NEUROFIBRILS 475 



3. Pass through equal parts of acetone and xylol into pure 

 xylol till clear. Imbed in paraffin. Cut at 5 ju. ; fix to slides. 



4. After dissolving out the paraftin with xylol, put the slides 

 in absolute alcohol for twenty-four hours. 



5. One per cent, acetic anhydride in absolute alcohol twenty- 

 four hours. 



6. Wash in distilled water and stain in 1 in 3000 toluidin blue 

 for one hour at 55° to 60° C. 



7. Wash in water and treat with 4 per cent, ammonium 

 molybdate, as in Bethe's method. 



8. Wash again, dehydrate in alcohol, clear in xylol, and mount 

 in Canada balsam. 



1010. DoNAGGio's Methods {Riv. Sper. Freniatr., xxx, 1904, 

 p. 397, and xxxii, 1906, p. 394). There are five methods of 

 Donaggio. By the first two, pieces are stained in bulk before 

 imbedding, but results are not so good as by the other three, 

 the most important of which is Method III. 



Method I. Fix in saturated solution of mercuric chloride ; 

 wash this out first with water to which a little tincture of iodine 

 has been added, and then in distilled water for two to three 

 hours. Place in pyridin for forty-eight hours, changing once, 

 and, without washing, stain for forty-eight hours in 1 in 10,000 

 or 1 in 15,000 solution of thionin blue in distilled water. The 

 pieces should be kept floating in the stain by being fixed to 

 pieces of cork by one corner by means of paraffin wax. The 

 stain should be changed once during this time. Pass directly into 

 4 per cent, ammonium molybdate solution containing 4 drops of 

 pure HCl for each 100 c.c, and leave in this for twenty-four hours. 

 Wash out in water for twelve hours, pass through alcohol and 

 xylol to paraffin. Cut sections, mount without further staining 

 after removal of paraffin. 



Method III. Good for spinal cord, pons, medulla oblongata, 

 spinal and sympathetic ganglia. Thin slices of tissues are fixed 

 for five to six days in pure pyridine changed at least once, and 

 then treated with repeatedly changed distilled water until the 

 pyridine has been entirely eliminated. The surfaces of pieces are 

 smoothed by means of a sharp razor, and the pieces brought for 

 twenty-four hours into 4 per cent, ammonium molybdate to which 

 4 drops of hydrochloric acid have been added. After a quick 

 wash, they are rapidly dehydrated in 95 per cent, and absolute 

 alcohols, and imbedded in paraffin. The sections, which must be 

 rather thin (3 to 6 fj.), are brought through xylol, absolute and 

 95 per cent, alcohols into distilled water and here washed. 



This is the crucial point of the method because, by washing, 

 ammonium molybdate becomes extracted from the sections, 

 and the success of the subsequent staining depends almost entirely 

 on carrying out the extraction up to the right point. The only 



