Acton . — Observations on the Cytology of the Chroococcaceae . 437 
The results described above are based almost entirely on work done 
on filamentous forms. On comparing these with my own results, it appears 
that the structure of the cell as described by Guilliermond most nearly 
approaches the condition in the Chroococcaceae. 
III. Technique. 
The method of fixation employed naturally depended on the material 
to be examined. Both wet and dry methods were used. 
In the case of gelatinous forms like Aphanothece and Gloeocapsa wet 
smears were made on the slide and fixed and stained without drying. 
If fixed in bulk, the material was afterwards embedded and cut with 
a microtome, as the fixing reagents caused coagulation of the gelatinous 
material, making it difficult to spread out on the slide. 
Forms like Chroococcus were usually fixed by allowing a drop of the 
material to almost dry on the slide, and then fixing and staining in the 
ordinary way. These preparations were usually fixed in absolute alcohol. 
There was very rarely evidence of shrinkage or of plasmolysis in specimens 
fixed in this way, except in the earlier preparations of Chr. macrococcus , 
probably because the films were never allowed to become absolutely dry, 
and also possibly because the tough envelope afforded some measure 
of protection to the cell. Wet methods were always used as a control. 
Chroococcus turgidus and Chr. macrococcus were found in such enormous 
quantities among the flocculent material from certain sphagnum bogs that it 
was possible to fix in bulk, and also to embed and cut microtome sections of 
the material. 
The fixing reagents used were absolute alcohol, Flemming’s weak 
chrom-osmium-acetic, alcoholic-picric, alcoholic sublimate, and per cent, 
formalin. 
The thick outer covering of the cell proved a very great obstacle 
to satisfactory staining. Many stains, e. g. safranin and gentian violet, 
stained the membrane intensely, preventing examination of the cell contents, 
while others only penetrated with difficulty. 
The stains that gave the best results were Loeffler’s methylene blue, 
Delafield’s haematoxylin, and iodine-green-fuchsin as used by Kohl (’03). 
This combination stain was very difficult to manipulate, as the exact 
proportion of the two solutions required seemed to vary with the condition 
of the material and the species under investigation. Repeated trials had to 
be made in each case, but when the right proportions were found the results 
were excellent. It was never found to be profitable to take back through 
the stains, if overstained with one or the other reagent. With Loeffler’s 
methylene blue and Delafield’s haematoxylin, preparations were overstained 
and the excess stain washed out. 
