ON TECHNIQUE 55 



a colorless space in the film, this is not true in older cultures. Eisen- 

 berg has noted that with the Gram-negative bacteria there frequent- 

 ly appears a colored spot in the middle of the cell, which he inter- 

 prets as being due to plasmolysis, the shrinkage of the protoplasm 

 to the poles of the cell leaving a concavity in the center which be- 

 comes filled with dye solution; such colored portions were not ob- 

 served with Gram-positive organisms which could not be readily plas- 

 molyzed. I have also observed this, but in addition have noted that in 

 B. megatherium blue stained granules appeared in the cell during the 

 period of spore formation, corresponding in size and position with 

 the sporogenous granules which could be demonstrated in prepara- 

 tions stained with methylene blue, and also that stained granules 

 appeared in the preparations of the diphtheroid organism described 

 in Chapter VI, corresponding with the volutin granules demonstrated 

 by Ponder's stain. It would appear, therefore, that such structures 

 are stained by the Congo red with the technique employed. It is 

 hard to understand why such intensely basophilic granules should 

 stain with an acid dye like Congo red, while the remainder of the 

 protoplasm remains unstained. Ernst has also observed that the 

 granules which bear his name are stained by Collargol (which, ac- 

 cording to Eisenberg, behaves like Congo red), free granules con- 

 tained within vacuoles in mold mycelium becoming stained a dark 

 brown. There is, then, no question but that these colloidal nega- 

 tively charged dyestuffs can penetrate within the cells. 



With increasing age of the cultures it is also found that some of 

 the cells become uniformly stained throughout by the Congo red. 

 Because these stained cells become increasingly more numerous with 

 increasing age of the culture, I have concluded that they are dead 

 cells. But cultures killed by formalin or other fixative solutions do 

 not become stainable by the Congo red, and cultures killed by heat- 

 ing to boiling show only a faint staining after some time. Cul- 

 tures killed by heating to 60 degrees, however, show all of the cells 

 distinctly stained after standing several hours following the heat- 

 ing. It would appear, therefore, that the mere death of the cells 

 does not cause them to become stainable, but that the change in their 

 reaction towards Congo red is due to some process which occurs after 

 death, i. e., to beginning autolysis. 



