ADDITIONAL NOTE ON WHOOPING-COUGH BACILLUS. 485 



surface. It is therefore well to make cultures in a flask with a 

 large flat bottom and to employ not more than a centimeter deep 

 of fluid. An excellent culture medium is made by a mixture of 

 1 per cent pepton bouillon containing 1 per cent glycerin and equal 

 parts of horse serum, the latter preferably heated for three-quarters 

 of an hour to 57 degrees. Under these conditions the organism grows 

 almost without clouding the fluid ; in 4 or 5 days the bottom of the 

 flask is covered with a whitish, slightly viscous, and rather thick 

 sediment. The supernatant fluid becomes more cloudy, and the 

 deposit less coherent if rabbit serum is employed instead of horse 

 serum, as the latter has a certain agglutinating property for the 

 organism. 



If, instead of using normal horse serum (57 degrees) for the 

 culture medium, we use the serum of a horse that has been im- 

 munized against the organism (also heated to 57 degrees), growth 

 still takes place. The bacteria, however, are more agglutinated 

 and show a more abnormal appearance; they grow in the form of 

 strepto-bacilli, or may even appear as streptococci in long chains, 

 their appearance, in short, being quite abnormal and resembling 

 in no particular the morphology on solid media. 



Immunization of a horse gives rise to an extremely agglutinating 

 serum.* If a two-day growth of culture on blood agar is sus- 

 pended in 2 or 3 c.c. of salt solution, and the organisms allowed to 

 diffuse in the suspension without much agitation, we obtain a cloudy 

 fluid of homogeneous and almost colloidal appearance. This emul- 

 sion is agglutinated by traces of the active serum; for example, agglu- 

 tination takes place on the addition of 0.002 of a cubic centimeter of 

 serum to 1 c.c. of the emulsion. Too great an excess of agglutinin 

 inhibits the phenomenon; the whooping-cough bacillus, indeed, 

 may serve as a good example to study this inhibiting power of 

 an excess of serum, which has already been noted by various 

 observers. 



The various strains of whooping-cough bacilli obtained from 

 different cases of the disease agglutinate variably. We find, for 

 example, that our horse serum agglutinates the organism that was 



* Our horse received in 15 successive injections, for the most part subcutane- 

 ously, but some intravenously, a total of about two liters and a half of rich fluid 

 culture (glycerinated bouillon and horse serum) . 



