Vol. 129, No. 1 Printed in U.S.A.
JoURNAL OF BACTERIOLOGY, Jan. 1977, p. 97-100
Copyright 0 1977 American Society for Microbiology
Fractionation of Saccharomyces cerevisiae Cell Populations by Centrifugal Elutriation C. N. GORDON* AND S. G. ELLIOT1 Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717
Received for publication 24 September 1976
An exponential population of Saccharomyces cerevisiae cells was fractionated by centrifugal elutriation, using water as the elutriating liquid. Evidence that the population had been fractionated according to age in the cell cycle was obtained by examinig the fractions for their size distribution, their microscopic appearance after Giemsa staining, and their ability to initiate synchronous growth. A number of procedures have been employed for obtaining synchronous cultures of the yeast Saccharomyces cerevisiae for use in studies of the cell cycle (3). Generally, synchronization procedures are subject to the criticism that the synchronization process may introduce distortions in the normal sequence of events in the cell cycle. In addition, some techniques use only a small percentage of the original cell population in growing the synchronous culture and, consequently, the yield of cells at any particular stage of the cell cycle is low. Sebastian et al. carried out large-scale fractionations of exponential cultures of S. cerevisiae by velocity sedimentation through sucrose gradients in a rotating-seal zonal rotor (7). Similar fractionations of the fission yeast Schizosaccharomyces pombe were carried out on nonliving cells by Wells and James in a reorienting gradient zonal rotor (8). These methods have an important advantage over synchronous growth in that cell cycle measurements are made on cells that have been fractionated from a condition of balanced growth, thus aVoiding possible artifacts induced by the synchronization proc-
obtained can be greatly increased by choosing smaller incremental increases in flow rate. Second, the fractionation can be carried out in virtually any aqueous solution of uniform density. In this paper we describe the fractionation of an exponential yeast population by centrifugal elutriation with water as the elutriating liquid. Our results indicate that elutriation separates the yeast cell population into fractions representing successive stages of the cell cycle, and is an effective method of obtaining large numbers of cells for cell cycle studies. MATERIALS AND METHODS Organism. S. cerevisiae SKQ2n, a prototrophic diploid with the genotype a/a, adel/+, +/ade2, +/
hisl was used. It was obtained from Brian Cox, Botany School, South Parks Road, Oxford, OX1 3RA, United Kingdom. Growth and harvest of cells. Cells were grown in YM-1 medium (1) to a cell density of 1.5 x 107 to 3.0 x 107 cells/ml at 23°C on a rotary shaker. Growth was retarded by chilling to 0°C, and the cells were centrifuged and washed with water at 4°C. The cells (3 x 109) were suspended in 10 ml of ice water and ess. in an ice bath were sonically treated while Centrifugal elutriation ("counterflow centrif- for 10jacketed s in a Biosonik Sonifier (Bronwill Scientific ugation") was used as a method of separating Inc., Rochester, N.Y.) at half the maximal setting. biological particles nearly 30 years ago (5), but The sonically treated cell suspension was then kept a rotor suitable for general use has only re- in ice until it was loaded into the elutriator rotor. cently become commercially available. In this Fractionation by centrifugal elutriation. A Beckprocedure, the force tending to sediment parti- man JE-6 elutriator rotor was driven in a Beckman cles in a centrifugal field is counterbalanced by J-21 centrifuge. The centrifuge was modified for elua liquid flowing in the opposite direction. A cell triation and equipped with a stroboscope assembly the manufacturer (Beckman Instruments, Inc., population can be fractionated into discrete size by Alto, Calif.). The pump used in the system was classes by incrementally increasing the flow Palo a Masterflex 745 equipped with a model 7014 pump rate while maintaining the centrifuge rotor at a head (Cole-Parmer Instrument, Co., Chicago, Ill.). constant speed (6). Centrifugal elutriation has To allow greater sensitivity in determining the two important advantages over zonal centrifu- pump flow rate, the one-turn potentiometer speed gation. First, the rotor has a much longer effec- control was removed and substituted with a 10-turn tive path length, and the number of size classes control (Beckman helipot 7276R50L.25) and a sepa97
