48 AN INTRODUCTION TO THE STUDY OF VIRUSES 
frozen, otherwise the virus is thrown out of solution and will not 
re-dissolve. 
Finally, a short description is given of the purification of a bacterial 
virus, the T, or P.C. bacteriophage of Escherichia coli. The method 
is that of Hook, Beard et al. (1946) and quotation from their work is 
freely made. The purification was carried out on phage-cultured 
bacteria grown both in broth and synthetic media. 
In the production of the phage for concentration and purification, 
batches of 7-5 to 151, distributed in 1500 ml volumes in 2-1 Florence 
flasks proved convenient. Seed inoculation of bacteria for the large 
volumes was prepared in 50 ml of the respective media in 125-ml 
Erlenmeyer flasks inoculated with o-1 ml of an 18-24 hour broth 
culture of Escherischia coli and incubated at 37°C for 18 hours. Stock 
phage containing 10° lytic units per ml was added in the ratio of one 
phage particle to 400-700 bacteria, and the flask was shaken and allowed 
to stand for 5-7 minutes. To each 1500-ml volume of medium 
previously warmed to 37°C, there was added 30 ml of the phage- 
bacteria suspension. 
The flasks were incubated for 8 hours at 37°C with vigorous mixing 
by hand every 15 minutes. The individual batches of nutrient broth 
cultures were pooled immediately and stored in the ice-box for 7 to 
14 days before processing. The nutrient broth cultures usually were 
completely lysed at the end of 8 hours but the synthetic medium 
cultures took longer. 
Whilst the phage can be readily concentrated by the centrifugation 
of freshly-lysed nutrient broth cultures of 8 hours’ incubation, purifi- 
cation at this stage is very difficult. The fresh lysates contain much 
mucoid material and the phage particles are apparently trapped in, 
or coated with, the slimy substance. As a result, efforts to remove this 
substance by filtration through candles or slow-speed centrifugation 
are liable to result in the loss of 60-80 per cent of the phage activity. 
This difficulty can be got over by allowing the lysates to stand in 
the refrigerator for 7-14 days. During this period a granular or 
flocculent precipitate will sediment, taking down with it much of the 
slimy substance. Elimination of bacteria and bacterial debris, as well 
as the flocculated mucoid material, can now be accomplished by low- 
speed centrifugation in the Sharples centrifuge. Later work, however, 
has shown that filtration of the broth lysates through 1o-in. 6- or 7-lb 
Mandler candles removes the extraneous material with less loss of 
virus than does the centrifugal clarification. 
