EXPERIMENTAL MYCOLOGY 5, 140-147 (1981)

Changes in Lipid Composition during Starvation and Germ-Tube Formation in Candida albicans

SOMASUNDARAM,' PATRICK A. SULLIVAN, AND MAXWELLG. SHEPHERDS

Department of Biochemistry, University of Otayo, P.O. Box 56, Dunedin, New Zenland

Accepted for publication November 7, 1980

SUNDARAM, S., SULLIVAN, P. A., AND SHEPHERD, M. G. 1981. Changes in lipid composition during starvation and germ-tube formation in Cundidu ulbicans. Experimentul Mycology 5, 140- 147. The changes in the lipid components of Cundidu ulbicuns have been determined during growth, starvation, and germ-tube formation. r4C-Labeled cells were used to determine the extent of synthesis and degradation of the different lipid fractions. On a dry weight basis the percentage of total lipid increased from 18% for blastospores to 22% after starvation but decreased to 11% after 4 h of germination. The major components of the lipid fraction were sterols (36652%) and phos- pholipids (28-42%). The free and esterified sterol fractions both increased approximately 45% during starvation. The free sterol content continued to increase over the initial stage of germ-tube formation, then decreased with time. The sterol ester fraction decreased throughout germination to the concentration found in growing blastospores. The changes in specific activity (cpm/pg sterol) of these fractions indicated that sterol esters were precursors for free sterols during germination. The total phospholipid fraction increased during starvation but there was a decrease in both the cellular concentration (60%) and the specific activity during germination. There were only minor changes in the relative concentrations of phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine which indicated coordinate synthesis and degradation of these compo- nents. Free fatty acids and triacylglycerides are only minor components of the total lipid pool.

INDEX DESCRIPTORS: Cundidu ulbicans; germ tubes; lipids; germination; sterols; phospholipids.

A method for the synchronous produc- tion of germ tubes from starved blasto- spores of Candida albicans has recently been described (Shepherd et al., 1980a). In this system germ tubes appear after 2 hours at 37°C. During germ-tube formation there is extensive RNA and protein synthesis but no DNA synthesis occurs (Shepherd et al., 1980a). Although there are reports on changes in the carbohydrate metabolism in the different morphological forms of C. al- bicans (Chattaway et al., 1973) and during germination (Chiew et al., 1980) there is no information available on the changes in lipid content of C. albicans during germ- tube formation. We describe the results of such an investigation in the following re- port. It is well established that growth con-

’ Present address: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wise. 53226.

2 To whom all correspondence should be addressed.

ditions affect the lipid composition of yeast and fungi (Jollow et al., 1968; Gordon et al., 1971; Hossack et al., 1973; Long and Coe, 1974). The lipid composition of the plasma membrane (Marriott, 1975) and cell walls (Chattaway et al., 1968) of C. albicans has been determined. Marriott (1975) reported marked differences between the phos- pholipids, free and esterified sterols, and total lipids of membranes from yeast and mycelial forms.

MATERIALS AND METHODS

Growth Conditians and Germ-Tube Formation

C. albicans (ATCC, 10261) was grown as a yeast batch culture at 28°C using the growth conditions and medium described previously (Shepherd and Sullivan, 1976) except that the carbon source was 0.2% (w/v> glucose plus 0.1% (w/v) sodium ace-

0147-5975/81/020140-08$02.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

140

LIPID COMPOSITION CHANGES IN Candida a~birrins 141

tate. C. albicans does not grow on acetate as a sole carbon source. A 100-ml culture, in a 500-ml Erlenmeyer flask was grown for 18 h and then transferred to 900 ml of fresh medium in a 2-liter Erlenmeyer flask in which the acetate was labeled (0.1 $X/ml of [U-14C]acetate). Sodium [U-14C]acetate (57.4 mCi/mmol) was obtained from the

adiochemical Centre, Buckinghamshire, United Kingdom. This suspension was grown at 28°C (gyratory shaker, 200 rpm) for 18 h. The cells were harvested and washed three times by resuspending and centrifuging in distilled water. The cell sus- pension was then aerated (100 cm3/min/100 ml yeast suspension) for 24 h and washed two times with distilled water. These cells are hereafter referred to as starved yeast cells. Starved cells were germinated using ClicNAc3 (2.5 rn~) as the inducer and in- cubating cell suspensions (6 x lo7 cells/ml) with shaking (200 rpm) at 37°C (Shepherd et al., 1980a).

