PATHOGENIC PROTOZOA OF THE CLASS SARCODINA 417 



Staining Methods. The organisms stain rather readily with the 

 usual laboratory stains, but these are of little value in the differ- 

 entiation of the parts of the cell and in separating species. Wright's 

 stain in favorable specimens, if carefully used, gives the best 

 differentiation of the parts of the cell. The organism in tissues 

 is best stained by Heidenhain's iron-hematoxylon or Borrel's 

 stain. The smears should be fixed from fifteen to twenty minutes 

 in alcohol and ether, equal parts, for all stains but Wright's. For 

 the latter no fixation is required. 



Methods of Isolation and Cultivation. Amebae commonly 

 use bacteria and related organisms as food. A culture of amebse 

 must provide for the growth of bacteria, but this growth must not 

 be so luxuriant as to overgrow and eliminate the amebse. The 

 agar medium recommended by Musgrave and Clegg may be used. 

 This contains agar, 20 gm.; NaCl, 0.03 to 0.05 gm.; beef-extract, 

 0.3 to 0.5 gm.; water, 1000 c.c., made 1 per cent, alkaline to 

 phenolphthalein. Variations in the materials used are sometimes 

 necessary for some specialized saprozoiites. This medium is 

 melted, poured into a sterile Petri dish, and allowed to solidify. 

 The surface of the medium is streaked with the material containing 

 the amebae. The bacteria and amebae will usually both multiply 

 if the plate be kept at a suitable temperature. Two operations are 

 necessary to secure a culture in which it is known that all of the 

 amebse are of one species and all of the bacteria of one kind. The 

 mixed culture is placed upon the medium in the center of the 

 Petri dish. Concentric circles of the organism with which it is 

 desired to grow the ameba are placed about this. The amebae, 

 as they crawl through the successive circles, gradually lose the 

 original organisms with which they started and come to feed on the 

 one kind. After one or more transfers a growth of the amebae 

 may be secured with the one species of organism desired. It is not 

 always possible to secure growth with every species of organism, 

 as the different amebae have been found to require various species 

 of bacteria. 



In order to secure a culture containing a single kind of ameba, 

 it is necessary to isolate a single individual. Ordinarily this may 

 be accomplished by examination of the surface of an agar culture 

 until an isolated ameba is found, and this is then transferred to a 



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