24 AIDS TO BACTERIOLOGY 



on appropriate medium, and three or four ' streak ' or 

 rather ' slant ' cultures made, the whole of the surface 

 of the slanted medium being used. About 1 c.c. of sterile 

 salt solution (O'l per cent.) is poured into one of the tubes, 

 and by rubbing up with a sterile platinum loop the growth 

 is detached and forms a milky emulsion. This is poured 

 into a sterile test-tube or flask and the culture tube is 

 rinsed out with a few more drops of the salt solution. The 

 process is repeated with the other slants, and their emul- 

 sions added to the first, so that a total volume of about 

 5 c.c. is obtained. The masses present in the emulsion 

 have to be thoroughly broken up, and the bacterial content 

 ascertained by Wright's method: A small definite volume 

 of the emulsion is mixed with an equal volume of blood, and 

 smears made on slides are stained with one of the blood- 

 stains. The relative numbers of red cells and bacteria 

 are determined. Human blood, if from a male, contains 

 five million red cells per cubic millimetre, or a thousand 

 times this number per c.c. A calculation therefore gives 

 the number of bacteria per c.c. The emulsion is diluted to 

 a strength suitable for administration, with sterile normal 

 saline containing 0-5 per cent, carbolic acid, and sterilised 

 for an hour or an hour and a half in a water- bath at 56 

 to 60 C. This will not always suffice for sterilisation. 

 In such cases a further sterilisation at 60 C. for one hour, 

 twenty-four hours later, is to be preferred to a single 

 sterilisation at a higher temperature. With some of the 

 cocci a temperature of 65 or even 75 C. is necessary. 

 Prolonged heating or the use of too high a temperature 

 lowers the activity of the vaccine. Before use a subculture 

 must be made from the vaccine to prove its sterility, and 

 all through the process of preparation rigorous sterility of 

 apparatus, diluting solution, etc., must be maintained. 



A main cause of failure in vaccine treatment lies in the 

 selection of the wrong organism. This is liable to occur 

 with the colon bacillus, of which there are said to be 150 

 varieties, so that the infecting organism may not be 

 matched even in a polyvalent vaccine. It is often difficult, 

 sometimes impossible, to say at a first examination which 

 of the organisms found is the eetiological agent, and when a 

 vaccine fails a further examination is necessary. Perhaps 

 two or three bacteria are responsible, and the vaccine has 

 only been prepared from one, with the result that the 



