130 CULTURE MEDIA AND THEIR STERILIZATION 



Such a bouillon is designated as a + 1.5 or + 1.0 bouillon. Its 

 reaction is acid toward the phenolphthalein indicator but alkaline 

 toward litmus. 



An easier method for preparing the nutrient bouillon is the fol- 

 lowing: Take 4 to 6 grams of meat extract, 10 grams of dried beef 

 peptone, 5 grams of common salt, dissolve in enough water to make 

 1000 c.c. Boil well and neutralize with caustic soda solution, then 

 filter clear, distribute to test-tubes, and sterilize as before. 



Gelatin. 1. Prepare 1000 c.c. of a clear, faintly alkaline nutrient 

 bouillon. Heat. 



2. Dissolve 100 grams of best clear French gelatin in the hot solu- 

 tion. This again makes the solution quite acid. 



3. Add caustic soda solution until the mixture becomes faintly 

 alkaline to litmus. 



4. Prepare a funnel with a double paper filter through which 

 boiling hot water has been poured. When the latter has drained off, 

 filter the gelatin through the hot filter into a flask. This must be 

 done in a warm room, as otherwise the filter is likely to cool, and the 

 gelatin may set in it. (If necessary, redissolve the gelatin over a 

 water bath, not over an open flame.) 



5. While still warm, distribute the filtered gelatin into sterile test- 

 tubes, about 10 c.c. to each. 



6. Sterilize by fractional sterilization, as already described, for three 

 or four days in the steam sterilizer (not the autoclave). During the 

 intervals keep in a warm place, but the temperature must not be so 

 high as to cause the gelatin to remain fluid. 



7. After the last sterilization in the steam sterilizer, keep the tubes 

 in an upright position so that the gelatin sets in a cylindrical mass 

 (not with a slanting surface). 



A good gelatin culture media must be perfectly transparent. 

 Agar-agar. 1. Prepare 1000 c.c. of clear, faintly alkaline, nutrient 

 bouillon. 



2. Take 15 to 20 grams of agar-agar and cut the long strips into 

 small pieces, the smaller the better. Soak for from twelve to twenty- 

 four hours in cold water. Drain the water off through a cloth and 

 wring out the swollen mass, so as to remove as much water as 

 possible out of the agar-agar. 



3. Add the agar-agar to the bouillon and heat until the former is 

 entirely dissolved. The heating may be carried on in an autoclave, 

 a steam sterilizer, or over an open flame. In the latter case the mix- 

 ture must be stirred continually so that the agar-agar does not burn. 



4. When solution is completed, test the reaction again. As a rule 

 it does, not change, but remains slightly alkaline, as it was originally. 



5. Filter clear. This is the most difficult and tedious process in 

 the preparation of a good agar. Filtration is very slow and sometimes 

 requires a number of days. The process must be carried on in the 

 autoclave, the steam sterilizer, or through a double jacketed copper 



