METHODS FOR OBTAINING SPORULATION 151 



medium for about 48 hours with frequent transfers. For this, beer 

 wort is generally used. It may be necessary for the rejuvenating 

 medium to contain some special substance which will stimulate the 

 formation of ascospores. 



The best method by which to make the yeast sporulate is to sub- 

 ject it to a period of inanition. Under such conditions the yeast, 

 rinding it impossible to vegetate, forms 

 ascospores. The method devised by Engel 

 and perfected by Hansen is most satis- 

 factory; it consists of placing a block of 

 plaster of Paris in the beer wort. This 

 plaster of Paris is mixed with three parts "^; 



of water and molded into a cylinder or Fig g^lc^^ Dish 

 truncated cone. It is important that the Used for Sporulation. 

 surface be smooth. 



The conditions for sporulation have been mentioned in a former 

 chapter. It is known that certain factors are indispensable: the free 

 access of air, favorable temperature, a certain degree of humidity, 

 a medium which is not too acid, not too alkaline with favorable con- 

 centration. In order to realize these conditions, the block of plaster 

 of Paris is placed in a dish in the bottom of which is a little distilled 

 water. (Fig. 63.) Enough water ought to be in the dish so that 

 about half of the block is covered. The water 

 ought never to cover the block, for the block will 

 absorb sufficient to support the development of the 

 yeast which is placed upon it. In this way the 

 yeast will find just those essentials which are neces- 

 sary for its growth. The dish is closed by a cover 



64, Hansen's ^ n suc ^ a wa ^ ^ a ^ there is full circulation of air. 



Flask for Cultur- The apparatus, thus prepared, is sterilized in the 



autoclave at 115 C. for a half hour. 



The rejuvenated yeast is placed on the block of plaster of Paris. 

 This operation is a very delicate one. If the yeast has been cultured 

 in a liquid medium, the cells may be filtered out. By means of a sterile 

 platinum wire some of the cells are then transferred to the surface 

 of the block. If the culture of yeast is in gelatin, it may be trans- 

 ferred directly to the block. The dish is then covered and put in the 

 incubator at a temperature depending on the yeast under examina- 

 tion. It has been stated that each yeast has an optimum tempera- 

 ture at which it sporulates, which is generally between 25 and 30. 

 At the end of thirty hours most of the cells will have sporulated. 

 To avoid the easy infection of the dish by -bacteria, Hansen has 

 devised a special flask which has received the name of the '* Hansen 



