PREPARATION OF MEDIA 81 



of blood pigment in the meat used, and according, to the 

 length of time it is steamed or boiled, i. e., according to the 

 amount of material precipitated out. After filtering, dis- 

 tribute the bouillon in tubes and flasks and stand them in a 

 wire basket for sterilization. Sterilize them by boiling in a 

 closed water bath or steaming in the Arnold's steam steril- 

 izer for 30 minutes,* the time to be computed from the time 

 the water boils or the temperature in the steamer reaches 

 99. The flasks of bouillon should be boiled or steamed for 

 20 minutes on each of the two succeeding days (certain 

 anaerobic bacteria may not be destroyed by this treatment). 

 When they have cooled the outside of the tubes should be 

 carefully wiped with a moist cloth and placed in an incubator 

 for twenty-four to forty- eight hours. Then carefully ex- 

 amine them, and if any of the tubes are contaminated, that 

 is, if the liquid is clouded or has a membrane on the surface, 

 they must be rejected. Label the others and store. 



Preparation of sugar free bouillon. Bouillon prepared 

 by the ordinary method usually contains small quantities of 

 muscle sugar. To eliminate this the following method has 

 been recommended. 1 Beef infusion is inoculated in the even- 

 ing with a rich fluid culture of some acid-producing organism 

 (B. coli) and placed in the incubator. The next morning the 

 white of an egg is added, and the infusion is boiled and 

 filtered. Peptone and salt are added as usual. It is boiled, 

 filtered again, distributed in tubes or flasks, as desired, and 

 sterilized and distributed in flasks the same as bouillon. 



Glycerin bouillon. After filtration, 3 to 5 per cent of 

 glycerine is added to the peptone bouillon and sterilized. 

 This medium is used especially for the growth of tubercle 

 bacteria in which case it is titrated to +1.3 to phenopthalein. 



1 Smith. Jour. Exper. Med., Vol. II. (1897) p. 543. 



* Media can be quickly sterilized by means of the autoclave 

 where the temperature is raised from 110 to 115 C. While this 

 method is quick and convenient, the high temperature seems to be 

 detrimental to media for the growth of certain pathogenic bacteria. 

 The autoclave, however, is quite extensively used. 



