Yeast methods

March 16th, 2011

 

Maintenance and storage of cultures
Physiological testing using microplate technology
Media for Isolating, Cultivating, and Identifying Yeasts
Uses of the media
Media to use for
Instructions for reviving freeze-dried yeast cultures

Maintenance and storage of cultures

Ascomycetous yeasts are maintained on glucose-peptone-yeast extract agar (GPYA).
Many basidiomycetous yeasts do not survive well on the glucose-peptone medium, although they grow well on it. Such yeasts are kept on potato-dextrose agar (PDA). Most strains are stored at temperatures between 4 and 12°C and subcultured at intervals of c. 6 months. Some yeasts, for instance Arxiozyma and Malassezia, have to be subcultured every month. Dekkera and Brettanomyces produce excessive amounts of acetic acid, therefore 2% of calcium carbonate is added to the medium to neutralize the acid. Nevertheless, these yeasts still need to be subcultured every two months.
Cultures are frozen in either liquid nitrogen or a mechanical freezer at temperatures between -80° and -135°C for long-term storage. Freezing gives good results at the Centraalbureau voor Schimmelcultures. Cultures of all strains held are frozen and are successfully kept at -80°C and in liquid nitrogen. The preparation of cultures for freezing is simple and quick. The general method used is as follows: short lengths of polypropylene drinking straws are sealed at one end, labelled with a black felt-tipped pen (e.g. Pentel Permanent Marker), and sterilized in the autoclave at 121°C for 15 min. The strain to be frozen is grown for about 24 hr in 3.0 ml of liquid medium on a shaker before adding 1.0 ml of a 60 % solution of glycerol in water. An amount of the resulting suspension is pipetted into the straws sufficient to half fill them. The straws are then closed by clamping the open ends in the jaws of a sealing machine for plastic packages. The cultures are then either frozen at approximately -30°C for between 30 and 60 min before being placed in the storage tank under liquid nitrogen, or put directly into a freezer cabinet at -80°C. Sterile plastic ampoules suitable for use in liquid nitrogen can be bought but are more expensive and take up more storage space.

Physiological testing using microplate technology

Preparation of microplates

For the composition of the media, see below. Media are sterilized by heating or filtration prior to their addition to sterile microplates. Alternatively, filled and sealed microplates can be sterilized by gamma irradiation at 4 KGRay. This latter option should be favored if a gamma radiation facility is available. The wells of the assimilation and growth microplate wells (Nunc, 96 wells, flat bottom) are filled with 100 µl of the media described in the above table. Microplates are sealed by heat (polypropylene-aluminum sealing foil) and can be stored at -18°C or lower temperatures for more than one year.

Inoculation and incubation of microplates

Fifty µl of inoculum (MacFarland standard # 2 diluted by a factor of 10) is introduced into each well using a multi-channel (8 or 12 channels) pipette. Place a loose cellophane (do not seal since air should be able to circulate) on the microplate to avoid desiccation of the wells. Replace the cover of the microplate on top. The microplate is incubated at 25°C (for most of the strains) for 3 to 10 days. Agitation of the microplates during incubation is not required.

Test reading

Microplates are properly shaken (with a micoplate shaker) just before automatic reading using a microplate reader. Absorbance values at 405 nm are transferred by cable (RS-232 through a serial port) to the computer and transformed by the BioloMICS software into negative, weak or positive results. The results of every test are transformed independently.

Preparation of microplates

Media composition and positions in the assimilation microplate used at the CBS. Basal medium for carbohydrates assimilation tests (BMC): demineralized water 100 ml, yeast nitrogen base (Difco) 1.0 g. Basal medium for nitrogen compounds assimilation tests (BMN): demineralized water 100 ml, yeast carbon base (Difco) 1.77 g. Basal medium for growth testing without some vitamin compounds (BMV): demineralized water 100 ml, vitamin free yeast base (Difco) 2.52 g.

