40 



Journal of Applied Microscopy. 



to do. Before giving these methods in 

 detail, I wish to give a few general 

 directions which should be borne in mind 

 in hardening tissues by any one of the 

 methods to be mentioned. 



A. It is not a good plan to wash the 

 tissues in water before placing them in 

 the hardening fluid, certainly not to 

 place them in the water for the purpose 

 •of "soaking out the blood." 



B. Do not handle the tissues more than 

 is necessary. 



C. Do not harden large pieces if it is 

 desired to make sections of the tissues 

 hardened. 



It is always a good rule to have one 

 diameter of the piece of tissue to be 

 hardened not m.ore than one-fourth of 

 an inch in thickness. If it seems desir- 

 able to harden larger pieces, to show for 

 instance the relation of a growth to the 

 surrounding tissues, a number of par- 

 allel cuts, about one-quarter of an inch 

 apart and passing nearly through the 

 tissue, may be made. The parts may 

 then be separated when placed in the 

 hardening fluid without destroying their 

 relative position. Small pieces are to 

 be used because most of the hardening 

 fluids do not penetrate readily. They 

 harden well the outer portion of a piece 

 of tissue, without hardening, or at least 

 not for a long time, the middle portion. 



The three hardening reagents to be 

 mentioned are: 



1. Alcohol. 



2. Formalin. 



3. Zenker's fluid. 



1. Alcohol. — As a general hardening 

 fluid, there is perhaps none which is so 

 universally applicable as alcohol. Small 

 pieces, not to exceed one-eighth to one- 

 quarter of an inch in diameter are to be 

 placed at once into ninety-five per cent, 

 alcohol. The volume of alcohol used 

 ought to be about twenty times as great 

 as the tissue to be hardened. If less is 

 used it should be renewed at the end of 

 a few hours. It is also advisable to 

 place a small quantity of absorbent cot- 

 ton in the dish or bottle containing the 

 hardening fluid. The alcohol used for 

 hardening the tissues should be renewed 

 every day for the first three days, and 

 if the pieces are not large, they will be 

 well hardened in four or five days, and 

 may be prepared for further manipula- 

 tion. (Embedding to be described in the 

 next article of this series.) Or, if this 

 is not possible, they should be trans- 

 ferred to eighty per cent, alcohol, in 

 which they may be stored away for 

 future use. 



2. Forinalin. — Formalin is a forty per 

 cent, aqueous solution of formaldehyde 

 gas. As a fixing or hardening reagent 



it is best used in a four per cent, solu- 

 tion, which is prepared by mixing ten 

 parts of formalin with ninety parts of 

 water. This fixative, which has been 

 in general use only a few years, seems 

 destined to have a very wide applica- 

 tion. It penetrates tissues very readily 

 and preserves the elements quite 

 well. Pieces not more than one-quarter 

 of an inch in thickness are well hardened 

 in about twenty-four hours. This hard- 

 ening fluid should be kept in a well stop- 

 pered bottle, as it evaporates quite read- 

 ily; fifteen to twenty times the volume 

 of the tissue should be used. After 

 hardening, that is, at the end of twenty- 

 four hours, the tissues may be placed 

 in eighty per cent, alcohol in which they 

 remain until further needed. 



3. Zenker's Fluid. — One of the best 

 hardening fluids for general work is the 

 solution known as Zenker's fluid, it has 

 the following composition: 



Bichromate of Potassium 5 parts 



Sulphate of Sodium 2 parts 



Bichlorid of Mercury 10 parts 



Distilled "Water 200 parts 



Glacial Acetic Acid 10 parts 



It is prepared by pulverizing the first 

 three ingredients in a mortar and dis- 

 solving them in the water; the solution 

 takes place more readily if the water 

 has been heated. This part of the above 

 solution may be kept on hand without 

 deterioration. To it the proper propor- 

 tion of the glacial acetic acid is to be 

 added just before the solution is to be 

 used. The pieces of tissue to be hard- 

 ened should not be over one-quarter of 

 an inch thick; they are well hardened 

 in about twenty-four hours. The tissues 

 thus hardened are now to be washed 

 in flowing water for about twenty-four 

 hours. This is most easily accomplished 

 by placing the bottle containing the 

 tissues under the tap of the sink or 

 wash-basin, and allowing a small stream 

 of water to run into it. To prevent the 

 tissues from being washed out of the 

 bottle, I have found it convenient to tie 

 a piece of mosquito netting over the 

 mouth of the bottle. After this thorough 

 washing the tissues are preserved in 

 eighty per cent, alcohol. This hardening 

 fluid, like all others containing bichlor- 

 id of mercury, has the disadvantage of 

 often leaving a precipitate of mercury 

 in the tissues. This may usually be 

 removed by adding to the alcohol into 

 which the tissues are placed after the 

 washing, a small quantity of the tinc- 

 ture of iodine. This forms with the mer- 

 cury in the tissues which usually appears 

 in the form of needle-shaped crystals, 

 a soluble and colorless compound 

 which is indicated by the fact 

 that the iodine color disappears from 



