LIFE HISTORY OF AMCEBA PROTEUS LEIDY. 343 



PREPARATION OF CULTURES. 



The material from which the Amcebce herein described were 

 reared was obtained from a pool in a cattail marsh, in about 

 two feet of water among decaying lily pads and ceratophyHum. 

 The thin glutinous deposit upon the bottom and especially on 

 the lily pads which had fallen to the bottom was found to be rich 

 in Amoeba proteus. In the laboratory the material was dis- 

 tributed into several battery jar aquaria after having been 

 filtered through cheese cloth to remove the larger creatures. 



An immediate examination of the material showed that beside 

 the active proteus, radiosa, gultula, and Umax, there were many 

 encysted protozoa. One encysted form which appeared in large 

 numbers, and which confusingly resembles some of the smaller 

 encysted Amcebce was Vorticella microstoma (Fig. 4). Under 

 ordinary magnification its distinguishing feature, the crescentric 

 nucleus, is not visible. With the 1.8 mm. objective it becomes 

 faintly discernible, but it is best seen, and the cyst is indubitably 

 identified apparently only when stained. Methyl green and 

 iodine gave the best results. 1 



After some weeks had elapsed proteus and radiosa appeared in 

 large numbers. The material was now transferred to a dozen 

 small petri dishes and kept in a constant temperature of about 75 

 degrees Fahr. After a space of a fortnight there was begun the 

 transfer of inoculation of Amoeba proteus to 4 cm. stender dishes, 

 furnished with straw infusion or oak leaf infusion, and free from 

 all protozoan forms. The infusions were prepared by boiling the 

 straw or leaves for several hours, and decanting off the dark 

 brown liquor, to be diluted to the optimum strength. A slimy 

 scum formed upon the surface of the infusions after a few days 

 time, which when stirred up and caused to sink to the bottom 

 furnished a nutritive substance upon which the Amcebce throve. 

 From the stender dishes individual Amcebce were removed from 

 time to time and kept in shallow cells sunk in slides ot unusual 

 thickness. The slides employed were furnished with a device 



1 Methyl green stain: Saturated alcoholic solution methyl green, 3 parts; 

 2 per cent, aqueous solution acetic acid, i part; water 3 parts. Iodine stain: 

 saturated alcoholic solution iodine, i part; water, 2 parts. For various protozoan 

 stains, see Hausman, "Fresh Water and Marine Gymnostominan Infusoria" (in 

 press). 