98
J. BACTERIOL.
GORDON AND ELLIOTT
rate on-off switch. The flow system was set up in the following way. The receptacle of a 100-ml "Yale Luer-Lok" syringe (Becton-Dickinson and Co., Rutherford, N.J.) was interposed between the pump inlet and the water reservoir by means of a threeway bypass valve and Silastic tubing. The syringe contained the cell suspension to be loaded into the rotor. With the pump running continuously, manipulation of the bypass valve allowed direct flow from the reservoir into the rotor or, alternatively, loading G) 0) of the cell suspension into the rotor. Elutriation was done at a centrifuge well temperature of 4°C while the water reservoir and collection flasks were maintained in ice baths. The length of CL connecting tubing was kept to a minimum. Water flow through the rotor was started and the rotor was brought to a speed of 3,000 rpm. Care was taken to remove all air bubbles in the system before the cells were loaded. The chilled and sonically treated cell suspension was placed in the loading syringe, and then the flow rate was brought to 9 ml/min. The cells were loaded at this flow rate and then flushed with 200 ml of water. Next, 200-ml fractions were Cell Diameter(microns) collected. The first fraction was collected at 11 ml/ distributions of cells in fractions obFIG. 1. Size min, and successive fractions were obtained with increments of 1 ml/min. At a flow rate of 27 ml/min, tained from the elutriator rotor. An exponential culvirtually all of the single cells were removed from ture of yeast cells was fractionated by centrifugal elutriation, and 16 fractions were obtained by succesthe rotor. Analysis of fractions. To each fraction, NaCl was sively increasing the flow rate in increments of1 ml/ added to 0.1 M and formaldehyde was added to 0.4%. min. The fractions were fixed with formaldehyde and The cells were collected by centrifugation and sus- centrifuged, and the size distributions were deterpended in 0.9% NaCl at a cell density of 106 cells/ml. mined in a Coulter counter. The four fractions shown The size distribution of the cells in each fraction was were obtained with the following flow rates: (A) 11 determined in a model T Coulter counter that had ml/min; (B) 15 ml/min; (C) 23 ml/min; and (D) 26 been calibrated with latex spheres of average parti- ml/min. cle diameter 3.49 ,um. Samples of the fractions were prepared for microscopic observation by a modified tions obtained from the elutriator rotor is also Giemsa staining procedure (2). To demonstrate synchronous growth in fractions shown. At the lower flow rates (11 to 13 ml/ elutriated at 11 and 25 ml/min, formaldehyde was min), the population consists predominantly of omitted and the centrifuged fractions were sus- unbudded cells. Budded cells, in which the nupended in YM-1 medium at a cell density of 3 x 106/ cleus has not yet migrated into the bud neck, ml. The cell suspensions were incubated at 23°C, constitute the major component in fractions and 0.1-ml portions were removed at the indicated elutriated at flow rates of 16 to 18 ml/min. Cells times and placed in 0.9% NaCl containing 0.5% in which the nucleus is in the act of migration formaldehyde. After sonic dispersion for 10 s, cell reach a peak in the fraction elutriated at 21 ml/ numbers were determined in a model F Coulter min. Finally, the fractions obtained at flow a
counter.
