INVERTEBRATES 597 



Kenyon (Ttifts Coll. Stud., No. 4, 1896, p. 80) fixes Pauropoda in 

 Carnoy's acetic alcohol and chloroform, § 90, cuts them in two for 

 staining, etc., and imbeds in celloidin followed by paraflin. 



Hennings (Zeit. wiss. Mik., xvii, 1900, p. 311) takes— Nitric acid 

 16 parts, chromic acid of 0-5 per cent. 16 parts, sublimate saturated in 

 60 per cent, alcohol 24 parts, picric acid saturated in water 12, and 

 absolute alcohol 42, fixes for twelve to twenty-four hours, and washes 

 out with iodine alcohol. He says that this mixture not only fixes, but 

 softens chitin enough to allow of paraffin sections being made through 

 hard parts. 



Hamann {Sitz. Xatunv. Freunde Berlin, 1897, p. 2) fixes small 

 Tracheata in 10 per cent, formol and finds the chitin sufficiently soft 

 for sections to be made. 



Van Leeuwen (Zool. Anz., xxxii, 1907, p. 318) takes for larva? of 

 Hexapoda 12 parts of 1 per cent, solution of picric acid in absolute 

 alcohol, 2 of chloroform, 2 of formol, and 1 of acetic acid. 



HoLLANDE {Arch. d'Anat. mic, xiii, 1911, p. 171) takes 12 parts of 

 saturated solution of picric acid in formol of 40 per cent., 54 of absolute 

 alcohol, 3 of benzene, and 1 of nitric acid, and finds that this fixes 

 quickly enough not to make chitin too hard. 



NuTTALi., Cooper and Robinson {Parasitology, 1908, i, p. 163) fix 

 for a few minutes in hot picro-sulphuric acid. 



1183. Methods for Clearing and Softening Chitin. The methods 

 of Loos have been described § 587, those of Hennings and 

 Hamann last §. 



In recent years the tendency has been to attack the problem 

 of chitin as follows : (a) To avoid higher strengths of ethyl 

 alcohol, substituting instead one of the other alcohols, see § 124. 

 {b) To do the whole imbedding in dioxan, § 130. (c) To make 

 the wax very hard by adding ceresin, § 177. {d) To use the 

 proprietary substance diaphanol, § 1184. Such insects as aphids 

 can be cut easily by avoiding ethyl alcohol and xylol, as made 

 possible by a and b, while insects more thickly chitinised can be 

 treated in fresh or properly kept diaphanol. See also remarks 

 on hardness in tissues in §§ 171. 172. 



1184. Diaphanol is best used on animal and plant tissues in 

 glass-stoppered bottles at room temperature in diffused daylight. 



The fixed and well-hardened tissues are rinsed in 63 per cent, 

 alcohol and then placed in diaphanol till they are perfectly bleached 

 and softened. In case of discoloration of the diaphanol the process 

 must be repeated. The tissues are now placed directly in 63 

 per cent, alcohol. After the tissues have been well hardened in 

 alcohol, they are then transferred through tetralin into paraffin. 

 It is essential to pierce or cut the objects in different places prior 

 to all procedures. All hardened objects produce COg, which 

 must be given an avenue of escape. 



Tetralin which is recommended for clearing diaphanol specimens 

 is tetra-hydro-naphthalene, an imperfectly saturated hydro- 

 carbon liquid, used in commerce as a solvent of resins, etc. It is 

 quite possible, but not so good, to use instead benzol, or the 



