APPENDIX. 



407 



Depending upon the object in view and the age of the animal, the tissue 

 remains in the fixing fluid from 2-15 days. If foetal or very young, 23 

 days suffice for the neuroglia-cells, 3-5 days for the nerve-cells, and 5-7 

 days for the collaterals. Older tissues require longer immersion, adult brain 

 being left in the fluid from 8-15 days. After rinsing for a few moments 

 with distilled water and quickly drying off with filter paper, the pieces, not 

 over .5 cm. thick, are placed in i p.c. aqueous solution of silver nitrate. In 

 this they remain 2-3 days or longer, during which a dark precipitate ap- 

 pears. In order to determine the probable success of the impregnation, free- 

 hand sections are cut and examined in 95 alcohol under the microscope. If 

 satisfactory, the tissue is dehydrated in absolute alcohol, embedded in cel- 

 loidin as rapidly as possible, and cut in 95 alcohol into sections not too thin. 

 The sections are passed to absolute alcohol for thorough dehydration, then 

 to creosote for 10 minutes, and, after remaining 5 minutes longer in xylol, 

 finally placed on the slide, dried rapidly with filter paper, and covered with 

 a large amount of balsam. The slide is now heated carefully over a flame 

 until the balsam will set as soon as cool. Before this occurs, however, a 

 heated cover-glass is applied and the section permanently mounted. This 

 manipulation, suggested by Huber, insures the removal of all moisture and 

 the probable permanency of the preparation, thus avoiding the unsatisfactory 

 plan of keeping the specimens covered with only balsam and unprotected by 

 a cover-glass. 



Should the preliminary examination disclose insufficient silver deposit, 

 as is frequently the case, the block of tissue is returned to the bichromate- 

 osmic solution for 2-3 days and again subjected to the silver solution. If 

 necessary, this procedure may be repeated a number of times with the same 

 tissue until, perchance, the results are satisfactory. This may be regarded to 

 be the case, if the desired nervous elements appear as sharply defined dark 

 figures on a light, almost colorless background. Even in otherwise success- 

 ful preparations, many parts of the section may be almost useless owing to 

 disturbing deposits of silver-precipitate. Notwithstanding its uncertainty, 

 the Golgi method yields such remarkable demonstrations of the nervous ele- 

 ments, that really successful results amply repay perseverance. 



Gold Staining. The gold-chloride method is useful to demonstrate 

 nerve-endings, such as the motor plates in striated muscle. Small pieces of 

 fresh tissue, not over 5 mm. in dimension, are treated with the gold solution 

 prepared as follows: 4 parts of i per cent, aqueous solution of gold-chloride 

 and i part of formic acid are heated to boiling three times. The tissue is 

 placed in this solution when cool for i hour, in the dark. It is then rinsed 

 in distilled water for half a minute and transferred to diluted formic acid ( i 

 part acid to 4 parts distilled water) and allowed to stand in the light, but 

 not direct sunlight, 18-48 hours. By the end of this period the exterior of 

 the tissue has acquired a dark violet color. Small fragments may be teased 

 in glycerine and if successfully stained may be mounted permanently in the 

 same ; or, if desirable, the entire piece may be hardened in ascending alcohols, 

 sectioned and mounted in balsam. The use of steel instruments during these 

 manipulations must be avoided, thin glass rods of suitable size being sub- 

 stituted. Admirable preparations, showing nerves and connective tissue cells, 

 may be made from the corneae of small animals, the tissue being separate^ 

 into thin lamellae before mounting. 



Silver Staining. Staining with argentic nitrate, as contrasted with 

 Golgi impregnations, is employed especially to differentiate the cell-boun- 

 daries of endothelium, since by its employment the intercellular cement sub- 



