INTESTINAL FLAGELLATES IN TISSUE CULTURE i6i 



of the Locke-Lewis are placed on a coverglass which has been care- 

 fully cleaned and flamed. This is then inverted over the hollow of 

 a depression slide which has previously been ringed with salvoline. 

 This ring of salvoline seals the culture and also raises it well 

 above the depression of the slide. The cultures are then incubated 

 at 35°-37° C. for two or three days. By this time a good growth 

 of cells has appeared from the explanted tissue and the cultures 

 are ready for the introduction of the protozoa. 



Sometimes the protozoa die soon after being introduced into 

 the hanging drop of the tissue culture. The drop must not be too 

 large or else the protozoa will remain in its lower part and never 

 come in contact with the tissue cells which are in the upper part 

 of the drop adhering to the coverslip. On the other hand, if the 

 drop is too shallow or if the protozoa wander to the edge of it, 

 they soon die. This may be because substances in the medium which 

 are injurious to the protozoa collect on the surface or at the edge 

 of the drop. It is quite certain that it is not due to the accumula- 

 tion of carbon dioxide and waste products nor to the lack of 

 oxygen, for when the protozoa were put on an ordinary slide 

 and the coverglass sealed with salvoline they lived at least twenty- 

 four hours and in one case they were found alive on the third 

 day. The number had increased, showing that they had divided. 



The problems open for solution by this method are unlimited 

 since it appears that nearly all sterile tissues whether the animal 

 be embryonic or adult can be cultured by the careful, resourceful 

 worker. Barta and Petrovits (1928) have recently grown forty 

 dififerent sterile tissues from adult rabbits (six to twelve months 

 old). Since most animals have parasites the problems are numer- 

 ous. The important things to do are : 



(i) to get pure cultures of protozoa, 



(2) to grow the tissue from the host in vitro, 



(3) to introduce the protozoa into the tissue culture and watch, 

 hoping that the protozoa will be able to live in the tissue culture. 



