490 NEUROGLIA AND SENSE ORGANS. 



920. ACHUCARRO'S Tannin Method and DEL Rfo-HoRTEGA's 

 Modifications. The methods described in this paragraph can be 

 considered as the direct outcome of various efforts at modifying 

 the Bielschowsky method for sections ( 840) in such a way as to 

 obtain a neuroglia stain. As a matter of fact, they all stain both 

 neuroglia cells (astrocytes) and connective tissue elements. In 

 other words, they are not elective, and may be used for the study 

 of reticular tissue in non-nervous organs, as well as of other 

 histological details in nervous and non-nervous tissues. 



PERUSINI'S modification of BIELSCHOWSKY'S method (N enrol. 

 CentrbL, xxix, 1910, p. 1256) should be first remembered. Pieces 

 of fresh material were fixed in Weigert's formalin-copper acetate- 

 chromium fluoride mixture for neuroglia stain ( 910), cut by the 

 freezing method, and stained as by Bielschowsky 's method for 

 sections, without pyridine treatment. Achucarro did the same, 

 except for silvering by Ramon y Cajal's reduced silver process. 



ACHUCARRO'S tannin method (Bol. Soc. Espan. Biol., Madrid, 

 1911, p. 139) consisted in putting sections made from frozen formol 

 material into a cold-saturated solution of tannin and warming this 

 until vapour arose. Without waiting for the tannin to become cool 

 again, the sections were, one by one, quickly rinsed in water and put to 

 stain for about ten minutes into three successive glass dishes, each 

 containing 10 c.c. of distilled water and 6 to 8 drops of Bielschowsky 

 ammoniacal silver nitrate- and-oxide bath, prepared beforehand, as 

 described in 841. As soon as they turned dark yellow, they were 

 transferred into 10 per cent, formalin, and, after about ten minutes, 

 washed, dehydrated and mounted. 



The results obtained by such a method were rather uncertain 

 and Achucarro himself felt the necessity of modifying it in the 

 following way, published by DEL Rio-HoRTEGA (Trab. Lab. Invest. 

 BioL, Madrid, xiv, 1916, p. 181) : (1) Fix pieces, 2 to 3 mm. thick, 

 for two or three days in formalin neutralised with ammonia. 

 (2) Make sections of 10 /ot, and mordant them in 10 per cent, tannin 

 until vapour arises. (3) Without waiting for the tannin to become 

 cool, wash the sections in distilled water alkalised with a few drops 

 of ammonia until they have again acquired their flexibility. 

 (4) Treat them with the diluted ammoniacal silver nitrate solution 

 as described above, but adding only 2 or 3 drops of it to every 10 c.c. 

 of distilled water. (5) Reduce in 20 per cent, formalin, either 

 neutralised as for fixing, or (according to Del Rio-Hortega) contain- 

 ing an excess of ammonia, say, 6 to 8 drops to every 10 c.c. of 

 20 per cent, formalin. 



