32 THE BACTERIOPHAGE 



been made, sometimes two or three passages on a single day, 

 introducing in each transfer about 0.001 cc. of the last tube lysed 

 into a fresh suspension. After more than 1500 passages the lysed 

 suspension of the last tube was as active, even more active, than 

 the filtrate which served to start the phenomenon in the first 

 tube of the series. 



A suspension once lysed does not contain any living bacteria. 

 On the other hand, the amount of bacteriophagous ultrami- 

 crobes introduced to start the lysis is increased, since the new 

 lysate is as active as was the preceding one. The lysed suspen- 

 sion has become what can be called, literally, a culture of the 

 bacteriophage. 



It has been previously stated that the inoculation of agar with 

 a bacterial suspension to which has been added a small amount 

 of fluid containing the active principle gives a bacterial culture 

 studded with clear areas. This observation suggests a means 

 of determining with approximate exactness the phenomenon of 

 multiplication of ultramicrobes, since each area undoubtedly 

 indicates the point at which, during the inoculation, there was 

 deposited an active element, — an ultramicrobe. It is only neces- 

 sary to work with measured quantities to ascertain the number 

 of active germs in a fluid. 



From an abundance of experiments a single one showing the 

 method of counting is taken. To avoid repetition it may be 

 stated that "suspension of B. dysenteriae Shiga" is to be under- 

 stood. 



A series of tubes is prepared, once for all, by any standard pro- 

 cedure, containing B. dysenteriae Shiga suspensions of the follow- 

 ing counts: 100, 200, 250, 300, and 400 million bacilli per cubic 

 centimeter. These suspensions are stabilized by the addition 

 of a small amount of formol and the tubes are sealed with the 

 blowpipe. 



The suspension to be subjected to lysis should be taken by 

 preference from a young growth on agar. A concentrated sus- 

 pension is prepared by adding one or two cubic centimeters of 

 bouillon to the agar slant and allowing the tube to remain 

 inclined for a few minutes in such a way that the whole bacterial 

 growth is under the fluid. With shaking, a perfect suspension 



