404 CLEGG. 



of the acid-fast organism to have a good growth of amcebse for the original 

 inoculations. 



In the cultivation of amoebfe the following medium, as described by 

 Musgrave and Clegg,^ was used: 



-Agar 20. 



Sodium chloride 3.0 



Extract of beef 3.0 



Water . 1,000. 



This is prepared in the same manner as ordinary nutrient agar, the 

 finished product being made 1 per cent alkaline to phenolphthalein. 



The medium is placed in tubes and sterilized. Befoi-e inoculating 

 with the material containing the amosbse the medium is melted and 

 poured into sterile Petri dishes and allowed to harden. The material con- 

 taining the amoebse is then streaked over the surface of the plate. In 

 cultivating amoebae from water and other external sources, 100 to 500 cubic 

 centimeters of water, or the same amount of an aqueous solution or sus- 

 pension of other substances is collected in sterile flasks to which is added 1 

 cubic centimeter of alkaline bouillon for every 100 cubic centimeters of 

 the sample. The flask is then set aside for from twenty-four to forty- 

 eight hours, when an examination will usually show the presence of 

 amoebae on the surface of the liquid. A loop of this culture is then 

 streaked over the plates already referred to. The plates are then kept 

 at a temperature not to exceed 37° C. The temperature used for in- 

 cubation must be regulated according to the growth of the symbiotic 

 bacteria; if the latter are found to grow so profusely at a given degree 

 as to interfere with the development of the amcebse, then a lower tem- 

 perature must be used. After incubating twenty-four to forty-eight 

 hours the amoebse may be detected by examining the plates under the 

 low power of the microscope. After two or three days' growth of the 

 amcebse upon agar, transplants are made to fresh plates of the same 

 medium. When a suitable growth of the amoebge and a mixed culture 

 of bacteria has been procured, the next step necessary is the isolation of 

 the amoeba with one kind of bacteria. This may be accomplished in 

 the following manner: The sterile medium for cultivating amcebse is 

 melted and poured into sterile Petri dishes and allowed to harden. 

 "With a platinum loop several rings of a pure culture of the organisms 

 with which it is desired to grow the amoeba are made on the surface of 

 the hardened agar, and a small smear inoculation of the mixed culture of 

 the amoeba is placed on the middle of the smaller or central bacterial 

 ring. If the usual precautions have been taken, most amcebse, as they 

 multiply, will generally spread rapidly over the plate and, in passing 

 through the bacterial rings, will lose to a certain extent the bacteria with 



*Pul. P. I. Bur. 8ci. Biolog. Lab. (1904), No. 18, 9-78. 



