INTEGUMENT OF NECTURUS MACULOSUS 491 
sections of fixed and fresh material also proved helpful. The 
fresh material was examined in glycerin or normal salt solution. 
For the determination of the general distribution of the various 
elements, relatively large strips of skin were dissected off, fixed, 
cleared, and mounted. 
Little difficulty was encountered in the preparation of serial 
sections. Some trouble arose due to overhardening, but it did 
not become necessary to resort to the use of celloidin. The 
hardening was avoided by using small amounts of tissue and 
reducing to a minimum the length of treatment in the stronger 
alcohols, xylol, and paraffin. Pieces of integument, 5 to 8 mm. 
square, cleared of all underlying muscle, were placed in Kleinen- 
berg’s fluid from two to two and one-half hours and then washed 
in 70 per cent alcohol for twenty-four hours. After the excess 
fixing fluid had been removed, the tissue was dehydrated, being 
left in 90 per cent alcohol for twenty minutes and in absolute 
alcohol for twenty-five minutes. This seldom failed to give 
complete dehydration, and clearing in xylol was accomplished 
in about fifteen minutes. Good infiltration was then obtained 
by using successively soft and hard paraffin, leaving the tissue 
twenty minutes in each. 
A large number of stains were used, separately and in combina- 
tion. Ehrlich’s or Delafield’s haematoxylin, contrasted with 
eosin or some common plasma stain, was used for ordinary 
routine work. Other stains, such as Heidenhain’s iron haema- 
toxylin, Weigert’s resorcin-fuchsin for elastic tissue, haematoxylin 
counterstained with Van Gieson’s mixture, and Mallory’s con- 
nective-tissue stain, were found useful in the differentiation of 
special parts of the integument. Thionin was generally employed 
as a specific stain for the mucous secretion, but a slight over- 
staining in either Delafield’s or Ehrlich’s haematoxylin followed 
by ‘bluing’ in an ammoniacal solution, was usually sufficient to 
color the contents of the slime glands. In the study of the nerves 
of the glands and epidermis two methods were relied upon, 
namely, the silver nitrate-hydroquinone method of Cajal and 
Ranson’s pyridin-silver nitrate- eae acid method. The last 
named gave the better results. 
