152 METHODS OF CULTURE AND ISOLATION 



flask." (Fig. 64.) It is a cylindrical flask fitted with a glass top 

 ground on with emery. This is pulled out and plugged with a bit of 

 cotton. A side tube is closed with a piece of rubber tubing. A cylin- 

 drical block of plaster is put into the flask about which is placed 

 bouillon. The apparatus is sterilized at 115 in the autoclave. The 

 yeast is introduced into the flask in the usual manner. 



Gorodkowa 1 has recently proposed a new method which has the 

 advantage of being less complicated than the method of Engel-Han- 

 sen. It seems to give equally good results. It consists simply in 

 inoculating a gelatin mixture with cells of the active young yeast. 

 This medium is prepared as follows: 



Distilled water 100 grams. 



Gelatin 1 " 



Meat bouillon 1 " 



Sodium chloride 0.5 " 



Glucose 0. 25 " 



The yeast develops quite rapidly after inoculation and the small 

 amount of glucose is not sufficient to assure its nutrition for a long 

 time. Thus, sporulation is stopped at the end of two or three days. 

 This medium was utilized by Guilliermond for many yeasts with quite 

 gratifying results. 



Many other methods, founded on somewhat the same principles, 

 have been perfected to demonstrate ascospores. Some consist of put- 

 ting the yeast on blotting paper in distilled water or on pure gelatin. 

 (Wasserzug.) Yeast water will also allow ascospore formation which is 

 not sufficient to assure the nutrition of the yeast. It soon finds it- 

 self reduced to a condition which makes it sporulate. All sorts of 

 liquids placed in extremely thin layers are sufficient to make a yeast 

 sporulate if the food is quickly exhausted. 



Yeasts will also sporulate on solid media (nutrient agar or gela- 

 tin). Rees has shown that slices of carrot make a good substrate for 

 this purpose. The great majority of yeasts form ascopores after 

 from 6 to 8 days, sometimes before. They grow actively for a few 

 days and then budding slows up probably on account of an accumula- 

 tion of toxic substances in the medium and sporulation begins. This 

 method is to be recommended for cytological investigations, for it 

 permits observations from the germination of an ascospore to the 

 formation of a new ascospore. It facilitates the fixations which are 

 necessary by making it easy to cut out a piece of the carrot on which 

 the yeast is growing and placing it in the fixing bath. Guilliermond 

 used this procedure with success in his investigations on the copula- 



1 Gorodkowa. Ueber das Verfahren rasch die Sporen von Hefepilzen zuge- 

 winnen. Bull. Jard. Bot., Petersburg, Vol. 1, 1908. 



