448 BACTERIOPHAGES 



plates. Incubate overnight at 37 ° C. and examine plates for 

 "plaques," small or large circular areas of lysis in the film of 

 bacterial growth. It is well to plate at least 4 serial 1:10 

 dilutions of the filtrate, since direct plating of a high concentra- 

 tion of virus may result in lysis of all bacteria and absence of 

 plaques, a result often confusing to the uninitiated. 



b. By lysis of broth culture. Inoculate 10 ml. of broth with 

 0.05 ml. of visibly turbid culture of host organism and with 

 0.1 ml. of the filtrate to be tested for phage. Incubate at 37° 

 C. and examine hourly for turbidity, comparing with a control 

 culture inoculated with bacteria but not with filtrate. If tur- 

 bidity does not develop or if lysis occurs after turbidity has de- 

 veloped, phage is present and mav then be tested for by plating 

 broth dilutions of the culture as described in (a) . 



Once plaques have been obtained on an agar plate, these 

 may be picked and replated twice to insure a pure culture of 

 a single phage type. The plaque is stabbed with a sterile 

 platinum wire and the wire rinsed in 1 ml. of sterile broth. 

 Several dilutions of the broth are then plated as in (a). 



It should be mentioned that a phage which brings about com- 

 plete lysis of broth cultures of bacteria may produce very tiny 

 plaques, may have a low efficiency of plating or a slow adsorp- 

 tion rate or may be otherwise unsuitable for quantitative work 

 by plaque-counting techniques. Also, a phage which produces 

 beautiful plaques may not bring about lysis in broth cultures, 

 may yield only low titer stock or be difficult to work with in 

 other ways. The method of isolation may in part determine the 

 properties of the phage isolated, through selection of variants. 

 In fact, repeated subculture of a phage in fluid media using 

 large inocula is a classic method of increasing phage "virulence," 

 probably through selection of mutants with the desired proper- 

 ties. 



After isolation, the phage may be characterized by host 

 range characteristics, immunologic properties, plaque morphol- 

 ogy, and electron microscopic appearance. 



In general, a bacterial virus should be looked for in the natural 



