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Induction of synchronous cell division in mass cultures of Tetrahymena piriformis
Experimentul
Cell Research,
6, 221-227
221
(1954)
INDUCTION OF SYNCHRONOUS CELL CULTURES OF TETRAHYMENA 0. Laboratory
of
SCHERBAUMl
and
Zoophysiology,
University
Received
October
DIVISION IN MASS PIRIFORMIS
E. ZEUTHEN
of Copenhagen,
Denmark
2
22, 1953
the ultimate scope of making the mitotic cycle accessible to the study with standard chemical and physiological techniques, we have during the past year endeavored to produce synchronism of cell division in mass cultures of Tetrahymena piriformis, Lwoff’s strain. Tetrahymena was selected because it can be grown in pure culture in proteose-peptone media and-sole among animal cells-also in fully defined media as reported by Kidder and Dewey (4). One possible way of synchronizing the growth activities of this organism would be to place a barrier at a certain point in the cell cycle, say by using inhibitors known or suspected to inhibit specific phases. Colchicin and Aminopterin were tried, with meso-inositol (5) and folic acid, or folic acid derivatives, as suggested releasing agents. We failed because the inhibitors proved non-toxic, as earlier reported (3, 4). The effect of temperature has been tried, however, starting out from a different theory. Ephrussi (2) many years ago reported that the Qlo is different for the different phases of cell division in the sea urchin egg. It was, therefore, considered that exposure to lowered temperature, for a time shorter than the mitotic cycle at the low temperature, with subsequent return to optimum temperature, might tend to bring closer separate stages having different Qlo’s. After the lapse of another cycle at optimum temperature repetition of the treatment should tend to make the group larger, etc., till synchronism in the whole culture had been produced. Indeed, shifts of temperature 28”+ 7” C had some effect, but disturbing factors seemed to interfere. These experiments, however, led the way to successful experiments in which the temperature was periodically raised to sublethal levels. This paper reports such experiments. We are not yet going to offer full interpretation of our findings; we think, however, that the results are best understood, not on the basis of different Qlo’s as discussed above, but on WITH
1 Supported 2 Address:
by a grant of “Statens Almindelige Juliane Maries Vej 32, Copenhagen.
Videnskabsfond”.
Experimental
Cell Research
6
222
0. Scherbaum
and E. Zeuthen
the basis of the assumption that subIetha1 temperatures act by blocking -with a considerable recovery time at optimum temperature-a specific step in the cell cycle. EXPERIMENTS The cells were grown as pure cultures in 2 per cent proteose peptone (Difco)+ 1 per mille liver fraction L (Wilson Laboratories); salts were added according to Kidder and Dewey (4). Under conditions of ample supply of oxygen, in single cell cultures grown in capillaries on 0.5-1.5 ~1, medium population densities may reach 1.2 million cells/ml on this medium (personal communication from Mr. H. Thormar). However, there is evidence that true exponential multiplication does not continue beyond population densities of about 100,000 cells/ml or perhaps less. In the experiments to be reported we have therefore worked with very dilute cultures (5,000-50,000 cells/ml), offhand assumed to be in the log-phase of growth. We used two types of culture flasks: either salt cellars (1 ml culture medium, covering 4.5 cm*, average depth 2.2 mm, bottom slightly hollow) or 1/B 1, flat-bottomed flasks (10 ml culture medium covering 57 cm 2, depth 1.75 mm). In the salt cellars the glass walls are thicker (5-6 mm) than in the big flasks (about I mm). No shaking was used. The flasks were dipped into a water bath which was regulated to k 0.1” C. The temperature of the bath could be shifted up and down by the use of a signal watch which at predetermined times switched the current from one thermoregulator to another. Adjustment of the temperature