422 PATHOGENIC MIRCOORGANISMS 



inoculations upon neutral bouillon should be made each day, so that a 

 young broth culture may always be on hand to furnish actively motile, 

 evenly distributed bacteria. These bouillon cultures may be grown 

 for from six to eight hours at incubator temperature or for from twelve 

 to eighteen hours at room temperature. The temperatures at which 

 the broth cultures are kept must depend, to a certain extent, upon the 

 peculiarities of the typhoid bacillus employed, since some strains are 

 rather more actively motile and furnish a more suitable emulsion if kept 

 at a temperature lower than 37.5 C. A false clumping in the broth 

 cultures due to a too high acidity of the bouillon or a too prolonged 

 incubation, must be carefully guarded against. It is also possible to 

 use for this test an emulsion of typhoid bacilli prepared by rubbing up a 

 small quantity of a young agar culture in salt solution. Uniformity 

 in the preparation of broth cultures or of emulsions should be observed, 

 since the quantitative relationship between typhoid bacilli and agglu- 

 tinins will markedly affect the completeness or incompleteness of the 

 reaction. In high dilutions an excess of typhoid bacilli may bring about 

 complete absorption of all the agglutinins present, without agglutinat- 

 ing all the microorganisms. 



The blood of the patient to be used for a Widal test may be obtained 

 in a number of ways. The most convenient method is to bleed the pa- 

 tient from the ear or finger into a small glass capsule, in the form of that 

 used in obtaining blood for the opsonin test, or into a small centrifuge 

 tube. About 0.5 to 1 c.c. is amply sufficient. These capsules or tubes, 

 after clotting of the blood, may be placed in the centrifuge which in a 

 few revolutions will separate clear serum from clot. . The dilutions of 

 the serum are then made. It is best to use sterile physiological salt solu- 

 tion as a diluent, but neutral broth may be used. The dilutions may be 

 made either by means of an ordinary blood-counting pipette or by means 

 of a capillary pipette upon which a mark with a grease pencil, made 

 about an inch from the tip, furnishes a unit of measure, and upon 

 which suction is made by means of a rubber nipple. It is convenient 

 to have at hand a small porcelain palette such as that used by painters, 

 in which the various cup-like impressions may be utilized to contain the 

 various dilutions. Dilutions of the serum are made, ranging from 1 : 10 

 to 1 : 50. A drop of each of these dilutions is mixed with a drop of the 

 typhoid culture or emulsion upon the center of a cover-slip and the cover- 

 slip inverted over a hollow slide. A control with normal serum and 

 the same culture should always be made and also one with the culture 

 alone to exclude the possibility of spontaneous clumping. Mixture 



