Cultivation 43 1 



I. Dahliaviolet i II. Methylgreen i 



Alcohol (90 per cent.) lo Alcohol (go per cent.) lo 



Water to loo Water to loo 



Of the solutions, one part of I is mixed with three of II and the fixed spread 

 stained for two minutes without warming. 



The Neisser* method of staining the diphtheria bacillus, to show 

 the metachromatic granules, is as follows: 



The prepared cover-glass is immersed for from two to three 

 seconds in 



Alcohol (96 per cent.) '. 20 parts 



Methylene blue i part 



Distilled water 950 parts 



Acetic acid (glacial) 5° parts 



Then for three to five seconds in 



Bismarck brown i part 



Boiling distilled water 500 parts 



Parkj found that neither the Neisser nor the Roux stain gave any 

 more information concerning the virulence of the bacilli than the 

 LofBer alkaline methylene blue. 



The bacilli stain well by Gram's method, which is excellent for 

 their definition in sections of tissue, though Welch and Abbott found 

 that Weigert's fibrin method and picrocarmin gave the most beau- 

 tiful results. 



Cultivation. — The diphtheria bacillus grows readily upon all 

 the ordinary media, and is very easy to obtain in pure culture, plates 

 not being necessary. It is almost purely aerobic. It grows at tem- 

 peratures ranging from 2o°C. to 4o°C., the optimum being 37°C. 

 To secure it a sterile swab or a platinum loop is introduced into ■ 

 the mouth of a patient suffering from diphtheria, and brought into 

 contact with the false membrane, after which it is immediately rub- 

 bed over the surface of a tube of Loffler's blood-serum mixture. 

 After twelve to eighteen hours in the incubator, the diphtheria bacilli 

 win usually be found to have outgrown all other micro-organisms, and 

 appears in scattered, rounded, cream-colored colonies or as a conflu- 

 ent surface growth. Transplantation to other media for further 

 study in pure culture can usually be effected by transplanting a 

 colony. 



Colonies. — Upon the surface of gelatin plates the colonies attain 

 but a small size and appear to the naked eye as whitish points with 

 smooth contents and regular, though sometimes indented, borders. 

 Under the microscope they appear granular and yellowish-brown, 

 with irregular borders. Upon agar-agar and glycerin agar-agar the 

 colonies are slower to develop, larger, more translucent, without the 



* "Zeitschrift fiir Hygiene," 1897, xxiv, 443. 



t "Bacteriology in Medicine and Surgery," igoo. 



