124 THE BACTEBIOPHAGE 



In a culture of bacteriophage the lysins which effect the solu- 

 tion of the bacteria ought to remain in solution when lysis is com- 

 pleted. On the other hand, the ultramicrobe does not resist 

 treatment with alcohol. Therefore, in order to obtain lysin it 

 is only necessary to subject the culture of bacteriophage to the 

 classic procedure for the separation of diastases. 



If we mix one volume of bacteriophage culture (anti-dysentery) 

 with nine volumes of 96 per cent alcohol, after contact for 48 hours 

 the precipitate which is formed is well compacted and the super- 

 natant fluid may be decanted. The precipitate, which contains 

 the lysins admixed with all the substances of the medium precipi- 

 table by alcohol, is almost completely soluble in saline. 8 



Experiment XXXIII. Precipitate a culture of anti-dysentery bacterio- 

 phage with alcohol and dissolve the precipitate in a quantity of 0.8 per 

 cent saline equal to the original volume of the culture. Mix equal parts 

 of this solution and bouillon and add a B. dysenteriae suspension sufficient 

 to give a slight turbidity. As a control, prepare a tube containing an 

 equal volume of bacilli suspended in a medium half bouillon and half 

 saline. Place these tubes in an incubator at 37C. 



After twenty-four hours the control is turbid, the bouillon containing 

 the lysin is slightly cloudy. Plantings on agar from the two tubes give 

 normal bacillary growths. 



After forty-eight hours the control presents the same appearance and 

 agar inoculation gives a perfect growth. The culture containing the lysin 

 is slightly cloudy and inoculations on agar give only isolated colonies. A 

 count shows that there are 22 times less living bacilli in the last culture 

 than in the control tube. 



After three days the appearance is the same as after forty-eight hours. 



After four days the bacteria begin to develop a resistance to the action 

 of the lysin, the medium becomes cloudy and inoculations on to agar again 

 give a film of growth. 



At no time does one obtain on the agar the plaques character- 

 istic of the presence of the bacteriophage, and the action is not 

 continued in series. 



The alcohol precipitate therefore contaias a lytic diastase, 

 free of living ultramicrobes. The dissolving action, although 

 definite, is weak; but on the contrary as we will see later, 

 the lysin manifests itself by an extremely powerful opsonic action. 



8 This manipulation should be carried out aseptically, since filtration 

 of the fluid through a bougie considerably weakens its activity. 



