148 FIXATION 



almost invariably included, though exceptions can be quoted.^*®' ^^^ 

 Fixatives for chromosomes usually also contain chromium trioxide 

 (or acidified potassium dichromate). 



Many of the mixtures used for the purposes mentioned in the 

 last paragraph contain, in addition to acetic acid, one or more 

 coagulant fixatives and either formaldehyde or osmium tetroxide. 

 This applies, for instance, to Allen's B.15, Bouin, Brasil, Flem- 

 ming, Susa, Hermann, Karpechenko-Navaschin, and Sanfelice. 

 This trio of ingredients (acetic acid + coagulant + non-coagulant 

 fixative of ground cytoplasm) is so frequent in widely-used mix- 

 tures that the reasons for its success must be sought. Acetic acid 

 does not in itself prevent great shrinkage in final preparations 

 (p. 136), but it does prevent it during fixation. In mixtures it 

 presumably antagonizes the shrinking effect of other fixatives 

 while the latter are stabilizing the proteins in the unshrunken 

 state; it also prevents excessive hardening. Ross ^^^ showed that 

 mixtures have much less tendency than primary fixatives have to 

 produce a badly-shrunk final preparation. The primary spermato- 

 cytes of the snail, fixed in 4% formaldehyde, retain in balsam only 

 34% of their original volume, and this appears to be the primary 

 fixative that results in the least final shrinkage of any (with the 

 doubtful exception of osmium tetroxide). With chromium tri- 

 oxide at 0-75% the figure is 29% of the original volume; with 5% 

 acetic acid, 28%. Yet when these three primary fixatives are mixed 

 to produce Sanfelice, the final volume in the mounted paraffin 

 section is 66% of the original. Sanfelice gives less final shrinkage 

 than any other mixture studied by Ross. All the mixtures he 

 studied (including Bouin, Helly, strong Flemming, mercuric/ 

 acetic, and Zenker) gave less final shrinkage than any of the pri- 

 mary fixatives (except perhaps osmium tetroxide). 



This only explains in part why fixatives containing the trio are 

 so successful. Why should not acetic acid and one other ingredient 

 suffice? It seems that tissues are not readily infiltrated with paraffin 

 unless they have been made spongy to some extent by the inclusion 

 of a coagulant fixative. If, however, a coagulant is used alone, the 

 cytoplasm and nuclear sap tend to be transformed into rather 

 coarse meshworks. If formaldehyde or osmium tetroxide be in- 

 cluded, a compromise is reached: the protoplasm is more smoothly 

 fixed, but paraffin can still enter. 



The requirements of paraffin embedding have in the past to a 

 large extent controlled our choice of fixatives. The introduction 



