192 BOTANICAL GAZETTE [SEPTEMBER 
a o.3 gm. molecular sugar solution. This strength of solution is 
still low enough to allow the cells to become fully turgid, and was 
found satisfactory for a number of species. Doubtless the strength 
of solution required will vary somewhat with the species and with 
the condition of the material. ; 
KILLING AND STAINING.—After removal the embryos may be 
transferred to the killing fluid by means of a pipette with a 2 mm. 
opening. A good fixing agent is 6 per cent formalin in 50 per cent 
alcohol, and it is at the same time an excellent preservative in which 
they may be kept indefinitely, but aqueous formalin alone is not 
satisfactory. The embryos should be washed through several 
changes of water before staining, and may be transferred directly 
to water from the solution. The staining was done in saltcellar 
watchglasses with Delafield’s haematoxylin or safranin. The 
haematoxylin was used for most of the preparations and was 
diluted to one-half of its usual strength. The water is removed 
with a pipette and a few drops of the stain applied for 5—10 minutes, 
which will stain them very deeply. At this point one of the most 
difficult steps is encountered, namely, to prevent losing the material 
while the stain is being removed. It was found best to dilute the 
stain with water until the watchglass is full. The upper layers of 
the solution may now be removed without disturbing the embryos 
at the bottom, but great care must be exercised to prevent losing 
the embryos, and the material should be watched as the pipette 
is filled by holding it over an illuminated white surface, as on the 
stage of a block dissecting microscope. More water is added and 
the operation repeated until the liquid is clear. : 
The overstained embryos are now de-stained with acidulated 
water (about one drop of HCl per 200 cc. of water). The stain is 
extracted slowly and must be watched over a low power micro- 
scope. The de-staining should be continued until the cytoplasm 
is well differentiated from the nucleus in the embryonal cells 
at the tip, and the suspensor cells should still be slightly blue. 
Very thorough washing is necessary to remove all traces of the 
acid or the preparations will fade. If safranin is used, it is 
advisable to overstain and then extract the stain to the desired 
point. 
