THE AGGLUTINATION REACTION 299 



TECHNIC OF THE MICROSCOPIC AGGLUTINATION TEST (WET METHOD). THE 

 WIDAL REACTION IN TYPHOID FEVER 



(1) Dilute the patient's serum by placing one drop from a capillary 

 pipet in a small watch-glass and adding 19 drops of normal salt solution. 

 This gives a dilution of 1 : 20. Mix thoroughly. 



(2) With a 3 or 4 mm. platinum loop place a drop on a clean cover- 

 glass that is sufficiently thin to permit the use of an oil-immersion lens. 

 The loop is better than a capillary pipet because the drop it gives is 

 smaller, and when it is later diluted with an equal quantity of bacterial 

 emulsion, it is not too large and is easily manipulated. 



(3) With the same sterilized platinum loop add one loopful of a 

 twenty-four-hour broth culture of Bacillus typhosus to the drop of 

 diluted serum on the cover-glass. Mix gently and without spreading the 

 drop. This gives a final dilution of 1 : 40. 



(4) Edge a hanging-drop slide with vaselin, and invert the cover- 

 glass slide over the hollow portion in such a manner that the drop will 

 be suspended in its center. Care must be exercised not to spread the 

 drop, for if this occurs and the fluid flows around the margins of the 

 chamber a new preparation must be made. Inspect the slide, and add 

 vaselin, if necessary, until it is sealed tightly. By means of a grease 

 pencil label the slide with the name of the patient, the dilution, and the 

 time when the preparation was made. 



(5) Place 2 to 5 drops of serum dilution 1 : 20 in a second watch- 

 glass, and add an equal quantity of normal salt solution. Mix well. 

 This gives a dilution of 1 : 40. 



(6) Prepare a second slide by mixing a loopful of this dilution with 

 an equal sized loopful of culture. Mix gently. This gives a final dilu- 

 tion of 1 : 80. Mark the slide with the name, dilution, and the time. 



(7) Prepare a third slide by placing a loopful of culture on a cover- 

 glass and invert over a concave slide to which vaselin has been applied 

 in the usual manner. This is the culture control. Label the slide. 



(8) Place the slides in a dark place at room temperature and examine 

 at the end of an hour with the K or oil-immersion lens. 



(a) First inspect the control. The bacilli should not be clumped, 

 but should be motile, and preferably in the form of long slender rods. 

 (See Fig. 81.) 



(6) Examine the 1 : 40 and 1 : 80 dilution preparations: a positive 

 reaction is indicated by loss of motility and definite clumping (Fig. 83). 

 A few free motile bacilli may be seen, or a clump may be seen to move, 



