506 PATHOGENIC MICROORGANISMS 



of the organisms is constant, though occasionally slightly larger 

 individuals are encountered. They are usually grouped separately, 

 though occasionally in pairs, end to end. 



Compared with the influenza bacillus in morphology, the bacillus 

 of pertussis is more regularly ovoid and somewhat larger. It has, 

 furthermore, less tendency to pleomorphism and involution. 



Staining. The Bordet-Gengou bacillus may be stained with al- 

 kaline methylene-blue, dilute carbol-fuchsin, or aqueous fuchsin solu- 

 tions. Bordet and Gengou recommended as a staining-solution 

 carbolated toluidin-blue made up as follows: 



Toluidin-blue 5 gms. 



Alcohol 100 c.c. 



Water 500 c.c. 



Allow to dissolve and add 500 c.c. of 5 per cent carbolic acid in water. 

 Let this stand one or two days and filter. 



Stained by the method of Gram, the bacillus of Bordet and 

 Gengou is decolorized. 



Cultivation. Early attempts at cultivation made by the discov- 

 erers upon ordinary ascitic agar or blood agar were unsuccessful. 

 They finally obtained successful cultures from sputum by the use 

 of the following medium : 



One hundred grams of sliced potato are put into 200 e.c. of 4 per 

 cent glycerin in water. This is steamed in an autoclave and a 

 glycerin extract of potato obtained. To 50 c.c. of this extract, 150 

 c.c. of 6-per-cent salt solution and 5 grams of agar are added. The 

 mixture is melted in the autoclave and the fluid filled into test tubes, 

 2 to 3 c.c. each, and sterilized. To each tube, after sterilization, is 

 added an equal volume of sterile defibrinated rabbit blood or prefer- 

 ably human blood, the substances are mixed, and the tubes slanted. 



On such a medium, inoculated with sputum, taken preferably 

 during the paroxysms of the first day of the disease, colonies appear, 

 which are barely visible after twenty-four hours, 3 plainly visible 

 after forty-eight hours. They are small, grayish, and rather thick. 

 After the first generation the organisms grow with markedly greater 

 luxuriance and speed. On the potato-blood medium after several 

 generations of artificial cultivation, they form a grayish glistening 

 layer which, after a few days, becomes heavy and thick, almost 



3 Wollstein, Jour. Exp. Med., xi, 1909. 



