PICRO-NIGROSIN, A COMBINATION FIXATIVE AND STAIN 
FOR ALGAEi 
Otis F. Curtis and Reginald H. Colley 
Pfitzer (i) used a saturated aqueous solution of picric acid, to which 
he added a varying amount of an aqueous solution of nigrosin, to kill 
and stain minute forms, such as diatoms and small algae, under the 
cover glass. Freeborn (2), Pianese (3), and Johnston (4), and other 
zoological investigators have employed picro-nigrosin alone, or in 
combination with other stains, with excellent success, but there is 
little reference to its use by botanists. The writers, by independent 
study, have found it a valuable stain for filamentous algae and for 
younger tissues of forms with denser structure, and believe that the 
results of their experiments, which confirm Pfitzer's results in prac- 
tically every detail, are of sufficient interest to warrant this brief note. 
The combination fixing and staining solution, which may be used 
several times without apparent deterioration, was prepared by satu- 
rating a saturated aqueous solution of picric acid with Gruebler's 
water-soluble nigrosin. Material was placed in this solution for from 
three to twenty-four hours, rinsed in water to remove the excess 
stain, and run through the grade alcohols with half hour intervals, to 
glycerine, clove oil, Venetian turpentine or balsam. The general 
result, regardless of variations in the method after the material has 
been run through the alcohols, is a blue-black coloration of the nucleus 
and nucleolus, a faint blue in the pyrenoid and cytoplasm, an almost 
colorless chroma tophore, and a colorless cell wall. The stain is un- 
usually transparent, difTerentiating the nuclear structure in cells and 
tissues with dense cytoplasm. For immediate class use, algae were 
studied in 70 per cent alcohol or transferred to a mixture of alcohol 
and glycerine which served to clear fairly well and prevented the mount 
from drying up during the laboratory exercise. Some of the best 
results were obtained with material transferred from absolute alcohol 
to clove oil and examined in the latter. Excellent permanent mounts 
were made by following Chamberlain's Venetian turpentine method 
^ Contribution from the Marine Biological Laboratory, Woods Hole, Massa- 
chusetts. 
