M. DELBRUCK 647 



Synthetic Medium.— The bacteria and phage were also grown with aeration in a 

 synthetic medium, consisting of 



/-Asparagine 2 gm. 



Glucose 4 gm. 



Na2HP04 (anhydrous) 6 gm. 



KH2PO4 3 gm. 



MgS04 .05 gm. 



NaCl 0.05 gm. 



Distilled H2O 1000 cc 



The bacteria in this medium grow more slowly than in broth but attain a higher final 

 concentration. The phage also grow well on bacteria in this medium and cause lysis. 

 But the growth rates of both the bacteria and the phage are only approximately re- 

 producible with different batches of medium. These irregularities must be eliminated 

 before the medium can be used for quantitative studies. 



Bacteria transferred from this medium into broth grow at once. Transferred from 

 broth to this medium they require a period of about 24 hours of adaptation before 

 growth begins. 



The bacteria were therefore carried on slants of synthetic medium agar and trans- 

 ferred into broth only for the specific experiments. 



Stock phage was obtained by lysis in broth and filtration through Jena sintered 

 glass filters. No measurable decrease in titer in periods over 6 months (in contrast to 

 Pi, compare (1)). 



Phage assay by plaque count on Difco nutrient agar plates, as described previously 

 (1). The plaques are large (2 mm. diameter) and are countable after 4 hours incuba- 

 tion at 37°. 



Turbidity was determined by visual comparison with turbidity standards, in most 

 cases taken from the same culture. Such turbidity determinations are of course very 

 rough, but a refinement of technique in this respect did not seem profitable. The 

 turbidity in any event would not be proportional to the lysis, which in dense cultures, 

 as we have seen, is a complex phenomenon, in which changes in shape, size, and re- 

 fractive index occur. Each of these factors contributes to the turbidity change. 



Preparation of Phage Concentrates.— Y or the experiments with large excess of 

 phage over bacteria stock phage of very high titer were needed. These were obtained 

 in the following way. 



It was observed that lysates obtained from synthetic medium cultures (in contrast to 

 broth lysates) lost all their phage on filtration through Jena sintered glass filters. The 

 phage is not inactivated by the filter but simply adsorbed, and it can be eluted with good 

 yields by small volumes of distilled water. An example is given in Table I. 



These concentrates could be further concentrated by adding phosphate buffer to 

 restore the original salt concentration and repeating the adsorption-elution procedure. 



Filters of the coarser grade 4 were also tried and though effective gave less reliable 

 yields. 



Ground glass, silica, and fullers' earth were tried as adsorbents. These also gave 

 good adsorption but the elution was again often unsatisfactory. 



The phosphate buffer was replaced by 1 per cent MgCl2 solution and by a 1 per cent 



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