n8 Phylum Protozoa 



III. PARTIAL PURIFICATION OF CULTURES OF 

 BALANTIDIUM COLI 



It has long been realized that bacteria-free cultures of the intestinal 

 protozoan parasites of vertebrates would be extremely useful for the 

 study of the biology and pathogenicity of these organisms. Yet up to the 

 present time no such protozoan has been grown in culture free of bacteria 

 and in most cases it has been impossible even to free the Protozoa of the 

 numerous bacteria which naturally accompany them. Cleveland (1928), 

 however, succeeded in freeing the coprozoic organism, Tritrichomonas 

 fecalis of man, from bacteria and was able to grow it on heat-killed 

 bacteria. Glaser and Coria (1934) have recently applied one of their 

 migration techniques to Balantidium coli from swine and have been able 

 to free the organism of all bacteria except Bacillus coli. Largely as a 

 result of this partial purification, they have been able to maintain this 

 parasitic ciliate in better condition and for a much longer time without 

 subculturing than has heretofore been possible. 



They used a liquid medium consisting of 9 cc. of sterile Ringer's 

 (Schumaker, 193 1) solution, 0.5 cc. of sterile horse serum, and a sprinkle 

 of rice starch in each tube. Much better results were obtained with a 

 semi-solid medium. This is prepared by adding 25 cc. of 2% standard 

 nutrient agar, 1 gram of sterile rice starch and 12.5 cc. of sterile horse 

 serum to 250 cc. of sterile Ringer's solution warmed to 50 C. The pH 

 of the mixture is adjusted to 7.2 to 7.4 and 15 cc. amounts are transferred 

 aseptically to sterile tubes. 



"V" tubes containing 15 cc. of semi-solid medium were inoculated 

 (after being warmed to 37 C.) on the surface of one arm with material 

 scraped from crypts in the mucosa near the ilio-cecal valve of a pig. 

 The Balantidia migrated downwards within 24 to 48 hours at 37 C. and 

 could then be recovered in a sterile pipette inserted through the uninoc- 

 ulated arm of the "V" tube. The semi-solid nature of the medium checked 

 the spread of bacteria, which in a liquid medium would rapidly grow over 

 both arms of the "V" tube. The Balantidia were able to push their way 

 through the gelatinous semi-solid medium and it was found that in such 

 a medium a much higher percentage of positive initial cultures could be 

 obtained than in a liquid medium. Balantidia removed after the final 

 migration were again placed on the surface of one arm of a fresh "V" 

 tube and again permitted to migrate for 24 to 48 hours. This procedure 

 was repeated five times, after which the ciliates were further cultured in 

 ordinary tubes of semi-solid medium. Strains which would not originally 

 grow in the liquid medium could be adapted to this after varying lengths 

 of culture in the semi-solid medium. 



These methods failed to free Balantidium of Bacillus coli but they did 

 yield cultures which might be regularly transplanted at 8-day intervals 