Test Amount Position in microplate

 

Test

Amount

Position in microplate

N Controle

BMN

A1

C Controle

BMC

A2

C1 D-Glucose

BMC + 0.78 g

A3

C2 D-Galactose

BMC + 0.78 g

A4

C3 L-Sorbose

BMC + 0.78 g

A5

C4 D-Glucosamine

BMC + 0.78 g

A6

C5 D-Ribose

BMC + 0.78 g

A7

C6 D-Xylose

BMC + 0.78 g

A8

C7 L-Arabinose

BMC + 0.78 g

A9

C8 D-Arabinose

BMC + 0.78 g

A10

C9 L-Rhamnose

BMC + 0.78 g

A11

C10 Sucrose

BMC + 0.78 g

A12

C11 Maltose

BMC + 0.78 g

B1

C12 a,a-Trehalose

BMC + 0.78 g

B2

C13 Me a-D-Glucoside

BMC + 0.78 g

B3

C14 Cellobiose

BMC + 0.78 g

B4

C15 Salicin

BMC + 0.78 g

B5

C16 Arbutin

BMC + 0.78 g

B6

C17 Melibiose

BMC + 0.78 g

B7

C18 Lactose

BMC + 0.78 g

B8

C19 Raffinose

BMC + 0.78 g

B9

C20 Melezitose

BMC + 0.78 g

B10

C21 Inulin

BMC + 0.78 g

B11

C22 Starch

BMC + 0.78 g

B12

C23 Glycerol

BMC + 0.78 ml

C1

C24 Erythritol

BMC + 0.78 g

C2

C25 Ribitol

BMC + 0.78 g

C3

C26 Xylitol

BMC + 0.78 g

C4

C27 L-Arabinitol

BMC + 0.78 g

C5

C28 D-Glucitol

BMC + 0.78 g

C6

C29 D-Mannitol

BMC + 0.78 g

C7

C30 Galactitol

BMC + 0.78 g

C8

C31 myo-Inositol

BMC + 0.78 g

C9

C32 D-Glucono-1,5-lactone

BMC + 0.78 g

C10

C33 2-Keto-D-Gluconate

BMC + 0.78 g

C11

C34 5-Keto-D-Gluconate

BMC + 0.78 g

C12

C35 D-Gluconate

BMC + 0.78 g

D1

C36 D-Glucuronate

BMC + 0.78 g

D2

C37 D-Galacturonate

BMC + 0.78 g

D3

C38 DL-Lactate

BMC + 0.78 g

D4

C39 Succinate

BMC + 0.78 g

D5

C40 Citrate

BMC + 0.78 g

D6

C43 Propane 1,2 diol

BMC + 0.78 ml

D7

C44 Butane 2,3 diol

BMC + 0.78 ml

D8

C45 Quinic acid

BMC + 0.78 g

D9

C46 D-glucarate

BMC + 0.78 g

D10

C47 D-Galactonate

BMC + 0.78 g

D11

C48 Palatinose

BMC + 0.78 g

D12

C49 Levulinate

BMC + 0.78 g

E1

C50 L-Malic acid

BMC + 0.78 g

E2

C51 L-Tartaric acid

BMC + 0.78 g

E3

C52 D-Tartaric acid

BMC + 0.78 g

E4

C53 meso-Tartaric acid

BMC + 0.78 g

E5

C54 Galactaric acid

BMC + 0.78 g

E6

C55 Uric acid

BMC + 0.78 g

E7

C56 Gentobiose

BMC + 0.78 g

E8

C57 Ethylene glycol

BMC + 0.78 ml

E9

C58 Tween 40

BMC + 0.78 ml

E10

C59 Tween 60

BMC + 0.78 ml

E11

C60 Tween 80

BMC + 0.78 ml

E12

N1 Nitrate

BMN + 0.11 g

F1

N2 Nitrite

BMN + 0.04 g

F2

N3 Ethylamine

BMN + 0.10 g

F3

N4 L-Lysine

BMN + 0.10 g

F4

N5 Cadaverine

BMN + 0.10 g

F5

N6 Creatine

BMN + 0.11 ml

F6

N7 Creatinine

BMN + 0.087 g

F7

N8 Glucosamine

BMN + 0.11 g

F8

N9 Imidazole

BMN + 0.047 g

F9

N10 D-Tryptophan

BMN + 0.10 g

F10

N11 D-Proline

BMN + 0.10 g

F11

N12 Putrescine

BMN + 0.10 g

F12

V1 w/o vitamins

BMV

G1

V2 w/o myo-Inositol

BMV + 300 µg calcium pantothenate, 3.0 µg biotin, 60 µg thiamine hydrochloride, 60 µg pyridoxine hydrochloride, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G2

