SPORULATION 3-4 



Water was added to give a final volume of 100 cc. The mixture was 

 steamed for 10 minutes and tubed. Tubes were sterilized at 15 lb. 

 for 20 minutes and slanted. Some strains of yeast produce spores 

 directly on the slants if allowed to grow for a few weeks. However, 

 if spores are needed sooner, transfer to gypsum is necessary. 



Engel's gypsum block method was replaced by the method of 

 Graham and Hastings. A mixture of plaster of Paris and water 

 (100 gm. of each) was poured into test tubes and solidified in a slant- 

 ing position. These slants were dried at 50°C. for 24 hours and 

 autoclaved. 



TECHNIQUE: About 1 cc. of sterilized water is poured over a 

 3 -day growth of yeast on the presporulation agar and allowed to 

 stand 10 minutes, then a thick suspension is made by stirring the 

 yeast cells around in this supernatant water, using the side of the 

 pipette. The yeast suspension is taken up in the pipette and poured 

 over the upper part of the gypsum slant. About 3 cc. of sterile wat- 

 er containing enough acetic acid to bring the pH down to 4 is pipetted 

 into the lower half of the gypsum slant. The inoculated gjrpsum 

 slants are incubated 1-2 days at 23^C. Spores may appear in 12 

 hours. 



FREE CELL FORMATION 



One of the imusual characteristics of sporulation is the phenom- 

 enon which Harper called ''free cell formation." It is the only case 

 in which a cell is cut out of cytoplasm leaving a residuum of cyto- 

 plasm. In this respect it is markedly different from cleavage which 

 divides a mass of multinucleated cytoplasm- into mononucleate cells 

 without leaving any residual cytoplasm. When ascospores of the 

 higher ascomycetes are cut out of cytoplasm the process is appar- 

 ently under the control of the centriole which sends out small fibres 

 resembling an umbrella frame. These eventually lead to the sepa- 

 ration of the spore cytoplasm from the epiplasm, and a wall is fin- 

 ally laid down around the spore cytoplasm. 



UNREDUCED ONE-SPORED ASCI 



When the sporulating cells are removed from the gypsum slant 

 the diploid cells which have been transformed into asci differ in the 

 number of spores which they contain. Asci containing 1, 2, 3, or 4 

 spores can be found. It is generally assumed that in sporulation 

 four original nuclei are produced after meiosis and that when fewer 

 than four spores are found one or more of the original nuclei degen- 

 erated preceding spore formation. If this view were correct the 1- 

 spored asci would result from a reduction division followed by the 

 degeneration of 3 nuclei. The following experiments indicate that 

 some 1-spored asci are produced by enclosing the intact, unreduced 



