REAGENTS 
23 
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 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 penetrated 
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 Muller’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 
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 desirable, and where this is not convenient the water must be 
changed frequently. 
About 8 or 10 hours should be long enough for filamentous algae 
and fungi, which are immediately penetrated by the water. It is a 
good plan to start the washing in the morning and let the material 
