APPENDIX 483 



The rough dilution is to take of the corpuscle-free serum to be 

 examined one drop. Dilute it with nine parts of neutral bouillon. 

 Mix on a slide or cover-glass a drop of this one-tenth diiutiou of 

 serum one or more drops of typhoid broth cultivation of eighteen to 

 twenty-four hours' growth. The serum and culture are thoroughly 

 mixed together in the trough of a hollow-ground slide, and a single drop 

 is taken, placed upon an ordinary clean slide, and a cover-glass super- 

 imposed ; or the mixture may be made on the cover-glass and super- 

 imposed on the slide. 



The measurement method is to dilute the serum by exact quantities, 

 giving say, a 10 per cent., a 1 per cent., and a O'l per cent, dilution ; or 

 three mixtures containing respectively 50 per cent., 5 per cent., and 

 0*5 per cent, of serum. The 50 per cent, dilution is made by adding 

 equal loopfuls of serum and of a typhoid broth culture on a slide or 

 cover-slip. The 5 per cent, is made by diluting 10 c.m. (measured by 

 graduated haematocytometer) of the serum, with 90 c.m. of the broth 

 culture in a small sterilised test-tube. After thoroughly mixing, one 

 loopful of this dilution (now 10 per cent.) is mixed with one of cultivation. 

 The 0-5 per cent, is made by first diluting 10 c.m. of the 10 per cent, 

 serum with 90 c.m. of sterile broth in a small test-tube, and then mixing 

 equal loopfuls of this diluted serum and of the broth culture. 



(c) The Typhoid Culture used should be one sub-cultured from a virulent 

 culture, and should be a broth or agar culture of about eighteen to twenty- 

 four hours ; and, if preferred, may be filtered before use to remove any 

 normally agglutinated masses of bacilli before commencing the test. 



(d) The Reaction. The reaction is positive if the bacilli have become 

 grouped together tightly into clumps (agglutination), leaving the field 

 between the clumps free from bacilli. Immotility will also be present. 

 The reaction time is half-aii-hour (see Plate 20). In his first experiments, 

 Widal used a test-tube in the following manner : The blood to be tested 

 is diluted by one part of it being added to fifteen parts of broth in a 

 test-tube. The mixture is inoculated with a drop of a typical B. typhosus 

 culture. The tube is then incubated at 37 C. for twenty-four hours, 

 after which it is examined. If the reaction be positive, the broth appears 

 comparatively clear, but at the bottom of the test-tube a more or less 

 abundant sediment will be found. This is due to the clumps of bacilli 

 having fallen owing to gravity. If, on the other hand, the reaction is 

 negative, the broth will appear more or less uniformly turbid. This 

 method is not as satisfactory as the one described. 



Some bacteriologists use two dilutions, 1 in 20 and 1 in 40, with a 

 time limit of one hour for each case. The reactions obtained are 

 interpreted as follows : Where both dilutions show clumping and loss of 

 motility at the end of the hour a diagnosis of "enteric fever" is made ; 

 but if the reaction is present only in the 1 in 20 dilution, a guarded 

 opinion is given and the case stated to be " probably enteric fever " ; if 

 both preparations are unchanged, the case is reported as "probably not 

 enteric fever." 



In the measured dilutions it may be said that if in half-an-hour there 



