442 Development and Vascularization of the Testis 
three days should be allowed, while for large objects the time should not 
be less than a week. Experiments with absolute alcohol in place of 
ninety-five per cent alcohol gave no better results, and its use is an 
unnecessary expense. The coagulation of the proteid occurs almost as 
quickly in one percentage as in the other. 
After the specimens have been sufficiently shriveled they should be 
placed in one per cent potassium hydroxide. When a higher percentage 
is resorted to, so rapid is the action that the safety of the specimen is 
endangered, and it was the use of the strong solutions recommended by 
Schultze which caused the loss of much valuable material. In this 
weaker solution the tissues become transparent in from four to forty- 
eight hours, depending upon the size of the specimens. After sufficient 
clearing in this medium they should be transferred to twenty per cent 
glycerine, in which clearing continues and a certain amount of harden- 
ing occurs, rendering the tissues firm enough to permit of dissection. 
Should the specimens be as transparent as is desired, they may be re- 
moved from time to time to higher percentages of glycerine till at last 
they are permanently stored in pure glycerine. <A certain amount of 
shrinkage is noted in some organs after an immersion in this fluid for 
a year or more, but when the specimens are studied immediately after 
being cleared the measurements are practically the same as in the fresh 
tissue. The shrinkage which some observers have noticed is probably 
due to transferring the specimens too rapidly to higher percentages of 
glycerine. After some experimenting we have found that embryos hard- 
ened in formalin can be cleared also, though in this case 10 per cent 
potassium hydroxide is essential and the specimens must remain in this 
solution for several weeks or months. 
Blood pigment in some instances will not be entirely removed by this 
process alone and in organs, such as the kidney, transparency can some- 
times only be obtained by a secondary treatment. After passing through 
the one per cent potassium hydroxide as outlined above and being placed 
in twenty per cent glycerine, the specimens containing the objectionable 
pigment are treated with equal parts of fifty per cent ammonium 
hydroxide and one per cent potassium hydroxide. In this solution there 
is comparatively little danger to the specimen on account of the harden- 
ing produced by the twenty per cent glycerine. Indeed, in cases where 
it is deemed advisable for any reason to stop the clearing action, or it 
is found to be more convenient to continue the process at some future 
time, the objects may be removed to this twenty per cent glycerine and 
retained in this medium until a more fitting time when much higher 
OO 
