BOUILLON, AGAR AND MILK. 239 



If the material used is a solid growth, it is advisable to 

 transfer a minute amount of this to a tube of bouillon 

 (10 c.c). With a sterilized wire, a loopful of this suspension 

 is transferred to a second tube of bouillon. The wire is again 

 sterilized, and, a loopful of this second dilution is trans- 

 ferred to a tube of freshly inclined agar and carefully spread 

 all over the surface. The same wire, not sterilized, is then 

 rubbed over the surface of a second tube of inclined agar. 

 The inoculated agar tubes should be placed in a horizontal 

 position in the incubator for 18 to 24 hours. Perfectly iso- 

 lated colonies will thus be obtained. 



The method of isolating colonies by means of agar plates 

 will be described in the next chapter. Esmarch roll-tubes 

 (p. 181), can be made with agar in the same manner as with 

 gelatin. The tubes should be rolled in ordinary tap-water 

 and not in ice-water. They should then be set aside in a 

 cool place, in a horizontal position, in order to allow the 

 agar to adhere firmly to the glass. 



Milk. 



This should be obtained as fresh as possible, before the 

 reaction has become acid. It is then filled into sterile 

 tubes and sterilized by exposure to steam for 30 minutes on 

 each of three successive days. It can be sterilized in the 

 autoclave at 110°-115° for 10-15 minutes. Too much heat 

 should be avoided, since this may cause an alteration of the 

 milk. It should be remembered, however, that milk is dif- 

 ficult to sterilize because of the presence of resistant organ- 

 isms, and, above all, because of the protection afforded by 

 the fat. Hence, it is advisable to place the milk tubes in 

 the incubator at 37° for 1-2 days in order to make sure of 

 the sterility of the medium. 



In the method as described, whole milk is employed, 

 and hence, on standing, a thick layer of cream rises to the 

 surface. This can be avoided by employing centrifugated 



