42 THE BACTERIOPHAGE 



Experiment III. Cultures of B. dysenteriae Shiga, aged fifteen days 

 and eighteen hours respectively, are centrifugalized. The sediment from 

 the first culture is suspended in the supernatant fluid of the second, and the 

 sediment of the second culture in the fluid of the first. Both suspensions 

 thus formed are inoculated with a drop of a culture of the bacteriophage. 

 The suspension consisting of "old" bacilli and "young" medium remains 

 turbid ; that of "young" bacilli and "old" medium becomes perfectly limpid 

 after seven hours. 



Thus, while the products of bacterial metabolism are not in- 

 hibitory for the lytic process, the products of lysis, as we will 

 see, exert quite a different action. These products are the result 

 of the activity of the ultramicroscopic bacteriophage, and as 

 such, they impede its activity. 



THE INFLUENCE OF THE MEDIUM 



It is evident from the foregoing experiments that the true cul- 

 ture medium of the bacteriophage is the living bacterium. The 

 nature of the fluid in which the bacteria are suspended is without 

 direct influence upon the culture of the bacteriophage, provided 

 only the bacteria remain living in it throughout a sufficient period 

 of time and provided it does not alter the constitution of the 

 bacterial cell. Experiment confirms this statement. 



The only additional condition regarding the medium is that it 

 be alkaline in reaction. Lysis will not take place in a medium 

 of acid reaction. 6 



Experiment IV. Peptone water (containing 25 grams of Chassaing pep- 

 tone and 5 grams of NaCl per liter) is neutralized to phenolphthalein. The 

 medium is then frankly alkaline to litmus. It is then distributed into 

 tubes, 10 cc. to each. Hydrochloric acid is added to each tube in dilutions 

 to form an increasing scale of acidity. All of the tubes are inoculated with 

 a concentrated suspension of Shiga, sufficient to yield a normal suspension 

 of 250 millions per cubic centimeter. Finally, each tube is inoculated with 

 0.001 cc. of a culture of the bacteriophage. After twenty-four hours the 

 numbers of bacteriophage in the several tubes are determined by the 

 method previously described. 



6 Certain commercial peptones contain glucose in appreciable quantity, 

 hence their use may be attended by failure. 



