AMOEBAE VERSUS TISSUE CELLS 6 



of the fibroblasts lived twenty-four hours after the introduction 

 of the amoebae and the bacteria into the tissue culture, though 

 most of the fibroblasts pulled in their processes and contracted 

 into small oblong masses within a few hours after the introduction 

 of the amoebae and bacteria. Later they went to pieces, while 

 the amoebae moved about quite normally in this new medium 

 (Locke-Lewis), and lived for over two weeks, when the experiment 

 was discontinued. 



REACTIONS TO STAINS 



Neutral red 



In trying out the effect of vital stains on the cells it was found 

 that a very weak solution of neutral red was sufficient to give an 

 excellent stain. When neutral red 1 to 10,000, the proportion 

 used for tissue-culture cells, was used with the amoebae, they 

 would immediately round up, stain red, and die. This solution 

 was sometimes allowed to dry on a cover-slip, which was then 

 put down on the drop of Locke or normal salt solution containing 

 the amoebae. The neutral red would gradually diffuse into the 

 medium and stain the neutral-red granules and vacuoles of the 

 amoebae without immediately killing them. Experiments with 

 neutral red of different strengths were made; 1 to 40,000, 1 to 

 60,000, and 1 to 80,000 in Locke solution were all good and not 

 sufficiently strong to kill the animals. Amoebae were kept in 

 hanging drops of Locke solution with neutral red 1 to 40,000 for 

 nine days; at the end of this time they were moving slowly and 

 had a few neutral-red granules. In another set of experiments 

 the amoebae were kept in Locke solution with neutral red 1 to 

 80,000 for twenty-four hours. At the end of this time most of 

 them had lost the stain, so Locke with neutral red 1 to 60,000 was 

 added, and they continued active with stained neutral-red gran- 

 ules for seven more days, when the experiment was discontinued. 

 This is especially interesting, as in tissue cultures the disappear- 

 ance of the color from the neutral-red granules was always taken 

 as an indication that the cells were dead. With the amoeba 

 after the color has disappeared the granules can be restained with 

 neutral red and the amoeba is still normally active. 



