THE BACILLUS AXD THE BACTERIOLOGY OF DIPHTHERIA 191 



If the colonies develop deepen the substance of the agar they are 

 usually round or oval, and, as a rule, present no extensions; but if near 

 the surface, commonly from one, but sometimes from both sides, they 

 spread out an apron-like extension, which exceeds in surface area the 

 rest of the colony. When the colonies develop entirely on the surface 

 they are more or less coarsely granular, and usually have a dark centre 

 and vary very much in their thickness. The colonies from some are 

 almost translucent; others are thick and almost as luxuriant as the 

 staphylococcus. The edges are sometimes jagged, and frequently shade 

 off into a delicate lace-like fringe; at other times the margins are more 

 even and the colonies are nearly circular. With a high-power lens the 

 edges show sprouting bacilli. The colonies are gray or grayish-white 

 by reflected light, and pure gray with an olive tint by transmitted light. 



The growth of the diphtheria bacillus upon agar presents certain 

 peculiarities which are of practical importance. If a large number of 

 the bacilli from a recent culture are implanted upon a properly prepared 

 agar plate a certain and fairly vigorous growth will always take place. 

 If, however, the agar is inoculated with an exudate from the throat, 

 which contains but few bacilli, no growth whatever may occur, while 

 the tubes of coagulated blood serum inoculated with the same exudate 

 contain the bacilli abundantly. Because of the uncertainty, therefore, 

 of obtaining a growth by the inoculation of agar with bacilli unaccus- 

 tomed to this medium, agar is not a reliable medium for use in primary 

 cultures for diagnostic purposes. A mixture composed of two parts of 

 a 1.5 percent, nutrient agar and one part of sterile ascitic fluid makes 

 a medium upon which the bacillus grows much more luxuriantly, but 

 not so characteristically. The mixture is made by adding ascitic fluid , 

 warmed to about 45 to 50 C., to the tubes containing the melted agar 

 cooled to 60 C. After shaking, the Petri plates are filled. 



Isolation of the Diphtheria Bacillus from Plate Cultures. Nutrient 

 plain or glycerin-agar is the medium employed to get by plating methods 

 a pure culture from the original serum tube. The agar should be freshly 

 melted and poured in the Petri dish for this purpose. After it has hard- 

 ened, the layers in a number of plates are streaked across with bacteria 

 from colonies on the serum culture, which appear in size and color like 

 the diphtheria bacilli. Other plates are made from a general mixture 

 of all the bacteria, selected, as a rule, from the drier portion of the 

 serum. Other plates are inoculated from the pellicle of an ascitic broth 

 culture. The plates are left in 'the incubator for about sixteen hours at 

 37 C. In the examination of the plates one should first seek for typical 

 colonies, and then later for any that look most nearly like the character- 

 istic picture. Diphtheria colonies are very apt to be found at the edges 

 of the streaks of bacterial growth. 



GROWTH IN BOUILLON. The diphtheria bacilli from about one- 

 half the cultures grow readily in broth slightly alkaline to litmus; the 

 other cultures grow very feebly. The characteristic growth in neutral 

 bouillon is one showing fine grains. These deposit along the sides and 

 bottom of the tube, leaving the broth nearly clear. A few cultures in 



