INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM 



are 40 by 100 van, that is to say, a volume of approximately .9 X 10"^ ii^. Let us 

 suppose that the polyhedral bacteriophages completely fill the entire diplo- 

 bacillus, which could then contain no more than 66,000 bacteriophages. But, in 

 any case, the maximum number of bacteriophages produced by any single 

 bacterium never seems to surpass 1000. 



One can consider, without much chance of error, that in a viable lysogenic 

 bacterium the space occupied by the bacteriophages must not surpass 50% of 

 the total bacterial volume. An average viable lysogenic B. megaterium diplo- 

 bacillus could thus contain maximally 33,000 bacteriophages. This figure is 

 significantly lower than the value of 524,288 which represents the minimum 

 number of particles which must have been present in the original diplo-bacillus 

 in order to sustain the hypothesis of the perpetuation of the lysogenic function by 

 a simple distribution of pre-existing exogenous particles. This hypothesis thus 

 can be rejected. 



The hypothesis, according to which lysogeny perpetuates itself by an endo- 

 microbial route without the necessary intervention of exogenous bacteriophages, 

 seems to us, for the moment, the only one capable of accounting satisfactorily for 

 the experimental facts. The problem of the form in which the lysogeny perpet- 

 uates itself shall be discussed in the course of this work. Another conclusion can 

 be drawn from our experiments. One knows (J. Bordet and J. Renaux, 1928; 

 F. Burnet and M. McKie, 1929a,b) that all colonies issuing from a lysogenic 

 culture are lysogenic. Our experiments confirm and validate these data: every 

 diplo-bacillus from a population of B. megaterium 899 isolated by means of the 

 macromanipulator gives rise to a lysogenic colony. 



V. Lysozyme Lysis Does Not Liberate Bacteriophage 



F. Burnet and M. McKie (1929a), F. Burnet and D. Lush (1936) observed that 

 lysis by a bacteriophage X of a lysogenic bacterium sensitive to this bacterio- 

 phage X and normally producing a bacteriophage Y does not liberate the 

 bacteriophage Y. F. Burnet and M. McKie understood the whole significance of 

 this phenomenon as early as 1929 and concluded that all bacteria of a perma- 

 nently lysogenic strain possess in their hereditary constitution some unit which is 

 potentially capable of liberating phage. Burnet and McKie found, nevertheless, 

 that a small proportion (about 1%) of the bacteria, of a 24-hour-old agar culture 

 harbors bacteriophages which can be liberated by treatment with distilled water. 

 E. and E. Wollman and A. Gratia (1936), independently and within a few weeks of 

 each other, made a similar observation: lysozyme lysis of lysogenic B. megaterium 

 899 does not liberate bacteriophages. E. and E. Wollman found one bacterio- 

 phage for several thousand bacterial colonies and concluded that the bacterio- 

 phage exists in two different forms, phases, or states: the "mature" bacterio- 

 phage which one could call corpuscular, or, more simply, bacteriophage-virus as 

 it appears in the free state in the culture medium, and the non-virulent, latent 

 "intracellular bacteriophage" as it exists inside lysogenic bacteria and which 

 we call probacteriophage. Furthermore, it is known that it is impossible to 

 liberate by lysozyme lysis bacteriophages adsorbed to living B. megaterium cells. 



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