TECHNIC. 443 



form and alcohol into water, where they are allowed to remain for at least 

 six hours ; or they may be washed in water, placed for one minute in i % 

 formic acid, again washed in water, immersed for twenty-four hours in a 

 i <f solution of gold chlorid, rinsed in water, and finally placed in a i / 

 formic acid solution and exposed to the light. For the latter purpose 

 glass tubes are employed in which the slides are placed obliquely, with the 

 sections downward. A uniform illumination of the section with "as 

 intense a light and low a temperature " as possible are conditions indis- 

 pensable to the attainment of successful results. The sections are then 

 again washed in water and mounted in glycerin or syrup of acacia, or in 

 Canada balsam after being dehydrated. Thin membranes are stretched 

 upon small frames of linden wood especially prepared for this purpose. 

 Their further treatment is then the same as that of sections fixed to the slide. 

 If successful, the nerve-fibrils appear dark violet to black. The large 

 ganglia in the spinal cord of lophius, the calf, etc., are especially recom- 

 mended as appropriate vertebrate material. 



Bethe (1900) has recommended the following method for staining 

 neurofibrils and Golgi-nets in the central nervous system of vertebrates: 



The perfectly fresh tissue is cut in thin lamellae, varying in thickness 

 from 4 to 10 mm. These are placed on pieces of filter-paper and 

 then in 3 to 7-5% nitric acid, in which they remain twenty-four hours. 

 From the hardening fluid the pieces of tissue are transferred into 96% 

 alcohol, where they remain for from twelve to twenty-four hours. They 

 are then placed in a solution of ammonium -alcohol, ammonium (sp. gr. 

 0.95 to 0.96), i part; distilled water, 3 parts ; 96% alcohol, 8 parts, 

 in which they remain for from twelve to twenty-four hours. The temper- 

 ature of this solution should not exceed 20 C. The tissues are then 

 placed for from six to twelve hours in 96% alcohol, and next in a hydro- 

 chloric acid-alcohol solution, concentrated hydrochloric acid (sp. gr. 

 1.18 37%), i part; distilled water, 3 parts; and 96% alcohol, 8 to 12 

 parts, in which they remain for several hours. The temperature of this 

 solution should not exceed 20 C. The tissues are then again placed in 

 96% alcohol for frofri ten to twenty-four hours, and next in distilled water 

 for from two to six hours. The tissues are now placed for twenty-four 

 hours in a 4% aqueous solution of ammonium molybdate. (This solution 

 should be kept at a temperature varying from 10 to 15 C., if it is de- 

 sired to stain the neurofibrils ; or at a temperature varying from 10 to 

 30 C., if it is desired to bring out the Golgi-nets.) After the ammo- 

 nium molybdate treatment, the tissues are rinsed in distilled water, placed 

 in 96% alcohol for from ten to twenty- four hours, then in absolute alco- 

 hol for a like period, cleared in xylol or toluol, and imbedded in par- 

 affin. Sections having a thickness of 10 // are now cut and fixed to slides 

 with Mayer's albumin-glycerin. Since the various solutions used in the 

 fixation and further treatment of the tissues do not act with the same in- 

 tensity on all parts of the piece of tissue to be studied, and since the differ- 

 entiation and staining depend on a relative proportion (not yet fully de- 

 termined) of the mordant (ammonium molybdate) and the stain in a 

 given section, it is advised by Bethe to cut large numbers of sections and 

 fix to each slide about three sections from different parts of the series. 

 After fixation of the sections to the slide the paraffin is removed with 

 xylol ; and they are then carried through absolute alcohol into distilled 

 water, in which, however, the sections remain only long enough to re- 



