456 BACTERIOPHAGES 



Procedure. Ordinary agar plates are spread with a mixture 

 of heavy broth culture of £". coli and enough phage to give barely 

 confluent lysis when the plaques are completely developed, 

 i.e., 10''~10'' phage particles/plate. After incubation for 

 12-18 hr., about 3 ml. of broth is added to each plate and al- 

 lowed to stand for 15-20 min. The broth is then decanted and 

 centrifuged to remove any bacteria, agar or other debris. Phage 

 stocks prepared thus should have 10^^-10^^ infectious particles/ 

 ml. This high concentration is not surprising since a single 

 plaque 2 mm. in diameter may contain between 10^ and 10^ 

 recoverable phage particles (T. F. Anderson, 1948b) and con- 

 fluent lysis requires 10^-10*^ plaques/plate. 



Plates prepared by the agar layer technique can be used in 

 this way also. Four plates were made using 10^ T5 phage 

 particles/plate and incubated overnight. Five ml. of broth 

 was added to each plate, the soft agar layer was scraped oflf 

 with a glass rod, and the agar layers shaken in a flask with the 

 20 ml. of broth. After 30 min. extraction with occasional 

 shaking, the agar and bacterial debris were sedimented at low 

 speed in a centrifuge. Yield was 15 ml. of a T5 stock with 

 8 X 1011 particles/ml. 



[Some of the variables involved in the production of high 

 titer stocks of the T phages by the plate method are discussed 

 in a paper by Swanstrom and Adams (1951).] 



Filtration of Phage Stocks 



For most purposes it is desirable to use phage stocks which 

 are free from bacteria, molds, and insoluble debris. The first 

 step in such purification should be centrifugation at moderate 

 speed to remove most of the bacteria and larger particulate 

 matter. Another method of removing insoluble materials, 

 which is particularly applicable to large batches of bacterial 

 lysate, is to filter the lysate through a pad of filter paper pulp on 

 a Buchner funnel. Better filtration is often obtained if a layer 

 of infusorial earth, filter cell, or fine sand is placed above the 

 paper pulp. After clarification by either centrifugation or 



