ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 315 



3. Grades of alcohol from 35 p.c. slowly by the siphon capillary drop 

 method to avoid shrinkage. Xylol, to which paraffin is added as lit 

 dissolves. 4. Embed in rather hard paraffin of best quality. 5. Thin 

 sections, not over 8 ,u, preferably 4 to 6 //.. 



Vom Rath's method is not specific for the neurofibrils, which are 

 nevertheless deeply stained. Cell boundaries are shown with special 

 distinctness and shrinkage is slight. The process is advantageous in 

 demonstrating cell relations in the stages when nervous connexions of 

 tube and somite are effected. 



Flernming's stronger formula gives excellent fixation of Selachian 

 embryos, but does not allow the use of pyrogallic acid for subsequent 

 staining. Fixation seems quite as faithful as in Vom Rath preparations, 

 but cell boundaries are not so distinct as in the latter. Iron-hematoxylin 

 gives the best stain, subsequent to the use of Flernming's fluid, but it is 

 necessary to paint the sections with 0'5 p.c. celloidin in order to pre- 

 vent their loss in staining on the slide. 



For the specific purpose of demonstrating the neurofibrils Cajal's 

 method has given uniformly satisfactory results, which appear somewhat 

 less refined than those obtained by the Bielschowsky-Paton process. The 

 Cajal method is as follows : — 



1. Fix in absolute alcohol and 1 p.c. ammonia for forty-eight hours. 

 2. Wash for one-half to three minutes in distilled water. 3. Pyridin 

 for twenty-four hours. 4. Distilled water— many changes — for twenty- 

 four hours. 5. A 2 p.c. aqueous solution of silver nitrate for three 

 days at 35° C. in the dark. 6. Rinse in distilled water. 7. Four p.c. 

 pyrogallic acid in 5 p.c. formalin for one to two days. 8. Paraffin 

 sections. 



The Simarro-Cajal silver reduction method, following fixation in 

 70 p.c. pyridin, which has given such splendid results when applied to 

 Mammal and other Amniote embryos, has proved a complete failure in 

 the case of Squalus embryos. 



Excellent results in the differentiation of the neurofibrils have 

 followed the use of the molybdic-acid hematoxylin process as developed 

 by Held (1909). Tissues may be fixed by various methods, including 

 Zenker's fluid and Rabl's picro-sublimate. The stain is effected by a 

 solution of molybdic acid in a 1 p.c. solution of hematoxylin in 70 p.c. 

 alcohol. The stain is better after months or years. Immediately 

 before use, several drops of this tincture — depending on the strength 

 wanted — are dissolved in distilled water, and the sections are stained 

 warm on the slide at 50° C, or for a longer time cold. The sections 

 may be stained directly, or they may be mordanted in iron-alum. 



The neurofibrils are differentiated by the Bielschowsky-Paton process, 

 but, like the Cajal method, this does not demonstrate the fibrils within 

 the neuroblast ceil in the earliest stages of histogenesis. By this method 

 the neurofibrils are stained a dark brown or black, while other tissues 

 are light brown or yellowish brown. In the process only tested distilled 

 water, and absolutely clean glass-ware and glass or bone spatulas — no 

 metal — should be used. 



1. Fix and keep embryos in 10 p.c. formalin, neutralized or made 

 slightly alkaline with magnesium carbonate. 2. Wash in running tap- 



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