ERIK ZEUTHEN AND OTTO SCHERBAUM 



remarks clearly indicate the possibility of inducing synchronous cell divisions by con- 

 trolling the chemistry of the medium. Nevertheless, in view of our present ignorance 

 as to the best way of making such an approach, we decided on changes of temperature 

 as a possible way of changing the relative concentration in the cells of a great number 

 of metabolic intermediaries, which might include one or more agents necessary to 

 enable the cell to pass from one phase of the cell cycle to the next. 



If in a mass culture of a micro-organism there is a random distribution of all 

 stages in the cell cycle, we shall find that as long as the organism is growing exponen- 

 tially a constant fraction of the cells will be in division. We observe a steady state in 

 which there is an equilibrium between cells entering and cells leaving the division 

 stage. Transitory changes in the number of dividing cells divided by the total num- 

 ber of cells, termed hereafter the 'division index', may indicate either that for some 

 reason the time which the cells spend in division changes relative to the time 

 occupied by the whole cycle, or that cells have accumulated into groups which tend 

 to divide together and possibly pass through one or more full cycles together. 



In our search for group formation we have accepted the division index as our guide, 

 division indicating all stages from onset of furrowing until separation of the two cells. 

 Decision between the two possible interpretations can only be made if multiplica- 

 tion is estimated by actual cell counts. 



Let us then accept as a working hypothesis that some phase in the cell cycle has a 

 higher temperature coefficient than any other. Indeed, Ephrussi's work (1926) on 

 dividing sea-urchin eggs had indicated a case where this is so. Such a phase will be 

 more sensitive to temperature changes than any other. With lowering of the tempera- 

 ture cells in this phase will tend to drop behind and unite with cells behind them in 

 the cycle, whereas with increase in temperature they will tend to catch up with cells 

 in front. In both cases we should get group formation, detectable only at the time 

 when the group passes through the division stage. If experiments were to confirm 

 our working hypothesis thus far, the next step would then be to transfer the culture 

 back to the first temperature, after a time shorter than the duration of the cell cycle 

 at the new temperature. When our group was again in the sensitive phase the tem- 

 perature would again be shifted. For every such periodic change in temperature the 

 original synchronized group should become larger, and in the end the whole culture 

 should become synchronized. The first part of our working hypothesis was confirmed 

 by experiment; the second was never put to serious test. It is mentioned here because 

 a number of our experiments might otherwise seem curious. 



THE EXPERIMENTAL ORGANISM 



For our experimental organism we chose the ciliate protozoon Tetrahymena 

 pyriformis, because it grows readily on dissolved and fully defined media, as reported 

 by Kidder and Dewey (1951). So far, however, we have grown our cells in 2 per 

 cent, proteose peptone (Difco) plus 1 per mil. liver extract (Wilson Lab.) with salts 

 as in Kidder and Dewey's synthetic medium A. The cultures were shaken and aerated 

 with a flow of air which was passed over them. Each culture contained 150 ml. of 

 medium and the total synchronized population represented about 100 mg. wet 

 weight of cells. 



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