90 



with others the hardening action is a minimum, e. g., aqueous solu- 

 tions of picric acid. The hardening action of the fixer is generally 

 supplemented by the subsequent use of alcohols of increasing 

 strengths (50% to absolute), as well as in preparation for the paraffin 

 and collodion methods of imbedding. In fact, with modern meth- 

 ods of imbedding excessive hardening of the tissue is not necessary 

 and indeed often should be avoided as affecting the cutting quality 

 of the tissue. Tissue after fixation has been completed may be 

 stored in 82% or 95% alcohol, or (better) imbedded at once. 



FIXERS. 



J 22. Osmic acid. A very useful as well as expensive reagent and some- 

 what difficult to use. It is generally employed as a i&er in conjunction with 

 other reagents, as in the mixtures below {?§ 23 and 24). When used alone as 

 a fixer weak solutions are generally best — ris~i%. It penetrates slowly 

 and it "over-fixes" cells very easily, obscuring detail and giving the parts a 

 homogeneous, glassy appearance. Over-fixed cells cannot be stained, or with 

 great difficulty. More or less blackening of the protoplasm also occurs. It 

 will be used in this course chiefly to demonstrate fat, which is blackened by 

 it, and the zymogen granules of pepsin and trypsin, which it preserves and 

 browns slightly. 



Fix small (about }4 c c.) pieces of tissue in 1% osmic acid for 6-12 hours, 

 wash well in water (running or changed frequently) for 12-24 hours, and place 

 in 67% and &2% alcohols. It is somewhat difficult to prevent pure osmic acid 

 of this strength from over-fixing the tissue, and cell detail is generally lost, 

 though the form of cells is well preserved. 



I 23. Hermann's fluid. Formula : 1% aq. sol. platinic chlorid, 15 parts ; 

 2% aq. sol. osmic acid, 4 parts ; glacial acetic acid, i part ; or you may take 

 10% aq. sol. platinic chlorid, 3 parts ; 1% aq. sol. osmic acid, 16 parts ; glacial 

 acetic acid, 2 parts ; water, 19 parts. This is generally recognized as the fine.it 

 fixer known, and it is also the most expensive. The form and structure of 

 cells are well preserved. It should only be employed, however, with very small 

 pieces of tissue, and is to be used especially when cell structure is to be 

 studied. Fat and the myelin of nerve fibers are stained black. 



Fix in this 1-24 hours (or longer — days or weeks are used by some), wash 

 well in water (running or frequently changed) 2-24 hours, and then place in 

 67% and 82% alcohols, 12-24 hours in each. In using this fluid, the smaller 

 the pieces taken the better the fixation will be, and in order that it be possible 

 to obtain a good stain afterwards tissue should not be over-fixed and the fixer 

 should be thoroughly washed out. If there is a blackening of the tissue, or a 

 precipitate in it, both may be removed by treatment of the sections on the 

 slide with a 10-20% solution of hydrogen dioxid in 67% alcohol. Employ 