in the bath always occurred with some delay (8 min. for an increase from 28” to 34” C, and 12 min. for a drop from 34” to 28” C), and within the cultures themselves the delay was in addition influenced by the amount of culture medium and by the thickness of the glass. The figures corresponding to those given for the bath were 20, resp. 17 minutes for the salt cellars, and 15 resp. 14 minutes for the 1/e 1 flasks. Usually, cultures were inoculated from stationary phase cultures (kept at 23-24” C) and grown at 28” C; after the end of a lag the division index was followed; it represents the ratio between number of cells in division and number of cells not dividing. As division cells were counted all stages from the onset of cytoplasmic constriction to separation of the daughter cells. The division index was determined on 100 ~1 samples fixed in 5 per cent formaldehyde; every point represents the counting of about 300 cells. We found the division index to vary from about 0.05 to about 0.10. Single cells growing in 0.5-1.5 ~1 of the medium here used were observed to divide every 135 minutes at 28” C ( 2 20 min.). Division times of 6.8-12.5 min. have been reported (1). The division index calculated from the two set of data is 0.05-0.095, which corresponds nicely with our observations (cf. Fig. 1). Upon transfer from 28”-29.5” C (which shall hereafter, without special qualification, be called “optimum temperature”, cf. however (6)) to a sublethal temperature (32”-34” C).) the division index tends to decrease and soon it gets very low. However, at the high temperature there is a partial or a complete block for growth (6). So we suggest that sublethal temperature Experimental
Cell Research
6
Synchronous
cell division
prevents growth (e.g. syntheses), division, but it does not prevent completion.
Fig. 1. Division index in a culture of Tetrahymena pirilormis treated for 6r/, hours with intermittent heat shocks. The uppermost curve shows the switch of the temperature as indicated by signals on the watch, respectively transfer into bath (1 h) and removal from bath (8 h). Proteose - peptone culture, 10 ml in flask. Total initial population about 400,000.
a%
it also prevents new cells from entering a a division, once initiated, from running to
I
I
U-
I
II-$ .r u-
1; -.I Ltl .tzl d .I; 21 I I 1
.iu2 a,6 a-
223
in Tefrahymena
I I
Jd,Z $j
i’
,i
;
i
F’ %I 21 &.I hl Ii
. ,?
3
I,
?J!L z
6
I
0
9
. M
. n
72
9.3
However, we expose cells to sublethal temperatures only for a short time and then, for another short period, we transfer them to the optimum temperature. The cells then resume growth but they do not, within this short time, begin to divide. Before they recover the mechanisms which in normal cells operate to switch cells from synthesis (growth in mass) to division they are exposed to another short temperature shock and thereafter again transferred to optimum temperature and so on. With this treatment of intermittent heat shocks (we might say: “intermittent fever”) the cells grow bigger and bigger (cf. Fig. 2 a, b). After several hours they are returned to constant optimum, or usually just to room temperature (the dishes were placed under a binocular microscope for continuous observation). About 1 1/2 ( + 10 minutes) hours at this temperature a burst in division activity was observed (Fig. 2~). As many as 85 per cent of the cells may be in division simultaneously. From now on, through a few mitotic cycles, the divisions appear to be synchronized (cf. Fig. 1). We have observed three successive peaks, with respectively 85, 83 and 64 per cent of the cells dividing simultaneously. In between there are minima in which only about 2 per cent of the cells are in division.1 The peaks appear to be closer in time than indicated by the duration of the normal mitotic cycle (at 24” C: 1.7 hours, as compared with a normal of 2.5-3 hours). 1 Here we may add that in the experiments with lowered peak in division activity, but after this division rate returned, an untreated culture, e.g. no rhythmicity was induced.
