真菌のプロトプラストの再生過程におけるグラム反応に関する研究
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概要
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Protoplasts of Geotrichum candidum were cultivated in agar layers and changes in their morphology and Gram reactions during cultivation were examined. The procedures used were the thin-layer-agar plating method of Fukui et al. (1969) for the former and a simplified Gram stain for the latter. The protoplasts, Staphylococcusd aureus and Escherichia coli were embedded together and treated with 6% glutaraldehyde containing 0.75 M NaCl for 1-24 hr. Then, the material was treated successively with 0.2% crystal violet, Lugol solution, dehydrating solution and safranin for 60, 60, 60-90 and 20-30 sec, respectively. The extent of the Gram positive reaction in protoplasts was measured by comparison with that in the other representative bacteria embedded close to the protoplasts. Studies were also made on the Gram reactions of regenerating protoplasts with the following results. 1. Protoplasts were completely gram-negative before cultivation. 2. A gram-positive substance first appeared on the growing point of protoplasts, but the mother cell remained gram-negative. Thus, when the regenerating cell became septate, the daughter cell was gram-positive whereas the parent cell was still gramnegative. This agrees well with the finding (Fukui and Ota, 1973) that the osmoresistant glucan layer is not involved in the gram reaction. 3. The gram-positive substance was also seen in the center of the regenerating protoplasts. This fact suggests that both the outer layer and the physiological state of the bacteria may influence the Gram reaction. 4. The Gram reaction could not be observed by electron microscopy because the dye was lost in the embedded medium. This does not agree with the report of Tchan in 1963 of intracellular dye in cells of gram-positive bacteria under an electron microscope.
- 1976-01-31