CULTURE-MEDIA. 67 



cates the more or less arbitrarily adopted neutral point. Repeat this procedure 

 with three different portions of bouillon, and determine the average amount 

 of -^--jj- sodium hydroxide required. It is now possible to calculate the amount 

 of -y- sodium hydroxide needed to neutralize the whole quantity of bouillon. 

 This should be added. The bouillon should then be boiled for ten minutes, 

 and again titrated. It will usually be found acid. The deficiency should be 

 corrected by adding the necessary amount of -- sodium hydroxide. It should 

 be boiled again, and again titrated, and any deficiency made good. It is rarely 

 necessary to repeat the process, except to determine that the neutral point has 

 been reached. After neutralizing it is boiled thirty minutes and filtered. 

 Enough -y- hydrochloric acid or sodium hydroxide is added to give the degree 

 of acidity or alkalinity desired. It is then sterilized. 



An acid reaction may be denoted by +, an alkaline by . The degree of 



acidity or alkalinity may be indicated by the amount of - solution required 

 to render the medium neutral to phenolphthalein, thus + i.oo signifies that a 

 medium is acid, and requires i.oo per cent, of -y- sodium hydroxide to 

 neutralize it. 



A reaction of -f- i.oo is recommended as the optimum. There is much dis- 

 agreement as to what reaction is most favorable for the growth of the majority 

 of species of bacteria. Even + 0.5 may be better for some bacteria. In any 

 case the degree of reaction should be noted in descriptions. 



Bouillon may be modified by the addition to it of other sub- 

 stances, the most important of which are glycerin (6 per cent.) 

 and sugars, as dextrose,* saccharose or lactose ( i per cent.). 

 It is better to sterilize media containing sugars in the steam 

 sterilizer by the fractional method rather than in the autoclave, 

 for the reason that at the high temperature of autoclave decom- 

 position of the sugars may occur. 



Sugar-free Bouillon. Ordinary bouillon often contains some muscle-sugar, 

 which is objectionable if fermentation tests with lactose or saccharose are 

 to be made. Muscle-sugar must also be removed from the beef-juice in 

 . growing diphtheria cultures for the production of antitoxine. To secure bouillon 

 free of sugar, the water is added to the finely chopped beef as in other cases, but 

 it is then inoculated at once before any further preparation with a culture of 

 B. coli communis and allowed to stand in the incubator for twelve or fifteen hours. 

 Early next morning it is boiled, filtered, peptone and salt added, and the 

 bouillon is prepared as usual. f The medium should be tested for the presr 



*Dextrose is the principal ingredient of commercial grape-sugar or glucose 

 and should be obtained in a pure condition. 



fSee Theobald Smith. Journal o/ Experimental Medicine. Vol. II., 1901, p. 546. 



