METHODS FOR MICROSCOPIC STUDY OF BACTERIA 203 
Benians^ has studied the factors underlying]; the principle of the Gram 
stain and has drawn the following conclusions with reference to it: 
1. The factors underlying Gram's stain are inherent in the physical 
structure of the bacterial cell, and are not conferred on it by the 
application of the mordant, although they are brought out more easily, 
as regards both positive and negative characters, in that way. 
2. There is nothing in the nature of a chemical fixation of the com- 
pound dye to the bacterial substance of those organisms that retain 
the stain when the cell is intact, and the amorphous bacterial sub- 
stance of either positive or negative organisms shows no marked 
difference as regards the extraction of the dyes by alcohol. 
3. The effect of the mordant is to dissociate the dye from its adsorp- 
tion compound with the tissues, forming with it a large compound- 
molecular body, which in alcoholic solution does not easily pass out 
of the Gram-positive bacteria. 
4. The capacity for retaining the compound dye in Gram-positive 
cells is related to the physical characters of the cell, and is chiefly 
dependent on the structure and integrity of the limiting membrane. 
5. The essential character of the Gram-positive cell membrane is 
that it does not readily permit the contained large, compound dye- 
iodine molecule in alcoholic solution to pass through it. 
6. It is probable that the imbibition of water from weak alcoholic 
solutions alters the permeability of the membrane of the Gram-positive 
cell, and also permits the dye to escape under these conditions. 
7. The Gram-negative bacteria here examined are of two types as 
regards their cell membranes: (a) Those that allow the attachment 
and probable permeation of methyl-violet and victoria blue; and {b) 
those to which these dyes are only peripherally adsorbed. The 
former, represented by the gonococcus, approximate to the Gram- 
positive organisms insofar as they contain the dye, but the absence of 
the specific cell membrane does not permit of the retention of the dye 
when alcohol is applied. The latter are represented by the coliform 
bacilli. 
8. Gram's stain, while it gives evidence of a certain type of cell 
membrane in the Gram-positive bacteria, is likely to be misleading 
if it is assumed that all those bacteria that fail to retain the stain 
are necessarily similar in structure to each other. It is more likely 
that the apparently permeable Gram-negative bacteria have more 
in common with the Gram-positive than the impermeable Ciram- 
negative type. 
Preparation of Gram Stain: 
Solution A — Saturated aqueous solution of anilin^ 90 cc. 
Saturated alcoholic solution of gentian violet . . 10 cc. or 
Five per cent aqueous solution of carbolic acid ... 90 cc. 
Saturated alcoholic solution of gentian violet . . 10 cc. 
1 Jour. Path, and Bacteriol., Cambridge, 1920, 23, 401. 
'^ Three cc. of anilin oil are shaken for several minutes in 100 cc. of distilled water. 
The solution is filtered through filter paper to remove the undissolved anilin. 
