352 



INFECTION AND RESISTANCE 



We do not think that any satisfactory substitute for careful isolation 

 by plating has been devised. After a pure culture of the organism 

 has been obtained this is grown on relatively large surfaces of agar, 

 glucose-agar, or ascitic agar, as the case may require. These culti- 

 vations may be made in Kolle flasks or, as Wright and 

 others have suggested, on large agar surfaces obtained 

 when the culture medium is allowed to harden in a 

 square 3-oz. medicine bottle laid on its side. Any de- 

 vice of this kind in which a large surface of agar is 

 exposed may be used. 



After suitable growth of the micro-organisms has 

 taken place, 24-48 hours, the growth is gently washed 

 off with 10 c. c. or more of sterilized salt solution. 

 Care must be taken to do this in such a way that no 

 agar is drawn away with the emulsion. The thick 

 emulsion so obtained is removed from the culture bottle 

 with sterile nipple pipettes or Pasteur pipettes and 

 transferred to a sterile thick-walled test tube into which 

 glass beads have been placed. By drawing out the 

 neck of this test tube in the flame a glass capsule is 

 formed, in which we now have our so-called stock 

 emulsion. (See figure.) 



The next thing to be done is to standardize this 

 OF S TEST TUBE stock emulsion, or, in other words, determine approxi- 

 TO HOLD STOCK mately the number of bacteria to the cubic centimeter. 

 There are a number of methods by which this can be 

 done. 



The method most extensively used by Wright and 

 his followers was that in which the bacteria are counted 

 against red blood cells. The bacteria in the capsule 

 are shaken thoroughly with glass beads so that clumps 

 lary tip until may be broken up and even distribution obtained. A 

 it has cooled little of the emulsion is then put into a clean watch 

 it 'may^crack glass, a step which can be accomplished most easily by 

 when quickly breaking off the tip of the drawn-out part of the cap- 

 cooled.) sule. tilting it very gently and heating the closed end 

 over a small flame, so that some of the emulsion will be 

 driven out by the expanding air. With a nipple pipette marked 

 about an inch from the tip, as in the taking of an opsonic index, a 

 little of the emulsion is drawn up. This is placed into another clean 

 watch glass and is mixed with about 2 volumes of salt solution and 

 one volume of blood from the finger, these quantities being measured 

 with the same nipple pipette. We then have a mixture in which, 

 in a total of 4 volumes, there are equal parts of blood and of bac- 

 terial emulsion. After this emulsion has been thoroughly mixed by 

 drawing in and out through the nipple pipette smears are made on 



VACCINE 

 EM UL s ION 

 FROM WHICH 

 D I L U T I ONS 

 ARE MADE. 



(It is well to 

 keep 

 open 



