156 



METHODS OF CULTURE AND ISOLATION 



cubated under -sterile covers. The gelatin solidified and the cells 

 which were contained in it developed into colonies where they lodged. 

 Macroscopic examination of the color of the colonies together with 

 the microscopic appearance of the cells gave assurance that pure cul- 

 tures had been obtained. 



This method has been very well adapted to the yeasts by Hansen. 

 He has replaced the plates of glass by moist chambers and Bott- 



cher chambers which permit the 

 development of the colonies to be 

 followed by the microscope. 



The ordinary moist chamber 

 (Fig. 65) consists of an ordinary 

 slide, in the middle of which is a 



Fig. 65. Ordinary Moist Chamber. 



depression, covered by a cover 

 slip. On this is suspended a drop 

 of the nutrient solution containing a few cells. The cover slip is 

 sealed with vaseline. 



Bottcher's chamber, or the chamber of Van Tieghm and Lecom- 

 mier, is made up of a glass ring sealed to the slide with Canada 

 balsam. (Fig. 66.) A little water is placed in this little chamber formed 

 by the ring, to maintain the proper humidity. On the top of this 

 ring is placed a cover slip from which is suspended a drop of solu- 

 tion which contains the yeast cells. Vaseline is used to fasten the 

 cover slip to the glass ring. It 

 is necessary to sterilize the ap- 

 paratus before using by passing 

 through the flame. Precautions 

 must also be observed to pre- 

 vent the ingress of extraneous 

 microorganisms. This apparatus 

 is very convenient since it allows continued observations of the cell 

 for many days (8 or more). 



Hansen's procedure for isolating the cells is to place a drop of 

 gelatin on a cover slip which is ruled into 16 numbered squares. 

 This is then placed over a Bottcher moist chamber. As soon as the 

 gelatin has solidified by cooling, the number of cells in it is counted 

 with the aid of the microscope. This operation is facilitated by the 

 rulings which allow the enumeration of each cell. The number of 

 squares occupied by the cells is determined, after which the apparatus 

 is incubated at 25. By means of microscopic observations at regular 

 intervals, it is easy to follow the multiplication of the cells as they 

 increase to form colonies. Pure cultures may be secured by inoculat- 

 ing a flask of media with one of these colonies. 



Fig. 66. Bottcher's Moist Chamber. 



a, Cover glass; b, droplets of nutrient material; 

 c, glass ring; d, water. 



