ISOLATION OF MICROORGANISMS 63 



in determining fermentation if this character is to have meaning in the separa- 

 tion of species. In the following discussion of methods, an attempt will be 

 made to point out some of the sources of error and objections raised to each 

 method. As in many other organic reactions, details of method often pro- 

 foundly influence the point of equilibrium. 



Lindner's Microfermentation Method. — A hollow-ground microscopic slide 

 is flamed and filled with sterile water. A relatively large number of yeast 

 cells is suspended in the water and a pinch of the sugar to be tested is added. 

 A cover glass is carefully lowered so as to exclude any air bubbles and sealed 

 in place with lanolin or vaseline. This is placed in an incubator for 24 hours 

 at the optimum temperature and the presence of bubbles (supposedly of car- 

 bon dioxide) noted. In this method it is assumed that there will be com- 

 paratively little growth and consequent production of CO2, due to absence 

 of oxygen and the lack of nutrients. The amount of gas should be roughly 

 proportional to the amount of the inoculum. If the organism ferments very 

 slowly and a larger quantity of inoculum is used, the amount of glycogen trans- 

 ferred with the yeast ceUs and that diffusing out of the dead cells may be 

 sufficient to give gas production. Since the sugar is not sterilized, there is 

 always a possibility of introducing some fermenting bacterium. Also there 

 is a possibility that the seal is not tight and that subsequent evaporation may 

 give the appearance of gas production or may allow the gas to escape. Changes 

 of temperature or of atmospheric pressure may also give erroneous results. 

 Guilliermond modified the method by filling a Van Tieghem cell with a rela- 

 tively large volume of liquid, and dissolving the sugar in a definite concentrar 

 tion in a yeast decoction. This modification presupposes that the yeast may 

 grow, and consequently the COg may be partly the result of respiration rather 

 than fermentation in the strict sense of the word, especially as there may be 

 dissolved oxygen in the liquid. 



The Fermentation Tube Method. — In this method, bent tubes of varying 

 pattern, perhaps originally used by Einhorn in 1885 but introduced into mi- 

 crobiology by Theobald Smith in 1890, are filled with a sugar solution and 

 sterilized. The amount of liquid should be sufficient to slightly more than fill 

 the long arm but not so much as to wet the plug. This is inoculated with 

 the organism and set aside in an incubator until the organism has grown long 

 enough to develop gas. If the long arm is graduated, the volume of gas pro- 

 duced may be noted. Since the surface of the liquid in the long arm is not 

 exposed to free oxygen, strict anaerobic conditions are maintained. Also, 

 if the organism is strictly aerobic, it grows only in the short arm, and there 

 is not enough growth in the long arm to show fermentation, even if the organ- 

 ism is capable of producing it. On the other hand, since the carbon dioxide 

 is quite soluble in the solution, small amounts are dissolved and do not show 

 up. There is also the problem as to the source of the gas, whether it is from 

 fermentation or respiration. Aichelburg (1932) reports that gas production 

 in fructose shows on the third day while glucose shows on the first day. 



