82 STAINING TECHNIQUE 
Gram. positive bacteria forms a compound between the protoplasm 
of the cell and the dye which is insoluble in alcohol. In the Gram 
negative bacteria this compound is soluble in the alcohol and broken 
up. Kruse (1910) attempted to cxplain this stain by stating that the 
Gram positive bacteria are more resistant to autolysis, solution in 
strong alkali, etc., than the Gram negative bacteria. Hisenberg (1909) 
assumed that the differential Gram staining rested upon the presence 
of some special compound such as unsaturated fatty acids or phos- 
phatids in the cell membrane. These formed a compound with iodine 
which renders the cell wall impermeable to alcohol. 
Dreter, Kriegler and Walker (1911) in seeking an explanation for 
this stain proposed the presence of lipoidal substances in the Gram 
positive bacteria which bind the pararosaniline compounds. This, 
however, has little foundation according to our present knowledge. 
Brudny (1908) regarded differences in the permeability of the cell 
membrane as the important factor. With the Gram positive bac- 
teria the membrane is more easily penetrated by the iodine solution. 
The structure of thése bacteria is probably “ looser’”’ and the stain 
gains an easy entrance. The Gram negative bacteria, however, are 
not penetrated and no deposit of the dye takes place on the interior 
of the cell. Benians (1912) places the explanation of this stain on a 
definite cell membrane. This prevents the removal of the dye by the 
alcohol while Hottinger (1916) explains it from the standpoint of col- 
loidal chemistry. He states that the relation of the bacteria to this 
stain depends upon the degree of dispersion of the nucleoproteins. In 
the Gram negative bacteria, the stained nucleoproteins are so widely 
dispersed that they are not seen. In the Gram positive bacteria the 
stained nucleoproteins form a coarse emulsoid. Gram positive bacteria 
are sald to become Gram negative when the degree of dispersion is 
mcreased by the action of proteolytic enzymes. Gram positive bac- 
teria resist ferment action longer than Gram negative. Weinkopff 
(1911) in studying tryptic digestion of bacteria concluded that the 
difference between the Gram positive and Gram negative bacteria 
rested in the penetrability of cell protoplasm. Dreter et al. (1912) 
extracted a substance from Gram positive bacteria (staphylococci) 
which when applied to Gram negative B. colt made them Gram positive 
in many cases. When this ether extract was dried on a slide, the 
residue took the Gram stain. This investigator also treated B. coli 
with lecithin which made them Gram positive. That the Gram nega- 
tive bacteria are structurally different from the Gram positive bacteria 
seems to be indicated by the work of Larson, Hartzell and Diehl (1918). 
