Methods of Cultivation and Investigation. 



a. Cultivation and Isolation. 



Where many hundred strains of bacteria have to be kept under observation for years 

 together, it requires no littlework merely to keep their vitality in some measure unimpaired. 

 At first, when we were unable to regulate the temperature during the summer months, 

 this was a serious difficulty, and it was only by experience acquired in the course of years 

 that we were able to master the situation. And even now, certain pathogenic streptococci, 

 as well as some divergent rod forms, still cause trouble. The method of preservation is 

 not the same for all species, but must be adapted to their particular demands. 



As regards the lactic acid bacteria of the dairy industry, it might seem natural to 

 propagate them by transference from milk to milk. Unless very lo"w temperatures can be 

 maintained, however, it will be necessary to repeat this process each week, as otherwise, 

 the bacteria will be killed off by the acid formed. We have therefore only employed this 

 method for the more specific milk bacteria. If chalk be added to the milk, and the cultures 

 shaken regularly, the bacteria can retain their qualities unaltered for several months. 

 This, however, necessitates the use of large flasks, as the chalk cannot be shaken up in 

 ordinary test tubes, so that method is only practicable where but a few cultures have to 

 be dealt with. The employment of large flasks, moreover, increases the danger of infection 

 from the air during inoculation. We have used this method to preserve the power possessed 

 by certain species of forming slime in milk; a quality which is very soon lost on solid sub- 

 strates. 



For preservation of the bacteria we have as a rule employed agar stab cultures in 

 Freudenreich flasks. Such cultures present, in reality, the same advantages as milk 

 mixed with chalk, as the acid, which is only formed in the stab, is thence difîused 

 into the remaining agar mass. Although the true lactic acid bacteria only exceptionally 

 grow in sugar-free broth, they will nevertheless all thrive in sugar-free agar^), which shows 

 that the agar, after sterilisation, contains some soluble carbohydrates. The growth 

 is furthered, however, by the addition of sugar, though in the case of keeping 

 cultures, this should be restricted to 14 % grape sugar, thereby prevent- 

 ing the formation of more than % acid. In order further to keep down 

 the hydrogenion concentration, the substrate should be rich in buffers 

 such as amino-acids and phosphates. Prior to sterilisation, the substrates were 

 neutralised as exactly as ])ossible to litmus paper. For nitrogen food, we used at first 2",, 



') The growth of tlie susar loving thci iii()l):ictcria is. however. e.\tremelv slight. 



