APPENDIX 459 



filtration through paper pulp or a coarse Mandler filter is 

 placed in a large Florence flask and connected through an 

 efficient water-cooled condenser and receiving flask with an 

 aspirator or other vacuum pump. Such a system should be 

 designed to operate at an internal pressure of 20-30 mm. Hg and 

 should evaporate several liters of water an hour at a temperature 

 in the distilling flask of about 30 °C. . . . 



Example: In a typical preparation 1 liter of Tl phage lysate with a titer of 

 2 X 10'" particles/ml. was concentrated in vacuo to 118 ml. at a concentration 

 of 2.2 X lO'i particles/ml. with no loss of activity. Eighty ml. of this concen- 

 trate was spun for 2 hr. at 18,000 r.p.m. in the International refrigerated centri- 

 fuge. After the supernatant fluid was decanted and the Lusteroid tubes were 

 drained well on filter paper, the pellets were resuspended in 5 ml. of chemically 

 defined medium and spun at low speed in the angle centrifuge to remove in- 

 soluble debris. The discarded supernatant from the high speed centrifugation 

 contained 5.6 X 10'" particles/ml., while the 5 ml. of concentrate had an ac- 

 tivity of 3 X 10'^ particles/ml., a yield of 80 per cent. 



3. Another method of concentrating bacteriophage is by 

 ultrafiltration through a collodion membrane of a permeability 

 which will hold back the phage but permit fairly rapid passage 

 of water and solutes. The medium may be filtered through 

 these membranes at the rate of ^/t-^ liter/hr. and the phage 

 readily concentrated 100-fold. The method as applied to T2 

 phage was described in detail by Kalmanson and Bronfen- 

 brenner (1939) and is not included here because the methods 

 described above are more convenient and rapid. 



Gradacol membranes are convenient for concentration and 

 purification of phage. Use of a membrane of porosity sufficient 

 to retain the phage permits rapid concentration of large volumes 

 of phage stock while permitting removal of proteins and other 

 high molecular weight substances which will pass the mem- 

 brane. Use of Gradacol membranes has been thoroughly 

 discussed by Elford (1938). 



[4. Herriott and Barlow (1952) have described in detail a 

 method for the preparation and purification of T2 in large 



