LABELING CROWN GALL BACTERIA WITH P 32 FOR RADIOAUTOGRAPHY

Abstract

A method was described for labeling crown gall bacteria for radioautography with p32. The addition of 100 [mu]c of p32/ml of synthetic culture medium was found to be most satisfactory, providing the specific activity of the medium was adjusted to be in the 5 to 10 [mu]c/[mu]g P range. At this level the specific labeling achieved was roughly 1 to 2 X 10-8 [mu]c per bacterium; each bacterium contained approximately 1000 p32 atoms. Lowering the specific activity of the medium results in a lowered specific labeling of the bacteria. With increasing specific activity, the toxic effect of p32 becomes correspondingly more pronounced. At 5 to 10 [mu]c/ug P growth inhibition can be observed; however, neither the morphology nor the pathogenicity of the bacteria appears to be significantly affected. It could be shown that the toxic effect of p32 is primarily caused by p32 atoms assimilated by the bacteria, and probably results from a transmutation rather than a radiation effect. Once labeled bacteria start to grow in a nonradioactive medium, their growth is indistinguishable from unlabeled controls. The P content of the bacteria calculated on the basis of the P uptake from the medium was estimated at 2.5 x 10-9 [mu]g per bacterium for strain A6. This amounts to 0.3% P by wet weight. Labeled crown gall bacteria released large amounts of p32 to the medium when they were transferred to nonradioactive media.