192 The Fibrils of the Heart Muscle Cell of the Chick 
METHODS. 
A detailed study of the cytoplasmic structures of the heart muscle cell 
requires the use of a very high magnification. To secure good defini- 
tion, it was found necessary to make sections 3 p» in thickness. Sections 
of 4 to 10 » thickness were used for general structure and form of the 
cell. Sections were cut in paraffin. 
Of the different fixing agents used, none gave more satisfactory prepa- 
rations than Kolossow’s, 92, solution. None was found to surpass it 
in faithful preservation and differentiation of the cytoplasmic structures. 
The tissue is killed and hardened by treatment for 15 minutes with a 
1 per cent osmic acid solution. This is followed by immersion for the 
same length of time in a reducing mixture of pyrogallol and tannin. 
The tissue is next washed in a 0.25 per cent solution of osmic acid, and 
then in water. After being passed through graded alcohols, the tissue 
is cleared in xylol, and embedded in paraffin. This method followed by 
Delafield’s hematoxylin or methylene blue, counterstained by 0.5 per 
cent solution of acid fuchsin in 70 per cent solution of alcohol gave 
excellent results. The chromatin structures of the nucleus and the cyto- 
reticulum appear almost black, while the undifferentiated cytoplasm is 
red. Acid fuchsin alone without the nuclear stains also yields good 
preparations. 
One objection to Kolossow’s solution is that it does not penetrate 
rapidly enough, and as a result, especially in adult tissue, the structure 
of the cells in the interior could not be made out as well as that of those 
in the peripheral portions of sections. 
Another killing fluid which gave good results was Gilson’s mercuro- 
nitric fixing mixture, followed by iron hematoxylin for staining. This 
method produces more uniformly penetrated preparations, but does not 
show the cytoplasmic reticulum to such good advantage as the osmic 
acid method. 
Hermann’s platino-aceto-osmic mixture was also tried. After treat- 
ment with this solution the tissue is stained with alcoholic safranin for 
24-28 hours. The preparation is then treated with gentian violet ac- 
cording to Gram’s method. This method was not as satisfactory as 
either of the foregoing. 
Picrosulphuric-acetic acid was used with good results where general 
structure was the object sought. This fluid is made by adding a 5 per 
cent solution of acetic acid to Kleinenberg’s picrosulphuric acid solution. 
Treatment with the above is followed by staining successively with 
carmalum and Delafield’s hematoxylin. 
