132 FIXATION 



membrane is very clearly seen; the nuclear sap is finely granular; 

 the nucleolus is shrunken. 



Washing out. An insoluble precipitate (presumably of chromic 

 oxide, CraOg) tends to be formed in tissues if they are transferred 

 directly from potassium dichromate to aqueous ethanol. It has 

 already been mentioned that the salt is insoluble in absolute 

 ethanol. Potassium dichromate is therefore usually washed out in 

 running water. The experiments of Virchow ^^^ suggest that it 

 may be safe to transfer tissues directly from potassium dichromate 

 solution to 95% alcohol if light be excluded. Overton ^"^ advised 

 washing tissues in sulphurous acid after fixation in potassium 

 dichromate (see p. 109). 



Effect on dyeing. Seki *^^ claims that potassium dichromate 

 renders proteins and cytoplasm acidophil, but in fact cytoplasm 

 can be coloured quite strongly by certain basic dyes after the 

 action of this fixative. Chromatin is left strongly colourable by 

 basic dyes,^^ but it is not fixed in its original position within the 

 cell. Since the nuclear membrane is well fixed, the chromatin 

 cannot escape, but distributes itself almost at random within the 

 nucleus. This is the last fixative one would choose for studies of 

 chromosomes (unless acidified). 



Effects on the histological picture seen in paraffin sections. It was 

 on paraffin sections of root- tips of maize {Zea mays) that the Ameri- 

 can cytologist Zirkle first clearly demonstrated the effect of pH on 

 fixation by potassium dichromate. He showed that when the 

 hydronium-ion concentration was on the more acid side of a certain 

 range, the fixation-image was that of chromium trioxide; on the 

 less acid side the image was completely different. Simple solutions 

 of potassium dichromate fall on the less acid side. Zirkle put the 

 change-over range at pH 4-2 to 5-2, but Casselman, in a recent 

 careful study with mammalian tissues, put it at pH 3-4 to 3-8. 



By itself, potassium dichromate is a very poor fixative for paraf- 

 fin sections (grade V). Cellular aggregates shrink apart from one 

 another, leaving wide artificial spaces; cytoplasm is rather homo- 

 geneously fixed, but tends to shrink round the nuclei and some- 

 times even disintegrates partially, so that cells become separated 

 from one another; mitochondria are retained, though often some- 

 what rounded up ; red blood-corpuscles are swollen and irregular. 

 The shape of nuclei is fairly well retained ; the nuclear sap is homo- 

 geneously fixed without net-like coagulum, but may retract from 

 the membrane ; the nucleolus is shrunken and often surrounded by 



