io6 Annals Entomological Society of America [Vol.1, 



METHODS. 



The usual methods of dissection were employed in making 

 the preliminary studies of the nervous system. On account of 

 the transparency of the tissues, however, it was found necessary 

 to supplement the examination of fresh specimens with the study 

 of stained or fixed specimens in which the nerves were more clearly 

 differentiated. For such preparations alcoholic picric acid 

 solution has been used to very great advantage. It stains and 

 hardens the tissues rapidly, so that the preparation can be used 

 almost without delay. The specimens when immersed for a 

 longer time (2-3 hours) in water will destain, but they can be 

 restained any number of times desired. 



Alcoholic sublimate (Gilson's fluid) is very eft'ective in differ- 

 entiating the tissues. It must, however, be borne in mind that 

 sublimate is very corrosive to metals, and a black precipitate is 

 quickly formed, when metal pins or tools are used. 



In tracing certain nerves intra viicm staining with methylene 

 blue has been used to advantage, but since the different nerves take 

 up the stain very irregularly*, much time and patience has been 

 recjuired to secure satisfactory results. The living larva was 

 injected with approximately 1-2 cc. of the solution (|% methy- 

 lene l^lue in normal salt solution) . Half an hour later it was ether- 

 ized and opened along the back and then spread out on a sheet 

 of cork, exposing the visceral cavity. The still living tissues were 

 kept moist with the above solution until the desired results were 

 obtained. It sometimes requires 3-5 hours before certain nerves 

 take up the stain. Once stained they will retain it for only a 

 short time. By the application of a few drops of hydrogen 

 peroxide solution (H^O.,) the stain is intensified. 



It was found that for some features a process of maceration 

 could be used to great advantage. By soaking for several days 

 in water considerable amount of muscular tissue is loosened and 

 can be washed oft' easily by moving the preparation in water, 

 leaving the more resistent nerves exposed and distinct. Similar 

 results were brought about by the use of 5-10% nitric acid 

 solution. In either of these tw^o processes, the preparation must 

 be afterwards hardened in alcoholic picric acid. These methods 

 should first be used after a general study of the ner\'Ous system 

 has been made. 



* According to Ehrlich '86 (Biol. Centralb., VI, p. 214) the sensory nerves 

 are the first stained, while the motor nerves require longer time. 



