184 THE TISSUES. 



The tissues are then transferred to xylol and imbedded in paraffin, sec- 

 tioned, fixed to the slide or cover-glass with albumin fixative, and may 

 be double stained in alum-carmin or alum-cochineal. After staining in 

 either of these stains, the sections are thoroughly dehydrated and cleared 

 in oil of bergamot. The oil is washed off with xylol and the sections are 

 mounted in Canada balsam. 



In staining nerve-fibers with methylene-blue by local application 

 of the stain to the tissues, the tissues to be studied are removed from an 

 animal which has just been killed, divided in small pieces, and placed on 

 a slide moistened with normal salt solution. A few drops of a -$-$% to 

 yV% solution of methylene-blue in normal salt solution are added from 

 time to time sufficient to keep the tissues moistened by the solution, but 

 not enough to cover them. The preparations are examined from time to 

 time, under the microscope, to see whether the nerve elements are stained. 

 The length of time required for staining by this method varies. Some- 

 times the nerve elements are stained in half an hour ; again, it may re- 

 quire two and one-half hours ; on an average, about one hour. As soon 

 as the tissues seem well stained they are fixed as previously directed. 

 Dogiel has found that sympathetic ganglia and sensory nerve-fibers of the 

 heart removed from the human body several hours after death may be 

 stained by means of the foregoing method. 



In order to obviate the necessity for the low temperature of the pre- 

 vious method, Bethe (96) has recommended the following procedure : 

 According to the method of Smirnow and Dogiel, he first employs as a 

 preliminary fixing agent a concentrated aqueous solution of ammonium 

 picrate. In this he places the tissue, previously treated with methylene- 

 blue, for from ten to fifteen minutes. Without further washing the larger 

 objects are immersed in a mixture composed of ammonium molybdate 

 (or sodium phosphomolybdate) i gm., distilled water 20 c.c., and pure 

 hydrochloric acid i drop. The following mixtures jnay also be employed 

 for the same purpose : ammonium molybdate (or sodium phosphomo- 

 lybdate) i gm., distilled water 10 c.c., 2% solution of chromic acid 

 10 c.c. , and hydrochloric acid i drop ; or, for very thin gross specimens 

 or sections, ammonium molybdate (or sodium phosphomolybdate) i gm., 

 distilled water, 10 c.c., 0.5% osmic acid 10 c.c., and hydrochloric acid 

 i drop. Small objects are permitted to remain no longer than from three 

 quarters of an hour to one hour in either of the first two mixtures, and 

 not more than from four to twelve hours in the third. After fixing, the 

 specimens are washed with water, carried over into alcohol, then into xylol, 

 and finally imbedded in paraffin. Subsequent staining with alum-carmin, 

 alum-cochineal, or one of the neutral anilin dyes gives good results. 



A very promising method recommended by Meyer (95) consists 

 in injecting subcutaneously about 20 c.c. of normal salt solution contain- 

 ing from i% to 4% of methylene-blue into a young rabbit, and repeating 

 the operation in one to two hours. Within the next two hours the animal 

 usually dies and the central nervous organs are then removed and small 

 pieces fixed according to Bethe' s method. 



The method of Chr. Sihler may be recommended for demonstrating 

 the nerve-endings in striated muscle : Muscle bundles of the thickness 

 of a goose quill are first placed for eighteen hours in a solution composed 

 of acetic acid i vol., glycerin i vol., and \ C J C solution of chloral hydrate 

 6 vols., and then teased in pure glycerin. Afterward they are placed in a 



