IMPORTANT PATHOGENIC BACTERIA 119 



sus, previously heated for i hour at 6cf C, is used. 

 This process kills the organisms, but the toxins remain 

 active. The first injection is followed by another one 

 with dead cultures after four to five days, and after the 

 same intermission a culture of virulent bacilli is in- 

 jected. By this time the agglutinative power of the 

 blood is well developed. The animal is then bled in 

 the following manner: One of the ears is shaved, and the 

 skin is washed with alcohol. A small vein near the 

 border is opened, and the blood is collected in a sterile 

 glass vessel. If the animal is hvmg head down enough 

 blood can be collected in a short time. The blood is 

 placed in the ice chest, and the serum is collected after 

 separation. 



The method of procedure with serum obtained in 

 the above-described manner is as follows: 



a) Small quantities of the serum are diluted with 

 sterile salt solution (0.85 per cent) so as to represent 

 dilutions of i : 5, i : 25, and i : 50. 



b) A suspension of a 24-hour-old agar culture of B. 

 typhosus in salt solution is prepared. This suspension 

 should be uniform and not heavy. It is desirable to 

 filter the suspension through absorbent cotton or sterile 

 filter paper to remove clumps of bacteria. 



c) Three hanging-drop preparations are made by 

 mixing a loopful of this suspension with a loopful of the 

 three serum dilutions, respectively. The final dilutions 

 then are: i : 10, i : 50, and i : 100. 



d) Examine with the high power (dry lens), and 

 observe the clumping of the bacilli, preceded by the 

 loss of motility. 



e) Tabulate the results as to time and completeness 

 of reaction. 



