THE PREPARATION OF BOUILLON 9 



for three quarters of an hour and filtered hot. The nitrate 

 should be perfectly clear. The color will vary according to the 

 amount 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, distribute 

 the bouillon in tubes and flasks (see above), 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 sterilizer for 

 30 minutes, 1 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 bac- 

 teria 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 the incubator until the next 

 laboratory day. Then carefully examine 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 place them in the locker. 



1 The customary method of sterilizing culture media is to steam or 

 boil them for about 10 minutes on each of 3 consecutive days. This 

 was found very troublesome by the students, and, feeling that it was not 

 necessary, a long series of test experiments was made by Mr. R. C. Reed, 

 who found that I boiling or steaming for 30 minutes gave just as good 

 results as the customary 3 boilings. As the media are not used for 2 or 

 3 days after sterilization, during which time they are kept in an incu- 

 bator, the method is well suited to student laboratories, not for the 

 reason that it saves time in preparing the media, but it relieves the 

 congestion in the sterilizer and appreciably aids the student. When 

 sterilized by this method the media must not be inoculated for several 

 days after their preparation or until they have stood in an inctibator for 

 at least 18 hours to test their sterility. 



Media can be quickly sterilized by means of the autoclave when 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 certain pathogenic bacteria. The autoclave, however, is quite 

 extensively used. 



