Filamentous Cells of Escherichia coli Formed in the Presence of Mitomycin

The effects of mitomycin C on cell elongation of Escherichia coli B were studied. Filament formation was most marked in cultures treated with a moderate level (1 mug/ml) of the antibiotic, becoming less obvious at higher levels (10 mug/ml). Cells treated with a bacteriostatic concentration (0.1 mug/ml or less) of mitomycin C were also significantly elongated. The filamentous or elongated cells appeared to lack septa, since their spheroplasts were considerably larger than those formed from normal cells. The appearance of empty spheres also indicated some defects in the surfaces of the filamentous cells. Electron micrographs of the filaments revealed a characteristic difference in the arrangement of the nuclei in the filaments formed in the presence of low (0.1 mug/ml) and high (5 mug/ml) concentrations of mitomycin C. The filaments formed by the low level of mitomycin C had normal well-defined nuclear bodies distributed along the long axis, whereas those formed by the elevated level of the antibiotic contained smaller nuclei. The latter were characteristically confined to the center of the cells and did not extend out to the tips of the filaments.     

METHOD FOR COMBINED ULTRASTRUCTURAL AND BIOCHEMICAL ANALYSIS OF NEURAL TISSUE

No significant change was found in the electrolytes and lipids of the brain analyzed after glutaraldehyde fixation by perfusion of laboratory animals; such fixation also satisfactorily preserves neural tissues for electron microscopy. The brains of normal and tumor-bearing C3H mice, Wistar rats, and New Zealand rabbits were studied. Little difference was found in the dry weight and the content of sodium, potassium, total lipid and lipid fractions, and in the sulfate space (S³⁵O₄) between specimens from unperfused and perfused animals, whether normal or tumor-bearing. The results suggest the possibility of using selected regions of the nervous system, dissected after fixation, for chemical study and at the same time characterizing similar regions morphologically with the electron microscope.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.