V3 w/o Pantothenate

BMV + 1500 µg myo-inositol, 3.0 µg biotin, 60 µg thiamine hydrochloride, 60 µg pyridoxine hydrochloride, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G3

V4 w/o Biotin

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 60 µg thiamine hydrochloride, 60 µg pyridoxine hydrochloride, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G4

V5 w/o Thiamin

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 3.0 µg biotin, 60 µg pyridoxine hydrochloride, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G5

V6 w/o Biotin & Thiamin

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 60 µg pyridoxine hydrochloride, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G6

V7 w/o Pyridoxine

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 3.0 µg biotin, 60 µg thiamine hydrochloride, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G7

V8 w/o Pyridoxine & Thiamin

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 3.0 µg biotin, 60 µg niacin, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G8

V9 w/o Niacin

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 3.0 µg biotin, 60 µg thiamine hydrochloride, 60 µg pyridoxine hydrochloride, 30 µg  p-aminobenzoic acid, 0.3 µg folic acid, 30 µg riboflavin

G9

V10 w/o PABA

BMV + 1500 µg myo-inositol, 300 µg calcium pantothenate, 3.0 µg biotin, 60 µg thiamine hydrochloride, 60 µg pyridoxine hydrochloride, 60 µg niacin, 0.3 µg folic acid, 30 µg riboflavin

G10

O1 Cycloheximide 0.01%

Demineralized water 100 ml, D-glucose 1.5 g, yeast nitrogen base (Difco) 1.0 g, cycloheximide 0.0075 g

G11

O2 Cycloheximide 0.1%

Demineralized water 100 ml, D-glucose 1.5 g, yeast nitrogen base (Difco) 1.0 g, cycloheximide 0.075 g

G12

O3 Acetic acid 1%

100g glucose, 10g tryptone, 5g yeast extract, 1l deionized water. Cool the molten medium to approximately 45°C and add 1 ml of glacial acetic acid to each 100 ml

H1

O6 10% NaCl

10g NaCl, 5g glucose, 0.7g Difco Yeast Nitrogen base, 100ml deionized water

H2

O7 16% NaCl

16g NaCl, 5g glucose, 0.7g Difco Yeast Nitrogen base, 100ml deionized water

H3

O8 Growth at pH=3

Demineralized water 100 ml, D-glucose 1.5 g, yeast nitrogen base (Difco) 1.0 g, ajust pH with HCl 1N

H4

O9 Growth at pH=9.5

a. 10g Peptone, 6g       Yeast extract, 10g Glucose, 500ml deionized water

b. 400 mmol KCl, 40 mmol NaCl, 1800 mmol Na2CO3, 500 ml deionized water. Sterilize a and b seperately by autoclaving. Mix aseptically when cool and dispense in microplate.

H5

O10 Fluconazole

Demineralized water 100 ml, D-glucose 1.5 g, yeast nitrogen base (Difco) 1.0 g, fluconazole 0.8 g

H6

O11 Fluconazole

Demineralized water 100 ml, D-glucose 1.5 g, yeast nitrogen base (Difco) 1.0 g, fluconazole 6.4 g

H7

 

 

Media for Isolating, Cultivating, and Identifying Yeasts

Acetic-acid agar
100g glucose
10g tryptone
5g yeast extract
20g agar
1l deionized water
Cool the molten medium to approximately 45°C and add 1 ml of glacial acetic acid to each 100 ml, mix rapidly and pour into petri dishes.

Acidified media
To acidify agar media a determined amount of 1 N HCl is added after autoclaving. Add 7ml of acid to each litre of medium for a pH of approximately 3.8.

Actidione see Cycloheximide

Aqueous agar see Water agar

Canavanine-Glycine-Bromothymol blue medium
Stock solution A
300mg L-canavanine sulphate
100g glycine
10g potassium dihydrogen phosphate
10g magnesium sulphate heptahydrate
1l deionized water
either
10 drops Bejectal with vitamin C (Abbott, Chicago)
or
10mg thiamine hydrochloride
Adjust pH to 5.6 and sterilize by filtration.

