214 Appendix. 



and incubate for 24 hours at 20 C. Transfer from this 

 culture to a second tube of broth, and again incubate for 

 24 hours at 20 C. Transfer from this second culture to 

 a third tube of broth and incubate again for 24 hours at 

 20 C. Frorh this third broth culture make a gelatin 

 plate and incubate for 48 hours at 20 C. (This is to 

 prevent working with a possible mixed culture due to 

 accidental contamination.) From one of the colonies on 

 the gelatin plate transfer to a tube of slanted agar, incu- 

 bate at 20 degrees for 48 hours, and use this culture for 

 making subsequent inoculations in the various media. 



ADDITIONAL FORMULAE. 



LOEFFLER'S BLOOD SERUM. 



This medium consists of 3 parts blood serum and i part 

 of i per cent broth with reaction + 0.8. The serum 

 is obtained from fresh beeves' blood, which is collected 

 in sterile jars and allowed to stand for 24 hours in the 

 /refrigerator for coagulation. The serum is then drawn 

 off, filtered, and mixed with the dextrose broth in the 

 proportion above indicated. Hill finds that filtering the 

 serum through the coagulum obtained after adjusting the 

 reaction of the broth, gives a filtrate which is clear and 

 almost colorless (Hill, 1899). 



Tubes are filled with the mixture, placed in trays so 

 that the desired slant is obtained, and carefully heated 

 in a Koch coagulator containing cold water in the water- 

 jacket. This water is brought to a boil and kept boiling 



