ISOLATION AND PURE-CULTURE METHODS 25 



It is usually made directly from lean beef, and is an infusion of 

 the beef in twice its weight of water. To prepare it, 500 grams of 

 lean meat, as free as possible from fat, are chopped or ground 

 finely, and to this is added 1000 cc. of distilled water. It is then 

 placed in a cool place for twelve to fifteen hours, and occasionally 

 stirred, if possible ; or it may be placed in a water bath at 65 C. 

 and frequently stirred for a period of about one hour. By the latter 

 method the bouillon is said to contain some less desirable substances, 

 but it will often be found the more desirable process for students 

 who cannot be at the laboratory regularly. All of the juice possible 

 should be squeezed out of -the meat, and a hand press is frequently 

 used by bacteriologists to accomplish this more effectively. The 

 red liquor filtered off through cheese cloth is made up to one liter 

 with water, and then to it is added I o grams of peptone and 5 grams 

 of sodium chloride. It is then heated in the steam sterilizer for 

 one hour, or in the autoclave fifteen minutes, when a clear liquid 

 with a well-differentiated coagulum is to be seen. This is filtered 

 readily through filter paper. The bouillon will now be slightly acid 

 and should be neutralized or given the desired reaction with sodium 

 hydrate (see page 33). For ordinary purposes with fungi it may 

 be enough to use the litmus paper test, but special methods of 

 neutralization are essential in the most careful work either with 

 fungi or bacteria. These must be adhered to for accurate physio- 

 logical work, or for furnishing descriptions of the fungus on specified 

 media. If the bouillon is not perfectly clear, an egg also may be 

 used as with the plant decoctions, to effect clarification. The medium 

 is next sterilized and preserved. Prepared meat extracts are used 

 by some in making bouillon. 



Milk and litmus milk are only important with the bacteria. 

 Fresh milk alone should be employed, and from this the cream 

 should be removed either by the centrifuge or by standing. Litmus 

 milk is made by the addition of 2 cc. of a saturated solution of blue 

 litmus to each 100 cc. of milk. This is extremely sensitive to the 

 development of alkalis or acids during the growth of organisms. 



Synthetic liquid media, as they are termed, that is, media pre- 

 pared by the use of salts, carbohydrates and other substances of 

 known composition are more extensively used in physiological work. 

 There the specific purpose of the experiment should be depended 