Lipid Analysis

Growing, starved, or germinating cells (75 x 108) were suspended in 50 ml of 80% (v/v) aqueous ethanol and heated at 80°C for 15 min to destroy phospholipases (Let- ters, 1968). The extracts were then filtered

rough Millipore filters (1.2 pm) into round-bottomed flasks. Cell residues were washed from the filters and extracted two times with 50 ml of a chloroform/methanol mixture (211, v/v). During extraction the cell residues were shaken intermittently. The pellet was reextracted overnight at roam temperature under nitrogen with 50 ml of chloroform/methanol/concentrated

Cl (12416111, v/v). After centrifuging, this extract was neutralized with dilute KOH before being combined with the other ex- tracts. Each combined extract was con- centrated to dryness in vacm at 35°C. Pro-

(i Abbreviation used: GlcNAc. N-acetylglucos- amine.

tein-lipid complexes were broke ing absolute alcohol and co~ce~trat~~~ again. This procedure was repeated three times. Thee dried lipids were redissolved in 40 ml of ch~oroform/methanol (21 I ) v/v) and washed with 10 ml of dilute salt solution (Folch et al., 2957). The lipids were made up to known volumes before storing at -20°C.

Totall lipids were estimate chromate/sulfuric acid colorimet (Bragdon, 1951). Neutral lipids were sepa- rated by one-dimensional thin-layer chro- matography on silica gel plates using pe- troleum etheridiethyl ether/acetic acid (SO/ 2012, v/v) as described by Kates ( individual lipids were located vapor. Each band was scraped plate and separated from the silic chloroforl~/methano~/eth~r (l/l/l, v/v) (Kates, 1972).

were estimated with the &ie urchard reagent

al., 1959; Zlatkis et al., 1953). Triacyl- glycerides ere assayed with th tropic aci eagent (Bartlett, 1 triolein as a standard. The free fatty acid fractions were taken up in ethanolldiethyl ether (j/l, v/v) cresol red 005%, v/v) was a cator. So m hydroxide (5 ethanol was then used to titrate the a wbile a stream of nitrogen was ~~~~~~d through tbe liquid to allow for c~~t~~~~~s mixing.

The phospholipids were sepa two dimens,ions by thin-layer ch phy on silica gel-coated plates following solvent systems: (a) chBoro- form/methanol/water (66/30/4, v butanoliacetic acid/water (60/20/2 Spots were visualized by iodine vapor, The ninhydrin-positive phospholipids were de- tected by spraying the plates with a 0.2% (w/v) solution of ninhydrin in bu lowed by warming in an oven at 1 individual phospholipids were recovered from the plates and phosphate was mea-

142 SUNDARAM, SULLIVAN, AND SHEPHERD

sured (Bartlett, 1959a) after digestion with 0.4 ml of 70% (v/v) HClO,; identification was by comparison with known phospho- lipid standards.

Radioactive Lipids

All of the lipid separations were per- formed in duplicate and one sample was used for radioactive counting. Radioactiv- ity was determined in 10 ml of scintillation fluid (6 g of 2,5-diphenyloxazole (Sigma) per liter of Toluene) in a Packard 3300 liq- uid scintillation spectrometer.

Dry Weight Estimations of C. albicans Blastospores and Germinating Cells

One liter of germinating medium con- taining starved C. albicans blastospores, 6.0 x lo7 cells/ml, was separated into 200-ml portions in 500-ml Erlenmeyer flasks and each flask was shaken at 200 rpm and 37°C on a New Brunswick gyratory shaker. At hourly intervals the contents of one flask were filtered through a pre- weighed Millipore filter (1.2 pm). Each fil- ter was washed three times with 50 ml of distilled water and then dried to constant weight at 80°C. After cooling in a desic- cator, each filter was reweighed.

