REAGENTS 27 



e) Flemming's fluid (stronger solution). — 



. f One per cent chromic acid 45 c.c. 



\ Glacial acetic acid 3 c.c. 



B. Two per cent osmic acid 12 c.c. 



This formula has been very popular for cy tological work, and has been 

 highly recommended for chromosomes, centrosomes, achromatic struc- 

 tures, and mitotic phenomena in general. The fluid should be allowed 

 to act for from 24 to 48 hours, and the washing should be very thorough. 



Material should be in very small pieces | inch square, or in thin 

 slices I inch or less in thickness, for the fluid penetrates poorly. The 

 blackening due to the osmic acid may be removed by peroxide of hy- 

 drogen juvst before the slide is passed from the alcohol into the stain. 

 Harper and Holden, in their work on Coleosporium, recommended 4 

 hours on the slide in a 3 per cent solution of the peroxide of hydrogen. 

 Some prefer a stronger solution of the peroxide of hydrogen, even 20 

 per cent. The peroxide should be in water, if one is following it by an 

 aqueous stain, but may be in 50 per cent alcohol if it is to be followed 

 by an alcoholic stain. Yamanouchi has used chlorine for bleaching, 

 and the results are fully equal to those obtained with peroxide of hy- 

 drogen, and the chlorine is cheaper. Make the bleacher as follows: 

 Place some potassium chlorate crystals — a group about as large as a 

 grain of wheat — in the bottom of a 100 c.c. Stender dish; add one drop 

 of 25 per cent hydrochloric acid in water; immediately fill the Stender 

 full of 30 per cent alcohol and thus dissolve the fumes in alcohol. This 

 will bleach sections in 10 minutes, or even less. Wash in 30 per cent 

 alcohol 2 or 3 hours before staining. Trondle uses 1 per cent chromic 

 acid in water for bleaching; it is slow, requiring about 8 hours, but he 

 maintains that material stains better than after bleaching with perox- 

 ide of hydrogen. According to Miss Merriman, the linin in the nuclei 

 of onion root-tips is not so well preserved in this solution, but the 

 arrangements of the chromatin granules is brought out with greater 

 distinctness. Flemming's safranin, gentian violet, orange combination 

 gives excellent results after this reagent. 



/) Flemming's fluid (weaker solution). — 



(One per cent chromic acid 25 c.c. 



One per cent acetic acid 10 c.c. 



Water 55 c.c. 



B. One per cent osmic acid 10 c.c. 



