KKKMKSTAT10N ORGANISMS 



3. Make hanging-drop cultures of very dilute cultures (one 

 or two cells in the hanging drop) and study the process of bud- 

 ding. Make observations once or twice daily for three days. 



4. Make a set of drawings to illustrate the stages in the for- 

 mation of the bud. 



Exercise 139. Spore Formation in Yeast 



The essential conditions for spore formation are abundance of 

 oxygen and paucity of food substances. 



1. Prepare gypsum blocks. Mix gypsum (plaster of Paris) 

 with half its volume of water, stir thoroughly, and pour into a 

 paper cylinder mold to harden. The block 



should be about 4 cm. in diameter and 

 2 cm. high. When dry, remove the paper 

 and place the block in a stender dish cov- 

 ered with a loose glass cover or a Hansen 

 flask (Fig. 37). Sterilize the block, covered 

 as described above, in the dry sterilizer for 

 about an hour and a half at a temperature 

 no higher than 120 C. 



2. Inoculate a flask of sterilized wort with 

 a small quantity of good yeast, Incubate at 

 25 C. for twenty-four hours. At the expira- 

 tion of that time there should be a good 

 layer of yeast at the bottom of the flask. 



3. Draw off the supernatant liquid with 



a pipette. Transfer a small quantity of the yeast sediment to the 

 dry surface of a gypsum block. It is essential that the layer 

 of yeast on the gypsum block be very thin. Pour sterile distilled 

 water into the dish so that the gypsum block stands in water 

 two thirds of its height. Incubate at 25 C. 



4. Examine yeast from the gypsum block after eight to 

 fourteen days. Look for small globular ascospores in the cells. 

 How much of the cell do they occupy ? What is their arrange- 

 ment ? Does each spore have a cell wall of its own ? 



5. Make stained preparations using gentian violet or carbol- 

 fuchsin. 



Fi<;.37. Gypsum block 

 in a Hansen flask 



