BACTERIA IN CULTIVATION OF INTESTINAL PROTOZOA 113 



inhibit the growth of the bacteria more than that of the trichom- 

 onads, but, as Andrews (1926) states, this appears to be almost 

 a hopeless undertaking. Certainly most of the substances used 

 inhibited the growth of the trichomonads far more than the bac- 

 teria. All there was left to do then was to improve the methods 

 of cultivating trichomonads, so that they might be grown more 

 abundantly. 



"After experimentations with many types of media, it was 

 found that a medium composed of about one gram of Loeffler's 

 dehydrated beef serum to 100 cc. of 0.7 per cent sodium chloride 

 gives a fairly abundant growth of trichomonads. Buffering of the 

 medium sometimes increases the growth, depending on the bac- 

 teria that are dominant. This medium was made in great quan- 

 tities and was run through the large Berkefeld filters (V, No. i). 

 It was then run through another Berkefeld filter in a tubing 

 and sterilizing apparatus, which is illustrated in Fig. 11. This 

 apparatus was wrapped with paper and sterilized in the autoclave 

 each time it was used. It was possible to sterilize the medium 

 in this way without heating it and to fill tubes at the rate of two 

 to three hundred an hour. After being filled, the tubes were in- 

 cubated for four days at 36° C. to test their sterility. Far less 

 than one per cent of the tubes filled were contaminated in the 

 filling process. Four and five hundred tubes have been filled 

 many times without a single tube being contaminated. Hundreds 

 of liter flasks of the medium were also filled and sterilized in 

 the same manner as the tubes. 



"... Inoculations were made from tubes in which the organism 

 was growing abundantly to liter flasks of serum-saline medium 

 (prepared and sterilized as described above). The organism grew 

 very well in this large quantity of medium. The growth was 

 more abundant, however, when the flasks were filled so that the 

 fluid came up into the neck of the flask. The organism grew so 

 well that from one to two cubic centimeters of Tritrichomonas, 

 having the appearance of heavy cream, could be obtained when 

 the organisms in a liter were concentrated by centrifugalization. 

 It was discovered later, after a number of the washing experi- 

 ments, to be described presently were carried out, that the bac- 

 terial growth could be cut down greatly and without any decrease 

 in the number of trichomonads, by placing vaseline on the surface 

 of the fluid in the neck of the flasks. The lack of oxygen cut 



