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

Localization of tritium label in bacteria has been successfully demonstrated both at the intercellular level (1) and at the intracellular level (2). However the use of Kodak AR-10 (3) stripping emulsion gives rise to a complication in those experiments. The 2 # maximum range (4) of the/3-rays from tritium in this emulsion, plus the greater than 2 # thickness of the stripping emulsion itself, combine to allow a single point source to give photographic grains frequently located as far apart as 2.5 #. Since this distance corresponds to the length of a large E. coli cell, one cannot associate any grain pattern above a bacterium, or section of a bacterium, with any one portion of the specimen. To improve on the physical localization of label in whole cells, one might select a tracking emulsion, i.e. an emulsion giving photographic grains closely spaced along the paths of the particles, in this case the weak/3-rays from tritium atoms. One would then be able to trace each track to its origin (5). Alternatively, one might confine the photographic grains to a region near the point of origin of the emanating radiations by using thin films (9, 10). It is the latter approach which is utilized in this paper. From considerations of solid angle a fine grain emulsion (6) of 0.2 # thickness should give localization to well within 1 #. I t was found that Kodak V-1055, a non-tracking, x-ray gel emulsion similar to Kodak AR-10, can be easily converted to a thin film. With films of this emulsion, improved localization can be realized even for observations using the light microscope. Additional improvement can be achieved by viewing the radioautograph in an electron microscope. The improved optical resolution and the increased depth of field of the electron microscope (7) not only give better visualization of the structure under examination but facilitate the identification of the individual, small ( <0.2 /~) and often adjacent photographic grains. The appearance of such photographic grains in the electron microscope has already been described by Hall and Sehoen (8). The first work combining electron microscopy and radioautography is due to Liquier-Milward (5). By using a tracking emulsion he was able to demonstrate the in vitro incorporation of Co 6° into mammalian nuclei. Subsequently George and Vogt (10), using thin fihn tracking emulsion, were able to identify Pu 239 labeled dust particles. Similarly, O'Brien and George (9) by examining thin sections of individual yeast cells and superimposing thin photographic films localized adsorbed Po 182 on the cell walls. In the work presented here the localization of D N A labeled with thymidine-H a (11-13) was studied in E. coli three generations after the labeling had taken place (1). The method used was to examine in the electron microscope radioautographs of the cells using a thin film of Kodak V-1055 x-ray emulsion. In so doing it was possible to localize the labeled D N A in whole bacteria to regions <1 ~ in length and <1/~ #2 in area.