FAT AND MITOCHONDRIA IN CARDIAC MUSCLE 5 



ance of fat may be noticed when the blocks of tissue have re- 

 mained in the fixative only a few hours, at other times it occurs 

 only after several weeks or months, if at all. 



As a fixative I now employ formalin which has been neutralized 

 and distilled according to the method given by Mann ('02) in his 

 Physiological Histology, p. 88. A twenty per cent solution 

 of formalin is prepared and is rendered isotonic by the addition 

 of 0.75 gm. of sodium chloride to each 100 cc. of the fluid. With 

 short fixation in this solution the quantity of fat usually does not 

 difi"er from that seen in fresh tissue. Blocks are fixed for thirty 

 minutes to five hours and are then cut on the freezing microtome. 

 Frozen sections of fresh unfixed tissue are employed as controls. 



Siainirig. In this study I have employed all the fat stains in 

 common use but principally Herxheimer's alkaline alcoholic solu- 

 tion of Scharlach R., which stain usually shows much more fat 

 than the simple alcoholic solutions of the same dye. The latter 

 solutions sometimes fail to stain a large part of the fatty drop- 

 lets seen in the fresh unstained tissue. This is not the case with 

 Herxheimer's solution. Herxheimer's stain is a saturated solu- 

 tion of Scharlach R. in seventy per cent alcohol to which sodium 

 hydroxide, 2 gm. per 100 cc. has been added. It is essential 

 that care be taken to avoid precipitates. Also the excess stain 

 must be thoroughly washed out of the sections before the nuclei 

 are stained with dilute hematoxylin. For the details of this 

 method the reader is referred to Herxheimer's papers ('01-'02), or 

 to my former papers ('12). 



DIFFERENTIATION OF NEUTRAL FAT 



Among recent contributions to our knowledge of the technique 

 and chemistry of fat demonstration may be mentioned those of 

 Herxheimer ('01), J. Lorraine Smith ('06-'07-'10), Smith, Mair 

 and Thorpe ('OS), Smith and Mair ('10), Fischler ('04), Faure- 

 Fremiet, Mayer and Schaeffer ('10), Eisenberg ('10), Klotz ('06), 

 Aschoff ('09), Kawamura ('11) and Hanes and Rosenbloom ('11). 

 These observers have introduced a number of valuable staining 

 methods but more important still they have established a large 



