40 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH 



Adsorption of isotope to bacteria was usually measured by mixing the sample 

 in adsorption medium with bacteria from 18 hour broth cultures previously heated 

 to 70°C. for 10 minutes and washed with adsorption medium. The mixtures were 

 warmed for 5 minutes at 37°C., diluted with water, and centrifuged. Assays were 

 made of both sediment and supernatant fractions. 



Precipitation of isotope with antiserum was measured by mixing the sample in 

 0.5 per cent saline with about 10" per ml. of non-radioactive phage and slightly 

 more than the least quantity of antiphage serum (final dilution 1:160) that would 

 cause visible precipitation. The mixture was centrifuged after 2 hours at 37°C. 



Tests with DNase (desoxyribonuclease) were performed by warming samples 

 diluted in veronal buffer for 15 minutes at 37°C. with 0.1 mg. per ml. of crystalline 

 enzyme (Worthington Biochemical Laboratory). 



Acid-soluble isotope was measured after the chilled sample had been precipitated 

 with 5 per cent trichloroacetic acid in the presence of 1 mg./ml. of serum albumin, and 

 centrifuged. 



In all fractionations involving centrifugation, the sediments were not washed, and 

 contained about 5 per cent of the supernatant. Both fractions were assayed. 



Radioactivity was measured by means of an end-window Geiger counter, using 

 dried samples sufficiently small to avoid losses by self-absorption. For absolute meas- 

 urements, reference solutions of P^^ obtained from the National Bureau of Standards, 

 as well as a permanent simulated standard, were used. For absolute measurements 

 of S^^ we relied on the assays (±20 per cent) furnished by the supplier of the isotope 

 (Oak Ridge National Laboratory). 



Glycerol-lactate medium was chosen to permit growth of bacteria without un- 

 desirable pH changes at low concentrations of phosphorus and sulfur, and proved 

 useful also for certain experiments described in this paper. 18-hour cultures of sensitive 

 bacteria grown in this medium contain about 2 X 10^ cells per ml., which grow ex- 

 ponentially without lag or change in light-scattering per cell when subcultured in 

 the same medium from either large or small seedings. The generation time is 1.5 hours 

 at 37°C. The cells are smaller than those grown in broth. T2 shows a latent period 

 of 22 to 25 minutes in this medium. The phage yield obtained by lysis with cyanide 

 and UV-phage (described in context) is one per bacterium at 15 minutes and 16 

 per bacterium at 25 minutes. The final burst size in diluted cultures is 30 to 40 per 

 bacterium, reached at 50 minutes. At 2 X 10* cells per ml., the culture lyses slowly, 

 and yields 140 phage per bacterium. The growth of both bacteria and phage in this 

 medium is as reproducible as that in broth. 



For the preparation of radioactive phage, P^^ of specific activity 0.5 mc./mg. or 

 S^^ of specific activity 8.0 mc./mg. was incorporated into glycerol-lactate medium, 

 in which bacteria were allowed to grow at least 4 hours before seeding with phage. 

 After infection with phage, the culture was aerated overnight, and the radioactive 

 phage was isolated by three cycles of alternate slow (2000 G) and fast (12,000 G) 

 centrifugation in adsorption medium. The suspensions were stored at a concentration 

 not exceeding 4 )uc./ml. 



Preparations of this kind contain 1.0 to 3.0 X 10-^^ ^g s and 2.5 to 3.5 X 10-" 

 /xg. P per viable phage particle. Occasional preparations containing excessive amounts 

 of sulfur can be improved by absorption with heat-killed bacteria that do not adsorb 



88 



