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An Intestinal Content Cultivation Medium

I. Methods of Preparation and Use and Data Obtained in the Cultivation of Balantidium coli from the Pig¹

1. A simple method has been devised for using intestinal contents in the preparation of a culture medium. This report covers an investigation of the properties of an intestinal content medium prepared from the cecal contents of the pig and the results obtained in the use of the medium in the cultivation of Balantidium coli from the pig.

2. The medium is prepared as follows: one part intestinal contents is mixed with nine parts Ringer's solution. The resulting 10 per cent mixture is stirred, strained through a sieve to remove the coarse particles and then strained through a funnel lined with a pad of absorbent cotton. The liquid and the finest material only, such as bacteria, are obtained by this process.

3. In cecal content medium strains of balantidium were cultivated for as long as 113 days and then voluntarily discontinued. A strain cultivated for 113 days was subcultured seven times at intervals of from seven to 22 days. The cultures used in the series lived from nine to 34 days.

4. For some reason bacterial growth is self limited. In the ice box or even at room temperature the bacteria appear to be inactive. At 37.5° a moderate turbidity arises in tubes of the medium but bacterial growth is never sufficient to make subcultures necessary as in the case of serum medium.

5. The cultures die when the pH reaches an acidity of about 5.0. This pH is usually reached in a few days if the initial pH of the medium is below 6.0; if the initial pH is 7.0 or above it usually takes 15 days or more for the pH to drop to 5.0. The greatest length of life of a culture has been 34 days.

6. The limited bacterial growth made it possible to use the medium in large quantities. Flask cultures using 200 cc. of medium lived for as long as 34 days and attained a population as high as 8260 balantidia.

7. In medium autoclaved or filtered to kill or remove the bacteria, balantidium would not live until a living bacterial population had been established. Apparently living bacteria are essential for the growth and multiplication of balantidium.

8. The data obtained seem to justify the conclusion that: (a) Balantidium can obtain from the cecal contents all the essentials necessary for growth and multiplication. (b) Since serum or host tissues need not be added to the cecal content medium it seems questionable that balantidium needs to attack its host. (c) Living bacteria are essential for the growth and multiplication of balantidium. The question is raised whether balantidium penetrates and lives in the tissues of the host unless preceded or accompanied by living bacteria.

Received October 5, 1939.
¹ These investigations have been made possible by grants from the Coe Fund Committee of the University of Maine and generous contribution of facilities by Dr. F. R. Ramaley of the University of Colorado, Dr. E. E. Tyzzer of the Department of Comparative Pathology at Harvard Medical School and Dr. Wm. H. Cole, Director of the Mt. Desert Island Biological Laboratory. The writer is greatly indebted furthermore to Dr. E. E. Tyzzer and Dr. R. W. Hegner for advice and encouragement.