436 THE SALMONELLA GROUP 



pathogenic organism, immunization is most successfully carried out by 

 inoculating the animal thus 



1st day, - O'OOl c.c. of a 20-hour broth culture intra-venously 



15th day, - O'Ol c.c. 



29th day, - O'Ol c.c. 



and bleeding it on the 8th day after the last injection from the carotid after 

 ansesthetization. In this way it is easy to obtain a serum with a specific 

 titre of 1-20,000, and even of 1-40,000. 



An anti-paratyphoid B serum has little action on the typhoid, paratyphoid 

 A and gaertner bacilli, but agglutinates the aertrycke bacillus often in high 

 dilution, hence the confusion which has until recently existed between that 

 organism and the paratyphoid B bacillus. And the large amount of aertrycke 

 co-agglutinin in a paratyphoid B serum, and paratyphoid B co-agglutinin in 

 an aertrycke serum, is obviously the explanation of the " irregularity " of the 

 action of a " paratyphoid B " serum on a " paratyphoid B " bacillus. 



5. Absorption tests. 



By means of absorption tests the true nature of a serum or of an unknown 

 bacillus of the typhoid, colon, or paratyphoid groups is readily determined. 



In the case of a paratyphoid B. serum the whole of the specific agglutinin 

 as well as the co-agglutinins are removed by saturating with a paratyphoid B. 

 bacillus : whereas by saturating with for example the aertrycke bacillus only 

 the co-agglutinin for that organism is removed, leaving the specific agglutinin 

 intact. 



Similarly, in the case of an aertrycke serum all the specific agglutinin and 

 the co-agglutinins are removed by saturating with an aertrycke bacillus, 

 but by saturating with a paratyphoid B. bacillus only the co-agglutinin for 

 the paratyphoid B. bacillus is removed. 



It is due to these observations that the paratyphoid B. bacillus is dis- 

 tinguished from the aertrycke bacillus, and recognized to be an independent 

 species. 



Technique. 



Assume that a paratyphoid B serum has an agglutination titre of 20,000. 



1. Take O'l c.c. of the serum and add 4'9 c.c. of normal saline solution. This 

 will give 5 c.c. of a 1 in 50 dilution of the serum. Divide into two equal portions of 

 2-5 c.c. each. 



2. Take five large agar slope cultures (24 hours) of the paratyphoid B bacillus, 

 scrape off the growth with a platinum wire and add it to one portion of the diluted 

 serum. (The growth should not be washed off with the diluted serum.) 



3. Emulsify similarly in the second portion of the diluted serum five large agar 

 slope cultures of the aertrycke bacillus. 



4. Incubate both emulsions at 37 C. for 2 hours. 



5. Centrifuge the emulsions until the whole of the bacilli are precipitated and the 

 supernatant fluids are clear. 



6. Pipette off the supernatant fluids into separate sterile tubes. 



7. Test the agglutinating reactions of both fluids up to a dilution of 120,000 

 against both the paratyphoid B bacillus and the aertrycke bacillus, remembering 

 that the serum is already diluted 1 in 50. 



Before absorption the. serum agglutinated both the paratyphoid B. bacillus and 

 the aertrycke bacillus in high dilution (1-20,000 and 1-10,000). After absorption 

 with the paratyphoid B bacillus the titre for both organisms was reduced to about 

 1-100 to 1-400. After absorption with the aertrycke bacillus the titre for that 

 organism had fallen to about 1-100 to 1-400 while the titre for the paratyphoid B 

 bacillus was unchanged or only slightly reduced. 



The amount of bacilli to be added must necessarily vary according to the 

 titre ; the higher the titre the more bacilli. 