RESULTS The size distributions of four fractions obtained from the elutriator rotor and then fixed with formaldehyde are shown in Fig. 1. It is evident that there is an increase in the diameter of the cells elutriated with increasing flow rate. Evidence that the elutriation is effecting a fractionation of the population according to age in the cell cycle was obtained from microscopic examination of the fractions after Giemsa staining. In Giemsa-stained preparations, cells can be classified into one of four groups, shown schematically in the inset to Fig. 2. The distribution of the four basic cell types in the frac-
rates of 23 to 26 ml/min consist primarily of cells in which partition of the nucleus between mother and daughter cells has been completed. Remaining in the rotor were clumped cells representing less than 1% of the cells loaded into the rotor. These clumps may have been formed during growth, harvesting, and washing of the cells, and they were apparently not dispersed by the sonic treatment. Figure 3 shows growth curves obtained with fractions elutriating at 11 and 25 ml/min. The growth curve of the unfractionated culture is shown for comparison. It is evident that the fractions obtained by elutriation behave like synchronously growing cultures.
,..~ ,~
i~A 8tJ t y (! t, va, o
o
100
.
*
75
1
CD
/ /
y /r ° \\
C
V
99
CENTRIFUGAL ELUTRIATION OF YEAST CELLS
VOL. 129, 1977
~ 50
CL
within each fraction was due to an enrichment of the fraction by cells in comparable stages of the cell cycle. (iii) Fractions obtained from the elutriator rotor showed synchronous growth. An important advantage of this method over zonal centrifugation or other selection techniques is the wide choice of media available to carry out the fractionation. Although water was used in our experiments, the separation can be effected in any medium of uniform density, including growth medium. In addition, the effective path length of the rotor can be increased by using smaller incremental increases
/flow *in
rate. Under our conditions, flow rate
could be controlled to within 0.1 ml/min; however, smaller increments could be realized with
/more elaborate pumping systems.
dE a
The synchrony shown here (Fig. 3) is admittedly not as good as that obtained by Sebastian et al. for cells fractionated by zonal centrifuga-
0
25
12 13 14 15 16 17 18 19 20 21 22 23 24 25
Flow Rate(ml/mi) FIG. 2. Distribution of cells in fractions obtained from the elutriator rotor after Giemsa staining. The inset shows schematically how cells may be classified according to disposition of the nucleus after Giemsa staining: (0) unbudded cells, (0) budding cells in which the nucleus is in the parent cell, (A) cells in which the nucleus is in the act of migration between parent and daughter cell, and (a) cells in which the nucleus has partitioned itself between parent and daughter cell. Fractions obtained from the elutriator rotor at the indicated flow rates (abscissa) were stained with Giemsa, and the distribution ofthe four cell types were determined microscopically. At least 100 cells in each fraction were scored.
DISCUSSION
0
xS 10 E
c , U 2
_
200
100
TTime
300
(m i n)
FIG. 3. Synchronous growth of cells elutriated at
Our results show that centrifugal elutriation 11 ml/min (A) and 25 mllmin (0) and exponential is an effective method of separating an expo- growth of a portion of the unfractionated population nential yeast population into fractions repre- (0). The major portion of an exponential population senting successive stages of the cell cycle. This of cells was fractionated by elutriation at 4°C. Fracconclusion is based on the following lines of tions elutriating at 11 ml/min (largely unbudded nuclear cells in which cells) and is25complete) ml/min (largely evidence. () Successive fractions from the elu- partition and at 4°C were rotor triator tha were examinedintheCou ofF at 23°C. A portion that were examined in the Coulter inoculated into fresh mediumcentrifuged rotor triator counter showed an increase in average cell size the cells in the exponential population was not fracwith increasing flow rate. (ii) Microscopic ex- tionated but was kept at 4°C and then centrifuged amination of the fractions showed that the in- and inoculated into fresh medium at 23°C at the crease in average size of the cell population same time as the fractionated samples.
