquid samples, or small plastic bags for solids. (Whnlpack bags are suit-
able.) Collecting tools may vary according to the nature of the samples,
and may be a spoon or knife, alcohol, and swabs for resterilizing the col-
lecting tool, some kind of reaching pole and bottle handler for sampling
small, shallow bodies of water, or some type of standard collectmg bottle
for taking samples at depth, for deep lakes or seas.
3. Method
1. Collect specimensi n sterile containers. On returning to the laboratory (unless
a mobile laboratory is available), use the following procedure.
2. Weigh out samples into tubes of sterile water.
3. Streak or spread on plates of acidrfied malt extract agar or YM agar (Difco,
Detroit, MI), pH 3 7-3.8 (I), and incubate for 3-6 d at approx 25OC, or at
any temperature that is appropriate for the type of yeasts expected in the
sample. Examine plates at frequent intervals, as the agar may be over-
grown with filamentous fungi if left too long.
4. After incubation, examine colonies under the dissecting microscope, and
pick at least one of each colony type, but preferably more. Count any dis-
tinctive types (form or color) to determine their frequency in the total popu-
lation. If possible, determine the total colony count.
5. Restreak these colonies on malt extract agar or other appropriate medium,
determine their morphological characteristics (colony morphology,
Identification of Yeasts 3
vegetative cells, and spores), and maintain the cultures on any suitable
medium for more detailed study.
6. Attempt to identify isolates (see Note 2). For example, determine cellular
morphology, presence or absence of spores, and in sporulating cultures,
spore and ascus morphology. Also, determine fermentation and assimila-
tion patterns for carbon sources (1,3,4) and assimilation of nitrogen
sources. Attempt to find the species in a recognized key (3,4).
4. Notes
1. A few yeast speciesf rom specialized habitats require particular procedures
for isolation and handling.
2. Brettanomyces species form acid in quantity and die quickly in the com-
mon media. Addition of 0.5% calcium carbonate to yeast extract-glu-
case-peptone agar allows longer survival. Use of CaCOs in the isolation
medium allows detection of acid-forming species.C lear zones form around
the colonies.
3. Cyniclomyces guttulatus inhabits the rabbit digestive tract, and can be iso-
lated from rabbit feces. It requires an array of amino acids, or else yeast
autolysate in the isolation medium, an incubation temperature of 37”C,
and a CO* concentration of 15% v/v in the atmosphere. 1% Proteose pep-
tone, 1% dehydrated yeast autolysate, and 2% glucose, pH 3.5 (adjusted
with 1N HCl), sterilized in a flowing steam, is satisfactory. This species
sporulates at about 18”C, which is outside the temperature range permit-
ting growth, so sporulation medium (YM medium or similar) must be
inoculated with a heavy inoculum of vegetative cells, to obtain spores.
4. Osmotolerant species (Zygosaccharomyces, Candida mogii, Schizo-
saccharomyces octosporus) may require elevated concentrations of sugar
or NaCl in the isolation medium. However, they can usually be adapted
easily to the commonly used media, such as yeast extract-peptone-dex-
trose agar. Yeasts occurring in saline lakes, such as Metschnikowia
bicuspidata, when isolated from infected brine shrimp in saline lakes, may
require 2% NaCl in the isolation medium.
5. Identification may present some problems for the beginner. Detailed and
extensive protocols for determining the morphological and physiological
characteristics, used in identification of yeast species, are given in The
Yeasts, a Taxonomic Study, by N. J. W. Kreger-van Rij (4), and these
should be followed exactly. The guidance of an experienced yeast taxono-
mist is highly desirable.
The volumes by Barnett et al. are either older (1983) (3) or very expen-
sive, and are based only on physiological criteria, but contain a great deal
of useful information. A computer program is available from Barnett.
4 Spencer and Spencer
Methods of identification based on molecular genetics (DNA reassoci-
anon, characterization, of ribosomal RNA (rRNA) (7) and DNA (rDNA)
(5,6) and PCR studies of particular sequences of DNA (8), are extremely
useful for identification and also for determination of taxonomic position,
but are in the province of the specialist.
6. Formation of pseudohyphae is no longer considered significant as a taxo-
nomrc criterion, and the genus Torulopsis has therefore been merged with
Candida.
References
1. Phaff, H. J. and Starmer,W . (1987) Yeastsa ssociatedw ith plants, msectsa nd soil,
in The Yeasts, vol. 1, Biology of Yeasts (Rose, A. H. and Harrison, J S., eds.),
Academic, London, pp. 123180.
