CHAPTER XXVI. 319 



the fixative ; kill vertebrates by a blow on the head, or by pithing. 

 If for a study of brain, bleed, or anaesthetise in coal gas, less pre- 

 ferably chloroform or ether. Insects can be killed with cyanide 

 or xylol. 



Hints on removing Tissues and Cutting. Avoid pinching the 

 material with forceps, as this will introduce artifacts ; it is preferable 

 to remove tissue without recourse to dissection under tap-water or 

 salt solution ; for Kopsch techniques, quickly remove blood or 

 lymph, etc., from surface of material with aq. dest. before placing 

 in fixer ; for cutting tissue the best instrument is a new safety 

 razor blade stuck in a special holder made for the purpose, or in a 

 split penholder, or held by artery forceps. When working on 

 arthropods, it is best to dissect the organ from the animal, instead 

 of preserving the whole body ; surrounding fat, etc., should be 

 removed. See also 12 and 653. 



677. Chrome-osmium Techniques. Potassium bichromate or 

 chromium trioxide, used in watery solutions will not oxidise true fats 

 (see 768) in such a way that full vacuoles of the latter will appear 

 in finished sections prepared by routine methods ; but combinations 

 of such salts with osmium tetroxide provide fixatives which will 

 preserve almost all cell elements in finished sections. The basis of 

 all chrome-osmium techniques consists in a preliminary fixation of 

 small pieces of tissue, small embryos or eggs, in such a fluid as Benda, 

 Champy, Flemming-without-acetic acid, or Altmann, for from at 

 least twelve hours to a week. The osmic reaction is then, in some 

 methods (Kull, Benda), " set ' or strengthened by the reducing 

 effect of pyroligneous acid ; following this treatment is a further 



' chroming ' in 3 per cent, bichromate of potash, and, finally, a 

 thorough wash out under the tap. Material treated in this way is 

 generally perfectly preserved, and fit for selective staining. Arranged 

 below are chrome-osmium techniques of progressive intensity and 

 difficulty. 



4 



678. Modified " Flemmings " for Cell Inclusions. BENDA : 15 c.c. 

 chromic acid 1 per cent., 4 c.c. osmic acid 2 per cent., 3 to 6 drops 

 of acetic acid. MEVES : 15 c.c. of chromic of 0-5 to 1 per cent., 

 containing 1 per cent, sodium chloride, with 3 to 4 c.c. of 2 per cent, 

 osmic acid, and 3 to 4 drops of acetic (Encycl. mik. Techn., 1910). 

 GATENBY : Strong Flemming without acetic acid ; and same 

 solution diluted by one-half or one-third (Quart. Journ. Micr. 

 Sci., 1919). The presence of a small quantity of acetic acid is 

 always liable to introduce distortion, but less so among vertebrates 



