THE FROG 73 
the sections must be stained deeply with the Orange G, and the 
slides passed very rapidly to the higher alcohols, otherwise this 
stain will wash out in the lower alcohols. Staining on the slide 
with Delafield’s hemotoxylin and Orange G is a time-consuming 
process in case a large number of slides are to be prepared, but 
the practical certainty of getting good results, and the permanency 
of the stains, make this method decidedly to be preferred for 
most tissues of the frog. (b) A more rapid method, so far as 
the actual work is concerned, is to stain the material in toto in 
Grenacher’s borax carmine for a day or two, and destain for 
several hours, to remove the stain from the cytoplasm, in acid 
alcohol (0.25 per cent. hydrochloric acid in 70 per cent. alcohol). 
Be careful not to destain too long; the process should be stopped 
as soon as the object changes from a dull red to a slightly pinkish 
hue. Counterstain on the slide with Lyons blue to which suffi- 
cient picric acid has been added to change it to green. Be careful 
not to counterstain too deeply, and wash in at least one change 
of xylol. The counterstain gives remarkably good differentia- 
tion of the cytoplasmic structures, but it will fade in time. In 
order to stain the nuclei with sufficient intensity in the borax 
carmine, be careful to get a good preparation of this stain, and 
if necessary stain for two days, keeping the material in a warm 
place. The object should have an acid reaction. Certain objects 
(e. g., the stomach, intestine and kidney) after fixation in Zenker’s 
are sometimes very resistent to this stain; in such cases Mayer’s 
paracarmine may be substituted for borax carmine and the object 
washed for an hour or two in I per cent. acetic acid. 
As a general rule the paraffin method of imbedding and cut- 
ting is to be preferred. Paraffin with a melting-point of about 
52° C. is best for general use. Thin sections must be cut at a 
low room temperature, thick sections at a high room temperature. 
The sections are best cut with a Minot rotary microtome. Xylol 
is usually to be preferred as a clearing reagent, and Canada balsam 
for the final mounts. 
The following preparations are needed: 1. Stratum corneum. 
Shed epidermis (stratum corneum) may be found in the water 
in which living frogs are kept; this is usually satisfactory for 
study when preserved promptly in formalin. The material should 
