410 ISOLATION OF THE TYPHOID BACILLUS 



To identify an organism isolated on this medium Loeffler advises the use of a 

 so-called typhoid solution. 



Loeffler" s typhoid solution. This fluid is coagulated by the typhoid bacillus in 

 16-24 hours and floating on the coagulum is a clear green liquid. The colon bacillus 

 produces not an homogeneous coagulum but a greenish mass adhering to the sides 

 of the tube. 



The solution consists of : 



Distilled water, 100 c.c. 



Nutrose, gram. 



Glucose, - 



Peptone, 2 grams. 



Lactose, ------ 5 ., 



2 per cent, solution of chemically pure malachite green, - 1 c.c. 



Normal soda solution, - 1'5 ., 



Peabody and Pratt's technique. Sow the suspected fluid in broth con- 

 taining O'l per cent, of malachite green, incubate at 37 C. for 18 hours and 

 isolate on Conradi-Drigalski plates. 



10. Method based upon the use of China green. 



[Werbitzki recommends the addition of China green to agar (I'l-l'S c.c. 

 of a 0*2 per cent, solution per 100 c.c. of agar) for the purpose of restraining 

 the growth of the colon bacillus when attempting the isolation of the typhoid 

 and paratyphoid bacilli from such material as stools.] 



11. Methods based on the use of bile. 



The adjuvant properties of bile for the typhoid bacillus have been applied 

 to the detection of the bacillus in water and stools. Bile may be used alone 

 (either as such or in the form of bile salts) or mixed with malachite green. 

 Diinschmann's technique. Prepare the following medium : 



Distilled water, - 100 c.c. 



Agar, - 3 grams. 



Peptone, 3 



Lactose, - 3 , 



Gelatin, - 1 gram. 



Taurocholate of sodium, - 1 



Heat to 120 C. Filter and distribute the medium in tubes in quantities of 10 c.c. 

 After sterilization, add 1 c.c. of a sterile solution of litmus to each tube. 



To use the medium, pour the contents of four or five tubes into a similar number 

 of Petri dishes and with a Drigalski's spatula (p. 407) charged with the suspected 

 material and without recharging sow surface cultures on each dish. 



Jackson and Melia's technique. The suspected material is enriched by growing 

 in ox bile and the culture used to sow plates of Hesse's agar. 

 Hesse's agar. In a litre of boiling water dissolve : 



Liebig's extract, - 5 grams. 



Peptone, 10 



Sodium chloride, - 8'5 ,, 



Agar, - 30 



Heat to 120 C., filter, distribute in tubes (10 c.c. in each) and sterilize in the 

 autoclave. 



Method. Sow the suspected material in 5 c.c. of bile and incubate at 37 C. for 

 24 hours. Take eight test-tubes each containing 9 c.c. of sterile water : to the first 

 add 1 c.c. of the bile culture, to the second 1 c.c. of the first, to the third 1 c.c. of 

 the second and so on. Take eight tubes of Hesse's agar, liquefy the medium and to 

 one add 1 c.c. of the first dilution to the second 1 c.c. of the second dilution and so 

 on, and pour plates. 



Lceffler's technique. A mixture of bile and malachite green is used. Sow the 

 material on plates of Leuchs' nutrose-agar (p. 409) containing 3 per cent, of ox bile 

 and 1-9 per cent, of a 0*2 per cent, solution of malachite green. 



