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Synthetic and division rates of Euglena gracilis grown in batch cultures
Experimental
Cell Research 35, 69-76 (1964)
SYNTHETIC
AND DIVISION GROWN B.
Department
RATES OF EUGLENA
IN BATCH
W. WILSON2
of Zoology,
69
University
GRACILIS
CULTURES1
and B. H. LEVEDAHL of California,
Los Angeles, Calif.,
U.S.A.
Received June 20, 1963
THE biochemical
properties of the average cell from exponentially growing cultures have often been thought to be time invariant. Cells have been “ . . . considered to be rather fixed entities that can be dealt with much as a physicist deals with an atom or a molecule” [ 131. However, changes with time in the characteristics of exponentially growing bacteria [26], tissue culture cells [23, 251 and protozoa [4] have been described. Explanations for these changes fall logically into two categories; the synthetic rates of the constituents in question may alter due to changes in the composition of the medium, or these syntheses may proceed at constant rates which differ from the rates of cell division in the cultures. Buetow and Levedahl [4] studied the growth in batch culture of a colorless strain of the flagellate Euglena gracilis var. bacillaris. They found that ribonucleic acid, protein and dry weight of the cells decreased during logarithmic growth on acetate while the deoxyribonucleic acid content of the cells was unchanged. The present series of experiments was designed to further examine changes in the growth and synthetic ability of Euglena on media containing acetate, succinate or ethanol as sole carbon sources for growth.
MATERIALS
AND METHODS
Cultures of a streptomycin bleached strain, Sm-Li, of Euglena gracillis var. bacilwere grown on the defined Cramer-Myers salt medium [8] with acetate, ethanol, or succinate, added at 10 mm/l, as sole carbon sources for growth. All stock and experimental cultures were grown in the dark at pH 6.8 and 25°C. Cells from exponentially growing cultures were inoculated into 1 1 of medium contained in a 4 1 flask. Initial populations of less than 10,000 cells per ml were used. The cells had been cultured through several transfers in medium containing the carbon source to be studied before they were inoculated into the experimental flasks. The cells were
laris
1 This work was aided by a contract between the Office of Naval Research, Department of the Navy, and the University of California, Los Angeles, NR 120-336. 2 Present address Department of Poultry Husbandry, University of California, Davis, California. Experimental
Cell Research 35
B. W. Wilson
and B. H. Levedahl
sampled only during the exponential growth phase. Cell densities were counted with a Coulter electronic cell counter Model A [22]. Triplicate aliquots of 1 to 2 million cells were dried to constant weight at 105’C on previously weighed stainless steel planchets and their dry weights determined with a Werke-Sartorious microbalance. Total protein was determined on sextuplet samples using the Folin-Ciocalteau phenol reagent method of Lowry et al. [15]. Standard curves u-ere obtained with purified bovine serum albumen. Total carbohydrate was estimated by a modification of the anthrone method of McCready et al. [17] developed by Cook [5]. The values should be considered as estimates of the upper limit of the carbohydrate content of the cells. There has been much discussion concerning the accuracy of anthrone determinations on unpurified materials [I, 121 and it is possible that this method resulted in high values for the carbohydrate content of Euglena. Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were determined by a modification of the technique of Schmidt and Thannhauser [24] discussed in a previous publication [4]. RESULTS Table I contains the results of an experiment measuring the dry weight, protein, DKA, and RNA content of Euglena gracilis at different cell populations growing exponentially with acetate as the sole carbon source. These data confirm the findings of Ruetow and Levedahl [4j; the dry weight, protein, and RNA content per cell declined progressively during exponential growth and the DNA content per cell was relatively constant. The dry weight per cell declined more than 50 per cent as the cell number increased exponentially from 24,000 to 176,000 cells per ml. The protein content and the HNA content per cell declined ‘27 per cent. The DSA content did not decrease; it averaged 4.7 pugper million cells throughout this period. It should be noted ‘Ilu~,l:
I. Growth characteristics All
values
are expressed
of’ Euglena
cultured on acetate.
as pcg:‘106 cells &l S.D.
