[dupokte] LARVA OF SPHIDA OBLIQUA WALKER 227 



Picro-formol (P. Bouin) was used with good results as it fixes 

 and stains rapidly. The formula of this fixing agent is: — 



Picric acid, sat. aq. solution 75 parts, 



Formalin 25 " 



Acetic acid 5 " 



M alteration. For a study of the peripheral nerves it was found 

 advantageous to macerate in distilled water for several days, or in 

 dilute nitric acid for a shorter time. The muscular tissue could then 

 be teased out, leaving the integument and its nerves exposed, the latter 

 being more resistant than the muscles to the macerating action of the 

 water. Specimens thus treated were always hardened afterwards 

 in picro-formol. 



Staining. Several stains were tried with varying success. A 

 weak solution of Delafield's haematoxylin gave fair results but did 

 not prove satisfactory for the finer details. The only really satis- 

 factory stains of the nervous tissue were obtained with methylen blue. 

 The strength used was -5% in physiological salt solution. In most 

 cases where fresh specimens were used the nerves were stained intra 

 vitam. About 2 cc. of the staining solution were injected into the 

 living caterpillar. In from thirty to sixty minutes the insect was 

 etherized and opened in the usual manner. On opening the cater- 

 pillar it was usually found that the nerves were not uniformly stained. 

 Indeed it was extremely difficult ever to obtain a perfect stain of all 

 the nerve tissues. Ehrlich who first used the intra-vitam method of 

 staining nervous tissue, pointed out that the sensory nerves were 

 stained while the motor nerves remained colourless. The motor 

 nerves however will also stain if given sufficient time. The same 

 worker also states that methylen blue, in contact with reducing 

 agents in alkaline solution, is reduced to its colourless leucobase. 

 Because of this, tissues, after they have obtained their maximum 

 degree of coloration, lose the blue colour rapidly. These statements 

 explain the difficulty of obtaining a perfect stain. 



In several cases the caterpillar was killed and pinned before 

 staining. The stain was poured on the insect in the dissecting dish 

 and left for half an hour or longer. As long as the tissues were fresh, 

 I obtained a stain equally as good as by Ehrlich's intra-vitam method. 

 Indeed no advantage could be observed in the latter method over the 

 staining of the dead but fresh tissue. This holds good both for 

 material sectioned and for that stained for dissection. 



Specimens stained in methylen blue lost their colour very readily 

 in water and it was found necessary to permanently fix the blue 

 colour in the tissues. The method used was that devised by Bethe 



