Journal of Applied Microscopy. 365 



chromic acid, '2 cc. acetic acid, and '296 cc. water as a good formula for most 

 purposes. 



The time required for lixing undoubtedly varies with different plants, but 

 twenty-four hours may be considered a minimum even for the most delicate 

 objects. It is well known that zoologists allow fixing agents like Miiller's fluid 

 and Erlicki's fluid to act for weeks before the material is passed on to the next 

 stage, and it may well be questioned whether botanists have not made a mistake 

 in allowing the chromic solutions to act for so short a time. At present most 

 botanists recommend sixteen to twenty-four hours for material which is to be 

 imbedded in paraffin, but some recent experiments in my laboratory indicate 

 that material which has been in the fixing fluid for two or three days is better 

 able to withstand the subsequent processes. More rapid penetration and conse- 

 quently more immediate killing can be secured if the reagent is kept warm (30 

 degrees to 40 degrees C.) Since chromic acid has a tendency to render objects 

 hard and brittle, it is often better to use some other fixing agent if much difficulty 

 is anticipated'in the cutting. 



After fixing is complete, the reagent should be washed out with water. 

 Running water is desirable, and where this is not convenient the water must be 

 changed frequently. Any material should be sufficiently washed in sixteen to 

 twenty-four hours, but the time may be shortened about one-half by using luke- 

 warm water. 



Some of the formulae are as follows : 



a. Strong Chromo-acetic Solution. — 1 gr. chromic acid, 1 cc. glacial acetic acid, 

 98 cc. water. This solution has been used quite extensively in embryological 

 work upon the higher plants. 



b. Weak Chromo-acetic Soii/tion. — (Schaffner's formula) : 0.3 gr. chromic 

 acid, 0.7 cc. acetic acid, 99 cc. water. This has also been used in embryological 

 work. It causes little or no plasmolysis, but the chromic seems rather weak. 

 Difficult material, like Aster heads and ripe Capsella pods, cuts more readily after 

 this reagent than after the stronger solution. 



c. Fkfnmi/ig's Fluid. — (Weaker solution.) 



A. 1 per cent, chromic acid, 25cc. 

 1 per cent, acetic acid, lOcc. 

 Water, 55cc. 



B. 1 per cent, osmic acid, lOcc. 



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 gives better results than any other fixing 

 agent which has yet been thoroughly tested. It is especially recommended for 

 chromosomes, centrosomes, achromatic structures, and mitotic phenomena in 

 general. Material should be in very small pieces, one-eighth of an inch square 

 or in thin slices one-eighth of an 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. Flem- 

 ming's safranin-gentian violet-orange combination gives its most brilliant results 

 after this reagent. 



