NOTES

PREPARATION OF CELL WALLS OF YEAST

Present linowledge of the chemical composition of cell walls of yeast is still incomplete. Most of the studies of the cl~emical composition of the cell wall have been qualitative in nature and little is lcnown of the quantitative variations in wall coinposition that may occur in closely related organisms. Accordingiy, a chemical investigation of the cell walls of several yeasts was undertalien; results will be reported later. The method used to obtain isolated yeast cell walls is described in this report.

A number of methods have been used in order to obtain cell walls of yeast (1, 2, 3, 5). The clevelopment of techniques for brealtage of cells, and separa- tion of morphologically intact walls, has shown the complexity of cell walls of yeast species. In the present study, cell walls of a number of yeasts were isolated in a fairly pure state and their structures observed under the light and electron microscope. Yeasts were grown by the shalie-flasli culture in a medium of the following composition: sucrose, 50 g, Difco peptone 10 g, MgS04.7H20 3 g, and distilled water 1000 ml. Cultures were incubated a t 28" for 18 hours. After this time the resulting cells were harvested in a centri- fuge and washed three times with water. The washed cells were suspended in water (approximately 30 nlg dry weight per ml) and treated in a R'Iiclile disintegrator for complete brealtage. Ten illilliliters of the cell suspension was added to each cup of the nlachine and shalten with 10 ml of grade 12 Ballotini glass beads for various times at- room temperature. T o avoid en- zymic degradation of cell walls, the temperature of the flaslis containing the cell suspensions was nlaintained below 10". Breakage was followed by micro- scopic examination of Gram-stained material. Disintegration of a large number of yeasts proceeds slowly and a high proportion of brealiage was obtained only after 3 hours treatment.

After a high proportion of cellular breakage was achieved, cell walls were collected by differential centrifugation by the following procedure: the con- tents of the tubes were decanted into a sucrose solution (60% w/v), shaken, and left to stand for 5-10 minutes. Aftcr this time all the glass beads had sedimented and the supernatant was decanted into a centrifuge tube and spun down a t 1500 X g for 10 minutes. The sedimented material was resuspended in a 10% sucrose solution and spun a t 1500 X g. The latter treatment was repeated five times. The resulting sediment was resuspended in 1% (w/v) NaCl solution and centrifuged a t the same speed. This operation was repeated twice. Finally the residual cell walls were thoroughly washed in water until the final supernatant became clear. The residue comprised the required cell wall fraction.

In those cases in which unbrolten cells were still present the total cell wall preparation was resuspcnded in water, put in capillary tubes (3 X 10 mm), and centrifuged a t low speed (about 600 X g) for 5 minutes. By this modifica- tion the whole cells were deposited a t the bottoill of the capillary, cell walls remaining in the supernatant, which could be reilloved readily with a Pasteur pipette.

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142 C;\SADIAN JOURNAL OF MICROBIOLOGE'. VOL. 9. 1963

The cell-wall preparations obtained in the above manner are Gram-negative, whereas the whole yeast cells are Gram-positive, thus it is easy to distinguish any unbrolten cells in the preparations by a microscopical examination of stained films. i\/Iany fields of numerous preparations were examined and upon careful examination an occasional whole cell was detected. It is concluded that the inaterial isolated represents only the cell walls. Light micrographs of intact cells of Candida z~ti l is and their corresponding cell walls are shown in Figs. 1 and 2. Most of the cells appear to be damaged only a t one point.

This procedure was effective for all the species of yeasts tested. Under these conditions, three to five 30-minute treatments in the disintegrator were sufficient. A strain of Torulopsis aedis has beell shown to be very resistant not only to mechanical breakage but to enzymic digestion (4).

1. .ARONSON, J. i\'l. and MACIILIS, L. The chemical composition of the hyphal walls of the fungus Allomyces. Am. J. Botany, 46, 292-300 (1959).

2. B:\RTNICI~~-G:\I~C~A. S. and NICI~ERSON. W. 1. Isolation. coln~osition. and structure of rell-walls of 'hlarnentous and veast-lil;edfor~~ls of il'lz~cor ' roz~s i i . biochim. B i o ~ h v s . . , Acta, 58, 102-119 (1962).

3. FULLER, M. S. and BARSHAD, I. Cllitin and cellulose in the cell-walls of Rhisidiovtyces sp. Am. J. Botany, 47, 105-109 (1960).

4. GARCI.~-~\~IENDOZ.~, C. and VILLANUEVA, J. R. Production of yeast protoplast by a n enzyme preparation of Streptoi~tyces sp. Nature, 195, 1326-1327 (1962).

5. NoR'raco~E. D. H. and HORNE. I<. W. The che~nical com~osition and structure of the yeastcell wall. Biochem. 51, 232-236 (1952).

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FIG. 1. \\'hole cells of Calldidr~ rllilis as obscr\-etl under the phasc contrast microscopc. FIG. 2. Cell-\\.all prcpnrntion of C, zibilis obtailicd by treatnlerit in the Miclcle followed

by differe~~tial centrifugation.

Mendoza ant1 Villa~iueva-Can. J. Microbial.

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