BACTERINS OR BACTERIAL VACCINES 317 



agar. Make a morphological study of the organism. After 

 twenty-four hours wash off the growth from each tube 

 with 3 c.c. of sterile saline solution. 



6. Put the suspension all in one container, reserving 1 

 c.c. to be used in standardization. 



Note. In the hemocytometer method for standardizing bacterins 

 it is desirable to use a special hemocytometer with a counting chamber 

 0.02 mm. deep provided with a special cover-glass for counting bac- 

 teria, but if this is not accessible, an ordinary hemocytometer and 

 cover-glass as used for blood counting may be used. If the latter, 

 a 4 mm. objective must be used for counting. 



Using the diluting pipette of the blood counting apparatus the 

 suspension of bacteria is diluted to the desired dilution with Collison's 

 fluid made as follows: 



Hydrochloric acid, 2 cc. 



Mercuric chloride 1-500, 100 cc. 



Acid fuchsin, 1% aqueous solution enough to color to a deep 

 cherry red. 



Filter before using. 



The bacterial suspension is allowed to remain in the pipette eight 

 to ten minutes to stain, then thoroughly agitated by rotating the 

 pipette and the first few drops from the arm of the pipette discarded. 

 The mount is then prepared and the slide placed on the stage of 

 microscope which has been previously leveled, and the count made. 

 The count and calculations are made as for blood counting. 



7. Heat in a water bath at 60 C. for one hour. This 

 is usually sufficient to kill the bacteria, unless they are 

 spore producers. 



8. To test the sterility of the suspension after heating, 

 with a sterile loop make an agar streak and incubate for 

 twenty-four hours. If growth is obtained the culture 

 must be heated again. 



B. STOCK BACTERINS 



The procedure in the preparation of a stock bacterin 

 is the same as in the preparation of an autogenous bacterin, 

 except that the organisms used are from cultures kept in 

 stock for that purpose. 