temperature we found more or less, to the
Experimental
a low first average for
Cell Research
6
0. Scherbaum
and E. Zeuthen
Obviously, in the type of experiment reported there is a considerable number of variables which have not yet been examined in sufficient detail. In attempting to establish conditions of temperature treatment which will produce the highest possible degree of synchronism in the cultures we have made the following observations: Time of treatment: 6-10 hours. Shorter treatment tends to produce lower peaks of division activity and longer treatment (16 hours) results in expressed distortion of the cells. Temperature during treatment: so far we have obtained the best results if we let the temperature shift between 29” and 33” C (32.3”-33.7” C in the different cases) with a period of 1 hour, e.g. ‘I2 hour high, and 1/z hour low. Two-hour periods were rather ineffective, the division index was not considerably reduced during treatment, and not very high after. Thirty-minutes periods were more effective than 2-hours, but still inferior to l-hour periods. We have also tried to change the ratio: time at high temperature over time at low temperature, however, with no beneficial effects. Increasing the ratio tended to make the cells more distorted, decreasing the ratio resulted in cell multiplication during treatment and in less piling up of divisions. During the heat treatment the average cell size increases, e.g. about 3-fold in 7-8 hours. This is illustrated in the photomicrographs, Fig. 2 a, b, c. The treated cells (b) become big and, compared with the controls (a), relatively thick (pear-shaped). Also, they may become somewhat distorted. In addition to what has been said above we add that for a minimum of distortion to develop it may be essential that 1) the population density is so low (order 5,000-5O,OOO/ml) that true exponential multiplication would have been possible had we not exposed the cells to temperature treatment, that 2) oxygen is not limiting in any part of the culture, and 3) that the temperature changes are not too abrupt. Even if there are some distorted cells by the end of heat treatment, all cells assume a perfectly normal look after one or two cycles at optimum or room temperature. It should be commented that no-or at least very few-cells are killed either during or after the heat treatment. Actually, during treatment cell numbers increase very slowly, and about as much as could be expected from the very low, however real, division index found during treatment. The increase in cell size was followed by volume measurements of single cells in a compression chamber a.m. Scholander, Claff and Sveinsson (7). While the average cell volume increases as shown in Fig. 3, curve x , the relative size differences, initially observed, are retained. The average protein content was studied with an unconventional (and not yet well enough Experimental
Cell
Research
6
Synchronous
Fig. 2. Exp. 29. 4. 1953. Proteose - peptone culture, 10 ml in flask. Total initialpopulationll5,OOO cells, Individuals alive when pictures taken. culture 24” C) several hours after inoculation. h: cells treated 9 hours at 28” + 34” C (I/, hour high, 1/Z hour lo;‘): Photographed ‘iz hour after return to 24” C. c: same after 100 minutes at 24” C.
cell division
in Tetrahymena
225
w
2i L-l ioop Experimental
Ceil Research
6
226
0. Scherbaum
and E. Zeuthen
checked) method in which the amount of fixative (for 3-5 days, maximum weight after 3 days) in 3 acid was measured as the reduced weight of the diver balance (8) floating in the fixative was used.
‘1
0
2
*
6
8
,o
,2
,4
picked up by cells fixed per cent phosphotungstic fixed cells. A Cartesian The growth during heat
Fig. 3. Reduced weight (RW) per cell, after 7-8 days of fixation in 3 per cent phosphotungstir acid. RW measurements carried out in the fixative (density: 1.0226, water at same temperature 1.0000). RW of unfixed cells not measured; however, cells fixed for 4 hours weigh only 40 per cent of same cells fixed for three days or more. x cell volumes measured as area x height in a compression chamber, height 10 I*.