2 Wickerham, L. J. (195 1) Taxonomy of Yeasts. Techn. Bull. 1029, US Department
of Agriculture, Washington, DC
3. Barnett, J. A., Payne, R. W., and Yarrow, D. (1983) Yeasts: Characteristics and
Identijkat~on, Cambridge University Press, Cambridge, UK.
4. Kreger-van Rij, N. J. W. (1984) The Yeasts* A Tuxonomic Study, Elsevier,
Amsterdam.
5. Lachance, M.-A. (1988) Restriction mapping of rDNA and taxonomy of Kluyvero-
myces van der Walt emend. van der Walt. Yeast S(Specia1 Issue), S379-S383.
6. Shen, R. and Lachance, M.-A. (1993) Phylogenetic study of ribosomal DNA of
cactophthc Pichia species by restriction mapping. Yeast 9,3 15-330.
7. Yamada, Y., Maeda, K., Nagahama, T., Banno, I., and Lachance, M.-A. (1992)
The phylogenetic relationships of the genus Sporopachydermiu Rodriguez de
Miranda (Saccharomycetaceae) based on the partial sequences of 18s and 26s
rtbosomal RNAs. J, Gen. Appl. Microblol. 38, 179-l 83.
8. Fell, J. W. (1993) Rapid identification of yeast species using three primers in a
polymerase cham reaction. Mol. Murlne Biol. Biotechnol. 2(3), 174-180.
CHAPTER2
Maintenance and Culture of Yeasts
John F. T. Spencer and Dorothy M. Spencer
1. Introduction
Yeasts generally are very easy to maintain, store, cultivate in almost
any size of batch, and isolate from most habitats, but if a few elementary
methods are understood, their isolation, storage,a nd culture, for day-to-day
use in the laboratory is greatly simplified. Furthermore, the same methods
can be used for maintenance of a stock collection for use in a production
plant in a yeast-basedin dustry. Being a group of unicellular organisms,y easts
can be handled in the same way as most bacteria, except that their nutri-
tional and environmental requirementsa re, with few exceptions, much sim-
pler. The techniquesu sed will be consideredu nder the following headings:
1. For short-term maintenance, for daily use. Rich, undefined media, such as
yeast extract-peptone-glucose agar, YM agar, or malt agar, are commonly
used (see Section 3.);
2. For medium-term storage;
3. For long-term preservation, as in a culture collection, where immediate
access is less important, but maintenance of the characteristics of the spe-
cies and strain is the primary objective; and
4. Methods for cultivation of yeastsi,n small-,m edium-, and large-scale volumes.
2. Materials
2.1. Growth Media (in g/L)
1. Malt agar: dehydrated malt extract (30 g), mycological peptone (0.5 g),
agar (15 g).
2. MYYGP( YM) agar: dehydrated malt extract (3 g), dehydrated yeast extract
(3 g), peptone( 5 g), glucose( 10 g), agar (15 g).
From+ Methods m Molecular Biology, Vol 53 Yeast Protocols
Edlted by* I Evans Humana Press Inc., Totowa, NJ
5
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
1O X 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.
Maintenance and Culture of Yeasts 7
The commercially prepared media are available without amino acids,
without vitamins, or without either, if desired, to allow greater flexibility
in making special-purpose media.
Cultures may be stored in the above media in screw-capped tubes or
bottles, such as McCartney bottles, though ordinary culture tubes with
cotton plugs or plastic caps are satisfactory for periods of 3 or 4 wk or
less. Cultures can be stored on Petri plates, on similar media, in the
refrigerator, for a few weeks, if they are kept from drying out by placing
in plastic bags or plastic boxes with tight-fitting covers.
2.2. Sporulation Media (2)
These are included, since the production of spores, by various yeast
species, is an important type of culture of yeasts on solid or liquid media,
in some investigations.
1. McClary’s medium (g/L): potassiuma cetate( 10 g), yeast extract (2.5 g),
glucose( 1. Og ).
2. Raffinose-acetatem edium: raffinose (20 g), potassiuma cetate( 10 g).
3. Sodium acetatem edium: sodium acetate( 5 g). Adjust pH to approx 6.8
with HCl.
The describedm edia aret hosem ost often usedf or sporulationo f strains
of Saccharomyces cerevisiae. They may be used asl iquid media or solidi-
fied by addition of 1.5% (all percentagesa re w/v, unless otherwise indi-
cated) agar.
