412 ISOLATION OF THE TYPHOID BACILLUS 



14. Methods based on the agglutination of the typhoid 



bacillus. 



Chantemesse, Windelbandt, Schepilewsky have made use of the agglutinat- 

 ing properties of antityphoid serum for isolating the typhoid bacillus. This 

 method is very delicate and permits of the isolation of the bacillus from 

 mixtures in which it is present in great dilution. - 



Windelbandt' s technique. To 10 c.c. of sterile broth add 1 c.c. of the water under 

 examination. Incubate the mixture for 35 days. By this time the growth is very 

 abundant, the broth is cloudy and the surface covered with a pellicle. Remove the 

 surface growth and add to the remainder a few drops of a powerfully agglutinating 

 anti-typhoid serum. The agglutinated typhoid bacilli fall to the bottom of the 

 tube. Centrifuge the broth culture, collect the deposit and dilute it with a little 

 normal saline solution. Sow litmus-lactose-agar plates with the diluted deposit. 



Chantemesse' s technique. A simple method devised by Chantemesse consists in 

 adding 30 grams of peptone to 1 litre of the suspected water, neutralizing and 

 incubating for 20 hours. If little clumps form filter through paper and add anti- 

 typhoid serum to the filtrate. After standing for 2 hours decant the liquid, filter 

 the deposit through paper, and sow the clumps retained on the filter on Chantemesse' s 

 agar (p. 407). 



Altschiiller's technique. Altschliller adds peptone and salt 'to the suspected water 

 and incubates for 24 hours. Ten c.c. of the culture are now transferred to a test- 

 tube the lower end of which is drawn out and opened and attached to a piece of 

 india-rubber tubing closed by a clip. A few drops of a typhoid immune serum are 

 added and a precipitate is soon formed which collects in the narrow drawn-out part 

 of the tube. By releasing the clip the deposit can be run into a tube containing 

 sterile peptone water. The mixture is shaken and then incubated at 37 C. The 

 typhoid bacillus grows rapidly and is unaccompanied by other organisms. 



[15. MacConkey's media.] 



[The basis of MacConkey's media consists of a stock solution composed of : 

 Sodium taurocholate (commercial), - 0'5 gram. 



Peptone (Witte), - - 2'0 grams. 



Distilled water, - 100 



For liquid media there is added to this stock solution 0'5 per cent, of a 1 per cent, 

 solution of neutral red and 0'5 per cent, of glucose or 1 per cent, of the other carbo- 

 hydrates or alcohols, as the case may be, and the medium is distributed into Durham's 

 fermentation tubes and sterilized in the steamer for 10 minutes on each of two days, 

 great care being taken not to overheat the medium. If it be thought advisable 

 white of egg may be used to clear the medium. 



[Bile-salt-agar is made by dissolving 1*5-2 per cent, agar in the stock solution. 

 This is best done in the autoclave. The medium is cleared with white of egg and 

 filtered. After filtration the. same amount of neutral red is added as in the case of 

 the liquid media (MacConkey). 



[A consideration of the fermentation reactions of the various organisms 

 shows that by the use of certain carbohydrates or alcohols either alone or in 

 combination organisms can be separated by means of colour reactions. Mac- 

 Conkey's medium forms a most useful nutritive medium to which to add 

 these substances.] 



SECTION II. THE IDENTIFICATION OF THE TYPHOID AND 

 COLON BACILLI. 



(i) An organism may be suspected to belong to the typhoid-colon group 

 if it have the following characteristics : 



1. A bacillus with rounded ends, generally motile, decolourized by Gram's 

 method, having no capsule [and not forming spores]. 



