286 LEO LOEB AND KENNETH C. BLA\< HARD. 



unstained piece, each respected the area of the other, but a 

 number of red cells grew into the unstained area in a direction con- 

 trary to the centrifugal direction of the majority of unstained 

 cells, and some of the latter grew similarly into the red area. 

 Sometimes the cells moved until they reached the other piece and 

 finding here resistance they turned around and moved back in the 

 direction of their own piece. The cells were able to extend in the 

 strange area and generally underwent the same changes as in 

 their own territory. On the whole, the cells wandering out from 

 the unstained piece remained unstained and did not take up 

 neutral red, w T hich may have become dissolved in the solution 

 surrounding the pieces; but in a few instances, it is probable 

 that such a secondary staining may have occurred in a few 

 cells. 



2. In the second method, we allowed tissue to grow out into 

 the surrounding fluid, according to the cover glass tissue culture 

 method, and after a sufficient layer of tissue had thus had a 

 chance to form, we poured off the fluid surrounding the cells and 

 replaced it by an isotonic solution of neutral red in sodium 

 chloride. After this staining solution had acted upon the cells 

 for from one to several minutes, we replaced it by a new solution 

 which was free from stain. We have discussed the results thus 

 obtained in another connection 3 and w r e shall here merely sum- 

 marize some of our observations. 



Almost instantaneously the neutral red penetrates into the 

 cells and stains the granules red brown. In case the tissue had 

 previously grown out in Limulus serum, the granules of the 

 amcebocytes stain more deeply than if the tissue had grown out in 

 a solution of sodium chloride. 



Gradually the granules begin to lose their stain; instead of 

 adhering to the granules, the stain begins to collect in the interior 

 of the cells in the form of droplets or particles which are identical 

 with those described above. If we add a weak acid (n/iooo HC1) 

 to the outgrown and previously stained tissue, the granules lose 

 their stain almost immediately. Furthermore, very soon the 

 cells contract in this medium and cease to show amoeboid 

 activity. If we replace the acid by a weak alkali, the granule 

 stain usually returns at least in the peripheral cells and a typical 



3 Am. Journ. Physiol., 1924, Vol. 67, 526. 



