PARAMECIUM IN PURE CULTURES OF BACTERIA 427 



gain the supremacy in fermenting, putrefying, or other abnormal 

 infusions. Under abnormal infusions are included all which for 

 any reason are unfavorable for continued growth or existence of 

 Paramecium and other infusoria. These unfavorable conditions 

 are probably due to bacteria directly or indirectly and the type 

 of bacteria producing such conditions should be known. 



One of the first things to determine in a study of healthy infu- 

 sions is the source of the bacterial infection and obviously there 

 are three possibilities. A given organism may have l^een present 

 on the hay and by this means gained entrance to the infusion. 

 It may have been present in the water used for starting the 

 infusion, or it may have settled in from the air. To secure the 

 forms present on hay, in the water or air, cultures were made as 

 follows : The standard hay, infusion used by Jennings and Hargitt 

 ('10) was made by allowing 10 grams of chopped hay to macerate 

 in a liter of tap-water. This was then sterilized in an autoclave 

 to kill all the bacteria. By pulling out the cotton plug from 

 the flask, after the fluid was cool, inoculation from the air was 

 made possible. A second flask was prepared in the same way 

 except that 100 cc. less of water 'was used. After having been 

 sterilized and cooled 100 cc. of tap water was added to furnish 

 the source of inoculation. The addition of this amount of water 

 made the solution standard. The third culture was made by 

 leaving out a little of the hay till after the sterilization of the 

 liquid. When the remaining hay was added the inoculation from 

 haj^ was accomplished and this medium was brought to the 

 standard. The three flasks contained the same amounts of 

 water and of hay, the water and hay being identical; they dif- 

 fered, therefore, in the source of bacterial inoculation. Since 

 the flasks were plugged with cotton, after having been made 

 and inoculated, any subsequent differences observed would clearly 

 be due to the source of inoculation. 



These cultures were analyzed at the end of a few days, at the 

 end of a few weeks, and again after four months had elapsed. 

 In the analysis the ordinary technic of making plates was fol- 

 lowed, the infusion being diluted with isotonic salt solution to 

 prevent crowding of the colonies of bacteria on the agar plates. 



