11 



is generally supplemented by the subsequent use of alcohols of increasing strengths 

 (50% to absolute, 99%), as well as in preparation for the paraffin and celloidin 

 methods of imbedding. In fact, with modern methods 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 com- 

 pleted may be stored in 82 or 95% alcohol, or (better) imbedded at once ( 47). 



Alcohols. 50%, 67%, and 82% alcohols form a series of increasing strengths 

 sufficient for most purposes. They may be prepared from 95% alcohol by taking 

 (a) for 50% alcohol; 95% alcohol 1 part, water 1 part; (b) for 67% alcohol; 

 95% alcohol 2 parts, water 1 part; (c) for 82% alcohol, 95% alcohol 5 parts, 

 water 1 part. Dilutions of other strengths may easily be prepared as desired 

 from 95% alcohol. 95% (94%) alcohol and absolute alcohol are necessary in 

 imbedding by the paraffin and celloidin methods ( 49-). 



10. Stock Solutions. It is advantageous to have on hand strong solutions 

 of the chemicals employed as fixers and stains. Where feasible, 10% solutions 

 are most c6nvenient. The following are the more important: In aqueous 

 solution; 10% potassium dichromate, 10% copper dichromate, 10% chromic 

 acid, 10% platinic chlorid, 40% formaldehyde (formalin), 4% sodium sulphate, 

 4% copper sulphate, 2% osmic acid, saturated solution of mercuric chlorid, 

 saturated solution of picric acid, 95% alcohol, absolute alcohol, etc., as well as 

 the strong acids, stock staining solutions, etc. 



FIXERS 

 i 



11. Mercuric chlorid. One may employ (a) a saturated solution in 

 water or (b) a saturated solution in normal salt solution, with 1 to 5% glacial 

 acetic acid. Water will dissolve about 5%, normal salt solution about 12% 

 of the mercuric chlorid. This is a good fixer, especially when the piece is small. 

 It fixes as soon as it penetrates and is apt to make tissue brittle if it is left too 

 long. Staining after it is brilliant. The larger percentage of acetic acid is, 

 perhaps, to be preferred for most histological objects. 



Fix the fresh tissue l /2 to 24 hours according to the size of the piece. Remove 

 to 67% alcohol for 1 or 2 days, 82% alcohol several days, changing often. The 

 82% alcohol should contain enough tincture of iodin to give it a yellow color, 

 and fresh tincture added or the alcohol changed when the yellow color of the iodin 

 in the alcohol is lost. As long as the alcohol is decolorized, washing should be 

 continued, since it is important that the mercuric chlorid be all removed from the 

 tissue; otherwise precipitates will form in the preparation after it is mounted 

 or before, and spoil the result. Wash out in alcohol thoroughly and carefully. 

 Almost any stain may be employed after a mercuric chlorid fixation. 



12. Zenker's fluid. Formula: Potassium dichromate, 2.5 grms. ; sodium 

 sulphate, 1 gram; mercuric chlorid, 5 grms.; water, 100 c.c.; and add before 

 using, glacial acetic acid, 5 c. c. A stock solution, without the acetic acid should 

 be kept on hand. This is a well balanced fixer; the potassium dichromate seems 

 to check the brittleness that the mercuric chlorid would cause and improves the 

 fixation of the cytoplasm while the mercuric chlorid and acetic afford a good 

 nuclear fixation. It is distinctly better than mercuric chlorid; staining after it, 

 however, is apt to be a little more difficult and not as brilliant as with mercuric 



