BACTERIA IN CULTIVATION OF INTESTINAL PROTOZOA 117 



ing procedure was fairly well perfected that the number of bacteria 

 could be reduced so low. In other words, in the earlier washing 

 experiments the trichomonads were not washed so free of bac- 

 teria. Only three or four out of a hundred tubes inoculated would 

 fail to show bacterial growth. . , . Now . . .it was possible to 

 be certain that inocula containing from ten to fifteen trichomonads 

 each could be placed in one hundred tubes, seventy to ninety 

 of which contained no bacteria." 



After this tedious and time-consuming method was perfected, 

 attempts were made to grow the trichomonads in pure suspensions 

 of heat-killed bacteria in the serum-saline medium and in many 

 other media. The protozoa failed to grow in any of these media, 

 but a chance contamination of one tube by an obligate psychro- 

 phylic bacterium made it possible for Dr. Cleveland to test 

 trichomonads at 36° C. with living bacteria instead of those killed 

 by heat, and still be sure he was dealing with pure cultures. 

 TritricJioiiionas grew exceedingly well with this psychrophyhc 

 organism at room temperature, but this bacterium would not grow 

 at a temperature above 34° C. while the trichomonads grew well 

 at room temperature or at 36° C. All that was necessary, therefore, 

 was to inoculate sterile media with trichomonads from the stock 

 culture and with the bacterial organism to be tested and incubate 

 at 36° C. The psychrophyhc organisms from the stock culture 

 died in four to hve days and the trichomonads were then in a 

 pure culture of bacteria growing at 36° C. 



PROBLEMS AND SUGGESTIONS 



This technique may be applicable to similar studies on other 

 species of trichomonads, if cultures can be induced to grow at 

 temperatures lower than 37.5° C. The availability of obligate 

 psychrophyhc bacteria is not known to the writer. Cleveland (1928) 

 states that only three or four species are known. 



Methods for studying other species of intestinal protozoa can 

 only be suggested since they have not been experimentally tested. 

 C hilomastix, an intestinal flagellate that forms cysts, may be 

 grown in a medium containing one part human blood serum and 

 four parts Locke's solution (Boeck, 1921). Locke's solution may 

 be autoclaved and the serum filtered to insure sterility or the 

 medium may be mixed and fihered as described for Trichomonas 

 medium. Certain species of Chiloniastix may also be satisfactorily 



