MECHANISM OF COMPETITION IN YEAST CELLS 65 



flasks were closed by cotton wool stoppers. The experiments made 

 in the flasks will be described in this book as "aerobic" and those in 

 test tubes as "anaerobic." 



(3) An inoculation of yeast cells was made into the sterilized nutri- 

 tive medium. Special attention was given to the standardization 

 of the inoculating material, for in order to obtain exact and compara- 

 ble results the inoculating cells had to be in a certain fixed physiologi- 

 cal condition. Cells for inoculation were always taken from test 

 tubes where the growth was just finished. For an anaerobic inocula- 

 tion of Saccharomyces cultures 48 hours old (at 28°C.) were used, 

 whilst the slow-growing Schizosaccharomyces for an anaerobic inocu- 

 lation was taken at the age of five days at 28°C. Before inoculation 

 the contents of the test tube was shaken, and a fixed number of drops 

 of the liquid was introduced into the nutritive medium by means of 

 a sterilized pipette. It was also necessary that an equal initial 

 quantity of each species or, in other words, equal initial masses should 

 be inoculated. It was found that in anaerobic test tubes intended for 

 inoculation a mass of yeast in a unit of volume of the nutritive liquid 

 is two and a half times smaller in Schizosaccharomyces than in Sac- 

 charomyces. Therefore in order to inoculate an equal initial quantity 

 two drops of uniform suspension of Saccharomyces and five drops of 

 Schizosaccharomyces were always introduced. In the case of a mixed 

 culture, two drops of the first species plus five drops of the second 

 were taken. 2 We must prepare a perfectly uniform suspension of 

 seed-yeast and the inoculation itself must be carried out rapidly so as 

 to avoid possible errors from a settling of yeast cells in the inoculating 

 pipette. This circumstance was pointed out by Richards ('32) and 

 Klem ('33). All the experiments were carried out in a thermostat at 

 a temperature of 28°C. 



(4) After inoculation it was necessary to study the growth of 

 number and mass of yeast cells, and on the other hand to trace and 

 to evaluate the changes in the factors of the medium. The counting 

 of the number of yeast cells per unit of volume does not present any 

 difficulty and for this purpose the Thoma counting chamber is usually 

 employed. In our experiments three test tubes (or flasks) of the 



2 A very strict equality of the masses of two species sown is not absolutely 

 necessary. It is only important that the very same quantity of each species 

 should be introduced into the mixed population and into the separately grown 

 culture. This is very easy to do with our mode of inoculation. 



