METHOD OF ISOLATION 49 



each about 8 or 10 c.c. of the medium, taking great care that none 

 of it touches the sides of the tube near the mouth. Plug each 

 tube with cotton wool and heat them all in the steam sterilizer 

 on three successive days for twenty minutes at a time, taking 

 care to have the water boiling before the tubes are placed in 

 the sterilizer. (The medium will keep a long time if stored away 

 from dust, especially if the plugged mouths of the tubes are 

 covered with indiarubber caps.) 



Ex. 18. Pour 250 to 300 c.c. distilled water into a flask, 

 lightly plug the opening with cotton wool, and boil for twenty 

 minutes on three successive days. 



When the water is cool put into it i c.c. of the liquid contain- 

 ing the bacteria which are to be isolated. Take J c.c. or less 

 of the dilute mixture with a sterilized pipette and transfer it to a 

 nutrient gelatine tube, the contents of which have been liquefied 

 by placing the end of the tube in warm water ; mix thoroughly, 

 not by shaking but by turning the tube and tilting it so as to 

 avoid bubbles of air in the medium, and then pour it into a 

 sterilized Petri dish. Cool rapidly by placing the Petri dish on 

 a glass plate or on ice for a minute or two ; then leave it in 

 a warm room and examine every day for colonies of bacteria. 

 They will appear as spots of varied colour and form on the 

 surface or within the substance of the gelatine medium. 



Ex. 19. Instead of making a dilute solution, as explained 

 above, of the liquid in which the bacteria to be isolated are 

 found, the following plan may adopted. Melt three or four 

 tubes of nutrient gelatine in warm water at 30 to 35 C. ; 

 label them i, 2, 3, and 4 respectively. Take up a drop of 

 the liquid containing the bacteria with a sterile platinum loop 

 and transfer it to the tube labelled i. Mix carefully and 

 transfer a drop of the mixture to tube 2 ; from tube 2 transfer 

 a drop to tube 3, and from 3 to 4 ; mix carefully each time 

 and sterilize the platinum loop each time before use by heating in 

 the flame. Then pour the contents of each tube into four sterilized 

 Petri dishes and leave them in a warm room to incubate. 



