408 ISOLATION OF THE TYPHOID BACILLUS 



to litmus paper : autoclave for an hour at 120 C., filter in the steamer and sterilize 

 for a quarter of an hour at 115 C. Prepare 



Litmus solution (Kahlbaum), - - 300 c.c. 



Lactose, - - - 30 grams. 



Sterilize at 100 C. for 15 minutes. 



Mix the agar and litmus solution while they are both hot. If the colour of the 

 litmus indicate that the medium is acid add sufficient 10 per cent, soda solution 

 to render it faintly alkaline and then a further 4 c.c. of warm 10 per cent, solution 

 of sodium hydroxide. Lastly, add to the mixture, 20 c.c. of a hot sterile (O'l per 

 cent.) solution of crystal-violet B, Hochst. 



(/3) Mode of use. Pour the Conradi-Drigalski agar carefully, without contaminat- 

 ing it, into large Petri dishes (1,5-20 cm. in diameter). Sow the suspected material 

 on the surface of the agar (vide ante Chantemesse's method). Incubate the plates 

 at 37 C. The typhoid bacillus gives blue transparent colonies and the colon bacillus 

 red opaque colonies. 



Hagemann's medium. 

 This is a modification of the preceding. 



Liebig's extract (Lemco), - 10 grams. 



Peptone (Witte), - - 10 



Salt, - 3 



Water, - 600 c.c. 



Boil. Add 500 c.c. of milk. Boil and dissolve 20 grams of agar in the hot liquid. 

 Heat to 120 C. in the autoclave for half an hour. Filter in the steamer and dis- 

 tribute in Erlenmeyer flasks. Sterilize. When required for use, liquefy the agar, 

 make slightly alkaline with soda solution, add a few cubic centimetres of litmus 

 and finally three drops of a 1 per cent, alcoholic solution of crystal violet. 



7. Method of Endo. 



The principle of the method depends upon the fact that if sulphite of sodium 

 be added to agar containing fuchsin the medium is decolourized, and if the 

 decolourized medium be sown with the colon bacillus the acids produced by 

 the organism restore the colour of the fuchsin and the colonies of the organism 

 acquire a red colour, while under similar conditions the colonies of the typhoid 

 and paratyphoid bacilli are colourless. 



The agar medium of Endo is used in exactly the same way as Chantemesse's 

 and Conradi-Drigalski's agar. It is very easy to prepare and gives good 

 results. 



Prepare a litre of peptone broth in the ordinary way, add 30 grams of agar and 

 dissolve in the steamer. Filter. Make absolutely neutral to litmus paper then add 

 10 c.c. of a 10 per cent, solution of sodium bicarbonate. * 



Add 10 grams of chemically pure lactose, and 5 c.c. of a filtered saturated alcoholic 

 solution of fuchsin which imparts a red colour to the medium. Now add 25 c.c. of 

 a freshly prepared 10 per cent, solution of sodium sulphite. Decolourization com- 

 mences at once and is complete after sterilization. Distribute in quantities of 15 c.c. 

 in tubes, sterilize at 115 C. and store in the dark. When required for use melt 

 the agar and pour into Petri dishes. 



After incubating for 15 hours at 37 C. colonies of the colon bacillus on this 

 medium have red centres and after 24 hours are entirely red with a greenish 

 iridescence. 



8. Methods based on the use of caffeine. 



As already mentioned (p. 375) Roth has shown that the addition of 0'5 per cent, 

 of caffeine to culture media checks the growth of the colon bacillus but does not 

 interfere with that of the typhoid bacillus. This fact has been successfully applied 

 by Roth, Hoffmann and others to the isolation of the typhoid bacillus from water 

 and stools. According to Courmont and Lacomme however, the method is uncer- 

 tain since some strains of the typhoid bacillus do not grow on caffeine- containing 

 media. The results should always be controlled by some other method. 



