110 
METHODS IN PLANT HISTOLOGY 
must not be washed out with water, but with alcohol, whether the 
picric acid be in aqueous or alcoholic solution. When washing with 
water, running water is best, and where this is not convenient the 
water should at least be changed frequently. The washing-out 
process usually requires about 24 hours. 
HARDENING AND DEHYDRATING 
After the material has been washed, it is necessary to continue 
the hardening and also to remove the water. Alcohol is used almost 
entirely for these purposes. It completes the hardening and at the 
same time dehydrates, that is, it replaces the water in the material, 
an extremely important consideration, for the least trace of moisture 
interferes seriously with the infiltration of the paraffin. 
The process of hardening and dehydrating must be gradual; 
if the material should be transferred directly from water to absolute 
alcohol, the hardening and dehydrating would be brought about 
in a very short time, but the violent osmosis would cause a ruinous 
contraction of the more delicate parts. In recent years, cytologists 
have been making the dehydration process more and more gradual. 
Twenty years ago most workers began the dehydration process with 
35 per cent alcohol and used the series 35, 50, 70, 85, 95, and 100 per 
cent alcohol. Some placed an intermediate grade between water and 
35 per cent alcohol. If plasmoylsis—the tearing away of the proto¬ 
plast from the cell wall—was avoided, the series was thought to be 
sufficiently gradual; but a series which may avoid plasmolysis may 
not be adequate if one is to study the finer details of cell structure. 
The following series is recommended: 2|, 5, 7§, 10, 15, 20, 30, 40, 50, 
70, 85, 95, and 100 per cent. There is no particular virtue in the 
fractions: it is convenient to make 10 per cent alcohol, dilute with 
an equal volume of water for the 5 per cent, and dilute the 5 per cent 
with an equal volume for the 2\ per cent. It will be noted that the 
series begins with very close grades and that the intervals are gradu¬ 
ally increased. The claim is that by beginning with very weak 
alcohols in close grades, more perfect dehydration can be secured 
at the end of the series. Various devices, like constant drip and 
osmotic apparatus, have been proposed to secure a more gradual 
transfer, but it is very doubtful whether these possess any real advan¬ 
tages. It is not necessary to use a large amount of alcohol: 2 or 3 
times the volume of the material is sufficient. 
