80 PEELIMINAKY COLD STOEAGE STUDIES. 



It was found that the more rapid methods now much used, and 

 which serve admirably for the study of many ordinary pathological 

 and normal tissues, will not serve for the tracing of the changes sus- 

 tained by the muscle during periods of cold storage. 



The sections were cut as thin as possible — usually about 5 microns 

 in thickness. The longer the fowl remains in storage, apparently, the 

 more difficult it is to obtain thin sections of the tissue, there being a 

 tendency to crumble, which makes it necessary to cut thicker sections, 

 and even then they are exceedingly difficult to get. 



A number of stains have been tried for the differentiation of the 

 tissue elements. As a general routine stain Erlich's acid hsematox- 

 ylin, followed by eosin, is very satisfactory. Not only does this differ- 

 entiate the nuclei and cytoplasm, but it also stains bacteria. The 

 color of the latter is that of the nucleus. Hence, unless the organisms 

 are morphologically very distinct, or in such masses that their identifi- 

 cation is comparatively simple, such staining will not serve as a 

 method of differentiation, the long, slender nuclei of the muscle and 

 of the connective tissue being readily confounded with bacilli. 



After many trials of the ordinary routine methods of staining, most 

 of which were distinctly unsatisfactory in various ways, Borrel's stain 

 as modified by Coca a was finally adopted. This process as used con- 

 sists of the following steps: 



(1) Fixation in Orth's fluid. 



(2) Paraffin sections. 



(3) Removal of the paraffin by xylol, and of the xylol by 95 per cent alcohol. 



(4) A concentrated aqueous solution of magenta red (Griiblers) for 30 minutes. 



(5) Rinsing in water. 



(6) Ten to fifteen minutes in indigo carmine (Griiblers) 0.75 gram, saturated aqueous 

 picric acid 100 cc, and distilled water 100 cc. 



(7) Differentiation in 95 per cent alcohol until the excess of the magenta red is 

 removed, which may require 10 minutes and is indicated by the failure of the alcohol 

 to color pink. Occasionally this treatment does not give a sufficient brilliancy to the 

 nuclei, which should be a clear carmine red. When such is the case the slides are 

 flooded for about 1 minute with magenta red, which is then quickly washed off with 

 95 per cent alcohol, just enough absolute alcohol following this for complete dehydra- 

 tion. 



(8) Clearing in carbolic acid and xylol, as quickly as possible. If the dehydration 

 with the absolute alcohol has been complete two floodings of the slide ought to suffice 

 for the clearing. 



(9) Mounting in xylol balsam. 



This process can be carried through without any difficulty on the 

 slide, and a number of sections can be stained and differentiated at 

 the same time. 



The muscle fibers are a brilliant green; connective tissue fibers are 

 blue; the nuclei are clear carmine red, and bacteria are also red, but 

 have usually much more of the purplish tinge than do the nuclei. 



« An Improved Method of Staining Fibroglia, Myoglia, Myo-fibrillae of Striped 

 Muscle, etc., U. of P. Medical Bulletin, June, 1907, p. 60. 