hours
also the macronuclear volume increases treatment is not only cgtoplasmic; very considerably. After recovery from heat treatment, e.g. at optimum or room temperature, the cells tend to become smaller with each new maximum of division. That is, now the situation is reversed relative to the one during heat treatment: cell divisions are outbalancing the growth activities. For Tetrahymena it has previously been reported (9) that big cells can be harvested from stationary phase cultures. Upon transfer to fresh medium these cells resume growth, but they divide faster than they double in mass, with the result that in the early generations cells get smaller and smaller. Carefully traced growth curves for single cell cultures (9) were interpreted to indicate that the growth in mass (mass measured by the amount of respiration!) does not keep up with the divisions because the initial big cell grows exponentially \vith only part of its body; the remainder does not grow at all Experimental
Cell Research
6
Synchronous
cell division
in Tetrahymena
227
and becomes diluted away upon the increasing number of cells in the progeny. It is a situation like this which we believe pertains to the giant cells produced by the heat treatment. SUMMARY
This preliminary report shows that, using intermittent heat treatment of 85 per cent of the cells can be induced to mass cultures of Tetrahymena, undergo division simultaneously. Three successive maxima of divisions have been observed. The cell populations used represent total cell numbers of anything between 5,000 and 1/2 million corresponding to dry weights of the order 0.03 to 5 mg. The work is continued with attempts to synchronize much larger quantities of material, first on proteose-peptone media, then on synthetic media. REFERENCES 1. BROWNING, I., VARNEDOE, N. B., SVINFORD, L. R., J. Ce!hzlar Comp. Physiot., 39, 371 (1952). 2. EPHRUSSI. B.. Protoplasma, I, 105 (1926). 3. HALBER&AE&R, L., and. BACK, A., Nature, 152, 275 (1943). 4. KIDDER. G. W.. and DEWEY, V. C., Biochemistry and Physiology of Protozoa. Ed. by A. Lwoff, P. ‘323, 1951. 5. MURRAY, M. R., DE LAM, H. H., and CHARGAFF, E., Expfl. CeZl Research, 2, 165 (1951). 6. PHELPS, A., J. exptl. Zool., 102, 277 (1946). 7. SCHOLANDER, P. F., CLAFF, C. L., and SVEINSSON, S. L., Biol. Bull., 102, 157 (1952). 8. ZEUTHEN, E., Compt. rend. Lab. Carkberg, SCr. chim., 26, 243 (1948). 9. -J. Embryot. and exptf. Morphol., 1, in press (1953).
Experimental
Cell Research
6
Cell Research,
6, 221-227
221
(1954)
INDUCTION OF SYNCHRONOUS CELL CULTURES OF TETRAHYMENA 0. Laboratory
of
SCHERBAUMl
and
Zoophysiology,
University
Received
October
DIVISION IN MASS PIRIFORMIS
E. ZEUTHEN
of Copenhagen,
Denmark
2
22, 1953
the ultimate scope of making the mitotic cycle accessible to the study with standard chemical and physiological techniques, we have during the past year endeavored to produce synchronism of cell division in mass cultures of Tetrahymena piriformis, Lwoff’s strain. Tetrahymena was selected because it can be grown in pure culture in proteose-peptone media and-sole among animal cells-also in fully defined media as reported by Kidder and Dewey (4). One possible way of synchronizing the growth activities of this organism would be to place a barrier at a certain point in the cell cycle, say by using inhibitors known or suspected to inhibit specific phases. Colchicin and Aminopterin were tried, with meso-inositol (5) and folic acid, or folic acid derivatives, as suggested releasing agents. We failed because the inhibitors proved non-toxic, as earlier reported (3, 4). The effect of temperature has been tried, however, starting out from a different theory. Ephrussi (2) many years ago reported that the Qlo is different for the different phases of cell division in the sea urchin egg. It was, therefore, considered that exposure to lowered temperature, for a time shorter than the mitotic cycle at the low temperature, with subsequent return to optimum temperature, might tend to bring closer separate stages having different Qlo’s. After the lapse of another cycle at optimum temperature repetition of the treatment should tend to make the group larger, etc., till synchronism in the whole culture had been produced. Indeed, shifts of temperature 28”+ 7” C had some effect, but disturbing factors seemed to interfere. These experiments, however, led the way to successful experiments in which the temperature was periodically raised to sublethal levels. This paper reports such experiments. We are not yet going to offer full interpretation of our findings; we think, however, that the results are best understood, not on the basis of different Qlo’s as discussed above, but on WITH
1 Supported 2 Address:
by a grant of “Statens Almindelige Juliane Maries Vej 32, Copenhagen.
