Page:Proceedings of the Royal Society of London Vol 60.djvu/209

Rh extruded sexual products, the female receptacles were placed in seawater, and after the complete liberation of the oospheres, a few male branches with ripe antherozoids were first placed in a capsule of sea water until it became turbid owing to their number. If on examination the antherozoids proved to be active, small quantities were added to the vessels containing the oospheres. The latter were then fixed at intervals of five minutes during the first hour, and then at intervals of fifteen minutes, up to six hours after the addition of the antherozoids. After that, samples were killed at longer intervals up to three days, and this was continued till we had material fixed at all stages for the first fortnight. At first we used sea water in which to keep the embryos growing, but a proper solution of Tidman’s sea salt was found to answer quite as well.

For fixing, we tried the following reagents—chrome alum, picric alum, Mann’s picro-corrosive, corrosive sublimate, and acetic acid; these were all dissolved in sea water, absolute alcohol, Flemming’s and Hermann’s solutions, and the vapour of osmic and formic acids. The Flemming’s (strong formula) and Hermann’s solutions were diluted with equal parts of sea water. The first three fixatives were unsuccessful, acetic-corrosive yielded fair nuclear figures, but the material proved very brittle, and the spores were somewhat distorted. A portion of the cytoplasm was disorganised and the polar radiations were not preserved. Absolute alcohol fixed the oospheres and newly fertilised spores without distortion, but was useless for all other stages. Vapour fixing with osmic acid succeeded better than any of the preceding reagents but was greatly inferior to either Hermann’s or Flemming’s solutions in preserving the protoplasmic structure in an unaltered state.

After the material had been fixed it was dehydrated and passed in the usual way into paraffin, the temperature of which was not allowed to exceed 50° C., and it was then cut with the microtome. ie sec ions were stained with Heidenhain’s iron-hsematoxylin, with Flemming s triple stain, and a large number of other dyes. The results which were compared carefully, led us to rely chiefly on the two staining processes mentioned, but at the same time we often obtained valuable preparations with other staining reagents as well.

In spite of repeated attempts, we have not succeeded in observing the first nuclear division in the oogonium, but the later ones hav^e been seen both m Fucus vesiculosus and in F. platycarpus, in which eight oospheres are formed. Oltmanns asserts that in o e c r n ^ 7 T ° ° !Pliert S ^ form ed, eight free nuclei and d > T, earh er Stage’ b u t th a t four of ^ e s e ultim ately abort, to on fi ueC° me CT r6S ° f 0611 form ation- O ur observations tend a fifth oT h m 5 “® r PeCt’ but We f°Und that in some case« fifth oosphere, smaller than the rest, was occasionally differentiated