RESULTS

Total Lipids

Previous studies (Shepherd et al., 1980a, b) showed that consistent, high yields (85%) of germinated cells were obtained with starved blastospores. In the present study blastospores were 14C-labeled during yeast growth, starved for 24 hours by aerating blastospores suspended in water, and then germinated by incubation in a buffered GlcNAc medium. The lipids of late- exponential-phase blastospores, starved blastospores, and germinating cells were analyzed and the results have been pre- sented in terms of changes in concentration and specific activity. The dry weight of the cells decreased over the 24-hour starvation period. When germination was initiated the

GlcNAc was metabolized (Shepherd et al., 1980a, b) and the dry weight almost doubled (Fig. 1). During this period of development the specific activity of the cells decreased by almost 50% (Fig. I). Table 1 shows the total lipid content during growth, after star- vation, and during germination. Synthesis of lipids continued during starvation and re- sulted in a doubling of the lipid content per cell. The lipid that accumulated during starvation of the yeast cells was rapidly utilized during germination. After 4 hours of germination 50% of the lipid had been used. Figure 2 shows a comparison of the changes in total lipid and specific activity of the total lipid fraction.

While the total lipid content per cell in- creased during starvation there was no change in the specific activity. These data indicate that the lipids synthesized during starvation were from a uniformly labeled pool of precursors. During germ-tube for- mation the specific activity of the total lipids decreased and was accompanied by a decrease in the lipid content per cell.

Sterols

Free and esterified sterols are major components (up to 50% w/w) of the total lipids (Table 1). During starvation there was a large increase in the total sterol content

FIG. 1. Changes in the dry weight (u) and “C-label (0) of whole cells of C. albicurzs during starvation and germination. The results are the means of duplicate determinations.

LIPID COMPOSITION CHANGES IN Candida ~lfhicans

Frc. 2. Changes in the total lipid (B) and specific FIG. 3. Changes in the sterol content during starva-

activity of the lipid fraction (e) during starvation and tion and germination: concentration of free (

germination. The results are the means of duplicate esteritied (e) sterols. The results are the means of do-

determinations, plicate determination.

per cell. The esterified sterols were rapidly utilized during germination and the cell content returned to that found in growing blastospores (Fig. 3). The cellular content of free sterols continued to increase during the first hour of germination but by 4 hours the cellular concentration had returned to that found in growing blastospores (Fig. 3). The changes in the labeling pattern of sterols were complex (Fig. 4). There was a decrease in specific activity of the sterol

esters throughout germination. In contrast, there was no change in specific activity of the free sterol pool for 2 hours but then the specific activity decreased rapidly.

Phospholipids

The other major lipid fraction (30-4096 w/w) in C. albicans is phospbolip~ds (Table 1). During starvation the phosp content per cell increased (Fig. 5) germination was initiated there was decline in ~hos~holipid content o

TABLE 1 Lipid Composition of C. ulbicuns during Growth, Starvation, and Germ-Tube Formation’!

Composition (&mg)

Free Time Total fi3Btiy

(h) lipid Phospholipids Sterols Triglycerides acids -

Growth 4 105 39 49 10.5 I.1 8 110 45 51 11 1 .o

12 120 51 50 10.0 0.9 18 175 49 55 9.5 0.8

Starvation 24 220 83 98 11.5 1.2

Germination 1 178 61 89 11.5 1.2 2 140 49 73 12 1.2 3 125 38 60 11 1.2 4 110 34 40 10.5 1.1

U The data are the average values from at least three different experiments with variations of less than 10%~. Phospholipid values were derived by multiplying the nanomoles of lipid phosphate by 750. Sterol values are ?he sum of free and esterified sterols. The free fatty acid values were obtained by multiplying the nanomoles of free fatty acids by 150.