4. Gorodkowa agar: Glucose (1 g), peptone( 10 g), NaCl(5 g), agar (20 g).
Glucose (2.5 g) and meat extract (10 g) are some times recommended for
Debaryomyces spp.
5. Vegetableju ice agar:E qualw eightso f potatoesb, eetsc, arrotsa, ndc ucumbers
are washed (not peeled),g rated, and autoclavedf or 10 min and pressed
through cheesecloth. Dried yeast (2%) and agar (2%) are added and the
medium is melted, dispensed, and autoclaved.
Wickerham (1950) recommendeda commercial vegetablej uice prepa-
ration (V8 juice, 350 mL), diluted in water (350 mL) containing 5 g of
moist yeast and 14 g of melted agar, and the pH adjusted to 6.8. The melted
medium is dispenseda nd sterilized.
6. Dilute V8 agar. Commercial V8 juice is diluted 1: 1 with demineralized
water and adjusted to pH 5.5 with NaOH. Further dilutions are made to
1:2, 1:9, 1: 19, and 1:29, agar is addedt o these dilutions, melted, and the
medium is dispenseda nd sterilized.
This medium is used to induce sporulation in Metschnikowia species
and other yeastst hat do not sporulateo n other media.
8 Spencer and Spencer
7. Potato agar: Peel and grate 100 g of raw potatoes and soak in 300 mL of
tapwater for several hours at 4OC. Filter the liquid through cloth, and auto-
clave for 1 h at 15 psi. To prepare the agar, add 230 mL of the extract to
770 mL tapwater containing 20 g glucose and 20 g agar. Dissolve the agar
by heating, dispense the medium, and autoclave for 15 min at 15 psi.
8. Corn meal agar: Soak 12.5 g yellow corn meal in 300 mL of water, with
stirring, in a waterbath at 60°C for 1 h and recover the liquid by filtering
through paper. Make up the volume to 300 mL and add 3.8 g of agar.
Autoclave the material for 15 min at 15 psi, filter the hot liquid through
absorbent cotton wool, dispense, and sterilize for 15 min at 15 psi.
9. Vegetable wedges:W ith a cork borer, cut cylinders, about 1- cm diameter, from
carrot, potato, beet, cucumber, or turnip, afier thorough washing. Cut wedges
obliquely through them. Rinse the wedges again, place them in culture tubes
with a little water to prevent drying out, and autoclave for 15 min at 15 psi.
10. Gypsum blocks: Make by mixing eight parts of gypsum with three parts of
water. Cast the paste into small cylinders or cones, 3-4 cm high and allow
to set. Place the blocks in covered jars or dishes and hold at 1 lO-120°C for
2 h. Before use, add sterile water or a solution of a mannitol-phosphate
(18 mL of OS-2.0% mannitol + 2 mL of 5% K@PO,), 1 cm deep, to the
dishes. Spread a thin layer of freshly harvested yeast cells on the gypsum.
Or, prepare gypsum slants by pouring the paste into sterile, plugged tubes,
slant them, and harden them by holding at 50°C for 24-48 h.
11. Rice agar: Put 20 g of white rice into 1 L of tapwater and bring to the boil.
Let simmer for 45 min m a covered pot, and then filter the liquid through a
cloth. Let cool (discard the solids), make up the volume to 1 L, and add
20 g agar. Melt the agar by steaming, dispense in tubes or bottles, and
autoclave for 15 min at 15 psi.
12. Oatmeal agar: Add 4 g of oatmeal to 100 mL of tapwater, boil for 1 h, and
filter through cheesecloth. Discard the solids, make up the filtrate to
100 mL, and add 1.5 g of agar. Melt the agar, mix well, and dispense in
tubes. Autoclave 15 min at 15 psi.
2.3. Media for Germination
of Teliospores of Basidiomycetous Yeasts
1. Hay infusion agar: Add 50 g of decomposing hay to 1 L of demineralized
water, autoclave for 30 mm at 15 psi, and filter off the solids. Dissolve 2 g
of K&IP04 and 15 g of agar in 1 L of the filtrate, adjust the pH to 6.2,
dispense the material in tubes or bottles if desired, and autoclave for
20 min at 15 psi.
2. Sucrose-yeast extract agar: Add 1 g of KH2P04, 0.5 g of MgS04*7H20,
0.1 g of CaC12, 0.1 g of NaCl, 5.0 pg of biotin, 20 g of sucrose, 0.5 g of
Maintenance and Culture of Yeasts 9
yeast extract, and 40 g of agar to 1 L of demineralized water, and dissolve
these constituents by heating. Autoclave the medium for 15 min at 15 psi.
