VITAL STAINING OF AMGEBOCYTE 

 TISSUE OF LIMULUS. 1 



LEO LOEB AND KENNETH C. BLANCHARD. 



In our analysis of the factors underlying tissue formation, 2 it 

 was found necessary to make use of vital staining in order to 

 elucidate certain effects of environmental conditions on the cells 

 and on amoeboid movement. 



In the course of these investigations we made some observa- 

 tions on the staining of the amoebocyte ; these observations may 

 contribute to the understanding of the manner In which vital 

 stains enter cells and are fixed or held back within the cells. 



I. Neutral Red. Two methods were used: (i) In the first one 

 we prepared amcebocyte tissue in small stender dishes and re- 

 placed the supernatant serum by a solution of neutral red in a 

 n/2 NaCl solution of the strength of i 4000. After the tissue had 

 been acted on by the staining solution for from one to several 

 hours and had taken on a red color, it was used for tissue culture 

 experiments. The specimens were kept for variable periods of 

 time, usually in the icechest, but sometimes in the room, and 

 examined daily during the next two or three or more days. The 

 cells grew out of the piece in a centrifugal direction and gradually 

 extended in the manner described in previous papers. 2 The 

 pieces stained with neutral red either grew about as well as the 

 unstained control pieces, or in some cases they seemed to grow 

 slightly less well. In the amoebocytes, which had emigrated 

 from the piece, the neutral red stain was usually localized in one 

 or several droplets or particles in the interior of the cell. This 

 condition applied to the cells which had spread out in a hyaline 

 condition as well as to the more contracted granular cells. How- 

 ever, in some cases there were still some stained granules visible 

 and occasionally we could observe such a granule, which had 



1 From the Department of Comparative Pathology, Washington University, 

 St. Louis, and from the Marine Biological Laboratory, Woods Hole. 



2 Leo Loeb, Washington University Studies, 1920, VIII., 3. Science, 1919, I., 

 502. American Journ. Physiol., 1921, LVL, 140. Sicence, 1922, LVL, 237. 

 Leo Loeb and K. C. Blanchard, Amer. Journ. Physiol., 1922, LX., 277. 



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