HYPOTHESES CONCERNING NATURE OF BACTERIOPHAGE 321 



transplants of experimentally produced secondary cultures), one of 

 bacteriophage-staphylococcus, the other of bacteriophage-coli, by trans- 

 ferring them en bloc on agar about once a month. 



After two years, the cultures have an absolutely normal appearance, 

 but filtrates from a transfer made in bouillon invariably cause bacteri- 

 ophagy of a pure culture of the homologous susceptible bacteria. But it 

 has been impossible to obtain ultrapure colonies by agar isolations. 

 With these old secondary cultures all colonies give cultures from which 

 may be isolated a bacteriophage, showing thus that every bacterium in 

 the mixed culture is contaminated. In order to derive an ultrapure 

 bacterium from these it has been necessary to have recourse to the 

 method described above, namely, effecting a series of cultures at a 

 limiting temperature in a bouillon acidified to the growth limit. After 

 this treatment an ordinary isolation on agar has yielded ultrapure col- 

 onies. The symbiosis becomes more and more intimate, therefore, as 

 time passes, but a separation is always possible. 



Could it not be assumed that the ''bacteriophage-autolysin" exists 

 in all bacteria even though in all of them it can not be demonstrated? 

 The fact that bacterial strains can be purified already shows that this 

 can not be true, but it is possible, in still another manner, to show that 

 such an interpretation is merely a fabric of words. Subject a bacter- 

 ial culture to the action of a virulent bacteriophage. Bacteriophagy 

 takes place and this dissolution, say the adherents of the autolytic the- 

 ory, is brought about through the action of an autolysin contained in 

 the bacteria. If this explanation is correct, a normal culture of this 

 same bacterium should contain it, for not to admit this is to imply that 

 bacteriophagy is caused by something other than an autolysin. This 

 question may be investigated by a method which leaves no question as 

 to the significance of the results obtained. 



Jaumain^^^ has recently described a procedure which leads to the 

 complete autolysis of several bacterial species, of the staphylococcus in 

 particular. He prepares a bouillon suspension of bacteria removed from 

 an agar slant and introduces this suspension into a test tube which is 

 then sealed in the flame, making every effort to have as little air as pos- 

 sible remain in the sealed tube. 



Several times I have attempted to perform experiments leading to a 

 complete autolysis in order that I might compare a true autolysis with 

 the autolysis induced by the bacteriophage, but my plans never suc- 

 ceeded, for the simple reason that, under natural conditions, autolysis 

 does not take place with the species of bacteria for which I have up to 



