424 THE DIPHTHERIA BACILLUS GROUP 
or less pointed at the ends. Occasionally one end only is thickened, 
giving rise to a long, somewhat wedge-shaped rod. The distinctive 
morphology is best seen in eighteen- to twenty-four-hour gro^^•ths on 
Loffler's coagulated blood serum; organisms from growths on agar 
are more uniform in appearance. Diphtheria bacilli observed directly 
in diphtheritic membranes are also less pleomorphic than those from 
blood serum cultures. The organisms occur singly or in pairs, very 
uncommonly in short chains. The size is very variable, ranging from 
0.3 to 0.8 micron in diameter and from 1.5 to 6 microns in length. 
The organism as ordinarily seen in diphtheritic membranes is about 
0.6 micron in diameter and about 2 to 3 microns in length. Branched 
forms are occasionally seen, particularly in the pellicle which forms on 
old plain broth cultures. 
The stainable substance of the organisms is not uniformly dis- 
tributed, but occurs in somewhat irregular concentration, gi^'ing rise 
to three rather distinct types of bacilli: the granular, the barred, and 
the solid. 1 Metachromatic granules (Ernst-Babes granules) are also 
present, and, according to Williams, the diphtheria bacillus reproduces 
by fission at one of these granules. It was originally supposed that 
the metachromatic granules were only found in virulent strains and 
that the non-A'irulent strains had no granules. Neisser^ invented a 
stain which brings out these granules very sharply.'' It is now known 
that the granules are not necessarily related to virulence, conse- 
quently the Xeisser stain is rarely used. Diphtheria bacilli stain 
well with the ordinary anilin dyes and very characteristically with 
Loffler's methylene blue. With methylene blue the granules above 
mentioned are brought out very sharply, and it is observed that these 
granules exhibit the phenomenon known as metachromatism, that is, 
they stain mahogany-red while the rest of the organism stains blue. 
The granules may be seen also with dark-field illumination. The 
Albert stain (see page 201) is the best for demonstrating these granules, 
however. Diphtheria bacilli are Gram-positive, but prolonged washing 
with alcohol removes the Gram-positive stain. Cultures prepared 
directly from diphtheritic membranes stain more uniformly than 
organisms obtained from cultures on Loffler's blood serum. 
1 Westbrook, Wilson and McDaniel: Jour. Boston Soc. Med. Sci., 1900, 4, 75; 
Trans. Assn. Am. Phys., 1900. 
2 Ztschr. f. Hyg., 1897, 24, 443. 
' The stain is prepared in the following manner: 
A. ^Methylene blue (Griibler's) 1 gm. 
Alcohol, 96 per cent 20 cc. 
Glacial acetic acid 50 cc. 
Distilled water 950 cc. 
B. — Bismarck brown ... 1 gm. 
Distilled water 500 cc. 
The smear, fixed in the flame in the usual manner, is covered with solution A for 
three to five seconds, washed in water, then covered with B for three to five seconds. 
After thorough washing in water, the preparation is ready for microscopic examination. 
The granules are stained blue, the bodies of the bacilli brown. 