144 SUNDARAM, SULLIVAN, AND SHEPHERD

-2L 0 I 2 3 k Ilmelhl

FIG. 4. Changes in the specific activity of sterols during starvation and germination: specific activity of free (@) and esterified (m) sterols. The results are the means of duplicate determinations.

first 2 hours and after 4 hours the phos- pholipid content was similar to that found in growing blastospores (Table 1 and Fig. 5). The specific activity of the phospholipid fraction remained constant during starva- tion and over the first 2 hours of germ-tube formation; between 2 and 4 hours of germi- nation, however, there was a 50% decrease in the specific activity.

The major components of the phos- pholipids are phosphatidylcholine, phos- phatidylserine, and phosphatidyletha- nolamine (Table 2). Although there were substantial changes in phospholipid con- tent per cell during starvation and ger- mination (Fig. 5), there were only small changes in the relative proportions of the phospholipid components (Table 2). The

fime(hl

FIG. 5. Changes in the phospholipid specific activ- ity (0) and cellular content (m) during starvation and germination. The results are the means of duplicate determinations.

relative amount of the sphingolipid fraction increased during growth and starvation and then fell during germ-tube formation.

Free Fatty Acids and Triacylglycerols

Free fatty acids are only a minor con- stituent of the total lipids and accounted for less than 1% on a weight basis (Table 1). There was an increase in the free fatty acid content per cell during starvation but the relative content of this fraction did not change during germination (Fig. 6). Tri- acylglycerols accounted for 5- 10% (w/w> of the total lipids and during growth, star- vation, and germination there was no change in the triacylglycerol content per cell (Table 1 and Fig. 7). During germina- tion there was a rapid and extensive de- crease in the specific activity of both free fatty acids and triacylglycerols.

DISCUSSION

Shepherd et al. (1980a) have described a method for the synchronous production of germ tubes from starved blastospores of C. albicans and we have used this system to study changes in lipid composition during growth, starvation, and germination. It should be emphasized that during starva- tion and germ-tube formation there is no change in the cell number and that the ex- ogenous GlcNAc used to induce germ-tube formation is a potential precursor for lipid synthesis. The 50% decrease in specific ac- tivity of the 14C-labeled cells during germi- nation reflects the rapid uptake and me- tabolism of GlcNAc (Shepherd et al., 1980a, b). Starving the yeast cells caused an increase in the lipid content of the cells but there was no change in the specific activity of the lipid fraction. This indicates that the lipid synthesized during starvation was de- rived from other endogenous 14C-labeled precursors. Both the, specific activity and the cellular lipid content decreased during germ-tube formation. The decrease in spe- cific activity indicates that new lipid was synthesized. from ,the exogenous carbon source, GlcNAc, whereas the decrease in

LIPID COMPOSITION CHANGES IN Candidu albicans 145

TABLE 2

Phospholipid Composition of C. albicans during Growth, Starvation, and Germ-Tube Formation”

Component

Phosphatidyl- inositol

Phosphatidyl- serine

Phosphatidyl- choline

Sphingolipid Phosphatidyi-

ethanolamine

4h

12

18

35 4

31

Growth Starvation Germination

8h 12 h 18 h 24 h lh 2h 3h 4h --

7 5 6 9 13 16 5 10

23 24 21 18 18 16 17 17

35 30 45 35 36 43 56 46 5 6 10 13 7 4 4 3

30 33 17 22 2s 21 18 21

” The figures given are percentages and were determined as described under Materials and Methods. The va!ues shown are the means from at least three different experiments with variations of less than 10%.

lipid content indicates rapid turnover of the lipid pool.

