HISTOLOGICAL EXAMINATION 189 



subsequently transferring to solutions of increasing strength not only takes 

 more time but yields only moderate results. 



To fix in alcohol, place the tissue (about 1 cm. cube) in 25-30 c.c. of absolute 

 alcohol : renew the alcohol after 3 hours and again after 24 hours. The 

 tissue is then fixed, but it is found to stain better if left in the alcohol for 

 3 days. Tissues should not under any circumstances remain in absolute 

 alcohol for more than a week at the outside, and if it is not convenient to 

 use them then they should be transferred to 90 per cent, alcohol. 



The tissue should be suspended in the fluid or laid on a piece of wool at the 

 bottom of the bottle to ensure its hardening uniformly. 



2. Formalin. Formalin is an excellent hardening agent ; it does not 

 interfere with any of the staining methods and is particularly valuable for 

 tissues which are to be cut by the freezing process. The best solution is 

 the following : 



Commercial formalin, - 10 c.c. 



Distilled water, - 90 



The tissue is fixed in about 6-8 hours ; but better preparations are obtained 

 if the formalin is allowed to act for 24-48 hours. 



If frozen sections are to be cut the tissue is used straight out of the 

 formalin : but before embedding in paraffin, transfer the tissue first to 90 per 

 cent, alcohol, then to absolute alcohol, leaving it for 24 hours in each solution. 



3. Corrosive sublimate. Corrosive sublimate is another most useful harden- 

 ing agent, and can be used whatever method of staining is to be subsequently 

 employed. It can be used as a cold saturated solution 1 but it penetrates 

 and fixes the tissue better when acidified with acetic acid. 



Acid sublimate (Mayer}. 



Saturated aqueous solution of corrosive sublimate, - - 100 parts. 

 Glacial acetic acid, - - - 1-3 



Allow 20-30 c.c. for each piece of tissue. The solution penetrates well and 

 rapidly, so that the pieces may be relatively large (cubes of 2 cm.). Leave 

 in the acid solution for at the most 12 hours. The tissue is then white and 

 opaque. Wash in running water for an hour (this is not absolutely essential). 

 Transfer to 100 c.c. of 70 per cent, alcohol containing xv-xx drops of tinc- 

 ture of iodine and leave for 24 hours to remove the excess of perchloride and 

 prevent the deposition of crystals in the tissue. Transfer to 80 per cent, 

 alcohol for 24 hours and finally to 90 per cent, alcohol for a similar period. 



It must be remembered of course that perchloride of mercury acts on metal 

 instruments, so that in removing tissues from perchloride solutions horn, glass or 

 wood spatulas must be used. 



4. Flemming's solution. For purposes of bacteriological examination the 

 diluted solution is better than the concentrated. 



(a) Diluted solution. 



1 per cent, aqueous solution of chromic acid, 25 volumes. 



1 per cent. r , osmic acid, - , - 10 



1 per cent. ,, acetic acid, - 10 



Distilled water, - 55 



(b) Strong solution. 



1 per cent, aqueous solution of chromic acid, - - 15 volumes. 



2 per cent. ,, osmic acid, 4 

 Glacial acetic acid, 1 volume. 



1 Cold water dissolves about 6 '6 per cent, of perchloride of mercury. A saturated 

 solution is easily prepared by dissolving 70 to 75 grams of perchloride in 1 litre of distilled 

 water in the warm : filter while warm, and, as the solution cools, white needles crys- 

 tallize out at the bottom of the vessel ; pour off the supernatant liquid which is then 

 ready for use. 



