174 VETERINARY BACTERIOLOGY 



slanted. Streaks of the organism are made the full length of the 

 slant and incubated twenty-four hours at blood-heat. The water 

 of condensation is removed without disturbing the growth, and 

 100 c.c. of sterile physiological salt solution added. The growth 

 is carefully scraped from the surface with a long sterile glass rod; 

 the suspension is placed in a sterile flask and shaken thoroughly. 



Standardization of the Vaccine. Before injections are made, 

 the number of bacteria present per given volume of the suspension 

 must be known in order that accurate dosage may be determined. 

 The standardization may be effected by direct count or by use of 

 the nephelometer. In the first method, equal parts of the bac- 

 terial suspension and of defibrinated human blood are thoroughly 

 mixed, a smear prepared, and stained with a blood-stain or carbol- 

 thionin. The number of red blood-cells on several fields and the 

 number of bacteria on these fields are counted (see Fig. 79) . The 

 number of red blood-cells may be taken as 5,000,000 per cubic 

 millimeter. The number of bacteria per cubic millimeter will be 

 found by the following proportion: 



5,000,000 : x : : number red blood-cells per field : number bacteria per field. 



Suppose 20 red blood-cells per field, and 50 bacteria, the ratio 

 becomes 



5,000,000 : x :: 20 : 50 or x (number of bacteria per cubic centimeter) = 



12,500,000. 



To convert the number of bacteria per cubic centimeter, multiply 

 by 1000, which in this case would give 12,500,000,000. It is cus- 

 tomary to dilute the bacterial suspension so that each cubic cen- 

 timeter will contain a number of bacteria that is readily reckoned, 

 usually 50,000,000 per cubic centimeter. 



The nephelometer is an instrument described by McFarland, 

 in which tubes containing varying amounts of barium sulphate 

 in suspension in water are observed side by side with tubes of equal. 

 size containing the bacteria. After the tubes containing barium 

 sulphate have been compared as to opacity with tubes containing 

 known numbers of bacteria per cubic centimeter, they may be 

 used in determining the number present in other bacterial suspen- 

 sions. 



