ISOLATION OF BACTERIA tN PURE CULTURE 541 



is evident that we must feel perfectly sure on this point, consid- 

 ering that the area within the circle is the only one examined. 

 However, it seems difficult to understand how one can be sure 

 that the small droplet keeps within the etched circle, as a small 

 liquid layer will form between the coverslip and the agar, the 

 moment these two are directly applied to each other, which will 

 immediately make the droplet invisible. The possibility will like- 

 wise always exist, of various currents arising between coverslip and 

 agar, both when the slip is placed and removed; and, even though 

 the growth of a colony from one single cell has been observed 

 ^vdthin the circle, this may become contaminated from a small 

 colony immecUately outside the circle, the moment the cover- 

 slip is removed. As a rule, there is little chance of this happen- 

 ing, but it does compromise the reliability of the method. 



Moreover, there is the question of the power of certain motile 

 bacteria to move on the uncovered agar plate. The possibility 

 of such active motilit}' on the part of the organisms is, of course, 

 increased by the placing of the coverslip on the agar, by which 

 a small liquid-filled space is formed. 



For his second method Hort emploj^s a medium prepared in a 

 similar way. A highly chluted emulsion of bacteria is spread 

 over the agar plate by means of a glass rod, the inoculated plate 

 being now covered ^^ith a thin sterile strip of celluloid, which 

 has been previously perforated. Small sterile covershps are 

 placed over the holes so as to form minute moist chambers. 

 These are now searched with a high-power dry lens and the cham- 

 bers containing one single cell only, are marked. The examina- 

 tion is now continued as described above. (It is not evident 

 from Hort's treatise, whether the coverslips are removed during 

 the examination under the microscope; if not, one would think 

 that the dew on the coverslip would be obstructive.) The col- 

 onies ha\ing reached an appropriate size, subcultures are pre- 

 pared by means of a special apparatus consisting of a tube with 

 a needle adjusted in a special way and screwed on to the nose-piece 

 like an objective. This method is reliable, but as Hort himself 

 says in his final remarks: "In conclusion it is necessary to point 

 out that cultivation of bacteria from single cells is, even when 



