84 Phylum Protozoa 



them for some time in pedigreed or clone cultures to be sure you have 

 the right species. To obtain these amoebae in clone cultures proceed 

 as follows: 



With a sterile capillary pipette pass an amoeba through three or four 

 changes of sterile seawater in sterile depression slides. From the last 

 depression slide transfer it to a small drop of sterile wheat infusion in 

 seawater (prepared by boiling about 30 grains of wheat in 100 cc. of 

 seawater for about two minutes). Now with sterile vaseline seal the 

 coverslip over the depression slide with the drop hanging down from 

 the under side of the coverslip. Prepare several hanging-drop clone 

 cultures in this way. When several amoebae have developed, sterilize 

 the upper surface by washing with absolute alcohol. Allow the alcohol 

 to evaporate completely, break the seal with sterile forceps, remove the 

 coverslip and drop it with the amoeba-side up into sterile seawater in a 

 sterile petri dish. Add 6 grains of wheat sterilized by autoclaving 15 

 minutes at 15 pounds' pressure. The amoebae will remain attached to 

 the coverslip and thus will easily be found when desired. They will 

 become abundant within a week. If evaporation is prevented, the 

 amoebae will remain viable for weeks. The cultures, however, come to 

 a condition of maximum population in about seven days. They should 

 be subcultured every two weeks. 



To subculture the pure clones sterilize wheat, petri dishes, seawater, 

 and inoculating pipettes; place seawater and 6 grains of wheat, in a 

 petri dish ; then with the pipette draw some of the amoebae up from the 

 bottom of an old culture and add them to the new culture medium. 

 Growth will proceed as before. 



These amoebae feed on bacteria; therefore there is little difficulty in 

 obtaining food organisms. Sufficient bacteria, except in rare cases, are 

 carried with the amoeba to inoculate the new cultures even after four 

 or five washings. 



The salt content of cultures. F. mira is remarkable in its ability to 

 adapt itself to great variations in the salt content of its medium. It may 

 be cultured readily and indefinitely in the following modification of the 

 artificial seawater of McClendon, Gault, and Mulholland (1917): 

 Substance • grris. 



CaClo 1.220 



MgCl 2 .6H 2 5.105 



MgS0 4 .7H 2 7.035 



KC1 0.763 



NaCl 28.340 



NaBr.2H 2 0.082 



NaHC0 3 0.210 



Distilled water 1000 cc. 



Butts (1935) has made a study of the effects of salts on the growth 



