GRUNBAUM-WIDAL REACTION 387 



be a typhoid-agglutinating serum the bacilli will be found agglutinated together 

 in more or less large masses.] 



(/3) Another method. Into a small conical glass vessel introduce 10-100 

 drops of the culture and 1 drop of the serum. Place a drop of the mixture 

 on a slide and cover with a cover-glass. Examine with a high power dry 

 lens. If the serum has the power of agglutinating the typhoid bacillus, 

 masses of agglutinated bacilli will be seen and among them a greater or 

 smaller number of non-agglutinated bacilli. The reaction is still more 

 distinct if the preparation be examined after 15 or 20 minutes, for compact 

 islets of agglutinated bacilli will then be visible under the microscope. When 

 the agglutinating property of the serum is small the reaction may only appear 

 after the lapse of 40 minutes or an hour. The appearance is quite charac- 

 teristic and renders mistakes- impossible. Agglutination is assisted by a 

 slight drying at the edge of the drop between the slide and the cover- 

 glass. 



Whole blood. The whole blood may be used for the reaction, but before examining 

 the preparation under the microscope time must be given to allow most of the red 

 cells to settle, since the presence of a large 

 number of cells detracts from the sharp- 

 ness of the reaction. The method is there- 

 fore no quicker than the serum method. 



Method of staining. The preparation 

 may be stained so as to render the masses 

 more distinct and preserved for future 



use: for this purpose the folio wing technique, _ 



described by Guillemin, gives good results. 



Mix 1 drop of the whole blood with 9 

 drops of sterile broth, and add 1 drop of 

 this to 2-5 drops of a culture of the 

 typhoid bacillus. Spread a large drop of 

 the mixture on a slide : place in a moist 

 chamber for an hour or two : dry slowly, 

 fix in alcohol-ether, treat with 10 per cent, 

 acetic acid to dissolve the red cells, wash, 

 stain with dilute carbol-fuchsin, wash and 



C ^ r ' FIG. 220. Agglutination of the typhoid bacillus 



2. Reaction with dried blood.-The ^VS serum< Jenner>S ^ ( C - 2> 

 blood collected and dried as already 



described is dissolved immediately before use in a drop or two of water. The 

 solution is added to 10-50 drops of a broth culture of the typhoid bacillus 

 contained in a conical glass vessel. It is left for a moment to allow the red 

 cells to settle and then examined as before. 



3. Reaction with dead bacilli. The phenomenon of agglutination is not 

 dependent upon any vital reaction of the bacilli, since it can be demon- 

 strated with dead organisms. This fact may in certain circumstances be of 

 practical value, because a recent culture of the typhoid bacillus is not always 

 immediately available with which to perform the reaction, and to obtain 

 one may involve a delay of 12 hours or so. In such a case a dead culture may 

 be used, since experience has shown that such a culture retains its sensitive- 

 ness towards an agglutinating serum for several weeks. The following 

 technique may be adopted in the preparation of a dead culture for this purpose 

 (Widal and Sicard). 



A sixteen- or twenty-four-hour culture of the typhoid bacillus is examined micro- 

 scopically to test its purity. Formalin in the proportion of 2 drops to 15 c.c. of culture 

 is added to kill the bacilli, which become as it were embalmed. Care must be taken 

 to cover the cotton- wool plug of the vessel containing the culture with an india- 