100
GORDON AND ELLIOTT
tion (7). However, Sebastian et al. fractionated the cells at room temperature, whereas our experiment was carried out in the cold and nonuniform recovery from cold shock may have worsened the degree of synchrony. Low-temperature fractionation was chosen because we wished to fractionate the entire population of cells. This process takes 2 to 3 h under our conditions of flow rate and centrifuge speed, and a low temperature was used to minimize growth changes during this period. However, elutriation of a particular size class can be performed rapidly at room temperature. For example, suppose one wished to obtain the fraction of cells that elutriates at 21 ml/min. After the cells were loaded, the flow rate would be immediately raised to 20 ml/min, and all cells elutriating at flow rates of 20 ml/min or less would be flushed out of the rotor. The flow rate would be raised to 21 ml/min, and the cells in this size class would be obtained as a discrete size class. In theory, the fractionation process could be shortened by collecting smaller volumes and by the use of higher centrifuge speeds and faster flow rates. However, we have not determined what, if any, effect these variables would have on the quality of the separation. Cell cycle studies were performed on cells that were harvested during balanced growth and then fixed with trichloroacetic acid prior to fractionation by zonal centrifugation (8). In this case, the length of time required to carry out the separation does not affect the resolution of the system because the cells cannot exhibit growth changes during the fractionation process. Elutriation would be ideally suited for this purpose because of the fine degree of separation obtained by using small incremental increases in flow rate. Thus, events occupying relatively short periods in the cell cycle can be studied. Since both zonal centrifugation and centrifugal elutriation separate cells on the basis of sedimentation rate, some comparisons of the two systems are in order. Intrinsic variation in the basic parent cell size among the members of a yeast population would result in a poorer fractionation with both methods. Size heterogeneity among a yeast population arises because of divisional age-as cells get older (i.e., acquire more bud scars), they become larger (4). Thus, any exponential yeast cell population will show some degree of size heterogeneity, and this places a basic limitation on the extent
J. BACTERIOL.
to which a population can be fractionated according to cell cycle age. Since size heterogeneity is also a strain-dependent phenomenon, optimum separation on the basis of cell cycle age will be obtained for strains that show high degrees of size uniformity. Both methods have additional physical characteristics that further impair the efficiency of fractionation of a yeast cell population. In elutriation, a Coriolis or streaming effect tends to make the removal of small particles from the rotor less efficient than the removal of larger particles (6). In zonal centrifugation, resolution is impaired by diffusion of the starting zone (8) and possibly by the pushing of small cells into the gradient by the larger cells as they begin migration (7). Because ofthese limitations, neither method can achieve a perfect separation of cells on the basis of cell cycle age. Elutriation, because of its advantages of long path length and wide choice of solvents, is a useful addition to existing methods of obtaining cell cycle separations of yeast by size selection. ACKNOWLEDGMENTS We wish to thank C. McLaughlin for his comments and suggestions, James Cladek for assistance with some of the fractionations, and 0. M. Griffith, Bob Lopes, and Bill Cassel of Beckman Instruments for aid and advice in setting up and operating the elutriation system. This work was supported by grant BMS 73-06847 A01 from the National Science Foundation. S.G.E. is supported by Public Health Service grant CA 10628 (awarded to C. McLaughlin) from the National Cancer Institute. LITERATURE CITED 1. Hartwell, L. H. 1967. Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93:1662-1670. 2. Hartwell, L. H. 1970. Periodic density fluctuations during the yeast cell cycle and the selection of synchronous cultures. J. Bacteriol. 104:1280-1285. 3. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 4. Hayashibe, M., N. Sando, and N. Abe. 1973. Increase in cell size as a measure of growth of Saccharomyces cerevwiae. J. Gen. Appl. Microbiol. 19:287-303. 5. Lindahl, P. E. 1948. Principle of a counter-streaming centrifuge for the separation of particles of different sizes. Nature (London) 161:648-649. 6. McEwen, C. R., R. W. Stallard, and E. T. Jukos. 1968. Separation of biological particles by centrifugal elutriation. Anal. Biochem. 23:369-377. 7. Sebastian, J., B. L. A. Carter, and H. 0. Halvorson. 1971. Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol. 108:1045-1050. 8. Wells, J. R. and T. W. James. 1972. Cell cycle analysis by culture fractionation. Exp. Cell Res. 75:465-474.
