CHAPTER XXXII. 411 



subsequent differentiation with a 10 per cent, solution of anilin 

 oil in 96 per cent, alcohol. The present form of the method was 

 published in 1894 (N enrol. CentrbL, xiii, p. 507) ; but NISSL con- 

 tinued to introduce into it slight modifications, as one understands 

 from many of his papers, to which due attention was paid when 

 preparing the following account. It must be added here that 

 Nissl's method has been, and still is, extremely useful for the study 

 of nervous tissue under various physiological and pathological 

 conditions, and that it stains, when properly carried out, not only 

 the tigroid substance and the basophil parts of nuclei of nerve cells, 

 but also the nuclei and certain parts of the cytoplasm of neuroglia 

 cells and connective tissue elements normally or abnormally present 

 in the nervous tissue. 



826. NISSL'S Methylene-blue Method. Not too small pieces of 

 fresh tissue are fixed in 96 per cent, alcohol and hardened therein 

 for a few days. They should not be allowed to fall to the bottom of 

 the bottle, but kept floating by means of some filter paper or cotton 

 wool. The alcohol must be in large quantities in proportion to 

 the number of pieces, and repeatedly changed. The pieces are cut 

 without embedding and the sections collected in 96 per cent, alcohol, 

 from which they are directly floated on some stain filtered into a 

 watch glass at the moment of using it. The stain should be at least 

 three to four months old, and shaken at the moment of filtering the 

 quantity needed. It is prepared by carefully dissolving 1 -75 grms. 

 of Venetian soap in 1 litre of distilled water and adding to it 3 -75 grms. 

 of methylene blue (B patent). It is a good practice to vigorously 

 shake the bottle from time to time, and to re-filter into the same 

 bottle the amount of stain left in the watch-glass after staining one 

 or more sections. 



The watch-glass containing the stain with the section floating 

 on it is warmed carefully over a flame until small bubbles rise to 

 the surface. The section, which should not have fallen to the 

 bottom of the watch-glass, is immediately transferred into a mixture 

 of 10 parts of anilin oil and 90 parts of 96 per cent, alcohol, and as 

 soon as no more colour is given off (it often takes only some seconds), 

 it is lifted on to a slide, pressed with smooth filter paper, and cleared 

 with a few drops of pure anhydrous cajeput oil. Care should be 

 taken not to dry the section excessively with the filter paper and to 

 pour the cajeput oil on to the section very quickly. 



The cajeput oil not only clears the section, but stops the 

 differentiation ; it is, therefore, advisable to renew it after a little 



