432 Marine Microbiology 



in Figure 2. Repaske's procedure thus sliovved promise of yielding 

 "protoplasts" and conditions for this were examined. 



Most rapid lysis (15 to 20 min. at 37 C) took place with 

 SO^g/ml. lysozyme + 400Aig/ml. EDTA at 37 C in dilute (about 

 1 mM) potassium phosphate buffer. Alteration of the absolute 

 amounts of lytic agents, or their ratio, slowed lysis. The process 

 had a broad pH optimum about 7.6 but was little slower at values 

 of 6.0 and 8.7; within the range 0.3 to 1.3 mg dry wt. 

 cells/ml the rate of lysis was little influenced by the cell concen- 

 tration.The EDTA concentration routinely used was 1.08 mM; 

 antagonism of lysis by metal ions was tested using the following 

 salts at 4 mM; MgS04, CaCL Fe(NH4)2(S04)2, NH4 - moly- 

 date, ZnS04, CuS04, MnS04, NH4-vanadate, CoCL. Ca, Mg, Mn 

 or to a lesser extent Fe antagonized lysis; the other materials had 

 no observable effect. 



Concentrations of sucrose, phosphate buffer or NaCl in the 

 region of 0.5 to 1m prevented lysis, and the organisms retained 

 their vibrio form. Nevertheless, the cells had become osmotically 

 fragile because they lysed rapidly when transferred to distilled 

 water or dilute buffers. Lysis, observed under the phase con- 

 trast microscope, involved first a swelling of the vibrio until 

 it became a symmetrical sphere, then an abrupt breakage of the 

 sphere with loss of contrast and formation of a flat, circular 

 "ghost." These bacteria, therefore, though rendered osmotically 

 fragile by treatment with the lytic agents, formed in these 

 conditions neither true protoplasts nor spheroplasts. Sufficient of 

 the cell wall remained to retain morphological rigidity in isotonic 

 media, but not to survive osmotic shock. These osmotically fragile 

 vibrios will be referred to as "O.F.V.s." 



O.F.V.s could thus be prepared in nearly 100 per cent yield. 

 Batches of organisms from 100 ml cultures of strains Hilden- 

 borough, its salt-adapted variant and El Agheila Z were harvested 

 from the stationary phase of growth by centrifugation and sus- 

 pended at turbidities equivalent to 0.1 to 0.6 mg dry wt. cells/ml 

 in NaCl solutions of various molarities. The optical densities were 

 then measured; they were lowest with dilute salt solutions in 

 accordance with Mager's et al. ( 12 ) optical effect. Lysozyme and 

 EDTA were then added in a volume of water small enough not 



