648 GROWTH OF PHAGE AND LYSIS 



NaCl solution. Both were as effective as phosphates in causing the phage to be ad- 

 sorbed by the filter. 



Finally various concentrations of phosphate buffer were tried in order to deter- 

 mine the lower limit at which adsorption would take place. It was found that reduction 

 of the buffer concentration to one half practically eliminated the adsorption. 



By this method several concentrates were obtained with titers between 10"/cc. 

 and 10^'^/cc. These concentrates were clear in transmitted light and showed intense 

 blue Tyndall scattering. When kept in the ice box they showed no measurable decrease 

 of titer over periods of more than 5 months. 



Standard Cullural Conditions. — The cultural conditions of the bacteria were stand- 

 ardized in the following way. A stock culture was kept going for about a week by 

 daily transfers into fresh synthetic medium. At the time the transfer was made, the 

 culture contained about 2.5 X 10^ bacteria /cc. These 24 hour aerated bacteria were 

 used for phage assay. They will be referred to as the stock culture. 



TABLE I 



Adsorption and Elution of Phage Jrom Jena Sintered Glass Filters 



Total phage 



25 CC. fresh lysate 1.4 X W> 35 X lOi" 



Filter lysate through Jena sintered glass filter grade 5 



on 3, filtrate 0.001 X lO" 0.025 X W 



Follow with 2 cc. distilled water 6.4 X W> 12 .8 X W 



Follow with 5 cc. distilled water 0. 19 X W 0.95 X 10" 



At the time of the transfer of the stock culture a sample of 0.1 cc. was also transferred 

 into 25 cc. broth and aerated at 37°. This culture was used for growing the phage, 

 and will be referred to as the experimental culture. 



Fig. 2 shows the growth of this experimental culture as determined by colony counts 

 in 7.5 minute intervals. It is seen that the bacteria do not start to divide in the first 

 90 minutes and divide at maximum rate after 3 hours. It can therefore be used to 

 study phage growth either on bacteria that are small and have a long period of growth 

 in size without divisions ahead of them, or on bacteria that have attained their maximum 

 average size and divide at a maximum rate. 



Microscopic Observations 



We have made some microscopic studies of the lysis of our bacteria under 

 various conditions, both in hanging drop preparations and on nutrient agar 

 plates. The hanging drop preparations have the advantage that one can 

 study changes in mobility of the bacteria and also that the bacteria are 

 subjected to more uniform conditions, while on the agar plate one can 

 follow the history of individual bacteria over a stretch of time. These 

 observations were made at room temperature, the time schedule is therefore 

 retarded in comparison with the growth curves at 37°C. 



Great differences in behavior were found depending on whether the bac- 



62 