JoURNAL OF BACTERIOLOGY, Jan. 1977, p. 97-100
Copyright 0 1977 American Society for Microbiology
Fractionation of Saccharomyces cerevisiae Cell Populations by Centrifugal Elutriation C. N. GORDON* AND S. G. ELLIOT1 Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717
Received for publication 24 September 1976
An exponential population of Saccharomyces cerevisiae cells was fractionated by centrifugal elutriation, using water as the elutriating liquid. Evidence that the population had been fractionated according to age in the cell cycle was obtained by examinig the fractions for their size distribution, their microscopic appearance after Giemsa staining, and their ability to initiate synchronous growth. A number of procedures have been employed for obtaining synchronous cultures of the yeast Saccharomyces cerevisiae for use in studies of the cell cycle (3). Generally, synchronization procedures are subject to the criticism that the synchronization process may introduce distortions in the normal sequence of events in the cell cycle. In addition, some techniques use only a small percentage of the original cell population in growing the synchronous culture and, consequently, the yield of cells at any particular stage of the cell cycle is low. Sebastian et al. carried out large-scale fractionations of exponential cultures of S. cerevisiae by velocity sedimentation through sucrose gradients in a rotating-seal zonal rotor (7). Similar fractionations of the fission yeast Schizosaccharomyces pombe were carried out on nonliving cells by Wells and James in a reorienting gradient zonal rotor (8). These methods have an important advantage over synchronous growth in that cell cycle measurements are made on cells that have been fractionated from a condition of balanced growth, thus aVoiding possible artifacts induced by the synchronization proc-
obtained can be greatly increased by choosing smaller incremental increases in flow rate. Second, the fractionation can be carried out in virtually any aqueous solution of uniform density. In this paper we describe the fractionation of an exponential yeast population by centrifugal elutriation with water as the elutriating liquid. Our results indicate that elutriation separates the yeast cell population into fractions representing successive stages of the cell cycle, and is an effective method of obtaining large numbers of cells for cell cycle studies. MATERIALS AND METHODS Organism. S. cerevisiae SKQ2n, a prototrophic diploid with the genotype a/a, adel/+, +/ade2, +/
hisl was used. It was obtained from Brian Cox, Botany School, South Parks Road, Oxford, OX1 3RA, United Kingdom. Growth and harvest of cells. Cells were grown in YM-1 medium (1) to a cell density of 1.5 x 107 to 3.0 x 107 cells/ml at 23°C on a rotary shaker. Growth was retarded by chilling to 0°C, and the cells were centrifuged and washed with water at 4°C. The cells (3 x 109) were suspended in 10 ml of ice water and ess. in an ice bath were sonically treated while Centrifugal elutriation ("counterflow centrif- for 10jacketed s in a Biosonik Sonifier (Bronwill Scientific ugation") was used as a method of separating Inc., Rochester, N.Y.) at half the maximal setting. biological particles nearly 30 years ago (5), but The sonically treated cell suspension was then kept a rotor suitable for general use has only re- in ice until it was loaded into the elutriator rotor. cently become commercially available. In this Fractionation by centrifugal elutriation. A Beckprocedure, the force tending to sediment parti- man JE-6 elutriator rotor was driven in a Beckman cles in a centrifugal field is counterbalanced by J-21 centrifuge. The centrifuge was modified for elua liquid flowing in the opposite direction. A cell triation and equipped with a stroboscope assembly the manufacturer (Beckman Instruments, Inc., population can be fractionated into discrete size by Alto, Calif.). The pump used in the system was classes by incrementally increasing the flow Palo a Masterflex 745 equipped with a model 7014 pump rate while maintaining the centrifuge rotor at a head (Cole-Parmer Instrument, Co., Chicago, Ill.). constant speed (6). Centrifugal elutriation has To allow greater sensitivity in determining the two important advantages over zonal centrifu- pump flow rate, the one-turn potentiometer speed gation. First, the rotor has a much longer effec- control was removed and substituted with a 10-turn tive path length, and the number of size classes control (Beckman helipot 7276R50L.25) and a sepa97
