PURIFICATION AND ISOLATION OF YEASTS 155 



dilution and added five drops to five flasks of sterile milk. Some of 

 the flasks remained sterile while others seemed to possess a pure cul- 

 ture of lactic acid bacteria. These probably came from a single cell. 

 Pasteur made the first application of this method to the purification 

 of yeasts. He dried a small amount of yeast and after reducing it 

 to a powder mixed it with plaster of Paris. He allowed this mix- 

 ture to fall from a great height to make a dust. At this moment, 

 he opened flasks of sterile media. Some of the dust particles carry- 

 ing yeast cells fell into the flasks and in this way gave him pure cul- 

 tures. 



The dilution method is infinitely more certain than the physio- 

 logical method. However, it does not yield absolutely sure results 

 but only probabilities. How is it possible to affirm that a pure cul- 

 ture secured by this method came from a single cell? In spite of its 

 fundamental imperfection, however, Hansen has devised two steps 

 founded on the same principle, which allows the necessary accuracy. 



These were perfected in 1881 by Hansen. 1 Part of the yeast 

 culture is placed in a flask and is diluted with distilled (sterile) 

 water. After shaking this flask the cells will be separated uniformly 

 in the water. A drop of this liquid is taken and the cells 

 counted under the microscope. A drop may be placed under a 

 cover glass for examination. Let us suppose that there are 10 cells 

 in the drop. If such a drop is put into a flask and diluted with 20 

 c.c. of sterile water, each cell should be separated after shaking. If 

 one cubic centimeter of this liquid should be put into twenty sterile 

 flasks, theoretically one-half of the flasks should show yeast growth. 

 Practically, the results are somewhat different, for it is improbable 

 that all of the flasks received a single cell. Hansen overcame this 

 difficulty by shaking the flasks vigorously to separate the cells and 

 to distribute them equally in all of the dilution water. The flasks were 

 allowed to remain quiet until the yeast had developed into colonies. 

 These could be seen on the bottom of the flasks. The number of 

 colonies which developed gave some indication of the number of single 

 cells which were present. 



Hansen's second method consisted in the employment of a solid 

 medium. Gelatin or agar was used. He secured his idea from Koch's 

 work which today is always used. Koch's procedure involved the 

 mixing of a part of the culture in a large amount of sterile water. A 

 drop of this mixture was introduced into a flask of gelatin at 30. 

 This was well shaken to separate the microorganisms in the medium. 

 This gelatin was then poured out onto plates of glass which were in- 



1 Hansen, E. C. Chambre humide pour la culture des org. microscop. Comp. 

 Rend. lab. de Carlsberg, 3, 1881. 