Timea hi-
Cell no. cells/ml
28
29,400
2510 k 30
45
47,300
2440 i 30
539 + 25
56 61
60,400 73,600
21301-30 1230 ‘1- 50
51Ok12 426’51
4.44io.24
1310230
425 + 56
4.62 ? 0.22 4.56 + 0.41
22.5 + 0.2
117oi30
411+41
5.14 k 0.57
21.6?1.0
76
117,000
78
122,000
85
176,400
a Time
Experimental
measured
Dry
from
Cell Research 35
weight
inoculation
Protein
DXA
RNA
563 + 68 30.0 i 0.2
Euglena growth studies
71
that Euglenu increase exponentially to populations of more than 300,000 cells per ml under these conditions (Fig. 1). The data presented in Table II indicate that similar events occurred when Euglenn cells were grown with succinate as the sole carbon source. The cells increased exponentially from 31,000 to 217,000 cells per ml (Fig. 2), while
Fig. 2.
Fig. 3.
Fig. l.-Logarithmic growth and synthesis by Euglena utilizing acetate as the sole carbon source. Each substance is expressed in terms of concentration per ml of culture. Doubling times are given in parentheses. Fig. P.-Logarithmic growth and synthesis by Euglena utilizing succinate as the sole carbon source. Each substance is expressed in terms of concentration per ml of culture. Doubling times are given in parentheses. I;ig. S.-Logarithmic growth and synthesis by Euglenu utilizing ethanol as the sole carbon source. Each substance is expressed in terms of concentration per ml of culture. Doubling times are given in parentheses.
the weight per cell progressively decreased 36 per cent. The protein content decreased approximately 17 per cent, the carbohydrate material decreased 44 per cent and the RN4 content per cell decreased 37 per cent. The DNA content was relatively constant, averaging 4.4 pg per million cells. The data in Table III were obtained from cells grown upon ethanol as the sole carbon source. During the period of exponential growth the dry weight per cell decreased 41 per cent. The largest decrease occurred in the early stages of exponential growth. The protein content per cell decreased 25 per cent by the time the cells had reached 237,000 cells per ml. The carbohydrate content per cell decreased 37 per cent and the RNA content decreased 25 per cent by the time the cells had reached 348,000 cells Experimental
Cell Research 35
B.
72
W.
Wilson
and
B.
H.
Levedahl
per ml. The DNA content remained relatively unchanged tial growth, averaging approximately 3.7 pg per million population seen in Table III was higher than in the because Euglenn increase exponentially to higher cell on this carbon source than they do in acetate or succinate TABLE
II.
Growth
chcu-ncteristics
LU1 values Time’ hr
Cell no. cells/ml
36
31,200
47
45,300
54
55,300
56
60,000
60 63
Dry
weight
are expressed
Protein
10
538k15
68,000 74,600
1350+50
504+10
1140*30
98,000 114,000
81
135,000
83
142,000
84 89
cultured
cells +l
Carbohydrate
on succinute.
S.D.
DNA
RNA
4.32kO.16
805 I22 856 + 42
515 +- 34
71
as pg/106
836 f 56 2270+
2150 i 80 -
76
of’ Euglena
throughout exponencells. The final cell two preceding tables populations (Fig. 3) containing media :3].
4.37 Ik 0.77
39.4 i 6.7
750 + 52 463+34
718k52
4.42 i 0.09
457 k 56
639 C 40
4.22 310.32
29.0 It 1.4
629 + 37
4.88 It 0.48
27.6 -i- 1.7 -
151,000
557+15
4.11 k 0.51
25.6 i 1.5
175,000
536+14
4.42 i 0.10
24.1 k1.1
94
202,000
96
217,000
451 k 24 -
a Time
measured
960 I 90 1060 -’ 20
850 -t 20 from
460 III 37
444 + 34
inoculation.
