194 Cultivation of Micro-organisms 



cold. The jelly, which solidifies between 40 and 5oC., cannot 

 again be melted except by the elevation of its temperature to the 

 boiling-point. The culture-medium made from agar-agar is nearly 

 transparent. In addition to its ability to liquefy and solidify, it 

 has the advantage of remaining solid at comparatively high tem- 

 peratures so as to permit keeping the cultures grown upon it at the 

 incubation temperature, i.e., 37C., at which temperature gelatin 

 is always liquid. 



The preparation of agar-agar is commonly described in the text- 

 books as one "requiring considerable patience and much waste of 

 filter paper." In reality, it is not difficult if a good heavy filter paper 

 be obtained and no attempt made to filter the solution until the agar- 

 agar is perfectly dissolved. 



It is prepared as follows: To 1000 cc. of bouillon made as described 

 above, preferably of meat instead of beef-extract, 10 to 15 grams of 

 agar-agar are added. The mixture is boiled vigorously for an hour 

 in an open pot over the direct gas flame or in the double boiler with 

 saturated calcium chlorid solution in the outside pot. After being 

 cooled to about 6oC., and after the correction of the reaction by 

 titration, an egg beaten up in water is added, and the liquid again 

 boiled until the egg-albumen is entirely coagulated. 



After the second boiling and the replacement of the volatilized 

 water, the agar-agar is filtered through a carefully folded pharma- 

 ceutic filter wet with boiling water. It may expedite matters to 

 pour in about one-half of the solution, keep the remainder hot, and 

 subsequently add it. 



The formerly much employed hot-water and gas-jet filters are un- 

 necessary. If properly prepared, the whole quantity will filter in 

 from fifteen to thirty minutes. 



Ravenel* prepares agar-agar by making two solutions, one repre- 

 senting the meat-infusion, but twice the usual strength, the other 

 the agar-agar dissolved in one-half the usual quantity of water* 

 The agar-agar is dissolved by exposure to superheated steam in the 

 autoclave, after which the two solutions are poured together and 

 boiled until all of the albumins are precipitated. The coagulation 

 of the albumins of the meat-infusion serves to clarify the agar-agar. 



If agar-agar is to be made with beef-extract, the bouillon should 

 be made first and filtered when cold, to exclude the uratic salts which 

 otherwise precipitate in the agar-agar when cold and form an un- 

 sightly cloud. 



The finished agar-agar should be a colorless, nearly transparent, 

 firm jelly. It is dispensed in tubes like the gelatin and bouillon, 

 sterilized by steam, either by the intermittent process or in the auto- 

 clave, and after the last sterilization, before cooling, each tube is 

 inclined against a slight elevation, so as to permit the jelly to solidify 

 obliquely and afford an extensive flat surface for the culture. 

 * "Journal of Applied Microscopy," June, 1898, vol. I, No. 6, p. 106. 