Bromothymol-blue solution
0.4g sodium bromothymol blue
100ml deionized water
Sterilize by filtration.

Bromothymol agar
20ml bromothymol blue solution
20g agar
880ml deionized water
Cool the 900ml of bromothymol agar to about 55°C after autoclaving and add 100ml of stock solution A. Mix well and pour into tubes or plates.

Carbon base-urea agar
11.7g Difco Yeast Carbon Base
0.2g acid fuchsine
20g agar
800ml deionized water
200ml filter-sterilized 10% solution of urea
Add the urea solution to the other ingredients after they have been sterilized and cooled to approximately 50°C.

Chalk agar
20g glucose
10g finely powdered calcium carbonate
5g peptone
20g agar
1l yeast extract
Sterilize by autoclaving and gently agitate to keep the chalk in suspension while the agar is setting.

Chloramphenicol
0.1-1.0g chloramphenicol
1l medium
It can be added to the medium before autoclaving.

Christensen urea agar
1g peptone
5g sodium chloride
2g dihydrogen phosphate
0.012g phenol red
1l deionized water
Adjust the pH to 6.8, and then dissolve
20g agar
Sterilize by autoclaving at 121°C for 15 minutes, then add
100ml filter sterilized 20% solution urea

Corn-meal agar
15g agar
1l maize infusion
Powdered products are available from Oxoid and Difco.

Corn-meal + Tween agar
1l corn-meal agar
10ml tween 80

Custers agar
50g glucose
5g finely powdered calcium carbonate
20g agar
1l yeast extract
Agitate gently while the agar is setting to keep the chalk in suspension.

Cycloheximide 0.01% medium
0.1g cycloheximide
2.5ml acetone
6.7g Difco Yeast Nitrogen Base
10g glucose
100ml deionized water
Dissolve the cycloheximide in the acetone and the other ingredients in the water. Mix the solutions and filter sterilize. To use, add 0.5 ml aseptically to 4.5 ml of sterile water.

Cycloheximide 0.1% medium
1g cycloheximide
2.5ml acetone
6.7g Difco Yeast Nitrogen Base
10g glucose
100ml deionized water
Dissolve the cycloheximide in the acetone and the other ingredients in the water. Mix the solutions and filter sterilize. To use, add 0.5 ml aseptically to 4.5 ml of sterile water.

Cyniclomyces medium
Agar
10g yeast autolysate
40g glucose
10g proteose peptone
20g agar
1l deionized water
Sterilize 100ml amounts in bottles by autoclaving.
Melt the agar, cool to approximately 45°C, then adjust the pH with 1N HCl (add approx. 4.5ml to 100ml of agar) to between 3.5 and 4.5 and pour into petri dishes.

Broth
10g yeast autolysate
40g glucose
10g proteose peptone
1l deionized water
Adjust the pH with 1N HCl to approximately 4.0.

D-20 medium
6.7g Difco Yeast Nitrogen Base
1g yeast extract
1g malt extract
20g agar
1l deionized water

Dilute V8 agar
1 can V8 Vegetable Juice (Campbell Soup Co.)
Dilute juice with equal volume of water and add sodium hydroxide to bring pH to 5.5. Filter through Whatman No. 1 paper then dilute 1:9 and 1:19 before adding 2% of agar.

Diphenyl solution
1g diphenyl
100ml 95% ethanol
Add 10 ml of the solution aseptically to each litre of molten medium after it has cooled to approximately 45°C. Inhibits growth of moulds.

Fermentation medium
1l yeast extract
20g test sugar
(40g in the case of raffinose)
Some laboratories also add 7.5 g of peptone and use bromothymol blue as pH indicator. Dispense medium in tubes or bottles containing a small inverted tube (Durham insert). The insert should be full of medium after autoclaving.

Fowell acetate agar
5g sodium acetate (trihydrate)
20g agar
1l deionized water
Dissolve the acetate in water and adjust pH to between 6.5 and 7 before adding the agar.