The synthetic rates of Euglena grown upon these substrates were calculated by determining the increase in the cell constituents per ml of culture fluid. This was done by multiplying the cell number per ml by the quantity per cell of the constituents under consideration and plotting the logarithm of the resulting value against the hours of growth of the culture. In this way, the rate of increase of cell number could be readily compared to the rates of the cellular constituents. Fig. 1 contains plots in the increase in cell number, dry weight, protein and DNA per ml of culture for acetate grown cells. The abscissa is expressed in arbitrary logarithmic units. Fig. 2 contains curves for the time course of the syntheses of cell materials of Euglena grown upon succinate. Fig. 3 illustrates the syntheses of cell material for Euglenrr grown upon ethanol. These data indicate that the Experimenfal
Cell Research
35
Euglena growth studies syntheses of cell materials proceeded at constant rates during the exponential increase in cell number of Euglena grown on all three substrates. The curves are log-linear; there was no evidence for drastic changes in the synthetic rates during the time periods investigated. Table IV lists the doubling times for the data plotted in the three figures. TAHLE III.
Growth characteristics All
values
are expressed
of’ Euglena as pg/lO’
cultured on ethanol.
cells *l
so.
Timea hr
Cell no. cells/ml
16 24
35,300
2360 + 10
552k7
974 k 26
3.17 F 0.69
27.4k1.3
44,500
259Ok
516539
825 + 63
3.54kO.50
26.2f0.3
Dry
weight
12
Protein
DSA
Carbohydrate
RNA
4.11 k 0.28
13
77,900
16601-00
523 + 18
67
156,000
1640+30
464k16
664 k 39
3.58 i 0.32
22.0 i: 0.7
77
207,000
445k16
732 + 45
4.64 k 0.61
23.6+
82
237,000
3.37 i 0.32
21.0F0.5
348,000
413110 -
831 f 30
95
1400+40
617?29
115
605,000
148OklO
3.57 * 0.30 -
20.6k1.4 -
a Time
measured
from
TABLE IV.
-
1.4
inoculation.
Cell number and cell constituent
Substrate
Acetate
doubling
Succinate
times in hours. Ethanol
Cell number
21
21
Dry
36
45
27
Protein
26
23
26
Carbohydrate
-
29
26
DNA
22
21
23
RNA
-
32
27
weight
24
The rates of syntheses of the cell constituents were slower than the rates of increase of cell number, especially for cells grown on acetate and succinate. DNA was the only cell material synthesized at rates comparable to the division rates of the cells. The results also indicate that ethanol-grown Euglena had synthetic rates which approached the rate of increase in cell number of the culture. Experimental
Cell Research 35
74
B. W. Wilson and B. H. Levedahl DISCUSSION
The evidence presented in this report is sufficient to indicate that Euglenn populations maintain relatively constant synthetic rates during logarithmic growth in batch culture, and that the progressive decrease in the dry weight, carbohydrate and RNA content per cell during this period is protein, due to the fact that these materials are manufactured at rates which are slower than the rates of division of the cells. In addition, these data show that batch culture growth methods may produce transient rather than stable protozoan cell populations. The machinery of the average cell was not replicated during one generation time. Synchrocells and synchronized photosynthetic Errglenrr cells exhibit nized Adasin “balanced” growth vvhen grown on the same basal medium [2, 5, 20, SO]. These sphchronized cell populations vvere grown in continuous culture under regimes whereby the cells were diluted with a fresh medium once each generation time, It is possible that continuous replenishment of the medium would lead to stable populations of euglenoid cells; cells with less than optimal synthetic rates might be gradually eliminated. However, one should not assume, a priori, that continuous culture techniques will automatically lead to the establishment of time invariant populations. *James [13], Powell [‘Ll], and Moser [la] have discussed this topic previously. The measurements made in this study, and the conclusions drawn from them, apply only to the average cell. Information concerning the specific changes in the amounts of material synthesized by the individual cells in the population during their life cycles cannot be readily inferred from these data. The fact that the cell materials were not replicated during the time intervals in which the populations of cells doubled does not imply that all