The Use of Radioautography and Electron Microscopy for the Localization of Tritium Label in Bacteria

The localization of DNA labeled with thymidine-H/sup 3/ was studied in Escherichia coli by electron microscopic examination of radioautographs of the cells using a thin film of Kodak V-1055 x-ray emulsion. In this way it was possible to localize the labeled DNA in whole bacteria to regions <1 mu in length and <1/2 mu /sup 2/ in area. When cells of E. coli 15 T/sup -/ were labeled with thymidine and allowed to grow for 3 generations, approximates 1/2 the cells showed radioactivity. Electron micrographs of such radioautographed cells showed the silver, ribbon-like grains, and the bacteria were also clearly visible. It was noted that labeling was present in only half of a cell, in cell regions <1 mu in length. Over 1/3 of the labeled 3rd generation cells showed this particular localization. Approximately 1/3 of the labeled cells showed a concentration of grains in the central region while less than 1/3 showed a random grain pattern. In contrast, fully labeled cells showed a consistently random grain pattern. This demonstration of the localization of each DNA structure in 1/ 2 of the cell supports the view that the chromosomes in E. coli are haploid, 2 in number, and physically separated.more » The results also showed that, by using Kodak V- 1055 emulsion, electron microscopy and radioautography can be simply combined for examining tritium-labeled bacteria. The localization of label to areas of the order of less than 1/2 mu /sup 2/ thus enables the examination of high specific activity, tritium-labeled biologicai preparations. (H.H.D.)« less