284 IMMUNOLOGICAL METHODS OF DIAGNOSIS 



diagnosis (as in plague, for example), or there are certain technical 

 difficulties which make it inapplicable (tuberculosis, cholera), while 

 in still others a diagnosis can be conveniently reached in an even 

 more direct manner (as by the isolation and cultivation of the 

 offending microorganism), etc. 



To give a general idea of the technical method of procedure it will 

 be best to describe the reaction as it is applied to the diagnosis 

 of typhoid fever, i. e., the Widal reaction proper. 



The Widal Reaction. TECHNIQUE. Microscopic Method. A small 

 amount of blood (5 to 10 drops) is collected in a little glass tube or in 

 one of the capsules pictured in Plate III. The serum is separated by 

 centrifugation and a drop diluted in the white mixing pipette accom- 

 panying the hemocytometric counting chamber in the proportion of 

 1 to 20. From this, subdilutions of 1 to 40, 1 to 80, 1 to 160 are pre- 

 pared with the aid of the same pipette, normal salt solution being 

 used as diluent in all cases. Four slides are then ringed with vaseline, 

 and into each little chamber a drop of the diluted serum is placed 

 together with a drop of a typhoid culture in bouillon, not more than 

 twenty-four hours old. The resultant dilutions will then be 1 to 40, 

 1 to 80, 1 to 160, 1 to 320. A cover-glass is adjusted so as to be in 

 contact all around with the vaseline, as also with the drop in the cen- 

 tral chamber. The specimens are immediately examined with the 

 middle power of the usual microscopic outfit (J B. and L.; No. 6 or 7 

 Leitz), and discarded if any large clumps of bacteria are seen. 

 Should this be the case, it is well to make new mounts with a culture 

 that has been centrifugalized for a minute or two, the supernatant 

 fluid only being used, in which no clumps will be found. If the 

 mount is satisfactory, it is set aside and reexamined at the expira- 

 tion of half an hour. If the reaction is positive, all the bacilli will 

 be found motionless at the expiration of this time, and gathered in 

 clumps of variable size (Fig. 15). This will be the case at least in 

 the lowest dilutions, while in the higher ones it may be necessary 

 to wait until another half-hour has expired. The higher the dilution 

 in which complete clumping may be obtained the greater is the 

 diagnostic significance of the reaction. Ordinarily, complete clump- 

 ing at the end of half an hour in a dilution of 1 to 40 is sufficient; if, 

 however, the question of paratyphoid enters into consideration, the 

 result in the higher dilutions should be considered. As the typhoid 

 and paratyphoid bacilli carry certain receptors in common, the 