of the cells in the populations behaved in similar manner. Cook and Cook [6: have shown that there is a large variation in the doubling times of single cells cultures, and several data removed from exponentially growing Euglena Lo, l-l, 191 indicate that the size distributions of flagellate cells have a greater range than would be expected in homogeneous populations of exponentially dividing cells. There is a strong possibility that these cell populations are inhomogeneous. The DNA content of Erlglena reported here may reflect such an inhomogeneity. Although the rate of DK’A synthesis was approximately the same as the division rate for cells grown on acetate, succinate, or ethanol, the average amounts of DNA per cell may differ from one experiment to another. Similar results have been obtained in other unpublished studies on Euglena from this laboratory. The ploidy of Euglenn cells is unknown; Experimental
Cell Research 35
Euglena
growth
studies
multinucleate Euglena can readily be produced with high temperatures or high light intensities [ 111. There may well be classes of cells within these exponentially growing populations which differ in their DNA content. This could still lead to an average DNA replication rate which is the same as the average division rate of the cells, particularly if the time course of DK\‘A synthesis determined the doubling time of the cells [lB]. I:vidence is mounting to shovv that many of the physiological characteristics of Euglentr associated with the steady state metabolism of the cells are drastically influenced by the carbon source upon which they are grown. For example, cells grown upon ethanol in a complex medium exhibit a respiratory adaptation to acetate within two hours of exposure to this substrate [lo:. Indeed, ethanol and acetate seem to compete for the same oxidative pathways [3, I)]. It has been shown that the effect of pH on the growth and respiration of Euglencz depended upon the carbon source present [‘LS]. However, the stoichiometry of the oxidation and assimilation of carbon is little affected by the conditions of growth or the age of the culture [27, 291. Regardless, vvith respect to the results of this investigation, one is led to consider that the Euglenrr cell plus its medium, rather than the cell itself, is the real unit of physiological activity of the growing culture.
SUMMARY
Cultures of a colorless strain of Euglena gracilis var. bacillaris were grown on media containing acetate, succinate or ethanol as the sole carbon sources for growth. The cells were sampled during the exponential growth phase and their dry weight, protein, carbohydrate, RNA and DSA contents were determined. All the cellular materials except DNA progressiveiy declined on a per cell basis during exponential growth. Hovvever, this vvas not due to any change in their synthetic rates during this time interval. The cellular contents vvere replicated at rates which were log-linear and unchanging when expressed on a per culture basis. However, these rates vvere slower than the rates of cell division in every case except the synthesis of DSA, and this lack of a correspondence between the synthetic and the division rates of Euglentr apparently leads to the decline in the cellular constituents during exponential growth. \Ve wish to thank these experiments.
Mrs. Anna Saudek for technical
assistance
during
Experimental
the course of
Cell Research 35
B. W. Wilson and B. H. Levedahl REFERENCES 1. ASHWELL, G., in COLO~~ICK and KAPLAX (eds.) lMelhods in Enzymology, Vol. 3, p. 73. Academic Press, New York, 1957. 2. BLUM, J. J and PADILLA, G. M., Ezpfl Cell Res. 28, 512 (1962). 3. BUETOW, D. E., Nature 190, 1196 (1961). 4. BUETOW, D. E. and LEVENDAHL, B. H., Gen. Microbial. 28, 579 (1962). 5. COOK, J. R., Ph.D. Thesis, University of California, Los Angeles, California, 1960. 6. COOK, J. R. and COOK, B., Exptl Cell Res. 28, 524 (1962). 7. COOK, J. R. and JAMES, T. W., ibid. 21, 583 (1960). 8. CRAMER, M. and MYERS, J., Arch. Mikrobiol. 17, 384 (1952). 9. DANFORTH, W. F., J. ProtozooI. 8, 152 (1961). 10. DANFORTH, W. F. and WILSON, B. W., J. Protozoa!. 4, 52 (1957). Il. GROSS, J. A. and JAHN, T. L., J. Protozool. 9, 340 (1962). 12. HAXSON, R. W., SCHWARTZ, H. S. and BARKER, S. B., Am. J. Physiol. 198, 800 (1960). 13. JAMES. T. W.. Ann. Rev. Microbiof. 15. 27 (1961). 14. LEVE~AHL, B: H. and WILSON, B. W.
