Chapter 3 

 SPORULATION 



PRESPORULATION MEDIUM 



Sporulation in yeasts was probably first studied by deSeynes 

 (1868), who reported that round spores found "in the surface of the 

 water" germinated to produce elongated cells in a mixture of wine 

 and water, but that these elongated cells produced spores again when 

 transferred to a more dilute medium. Rees (1869) found that when 

 yeast was planted on the cut surface of various vegetables (cooked 

 or raw), growth continued until the fourth day, when budding stopped. 

 On the fifth day the vacuoles in the cell disappeared and the proto- 

 plasm became coarsely granular. Spore formation occurred regu- 

 larly on the sixth day. Spores also appeared on the sixth day if fresh 

 yeast from beer vats or wine must was transferred to the cut sur- 

 face of carrot or potato. Rees did not find spores in old lagering 

 vats nor in compressed yeast, but washed yeast from these sources 

 placed in a beaker in a layer approximately 4 mm. thick and pro- 

 tected from dust sporulated abundantly in about three weeks. 



Engel (1872) devised the classic method of inducing yeasts to 

 sporulate by transferring the cells from a nutrient broth to plaster 

 of Paris blocks. Hansen (1883) used this method successfully. Gra- 

 ham and Hastings (1941) have improved the technique considerably 

 by making the gypsum slants in test tubes, thus reducing the danger 

 of contamination. Beijernick (1898) obtained yeast spores by trans- 

 ferring the suspensions to slants made from agar that had been fer- 

 mented and subsequently washed. He preferred this method to the 

 gypsum block, probably because less contamination was encountered. 



Hansen and Beijerinck considered it essential that the yeast cells 

 be well nourished before transferring them to the aerating surface, 

 but they did not specify any nutrients aside from beer wort or grape 

 must. They believed that sporulation occurred when the "well -nour- 

 ished" cells were deprived of all nutrients, and Hansen's blocks and 

 Beijerinck's purified agar were flooded with distilled water. They 

 agreed that starvation conditions and an abundance of oxygen were 

 essential. 



Gorodkowa (1908) developed an agar containing 1 per cent pep- 

 tone and 0.25 per cent glucose. On this medium sporulation occurred 

 after 3-4 days. She showed that on a similar medium containing 5 

 per cent glucose no spores were formed, indicating that the "star- 



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