Videnskabsfond”.
Experimental
Cell Research
6
222
0. Scherbaum
and E. Zeuthen
the basis of the assumption that subIetha1 temperatures act by blocking -with a considerable recovery time at optimum temperature-a specific step in the cell cycle. EXPERIMENTS The cells were grown as pure cultures in 2 per cent proteose peptone (Difco)+ 1 per mille liver fraction L (Wilson Laboratories); salts were added according to Kidder and Dewey (4). Under conditions of ample supply of oxygen, in single cell cultures grown in capillaries on 0.5-1.5 ~1, medium population densities may reach 1.2 million cells/ml on this medium (personal communication from Mr. H. Thormar). However, there is evidence that true exponential multiplication does not continue beyond population densities of about 100,000 cells/ml or perhaps less. In the experiments to be reported we have therefore worked with very dilute cultures (5,000-50,000 cells/ml), offhand assumed to be in the log-phase of growth. We used two types of culture flasks: either salt cellars (1 ml culture medium, covering 4.5 cm*, average depth 2.2 mm, bottom slightly hollow) or 1/B 1, flat-bottomed flasks (10 ml culture medium covering 57 cm 2, depth 1.75 mm). In the salt cellars the glass walls are thicker (5-6 mm) than in the big flasks (about I mm). No shaking was used. The flasks were dipped into a water bath which was regulated to k 0.1” C. The temperature of the bath could be shifted up and down by the use of a signal watch which at predetermined times switched the current from one thermoregulator to another. Adjustment of the temperature in the bath always occurred with some delay (8 min. for an increase from 28” to 34” C, and 12 min. for a drop from 34” to 28” C), and within the cultures themselves the delay was in addition influenced by the amount of culture medium and by the thickness of the glass. The figures corresponding to those given for the bath were 20, resp. 17 minutes for the salt cellars, and 15 resp. 14 minutes for the 1/e 1 flasks. Usually, cultures were inoculated from stationary phase cultures (kept at 23-24” C) and grown at 28” C; after the end of a lag the division index was followed; it represents the ratio between number of cells in division and number of cells not dividing. As division cells were counted all stages from the onset of cytoplasmic constriction to separation of the daughter cells. The division index was determined on 100 ~1 samples fixed in 5 per cent formaldehyde; every point represents the counting of about 300 cells. We found the division index to vary from about 0.05 to about 0.10. Single cells growing in 0.5-1.5 ~1 of the medium here used were observed to divide every 135 minutes at 28” C ( 2 20 min.). Division times of 6.8-12.5 min. have been reported (1). The division index calculated from the two set of data is 0.05-0.095, which corresponds nicely with our observations (cf. Fig. 1). Upon transfer from 28”-29.5” C (which shall hereafter, without special qualification, be called “optimum temperature”, cf. however (6)) to a sublethal temperature (32”-34” C).) the division index tends to decrease and soon it gets very low. However, at the high temperature there is a partial or a complete block for growth (6). So we suggest that sublethal temperature Experimental
Cell Research
6
Synchronous
cell division
prevents growth (e.g. syntheses), division, but it does not prevent completion.
Fig. 1. Division index in a culture of Tetrahymena pirilormis treated for 6r/, hours with intermittent heat shocks. The uppermost curve shows the switch of the temperature as indicated by signals on the watch, respectively transfer into bath (1 h) and removal from bath (8 h). Proteose - peptone culture, 10 ml in flask. Total initial population about 400,000.
a%
it also prevents new cells from entering a a division, once initiated, from running to
I
I
U-
I
II-$ .r u-
1; -.I Ltl .tzl d .I; 21 I I 1
.iu2 a,6 a-
223
in Tefrahymena
I I
Jd,Z $j
i’
,i
;
i
F’ %I 21 &.I hl Ii
. ,?
