FIXATION 2()7 



of the actions of the reagents noted above incUcates a reason for the 

 protoplasmic conditions observed in fixed tissues. We will now 

 indicate a few fixing fiiiids found useful in general work. 



Fixing Fluids.— Bouin's Fluid. — The fornuila is as follows: 75 

 parts saturated aqueous solution of picric acid; 25 parts formalin 

 (40 per cent formaldehyde); 5 parts glacial acetic acid. This is one 

 of the best general fixatives, and in it the effects of the three chemi- 

 cals are well balanced to give a fair preservation of cellular structure. 

 Chromosomes are especially well fixed. Glycogen, fat, chondrio- 

 somes, and the Golgi apparatus are not usually preser\ed. Tissues 

 may be left for long periods in it without harm, but twelve to 

 twenty-four hours is ordinarily an adequate time. There is little 

 or no hardening or shrinkage, and tissues will later stain well. 

 After fixation the tissue may be washed in 50 or 70 per cent alcohol 

 to get rid of the free fixati\e remaining. 



Flemmimfs Fluid.— The formula is 1 per cent aqueous solution 

 of chromic acid, 15 parts; 2 per cent aqueous solution of osmium 

 tetroxide, 4 parts; glacial acetic acid, 1 cc, or even less, to be added 

 just before using the fixati\'e. This solution is useful for fixing 

 chromosomes, chondriosomes, and fat. The amoimt of acetic acid 

 should be reduced to a few drops for better results with chondrio- 

 somes. The osmic acid fixes the fats and chondriosomes. The 

 acetic acid fixes the chromosomes and aids in preventing shrinkage. 

 The chromic acid fixes proteins and chromosomes. However, 

 fixation may be une\'en, part of the tissue being overfixed, part 

 properly fixed, and the inner portion of the tissue being underfixed. 

 Fixation should extend over a twenty-foin--hour period when acetic 

 acid is present to the extent of 1 part, but the period should be ex- 

 tended to four flays when the acetic acid is reduced and when it is 

 desired to retain chondriosomes. After fixation the tissues should 

 be washed for twelve hours in running water to remove all the free 

 fixative present before going ahead with the technique. 



Zenker s Fluid.— The formula is 2 grams potassium dichromate; 

 1 gram sodium sulphate; 5 grams mercuric chloride; 100 cc. water; 

 5 cc. glacial acetic acid. The acetic acid should be added just 

 before using the fluid. Tissue should be fixed for twelve hours. 

 Long fixation causes formation of crystals in the tissue. After 

 fixation the tissues should be washed in rimning water for several 

 hours, then transferred into 70 per cent alcohol to which iodine 

 has been added. The latter becomes clear, due to the extraction of 

 mercury salts from the tissue. Fresh iodized alcohol should be 



