ISOLATION OF BACTERIA IN PURE CULTURE 539 



become blurred under such conditions. The India ink will be 

 broken up, the new-formed elements pushing in beneath it; 

 even though the India ink be highly diluted, which facilitates 

 observation, the image will rapidly lose its sharpness. Our 

 objective being, in my case, partly to obtain single-cell cultures 

 from some atypical bacterial elements, and partly to trace their 

 development, it seemed natural, to attempt to do completely 

 without the India ink, the multiplication of the bacteria being 

 much easier to follow, the more dilute the India ink. When 

 this was done I admit being surprised at seeing how well the 

 organisms showed up as sharply defined, highly refractive ele- 

 ments, readily distinguishable from other chance particles or 

 impurities on the surface. 



Gelatin is however not a particularly suitable medium for 

 most bacteria, and growth at 37°C. covild not be observed in this 

 manner, so I tried whether the bacteria were as clearly visible 

 on an agar surface. In order to study this point, agar ^common 

 filtered broth agar) was melted, and by means of a coarse Pas- 

 teur pipette poured over a sterile slide. The bacteria were even 

 more readily discernible on this surface than on the gelatin. It 

 is difficult however to procure a perfectly level agar surface in 

 this maimer. This difficulty was overcome by abandoning the 

 pouring of the agar on the slide and, instead, excising the 

 medium out of an agar plate in a Petri dish by means of a knife, 

 pre\'iously sterilized in a flame and cooled, lifting the excised cube 

 of agar on the blade of the knife and depositing it on the steri- 

 lized slide to which it will immediately adhere. 



The essential conditions were now provided for tracing bac- 

 terial growth on a solid medium, especially if certain difficulties 

 could be overcome in regard to ensuring reliable and readily 

 obtained single-cell cultures. At this point in my investigation 

 a paper appeared in The Journal of Hygiene by Hort, in which 

 he describes a method, the underlying principles of which are 

 the same, namely, the observation of unstained bacteria without 

 a contrast on an agar surface, partly by means of oil-immersion 

 lenses, partly by a system of high-power dry lenses. Having 

 given a review of the usually employed methods of isolation, 



