The utilization of the three singly-C14-marked lactic acids by Escherichia coli.

As a preliminary to preparing bacteriophages marked with C14, it was desirable to ascertain which singly-labeled lactic acid, when used as a carbon source, gives the highest incorporation of radioactive carbon in the celLs of Escherichia coli. The results, while not unexpected, present several interesting features and serve to supplement the much more complete data obtained by Wiame and Doudoroff (1951) with another bacterium under rather different conditions (see the accompanying article). A synthetic medium containing sodium lactate as the energy source has been used widely in studies of the T-series of Escherichia coli bacteriophages (Adams, 1950). Since we have found that our usual glycerol medium gives better yields of T3 bacteriophage when fortified with casein hydrolysate, experiments on the utilization of lactates were conducted in the presence or absence of such a casein hydrolysate supplement, each set containing a control of the opposite type. The medium was prepared as usual (Adams, 1950). The radioactive lactates were available as zinc salts. These were converted to the free acid by treatment with washed "dowex 50"1 ion exchange resin in the acid form. Each sample was dissolved in 1 ml water, mixed with 1 ml wetpacked resin, and the resin separated using a low speed centrifuge. The resin was washed with two half ml portions of water. The combined solutions in 1.5 ml water were made up to 50 ml with inactive lactate medium, the pH adjusted, and the solution autoclaved as usual. The specific radioactivity of each of the final mixtures was determined (Dauben et al., 1947). Each culture was aerated with a simple bubbler in a test tube, and the effluent CO2 was collected in a scrubber of 1 N NaOH using an apparatus which passed the gas in a helical path. The bacteria were harvested at a concentration of 1 to 2 X 109 cells per ml, washed by centrifugation twice with a 1.5 per cent phosphate buffer (pH 7.0) and once with distilled water. This suspension was plated directly for radioactivity (Dauben et al., 1947). The CO2 in the scrubbers was precipitated as BaCO8 on aluminum discs (Calvin et al., 1949). The radioactivity of the samples was determined either with a proportional counter ("nucleometer") or with thin end window GM tubes, depending on the specific activity. The final supernatant fluid from each sample was tested to show that no significant activity remained.