LEO LOEB AXD KEXXETH C. BLAXCHARD. 



Effect of Heating Amcebocyte Tissue on the Extraction of Stain. 



In various experiments amoebocyte tissue was heated to a 

 temperature varying in different experiments between 60 and 75 

 for fifteen minutes. This temperature is sufficient to kill the 

 cells; at the same time the heated tissue becomes soft. If such 

 a heated tissue is stained with neutral red and pieces of the 

 stained tissue are extracted with acid, alkali and neutral solutions 

 of sodium chloride in the usual manner, stain is apparently given 

 off in all three solutions. However, if we centrifuge these 

 solutions, we find that in reality only the acid solution w r as 

 capable of extracting stain from the tissue. In the neutral and 

 alkaline solutions the stain had not actually been extracted, but 

 the soft state of the heated tissue had rendered possible, in the 

 three solutions, the distribution of fine particles of tissue, which 

 were stained and this suspension of stained particles of tissue 

 simulated a real extraction. Amcebocyte tissue killed through 

 heating behaves therefore towards the extraction of neutral red 

 stain like living tissue. 



The same results are obtained if we stain heated tissue which 

 had been exposed to acid or alkaline solutions previous to the 

 heating; again, only the acid extracts the stain. 



If previously heated amcebocyte tissue is stained with eosin, 

 instead of with neutral red, it is necessary to repeat the process of 

 washing the stained tissue in n/2 NaCl about 40-50 times in order 

 to remove a surplus of stain which adheres to the tissue. Tissue 

 thus prepared gives off the stain most readily in an alkaline 

 medium, somewhat less in a neutral, and no stain is given off in an 

 acid medium. The tissue behaves therefore in this respect like 

 living unheated tissue. An exposure of the tissue to acid or 

 alkaline solutions previous to the staining does not alter this result. 



Extraction of Stain from Stained Eggs. 



Unfertilized eggs of Asterias were stained with neutral red, 

 centrifuged and thoroughly washed with a solution of n/2 NaCl. 

 Samples, each of two cc., of such an egg suspension were treated 

 with an isotonic acid (w/iooo HC1) or isotonic alkaline (w/iooo 

 NaOH) solution. The suspension was shaken, centrifuged, the 

 supernatant fluid pipetted into test tubes, and the volume and 

 hydrogen ion concentration were made the same in all the tubes 