98
J. BACTERIOL.
GORDON AND ELLIOTT
rate on-off switch. The flow system was set up in the following way. The receptacle of a 100-ml "Yale Luer-Lok" syringe (Becton-Dickinson and Co., Rutherford, N.J.) was interposed between the pump inlet and the water reservoir by means of a threeway bypass valve and Silastic tubing. The syringe contained the cell suspension to be loaded into the rotor. With the pump running continuously, manipulation of the bypass valve allowed direct flow from the reservoir into the rotor or, alternatively, loading G) 0) of the cell suspension into the rotor. Elutriation was done at a centrifuge well temperature of 4°C while the water reservoir and collection flasks were maintained in ice baths. The length of CL connecting tubing was kept to a minimum. Water flow through the rotor was started and the rotor was brought to a speed of 3,000 rpm. Care was taken to remove all air bubbles in the system before the cells were loaded. The chilled and sonically treated cell suspension was placed in the loading syringe, and then the flow rate was brought to 9 ml/min. The cells were loaded at this flow rate and then flushed with 200 ml of water. Next, 200-ml fractions were Cell Diameter(microns) collected. The first fraction was collected at 11 ml/ distributions of cells in fractions obFIG. 1. Size min, and successive fractions were obtained with increments of 1 ml/min. At a flow rate of 27 ml/min, tained from the elutriator rotor. An exponential culvirtually all of the single cells were removed from ture of yeast cells was fractionated by centrifugal elutriation, and 16 fractions were obtained by succesthe rotor. Analysis of fractions. To each fraction, NaCl was sively increasing the flow rate in increments of1 ml/ added to 0.1 M and formaldehyde was added to 0.4%. min. The fractions were fixed with formaldehyde and The cells were collected by centrifugation and sus- centrifuged, and the size distributions were deterpended in 0.9% NaCl at a cell density of 106 cells/ml. mined in a Coulter counter. The four fractions shown The size distribution of the cells in each fraction was were obtained with the following flow rates: (A) 11 determined in a model T Coulter counter that had ml/min; (B) 15 ml/min; (C) 23 ml/min; and (D) 26 been calibrated with latex spheres of average parti- ml/min. cle diameter 3.49 ,um. Samples of the fractions were prepared for microscopic observation by a modified tions obtained from the elutriator rotor is also Giemsa staining procedure (2). To demonstrate synchronous growth in fractions shown. At the lower flow rates (11 to 13 ml/ elutriated at 11 and 25 ml/min, formaldehyde was min), the population consists predominantly of omitted and the centrifuged fractions were sus- unbudded cells. Budded cells, in which the nupended in YM-1 medium at a cell density of 3 x 106/ cleus has not yet migrated into the bud neck, ml. The cell suspensions were incubated at 23°C, constitute the major component in fractions and 0.1-ml portions were removed at the indicated elutriated at flow rates of 16 to 18 ml/min. Cells times and placed in 0.9% NaCl containing 0.5% in which the nucleus is in the act of migration formaldehyde. After sonic dispersion for 10 s, cell reach a peak in the fraction elutriated at 21 ml/ numbers were determined in a model F Coulter min. Finally, the fractions obtained at flow a
counter.
