CHAPTER V. 



THE MATERIALS AND METHODS USED IN THE CULTIVATION 



OF BACTERIA. 



THE methods for the artificial cultivation of bacteria are of funda- 

 mental importance in bacteriology. The study of the characteristics 

 of any bacterium requires that it be examined growing apart from all 

 others. In order to separate one species from others and to study its 

 morphological, biochemical, and cultural characteristics we have to 

 prepare a number of sterile, solid, and liquid media and employ them 

 in various technical ways. Before we can get a suitable growth of any 

 special variety of bacteria, we must have the substances necessary for 

 their growth present in the proper proportion and concentration. Dif- 

 ferent species of bacteria require very different foodstuffs, so that for 

 each kind the proper food must be found through experimentation. 



Preparation of Culture Media. The most commonly used media have 

 as their basis the watery extract of meat and peptone. The addi- 

 tion to this by Koch of gelatin gave us a transparent solid medium 

 which had, however, the objection of melting below the temperature 

 required for the growth of many pathogenic bacteria. Another sub- 

 stance of vegetable origin (agar) was found, which melted just below the 

 boiling point of water. This has been substituted for gelatin whenever 

 we desire to grow bacteria at temperatures above 20 C. or desire other 

 characteristics of the agar media. 



PREPARATION OF MEAT IXFUSIOX. One pound (500 grams) of finely 

 chopped, fresh, lean meat is macerated in 1000 c.c. of water and put in 

 an ice-chest for from eighteen to twenty-four hours; or it may be warmed 

 at a temperature not exceeding 60 C. for one hour. Any fat present is 

 skimmed off. The last traces can be removed by stroking the surface 

 with filter-paper. The infusion is now strained through a fine cheese- 

 cloth into a flask, and the remaining meat placed in a cloth and squeezed 

 by hand or in a press. The resulting fluid contains the soluble albumin, 

 the soluble salts, extractives, and coloring matter of the meat. This 

 meat extract is then exposed to live steam, either without pressure in the 

 Arnold steam sterilizer (Fig. 21) for thirty minutes, or, if the changes 

 produced by a temperature of 110 to 115 C. are not objectionable, in 

 the autoclave at one atmosphere of pressure for fifteen minutes, or 

 boiled over a free flame for ten minutes. During this process all albumins 

 are coagulated. While still hot the fluid is filtered through filter-paper 

 or through absorbent cotton, and the reaction is tested and sufficient 

 normal hydrochloric acid solution or sodium hydroxide added to give 

 it the desired reaction, which is for most bacteria slightly alkaline to 

 litmus. If in the boiling there has been any evaporation, sufficient 



