SINGLE-CELL CULTURES 81 



dish, solidified in the inspissator in the same manner as 

 for blood-serum tubes, and the coagulated film inoculated. 



For many purposes plates are unnecessary, the same 

 result being obtained by rubbing over the surface of two 

 or three tubes of sloping agar or gelatin successively the 

 once charged needle, straight or looped. In the second or 

 third tubes isolated colonies generally develop. 



The plate-culture method often fails if the organism 

 to be isolated forms but a small minority of the total 

 organisms present in the mixture ; the only alternative 

 then is to multiply the number of plates, which, however, 

 may entail great labour in their examination. 



Single-cell cultures. With large cells, such as yeasts, 

 it is not difficult to obtain growths from single cells by 

 making miniature plate cultures on ruled cover-glasses 

 and ascertaining where single cells are located in the film 

 by examining the preparation with a J or in. objective 

 (see Chapter XVI). But with the minute bacterial cells 

 this method is inapplicable. By the use of Burri's Indian 

 ink method, 1 however, single- cell cultures of bacteria can 

 be obtained. Fluid Indian ink is diluted with 6-10 

 volumes of distilled water and the mixture is sterilised 

 in the autoclave. Several loopfuls of this are deposited 

 in series on a sterile slide. The first drop is inoculated 

 with the culture which is being investigated, the second 

 drop is inoculated from the first, the third from the second, 

 and so on. A fine mapping-pen, sterilised in the flame, 

 is then dipped into the third, fourth, or fifth drops, and 

 the trace of Indian ink mixture so picked up is deposited 

 on a gelatin or agar plate. The droplet is covered with 

 a sterilised cover-glass and is examined with a J in. or 

 J in. objective, with a high eyepiece. An organism shows 

 up white on a black background. Many drops are de- 



1 Das Tuschverfahren (G. Fischer, 1909). Giinther Wagner's ink 

 Hanover) is recommended and is supplied by Griibler. 



6 



