APPENDIX 457 



filtration, the lysate is passed through a fine pore bacteriologic 

 filter to remove the few remaining bacterial cells and spores. 

 Filters such as Seitz, Berkefeld, Mandler, porcelain, or sintered 

 glass may be used. In general, Seitz filters are not satisfactory 

 because a large fraction of the phage is lost in filtration. Mand- 

 ler, porcelain, and Corning ultrafine sintered glass filters are 

 usually satisfactory. The pH of the medium and the salt 

 concentration are important controllable variables which 

 affect the filter-passing ability of viruses. Gradacol membranes 

 of a porosity which will retain bacteria but permit passage of 

 the virus can also be used for sterilization of phage stocks (El- 

 ford, 1938). 



[A simple method for the sterilization of phage stocks is to 

 add approximately 0.5 ml. of chloroform per 10-20 ml. of 

 the bacterial lysate, shaking vigorously a few times to saturate 

 with chloroform. After the chloroform has settled (or al- 

 ternatively is centrifuged) to the bottom, the stock is decanted 

 and aerated until it no longer contains chloroform. Although 

 this method has been used successfully with the T phages, it is 

 advisable to test the viability of any new phage strain under 

 these conditions.] 



Concentration and Purification of Phage 



A particularly valuable tool for the concentration and partial 

 purification of large quantities of viruses is the Sharpies super- 

 centrifuge. Stanley applied it to the large scale concentration 

 of tobacco mosaic (1942) and influenza (1944) viruses, and 

 Hook and co-workers used it for concentration of a number of 

 viruses including T2 bacteriophage (1946). The principles 

 involved in use of the Sharpies centrifuge for concentration of 

 viruses were discussed by Markham (1944). The following 

 description of use of the Sharpies centrifuge for concentration of 

 T2 phage is abstracted from the paper by Hook and co-workers 

 (1946). 



Procedures. 1. Freshly lysed nutrient broth cultures of T2 

 phage were stored in the refrigerator 7-14 days, during which 



