NEUROGLIA AND SENSE ORGANS 549 



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 jjl, 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 (accord- 

 ing to Del Rio-Hortega) containing an excess of ammonia, say, 

 6 to 8 drops to every 10 c.c. of 20 per cent, formalin. 



Del Rio-Hortega {op. cit.) found that the method could be 

 further modified, and usefully employed for the staining not 

 only of the neuroglia, but also of centrosomes of nerve-cells and 

 neuroglia cells, mitochondria, secretion granules, intra- epithelial 

 fibrils, reticular tissue, collagenous fibres, etc. The modifications 

 proposed by Del Rio-Hortega for these various purposes are 

 four in number, and known as the variants of Achucarro' s method. 



Modification I. Suitable for the staining of fibrous neuroglia 

 as well as for elastic membranes and connective tissue cells. 



(1) Fix tissues for no less than ten days in 10 per cent, formalin. 



(2) Make sections by the freezing method, and mordant them 

 for five minutes in 3 per cent, tannin kept at a temperature of 

 50° to 55° C. (3) Wash them in distilled water alkalised with 

 ammonia, and transfer them successively into three glass dishes, 

 each containing 1 c.c. of ammoniacal silver nitrate, prepared as 

 described in § 1022, and 10 c.c. of distilled water. (4) As soon 

 as they have taken a distinct yellowish-brown colour, wash them 

 in distilled water and reduce them in a 1 : 500 gold chloride solution 

 kept for twenty or thirty minutes at a temperature of about 

 40° to 45° C. (5) Fix with 5 per cent, sodium hyposulphite, 

 wash, dehydrate and mount as usual. 



Modification II. Good chiefly for reticular tissue and its histogenesis. 

 Material may be fixed either in 10 per cent, formalin or Bouin's fluid, 

 or alcohol ; if one or the other of these last two fluids has been used, 

 it is advisable to re-transfer pieces for a few days into a formalin solu- 

 tion. Sections should, as a rule, be made by the freezing method, but 

 pieces may also be imbedded in celloidin, this being dissolved after 

 cutting. The sections, however obtained, are mordanted for five 

 minutes at 50° to 55° C. or for fifteen to thirty minutes at 40° to 45° C. 

 in a 1 per cent, alcoholic solution of tannin. Stain as in Modification I ; 



