224 FARMYARD MANURE 



but later an almost pure culture of Urobatillus Pasteurii is 

 obtained, the large amount of urea (10 per cent.) inhibiting 

 the growth of most of the other organisms met with in the 

 soil. 



(b) Transfer a drop to a fresh flask of the medium, incubate 

 for ten days at 30 C., and examine as before. 



(c) Repeat the above, using garden soil which has been heated 

 to 90 C. for one hour instead of a fresh sample. 



Ex. 104. Prepare 250 c.c. urea-bouillon as above; infect with 

 garden soil and incubate at 22 C. 



Remove 20 c.c. of the solution with a pipette, and titrate with 

 normal hydrochloric acid. Repeat the titration of 20 c.c. drawn 

 from the flask every three days, and note the increasing amount 

 of acid needed for neutralization. The increased alkalinity is 

 due to the formation of ammonium carbonate. 22 c.c. of 

 normal HC1 neutralizes about i gram of ammonium carbonate. 



Ex. 105. Expose some urine in a cool, shady place to the 

 open air in a shallow vessel. Examine, after two or three days, 

 for bacteria present ; stain with gentian violet. 



Ex. 106. Prepare (i) 2 per cent, urea-gelatine; (ii) 2 per 

 cent, urea-agar. 



(i) Water . . . . , . 1000 c.c. 

 Lemco's extract (or beef broth) 5 gr. 



Witte's peptone , . . . 10 gr. 



Gelatine . . . . 100 gr. 



Urea, . , . . 20 gr. 



(ii) Same as above, using 1 5 gr. agar in place of the gelatine. 

 If acid neutralize with ammonium carbonate ; 

 sterilize and tube as usual. 



(a) Inoculate a tube of each with a drop of the liquid from 

 Exs. 103 and 105. Pour into Petri dish and incubate 



(i) The gelatine medium at 22 C. 

 (ii) The agar medium at 30 C. 



(b) Examine the organisms from the several kinds of colonies 

 on the plates. 



