VACCINE-THERAPY 73 



Having arrived, at a fair conclusion as to the causative 

 organisms, we proceed to cultivate them. An agar tube, 

 a blood-agar tube, gelatin tubes (two — slope and stab), 

 and one or two anaerobic tubes, should be inoculated 

 and incubated. Growth may be detected in from twelve 

 to twenty-four hours. But if any colonies fail to appear 

 within that period, incubation should be continued for 

 at least three days before the tubes may safely be said 

 to be sterile. When we get a mixed growth, we should 

 start to grow our bacteria on Petri dishes. A Roux 

 bottle is also useful for cultivating bacteria. A first pure 

 subculture is best for the preparation of a vaccine. A 

 careful microscopical examination should now be made of 

 the colonies for means of identification, noting purity, etc. 

 This being satisfactory, 5 c.c. of a O'l per cent, saline (NaCl) 

 solution is pipetted into the tube or bottle, and the whole 

 gently shaken. If some of the colonies are difficult to dis- 

 place, a bent sterile platinum wire or pipette should be used 

 to rake the surface of the medium, taking care, of course, 

 not to graze it. We now have a bacterial emulsion, which 

 should be poured into a sterile test-tube, and which in turn 

 should be heated about the middle in the blow-flame, and, 

 when sufficiently melted, drawn out, and the pointed end 

 sealed by the flame (as described on page 50, Fig. 23), and 

 placed, point upwards, in plasticine or in a tube-rack. The 

 emulsion should now be well shaken for ten minutes to 

 break down bundles or chains of bacteria. To assist in this 

 process, glass beads (sterile) are sometimes inserted also. 



Standardization. 



Having obtained our emulsion, we now proceed to 

 standardize it, and for this purpose several methods are 

 adopted. The writer has followed Wright's method, which 

 is as follows : A capillary pipette, 6 inches long, with a 

 rubber teat fixed to the open large extremity, is taken, and 

 marked \ inch from the end of the capillary extremity 

 with a grease pencil. This will indicate one unit volume. 