Glucose 50% agar
500g glucose
500ml yeast extract
13g agar
(The volume of yeast extract plus dissolved glucose is 630ml, the amount of agar to add is calculated from this). Sterilize by autoclaving at 115°C (10psi) for 10 minutes. If the medium is over-heated during autoclaving it turns brown and must be discarded.

Glucose 60% agar
600g glucose
400ml yeast extract
22.5g agar
(The volume of yeast extract plus dissolved glucose is 750 ml, the amount of agar to add is calculated from this). Sterilize by autoclaving at 115°C (10psi) for 10 minutes. If the medium is over-heated during autoclaving it turns brown, in this case it must be discarded.

Glucose-peptone-yeast extract broth (GPYB)
20g glucose
5g peptone
1l yeast extract
pH is not adjusted.

Glucose-peptone-yeast extract agar (GPYA)
20g glucose
5g peptone
20g agar
1l yeast extract
pH is not adjusted. Dispense 200ml amounts in bottles (for plates) or 5ml in 16ml diameter test tubes for slants.

Glucose 2%-yeast extract agar
20g glucose
20g agar
1l yeast extract

Glucose 1%-yeast extract agar
10g glucose
15g agar
1l yeast extract

Gorodkowa agar
1g glucose
5g sodium chloride
10g peptone
20g agar
1l tap water
A modified version of this medium contains 2.5g of glucose, and 10g of meat extract is substituted for the peptone.

Hay-infusion agar
50g decomposing hay
1l deionized water
2g potassium monohydrogen phosphate
15g agar
Autoclave the hay in the water for 30 minutes at 121°C and then filter. Dissolve the phosphate and the agar in the filtrate and adjust the pH to 6.2.

Killer-test medium
YM agar
0.05M citrate buffer pH 4.2
0.003%w/v methylene blue

Leeming & Notman agar (LNA)
10g Bacteriological peptone
5g glucose
0.1g yeast extract
8g ox bile, desiccated
1ml glycerol
0.5g glycerol monostearate
0.5ml tween 60
10ml whole-fat cow's milk or cream
12g agar
1l deionized water

Leeming & Notman agar Modified (MLNA)
10g Bacteriological peptone
10g glucose
2g yeast extract
8g ox bile, desiccated
10ml glycerol
0.5g glycerol monostearate
5ml tween 60
20ml olive oil
15g agar
1l deionized water

Maize infusion
42g maize
1l deionized water
Heat the maize in water to 60°C for 1 hour, filter through paper, then add water to restore the volume to 1 litre.

Malt agar (MA)
1l malt extract (10° Brix) (brewer's wort)
20g agar
Alternatively the following may be used
100g malt extract powder
20g agar
1l water

Malt 5% agar
50g malt extract powder (Difco)
20g agar
1l deionized water

Malt 2% agar
20g malt extract powder (Difco)
20g agar
1l deionized water

Malt extract (ME)
Unhopped brewer's wort
if this is not obtainable prepare wort as follows:
1kg malt
2.6l tap water
Stir the malt in the water at 45°C for 3 hours, then raise the temperature to 63°C for 1 hour and filter the infusion through a cheese cloth. Autoclave the filtrate at 110°C for 15 minutes then filter it through paper.
The wort is diluted to a density of 15° Brix and the pH is adjusted to 5.4.

Midgley Dixon agar
36g malt extract (Oxoid)
6g mycological peptone
20g ox-bile (dessicated)
10ml tween 40
2ml glycerol
2ml oleic acid
15g agar
1l water

McClary acetate agar
1g glucose
1.8g potassium chloride
8.2g sodium acetate (trihydrate)
2.5g yeast extract
15g agar
1l deionized water

Morphology agar (MoA)
35g Difco Yeast Morphology agar
1l deionized water

Niger-seed agar (1)
1g glucose
20g agar
200ml niger-seed infusion
800ml deionized water
Chloramphenicol and diphenyl are sometimes added to inhibit growth of bacteria and moulds.

Niger-seed infusion
40g ground or pulverized Niger seed (Guizotia abyssinica)
200ml deionized water
Autoclave the seed in the water for 10 minutes at 115°C (10psi) and filter the infusion through gauze.

