THE HISTOLOGICAL TECHNIQUE 5 



the briefest period of hardening, and that the cord possibly does not require quite the same 

 length of time that the basal ganglia and cerebrum do. 



(c) The stage of development of the tissue is an important element in the duration of 

 hardening. In general, the older the tissue, the longer the period of hardening. Embryonic 

 tissue is much more favourable than adult tissue. Good impregnations of nerve fibres cannot 

 be looked for in tissue in which the fibres are medullated. It is not entirely true that the 

 axis-cylinder of a medullated nerve fibre will never impregnate, but such fibres are decidedly 

 unfavourable. In an eight-months human embryo, for example, many of the axis-cylinders of 

 the pyramidal tracts, in some of the preparations, were apparently impregnated, while the 

 medullated tracts of the cord and medulla had simply the faint brown stain due to the osmic 

 acid used in hardening. This indicates that for the best study of the cord and medulla, 

 younger material must be used than is necessary for the higher centres. The medullation 

 of fibres is not the only cause of the difference between embryonic and adult tissue, however, 

 for some cells impregnate better in embryonic tissue. This is possibly due to the different 

 consistency of the embryonic tissue and also to the fact that its cells are smaller. 



(d) The kind of animal used is another factor to be taken into account. Cajal has 

 shown that in the mouse the elements of the cortex are very embryonic at birth, and that 

 the most favourable age is from the 8th to the 25th day after birth. In larger mammals 

 (e.g. the rabbit), he shows that this period is thrown back to between the ist and 8th days 

 after birth. This interesting tendency is still further exemplified in man ; for, as the atlas 

 shows, the cortical cells are well differentiated, though perhaps not in all respects completely 

 developed, one and two months before birth. Consequently, also, the period of hardening 

 would exceed that required for smaller mammals (with shorter gestation periods) of the same 

 age relative to birth. 



(e) A higher temperature naturally accelerates the hardening. The temperature of the 

 room in which these impregnations took place rarely varied much from 21 C. 



The best period of hardening in general for an eight-months human embryo, as can be 

 seen from the table below, is six days, though good results in the cortex were obtained before 

 this and also in tissue hardened eight days. Six days is also the best period for the medulla 

 and basal ganglia. While cells may be obtained in the cortex before six days of hardening, 

 six days is necessary to bring out the tangential fibres and Cajal cells. It is obvious, how- 

 ever, that it is best not to depend entirely upon any one period of hardening, but to transfer 

 pieces at various intervals of time into the silver nitrate. 



Impregnation. After the hardening, the next step is .to bring the tissue into the solution 

 of silver nitrate. The solution used is a f % in water, but a | % or a i % solution may be 