Experimental
Cell Research 35
Cell Research 35, 69-76 (1964)
SYNTHETIC
AND DIVISION GROWN B.
Department
RATES OF EUGLENA
IN BATCH
W. WILSON2
of Zoology,
69
University
GRACILIS
CULTURES1
and B. H. LEVEDAHL of California,
Los Angeles, Calif.,
U.S.A.
Received June 20, 1963
THE biochemical
properties of the average cell from exponentially growing cultures have often been thought to be time invariant. Cells have been “ . . . considered to be rather fixed entities that can be dealt with much as a physicist deals with an atom or a molecule” [ 131. However, changes with time in the characteristics of exponentially growing bacteria [26], tissue culture cells [23, 251 and protozoa [4] have been described. Explanations for these changes fall logically into two categories; the synthetic rates of the constituents in question may alter due to changes in the composition of the medium, or these syntheses may proceed at constant rates which differ from the rates of cell division in the cultures. Buetow and Levedahl [4] studied the growth in batch culture of a colorless strain of the flagellate Euglena gracilis var. bacillaris. They found that ribonucleic acid, protein and dry weight of the cells decreased during logarithmic growth on acetate while the deoxyribonucleic acid content of the cells was unchanged. The present series of experiments was designed to further examine changes in the growth and synthetic ability of Euglena on media containing acetate, succinate or ethanol as sole carbon sources for growth.
MATERIALS
AND METHODS
Cultures of a streptomycin bleached strain, Sm-Li, of Euglena gracillis var. bacilwere grown on the defined Cramer-Myers salt medium [8] with acetate, ethanol, or succinate, added at 10 mm/l, as sole carbon sources for growth. All stock and experimental cultures were grown in the dark at pH 6.8 and 25°C. Cells from exponentially growing cultures were inoculated into 1 1 of medium contained in a 4 1 flask. Initial populations of less than 10,000 cells per ml were used. The cells had been cultured through several transfers in medium containing the carbon source to be studied before they were inoculated into the experimental flasks. The cells were
laris
1 This work was aided by a contract between the Office of Naval Research, Department of the Navy, and the University of California, Los Angeles, NR 120-336. 2 Present address Department of Poultry Husbandry, University of California, Davis, California. Experimental
Cell Research 35
B. W. Wilson
and B. H. Levedahl
sampled only during the exponential growth phase. Cell densities were counted with a Coulter electronic cell counter Model A [22]. Triplicate aliquots of 1 to 2 million cells were dried to constant weight at 105’C on previously weighed stainless steel planchets and their dry weights determined with a Werke-Sartorious microbalance. Total protein was determined on sextuplet samples using the Folin-Ciocalteau phenol reagent method of Lowry et al. [15]. Standard curves u-ere obtained with purified bovine serum albumen. Total carbohydrate was estimated by a modification of the anthrone method of McCready et al. [17] developed by Cook [5]. The values should be considered as estimates of the upper limit of the carbohydrate content of the cells. There has been much discussion concerning the accuracy of anthrone determinations on unpurified materials [I, 121 and it is possible that this method resulted in high values for the carbohydrate content of Euglena. Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were determined by a modification of the technique of Schmidt and Thannhauser [24] discussed in a previous publication [4]. RESULTS Table I contains the results of an experiment measuring the dry weight, protein, DKA, and RNA content of Euglena gracilis at different cell populations growing exponentially with acetate as the sole carbon source. These data confirm the findings of Ruetow and Levedahl [4j; the dry weight, protein, and RNA content per cell declined progressively during exponential growth and the DNA content per cell was relatively constant. The dry weight per cell declined more than 50 per cent as the cell number increased exponentially from 24,000 to 176,000 cells per ml. The protein content and the HNA content per cell declined ‘27 per cent. The DSA content did not decrease; it averaged 4.7 pugper million cells throughout this period. It should be noted ‘Ilu~,l:
I. Growth characteristics All
values
are expressed
of’ Euglena
cultured on acetate.
as pcg:‘106 cells &l S.D.
