Observations on intravitam staining of centrifuged marine eggs.

Abstract

That living protoplasm cannot be stained has long been known but is not as yet generally appreciated. Because of the numerous cellular inclusions (granules, globules and vacuoles of various kinds) which do take up the dye, a cell stained intravitam may appear stained as a whole, but upon separating the inclusions from the protoplasmic matrix the latter will be found free from the dye. The relatively large eggs of certain marine invertebrates furnish excellent objects for demonstrating this fact, since by centrifugation the formed elements may be separated from the protoplasmic ground substance. If Arbacia eggs are centrifuged, the cell contents separate into four well-defined zones; the lipoid globules are massed at one pole, the pigment granules at the opposite pole; adjoining the pigment zone is a layer of granules of varying sizes, and between this and the mass of lipoid globules, a band of optically empty, homogenous cytoplasm. The width of these four zones depends, to a certain extent, upon the length of time the eggs have been subjugated to centrifugal force. In the egg of the clam, Cumingia, only three zones are formed (the pigment and granular zones do not separate).

Freshly obtained unfertilized Arbacia eggs were centrifuged in small haematocrit tubes; the zoned eggs were then placed in a 1:40,000 solution of neutral red or brilliant cresyl-blue in sea-water. It was found that only the pigment and granular zones took up the dye; the clear cytoplasm and the lipoids remained unstained. Identical results were obtained when the eggs were first stained and then centrifuged. Examination with dark field illumination showed the zoning lvith particular clearness, and brought out the neutral red stained granules with great brilliance. Arbacia eggs stained am1 centrifuged could be fertilized, and many developed to the gastrula stage.