Amoebidae 73 



differs from previous methods chiefly in the use of agar* and the slight 

 modification of the salt content of the culture solution. 



Prepare finger bowls by covering the bottom with a thin (1-2 mm.) 

 sheet of agar. This is done by pouring a warm, filtered, aqueous 0.75% 

 solution of powdered agar into each bowl. While the agar is still soft 

 imbed 5 rice grains, evenly spaced. The finger bowls and pipettes are 

 previously washed thoroughly in hot water, and the rice heated (10 

 minutes) in a dry test tube immersed in boiling water — two necessary 

 precautions against contaminating organisms. 



About 50 Amoebae, together with 10 cc. of the medium in which 

 they have previously been growing, are introduced into each bowl and 

 then 30 cc. of the general culture solution (Solution A) f are added. 

 Thereafter, every three days, 20 cc. of solution A are added to each 

 bowl until the total volume is 80-90 cc. 



When maximum growth has been attained and a culture shows signs 

 of waning, it may be replenished by adding 10 cc. of solution A and 1 

 rice grain (preheated). 



In a day or two after starting a culture the agar layer becomes de- 

 tached from the bottom of the vessel and the Amoebae grow in layers on 

 its upper and lower surfaces and also on the glass surface. 



In about 2 months, it is advisable to subculture by dividing the con- 

 tents of each bowl, exclusive of the rice-agar, into four parts, pouring 

 each into a freshly prepared finger bowl, and adding an equal quantity 

 of solution A. From here the procedure is the same as before. 



If the original source of Amoebae is limited, as is the case when they 

 are collected from the fieldj, it is necessary to modify the method 

 slightly, by starting the cultures in Syracuse dishes instead of finger 

 bowls. This apparently gives a better initial concentration of the 

 Amoebae and makes the change of culture conditions less abrupt. 



The Syracuse dishes are prepared with an agar film in which 2 rice 

 grains are imbedded. Introduce the available Amoebae with 4 cc. of 

 the water in which they were collected and 4 cc. of solution A. In suc- 

 cessful cases there will be a rapid proliferation and when 200-300 animals 



*The use of an agar layer on the bottom of the dishes was originally suggested by 

 Dr. R. Chambers for the purpose of anchoring the rice grains, about which the Amoebae 

 tend to congregate. Mr. M. Sheib, working for Dr. Chambers, has been very successful 

 with this method. The writer came upon it independently, and is of the opinion that the 

 augmented growth is due to the increased surface available to the Amoebae for securing 

 their prey, although some component, added via the agar, may be involved. 



fGeneral culture solution (Solution A) — NaCl 1.20 gms., KC1 0.03 gms., CaCU 

 0.04 gms., NaHC0 3 0.02 gms.; phosphate buffer solution having a pH 6.9-7.0, 50 cc; 

 distilled water to 1000 cc. For use dilute this 1:10. This solution maintains a fairly 

 constant pH of about 7.0 and serves well not only for Amoeba, but also for general use. 



Jin ponds from beds of Vaucheria, Hydrodictyon, from the under side of Castalia, 

 Lemna, and Spirodela leaves. 



