78 Phylum Protozoa 



satisfactory merely drop the coverslip into the culture bowl. Now cover 

 the cultures carefully with a glass cover or stack the finger bowls. In 

 either case be sure that the covers are sterile and chemically clean. It 

 is always wise to set up several cultures to insure against mishap. If 

 successful the cultures will become more or less abundant in from four to 

 six weeks. To subculture proceed as before except that it is not now 

 necessary to wash the Amoebae, unless observation has shown that they 

 have become contaminated with undesirable organisms. 



The salt content of culture media. While almost any uncontaminated 

 freshwater will support growth it was found by Pace (1933) during a 

 detailed study of the relation of salts to growth and reproduction that 

 a more certain way of obtaining the proper salt concentration for abun- 

 dant growth and reproduction is by using a synthetic spring water of a 

 definite composition and concentration determined by experiment to be 

 optimum for growth and reproduction. The following solutions are 

 recommended: 



(1) (2) 



NaoSiC>3 15 mg 100 mg 



NaCl 12 mg 12 mg 



Na 2 S0 4 6 mg 6 mg 



CaClo 6.5 mg 6.5 mg 



MgClo 3.5 mg 3-5 mg 



FeCl3 4 mg 4 mg 



Dist. Water 1000 cc 1000 cc. 



Sufficient HC1 to give a pH of 7.0 to 6.8. 



We shall designate (1) as "dilute artificial spring water" and (2) as 

 "concentrated artificial spring water." The difference between the (1) 

 and (2) solutions is in the concentration of sodium silicate. The first 

 solution is recommended when attempting to culture Amoebae recently 

 collected, since the concentration is nearer that of most natural fresh- 

 waters than is the second. However, when Amoebae have been cultured 

 in a dilute medium they may be transferred to the more concentrated 

 solution and more rapid growth and reproduction will be obtained. In 

 fact the second solution is a solution in which optimum growth and 

 reproduction was found to take place. A concentration between those of 

 these two solutions in which the sodium silicate is 25 mg. per liter in- 

 stead of 15 mg. or 100 mg. per liter, results in a much slower rate of 

 reproduction. No serious difficulty will arise if the total salt concentra- 

 tion of (1) is a little less or that of (2) is a little greater, but the con^, 

 centration of (1) must not be a little greater, or that (2) a little less 

 than that indicated. Reproduction is much better when sodium silicate 

 is present than when it is replaced by some other salt. Chalkley (1930) 

 and Hahnert (1932) give salt mixtures which allow good growth and 



