168 THE AMERICAN MONTHLY [September, 
teasing to remove it from other parts. The whole of any desired part of the 
organ thus separated, several courses of treatment are presented for our 
choice. The specimen studied and figured for the present article was treated 
with the specimen last described, and hence received precisely similar hand- 
ling. Whether it was best or not could only be determined by treating other 
portions of the same organ by other methods for comparison. This, if faith- 
fully pursued, would furnish valuable results, but the topic will be discussed 
and we may leave it now. Since I have already described one method (see 
page 83), that of hardening with corrosive sublimate, I will here detail a 
way which might have been followed in the preparation of the sections with 
perhaps better results than the one pursued. I mean the picric acid method. 
The picric acid series of reagents for hardening animal tissues contains 
four members which are in very commonuse. These are :—(1) Picric acid, 
or Kleinenberg’s picric acid; (2) picric and nitric acid; (3) picric and 
sulphuric acid; (4) picric and hydrochloric acid. The first of these is 
simply a saturated aqueous solution of picric acid. The second, third, and 
fourth are made by adding 2 parts of strong acid to 98 parts of the Kleinen- 
berg’s solution, filtering to remove the heavy precipitate and reserving the 
clear filtrate for use. In use a dilute solution of the picro-nitric acid, etc., is 
desirable—one part to two of distilled water is commonly employed. 
No unnecessary time should elapse after the killing of the animal before 
the tissue to be hardened is immersed in the hardening reagent, for death 
changes in the cell, and subsequent alterations, if operative for any considera- 
ble time before the structure is fixed by the hardening reagent, may produce 
false appearances in the sections. Cells to be well preserved should be in 
contact with the preservative reagent before they are themselves dead, to be 
killed by it. The amount of the fluid to be used varies with’ the size of the 
specimen. <A good rule is to use about fifteen times as great an amount as 
the size of the specimen. Pieces should remain in the picric fluids from 
three to six hours according to their size. They should be transferred to 
30% alcohol, to remain ten minutes, thence to 50” alcohol, to remain one-half 
hour, and thence to 70% alcohol, which should be removed every twenty- 
four hours until no yellow tint is imparted to the alcohol. This, for small 
pieces, will require about three or four changes. 
If it is desired to harden a tissue which is contained in a calcareous envel- 
ope and cannot be removed from that envelope, the picric acid combined 
with nitric or, particularly, hydrochloric, presents especial advantage, for 
the hardening takes place simultaneously with the decalcification of the soft 
parts. This will appear when treating of the eye of the cray-fish. 
The ‘liver’ will be hardened sufficiently, and cleared of the picric coloration 
after four days, when the other steps preparatory to sectioning may be taken. 
Kleinenberg’s hematoxylin or borax carmine are most commonly employed 
as staining fluids where the piece is to be stained before sectioning. Borax 
carmine is to be preferred because of the readiness with which any over-stain 
may be removed. It is described, with notes on its use, on page 83. Hama- 
toxylin, prepared after Kleinenberg’s manner, is made as follows :— 
Add crystallized calcium chloride in excess to 70% alcohol, draw off the 
saturated solution, add alum in excess, and let stand for one day; filter. To 
one volume of this filtrate add six-eight volumes of 70% alcohol. To this 
mixture add, drop by drop, a saturated solution of hematoxylin in absolute 
alcohol till a moderately dark purple results. The solution improves on 
standing. Tissues may be stained in piece in this fluid; for this purpose the 
fluid is diluted (with 70% alcohol) and the tissue remains in the fluid one or 
two days. Only a small piece of the fluid should be taken to secure even 
staining throughout the piece; any marks of the stain may be removed by 
