Studies on a Marine Parasitic Ciliate 367 



a ciliated protozoan was found to he present after two days. The 

 ciliates fed on the provided tissues and developed into small 

 populations. The cultures proved to be bacteria free. After trans- 

 fer into monolayer cultures of fish cells it could be observed 

 that the parasites devoured tlie fish cells within a few hours 

 and responded to this meal with further increase in numbers. 

 They grew as well on monolayer cultures of mammahan cell 

 lines which were obtained in Eagle's Tissue culture medium, 

 supplemented with 10 per cent human serum. It was decided 

 to study this parasitic ciliate, as such an organism and similar 

 parasites of marine animals may conceivably play an important 

 role in the transmission of viruses among animals in the sea. 



MATERIALS AND METHODS 



Carnivorous Feeding Method 



A. Passages of Stock Cultures. Tissue cultm-es of the human 

 cancer cell strain KB grown in milk dilution bottles in 10 ml 

 of Eagle's medium (with 10% calf serum) have been used for 

 weekly passages of two clonal strains of the ciliate. One day 

 before the actual transfer was made the ciliate culture and the 

 fresh KB feeder cultin-e were checked for sterility. To the KB 

 culture 0.5 ml of ciHate suspension was added. The cultures were 

 incubated at 20 C in a refrigerated incubator. To the date of 

 writing, tlie clones have been passed twenty-four times in this 

 fasliion. 



B. Quantitative Growth Experiments with Carnivoroiishj 

 Feeding Ciliates. Kimble screw cap tubes were seeded with 

 50,000 KB cells in one ml of Eagle's medium (with 10% calf 

 serum) and incubated at 37 C until a confluent monolayer of 

 cells had developed. These feeder tubes were changed from 

 growth medium to maintenance medium which contained 5 per 

 cent calf serum. A counted number of ciliates contained in 0.1 ml 

 of maintenance were then added to a set of KB tubes maintained 

 in 0.9 ml of medium and to a set of tubes with an equal amount 

 of medium alone. These experimental tubes were incubated at 

 20 C. At chosen time intervals, sister cultures of botli kinds 

 were fixed with one drop of 10 per cent formalin. The culture 



