CHAPTER XXXV. 491 



DEL RJ'O-HORTEGA (op. tit.) 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 Achficarro'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 841, 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 histo- 

 genesis. 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 solution. Sections should, as a rule, be made by 

 the freezing method, but pieces may also be embedded 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 ; reduce for half a minute in 

 20 per cent, formalin, neutralised by shaking with chalk ; wash, 

 dehydrate and mount. 



Modification III. Particularly good for collagenous fibres, but 

 also for neuroglia fibres. Proceed as in Modification II until the 

 sections are placed in the staining bath ; keep them therein until 

 brown ; reduce and fix as in Modification I. 



Modification IV (op. tit., xv, 1918, p. 375, note). Suitable for the 

 demonstration of the protoplasmic neuroglia. Frozen sections of 

 formalin material are treated for some minutes at 45 to 50 C. 

 with a mixture of tannin, 3 grms. ; ammonium bromide. 1 grm. ; 

 distilled water, 100 c.c. Wash and stain as in Modification I ; 

 reduce in 20 per cent, formalin neutralised with chalk ; tone with 



