VOL. 10 (1953) NUCLEIC ACID TRANSFER 433 



extensively beforeHheir constituents are used for synthesis of new phage particles. We 

 do not, however, think that this evidence excludes the possibility that under normal 

 conditions the infecting and reproducing particle remains essentially unbroken. We shall 

 return to this important point in the discussion. 



MATERIALS AND METHODS 



The phages T2, T3 andT4, and their common host E. coli, strain B/i, have been used: the latter 

 because contamination of cultures with phage Tr occurs in our laboratory. Most of the techniques 

 used in this study have been described in detail by Adams^. All cultures were grown at 37° C: cen- 

 trifugations were done in a Servall Angle centrifuge at 10° C. 



Media. The nutrient broth is an aqueous extract of minced meat enriched with i % peptone 

 and containing 0.02% Tween 80 and 0.5% NaCl: pH is adjusted to 7.4. For experiments with T3, 

 the concentration of NaCl was reduced to 0.05 % to obtain rapid adsorption. In this medium, 

 at 37° C the latent period of T4r is 22-23 minutes and the burst size about 150. ^^p-iabelled phage 

 was prepared using the synthetic g-medium described earlier (Maaloe and Watson*). 



Anti-sera. Rabbit anti-phage sera were prepared, using highly purified and concentrated phage 

 suspensions as antigens. All sera had k-values (Adams^) of 500-1000 when tested against phage 

 suspended in broth (of. Jerne'). These sera showed no agglutination of E. coli, strain B/i, in dilution 

 T to 10. Samples of anti-phage sera were absorbed with large amounts of live B/i, and used in parallel 

 with unabsorbed serum for phage precipitation : no evidence for the presence of antibacterial anti- 

 bodies was found. Serum against E. coli. strain B/i, was obtained after series of subcutaneous and 

 intravenous injections of heat-killed and subsequently of live cultures. This serum had no anti-phage 

 activity. 



X-ray technique. The X-ray source was a Holbeck-Beaudouin tube operating at 33 kv and 

 36 mA. A cooled molybdenum target produced radiation with an average wave-length of 0.9 A. 

 On the surface of the samples the intensity of radiation was 66,000 r.p.m. The irradiation was done 

 by Dr R. Latarjet on samples sent to Paris by airmail. No decrease in titer was observed in control 

 samples as a result of the shipment. 



Isotope technique. Carrier-free orthophosphoric acid was obtained from the Isotope Division 

 of the Oak Ridge National Laboratory, United States Atomic Energy Commission. Adenine labelled 

 with "C in position 8 was also used: this preparation was synthesized by Clark and Kalckar*, 

 and had a specific activity of 0.8 mC per mMole. All samples containing "C were evaporated to 

 dryness and self-absorption due to solids in the suspension medium was made uniform by diluting 

 into nutrient broth before counting. Variation between counts on duplicate ^^C samples was less 

 than 10%. The counting equipment was the same as previously described. 



Preparation of ^^P-labelled T4r. Washed B/i bacteria from a 24-hour broth culture were in- 

 oculated into 10 ml of g-medium containing 20 uC ^^p. After two and a half hours of aeration, the 

 bacterial density was about lo^ cells per ml, and the culture was then infected with about 10 T4r 

 particles per cell Aeration was continued, and 2-3 minutes before the onset of lysis 0.5 ml of un- 

 diluted antibacterial serum was added. If antibacterial serum is not added, the titer of a crude 

 T4r lysate will drop appreciably during the first 24 hours and a fraction of the remaining phages 

 will adsorb slowly. Both effects are presumably due to phage particles absorbing on bacterial debris 

 (Maaloe and Stent^). The antibacterial serum proved completely effective in blocking adsorption 

 of T3 and T4 on B/i, but was not fully effective for T2r+. T2r+ stocks may, therefore contain inactive 

 as well as slowly adsorbing particles even when antibacterial serum is used. It is possible to restore 

 infectivity and full adsorbability to these particles by diluting into distilled water for several hours 

 at 37° C: presumably because adsorbed particles dissociate from the debris at low salt concentrations 

 (Puck, Garen, and ClineI" and Hershey, personal communication). This treatment was first used 

 by Bertani (personal communication) to raise the titer of T2r+ broth lysates. 



The crude radioactive lysates were centrifuged at 5000 g for 5 minutes to remove bacterial 

 debris and at 12,000 g for one hour to sediment the phage. Three cycles of low and high speed 

 centrifugation reduced the concentration of inorganic ^-P by a factor of about 10*. Further puri- 

 fication was achieved by adding heat-killed resistant bacteria (B/3, 4, 7 heated to 58° C for i hour) 

 at a concentration of 5- lo^ per ml. After 30 minutes at 37° C, about 5% of the radioactivity had 

 adsorbed to the resistant cells, which were removed by centrifugation. A similar number of sensitive 

 B/i cells adsorbed 95-98% of the activity. An additional test of the purity of the virus stock was 

 obtained by precipitation with antiphage serum, as previously described; with anti-T4 serum, 94% 

 of the radioactivity was precipitated, while in a control sample in which T3 phage was precipitated 

 with anti-T3 serum, the precipitate contained less than 2% of the activity. 



Assuming that about 95 % of the ^^P in the final preparation was present as phage phosphorus, 

 the initial specific activity was io~^ counts per minute per particle. From this we can calculate 



References p. 442. 



106 



