544 CLASSIFICATION AND MORPHOLOGY OF AMEBA 



medium are necessary before this end is reached. The further 

 inoculations are made with amebae obtained from outside the largest 

 ring on the next preceding culture. 



This method is simple in execution, and the entire process may 

 be watched under the microscope by inverting the plate and using a 

 low power, according to the method employed in studying colonies of 

 bacteria. With a low power the wanderings of the amebae and even 

 their multiplication can be kept under observation. 



The ring-shaped smear of bacteria has several advantages over 

 one covering the entire surface of the plate. In the first place amebae 

 develop more rapidly by its use, and secondly, they lose the original 

 organisms much more rapidly than when moving constantly over a 

 bacterial substratum. 



Another feature which commends this method is the facility with 

 which it lends itself to a determination of the symbiotic value of a 

 given organism. If such an organism for any reason is not satisfactory 

 to the amebae they will not mix with or cross the bacterial rings. 

 In some instances, where the organism is particularly unfavorable, 

 the amebae, after wandering up to the inner margin of the first 

 ring, encyst, and no further progress is made. On the other hand, 

 where the antipathy is less marked, the progress is simply delayed 

 until the bacteria carried over in inoculating the amebae have mixed 

 with or crossed the ring, whereupon the amebae follow them. 



When amebae have been isolated and grown in pure culture with 

 a satisfactory symbiotic organism it is sometimes difficult to transfer 

 them to another. This is best overcome by first cultivating the pro- 

 tozoa for a short time on a mixed culture of the two organisms and 

 then isolating them with the desired one by the use of the method 

 described. Even by this means success is often doubtful and some- 

 times impossible of attainment. 



Among the bacteria with which amebae have entered into symbiotic 

 community as reported by various observers are the spirillum of 

 Asiatic cholera, typhoid bacillus, Bacillus coli, Bacilli fluorescens 

 liquefaciens and non-liquefaciens, Staphylococcus pyogenes aureus, 

 Bacillus pyocyaneus, Bacillus ruber, spirillum of Metchnikoff, various 

 other bacteria, and also yeast cells. 



Walker, starting out from pure mixed cultures, according to the 

 above method, has modified it in such a manner that he could study 

 the whole development under the microscope. He calls his device 

 the "hanging-plate culture," and prepares it as follows: A thin | inch 

 cover-glass is flamed and placed under a flamed watch-glass. With 

 a large platinum loop a large loopful of melted sterile agar is trans- 

 ferred to the sterile cover and spread in a uniform, thin, and circum- 

 scribed layer. This film of agar will solidify almost instantly, and it 

 is at once inoculated from a pure mixed ameba culture. The cover- 

 glass culture is then inverted over a concave slide which has been 

 flamed, cooled, and rimmed with vaselin. On such cover-glasses 



