22 Methods in Plant Histology 



proportions must be determined for the particular plant under 

 investigation. In observing the effect of the fixing one can determine 

 whether there is any noticeable plasmolysis or distortion, but whether 

 the fixing is thorough can be determined only by noting how the 

 tissues endure the subsequent processes. When the effect of the 

 reagent cannot be observed directly, it is well to make a freehand 

 section and thus determine whether plasmolysis takes place. It 

 is not safe to judge the action of a fixing agent by the appearance 

 of sections cut from material which has been imbedded in paraffin, 

 because shrinking of the cell contents often takes place during the 

 transfer from absolute alcohol to the clearing agent or during infil- 

 tration with paraffin, and sometimes even during later processes. 

 When there is doubt as to proportions, we should suggest 2 c.c. 

 chromic acid, 3 c.c. acetic acid, and 300 c.c. water as a good formula 

 for most purposes. 



A large quantity of the fixing agent is required and it cannot be 

 used again. The volume of the fixing agent should be at least 25 

 times that of the material to be fixed. We use about 50 volumes of 

 the fixing agent to one of the material. 



The time required for fixing undoubtedly varies with different 

 objects, but even a delicate object, like Spirogyra, which is pene- 

 trated immediately, should remain in the fixing fluid for 18 to 24 

 hours. Most botanists leave material like onion root-tips and lily 

 ovaries in the chromo-acetic acid about 24 hours. Some recommend 

 longer periods. Christman, in his work on rusts, left material for 

 three days in Flemming's fluid, a much more vigorous agent than 

 the chromo-acetic acid. We have often imbedded material which 

 had been in chromo-acetic acid for several days, and it seemed to 

 have suffered no injury. It is well known that zoologists allow 

 fixing agents like Mtiller'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. More rapid pene- 

 tration, and consequently more immediate killing, can be secured if 

 the reagent is kept warm (30 to 40 C.). The warming also shortens 

 the time required for fixing, but, for cytological work, it is quite 



