202 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
4. A large loop of the supernatant fluid is placed on a clean, warm 
slide, and allowed to evaporate spontaneously. Drying may be advan- 
tageously hastened in the 37° C. incubator. 
5. The mordant is added as follows: 1 part of stain and 4 parts of 
distilled water are mixed, allowed to stand one minute, and filtered 
directly upon the film. After one minute, w^hen a slight bronzing of 
the film should be seen, the stain is washed off with tap water. 
6. The film is flooded with carbol fuchsin and allowed to stand for 
five minutes. 
7. Wash dry, and examine. 
F. DifEerential Stains for Bacteria.^ — 1. Gram Stain.'^—A most 
important difterential method of staining bacteria is that of Gram. 
Bacteria may be divided into two groups: those which retain the 
initial stain — Gram-positive organisms— and those which fail to retain 
the initial stain but color with the counter stain— Gram-negative 
bacteria. 
It was believed formerly that the organisms which retained the 
initial stain— the Gram-positive bacteria— contained within their 
protoplasm, a substance of unknown composition which united 
chemically with gentian violet (or other pararoseaniline dye) and 
iodine to form a compound relatively insoluble in alcohol. Gram- 
negative bacteria did not contain this hypothetical substance, which, 
in association with the dye and iodine, was insoluble in alcohol. Treat- 
ment of the latter group with alcohol, therefore, would remove the 
gentian violet, leaving them unstained. In the unstained condition 
the organisms were colored with the second or counter stain. Subse- 
quent investigation has largely discredited this view. It has been 
showTi by Kruse^ that the cytoplasm of Gram-positive bacteria is 
more resistant to autolysis, to the action of trypsin, and to solution in 
dilute KOH than that of Gram-negative organisms, probabl}' because 
the cytoplasm of the former is less permeable to these various reagents 
than is that of the latter. Eisenberg* has advanced the hypothesis 
that Gram-positiveness is due to the lipoidal content of the cell mem- 
brane (ectoplasm) and specifically to unsaturated fatty acids and 
phosphatids. Jobling and Petersen^ also state that Gram-staining 
bacteria are richer in unsaturated fatty acids. The addition of iodine, 
according to this theory, through the formation of alcohol-insoluble 
combinations with the lipoids in the ectoplasm, renders the cell wall 
impermeable to alcohol and thus prevents removal of the dye which 
has already penetrated into the cell contents. 
1 For a discussion of various modifications of the Gram stain, see Hucker and Conn: 
Tech. Bull. No. 128, July, 1927; New York Agr. Exp. Sta.; also Steam, A. E. and E. W.: 
Jour. BacterioL, 1924, 9, 463, 479, 491. 
•^ Gram: Fortschr. d. Med., 1884, 2, 185. 
3 Miinchen. Med. Wchnschr., 1910, 57, 685. 
* Centralb. f. Bakteriol., orig., 1909, 49, 465; 51, 115; 1910, 53, 481 551; 56, 183. 
5 Jour. Exp. Med., 1914, 20, 321. 
