OLE MAAL0E AND KARL G. LARK 



the growth curves to be determined with satisfactory accuracy by direct colony 

 counts; the second point, the interaction with phage, has been very helpful in that 

 it has revealed periodic changes in susceptibility which, as will be seen later, appear 

 to reflect nuclear doubling. The last property is potentially valuable because bio- 

 chemical work, including tracer studies, can be carried out under well-controlled 

 conditions. 



We shall first examine the type of growth curve which can be obtained by sub- 

 mitting the culture to cyclic changes of temperature. The technical details of the 

 experiments will be described in a forthcoming publication (Lark and Maaloe, 

 1954); here a brief mention of the experimental conditions will suffice: aerated 

 broth cultures, not containing more than 20 to 40 million organisms per ml., were 

 used throughout; the temperature shifts were effected almost instantaneously by 

 transferring the cultures to tubes kept at the desired temperature and adding hot or 

 cold broth as required. No loss in viable counts has been observed following the 

 sudden cooling or heating. One temperature cycle, consisting of changing the 

 temperature from 25 to 37 C. and back to 25 C, involves a dilution of the culture 

 by a factor 1 93, which is balanced quite accurately by the increase in colony count 

 during the cycle. It is important to notice that colony counts can give very precise 

 results, even when two large dilution steps are needed between sampling from the 

 culture and spreading on agar plates, provided the experiment is designed in such 

 a way that 300-600 colonies can be counted per sample. Under these conditions and 

 with careful pipetting the standard deviation is about 5 per cent., which is close to 

 the sampling variation to be expected on the assumption that the suspended bacteria 

 are randomly distributed. 



Figure la shows the step-wise rise in colony count regularly obtained after 2-3 

 conditioning cycles of temperature shift. In this experiment alternating periods of 

 30 minutes at 25 C. and 6 minutes at 37 C. were employed. The third, fourth and 

 fifth cycles are represented by points corresponding to individual counts, and a 

 continuous curve below the points shows the growth to be expected if the rate of 

 division was always that characteristic of prolonged growth at the prevailing tem- 

 perature. This lower, theoretical curve is based on control experiments yielding 

 generation times of 45 to 50 minutes for growth at 25 C. and of 18 to 20 minutes 

 for growth at 37 C. Two characteristics of experiments of this type should be em- 

 phasized: the virtual absence of cell division during about 25 minutes of each 

 25 C period, and the near equality of the experimental and the theoretical genera- 

 tion times. Together these observations show that a considerable degree of phasing 

 or synchronization of cell division has been obtained, and that the temperature 

 regimen employed probably has not impaired the vitality of the cells. It may be 

 added that the steep portions of the experimental curves correspond to a division 

 rate about twice that normally found at 37 C. 



For convenience, all of the early experiments, including that of Figure \a, were 

 carried out with non-aerated cultures diluted beforehand to such an extent that 

 plating on agar could be made without further dilution. This procedure had to be 

 altered because the bacteriophage experiments as well as the cytological and bio- 

 chemical studies we wanted to carry out on synchronized cultures require densities 

 of at least 10 million organisms per ml. Systematic studies were therefore carried 



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