A. D. HERSHEY AND MARTHA CHASE 43 



heated to 80°C. for 10 minutes, at which temperature unadsorbed phage is 

 not sensitized to DNase. 



3. The DNA of phage adsorbed to unheated bacteria is resistant to DNase, 

 presumably because it is protected by cell structures impervious to the en- 

 zyme. 



Graham and collaborators (personal communication) were the first to dis- 

 cover the sensitization of phage DNA to DNase by adsorption to heat-killed 

 bacteria. 



The DNA in infected cells is also made accessible to DNase by alternate 

 freezing and thawing (followed by formaldehyde fixation to inactivate cellu- 

 lar enzymes), and to some extent by formaldehyde fixation alone, as illus- 

 trated by the following experiment. 



Bacteria were grown in broth to 5 X 10^ cells per ml., centrifuged, resuspended in 

 adsorption medium, and infected with about two P^Mabeled phage per bacterium. 

 After 5 minutes for adsorption, the suspension was diluted with water containing per 

 liter 1.0 mM MgS04, 0.1 mM CaCh, and 10 mg. gelatin, and recentrifuged. The cells 

 were resuspended in the fluid last mentioned at a concentration of 5 X 10^ per ml. 

 This suspension was frozen at -15°C. and thawed with a minimum of warming, 

 three times in succession. Immediately after the third thawing, the cells were fixed by 

 the addition of 0.5 per cent (v/v) of formalin (35 per cent HCHO). After 30 minutes 

 at room temperature, the suspension was dialyzed free from formaldehyde and cen- 

 trifuged at 2200 G for 15 minutes. Samples of P^^.j^beled phage, frozen-thawed, fixed, 

 and dialyzed, and of infected cells fixed only and dialyzed, were carried along as 

 controls. 



The analysis of these materials, given in Table III, shows that the effect 

 of freezing and thawing is to make the intracellular DNA labile to DNase, 

 without, however, causing much of it to leach out of the cells. Freezing and 

 thawing and formaldehyde fixation have a negligible efifect on unadsorbed 

 phage, and formaldehyde fixation alone has only a mild effect on infected cells. 



Both sensitization of the intracellular P^- to DNase, and its failure to leach 

 out of the cells, are constant features of experiments of this type, independently 

 of visible lysis. In the experiment just described, the frozen suspension cleared 

 during the period of dialysis. Phase-contrast microscopy showed that the cells 

 consisted largely of empty membranes, many apparently broken. In another 

 experiment, samples of infected bacteria from a culture in salt-poor broth 

 were repeatedly frozen and thawed at various times during the latent period 

 of phage growth, fixed with formaldehyde, and then washed in the centrifuge. 

 Clearing and microscopic lysis occurred only in suspensions frozen during the 

 second half of the latent peritad, and occurred during the first or second thaw- 

 ing. In this case the lysed cells consisted wholly of intact cell membranes, 

 appearing empty except for a few small, rather characteristic refractile bodies 

 apparently attached to the cell walls. The behavior of intracellular P^^ toward 

 DNase, in either the lysed or unlysed cells, was not significantly different from 



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