REAGENTS 23 



the tissues endure the subsequent processes. When the effect of the 

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

 tion 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 infiltration with paraffin, and 

 sometimes even during later processes. When there is doubt as to 

 proportions, we should suggest 2 g. 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 

 agents to one of the material. 



The time required for fixing undoubtedly varies with different ob- 

 jects, but even a delicate object, like Spirogijra, which is penetrated 

 immediately, should remain in the fixing fluid for from 18 to 24 hours. 

 Most botanists leave material hke onion root-tips and lily ovaries in 

 the chromo-acetic acid about 24 hours. Two days, or even 3 or 4 days, 

 does no damage, and we should prefer 48 hours rather than to use 

 less than 24 hours. 



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 Mliller'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 penetration, and consequently more imme- 

 diate killing, can be secured if the reagent is kept warm (30°-40° C). 

 The warming also shortens the time required for fixing, but, for cyto- 

 logical work, it is quite possible that the danger of producing artifacts 

 may be increased by the heat. 



After fixing is complete, all reagents containing chromic acid as an 

 ingredient should be washed out with water. Running water is desir- 

 able, and where this is not convenient the water must be changed fre- 

 quently. 



About 24 hours is long enough for complete washing in running 