3
I,
?J!L z
6
I
0
9
. M
. n
72
9.3
However, we expose cells to sublethal temperatures only for a short time and then, for another short period, we transfer them to the optimum temperature. The cells then resume growth but they do not, within this short time, begin to divide. Before they recover the mechanisms which in normal cells operate to switch cells from synthesis (growth in mass) to division they are exposed to another short temperature shock and thereafter again transferred to optimum temperature and so on. With this treatment of intermittent heat shocks (we might say: “intermittent fever”) the cells grow bigger and bigger (cf. Fig. 2 a, b). After several hours they are returned to constant optimum, or usually just to room temperature (the dishes were placed under a binocular microscope for continuous observation). About 1 1/2 ( + 10 minutes) hours at this temperature a burst in division activity was observed (Fig. 2~). As many as 85 per cent of the cells may be in division simultaneously. From now on, through a few mitotic cycles, the divisions appear to be synchronized (cf. Fig. 1). We have observed three successive peaks, with respectively 85, 83 and 64 per cent of the cells dividing simultaneously. In between there are minima in which only about 2 per cent of the cells are in division.1 The peaks appear to be closer in time than indicated by the duration of the normal mitotic cycle (at 24” C: 1.7 hours, as compared with a normal of 2.5-3 hours). 1 Here we may add that in the experiments with lowered peak in division activity, but after this division rate returned, an untreated culture, e.g. no rhythmicity was induced.
temperature we found more or less, to the
Experimental
a low first average for
Cell Research
6
0. Scherbaum
and E. Zeuthen
Obviously, in the type of experiment reported there is a considerable number of variables which have not yet been examined in sufficient detail. In attempting to establish conditions of temperature treatment which will produce the highest possible degree of synchronism in the cultures we have made the following observations: Time of treatment: 6-10 hours. Shorter treatment tends to produce lower peaks of division activity and longer treatment (16 hours) results in expressed distortion of the cells. Temperature during treatment: so far we have obtained the best results if we let the temperature shift between 29” and 33” C (32.3”-33.7” C in the different cases) with a period of 1 hour, e.g. ‘I2 hour high, and 1/z hour low. Two-hour periods were rather ineffective, the division index was not considerably reduced during treatment, and not very high after. Thirty-minutes periods were more effective than 2-hours, but still inferior to l-hour periods. We have also tried to change the ratio: time at high temperature over time at low temperature, however, with no beneficial effects. Increasing the ratio tended to make the cells more distorted, decreasing the ratio resulted in cell multiplication during treatment and in less piling up of divisions. During the heat treatment the average cell size increases, e.g. about 3-fold in 7-8 hours. This is illustrated in the photomicrographs, Fig. 2 a, b, c. The treated cells (b) become big and, compared with the controls (a), relatively thick (pear-shaped). Also, they may become somewhat distorted. In addition to what has been said above we add that for a minimum of distortion to develop it may be essential that 1) the population density is so low (order 5,000-5O,OOO/ml) that true exponential multiplication would have been possible had we not exposed the cells to temperature treatment, that 2) oxygen is not limiting in any part of the culture, and 3) that the temperature changes are not too abrupt. Even if there are some distorted cells by the end of heat treatment, all cells assume a perfectly normal look after one or two cycles at optimum or room temperature. It should be commented that no-or at least very few-cells are killed either during or after the heat treatment. Actually, during treatment cell numbers increase very slowly, and about as much as could be expected from the very low, however real, division index found during treatment. The increase in cell size was followed by volume measurements of single cells in a compression chamber a.m. Scholander, Claff and Sveinsson (7). While the average cell volume increases as shown in Fig. 3, curve x , the relative size differences, initially observed, are retained. The average protein content was studied with an unconventional (and not yet well enough Experimental
Cell
Research
6
Synchronous
Fig. 2. Exp. 29. 4. 1953. Proteose - peptone culture, 10 ml in flask. Total initialpopulationll5,OOO cells, Individuals alive when pictures taken. culture 24” C) several hours after inoculation. h: cells treated 9 hours at 28” + 34” C (I/, hour high, 1/Z hour lo;‘): Photographed ‘iz hour after return to 24” C. c: same after 100 minutes at 24” C.
