Generally, yeasts multiply as single cells that divide by budding (e.g., Saccharomyces cerevisiae) or direct division (fission, e.g., Schizosaccharomyces), or they may grow as simple irregular filaments (mycelium). The yeast Xanthophyllomyces dendrorhous (anamorph Phaffia rhodozyma) multiplies by budding when grown in optimal conditions (similar to S. cerevisiae, as seen in image d). However, we have found that under long hydric and nitrogen starvation conditions this yeast may produce multibudding cells (a, b, and c).

Description of images:
(a) Illustration of a X. dendrorhous cell showing bipolar budding taken at 8,000x magnification.
(b) Multibudding image of a X. dendrorhous cell taken at 7,000x magnification. Note how some buds were released leaving an edge coating scar on the mother cell surface.
(c) Image taken at 3,000x magnification showing multibudding cells and other X. dendrorhous structures that appear when stressed for long periods.
(d) Anamorphic X. dendrorhous cell at 10,000x magnification with regular budding (shown to compare how P. rhodozyma budding generally occurs).

Methodology tips:
Multibudding of X. dendrorhous occurred after stressing cells in sporulation agar (100 mM sorbitol, 25 mM ammonium nitrate agar) for at least 8 weeks (a, b, and c). Cells were checked on the dry agar using a light microscope. The agar was cut into small (1 mm) cubes and introduced into Eppendorf tubes. These cubes were washed twice in 20 mM phosphate buffer, pH 7.4, and kept on ice. After pouring off the washing solution, the cubes were exposed to the fixative solution (2% glutaraldehyde and 4% formaldehyde in phosphate buffer) overnight. To allow for fixation, the cubes were left undisturbed overnight at room temperature. As a precaution, the remainder of the work was conducted under the hood. The fixative was removed and a 2% aqueous osmium tetraoxide solution was added. Cubes were exposed to the osmium tetraoxide solution overnight. Then the osmium solution was removed and the cells were dehydrated by a succession of 10-minute ethanol treatments (20-40-60-80-100-100% (twice)). The last step involved a critical point dry process using carbon dioxide as a transitional solvent in a Samdri-780A (Tousimis Research Corporation, Rockville, Md.). Cubes were mounted on the scanning electron microscopy sample base and observed in the microscope.

Images were obtained using a Hitachi S-570 LaB6 scanning electron microscope. We acknowledge Philip Oshel at the Biological and Bio-Materials Preparation, Imaging, and Characterization Laboratory at the University Wisconsin-Madison for his expertise and technical assistance.

Additional background on P. rhodozyma and X. dendrorhous cell cycle, ecosystem, abilities, and phylogeny can be found in these references.

References.

1. Andrewes, A. G., and M. P. Starr. 1976. (3R,3’R)-Astaxanthin from the yeast Phaffia rhodozyma. Phytochemistry 15:1009-1011.

2. Fell, J. W., and G. M. Blatt. 1999. Separation of strains of the yeasts Xanthophyllomyces dendrorhous and Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis. Ind. Microbiol. Biotechnol. 23:677-681.

3. Golubev, W. I. 1995. Perfect state of Rhodomyces dendrorhous (Phaffia rhodozyma). Yeast 11:101-110.

4. Phaff, H. J., M. W. Miller, M. Yoneyama, and M. Soneda. 1972. A comparative study of the yeast florae associated with trees on the Japanese Island and on the West Coast of North America, p. 759-774. In G. Terui (ed.), Proceedings IV IFS: fermentation technology today. Society of Fermentation Technology, Osaka, Japan.