Timea hi-
Cell no. cells/ml
28
29,400
2510 k 30
45
47,300
2440 i 30
539 + 25
56 61
60,400 73,600
21301-30 1230 ‘1- 50
51Ok12 426’51
4.44io.24
1310230
425 + 56
4.62 ? 0.22 4.56 + 0.41
22.5 + 0.2
117oi30
411+41
5.14 k 0.57
21.6?1.0
76
117,000
78
122,000
85
176,400
a Time
Experimental
measured
Dry
from
Cell Research 35
weight
inoculation
Protein
DXA
RNA
563 + 68 30.0 i 0.2
Euglena growth studies
71
that Euglenu increase exponentially to populations of more than 300,000 cells per ml under these conditions (Fig. 1). The data presented in Table II indicate that similar events occurred when Euglenn cells were grown with succinate as the sole carbon source. The cells increased exponentially from 31,000 to 217,000 cells per ml (Fig. 2), while
Fig. 2.
Fig. 3.
Fig. l.-Logarithmic growth and synthesis by Euglena utilizing acetate as the sole carbon source. Each substance is expressed in terms of concentration per ml of culture. Doubling times are given in parentheses. Fig. P.-Logarithmic growth and synthesis by Euglena utilizing succinate as the sole carbon source. Each substance is expressed in terms of concentration per ml of culture. Doubling times are given in parentheses. I;ig. S.-Logarithmic growth and synthesis by Euglenu utilizing ethanol as the sole carbon source. Each substance is expressed in terms of concentration per ml of culture. Doubling times are given in parentheses.
the weight per cell progressively decreased 36 per cent. The protein content decreased approximately 17 per cent, the carbohydrate material decreased 44 per cent and the RN4 content per cell decreased 37 per cent. The DNA content was relatively constant, averaging 4.4 pg per million cells. The data in Table III were obtained from cells grown upon ethanol as the sole carbon source. During the period of exponential growth the dry weight per cell decreased 41 per cent. The largest decrease occurred in the early stages of exponential growth. The protein content per cell decreased 25 per cent by the time the cells had reached 237,000 cells per ml. The carbohydrate content per cell decreased 37 per cent and the RNA content decreased 25 per cent by the time the cells had reached 348,000 cells Experimental
Cell Research 35
B.
72
W.
Wilson
and
B.
H.
Levedahl
per ml. The DNA content remained relatively unchanged tial growth, averaging approximately 3.7 pg per million population seen in Table III was higher than in the because Euglenn increase exponentially to higher cell on this carbon source than they do in acetate or succinate TABLE
II.
Growth
chcu-ncteristics
LU1 values Time’ hr
Cell no. cells/ml
36
31,200
47
45,300
54
55,300
56
60,000
60 63
Dry
weight
are expressed
Protein
10
538k15
68,000 74,600
1350+50
504+10
1140*30
98,000 114,000
81
135,000
83
142,000
84 89
cultured
cells +l
Carbohydrate
on succinute.
S.D.
DNA
RNA
4.32kO.16
805 I22 856 + 42
515 +- 34
71
as pg/106
836 f 56 2270+
2150 i 80 -
76
of’ Euglena
throughout exponencells. The final cell two preceding tables populations (Fig. 3) containing media :3].
4.37 Ik 0.77
39.4 i 6.7
750 + 52 463+34
718k52
4.42 i 0.09
457 k 56
639 C 40
4.22 310.32
29.0 It 1.4
629 + 37
4.88 It 0.48
27.6 -i- 1.7 -
151,000
557+15
4.11 k 0.51
25.6 i 1.5
175,000
536+14
4.42 i 0.10
24.1 k1.1
94
202,000
96
217,000
451 k 24 -
a Time
measured
960 I 90 1060 -’ 20
850 -t 20 from
460 III 37
444 + 34
inoculation.