2.4. Materials for Medium-Term Storage
1. Storage on agar slants, liquid medium, or under mineral oil: Slants of yeast
extract-peptone-dextrose agar or bottles or tubes of yeast extract-peptone-
dextrose broth, as described previously; sterile mineral oil; pipeter with
sterile tips, set to deliver 5-10 mL of oil.
2. Drying on filter paper (3): Whatman (Maidstone, UK) No. 4 filter paper or
equivalent, cut into l-cm squares, wrapped m aluminum foil and auto-
claved; yeast cultures, grown in a heavy patch on a plate of YEPD agar, for
2-3 d at 30°C; suspending medium (evaporated milk, commercial, sterile);
sterile toothpicks (blunt-ended); desiccator.
2.5. Materials for Long-Term Storage
2.5.1. Lyophilization
1. Cultures grown on YEPD slants for about 48 h, at 25-30°C.
2. Suspending media: Skim milk (20%), DMSO (5%), sodium glutamate
(lo%), or inactivated horse serum no. 5, contammg 7.5% glucose, sucrose,
or inositol (Wellcome Reagents Ltd., Beckenham, UK). (Skim milk is prob-
ably better and is cheaper. Reconstituted skim milk powder may be used.)
3. Freeze-drying apparatus, if available.
4. Acetone + dry ice freezing bath, if a commercial freeze-drying apparatus
is not at hand.
5. Ampules, standard.
6. Filter paper labels, sterile, to fit inside the ampules. The labels are num-
bered, placed inside the ampules, the ampules are plugged loosely with
nonabsorbant cotton, and sterilized.
7. Sterile Pasteur pipets for filling ampules.
8. Small cotton plugs for ampules.
9. Torch, for sealing ampules under vacuum.
10. Where a commercial drier is not available:
a. Vacuum pump in good condition.
b. Acetone-dry ice bath, for freezing.
c. Manifold for attaching ampules for vacuum drying.
2.5.2. Storage Under Liquid Nitrogen
1. In ampules:
a. Plastic ampules, 2 mL, screwcap, sterile.
b. Inoculum: yeast cells, grown in liquid YM medium (Difco), grown at
25°C for 72 h with shaking, to a cell density of 106-lo7 cells/ml.
10 Spencer and Spencer
c. 10 or 20% v/v Cryoprotectant, sterile glycerol solution, 10% v/v DMSO,
YM broth (Difco), 5-l 0% hydroxyethyl starch.
d. Liquid mtrogen refrigerator, and also a domestic freezer (-30°C) if
available, or a cooling bath.
e. Dewar flask for transporting frozen ampules.
f. Racks for holding the ampules in the freezer, and other ancillary equipment.
2. In straws:
a Polypropylene “straws,” prepared from 2.5-cm lengths of drmkmg
straws of this material. (The sections are held tightly, 1 mm from the
end, in a pair of unridged forceps, and this end is held about 1 cm from
the flat flame of a fishtail burner. The polypropylene melts very quickly
and forms a seal. The melted part sets firmly m a few seconds when
removed from the heat and the end is thus sealed. The straws are auto-
claved at 121°C m a glass Petri dish.)
b. Inoculum and cryoprotectant are prepared as in method 1.
2.5.3. Freezing in Glycerol Solution (3)
1. Inoculum: yeast cells, grown for 48 h in YE, YM, or other normal medium,
to approximately 107-108cellslmL.
2. Cryoprotectant: glycerol, sterile.
3 Small culture tubes or vials, 3-4 mm diameter, sterile.
4. Freezer, capable of operation at -7O“C.
3. Methods
3.1. Medium-Term Storage
3.1.1. Storage on Agar Slants,
in Ltquid Medium, or Under Mineral Oil
Two tubes or slants of each culture are inoculated and incubated at
25°C for 48 h. One tube is reserved for starting a new stock culture when
required; the other is used for daily transfers as required. The tubes or
slants are closed tightly and refrigerated. If it is desired to keep the cul-
tures under oil, after incubation, the tubes are filled with sterile mineral
oil to a level just above the level of the agar and then refrigerated. The
cultures normally remain viable for 6-12 mo, although different species
have different storage lives.
3.1.2. Drying on Filter Paper (3)
1. Place three or four drops of sterile evaporated milk in separate locations m
a sterile Petri dish.