Niger-seed agar (2)
50g pulverized Niger seed
1l deionized water
Boil the seed in the water for 30 minutes. Filter through paper and restore final volume to 1 litre. Then add the following
10g glucose
1g KH2PO4
1g creatinine
15g agar
Streptomycin 40E/ml, Penicillin 20E/ml, and diphenyl solution are added when the medium has cooled to about 50°C .

Oatmeal agar
40g oatmeal
20g agar
1l tap water
Simmer the oatmeal in the water for 1 hour then filter through cheese cloth. Restore volume to 1 litre and dissolve the agar.

Olive oil
1 or 2 drops spread on the surface of media for cultivation of Malassezia spp.

PH10 medium
a. 10g Peptone
6g Yeast extract
10g Glucose
500ml deionized water

b. 400mmol KCl
40mmol NaCl
180mmol Na2CO3
500ml deionized water
Sterilize a and b seperately by autoclaving. Mix aseptically when cool and dispense 4ml amounts into test tubes.

Polyol agar
6.7g Difco Yeast Nitrogen Base
5g either ribitol or glucitol
15g agar
1l buffer

Potato-dextrose agar (PDA)
20g glucose
20g agar
230ml potato infusion
770ml deionized water

Potato infusion
300g well washed, grated or homogenized potato
900ml tap water
Soak the potato in water over night in a refrigerator. After filtering, autoclave the infusion for 1 hour at 110 C.

Rapid urea broth
Prepare according to the instructions on the container and dispense 0.5 ml amounts into sterile tubes. Store frozen until required.

Rice agar
20g agar
1l rice infusion
Commercial products are available but the results obtained with them and with an infusion of either polished or 'instant' rice are considerably inferior to those obtained with the medium prepared with an infusion of unpolished rice.

Rice infusion
20g unpolished rice
1l water
Simmer the rice in the water for 45 minutes, filter, and add water to restore volume to 1 litre. For good results unpolished rice must be used, the results obtained with white polished rice are inferior.

Sabouraud's glucose agar (SabG)
40g glucose
10g peptone
20g agar
1l water
The pH is adjusted to 7.0 before adding the agar.

Salt media
16g or 10g NaCl
5g glucose
0.7g Difco Yeast Nitrogen base
100ml deionized water
Dispense 4ml amounts in 16mm diameter tubes.

Sucrose-yeast extract agar
1.0g potassium dihydrogen phosphate
0.5g magnesium sulphate (heptahydrate)
0.1g calcium chloride
0.1g sodium chloride
0.5g yeast extract
20g sucrose
40g agar
5ug biotin
1l deionized water

V8 agar
350ml V8 Vegetable Juice (Campbell Soup Co.)
5g compressed yeast suspended in 10 ml of water
14g agar
350ml deionized water
Mix the yeast and V8 juice, adjust the pH to 6.8 and steam for 10 minutes. Adjust the pH the cool mixture to 6.8 again. Add the mixture to the agar which has been dissolved in the 350 ml of water.

Vegetable wedges
Cylinders about 1 cm in diameter are cut from washed vegetables with a cork borer or apple corer. The wedges are cut obliquely from these cylinders and put into test tubes with a little water. Vegetables which may be used are: carrot, beet, cucumber and turnip.

Vitamin solutions for growth tests
2000µg inositol
400µg calcium pantothenate
2µg biotin
400µg thiamine hydrochloride
400µg pyridoxine hydrochloride
400µg niacin
200µg para-aminobenzoic acid
2µg folic acid
100ml distilled water
Solutions are prepared with the following omissions:
1. all vitamins
2. inositol
3. calcium pantothenate
4. biotin
5. thiamine
6. biotin and thiamine
7. pyridoxine
8. thiamine and pyridoxine
9. niacin
10. PABA
11. no ommissions (complete medium for positive controls)
To use, add 0.5ml aseptically to 4.5ml of Difco vitamin-free basal medium in 16mm diameter tubes.
All glassware used in preparing these media must be thorougly cleaned with acid.

Water agar
20g agar
1l deionized water

Yeast extract-malt extract broth (YM broth)
3g yeast extract
3g malt extract
5g peptone
10g glucose
1l deionized water
The pH is not adjusted and ranges between 5 and 6. This medium is available from Difco.

