386 THE TYPHOID BACILLUS 



broken up by shaking the tube, while the broth remains perfectly clear. 

 In the control tube, on the other hand, the broth is cloudy and shows the 

 scintillating ripples characteristic of a growth of the typhoid bacillus. 



The reaction is not always so distinct as this. Sometimes the broth instead of 

 remaining clear shows an irregular turbidity, from which however the characteristic 

 watered-silk appearance is absent ; this turbidity is due to the precipitation of a 

 very fine powder each grain of which when examined under the microscope is seen 

 to be an agglomeration of bacilli. At other times the reaction may be quite charac- 

 teristic at first but after incubating for 18 or 24 hours the broth is turbid above 

 the precipitate. Naked eye appearances ought always to be supplemented by a 

 microscopical examination by means of which the small masses of bacilli may be 

 recognized if present and their structure defined. 



2. Add the serum to a twenty-four-hour broth culture of the typhoid 

 bacillus and incubate at 37 C. If the agglutinating power of the serum is 

 well marked characteristic changes will take place within a few hours ; the 

 culture is at first granular, but becomes gradually clear as the bacilli fall to 

 the bottom. When the serum is less powerfully agglutinating, clumps are 

 formed but the broth never becomes quite clear. Naked eye appear- 

 ances must, as in the previous case, always be controlled by microscopical 

 examination. 



B. Rapid method. Recommended. This method is quicker and more 

 sensitive than the foregoing and has the additional advantage of requiring 

 only a few drops of blood, an amount which can be easily obtained by prick- 

 ing the finger. It is therefore the method to be used in the majority of cases. 



A broth or peptone culture of the typhoid bacillus is required for the reac- 

 tion, and the greatest care should be exercised in the choice of a culture. 

 In the first place it is of course essential that it be pure : secondly the growth 

 must not be more than 24 hours old, because in old cultures clumps often 

 form spontaneously and these will falsify the results. Spontaneously formed 

 clumps may indeed be present even in twenty-four-hour cultures, so it is 

 always necessary to determine by microscopical examination immediately 

 before use that the chosen culture is satisfactory in this respect. A quantity 

 of culture sufficient for the investigation should be drawn up into a Pasteur 

 pipette and a little placed on a slide and examined under the microscope, 

 the remainder, if the sample is satisfactory, being used for the serum reaction. 

 To obviate the spontaneous formation of clumps in cultures it is better to 

 grow the organism in a 1 or 2 per cent, solution of peptone containing no 

 meat rather than in broth. 



The method is as follows. 



1. Reaction with the serum. [(a) Technique recommended,!. Take a 

 sterile Pasteur pipette, plugged with wool and fitted with an india-rubber teat 

 as shown in fig. 162, p. 241. Make a mark on the stem of the pipette. 



[2. Take up 9 volumes of the peptone water culture run them up into the 

 bulb and then take up 1 volume of the serum. Mix the culture and serum 

 thoroughly by repeatedly expelling on to a slide and aspirating. (Dilution 1.) 



[3. Take up 4 volumes of dilution 1 and 1 volume of serum. Mix. 

 (Dilution 2. 1 in 50.) 



[4. Take up equal volumes of dilution 2 and culture. Mix. (Dilution 3. 

 1 in 100.) 



[And so on, preparing the dilutions required. 



[Place a drop of each dilution on a clean cover -glass and invert the latter 

 over the cavity in a hollow-ground slide. Lute the edge with vaseline. Place 

 the preparations in the incubator at 37 C. Examine with a high power dry 

 lens at the end of half an hour and again at the end of an hour. If the serum 



