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Summary 1. Enzymes and related substances may be localized in cells either, (a) by separation and analysis of cell parts, or (b) by application of light absorption methods (with or without staining) to living cells or tissue sections. 2. Separation of cell parts has been achieved by microdissection, by centrifuga‐tion (sea‐urchin eggs), by sectioning (neurones or centrifugally stratified Amoebae) and by bulk centrifugation of tissue suspensions. Highly sensitive micromethods, based on titration, dilatometry, or manometry, have been evolved for measuring the enzyme content of small fragments of protoplasm. 3. Precautions must be taken to minimize separation artefacts. In the bulk centrifugation techniques it is only possible to draw satisfactory conclusions when an enzyme remains associated with certain granules after repeated washing with physiological solutions. Even if an enzyme is constantly associated with, for instance, the mitochondria, it may occur only in certain of them, for they are not necessarily all identical. It is important (a) to distinguish between enzyme activity under the conditions existing in the cell and the maximum activity shown by an extract under optimal conditions, (b) to ensure that the total quantity of enzyme in the fractions separated equals that of the whole cell or tissue, (c) to distinguish between the concentration of, for instance, peptidase in any fraction and the proportion of the total cell peptidase present in the fraction. 4. The investigations which have most nearly satisfied these requirements have led to the following conclusions: (a) In various nuclei, dipeptidase, alkaline and acid phosphatase, arginase, uricase and esterase have been detected, all in lower absolute amounts than in the cytoplasm and all except alkaline phosphatase, arginase and uricase in lower concentration. Chromosome‐like threads can be centrifuged from sperm and suspensions of somatic tissues; after removal of desoxyribonucleohistone, there remains a residual chromosome containing ribonucleic acid, alkaline phosphatase and a tryptophane‐containing protein as well as other components, (b) The major part of the cytochrome oxidase, of the succinic dehydrogenase and of several enzymes concerned in fatty acid oxidation and in the Krebs tricarboxylic acid cycle is present in the mitochondria in rat‐liver cells. Mitochondria contain neither dipeptidase nor catalase in sea‐urchin eggs and Amoebae, but in the latter they appear to be associated with amylase activity, (c) Microsomes (cytoplasmic particles 50–200 m/j. in diameter) have been found to contain dipeptidase, catalase, phosphatases, ribonuclease, about half the esterase and half the ribonucleic acid in adult liver cells, (d) Much of the catalase, carboxylase and lactic acid dehydrogenase in yeast, of the glycolytic activity in rat liver and of the ribonucleic acid in embryonic tissues and tumours remains in the supernatant after centrifugation for 1–2 hr. at 18,000 g., whether because these substances are freely dissolved in the protoplasm, or bec...

SUMMARY: Live rod-shaped bacteria incubated on a medium containing 3 yo urea grow but do not divide. They thus become much longer than dead bacteria lying among them, and the numbers of each kind can easily be counted in an electron microscope. This urea method gives accurate results and has several advantages over colony-counting methods. The significance of various interesting differences in the counts given by the two methods is discussed. Slope cultures at 18 hr. gave live counts of over 95 yo by the urea method.A common need in quantitative bacteriology is to discover what proportion of bacteria in n given sample is alive. The standard procedure is to make counts of the colonies which form when measured volumes of the sample, suitably diluted, are incubated in nutrient media. On the assumption that each colony arises from one live bacterium, the number of the latter in the sample can then be determined. Such counts made before and after some treatment will enable the resulting fall in viability to be measured. In order to find the actual percentage of live cells in a sample a total count of the bacteria in the measured volume must also be made. In studying the survival of bacteria during drying from tiny droplets or thin films, an accurate measurement of the volume of the samples becomes difficult or impossible. To overcome this problem a new method has been developed for determining the proportion of live bacteria in such tiny samples. This paper gives details of the method and typical results of a test of its accuracy.The method is based on the property possessed by urea in concentrations of about 3 yo ( 0 . 5~) of preventing the division of certain bacteria without inhibiting their growth (Wilson, 1906). Hence after incubation of suitable bacteria for a few hours on nutrient agar containing urea, the live cells have grown into giant forms which can readily be distinguished from the shorter dead organisms (Pl. 1, fig. 5 ) . The numbers of live and dead can then easily be counted in an electron microscope. EXPERIMENTALThe action of urea We have no information about the mechanism by which urea exerts its effect. We have, however, investigated its action on the growth of a paracolon bacillus over a range of concentrations of urea from 1 to 10 yo in nutrient agar medium.The bacteria were taken from 18 hr. slope cultures, suspended in broth, and grown on filmed electron microscope specimen grids as described below. 23G. Microb. XI

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