INTRODUCTION. 3 



tures were obtained by a kind of physiological separation, 

 to obtain which he fed his yeasts on food specially suited to 

 their nutrition more suited to their wants, in fact, than to 

 those of any micro-organisms with which they are usually 

 found associated and they were thus enabled to develop so 

 luxuriantly that they prevented the growth of almost all 

 other organisms, which might have found their way into the 

 nutrient medium along with them. These cultures were 

 comparatively pure, the presence of the small number of 

 other organisms not being sufficient to vitiate the main 

 general results. 



Klebs (1873) tried to obtain pure cultures by his fractional 

 method, which was simply the removal of those organisms 

 that vegetated most luxuriantly in any special fluid to another 

 flask, making special cultures of these, and so on until com- 

 paratively pure cultures were obtained. The difficulty, how- 

 ever, was that only the commoner forms could thus be 

 separated and even then they could not be relied upon as 

 being absolutely pure. 



This method (the "maas" method) is even now sometimes employed to 

 obtain pure cultivations of cholera bacillus. A drop of the discharge from 

 a cholera patient containing a large number of organisms besides the 

 specific cholera bacillus, when placed in broth which is so prepared as to 

 be specially suited to the nourishment of the cholera organism, soon 

 becomes almost a pure culture, as the cholera bacilli are able to multiply 

 in an extraordinary degree at their "optimum" temperature and to far 

 outnumber all the other organisms present, and it becomes a much easier 

 matter to obtain from the broth culture a pure culture of the "comma" 

 bacillus than directly from the cholera discharge itself. It may be said to 

 correspond to the passing of a mixed culture through an animal. Certain 

 organisms those that are " pathogenic " can grow luxuriantly whilst the 

 others make a much weaker struggle for existence. 



As soon as it became evident that no further steps of any 

 very great importance could be taken unless pure cultiva- 

 tions were used, various workers set themselves to devise 

 methods by which single organisms might be isolated, and 

 from which a "pure-bred stock" might be raised. Then 

 Roberts (1874) an< ^ Cohn (1876) obtained pure cultures of 

 different forms of spore-bearing bacilli, especially the bacillus 

 subtilis, by subjecting the fluid to the heat of 100 C., by 

 which all non-spore-bearing forms were killed off. This 

 method, however, has a very limited application, though 

 combined with other methods, it enabled Kitasato to obtain 



