CHAPTER XXXII. 437 



small, 1 per cent, silver nitrate will be sufficient. (3) Wash quickly and 

 reduce for eight to twenty-four hours in hydroquinone 1 to 2 grms., 

 formalin 15 c.c., distilled water 100 c.c., sodium sulphite 0-5 grm. 

 (4) Wash quickly, embed in paraffin or celloidin, or make sections 

 by the freezing method. (5) Tone and counterstain sections if 

 desirable. Dehydrate and mount as usual. 



Best results are obtained from vertebrates, preferably kittens 

 and young rabbits. The method may be applied to human 

 material, if available in a sufficiently fresh condition. From 

 invertebrates results are not so good, and rather uncertain, so that 

 Cajal advises a simple fixation in formalin or formalin-acetone, 

 followed by impregnation with silver nitrate, as by his reduced 

 silver methods for neurofibrils. 



848. DA FANG'S Cobalt Nitrate Modification (Proc. Physiol. Soc., 

 Journ. Physiol, liii, 1920 ; Journ. R. Micr. Soc., 1920, p. 157). 

 Small pieces of quite fresh tissues are fixed for six to eight hours at 

 room temperature in cobalt nitrate 1 grm., distilled water 100 c.c., 

 formalin 15 c.c. The solution can be prepared beforehand, and 

 keeps unaltered for months. The formalin need not be neutralised 

 unless strongly acid or containing free sulphuric acid, in which case 

 it is necessary to neutralise it by one of the usual methods. For 

 the fixation of embryonic organs and in all cases in which a 

 shrinkage of delicate tissues is to be feared, the quantity of the 

 formalin may be reduced to 10, 8, or 6 c.c. for every 100 c.c. of distilled 

 water. The time of fixation should be shortened to three to four 

 hours or even less in the case of very small pieces, such as spinal 

 ganglia of mice and rats, the pituitary body of the same animals, 

 etc. Pieces of spinal cord, cerebrum, cerebellum of adult animals 

 give better results if fixed for about eight to ten hours. The fixation 

 may be prolonged in special cases to twelve to twenty hours, but 

 should not exceed twenty-four hours. The fixation in an incubator 

 at a temperature varying between 25 and 37 C. has been attempted 

 with success in the case of tissues of adult subjects, but it leads to a 

 staining of both the internal apparatus and intracellular formations, 

 which, according to their morphology, are to be considered as 

 mitochondria. 



For the impregnation, Da Fano quickly washes the pieces in 

 distilled water, makes their surfaces smooth if necessary, and then 

 places them into 1 -5 per cent, silver nitrate in the dark for twenty- 

 four to forty-eight hours at room temperature. For very small 

 fragments, 1 per cent, silver nitrate may be used, whilst for pieces of 



