632 Davis.—Cytological Stic dies on Oenothera. 11. 
Should certain of these hybrids or others of later seasons prove of 
sufficient interest in the second generations this Woods Hole strain of 
biennis , which was constant in cultures of 1909, will be described in some 
detail. For present purposes, however, it will be sufficient to state that the 
strain is representative of the green-stemmed (lacking red dots), broader- 
leaved types of biennis with medium-sized flowers. The main stem grows 
to a height of about 1 m., and long side branches arise from near the base 
of the plant. The leaves on the lower portions of the stem are elliptical, 
the larger about 14 cm. long and 4 cm. broad. The inflorescence is 
characteristic of biennis in having bracts about § as long as the flowers. 
The petals are about 1*3 cm. long. 
Methods. 
Considerable attention was given during the summer of 1909 to the 
problems of fixation, especially to test the effects of Carnoys alcohol-acetic 
and chloroform-alcohol-acetic mixtures which Overton (’ 09 ) found so 
satisfactory for certain types. The chloroform-alcohol-acetic mixture 
caused such shrinkage, even when used for so short a time as ten minutes, 
that it was worthless for the stages of synapsis and later. The alcohol-acetic 
mixture was less violent in its action, but nevertheless proved for the same 
reason to be unsatisfactory for older cells where there is a large amount of 
cell-sap in cytoplasmic vacuoles. These two fluids of Carnoy have, 
however, a value for Oenothera in the study of presynaptic stages, where 
the cells are relatively small and the cytoplasm dense, because of the sharp 
staining quality of the chromatin with haematoxylin. 
The most satisfactory fixation was obtained with chrom-osmo-acetic 
fluids when due precautions were taken to facilitate rapid penetration. To 
attain this necessary end the anthers, freed from floral envelopes but still 
attached in a group to the ovary, should be thoroughly brushed with water 
before immersion in the fixing fluid. Satisfactory results were also obtained 
when such groups of anthers were dipped for a few seconds in Carnoy’s 
alcohol-acetic mixture before immersion in the chrom-osmo-acetic acid; 
in this procedure the former fluid appears to prepare the surface of the 
anthers so that the latter fluid is able to penetrate more quickly. Experience 
has shown that the formula known as strong Flemming (1 per cent, chromic 
acid 75 c.c., 2 per cent, osmic acid 20 c.c., glacial acetic acid 5 c.c.) gives 
somewhat better results than the weaker fluids used in the study of 
O. grandiflora (Davis ’ 09 , p. 552). There is another advantage in employing 
rather strong fluids since material appears to cut better after their use, 
perhaps because the acids may free the tissue from raphides. 
As in the previous study of Oenothera grandiflora iron-alum 
haematoxylin proved to be the most satisfactory stain. 
