RELATION BETWEEN THE ALGA AND FUNGUS OF A LICHEN. 499 
stained sections with orange acetone, is not so easily withdrawn from those 
cells that are entire. This difference in coloration is also noticeable when 
using an aqueous solution of methyl green on fresh material for, within 
a few seconds, the daughter cells become bright blue, as do the hyphe, while 
the older cells remain green, unless kept in the stain for several days. In 
this case there is no cutting of cells, they are simply teased out of the 
hyphal network ; the older cell-wall resists the entrance of the stain much 
more than does the thin newly-formed wall of the young cells. 
In introducing his new triple stain, 1908 (1), Bonney says : ‘ The process 
is as Follows :— 
Solutions. 
ie Se Ge ee EE ‘25 gramme. 
Pyronin Ey cee ie sn 1:0 a 
Distilled water... «Sates. =. agen: gene Ce. 
V.B.—Make the solution by heat, and filter. Label * Methyl-Violet- 
Pyronin Solution.’ 
“2. To 100 c.c. of Acetone slowly add, drop by drop, from a drop bottle, 
a 2 per cent. aqueous solution of Orange G. (made by heat and 
filtered). 
“ Whilst adding the solution of the stain, keep stirring and rubbing the 
mixture with a glass rod. When the fluid has attained a pale yellow colour, 
a faint cloudiness appears. Further addition of the stain increases this 
until a floceulent precipitate is formed. Continue the addition of the 
Orange G. drop by drop, and this precipitate presently redissolves. Imme- 
diately this has taken place, filter into a bottle and label * Orange Acetone.’ 
Method. 
“1, Fix material in Acetic Alcohol (Glacial Acetic Acid F.P. 14: 77-15? C. 
1 part, Absolute Alcohol 2 parts). Alcohol, or Sublimate Solution, 
Chromates and Formalin render the method useless unless subsequent 
to staining the sections are treated by oxidising agents. 
Stain for 2 minutes in the Methyl-Violet-Pyronin solution. 
Wash rapidly in water and wipe all the slide dry except the section. 
Flood the slide with Orange-Acetone. A colour-cloud comes out. 
Pour off and flood again—when no more colour comes out. 
5. Wash rapidly in pure Acetone, and 
_ 6. Transfer to Xylol and mount.” 
The whole process should not take more than five minutes, and probably 
bo 
ee 
= 
less. 
` In using this triple stain we found the best results were obiained by a 
slightly slower process than that suggested for animal tissues, thus the 
LINN. JOURN.—BOTANY, VOL. XLIV. 28 
