244 BOTANICAL GAZETTE [APRIL 
to obtain the same result that may be had with the ordinary triple 
stain when properly used. He soaks his sections on the slide in a 
2 per cent. aqueous solution of tannin for 10-60 minutes, washes for 
a minute in water, puts them into a 1.5 per cent. solution of tartar 
emetic for 5-15 minutes, and then stains them, after washing in 
water, for about 30 minutes in an aqueous solution of gentian violet. 
By this procedure he gets what he calls an inverse staining, in which the 
starch is stained a brilliant violet while the cytoplasm and chromatin 
remain unstained. In order to stain the chromatin he suggests a 
nuclear stain previously with paracarmin in toto. This lengthy and 
troublesome process is entirely unnecessary however, for the triple stain 
used after Flemming’s fixing solutions gives sharply defined starch 
grains stained a brilliant blue, which is permanent for years, as our 
slides in use in the laboratory show, while the chromatin is a red or 
violet. This reaction of starch in the triple stain has been noted not 
only by TimBeRLakE for pyrenoid starch, but also more recently by 
DENNISTON (6) of this laboratory for the starch of the higher plants. 
Methods 
While Closterium is one of the commonest of the fresh water 
algae in this region, it does not frequently occur so abundantly that _ 
a cytological study by means of sections can be made of it. For 
the past three years, however, the bottoms and sides of the lily banks 
in the university greenhouses, as well as the larger filamentous algae, 
such as Oedogonium, growing on the bottom of the tanks, have been 
covered during the summer months by an abundant and almost 
pure growth of C. Ehrenbergii Menegh., and during the last spring 
with a mixed growth of C. Ehrenbergii and C. moniliferum Ehrenb. 
The Closterium was so abundant in these tanks that all that was 
necessary was to shake some of the plants to which they were attached 
in a vial of water. The Closterium would sink to the bottom and 
form there a layer of considerable thickness. ‘The water could then 
be pipetted or decanted off and the fixing fluid added. Washing the 
plants and changing the alcohols and paraffins were all done in the 
same way, either by decantation or by pipetting off the liquid above 
them. About twelve hours after the change to 52° paraffin the vial 
was set in ice water to harden the paraffin. The vial was then br oken, 