RESULTS The size distributions of four fractions obtained from the elutriator rotor and then fixed with formaldehyde are shown in Fig. 1. It is evident that there is an increase in the diameter of the cells elutriated with increasing flow rate. Evidence that the elutriation is effecting a fractionation of the population according to age in the cell cycle was obtained from microscopic examination of the fractions after Giemsa staining. In Giemsa-stained preparations, cells can be classified into one of four groups, shown schematically in the inset to Fig. 2. The distribution of the four basic cell types in the frac-
rates of 23 to 26 ml/min consist primarily of cells in which partition of the nucleus between mother and daughter cells has been completed. Remaining in the rotor were clumped cells representing less than 1% of the cells loaded into the rotor. These clumps may have been formed during growth, harvesting, and washing of the cells, and they were apparently not dispersed by the sonic treatment. Figure 3 shows growth curves obtained with fractions elutriating at 11 and 25 ml/min. The growth curve of the unfractionated culture is shown for comparison. It is evident that the fractions obtained by elutriation behave like synchronously growing cultures.
,..~ ,~
i~A 8tJ t y (! t, va, o
o
100
.
*
75
1
CD
/ /
y /r ° \\
C
V
99
CENTRIFUGAL ELUTRIATION OF YEAST CELLS
VOL. 129, 1977
~ 50
CL
within each fraction was due to an enrichment of the fraction by cells in comparable stages of the cell cycle. (iii) Fractions obtained from the elutriator rotor showed synchronous growth. An important advantage of this method over zonal centrifugation or other selection techniques is the wide choice of media available to carry out the fractionation. Although water was used in our experiments, the separation can be effected in any medium of uniform density, including growth medium. In addition, the effective path length of the rotor can be increased by using smaller incremental increases
/flow *in
rate. Under our conditions, flow rate
could be controlled to within 0.1 ml/min; however, smaller increments could be realized with
/more elaborate pumping systems.
dE a
The synchrony shown here (Fig. 3) is admittedly not as good as that obtained by Sebastian et al. for cells fractionated by zonal centrifuga-
0
25
12 13 14 15 16 17 18 19 20 21 22 23 24 25
Flow Rate(ml/mi) FIG. 2. Distribution of cells in fractions obtained from the elutriator rotor after Giemsa staining. The inset shows schematically how cells may be classified according to disposition of the nucleus after Giemsa staining: (0) unbudded cells, (0) budding cells in which the nucleus is in the parent cell, (A) cells in which the nucleus is in the act of migration between parent and daughter cell, and (a) cells in which the nucleus has partitioned itself between parent and daughter cell. Fractions obtained from the elutriator rotor at the indicated flow rates (abscissa) were stained with Giemsa, and the distribution ofthe four cell types were determined microscopically. At least 100 cells in each fraction were scored.
DISCUSSION
0
xS 10 E
c , U 2
_
200
100
TTime
300
(m i n)
FIG. 3. Synchronous growth of cells elutriated at
Our results show that centrifugal elutriation 11 ml/min (A) and 25 mllmin (0) and exponential is an effective method of separating an expo- growth of a portion of the unfractionated population nential yeast population into fractions repre- (0). The major portion of an exponential population senting successive stages of the cell cycle. This of cells was fractionated by elutriation at 4°C. Fracconclusion is based on the following lines of tions elutriating at 11 ml/min (largely unbudded nuclear cells in which cells) and is25complete) ml/min (largely evidence. () Successive fractions from the elu- partition and at 4°C were rotor triator tha were examinedintheCou ofF at 23°C. A portion that were examined in the Coulter inoculated into fresh mediumcentrifuged rotor triator counter showed an increase in average cell size the cells in the exponential population was not fracwith increasing flow rate. (ii) Microscopic ex- tionated but was kept at 4°C and then centrifuged amination of the fractions showed that the in- and inoculated into fresh medium at 23°C at the crease in average size of the cell population same time as the fractionated samples.
