48 THE MICROSCOPIC PREPARATION. 



tissues. The method may be used on fresh tissues or on fixed tissues ; 

 the employment of fresh tissue is, however, more satisfactory. The tis- 

 sues to be impregnated are spread in thin layers, and immersed in a 

 0.5% to i c /c solution of silver nitrate for from ten to fifteen minutes; 

 they are then rinsed in distilled water and placed in fresh distilled water 

 or 70% alcohol or a 4% solution of formalin and exposed to direct sun- 

 light, where they remain until they assume a brown color. The sunlight 

 reduces the silver, in the form of fine particles which appear black on 

 being examined with transmitted light. The preparations thus obtained 

 may be examined in glycerin or dehydrated and mounted in balsam. 

 (See methods of injection for staining the endothelial cells of blood and 

 lymph vessels. ) 



Gold Chlorid Method. In gold chlorid impregnation the cells and 

 fibers of certain tissues are stained while the intercellular substances remain 

 uncolored. The coloration is obtained by a reduction of the gold (either 

 by sunlight or certain reagents formic acid, acetic acid, citric acid, oxalic 

 acid), in the form of very fine particles which impart to the tissues a pur- 

 plish-red color. This method is especially useful for bringing to view the 

 terminations of nerve-fibers, both motor and sensory ; however, it may also 

 be employed for staining other tissue elements. The method of gold 

 impregnation was introduced by Cohnheim and was used by him in 

 staining the nerve terminations in the cornea. It has received numerous 

 modifications since its introduction. The following may be mentioned : 



Cohnheim' s Method. Small pieces of muscle are placed in a \ c / r 

 solution of gold chlorid acidulated by a trace of acetic acid. In this 

 they become yellow (in from a few minutes to half an hour). They are 

 then rinsed in distilled water, placed in water slightly acidulated with 

 acetic acid, and kept in the dark. As a rule, the pieces will change in 

 color, becoming yellowish-gray, grayish-violet, and finally red, from one 

 to three days generally being required for this process. The parts best 

 adapted to examination are those in the transitional stage of violet to red. 

 This procedure has been subjected to innumerable modifications ; 

 of these, the most used are : ( i ) The method of Lowit : Small pieces are 

 placed in a solution of i vol. formic acid and 2 vols. distilled water 

 until they have become transparent (ten minutes). They are then placed 

 in a i cjc solution of gold chlorid, in which they become yellow (one-quarter 

 hour). They are now again placed in formic acid, in which they 

 pass through the same color changes as above. Finally, they are washed 

 and teased, or subsequently treated with alcohol and cut. (2) Kiihne 

 (86) acidifies with 0.5% solution of acetic acid (especially in the case of 

 muscle), then treats the specimens with a i c / solution of gold chlorid, 

 and reduces the gold with 20 to 25% formic acid dissolved in equal parts 

 of water and glycerin. (3) Ranvier (89) acidifies with fresh lemon juice 

 filtered through flannel, then treats with a 1% solution of gold chlorid 

 (quarter of an hour or longer), and finally either places the specimen in 

 water acidulated with acetic acid (i drop to 30 c.c. water) and subjects 

 it to light for one or two days, or reduces it in the dark, as in 

 Lowit's method, in a solution of i vol. formic acid and 2 vols. water. 

 (4) Gerlach uses the double chlorid of gold and potassium, but in weaker 

 concentrations than a i% solution, otherwise he continues as in the 

 method of Cohnheim. (5) Golgi (94) also uses the same double chlorid, 

 but acidifies with 0.5% arsenious acid, and then reduces in i r fo arsfni- 

 ous acid in the sunlight. 



