Electronmicroscopic Studies on Supravitally-Stained Granules Part 1. A New Method of Fixation for Dyes of Supravitally-Stained for Electronmicroscope and Observation of the Janus Green B Granules by this Methods

The auther successfully fixed stained granules of the supravitally stained bone marrow cells by the use of mercuric potassium iodide solution for quantative determination of NH(3). Insoluble complex of the dye can be produced by the reaction between dimethylamino rdaicals of the dye and mercuric potassium iodide. The fine structure of the protoplasma can be retained by using mercuric potassium iodide solution added an equal volume of 2% osmic acid solution (pH 7.4) for the fixation. Electronmicroscopic observation on the bone marrow cells stained supravitally with Janus green B and fixed by the solution just mentioned revealed that Janus green B granules appear as electron dense mass or rings having thick walls and it has been proved these granules are nothing but the mitochondria themselves, from the morphologic pictures of those less electron dense granules. The electron density of the granules should be given by mercury. As the reaction of the dye with mercuric potassium iodide should occur in both colored and non-colored leuco-Janus green as well, because the reaction has no relation with reduction or oxidation of the dye, then, the results, show that Janus green B stains mitochondria selectively, but on other elements of cytoplasma. Osmic acid is proven to have an ability to oxidize the leuco-Janus green B to a colored one in vitro, and the light microscopic observations of the cells treated with the fixatives just mentioned showed only the green mitochondria without any coloration of other cytoplasmic area. This again shows the Janus green enters into mitochondrira selectively. These observations refute the theory propored by Lazarow and Cooperstein in which they claim that the Janus green entors into the cytoplasma diffusely, but mitochondria only can be recognized as the stained granules by the oxydation of Janus green B. But, there are some of mitochondria remain without staining. Any other cytoplasmic elements are not stained by Janus green B as far as the cells the auther observed. As an exceptional case, in reticulocyte, Janus green stains endoplasmic reticulum but not the mitochondria, giving the picture of Substantia Granulo-Fiamentosa (SGF), themselves. This may mean the changes in chemical components of the organellae in reticulocytes as they are differentiated to synthesize haemoglobin.