Reagents 25 



Capsella pods, cuts more readily after this reagent than after the 

 stronger solution. 



c) Medium Chromo-Acetic Solution.- 



Chromic acid . 5 g. 



Glacial acetic acid 1.0 c.c. 



Water 100.0 c.c. 



This is a useful formula. The chromic is too strong for some 

 algae, but for fern prothallia and most liverworts the solution is 

 quite successful. 



d) Flemming's Fluid (stronger solution): 



f 1 per cent chromic acid 45 c.c. 



\ Glacial acetic acid 3 c.c. 



B. 2 per cent osmic acid 12 c.c. 



Keep the mixture A made up, and add B as the reagent is needed 

 for use, since it does not keep well. This fluid is quite expensive 

 on account of the osmic acid. For cytological work it has been very 

 popular, and it is especially recommended for chromosomes, centro- 

 somes, achromatic structures, and mitotic phenomena in general. 

 The fluid should be allowed to act for 24 to 48 hours and the washing 

 in water must be very thorough. 



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

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

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

 hydrogen just before the slide is passed from the alcohol into the 

 stain. Harper and Holden, in their work on Coleosporium, recom- 

 mended 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 hydrogen, 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; 



