FUNDAMENTAL METHODS OK ISOLATION 



Exercise 63. Isolation of a Pure Culture 



In order to get a pure culture from a mixture of bacteria such 

 as may be had in water, sewage, or commercial milk, proceed as 

 follows : Three tubes of sterile nutrient agar or gelatin are melted 

 in hot water and cooled to 

 about 42 C. Label the 

 tubes. A, B, and C. Select a 

 platinum inoculating needle 

 which has a loop 2 mm. in 

 diameter at the extremity. 

 Sterilize the needle by heat- 

 ing to redness in the gas 

 flame and, after allowing a 

 few seconds for it to cool, 

 dip the loop of the wire 

 into the liquid containing 

 bacteria. A film of liquid 

 is held in the loop at the 

 end of the wire. Inoculate 

 tube A with three loopfuls of 

 the liquid. Sterilize the wire 

 and place it in the holder. 

 Thoroughly mix the con- 

 tents of tube A. This must 

 be done without wetting the 

 cotton stopper. The result 

 can best be accomplished by 

 rolling the tube between the 

 palms of the hands, while 

 slanting it alternately right 

 and left. Tube A is called the First Dilution. After mixing, trans- 

 fer three loopfuls of tube A into tube B, using the same procedure 

 as before. Tube B is the Second Dilution. In the same way trans- 

 fer three loopfuls of B into (7, constituting the Third Dilution. The 

 Third Dilution usually contains few enough bacteria for the pur- 

 pose of isolation, but the Second Dilution should also be poured. 



FIG. 19. An inoculating room 



The interior walls are lined with linoleum and 

 can easily be washed with a weak solution 

 of bichloride. If inoculations are made in 

 such a room, the danger of contaminations is 

 greatly minimized 



