BACTERIOLOGY OF WHOOPING-COUGH 9 



more remarkable, since his bacillus grows readily on all ordinary 

 media. He very kindly sent ;ne a culture of his organism, which 

 I was thus able to compare with the various organisms found in 

 pertussis sputa of the cases reported in this paper, and in not one 

 instance have I been able to obtain an identical organism. It is an 

 entirely different organism from the influenza-like bacillus. 



Summing up, one seems justified in concluding that, with the 

 exception of Manicatide, probably all of the investigators, at least in 

 more recent years, have been dealing, either in pure or in impure 

 cultures, with the influenza-like bacillus first described by Spengler 

 and later by Jochmann. 



III. PERSONAL OBSERVATIONS. 



In undertaking a study of the bacteriology of whooping-cough, 

 my attention was naturally directed to the influenza-like bacilli. 

 In the examinations, however, careful observations were made upon 

 other organisms present with any degree of constancy. 



Technique and material. The sputum coughed up during a paroxysm was obtained 

 in a sterile Petri dish and examined as soon as possible both in smear and culture. As 

 a rule the particle of sputum was washed in several successive dishes of sterile water 

 to remove the contaminating mouth organisms. A part of this was then used for 

 smears and a part for culture. Two smears were made, one of which was stained 

 with Loeffler's methylene blue, and the other with Gram's stain. As a rule I found 

 it useful to counterstain the latter with a very dilute solution of carbolfuchsin. In 

 this manner the Gram-staining and non-Gram-staining organisms could be very 

 clearly differentiated, the latter, which are so numerous in pertussis sputum, taking 

 a faint red stain, while the former appear deep blue or black. 



The portion of sputum used for culture, after washing, was introduced into plain 

 broth and thoroughly agitated; four tubes of melted agar at a temperature of 43 C., 

 to which six drops of defibrinated blood had been added, were inoculated with varying 

 quantities of the washed sputum and then plated. In a few of the cases sputum could 

 not be obtained, and swabs of the throat were used. After 24 and also after 48 hours 

 the plates were carefully examined and the various organisms present recorded. 



Pigeon-blood agar was used in about one-half of the examinations. In this it 

 was found that the influenza-like organisms, as a rule, grew more abundantly, and the 

 colonies were larger. However, it is not essential, as they will develop on any hemo- 

 globin media. The pigeon-blood is obtained by passing the needle of a Luer syringe 

 into the heart of the bird at a point just above the large breast-bone under the left 

 wing. About 5 c.c. may be obtained at one time from the heart without injuring 

 the pigeon. This blood is at once defibrinated with a wire, and then may be used 

 immediately, or kept in the ice-box for future use. 



Bacteriology 0} the cases. The sputum of 61 cases of pertussis 

 have been studied in smear and culture. In most cases one specimen 



