A Staining and Dehydrating Procedure for the handling of Microörganisms

Publisher Website
Google Scholar
Abstract
A new staining and dehydrating procedure for microorganisms is presented. It is based on the observation of Atwood and Orinstein (1949) that azure A or thionin, in the presence of SO2, stains chromosomes intensely and apparently selectively. The staining process is as follows: Cover-slip smears are hydrolyzed in 1 N HCl at 60°C. for the previously ascertained optimal hydrolysis time. They are then briefly rinsed in distilled water and transferred to either of the two following stains, (1) 0.25% thionin (10 ml.) to which thionyl chloride (1 drop), as a source of SO2, is added; (2) 0.25% azure A (10 ml.) to which thionyl chloride (2 drops), as a source of SO2, is added. Neither of these stains decolorizes, i.e., forms a leuco base, in the presence of SO2. Staining is allowed to proceed for a minimum of two hours. Following staining, the preparations are rinsed in distilled water, drained on filter paper, and immediately immersed in absolute alcohol which has been previously chilled to about -50°C. by packing in solid carbon dioxide. Preparations are left in this freezing alcohol for at least twelve hours, during which time dehydration proceeds. They are then passed through absolute alcohol at room temperature, through two transfers of xylene, and mounted in one of the mounting resins. The principle of this dehydrating procedure is presented by Blank et al. (p. 193 of this issue). Distortion is avoided by instantaneous freezing. Two new modifications of cytologic methods useful in the study of micro-organisms are presented. The first of these consists of the selection of new stains which are apparently specific for deoxyribose-nucleic acid. The second consists of a new dehydration technic which permits the making of permanent, cleared, undistorted preparations. Both developments permit the definition of nuclear phenomena in bacteria and related organisms which have heretofore escaped observation.