1896-97-] THE GAMETOPHYTE OF BOTRYCHIUM VIRGINIANUM. 269 



of a slight depression in the moss. On removing some of the overlying 

 vegetation, numbers of the larger prothallia were easil}' obtained. It 

 required, however, careful sorting" of the peaty soil with the fingers to 

 secure the younger and more interesting stages. Nearly a week was 

 spent in working over about half the bed, the result being several 

 hundred examples in all stages of development, of the gametophyte and 

 attached sporophyte. Subsequently, in another season, a week was 

 spent on the spot, and all the plants which careful sifting of the soil 

 would yield, were removed. The second harvest amounted to over six 

 hundred specimens, by far the larger number of which, however, were 

 much too old for study. During the same summer, other and older 

 plants were found in rich woods about two miles back of Metis. In 

 the spring of 1896, additional discoveries were made in Foster's Flats, 

 below the Whirpool, on the Niagara River, and on the east branch of 

 the river Don, a few miles from Toronto. The last mentioned spot 

 proved rich in interesting examples of older stages of the attached 

 sporophyte. Most of these were removed last autumn (1897). 



III. 



One of the greatest difficulties in the way of the present research, 

 was the proper preservation of the prothallia. They are singularly 

 impermeable to fixing reagents on account of the thick external cuticle, 

 and must be cut at intervals with a razor, to allow the preserving 

 medium to penetrate. The presence of oil in large quantities in the 

 tissues, also renders aqueous fluids useless, as they scarcely make their 

 way in at all. A saturated solution of picric acid in thirty per cent, 

 alcohol, gave fairly good results ; but the best fixation was obtained by 

 using a mixture of three parts of a saturated solution of corrosive sub- 

 limate in ninety per cent, alcohol, and one part of saturated solution of 

 picric acid in the same menstruum, diluted with distilled water to 

 reduce the alcohol to thirty per cent, strength. The same reasons 

 which rendered the material hard to preserve, made it difficult to 

 embed. Paraffine was mainly used, and the most satisfying results 

 were obtained by infiltrating with benzole, in a vertical tubular dialyzer 

 with a chamois leather diaphragm, revolved slowly by means of clock- 

 work. It was found that the ordinary type of stationary dialyzer was 

 quite unsuitable for these very delicate objects. When the prothallia in 

 alcohol were placed in the top compartment, and the benzole below, the 

 osmosis was exceedingly slow ; and, if the position of the media was 

 reversed, the weight of the benzole carried it through too rapidly, and 

 injurious shrinkage was the result. The continued reversing of the 



