73 



5. Imbed in celloidin or paraffin. 



6. Stain and differentiate sections by the Weigert copper hema- 

 toxylin method ( 95). 



7. Mount in neutral or alkaline balsam. 



Large sections are usually best carried on as free sections. The 

 differentiation of the stain should be carefully watched and stopped 

 when the fibers are a rich dark blue on a yellow-brown-background. 

 The reagents used in dehydrating, clearing and mounting should 

 be neutral or alkaline, not acid. 



200. Pal's method may be used if it is desired to stain the 

 nerve cells subsequently. 



Fix and mordant, in the dichromate as above; omit the copper 

 mordantage; imbed and section, staining the sections with the strong 

 hematoxylin used in the Weigert method ( 199) until the sections are 

 a blue-black. 



Rinse the sections in tap water and differentiate by treating for 

 a short time (20 to 30 seconds) with a 1 /10% aqueous solution of 

 potassium permanganate and for a few seconds with a mixture of 

 1% oxalic acid and 1% potassium sulphite, equal parts. The action 

 will be very rapid and must be carefully watched. Wash the sec- 

 tions % hour in running water. * Counter-stain with a red stain 

 (eosin, erythrosin, carmine, etc.) if desired. 



201. Marchi Method. This method of staining differen- 

 tially degenerating myelinic nerve fibers depends upon the fact that 

 potassium dichromate (or chromic acid) is able to satisfy the re- 

 ducing power of myelin but does not oxidize the globules of free 

 fatty acid (?) formed in the degeneration of the myelinic sheath 

 of the fiber, which may be subsequently blackened by the reduction 

 of osmium tetroxid. Important points in the successful application 

 of the method are: (a) the length of time the degeneration should 

 be allowed to proceed before treating with potassium dichromate, 

 (b) the time in the dichromate mordant, (c) the time in the osmic 

 acid mixture (sufficient and complete penetration), (d) the preser- 

 vation in situ and final mounting of the osmicated fat granules; the 

 difficulties here are those of fat preservation in general ( 224). 



(a) The optimum will vary and must often be determined 

 experimentally: in general, for cold blooded animals; (Toad), 30 

 to 40 days; for mammals, 12 days. 



(b) 8 to 10 days in Muller's fluid or 3% potassium dichromate 

 is usually enough; a longer time does no harm. 



