SIXTY-TWO SPECIES OF FISHES FROM LAKE ERIE 
297 
Method II: Soak formalin-preserved specimens in distilled water for at least 
1 hour, and in 35 per cent alcohol for from 1 to 2 hours. Place for half an hour in 
70 per cent alcohol made alkaline by adding a drop or two of the following alkaline 
alcohol solution: 70 cubic centimeters absolute alcohol, 30 cubic centimeters distilled 
water, 1 cubic centimeter molecular solution of sodium bicarbonate. Stain with alco- 
holic alizarine solution diluted with an equal quantity of 70 per cent alcohol, to 
which is added one or more drops of alkaline alcohol solution until the color is a 
faint brown. Staining may require from 1 hour to a day or longer, depending upon 
the size and permeability of the specimen. Place in 70 per cent alcohol until color 
is washed out of flesh and left only in bones. Run slowly up to absolute alcohol. 
Clear in oil of cloves, oil of wintergreen, or xylol. 
Method III (most effective for transparency of vertebral column) : In order to 
make the stained skeleton distinct the use of potassium hydroxide is highly successful. 
This transparency method was recommended by Beale as early as 1853, and by 
Schultze in 1897. The technique of Schultze has been applied widely since by 
students of human embryology, (e. g., von Halvar Lundvall, (1905); Eben C. Hill, 
(1906); Franklin P. Mall, (1906).) More recently, excellent results have been 
obtained in the study of deep-sea fishes by Dr. William Beebe and Miss Gloria 
Hollister. 
The following modification of the Schultze and Lundvall methods has been 
used by the author in the present problem: Treat formalin-preserved specimens 
with 2 per cent potassium hydroxide to which has been added a few drops of aliz- 
arine solution (about 1 to 1,000) until bones are stained. The time varies from a 
few hours to a week, but usually one-half to one day is sufficient. Place in 1 per cent 
potassium hydroxide until color washes out of soft tissues. Place in glycerine and 
1 per cent potassium hydroxide (1 to 5) for 4 to 48 hours, or until tissues are quite 
clear. Transfer to higher percentages of glycerin at intervals of one day until 
perfectly transparent. 
It is urgent that distilled water be used in all solutions, including the formalin 
for hardening, since slight impurities may interfere with complete clearing. 
Hill (1906) was successful in rendering difficult embryological material trans- 
parent by using equal parts of 1 per cent potassium hydroxide and 50 per cent ammo- 
nium hydroxide for 5 to 72 hours, then 20 per cent glycerin for 48 or more hours, 
and ascending percentages of glycerin at intervals of 2 or 3 days. 
For staining Hill advocated Doctor Bardeen’s alum-cochineal method. Speci- 
mens without previous fixing in formalin, are placed in 95 per cent alcohol until 
shriveled, then stained for 24 hours in alumcochineal and cleared in 1 per cent potas- 
sium hydrate. 
ADDITIONAL METHODS OF STUDY 
The perfect way to identify a young fish with the adult is, of course, to secure 
a ripe female and male, artificially fertilize the eggs, and study the resultant develop- 
mental stages in the laboratory. The difficulty, however, of keeping most larval 
specimens alive and healthy in the ordinary laboratory for long after the yolk sacs 
are absorbed and the fish are actively feeding is great. Even when successful the 
artificially reared specimens are apt to be emaciated and their growth retarded. We 
can not duplicate exactly their normal conditions of life, and thus the chief source of 
young-fish material must be the lake itself. By extensive collecting over a period of 