cell division
in Tetrahymena
225
w
2i L-l ioop Experimental
Ceil Research
6
226
0. Scherbaum
and E. Zeuthen
checked) method in which the amount of fixative (for 3-5 days, maximum weight after 3 days) in 3 acid was measured as the reduced weight of the diver balance (8) floating in the fixative was used.
‘1
0
2
*
6
8
,o
,2
,4
picked up by cells fixed per cent phosphotungstic fixed cells. A Cartesian The growth during heat
Fig. 3. Reduced weight (RW) per cell, after 7-8 days of fixation in 3 per cent phosphotungstir acid. RW measurements carried out in the fixative (density: 1.0226, water at same temperature 1.0000). RW of unfixed cells not measured; however, cells fixed for 4 hours weigh only 40 per cent of same cells fixed for three days or more. x cell volumes measured as area x height in a compression chamber, height 10 I*.
hours
also the macronuclear volume increases treatment is not only cgtoplasmic; very considerably. After recovery from heat treatment, e.g. at optimum or room temperature, the cells tend to become smaller with each new maximum of division. That is, now the situation is reversed relative to the one during heat treatment: cell divisions are outbalancing the growth activities. For Tetrahymena it has previously been reported (9) that big cells can be harvested from stationary phase cultures. Upon transfer to fresh medium these cells resume growth, but they divide faster than they double in mass, with the result that in the early generations cells get smaller and smaller. Carefully traced growth curves for single cell cultures (9) were interpreted to indicate that the growth in mass (mass measured by the amount of respiration!) does not keep up with the divisions because the initial big cell grows exponentially \vith only part of its body; the remainder does not grow at all Experimental
Cell Research
6
Synchronous
cell division
in Tetrahymena
227
and becomes diluted away upon the increasing number of cells in the progeny. It is a situation like this which we believe pertains to the giant cells produced by the heat treatment. SUMMARY
This preliminary report shows that, using intermittent heat treatment of 85 per cent of the cells can be induced to mass cultures of Tetrahymena, undergo division simultaneously. Three successive maxima of divisions have been observed. The cell populations used represent total cell numbers of anything between 5,000 and 1/2 million corresponding to dry weights of the order 0.03 to 5 mg. The work is continued with attempts to synchronize much larger quantities of material, first on proteose-peptone media, then on synthetic media. REFERENCES 1. BROWNING, I., VARNEDOE, N. B., SVINFORD, L. R., J. Ce!hzlar Comp. Physiot., 39, 371 (1952). 2. EPHRUSSI. B.. Protoplasma, I, 105 (1926). 3. HALBER&AE&R, L., and. BACK, A., Nature, 152, 275 (1943). 4. KIDDER. G. W.. and DEWEY, V. C., Biochemistry and Physiology of Protozoa. Ed. by A. Lwoff, P. ‘323, 1951. 5. MURRAY, M. R., DE LAM, H. H., and CHARGAFF, E., Expfl. CeZl Research, 2, 165 (1951). 6. PHELPS, A., J. exptl. Zool., 102, 277 (1946). 7. SCHOLANDER, P. F., CLAFF, C. L., and SVEINSSON, S. L., Biol. Bull., 102, 157 (1952). 8. ZEUTHEN, E., Compt. rend. Lab. Carkberg, SCr. chim., 26, 243 (1948). 9. -J. Embryot. and exptf. Morphol., 1, in press (1953).
Experimental
Cell Research
6