588 PETER MITCHELL 



medium, when one breaks the cell wall and plasma membrane of bacteria 

 by methods that do not cause appreciable autolysis, are located within the 

 protoplast of the intact cell. Further, it has generally been assumed that 



TABLE II 



Fractionation of Escherichia coli 



Equivalent of 930 mg. dry \vt. organisms disrupted mechanically in standard saline 

 (0-17 M NaCl, 0-017 M KCl, 0-005 ^i MgClo) for 30 min. using the disintegrator of 

 H. Mickle as described by Mitchell and Aloyle [27], washed into centrifuge tubes with 

 c. 30 ml. standard saline at 2 and centrifuged at 2000 g for 30 min. 



Supernatant 

 centrifuged at 

 35 000 g 

 for I hr. 



Supernatant, Si 

 (48 ml.). 



Pad in laver, P3 



(2 ml.). 



Dispersed in 10 ml. 



standard saline. 



Morphology : aggregates 



of very small particles. 



Pad in 2 layers: Top, Pi (5 ml.). 



Bottom, P2 (i ml.). 



Pi, easily dispersed, made up to 10 ml. 

 in standard saline. 



Morphology: empty en\elopes, intact 

 and fragmented. 



P2, dispersed with difficulty, made up 

 to 10 ml. in standard saline. Extinction 

 at 700 m/x equivalent to 93 mg. dry wt. 

 intact cells. 

 Morphology: mainly intact cells. 



Of fraction Pi, 8 ml. returned to sonic 

 disintegrator for 15 min. Washed into 

 centrifuge tubes with standard saline 

 (total vol. 14 ml.). Centrifuged at 

 2000 g for 60 min. 



Supernatant, S2 Pad, P4 (2 ml.). 



(i2 ml.). Made up to 8 ml. 



in standard saline. 

 Morphology: as 

 Pi, but more 

 fragmented. 



The morphology of the fractions was exaniined by anoptral contrast microscopy of very 

 thin films of the untreated aqueous suspensions sealed between slide and coverslip w ith a 

 ring of vaseline. 



such "soluble", extracellular enzymes — like ^-galactosidase in Escherichia 

 coli [40, 41] — cannot be involved in catalytic activity at the surface of the 

 protoplast, or in membrane transport, because they are said to be " cryptic ", 

 or, in other words, enclosed behind the osmotic barrier component of the 

 plasma membrane [i]. Dr. Stephen and I have studied the "solubility" 

 and distribution of glucose-6-phosphatase activity in Escherichia coli 