The synthetic rates of Euglena grown upon these substrates were calculated by determining the increase in the cell constituents per ml of culture fluid. This was done by multiplying the cell number per ml by the quantity per cell of the constituents under consideration and plotting the logarithm of the resulting value against the hours of growth of the culture. In this way, the rate of increase of cell number could be readily compared to the rates of the cellular constituents. Fig. 1 contains plots in the increase in cell number, dry weight, protein and DNA per ml of culture for acetate grown cells. The abscissa is expressed in arbitrary logarithmic units. Fig. 2 contains curves for the time course of the syntheses of cell materials of Euglena grown upon succinate. Fig. 3 illustrates the syntheses of cell material for Euglenrr grown upon ethanol. These data indicate that the Experimenfal
Cell Research
35
Euglena growth studies syntheses of cell materials proceeded at constant rates during the exponential increase in cell number of Euglena grown on all three substrates. The curves are log-linear; there was no evidence for drastic changes in the synthetic rates during the time periods investigated. Table IV lists the doubling times for the data plotted in the three figures. TAHLE III.
Growth characteristics All
values
are expressed
of’ Euglena as pg/lO’
cultured on ethanol.
cells *l
so.
Timea hr
Cell no. cells/ml
16 24
35,300
2360 + 10
552k7
974 k 26
3.17 F 0.69
27.4k1.3
44,500
259Ok
516539
825 + 63
3.54kO.50
26.2f0.3
Dry
weight
12
Protein
DSA
Carbohydrate
RNA
4.11 k 0.28
13
77,900
16601-00
523 + 18
67
156,000
1640+30
464k16
664 k 39
3.58 i 0.32
22.0 i: 0.7
77
207,000
445k16
732 + 45
4.64 k 0.61
23.6+
82
237,000
3.37 i 0.32
21.0F0.5
348,000
413110 -
831 f 30
95
1400+40
617?29
115
605,000
148OklO
3.57 * 0.30 -
20.6k1.4 -
a Time
measured
from
TABLE IV.
-
1.4
inoculation.
Cell number and cell constituent
Substrate
Acetate
doubling
Succinate
times in hours. Ethanol
Cell number
21
21
Dry
36
45
27
Protein
26
23
26
Carbohydrate
-
29
26
DNA
22
21
23
RNA
-
32
27
weight
24
The rates of syntheses of the cell constituents were slower than the rates of increase of cell number, especially for cells grown on acetate and succinate. DNA was the only cell material synthesized at rates comparable to the division rates of the cells. The results also indicate that ethanol-grown Euglena had synthetic rates which approached the rate of increase in cell number of the culture. Experimental
Cell Research 35
74
B. W. Wilson and B. H. Levedahl DISCUSSION
The evidence presented in this report is sufficient to indicate that Euglenn populations maintain relatively constant synthetic rates during logarithmic growth in batch culture, and that the progressive decrease in the dry weight, carbohydrate and RNA content per cell during this period is protein, due to the fact that these materials are manufactured at rates which are slower than the rates of division of the cells. In addition, these data show that batch culture growth methods may produce transient rather than stable protozoan cell populations. The machinery of the average cell was not replicated during one generation time. Synchrocells and synchronized photosynthetic Errglenrr cells exhibit nized Adasin “balanced” growth vvhen grown on the same basal medium [2, 5, 20, SO]. These sphchronized cell populations vvere grown in continuous culture under regimes whereby the cells were diluted with a fresh medium once each generation time, It is possible that continuous replenishment of the medium would lead to stable populations of euglenoid cells; cells with less than optimal synthetic rates might be gradually eliminated. However, one should not assume, a priori, that continuous culture techniques will automatically lead to the establishment of time invariant populations. *James [13], Powell [‘Ll], and Moser [la] have discussed this topic previously. The measurements made in this study, and the conclusions drawn from them, apply only to the average cell. Information concerning the specific changes in the amounts of material synthesized by the individual cells in the population during their life cycles cannot be readily inferred from these data. The fact that the cell materials were not replicated during the time intervals in which the populations of cells doubled does not imply that all of the cells in the populations