OLE MAAL0E AND KARL G. LARK 



unorthodox procedure was adopted because we wanted to obtain fixation at a pre- 

 cise time relative to the time of the temperature rise and because it was important 

 that no cytological changes should occur between sampling and the application of 

 the fixing agent. After the process described above, impression prints were made on 

 cover slips, and hydrolysis, staining with a thionine-thionyl chloride solution, and 

 dehydration, were carried out as described by DeLamater (1952). The micrographs 

 obtained in this manner do not compare in beauty with those made by expert cyto- 

 logists using fixation in Os0 4 vapour; it is not known whether this is due to the 

 fixation process we were forced to employ or to our lack of experience. The micro- 

 graphs do show, however, that simultaneously with the rise in lysogenization fre- 

 quency the number of stained spots, or 'nuclei', per bacterium doubles. 



We shall continue to talk about these stained regions, in which the bacterial 

 desoxyribosenucleic acid (DNA) is concentrated, as nuclei, remembering that we 

 have no other way of defining a bacterial nucleus and that there are reasons to believe 

 that it does not constitute as well defined an organelle as does the nucleus of cells of 

 higher organisms (Birch-Andersen, Maaloe and Sjostrand, 1953). The distribution 

 of nuclei observed in the micrographs is as follows: while growing at 25 C, most 

 cells have two and a few have four nuclei; during the period between 5 and 10 

 minutes after raising the temperature to 37 C, while the lysogenization frequency 

 doubles, the fraction of cells containing four nuclei increases rapidly, so that by the 

 time when no further increase in lysogenization frequency is observed the majority 

 of the cells contain four nuclei and very few are found with only two. When the 

 culture has continued growing for a couple of generations at 37 C. the distribution 

 is again much like that observed before the temperature was raised; however, the 

 proportion of cells with four nuclei is a little larger than it was when growth took 

 place at 25 C. 



Up to now we have presented some rather heterogeneous experiments, one after 

 the other, without attempting to interpret the results. For practical reasons the ex- 

 periments have been described in the order of increasing complexity, ending with the 

 experiment in which cytological studies were included. Fortunately, these studies 

 gave a clear-cut answer which makes it natural now to examine all our findings in 

 the light of the cytological observations. These results may be summarized by stating 

 that upon raising the temperature of a Salmonella typhimurium culture which has 

 grown for some time at 25 to 37 ° C. nuclear division is induced in nearly all the cells. 



We shall now discuss possible implications of this finding from different points of 

 view: 



(1) It is natural first to consider what mechanism may be responsible for the 

 induction of nuclear division. The tentative explanation we have in mind is based on 

 general ideas similar to those put forward by Hotchkiss, who writes as follows 

 (Hotchkiss, 1954) : '. . . the biochemical processes of cell growth and division would be 

 disrupted in a systematic fashion by temporary exposure of a growing culture to a 

 temperature well below that at which these systems had achieved a steady-state 

 equilibrium. Certain of the enzyme systems should now be less able than others to 

 transfer an amount of substrate equal to that supplied them, and there would then 

 be a tendency for the metabolism to be selectively slowed or even temporarily halted 

 at certain points.' Concerning the synthesis of DNA which must precede nuclear 



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