HURWITZ, C. (Veterans Administration Hospital, Albany, N.Y.), C. L. ROSANO, AND J. V. LANDAU. Kinetics of loss of viability of Escherichia coli exposed to streptomycin. J. Bacteriol. 83:1210J. Bacteriol. 83: -1216J. Bacteriol. 83: . 1962.-The first effect of streptomycin on Escherichia coli B cells growing in nutrient broth is a decrease or cessation (depending on the concentration of antibiotic) of the rate of increase of viable cells. After this apparent bacteriostasis, a rapid decline in

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... le count begins. The length of time before onset of decline of viability is a first order function of the concentration of streptomycin between 0.1 and 2.0 ,ug/ml. Above 2.0 ,ug/ml, the lag in onset of loss of viability approaches asymptotically a value greater than 2 min. The length of the lag in onset of loss of viability is interpreted to be a measure of the rate of synthesis of the postulated streptomycin-initiated protein required for the later lethal action of the drug. Measurements of the effect of streptomycin on the rate of synthesis of protein by E. coli B cells show an immediate effect on the rate of protein synthesis, corresponding to the apparent bacteriostasis; this is followed by a later decrease in the rate of synthesis, which corresponds in time with the decline in viability. Washing exponentially growing E. coli B cells by centrifugation and resuspension in fresh nutrient broth makes the cells more resistant to streptomycin. The lag before the onset of decline in viability is prolonged, and the inhibition of protein synthesis is likewise affected. The washing procedure has no effect on total protein synthesis (as measured by incorporation of C14-leucine). Since synthesis of ,B-galactosidase is also delayed by the washing procedure, this effect is interpreted to mean that washing delays induced protein synthesis, but has no effect on the rate of total protein synthesis. In an accompanying communication (Hurwitz and Rosano, 1962a), evidence was presented in support of the thesis that a chloramphenicolsensitive, streptomycin-initiated protein synthesis is a necessary precursor for the lethal action of streptomycin. If a streptomycin-initiated protein synthesis is a necessary precursor of the lethal action of streptomycin, the kinetics of loss of viability of growing cells should show delineated phases corresponding to both the initiation and killing phases described in the preceding report. Studies of the kinetics of loss of viability and of the kinetics of inhibition of protein synthesis resulting from exposure of Escherichia coli to streptomycin were therefore undertaken to examine further the validity of the above thesis. In another accompanying report, the kinetics of accumulation of radioisotopic label from C14_ streptomycin will be discussed in a similar context (Hurwitz and Rosano, 1962b) . MATERIALS AND METHODS Suspensions of E. coli B were prepared by inoculating fresh nutrient broth with an overnight aerated culture, and by continuing aeration until the exponential growth phase was reached (about 2.5 to 3.0 X 108 cells/ml). The suspension was then rapidly concentrated by centrifugation and resuspension in one-half the original volume of fresh nutrient broth. This was done to conform with the procedures required for the isotopeincorporation studies reported in the following communication (Hurwitz and Rosano, 1962b) . After this treatment, the cells showed only a 5 to 10 min lag before re-entering exponential growth phase, which was then maintained for at least 60 min. Viable counts were made in quadruplicate by surface plating on nutrient agar. Protein synthesis was measured by the rate of incorporation of L-C'4-leucine into the hot trichloroacetic acid (TCA)-insoluble fraction of 1210 on May 9, 2020 by guest