476 NEUROFIBRILS 



way of ensuring this consists in proceeding by trials, which must 

 be repeated for every series of sections. Once the right amount 

 of washing has been decided upon, one can proceed to stain even 

 many shdes at the same time by means of a 1 : 10,000 solution 

 of thionine, to be freshly prepared every time from a less diluted 

 stock solution. 



The staining is a " progressive " one, and must be controlled 

 under the microscope. It generally takes about twenty minutes 

 to obtain it, at the end of which time the grey substance has a 

 red-purple tone whilst the white substance is bluish. If the 

 staining is right the preparations can be quickly washed, 

 dehydrated and mounted. But if the neurofibrils are not quite 

 sharply stained, the preparations can be " differentiated " for 

 another fifteen to twenty minutes in the ammonium molybdate 

 solution used for mordanting the pieces, or for ten seconds in a 

 diluted solution (1 : 10 to 1 : 20) of " pink salt " (C. Erha, Milano). 

 Preparations last only a. few months, but are sometimes of great 

 interest. See Da Fano, Ziegler's Beitrdge, xliv, 1908, p. 495. 



Method IV is particularly useful for the demonstration of 

 neurofibrils in the cells of the cortex cerebri and cerebelli. It 

 differs from Method III only as regards a preliminary fixation of 

 the pieces for twenty-four hours in a mixture of pyridine nitrate 

 10 grm., and pyridine, 100 c.c. ; they are then transferred for 

 another thirty-six hours into pure pyridine, proceeding as in 

 Method III. 



Method V may be used for the demonstration of both Nissl's 

 substance and neurofibrils. Pieces are fixed in a saturated 

 solution of corrosive sublimate ; after a day they are treated for 

 twenty-four hours with distilled water to which a few drops of 

 iodine tincture have been added, then for two to three hours with 

 pure distilled water ; and lastly passed for forty-eight hours into 

 pure pyridine, this being changed at least once. The rest as in 

 Method III. 



Method VII (for pericellular investments). Fix small pieces 

 of tissue in a saturated solution of corrosive sublimate for twenty- 

 four hours. Remove excess of sublimate by means of tincture 

 of iodine, 15 drops in 100 c.c. of water. Wash in distilled water 

 two to three hours. Place in pyridin, changing once, for thirty- 

 four to forty-eight hours. Remove pyridin by washing for 

 twenty-four hours in distilled water, repeatedly changed. Place 

 in 4 per cent, ammonium molybdate containing 4 drops of pure 

 hydrochloric acid for every 100 c.c. Wash in water for half to 

 one hour. Return the pieces to fresh pyridin, changing this 

 once, for thirty-six to forty-eight hours. Stain in 1 in 10,000 or 

 1 in 15,000 thionin for forty-eight hours, suspending them in the 

 solution as in Method I. Transfer the pieces, still attached to the 

 cork into the same acid solution of ammonium molybdate and 



