Variations in the individual generation times of Tetrahymena geleii HS

Experimenfal

Cell Research 16, 279-284 (1959)

NS IN THE IN F TETRAH D. M. PRESCOT epartment of Anatomy,

School of Medicine, University oj California Los Angeles, Cali., U.S.A.

at Los Arzgeks,

nn auerage generation time for a eel1 depen s entirely upon (I> the cekh’s genetically-determined capacity for synthesis and growth and (2) the environmental conditions provided for the cell’s activities. Thus, under a particular set of culture conditions and in the absence of any genetic alteration, the average multiplication rate must remain constant. With t e usual methods for the determination of the average multiplication rate (or erage generation time), eel1 numbers are counted directly or the cell density is measured by some indirect means. Culture growth curves so obtained consist, of course, of average values for whole cell populations. Because of their statistical nature, ata of this sort necessarily permit no comparison between the behaviom of the i~d~uid~~~ cells and the statistically-derived, auerc;lge cell. There is no indication, for-exdmgk, of-the degree of constancy with which eat cell adheres to the average generation time. When genetically identical cells share the same culture conditions, how much witl one individual generation time vary from another and from the average value? When any variation does exist, the explanation must be in other than direct genetic or environmental terms, i.e., fortuitous ine between cells such as variations in cell size, mitochondrial complement, etc. With accurate determinations of a large number o~~ndiv~~~a~ generation times the extent of the variation, under precisely controlled culture ~o~dit~o~~~ has been measured in a unicellular organism, Teirahymena geleii of these experiments, generation times have been compared fo which, at first thought, were considered likely to be the most nearly identical, i.e., sister cells derived from the same division Variations in individual generation times are of interest in themselves, but a~d~tioRal~y have a considerable bearing on the important problem, in. the study of the eel1 life cycle, of synchronizing mass population of cells and ne of the ex~e~ime~t~ sustainilng the synchrony once it has been achieved. Experimental

Cell Research 16

D. M. Prescott

280

reported here describes the effects of the variation in individual times on the subsequent multiplication pattern of a population menu initially in perfect division synchrony.

generation of Tetrahy-

METHODS

The HS strain of T. geleii was cultured under the optimal conditions for cell multiplication as they have thus far been determined: temperature, 32.0”+ 0.1%; pH, 7.30; proteose peptone concentration, 1.5 per cent (w/v); liver extract (Nutritional Biochemical Corp.), 0.1 per cent (w/v). The medium was made up with Pyrex distilled water, autoclaved, and stored at 4.0%. The above culture conditions support a generation time of Ill minutes. When multiplying cells are needed, a stock culture is serially transferred at least once every 24 hours to keep the organisms constantly in logarithmic multiplications. The technique used to measure average generation time has been fully described elsewhere [3]. Briefly, a small number of cells (initially about 80) are divided among 10 to 12 capillary culture pipettes such that each pipette contains initially 5 to 12 cells and about 40 ~1 of medium. The pipettes are kept at constant temperature and counts are made periodically with a dissecting microscope on the living cells in all pipettes. The total for all pipettes is taken as a reading for any particular time. It is usually sufficient to continue the counts through 3 generations, but counting can be continued with absolute accuracy until the total cell number reaches about 800. The method is simple and gives precisely repeatable results (see Fig. 1). The same type of capillary, culture pipette was used to determine individual generation times. A stock culture was first started with a very small inoculum of Tetrahymena such that, by the end of 14 to 16 hours, the cell density had risen to 30 cells per ml in logarithmic multiplication. Aliquots of 35 to 40 ~1 of the culture were drawn up into pipettes, and only those pipettes containing a single cell were kept 16OOj

Fig. I.-Five growth curves for small populations of T. g&ii HS in logarithmic multiplication and grown under optimal conditions. The curves were obtained over a seven-month period as a check on the constancy of the miltiplication rate. They demonstrate the exactness with which the average generation time can be determined.

Experimental

Cell Research 16

Generation

time in Tet~a~~rne~~

for observations. The minute of the completion of cytoplasmic fission was recorded as zero time for each particular cell, and the subseqilent generation times for the two daughter cells were determined to & 1 minute.

