On Fertilisation, and the Segmentation of the Spore in Fucus. 189 



extruded sexual products, the female receptacles were placed in sea- 

 water, 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, smalt 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 dis- 

 torted. 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. 

 The sections were stained with Heidenhain's iron-heematoxylin, 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 have 

 been seen both in Fucus vesiculosus and in F. platy carpus, in which 

 eight oospheres are formed. Oltmanns asserts that in Ascophyllum, 

 in which only four oospheres are commonly formed, eight free nuclei 

 occur at an earlier stage, but that four of these ultimately abort, 

 and do not become centres of cell formation. Our observations tend 

 to confirm him in this respect, but we found that in some cases 

 a fifth oosphere, smaller than the rest, was occasionally differentiated, 



P 2 



