1. It has previously been shown [by Dobell] that, in cultures of strain K 28c, Entamoeba histolytica could be maintained indefinitely and made to pass through all the stages of its life history.

2. It was also demonstrated that all non-encysting strains of E. histolytica could be induced to encyst in vitro by substituting their concomitant bacterial flora by that of strain K 28c.

3. In order to ascertain what part each constituent of the bacterial flora of K 28c played in the life of E. histolytica, a detailed analysis of this flora was carried out. It was found to comprise eight aerobic organisms (mostly of the B. coli group) and six anaerobes.

4. The properties of individual species and varieties of bacteria were studied by adding them to cultures of the amoebae or by hatching the cysts of E. histolytica in media previously seeded with the bacteria to be tested.

5. With the same end in view two methods were devised for producing simplified strains of E. histolytica associated with from one to four species of bacteria only. By treating the cysts with acriflavine, the mixed flora could be freed of the aerobic organisms, leaving only the spores of the anaerobes. The spore-bearing anaerobes, in their turn, could be eliminated by treatment of the mixture with gentian violet. By using these methods it was possible to produce cultures of ‘synthetic’ strains of E. histolytica accompanied by any desired combination of bacteria, whose properties it was desired to investigate.

6. The results of the earlier experiments suggested that the essential constituent of the flora of K 28c was B. coli, all varieties of which appeared to promote growth, induce encystation and excystation, and generally favoured the completion of the life-cycle of the amoebae in vitro, whereas the anaerobic bacteria present in the strain appeared to be inactive as regards encystation.

7. However, later experiments have demonstrated conclusively that, although the aerobic bacteria individually induced the cysts of E. histolytica to hatch and supported the growth of the amoebae indefinitely, only one species—the anaerobe N5—was capable of promoting encystation. As regards the aerobic bacteria, their chief role is to provide the anaerobic conditions necessary for the growth of the amoebae with N5 in culture.