Yeast extract-malt extract agar (YM agar)
3g yeast extract
3g malt extract
5g peptone
10g glucose
20g agar
1l deionized water
The pH is not adjusted and ranges between 5 and 6. This medium is available from Difco.

Yeast extract (liquid)
1kg compressed baker's yeast
5l deionized water
Mix ingredients and keep at 50°C for 24 hours, add the whites of 2 eggs to clarify the extract, shake well and filter.
Alternatively:
5g yeast extract powder
1l deionized water

All media are sterilized by autoclaving at 121°C (15psi) for 15 minutes unless otherwise stated.

Uses of the media

Acetic-acid agar
identification of Zygosaccharomyces

Acidified media
isolation, to reduce growth of bacteria

Canavanine-glycine-bromothymol blue medium
identification of varieties of Filobasidiella (Cryptococcus) neoformans

Carbon base-urea agar
Diazonium blue B test

Chalk agar
isolation and cultivation of Dekkera (Brettanomyces)

Chloramphenicol
additive to media to inhibit growth of bacteria

Christensen urea agar
test for hydrolysis of urea

Corn-meal agar
morphology, cells and filaments
sporulation, ascospores, teliospores, basidiospores
chlamydospores in Candida albicans & C. dubliniensis

Corn-meal+tween agar
chlamydospores in Candida albicans & C. dubliniensis

Custers agar
detection of acid production

Cycloheximide
identification
can also be used in selective media for isolation

Cyniclomyces medium
isolation and cultivation of Cyniclomyces guttulatus

D-20 medium
isolation

Dilute V8 agar
sporulation of Metschnikowia spp

Diphenyl
supresses growth of moulds

Fowell's acetate agar
ascospores

Glucose 50% & 60% agar
identification
isolation and cultivation of osmophilic and osmotolerant strains

Glucose-peptone-yeast extract agar and broth
isolation and cultivation

Glucose 2%-yeast extract agar
sporulation of Schwanniomyces spp.

Glucose 1%-yeast extract agar
sporulation of Metschnikowia & Wickerhamiella spp.

Gorodkowa agar
ascospores (particularly Debaryomyces spp.)

Hay-infusion agar
sporulation of basidiomycetes

Killer medium
testing strains for killer activity

Leeming & Notman agar
isolation and cultivation of Malassezia spp.

Malt agar & extract
cultivation
isolation
morphology

Malt 2% & 5% agar
ascospores, basidiospores, teliospores

McClary acetate agar
ascospores (particularly of Saccharomyces cerevisiae)

Midgley Dixon agar
isolation and cultivation of Malassezia spp.

Morphology agar
morphology

Niger-seed agar
isolation of Filobasidiella (Cryptococcus) neoformans

Oatmeal agar
ascospores

Olive oil
additive for Malassezia spp

PH10 medium
testing for growth at pH 10

Polyol agar
sporulation of Xanthophyllomyces sp

Potato-dextrose agar
cultivation
isolation
morphology (filamentation)
sporulation (ascospores, basidiospores, teliospores)

Rapid urea broth
testing for hydrolysis of urea

Rice agar
chlamydospores of Candida albicans
filamentation

Sabouraud's glucose agar
cultivation
isolation

Salt media
identification, particularly for discriminating Zygosaccharomyces rouxii from Z. mellis

Sucrose-yeast extract agar
sporulation of Filibasidiella neoformans

V8 agar
ascospores

Vegetable wedges
ascospores

Water agar
germination of teliospores

YM agar
sporulation
cultivation
isolation

YM agar + 2% NaCl
sporulation of Zygosaccharomyces rouxii

Media to use for:

Acetic-acid growth test
acetic acid 1% agar

Acid-production test
Custers agar

Ascospores
acetate agar
corn-meal agar
dilute V8 agar
dilute (1:10 & 1:20) YM agar
glucose 1% & 2%-yeast extract agars
Gorodkowa agar
malt 2% & 5% agars
malt agar + NaCl
oatmeal agar
potato-dextrose agar
vegetable wedges
V8 agar
YM agar
YM agar + 2% NaCL

Arthroconidia
There are no special media for inducing the formation of arthroconidia, they are usually seen on media used for testing filamentation and often on other media such as YM agar, malt agar, and GPY agar.

