CHAPTER XXXII. 417 



835. BETHE'S Molybdenum-Toluidine Blue Method (Ztschr. wiss. 

 Mikr., xvii, 1900, p. 13). Pieces of the central nervous system of 

 vertebrates are fixed for twenty-four hours in 3 to 7*5 per cent, nitric 

 acid, and then brought directly into 96 per cent, alcohol for a day or 

 longer. They are afterwards put for twelve to twenty-four hours 

 in a mixture of 1 part of ammonia (of sp. gr. -95) with 3 of distilled 

 water and 8 of 96 per cent, alcohol ; for six to twelve hours into pure 

 alcohol ; for twenty-four hours into a mixture of 1 part of concen- 

 trated hydrochloric acid, 3 of distilled water, and 8 to 12 of 

 alcohol ; for ten to twelve hours into pure alcohol ; for two to 

 six hours into water. They are now mordanted with 4 per cent, 

 ammonium molybdate, washed again, dehydrated and embedded in 

 paraffin. The sections, 8 to 10 ju, thick, are seriated on slides by 

 means of egg albumen, but without ivater, then passed through xylol 

 and alcohol and " differentiated," viz., covered with water poured 

 on the sections so as to form over them a layer 1 -5 to 2 mm. deep, 

 and put into an incubator at 55 to 60 C. for ten minutes. They are 

 then rinsed with water, covered with a 1 : 3000 solution of toluidine 

 blue, stoved for another ten minutes, rinsed with water, and lastly 

 treated with 96 per cent, alcohol till no more colour comes away. 

 After dehydration with absolute alcohol they are mounted in the 

 usual way. 



The method is also applicable to invertebrates for which other 

 fixing agents besides nitric acid are admissible, and the impregnation 

 with ammonium molybdate may be done on the sections instead of 

 previously on the uncut tissues. 



For LUGARO'S modification see Riv. pat. nerv. ment., x, 1905, p. 265. 



836. DONAGGIO'S Methods (Riv. Sper. Frenialr., 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 embedding, but results 

 are not so good as by the other three, the most important of 

 which is 



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 em- 

 M. 27 



