No. 7. DEPARTMENT OF AGRICULTURE. 191 



composition, the glycerin will also destroy bacteria and check de- 

 composition of the specimen. From the fresh brain tissue, smears 

 are usually made from the hippocampus major and cerebellum (see 

 Plate II). A piece 1mm. thick and several mm. in diameter cut 

 from the fresh surface exposed, after an incision is made through 

 the hippocampus major at right angles to its length, or of the cere- 

 bellum in which an incision has been made at right angles to the 

 convolusions, is jilaced upon a slide near one end. Instead of using 

 a cover slip, another slide is i^laced over the small piece of tissue and 

 gentle pressure is applied and the opposite ends of the slides are 

 moved toward one another. The smears are then placed in absolute 

 alcohol for two to five minutes, whereupon the alcohol is allowed to 

 evaporate and the smears then stained. The stain as recommended by 

 Van Gehuchten is used.* 



Loefi3ers alkalin methylene blue, one part. 



Distilled water, one part. 



Saturated alcoholic solution of fuchain added in 

 drops until the mixture has a purple tinge, or until 

 a metallic scum is seen on the surface. 



The mixture kept at a low temperature can be used for an un- 

 limited length of time but is apt to change quicklj' at room tempera- 

 ture, and for this reason a new batch of stain is usually made each 

 day, or as each specimen is prepared for examination, A smear 

 properly fixed upon a slide is taken up with a pair of forceps and 

 completely covered with stain. The slide is passed through the flame 

 of a Bunsen burner several times until steam arises from the heated 

 stain which is permitted to remain upon the smear for five to thirty 

 seconds. The smear is then washed in running water, and if the 

 color of the smear is blue where the brain tissue is most thick, and 

 red where the smear is thin, the slide is placed between filter paper 

 and dried. As soon as the slide is drv a search is made for large 

 nerve cells with a low power lens under the miscroscope. The pro- 

 toplasm of the nerve cells should be stained a light blue, the nucleus 

 a shade of purple and the nucleolus a dark blue. If the cells are 

 stained too deeply the stain may be weakened by the addition of 

 more distilled water or in heating the staining fluid on a smear for 

 a longer time, the intensity of the staining of the fuchsin will be 

 increased at the expense of the blue of the Loefflers alkalin methylene 

 blue. When a nerve cell is found properly stained it is examined 

 with an oil immersion lens. Negri bodies with this staining fluid 

 show the inner bodies a bluish black, and the structures around the 

 inner bodies a maroon red. They are found within the cell, outside 

 of the nucleus of the cytoplam in the nerve cells of sections, but not 

 infrequently in smear preparations, a few Negri bodies not within 

 the nerve cells are seen which have been forced out of the nerve cell 

 as the smear is made. In searching a smear for Negri bodies, only 

 those bodies within the nerve cells should be considered, 



Frothingham" in 190(j demonstrated his impression preparations 

 which he uses in preference to smears. Impressions of the tissue 



*Dr. John H. Engel, first assistant in the laboratory, who has been entrusted with the 

 examination of the smears during the past two jears finds the following formula of the above 

 mixture as a satisfactory stain for the average specimen: 



LoefHers alkalin methylene blue, - ,. 5 c. c. 



Distilled water, ._ 20 c. c. 



Saturated alcoholic solution of fuchsia i drops. 



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