Some Biochemical Differences 431 



(10), being essentially spheres with residual fragments of cell 

 walls attached. In most instances, however, these forms consti- 

 tuted only 10 to 20% of the population; the best yields were ob- 

 tained with 10"^ units penicillin/ml., 0.1 M sucrose and eighteen 

 to twenty hours incubation. Then up to 40 percent of the popula- 

 tion formed spheroplasts, the remainder being unchanged vibrios 

 showing no osmotic sensitivity. Such a yield was inadequate for 

 tlie purposes intended and this approach was abandoned. 



Preparation of Osmotically Sensitive Vibrios 



Repaske (17, 18) showed that lysozyme-insensitive bacteria 

 could be rendered sensitive by adding ethylendiaminetetraacetate 

 (EDTA) as well as lysozyme. Cells of the Hildenborough strain, 

 harvested from continuous culture and suspended in dilute buffer 

 lysed in the condition described by Repaske, thougli lysis was 

 slower (c 40 min. at 37 C) than with his organisms. Papain was 

 active in place of lysozyme; EDTA caused slow lysis on its 

 own. The lysed suspensions were mucoid and the viscosity was 

 reduced by adding ribonuclease. A typical lysis curve is given 



MIN. 



Fig. 2. Lysis of Dcsidfovihrio desidfuricans (HUdenhorough). Organisms 

 harvested from continuous culture were washed, suspended at 0.36 mg. 

 dry wt./ml. M/lOO KH.PO, (pH 6.9) were incubated at 37 C with 50 

 /ig/ml. lysozyme and 400 ml. EDTA and the changes in optical density in 

 blue light consequent on lysis were measured. Q control suspension, test. 