RESULTS

AND

CONCLUSIONS

jrhe average cell generution time .-The average cell generation time has been accurately established by a series of 21 separate growth curves. A few of these results are given in Fig. 1. These curves gave average generation times of 109 to 113 minutes with an overall average of Ill * I minutes. The standard deviation was less than i 2 minutes. The 21 growth studies were carried out over a period of 7 months as checks on the constancy of the cell multiplication rate through approximately 150 serial transfers and 3,OO generations. There would have been certain advantages to performing these experiments with the complete synthetic medium [I]$ but n~fo~t~nate~y such a medium was found to support a much slower multiplication rate, and t results were also quite variable (average generation times of 7 to 12 hour In recent attempts to improve the synthetic medium, cholesterol, added in the amount of 0.5 mg/l, essentially eliminated this variability and reduce average generation time to 5 hours and 20 to 30 minutes (at the pH optimum, 7.3; and temperature optimum, 32.5”C). L%e individual cell generation time.-The 766 individual generation ti measured make up the histogram in Fig. 2. The generation times are highly variable (standard deviation equals + 10 min.), extending over the broad range of 82 to 149 minutes. This represents a spread of 74 to 135 per cent of the average generation time (111 minutes). The main mass of the cells (70 per cent) falls into the 20-minute range between IO0 and 119 minutes (90 to 107 per cent of the average generation time). Thus 7 per cent of the eneration time. cells divide over a period equal to 18 per cent of the averag The distribution is also skewed slightly toward the longer generation times, and tbe mode, therefore, falls a few minutes below the mean. The 766 cells of Fig. 2 represent 383 pairs of sister cells. These pairs of according to a consistent pattern, one sister cell usually dividing in a shorter time than the average generation period, and the other usually requiring more than 111 minutes to reach division. This patter by the generation times for 28 typical pairs given in Table I. exceptions occurred (6 per cent) in which both cells divided either before or after the average generation time. The wide variation in individual generation times must reflect non-genetic Experimental

Cell Research 14

D. M. Prescoft

282

inequalities among the individual cells. In amoeba, for example, the generation time is inversely related to the initial daughter cell weight, i.e., the larger the cell at birth, the shorter the generation time [a]. In Tetrahymena, however, the difference in volume between daughter cells of a pair is normally Mean generarim rime, 111 minutes

I

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irriilii:jiiii:iiriiiiiiiiii. ...................:,:_:,:_,, iiiiiii/::ijjijji:j:j:i:i:l:i :j:j:i:::ljjjlijljjjj:i:i:l:i ...z... ._...._.:. :_:_ ::::::::::;:;:i::::::: :.:.:.: ... ._._._ :_:.:___._.__ ‘:.i:i:i:l.::i:i::::::.:.:.:.:::~~. --. . . . . -:.:.: . ..__ i)iiiljjii:j::::::::::.::::::::::::i:i:i:_:.

B L j

3w

i 20. $ 10 n

TABLE

Jerrohymeno geleri

HS

32.5”C pH 7.3

.......... ... .... ... :.,iiiiiiii_i:jj’jjjjiiiiiiiiiiiiiiii:i:~:~: .... iiiijjjijjiiijjiijjijiiiiijijiiijiiiiiii~~ ._..._., j:;; ‘.‘. .:....:::::::::::::::::::::::::j:j: . ..:_:.__..._(..._.._..,_,.,:j:: :;::i:/:i::::: . . . . . . . . -. :.: .._.._:.:.:. :.:_:.:_: .,:,:,: ig,. :::j $iiljjj:j:l::.j::::::::.:.:.:.:.:.:.:.: .,.,.,:,:,_ ii: F;; ::::::::::::_:.:.:..:::::::::::.::::::::::::::::~:~:~~~~:~:~:~:~:~:~: ““.‘.““‘:‘:‘::::-:.:~:::::::::i:j:i:::j~~::::::::~~~~:~:~:~:~:~:~.. .,,:, .::.:.::::::_:.:____...,..._.. :. ., ,._., .,..._....-... jj:i:i:_:.:_:.:.:.:_::.:.:.:.::::::::::.~:~.,. ._,, . :.:.:.>_-... _.. .I. _ ..,.,.... ...... . _._.._.. fio 90 100 110 120 IjO 140 Generarion time. minutes

I. individual

generation

Experimental

150

times in minutes (T. geleii HS).

for 25 pairs of sisfer cells

97 102

142 134

106

114

108 123

110

114

106

111

111

104 119

96 115

112 128

103 118

106 118

96 116

110 112

113

114

107 111

105 119

104 Ill

108 115

107 113

109 112

109 109

103

103 120

108 115

100

115 Average

too small completed, spherical apparent generation the failure

Fig. 2.-The histogram is a compilation of 766 individual generation times for T. geleii HS grown under optimal culture conditions. There is a slight skewing toward the longer generation times.