Ballistoconidia
corn-meal agar
malt agar
morphology agar
potato-dextrose agar
YM agar

Basidiospores
corn-meal agar
malt 2% & 5% agar
polyol agar
potato-dextrose agar
sucrose-yeast extract agar

Chlamydospores of Candida albicans & C. dubliniensis
corn-meal agar
corn-meal + tween agar
rice agar
rice agar + tween

Cultivation and maintenance
Cyniclomyces medium
GPYA for ascomycetes
PDA for basidiomycetes
GPYA + CaCO3 for Dekkera
Leeming & Notman agar for Malassezia spp.
YM agar

Cycloheximide resistance
0.01% & 0.001% cycloheximide media

CGB test
Canavanine-glycine-bromothymol blue medium
Diazonium blue B reaction
Carbon base-urea agar
YM agar

Endospores
There are no special media to stimulate the development of endospores, they are seen in old cultures on YM agar, malt agar, potato agar, corn-meal agar, and Gorodkowa agar.

Filaments
corn-meal agar
morphology agar
potato-dextrose agar
rice agar

Freezing
dimethylsulphoxide 10% (DMSO)
glycerol 10-20%
glycerol 10% + DMSO 5%
Freeze-drying
inositol 7.5% + glutamate 5%
skimmed milk 10%

Germ tubes of Candida albicans
egg white
blood serum
(see Ogletree et al. 1978 for survey of methods)

Growth tests for identification
acetic acid 1% medium
cycloheximide medium
glucose-peptone-yeast extract agar (temperature)
50% & 60% glucose media

PH10 medium
salt media
yeast nitrogen base + carbon test source
yeast carbon base + nitrogen test source
vitamin-free medium
vitamin-free medium + all vitamins except one

Isolating
Cyniclomyces medium
glucose-peptone-yeast extract
50% (40% or 30%) glucose agar
Leeming & Notman agar
malt extract and agar
Midgley Dixon agar
niger-seed agar
potato-dextrose agar
Sabouraud glucose agar
YM broth and agar
(possible additives:
acids to inhibit bacteria
antibiotics to suppress bacteria
diphenyl, rose bengal to inhibit moulds)

Lyophilizing, see freeze drying

Mould inhibitors
diphenyl
rose bengal
Osmotolerance
50% glucose agar
60% glucose agar
salt media

Starch production
Yeast nitrogen base + glucose

Teliospores
corn-meal agar
2% & 5% malt agar
hay infusion agar
potato-dextrose agar
sucrose-yeast extract aqar

Urea hydrolysis test
Christensen urea agar
Rapid urea test broth

Instructions for reviving freeze-dried yeast cultures

Yeast:

Pour the dry material into a tube or small flask containing 4 or 5 ml of sterile liquid medium and incubate on a shaker and inspect daily until growth can be seen. Streak a loopfull of cell suspension onto an agar plate and incubate.

The medium and temperature used by the CBS are shown on the label and can be found on this website by searching strains data.

Malassezia spp. and Oosporidium sp.

These species do not grow in liquid media.
Pour the dry material onto the surface of the agar in a petri dish, moisten the material with 1 or 2 drops of sterile medium, and streak over the surface.

 

 

 

Correspondance

General Address:

Visiting address: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands

Postal address: CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands

Phone: +31 (0)30 21 22 600

Fax: +31 (0)30 25 12 097


Fungal collection curator:

Dr. Gerard Verkleij

E-mail: g.verkleij@westerdijkinstitute.nl


Yeast collection curator:

Dr. Marizeth Groenewald

E-mail: m.groenewald@westerdijkinstitute.nl


Ordering strains:

Mrs. Francis Claus (sales)

E-mail: sales@westerdijkinstitute.nl


Mycobank and nomenclature:

Dr. Joost Stalpers and Ir. Gerrit Stegehuis

E-mails: j.stalpers@westerdijkinstitute.nl
g.stegehuis@westerdijkinstitute.nl


Software, websites and bioinformatics:

Dr. Vincent Robert and Kasper Luijsterburg

E-mail: v.robert@westerdijkinstitute.nl
k.luijsterburg@westerdijkinstitute.nl