INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM 



8.5 X 10^ M^ of fluid, corresponding to about half the volume of the drop: 110 

 bacteriophages. The total number of bacteriophages produced is thus about 220. 

 It is this observation which convinced us of the existence of a rapid lysis followed 

 by the appearance of bacteriophages. Subsequently, we have often observed 

 lysis followed by the appearance of bacteriophages. We have noticed that this 

 lysis does not leave any visible ghost and that it often occurs in less than one 

 second. It could thus very well pass unobserved in a drop which contained 

 several diplo-bacilli in the process of multiplication. 



We had thought (1949b) that we observed the liberation of bacteriophages 

 without lysis, but after having recognized the two types of lysis — slow and rapid 

 — and having analyzed the possible causes of error which can arise in the course 

 of experiments in microdrops, we have attempted experiments in which a very 

 small number of bacteria shall be present at the same time in a single microdrop. 

 Each time that this was possible, we have worked in such a manner that only a 

 single diplo-bacillus was present in a drop. It is only under these conditions that 

 one can be assured that bacterial lysis is not overlooked. 



We have also tried to achieve conditions under which a single diplo-bacillus, or 

 a single bacterium, lyses in one drop. In this way, one can sample the totality of 

 the microdrop, thus avoiding having to measure the dimensions of the micro- 

 pipette and calculating its volume and reducing the causes of error in the quanti- 

 tative determinations to a minimum. We took care to stir the drop repeatedly if 

 only a part of the drop was to be sampled. One will see in the following that no 

 liberation of bacteriophage was observed in the absence of bacterial lysis. 



Second Series of Experiments, a. A filament of 14 bacteria is washed and 

 transferred into a microdrop of peptone water at time h; at /i + 20 minutes, there 

 are 18 bacteria. A diplo-bacillus detaches itself and is transferred into a micro- 

 drop. Its two members lyse at /i + 40 minutes, at 40-second intervals. At 

 h -\- QS minutes, 8 bacteria of the original drop are transferred into microdrops, 

 where they lyse without leaving a trace in less than 10 minutes. There remained 

 8 bacteria in the original drop. Seven of them lysed under our eyes between 

 /i + 67 and h -\- 71. The residual bacterium is eliminated. All of the liquid is 

 then sampled. The plaques on the petri dish are almost confluent, impossible to 

 count. It looked as if there were about 2000 plaques on the plate. 



b. A filament of three bacteria is washed and brought into a drop A. At time 

 h + 45, there are about 4 bacteria. A sampling made then is negative: no 

 bacteriophages. At /i -f 60 minutes, there are 6 bacteria, only 4 at h -{■ 72. 

 These four residual bacteria are transferred with a minimum of fluid into one 

 drop B. All of drop A is sampled: 138 phages, corresponding to the lysis of two 

 bacteria. In drop B two out of four bacteria lysed between h -\- 72 and h -f- 79. 

 The two residual bacteria are transferred with a minimum of fluid into a drop C. 

 All of drop B is sampled, where two bacteria have lysed: 38 bacteriophages. 

 The two bacteria remain for 28 minutes in drop C. They are then transferred 

 into D. The total sampling of C does not reveal any bacteriophages. In drop D 

 the two bacteria disappear 4 minutes after their transfer: total sampling indicates 

 162 bacteriophages. One will notice that in drop C, where two bacteria remained 



322 



I 



