154 METHODS OF CULTURE AND ISOLATION 



Physiological method: This procedure rests on the fact that the 

 organisms in a mixed culture multiply unequally in the given medium 

 and at the given temperature. Certain species die or vegetate slowly; 

 they are finally eliminated by the most vigorous varieties. This is, 

 then, a selection by vital concurrence which results in the elimina- 

 tion of certain species by others. Thus, to separate bacteria from a 

 yeast a small quantity of acid is added to the culture medium (tar- 

 taric, lactic, hydrochloric or hydrofluoric). The bacteria prefer the 

 alkaline media, while the yeast finds the acid most favorable. The 

 yeast alone develops in this medium. If, on the contrary, one wishes 

 to secure the bacteria a little alkali is added to the medium. By 

 cultivating a mixture of yeasts in chemically different media and at 

 different temperatures, one is able to separate them. Some will find 

 one medium and temperature more favorable. 



This method, which has been employed by many of the early 

 workers, particularly by Pasteur and Cohn, is purely empirical and 

 does not give reliable results. One may never exactly know what to 

 expect with it. It is easy to suppose, for example, that one variety 

 may be temporarily eliminated for the time being by the development 

 of another form which finds the conditions more favorable; this last 

 variety, however, after developing rapidly, may finally be suppressed by 

 another form which has been dormant up to this time. Many vari- 

 eties may develop together if all of the conditions are favorable. 

 This was the case when Pasteur purified brewery yeast by this method. 

 He cultivated his yeast in a solution containing a little sugar to which 

 was added a small amount of tartaric acid. Later investigations by 

 Hansen showed that Pasteur had eliminated the bacteria associated 

 with the yeasts, but he had failed to effect a separation of the dif- 

 ferent varieties of wild yeasts, some of which caused the diseases of 

 beer. 



It is, then, impossible to secure reliable results by the physiolog- 

 ical method of dilution. It is valuable, however, in starting the 

 purification because it allows the bacteria to be separated from the 

 yeasts. The yeasts m'ay be only definitely separated by the proce- 

 dure which we shall now take up. 



Dilution Method for Separating Yeasts: This involves a mix- 

 ture of the microorganisms which one wishes to separate until the 

 cells are well isolated. 



Lister conceived this procedure for the separation of lactic acid 

 bacteria. He counted the number of bacteria in a drop of sour milk 

 under the microscope and from this computed the required amount of 

 distilled water which it would be necessary to add to this drop so that 

 a drop of the mixture would contain a single cell. He prepared such a 