Ballman and Chaffin (1979) have used [‘“Cjacetate incorporation as a measure of lipid synthesis during germ-tube formation and yeast growth in C. albicans. With the exception of phospholipids no quantitative data were provided on the relative amounts of the various lipids. The data from both the present study and that of Ballman and Chaffin (1979) showed that lipid synthesis occurred over the entire period of germ- tube formation and that lipid synthesis pre- ceded the morphological change. Although there is a paucity of information available on the changes in the lipid content of C. albicans during the yeast to mycelial tran- sition, the lipid composition of yeasts in-

hmelhl

FIG. 6. Free fatty acid content during starvation and development. Cells were analyzed for total amount (a) and specific activity (0). The results are the means of duplicate determinations.

eluding 6. atbicans is well doc~me~t~d~ The lipid composition of C, albicans changes with growth conditions. The lipid content (%, w/w) ranges from 34% (Nishi et al., 1973; Yamaguchi, 19743 to 3 (Davies and Denning, 1972). The low values were for blastospores grown in a ~~e~i~rn with a high glucose content whereas the value of 30% was for a mycelial couture grown in serum. Bianchi (1966) reported a lipid content of 12% for C. aibjc~~s. The lipid content found in the present stu varied between 11% in the growt and 22% after starvation.

The major lipid components in C. alki- cans are phospholipids and st sistent with previous studies ( Denning, 1972; Ballman and C these two frac . s accounted for 8O- of the total li Both ~bospho~i~~~ sterol content per cell increased with star- vation. Starvation of yeast cells is a neces- sary prerequisite for germ-tube formatj~~ in our experimental system (Shepherd et al., 1980a) and the changes that occur in steroli and phospholipid content may be ~rn~Q~a~~t for the induction of germination. There is an increasing bo y of evidence ind that intracellular vesicles are invol wall development for yeast budding (No- vick and Schekman, 1979) and germ-tune formation (Grove, 1978). Germ-tube for-

146 SUNDARAM, SULLIVAN, AND SHEPHERD

FIG. 7. Changes in the specific activity (0) and cel- lular content (m) of triacylglycerides during starvation and development. The results are the means of dupli- cate determinations.

mation in C. albicans must involve con- comitant development of plasma membrane and cell wall. It is well documented that free sterols are associated with the plasma membrane while sterol esters are as- sociated with cytosolic inclusions (Hossack et al., 1973; Parks, 1978). The changes in sterols may reflect the utilization of in- tracellular vesicles and synthesis of plasma membrane. During germination there was a decrease in both sterol ester content and specific activity. These data indicate exten- sive turnover of this fraction. The rate of degradation exceeded the rate of synthesis, taking into account the change in dry weight. The free sterol pool did not change in either content or specific activity during the first 2 hours of germination. Over this period the dry weight of cells increased 25% and thus indicated synthesis of free sterols. Since the specific activity remained con- stant free sterols were not derived from the exogenous GlcNAc. From these data we suggest that free sterols were derived from the sterol ester fraction. The decrease in content and specific activity of the free sterol fraction between 2 and 4 hours of germination correlated with the appearance of germ tubes and indicated turnover of the free sterol pool. The decrease in the phos- pholipid content during germination oc- curred in two phases: between 0 and 2

hours there was only a small change in spe- cific activity while the decrease in cell content can be accounted for by the in- crease in dry weight. This suggests that the phospholipid pool was relatively stable during this period. The decrease in specific activity between 2 and 4 hours indicates de nova synthesis and the change in cell con- tent suggests some turnover. The relative composition of the phospholipid fraction reported here is in agreement with the analyses of other workers (Ballman and Chaffin, 1979; Kates and Baxter, 1962; Nishi et al., 1973). The observation that there is little change in the relative abun- dance of each phospholipid indicates that there is coordinate synthesis and degrada- tion within the pool.

Free fatty acids and triacylglycerols are minor constituents of the total lipid fraction and during growth, starvation, and germ- tube formation there are only small changes in the content per cell of these components; there was, however, a rapid decrease in the specific activity of both these components during germination which indicates exten- sive turnover.

This study shows that the lipid content in C. albicans, particularly sterols, changes significantly during germination and these changes reflect the role of lipids in mem- brane and wall biogenesis.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the Medical Research Council of New Zealand. Soma Sundaram held a New Zealand University Grants Committee Postdoctoral Fellowship.

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