Amoebidae 77 



found that the heavy organic material will settle in a thick layer on the 

 bottom, and that the Amoebae will settle on top of this layer. They 

 then may be removed with a pipette to a shallow dish and examined. 



Amoeba proteus collected in this way may be cultured simply by 

 placing a suitable spring or pond water in finger bowls or other shallow 

 dishes to depth of about 2 cm., adding 3 to 4 grains of wheat, or 5 to 6 

 grains of polished rice, or 5 to 6 one-inch stems of timothy hay, and 

 then inoculating with Amoebae from the collection. The Amoebae in 

 these cultures will become very abundant in from 3 to 4 weeks. It 

 should be noted here that Dawson (1928) gives an elaborate method 

 for obtaining cultures from freshly collected material consisting of a 

 gradual dilution of the collected water with distilled water. This process 

 of dilution in our experience has not been necessary. Cultures obtained 

 from freshly collected material are likely to contain in addition to 

 Amoebae various organisms such as small Crustacea, rotifers, and va- 

 rious Protozoa. Since none of these organisms, except the cryptomonad, 

 Chilomonas, are necessary as food, and since others such as the Crustacea 

 probably feed on the Amoebae ; it is well to take steps to eliminate these 

 unnecessary organisms. A. proteus feeds on a variety of organisms, but 

 Chilomonas alone is entirely adequate. 



Freeing Amoeba cultures oj contaminating organisms and establish- 

 ing clone cultures. Sterilize some spring water by autodaving for 15 

 minutes at 15 pounds' pressure, or merely by bringing to a boil. Allow 

 to cool. Place sterile spring water in three or four sterile, chemically 

 clean Syracuse watch glasses, and a finger bowl. The water in the 

 finger bowl should have a depth of about 2 cm. Add to the finger bowl, 

 3 to 4 grains of wheat, 5 to 6 grains of rice, or 5 to 6 pieces of timothy 

 hay stems about 1 inch long. The wheat, rice, or hay should be auto- 

 claved dry 15 minutes at 15 pounds' pressure, or placed dry in a test 

 tube, then the test tube placed in a beaker of boiling water for about 15 

 minutes. Now with a sterile capillary pipette and under a binocular 

 microscope, select active Amoebae, pass them one at a time through the 

 dishes of sterile spring water. If it is desired to obtain a clone culture 

 (culture containing Amoebae all of which have descended from a single 

 parent) put only one washed Amoeba into the finger bowl culture. To 

 obtain merely a clean culture it is advisable to put into the finger bowl 

 culture 25 or more Amoebae. Now add to the culture in the finger bowl 

 containing washed Amoebae a drop of culture fluid containing only 

 Chilomonas [see pp. 59 and 63] and bacteria. The best way to do 

 this is to take small drops of the old culture fluid, examine them carefully 

 under high power selecting only those drops which show only Chilomonas 

 and bacteria to be present, rejecting all others. It is best to place these 

 drops each on a small sterile coverslip, and then if the drop is found 



