Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.

Exponentially growing cultures of E. coli were examined by quantitative radioautographic techniques to determine the distribution of labeled DNA, RNA, protein, and cell wall among the progeny cells of successive generations. It was found that DNA is in large structures, non-randomly distributed in the progeny. About one-half of the cells have four such structures and approximately one-half contain these four structures plus four smaller ones. These structures show remarkable stability. Fewer than 3.5 per cent of the large structures break in one division time. Protein, RNA, and cell wall are all distributed randomly among progeny cells. The number of units of each component that show random segregation must be 200 or more.

The purpose of this study was to localize deoxyribonucleic acid in a bacterial cell. Tritiumlabelled thymidine was employed to identify the D N A and radioautography was chosen as the method of detection. To insure a high percentage of labelling, a thymine-requiring strain of Escherichia coli, the strain 15 T -, was used (1). When whole cells are studied the localization of the label is limited by the resolution of the autograph procedure which, even for tritium, is about 1.5 micron, the same order of magnitude as the cell itself. This difficulty was overcome by studying the distribution of label among thin cross-sections of the cells. The thinness of the sections is then the determining factor in establishing the precise location of isotopic material in the cell. Materials and MethodsElectron Microscopy.--Cultures of E. cdi 15 Twere grown exponentially for 6 to 7 generations at 23°C. in M-9 synthetic medium (2) containing 4 micrograms of thymidine per ml. The cells were fixed for 15 minutes at room temperature in 1 per cent OsO4 buffered at pH 7.0 with acetate-veronal buffer and containing 0.6 ~ sucrose, followed by 4 hours in 10 per cent formaldehyde in the same buffer (3). They were then dehydrated and embedded according to standard techniques (4). Sections were cut on a PorterBlum mierotome and floated on a mixture of 20 per cent acetone and 1 per cent lanthanum nitrate in water (9). Lanthanum nitrate acts as a stain for certain cellular components and increases the over-all contrast. The sections were examined in an RCA EMU-3B microscope.Radioautography.--Smail cultures (0.05 ml.) were grown in M-9 medium containing tritium-labelled thymidine from Schwarz Labs, Inc. Approximately 0.05 ml. of 2 per cent agar at 40°C. was added to the culture, mixed thoroughly, and allowed to gel for a few minutes. ~; The authors wish to thank Dr. Harold J. Morowitz and Dr. Ernest C. Pollard for valuable suggestions and advice. § Received for publication, April 17, 1958. This procedure insured a good dispersion of the cells for subsequent radioautography. Fixation and embedding were carried out as described for electron microscopy. Examination in the electron microscope showed that the addition of the agar did not introduce morphological variations. Sections were cut at 0.25 micron, mounted on a clean glass slide, dried, and dipped into a 1 per cent solution of collodion in amyl acetate. The sections were then covered with a strip of Kodak autoradiographic emulsion and exposed for varying lengths of time (5). After photographic processing they were mounted in 50 per cent glycerin and covered with a thin cover slip. Without the collodion treatment the film became detached from the section during the final stages of processing. The amount of label present in a given cross-section of a cell was estimated by counting the number of exposed photographic grains associated with it, correcting for tracks whenever it seemed necessary. Phase contrast was employed in locating sections of ceils; bright field, for counting grains.

No abstract

No abstract