behaved in similar manner. Cook and Cook [6: have shown that there is a large variation in the doubling times of single cells cultures, and several data removed from exponentially growing Euglena Lo, l-l, 191 indicate that the size distributions of flagellate cells have a greater range than would be expected in homogeneous populations of exponentially dividing cells. There is a strong possibility that these cell populations are inhomogeneous. The DNA content of Erlglena reported here may reflect such an inhomogeneity. Although the rate of DK’A synthesis was approximately the same as the division rate for cells grown on acetate, succinate, or ethanol, the average amounts of DNA per cell may differ from one experiment to another. Similar results have been obtained in other unpublished studies on Euglena from this laboratory. The ploidy of Euglenn cells is unknown; Experimental
Cell Research 35
Euglena
growth
studies
multinucleate Euglena can readily be produced with high temperatures or high light intensities [ 111. There may well be classes of cells within these exponentially growing populations which differ in their DNA content. This could still lead to an average DNA replication rate which is the same as the average division rate of the cells, particularly if the time course of DK\‘A synthesis determined the doubling time of the cells [lB]. I:vidence is mounting to shovv that many of the physiological characteristics of Euglentr associated with the steady state metabolism of the cells are drastically influenced by the carbon source upon which they are grown. For example, cells grown upon ethanol in a complex medium exhibit a respiratory adaptation to acetate within two hours of exposure to this substrate [lo:. Indeed, ethanol and acetate seem to compete for the same oxidative pathways [3, I)]. It has been shown that the effect of pH on the growth and respiration of Euglencz depended upon the carbon source present [‘LS]. However, the stoichiometry of the oxidation and assimilation of carbon is little affected by the conditions of growth or the age of the culture [27, 291. Regardless, vvith respect to the results of this investigation, one is led to consider that the Euglenrr cell plus its medium, rather than the cell itself, is the real unit of physiological activity of the growing culture.
SUMMARY
Cultures of a colorless strain of Euglena gracilis var. bacillaris were grown on media containing acetate, succinate or ethanol as the sole carbon sources for growth. The cells were sampled during the exponential growth phase and their dry weight, protein, carbohydrate, RNA and DSA contents were determined. All the cellular materials except DNA progressiveiy declined on a per cell basis during exponential growth. Hovvever, this vvas not due to any change in their synthetic rates during this time interval. The cellular contents vvere replicated at rates which were log-linear and unchanging when expressed on a per culture basis. However, these rates vvere slower than the rates of cell division in every case except the synthesis of DSA, and this lack of a correspondence between the synthetic and the division rates of Euglentr apparently leads to the decline in the cellular constituents during exponential growth. \Ve wish to thank these experiments.
Mrs. Anna Saudek for technical
assistance
during
Experimental
the course of
Cell Research 35
B. W. Wilson and B. H. Levedahl REFERENCES 1. ASHWELL, G., in COLO~~ICK and KAPLAX (eds.) lMelhods in Enzymology, Vol. 3, p. 73. Academic Press, New York, 1957. 2. BLUM, J. J and PADILLA, G. M., Ezpfl Cell Res. 28, 512 (1962). 3. BUETOW, D. E., Nature 190, 1196 (1961). 4. BUETOW, D. E. and LEVENDAHL, B. H., Gen. Microbial. 28, 579 (1962). 5. COOK, J. R., Ph.D. Thesis, University of California, Los Angeles, California, 1960. 6. COOK, J. R. and COOK, B., Exptl Cell Res. 28, 524 (1962). 7. COOK, J. R. and JAMES, T. W., ibid. 21, 583 (1960). 8. CRAMER, M. and MYERS, J., Arch. Mikrobiol. 17, 384 (1952). 9. DANFORTH, W. F., J. ProtozooI. 8, 152 (1961). 10. DANFORTH, W. F. and WILSON, B. W., J. Protozoa!. 4, 52 (1957). Il. GROSS, J. A. and JAHN, T. L., J. Protozool. 9, 340 (1962). 12. HAXSON, R. W., SCHWARTZ, H. S. and BARKER, S. B., Am. J. Physiol. 198, 800 (1960). 13. JAMES. T. W.. Ann. Rev. Microbiof. 15. 27 (1961). 14. LEVE~AHL, B: H. and WILSON, B. W.
Experimental
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