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Sales:
robert@cbs.knaw.nl

Curator(s):
robert@cbs.knaw.nl

Address:

Uppsalalaan 8
3584 CT Utrecht
The Netherlands

Telephone + 31 (0)30 2122600
Telefax + 31 (0)30 2512097

Postal address:

P.O.Box 85167
3508 AD Utrecht
The Netherlands 

Citation(s)

Robert, V. and Szoke, S. 2006. BioloMICS Software. BioAware

Mycobank Yeast species database

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