Digby . — On the Archesporial and 
136 
Methods. 
After several seasons of repeated failures, Professor Farmer suggested 
the simple expedient of immersing the material in warm, weak spirit before 
placing it in the fixing fluid. This method proved to be a brilliant success, 
and the ensuing fixation was beautiful, with no apparent contraction or 
distortion. 
Fixing was done exclusively on warm ‘ growing * days at about noon. 
Small portions of the sporangiferous apex of the frond were plunged for 
a few seconds in warm (about 30° C.) 30 per cent, spirit. They were 
then speedily transferred to fixing fluid at the same temperature. A 
vacuum pump was attached to the bottle, but the material generally sank 
immediately without pumping. Rapid infiltration of the fixing fluid is 
apparently the secret of this almost perfect fixation. Wilson Smith ( 18 ) also 
tried moistening young sporangia in alcohol before placing them in the 
‘ killing’ fluid, but, although they sank at once, he reports that the fixation 
was so poor that they were useless for purposes of study. He does not 
mention the strength of the alcohol, nor whether it was warmed. 
The fixing reagents used in this investigation were strong Flemming, 
weak Flemming, strong Merkel, Hermann, strong chromic, and acetic 
alcohol. With acetic alcohol rapid penetration of the fixative occurs with- 
out the preliminary alcohol bath, but it is not a sufficiently fine fixative 
for critical examination. The tissue was left 15 minutes in acetic alcohol 
and then washed well with methylated spirit, or preferably with absolute 
alcohol. In the case of the other fixatives, the material was kept in the 
fluid until the following morning and then washed for four hours in running 
water. It is most important that the sporangia should not be submitted to 
any sudden change throughout the subsequent processes to the final 
embedding in paraffin wax. The material was therefore either put into 
10 per cent, glycerine and evaporated slowly to pure, or it was run up by 
a continuous dropping method devised by Mr. Tabor into absolute alcohol. 
Cedar-wood oil was found to be the most satisfactory clarifying reagent. 
After several changes of absolute alcohol, the oil was pipetted into the 
bottom of the bottle, and the material from floating in a layer of absolute 
alcohol became gradually impregnated by the oil and sank. After a change 
into fresh cedar-wood oil the bottle was placed in the embedding oven and 
shavings of paraffin wax gradually added. In an hour the material was 
transferred to pure paraffin. Four hours in melted paraffin, with three 
changes gave excellent results. Most of the sections were cut at 3 [x , as it 
was found that the thinnest sections were the most useful for critical 
phases. 
A variety of stains have been used, but of these Breinl, Flemming’s triple, 
Heidenhain, gentian violet and orange, proved to be the most useful. 
