68 RESEARCH IN PROTOZOOLOGY 



various temperatures, and inserting immediately a rubber 

 stopper. 



8. The determination of the most favorable temperature for 



growth of the organism in question. 



9. The determination of the correct intervals for making 



transfers. 



In discussing the approach to the problem of cultivating an 

 organism, for which the method has not previously been worked 

 out, it might be of interest to outline a plan recently used in 

 attempting to cultivate Opalina. In studying this outline, the reader 

 will note that the suggestions enumerated in the previous para- 

 graph were followed. In the case of Opalina, as in that of nearly 

 all protozoa, the first consideration is to obtain infected material. 

 Only about ten per cent of the tadpoles examined were infected 

 with Opalina. As exposure to air is harmful to most intestinal 

 protozoa, the plan of spreading the intestinal contents on a slide 

 for microscopic examination was abandoned early in the work. 

 It was found best, in order to save much labor as well as to pre- 

 serve the organisms in a fresh state, to place the unopened intestine 

 on a sterile slide and then to examine it under the low-power 

 microscope. In this way, uninfected animals were quickly dis- 

 carded. In the case of Opalina, it has been determined by previous 

 workers that the organism will live for days in concentrations of 

 salt solution ranging from one-half to one per cent. In our work, 

 it was thought best to determine just what salt concentration was 

 most favorable to Opalina. In order to do this accurately, the 

 method of Giffin and Sanford for testing the fragility of red 

 blood corpuscles were used, with this difference: in the fragility 

 test, concentrations of salt from 0.5 per cent down to 0.2 per cent 

 are used ; in this work with Opalina, concentrations from one per 

 cent down to o.i per cent were used. In this way, the most favor- 

 able salt concentrations were determined within fairly definite 

 limits. Having determined fairly accurately the correct salt con- 

 centration, the next step was to use this salt concentration with 

 varying hydrogen-ion concentrations. As the hydrogen-ion concen- 

 tration of the intestinal contents is rather definitely known, it was 

 not necessary to cover a wide range of variations. A pH of be- 

 tween 6 and 8 will cover the requirements of the majority of intes- 

 tinal protozoa. 