100
GORDON AND ELLIOTT
tion (7). However, Sebastian et al. fractionated the cells at room temperature, whereas our experiment was carried out in the cold and nonuniform recovery from cold shock may have worsened the degree of synchrony. Low-temperature fractionation was chosen because we wished to fractionate the entire population of cells. This process takes 2 to 3 h under our conditions of flow rate and centrifuge speed, and a low temperature was used to minimize growth changes during this period. However, elutriation of a particular size class can be performed rapidly at room temperature. For example, suppose one wished to obtain the fraction of cells that elutriates at 21 ml/min. After the cells were loaded, the flow rate would be immediately raised to 20 ml/min, and all cells elutriating at flow rates of 20 ml/min or less would be flushed out of the rotor. The flow rate would be raised to 21 ml/min, and the cells in this size class would be obtained as a discrete size class. In theory, the fractionation process could be shortened by collecting smaller volumes and by the use of higher centrifuge speeds and faster flow rates. However, we have not determined what, if any, effect these variables would have on the quality of the separation. Cell cycle studies were performed on cells that were harvested during balanced growth and then fixed with trichloroacetic acid prior to fractionation by zonal centrifugation (8). In this case, the length of time required to carry out the separation does not affect the resolution of the system because the cells cannot exhibit growth changes during the fractionation process. Elutriation would be ideally suited for this purpose because of the fine degree of separation obtained by using small incremental increases in flow rate. Thus, events occupying relatively short periods in the cell cycle can be studied. Since both zonal centrifugation and centrifugal elutriation separate cells on the basis of sedimentation rate, some comparisons of the two systems are in order. Intrinsic variation in the basic parent cell size among the members of a yeast population would result in a poorer fractionation with both methods. Size heterogeneity among a yeast population arises because of divisional age-as cells get older (i.e., acquire more bud scars), they become larger (4). Thus, any exponential yeast cell population will show some degree of size heterogeneity, and this places a basic limitation on the extent
J. BACTERIOL.
to which a population can be fractionated according to cell cycle age. Since size heterogeneity is also a strain-dependent phenomenon, optimum separation on the basis of cell cycle age will be obtained for strains that show high degrees of size uniformity. Both methods have additional physical characteristics that further impair the efficiency of fractionation of a yeast cell population. In elutriation, a Coriolis or streaming effect tends to make the removal of small particles from the rotor less efficient than the removal of larger particles (6). In zonal centrifugation, resolution is impaired by diffusion of the starting zone (8) and possibly by the pushing of small cells into the gradient by the larger cells as they begin migration (7). Because ofthese limitations, neither method can achieve a perfect separation of cells on the basis of cell cycle age. Elutriation, because of its advantages of long path length and wide choice of solvents, is a useful addition to existing methods of obtaining cell cycle separations of yeast by size selection. ACKNOWLEDGMENTS We wish to thank C. McLaughlin for his comments and suggestions, James Cladek for assistance with some of the fractionations, and 0. M. Griffith, Bob Lopes, and Bill Cassel of Beckman Instruments for aid and advice in setting up and operating the elutriation system. This work was supported by grant BMS 73-06847 A01 from the National Science Foundation. S.G.E. is supported by Public Health Service grant CA 10628 (awarded to C. McLaughlin) from the National Cancer Institute. LITERATURE CITED 1. Hartwell, L. H. 1967. Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93:1662-1670. 2. Hartwell, L. H. 1970. Periodic density fluctuations during the yeast cell cycle and the selection of synchronous cultures. J. Bacteriol. 104:1280-1285. 3. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 4. Hayashibe, M., N. Sando, and N. Abe. 1973. Increase in cell size as a measure of growth of Saccharomyces cerevwiae. J. Gen. Appl. Microbiol. 19:287-303. 5. Lindahl, P. E. 1948. Principle of a counter-streaming centrifuge for the separation of particles of different sizes. Nature (London) 161:648-649. 6. McEwen, C. R., R. W. Stallard, and E. T. Jukos. 1968. Separation of biological particles by centrifugal elutriation. Anal. Biochem. 23:369-377. 7. Sebastian, J., B. L. A. Carter, and H. 0. Halvorson. 1971. Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol. 108:1045-1050. 8. Wells, J. R. and T. W. James. 1972. Cell cycle analysis by culture fractionation. Exp. Cell Res. 75:465-474.
