A strain of E. histolytica with a mixed undetermined flora produced cysts for a period of three months and then ceased to do so. A second strain, growing with three separate species of bacteria, produced cysts in Erlenmeyer flask cultures for an indefinite period of time; a sub-strain developed by micro-isolation and seeded with Streptococcus NIH 563 and Escherichia coli communis failed to produce cysts.
Cysts of E. histolytica underwent marked loss of viability during storage for 19 days in physiologic saline at 10°C.
During the micro-isolation operation, exposure to a 1:5,000 solution of mercuric chloride in physiologic saline lowered the viability of cysts.
Cysts were freed by micro-isolation from bacteria and grown in pure culture with each of the following bacteria: Escherichia coli communis, E. c. communior, an actinomyces-like organism, Streptococcus NIH 563, Staphylococcus aureus, and Salmonella schottmuelleri. The cultural characteristics of the amoebae changed greatly with each change in the flora.
Ten cysts per tube were selected at random with the micro-isolation apparatus, freed from bacteria, planted into 564 tubes of the medium of Boeck and Drbohlav, and seeded either with Escherichia coli communis or Streptococcus NIH 563. Twenty per cent of tubes seeded with the latter bacterium developed amoebae but none seeded with the former became positive. Changes from the normal in morphological and cultural characteristics of the strain of E. c. communis were demonstrated and may have accounted for the results shown.
Twenty-five cysts per tube were selected by micro-isolation with the aid of the neutral red staining technique from lots of cysts having 75 per cent in the tetra-nucleate stage; the cysts were freed from bacteria and then seeded either with Streptococcus NIH 563 or an actinomyces-like organism. With one series of micro-isolations, the yield of positive amoeba cultures was as high as 80 per cent of the tubes planted.
Cysts of E. histolytica from fresh human stools were selected by micro-isolation technique, freed from bacteria and planted into Boeck and Drbohlav's medium with single species of bacteria. Amoeba cultures developed from these cysts.
The addition to the medium of Boeck and Drbohlav of bacteria killed at 56°C. and 100°C., or sterile filtrates of lyophilized bacteria, or products of the tryptic and pancreatic digestion of coagulated egg failed in each case to support growth of E. histolytica in cultures free from bacteria.
The help of Dr. A. E. Verder of the Division of Infectious Diseases in problems of bacteriology is gratefully acknowledged.