DETBltMIlSrATION OF AGE IN HONEY-BEES. 197 



sliced off and tlie head placed in a small tube of Petrunke- 

 witsch. fixing-fluid for about fourteen hours. After thoroughly 

 washing in 70 per cent, alcohol containing iodine, the head is 

 transferred to 90 per cent, alcohol for eome hours. It is then 

 easy to remove the remainder of the chitin from the posterior 

 part of the head, and to lift out the entire brain (PI. 11, figs. 

 1 and 2) with the subcesophageal ganglion and some of the 

 head-glands attached. After further washing the brain is 

 prepared for embedding and cut into serial sections as 

 required. 



Sagittal sections were cut of some brains, but generally 

 transverse sections are more useful. These are cut from 

 above downwards, starting through the three simple eyes. 

 Jiy leaving the optical pigment attached to the brain it is easy 

 to orientate it when embedded. 



For routine examination of individual bees transverse 

 sections 1 n thick are preferred, and they are stained with 

 Ehrlich's hsematoxylin and orange Gr. Many other stains 

 were tried. Among them carbolthionin was good for brain- 

 cells, and alcoholic carmine with picronigrosin as a counter- 

 stain was useful for picking out membranes and connective 

 tissue. Perfect fixation of the material immediately on death 

 is, of course, most important in order to exclude error arising 

 from post-mortem changes. 



Ohlmacher's fixative penetrated very well, but owing to the 

 large percentage of absolute alcohol (80) and chloroform (15) 

 present, all the lipoids of the cells were dissolved, producing 

 intense vacuolation throughout. Fleming's solutions, on the 

 other hand, were not sufficiently penetrating for the whole 

 brain. No doubt by employing some of the osmic acid 

 mixtures without acetic acid on small portions of the brain 

 ■or other ganglia and staining by the more elaborate methods, 

 much more interesting detai|"tnight be made out in the cyto- 

 plasm. I have not, however, myself felt justified in spending 

 time on these methods, since all that is required for the 

 pathological work undertaken for the Board of Agriculture 

 is to discover whether a crawling bee sent for examination is 



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