A method is described for determining the localization of alkaline phosphatase in the cells of E. coli B with the electron microscope. Enzyme activity, determined by deposition of inorganic phosphate, is located in the exterior layer of the cell wall.Electron microscopy is of great importance in the study of the relationship of enzymes and cell ultrastructures and this application has permitted the localization of some esterases and dehydrogenases (1,4,11). A number of similar experiments concerning dehydrogenase localization have been carried out on bacterial cells (2,6,9,14,15,16,18,19). A relationship between esterases and structures of the bacterial cell has not been studied by electron microscopy.The present paper concerns the distribution of alkaline phosphatase in the cells of Escherichia coli B. Materials a n d MethodsThe cells used were E . coli B. An 18-hour culture of E. coli B on nutrient agar was inoculated into a salts medium having limited inorganic phosphate (17) and grown for 24 to 48 hours. All the salts were recrystallized. Sodium P-glycerophosphate for determination of phosphatase was obtained from the Merck Company. No trace of free orthophosphate was found in this preparation.Coloration.-After growth, the cells were centrifuged and placed in a glycerophosphate medium (3) a t pH 9.0. The incubation was continued a t 37 OC for 10 minutes to 2 hours. After that the cells were again centrifuged and suspended in a 2y0 cobalt nitrate soiution, b u t in some experiments, suspension in cobalt solution was omitted. After 2-3 minutes the cell suspension in cobalt solution was centrifuged a t 3000 r.p.m. for 3 minutes and the supernatant was placed on formvar-coated grids for electron microscopy.Ultrathin sections.-The material obtained after cobalt nitrate treatment was fixed according to Ryter et al. (13), dehydrated in alcohols, and embedded in the mixture of butyl-and methyl-metacrylates (4: 1). The polymerization was performed a t 55' for 24-48 hours. The sections were obtained on the "Ultratome 4800" with glass knives and collected on formvar-covered copper grids strengthened with carbon. The material obtained was contrasted in 5yo uranyl acetate solution a t room temperature for 1 hour.Electron microscopy.-The preparations of whole cells and sections were looked over in UEMV-100B electron microscope a t 50 and 75 kV with a 40-p objective and recorded with photoplates MR. Electron optical magnification, 8000-62,000 X.

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The adenosine triphosphatase (ATPase) activity in Escherichia coli cells was investigated by a combination of electron microscopic and cytochemical methods. The activity is located in the cytoplasm and cytoplasmic membrane. After prolonged incubation in the substrate medium, the insoluble calcium phosphate was concentrated into several conglomerations and probably extruded from the cell. The possible mechanism of extrusion is also discussed.The location of ATPase in the membrane preparations was investigated by similar technique. The activity in the membranes is located in between the membrane subunits. The order of the subunits became well arranged after incubation with ATP.

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