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Studies on Entamoeba Histolytica

I. Effect of Hydrogen-Ion Concentration on Encystation of E. histolytica in Culture

1. Using a fluid medium consisting of buffered phosphate in sodium chloride solution, liver fluid, and rice starch as an encystment medium, a study has been made of the relation of hydrogen-ion concentration of the medium to the encystment of E. histolytica in culture.

2. It has been found that the optimum total salt concentration in the medium for growth and encystation of E. histolytica is M/30 phosphate and 0.4 per cent sodium chloride.

3. Utilizing the optimum phosphate and sodium chloride concentration, the optimum pH value of the medium for cyst production was determined. It was found that when the initial pH value of the medium was 6.8 to 7.0, and the pH values after 24, 36 and 48 hours of incubation were 6.7–6.9, 6.8–7.0 and 6.9–7.2 respectively, rich encystation was observed. The maintenance of a favorable pH range throughout the period of incubation is believed to depend not only on the initial pH value but also on the accompanying bacterial flora.

4. It appears under the condition of these experiments that in order to have a rich encystment, even in a medium of favorable pH value, the rate of growth of the amoebae must also be greatly accelerated. When abundant growth is reached, the majority will excyst.

5. Not all strains of amoebae give a rich cyst yield even when these two requirements are fulfilled. It is believed that all strains of amoebae have a tendency to encyst under favorable conditions, but much of this tendency may be lost on prolonged cultivation in a medium.

6. The liver extract favors the growth of amoebae, but the amount of liver extract required to make up the medium, within the ratio of 1:60 to 1:160 to that of the phosphate-salt solution, affected the encystment only through its effect on the pH values.

7. Amounts of encystment medium of 15–20 ml. seem to maintain a favorable pH value range better than smaller or larger amounts, and therefore induce better encystment.

8. On the basis of this study, a medium is proposed for encystment of E. histolytica in culture. It is composed of 15–20 ml. of M/30 phosphate buffered at pH 7.6, – ml. of liver extract fluid, and 2–3 loopfuls of rice starch. With a strain known to encyst in culture and having a good accompanying bacterial flora, rich encystment is to be expected in this medium. To assure good results, the initial pH of the medium prepared should be tested and should not be lower than 6.8. If the medium is to be stored for some time before use, the phosphate-salt solution should be buffered at pH 7.8, as the pH value decreases on storage.

Received April 28, 1942.
¹ The study represents a portion of an investigation presented in a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Public Health.