121

generation

100

time:

128

91 123

107

111 minutes.

to be measurable. A few minutes before cytoplasmic fission is the two forming daughter cells are, for a short time, perfectly and accurate volume measurements are possible. In spite of the volume equality, it seems likely, nevertheless, tbat the variation in times may represent inequalities between cells originating with of cell components (mitochondria, microsomes, etc.) to be divided Cell Research 16

Generation time in Tetrahymena

283

with exact equality at cell division. This is supported by the information on daughter cell pairs. While one cell of a pair divides early, presum cause of some metabolic advantage, the division of the other ce%l is by an approa+mafeIy equal extent. The skewing of the istogram in Fig. 2

Fig. 3.--The growth curve represents the multiplication pattern over 4 generations for a popuiation of 2.5 celIs in perfect division synchrony at the first population doubling. Because of the wide variation in individual generation times the synchrony is rapidly lost. The slopes at the iou-er right demonstrate the decrease in synchrony with each successive population doubling.

toward longer generation times, however, indicates that while a reduced endowment at division is reflected by a Longer period in order to reach division, the advantage gained by an increased endowment at division is slightly diminished by other limitations. In other words, the generation time can be lengthened more easily than it can be shortened. T is has been illustrated in amoeba, in which a doubling in the average daughter cell size reduces the generation time by about 40 per cent, whereas halving the daughter cell size increases the generation time by 60 per cent [a!, The efecf of cell variations on division synchrony.--In another series of 25 pipettes, each containing one cell initially, the observations were continued until the count rose to 16 per pipette. These data have been added together by using the first division in each pipette (from one to two cells) as the common zero time point. Thus, the curve in Fig. 3 describes the multiplication pattern for 50 cells which enter the interphase period nn perfect synchrony. The first division in Fig. 3 (from 25 to 50 cells) represents this initial, perfect Experimental

Cell Research 16

D. M. Prescoft

284

synchrony. By the next division (50 to 100 cells) the loss of synchrony is certainly marked, and the accumulated loss of synchrony by the next population doubling (100 to 200) is severe. In the rise from 200 to 400 cells, the divisions have become almost completely asynchronous, and the multiplication rate assumes an essentially continuous, constant value. The solid straight line represents the growth of a population which is dividing with perfect asynchrony. The rapid loss of synchrony with each succeeding generation time is summarized by the series of slopes marked in the lower right of Fig. 3. These slopes are taken from the experimental curve and correspond to the division pattern defined by about 80 per cent of the population at each of the successive population doublings. The perfect synchrony of the first doubling fades into complete asynchrony by the fifth doubling. The asynchronicity developed by the first division is not great enough to limit seriously the use of such populations for the study of the cell life cycle, but in general, it must necessarily cause some reduction in the precision of an experiment. Finally, although the frequently employed description of data in terms of the “average cell” may be convenient, it should be remembered that a relatively small proportion of cells may possess the properties described as average. In the above growth curves for T. geZeii (Fig. 1), the average generation time has been defined as 111 minutes, but, in fact, only about 4 per cent of the cell population actually conforms exactly to this particular value.

SUMMARY

The generation times for individual Tetrahymena are highly variable, ranging from 74 to 135 per cent of the average generation time. Only 4 per cent of the cells show the same generation time as the statistically-derived average cell. The variation may be due to inequalities between cells arising at division. Because of the spread in individual generation times, any division synchrony in a population of Tetrahymena is lost very rapidly. REFERENCES 1. EILLOTT, A. M., BROWNELL, L. E. and GROSS, J. A., ProfozooZogy 2. PRESCOTT, D. M., Exptl. Celf Research 11, 86 (1956). 3. ibid. 12, 126 (1957).

Experimental

Cell Research 16

1, 193 (1954).