HANSEN'S METHOD OF SINGLE-CELL CULTURE. 221 



more of those selected remains barren, either because it was already 

 dead at the commencement of the test or else was too feeble to 

 develop in the strong (10 per cent.) gelatin medium. The task of 

 seeking out the cells is troublesome, even for the experienced 

 worker. It may be greatly lightened by using cover-glasses cross- 

 etched in squares of about 2 mm. side, as recommended by Will, 

 and preferably marked with etched figures as advised by Alfred 

 Jorgensen. This is also desirable on account of the ease with 

 which it enables the position of the cells to be entered in the 

 note-book, and facilitates keeping a record of the observations. 

 When the marking is finished, the Bottcher cells are placed in a 



FIG. 160. 



Chamberland Flask. 



About one-fifth natural size. 



(After Hansen.) 



FIG. 161. Pasteur Flask, with Hansen's 

 improved modification by widening 

 the swan neck tube. About one-fifth 

 natural size. (After Hansen.) 



large double basin and left for about twenty hours in the 

 thermostat at about 20 C., by the end of which time the cells 

 that are capable of development will be actively forming colonies. 

 After another twenty-four hours the colonies will be visible to 

 the unaided eye, as small white dots. The selected colonies are 

 again examined under the low power, for the presence of 

 dangerous neighbours, and inoculations are made from each 

 colony into flasks already charged with sterilised nutrient medium 

 (wort, wine, must, &c.), another examination being afterwards 

 made to make sure that the selected colonies alone have been 

 drawn upon and that the adjacent ones are intact. When an 

 inoculating hook is used, it will be found more convenient to 

 transfer the cells to a Freudenreieh or Chamberland flask (Fig. 

 1 60), and then, after further reproduction, inoculate from this 

 with a loop into a Pasteur flask (Fig. 161), with a capacity of 

 about 250 c.c. and charged with about 100 c.c. of wort. On the 

 other hand, the inoculation can be conveniently effected into the 



