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Cysts of the NRS strain of E. histolytica were exposed to various concentrations of ozone in aqueous solution for varying time intervals and under varying conditions oftemperature, organic nitrogen, and pH. The criterion of survival of cysts so exposed was their ability to establish growth in culture compared with that of untreated controls.
In 8 preliminary experiments with varying 5-minute ozone residuals, the treated cysts were planted in numbers of 3,000 to 4,000 cysts per tube. Completely negative results were obtained with 5-minute residuals of 1.8 P.P.
. and above. With lower residuals some positive cultures were obtained. However, the method of planting cysts did not permit estimation of the amount of cyst survival in these instances.
In further experiments with 5-minute ozone residuals, both control and treated cysts were planted in series of decreasing numbers. Results with the control cysts indicated that the presence of an average of 10 cysts per tube almost invariably would result in a positive culture. In addition, growth expectancies were obtained when averages of 8 and 4 untreated control cysts were present per culture tube. These data were used as a basis for estimating cyst destruction when treated cysts were planted in numbers varying arithmetically from 2,000 to 125 cysts per culture tube. The results of 15 experiments indicated a cyst destruction of 98 to more than 99 per cent with 5-minute ozone residuals of 0.3 P.P.. and above, resulting from ozone applications of 0.7 P.P.. and above.
In experiments designed to evaluate the importance of contact time, cyst samples were deozonized 
, 1, 3, 5, and 10 minutes after application of ozone ranging from 0.3 to 0.8 P.P.. For the evaluation of the results of these experiments, another method of planting cysts in decreasing numbers was employed, in which the numbers varied logarithmically. The results of 18 experiments indicated that the prolongation of the exposure period beyond 1 minute did not effect a measurable, consistent increase in cyst destruction. It was estimated that 96 to more than 99 per cent of the cysts were destroyed after 1 minute in all experiments, the greater variation in percentage destruction occurring between experiments rather than between the exposure periods of a single experiment.
The following variations in experimental conditions were evaluated with regard to their possible influence on the cysticidal action of ozone; Temperature, 10° to 27°C.; pH in the 6.5 to 8.0 range; distilled water compared to tap water; and organic nitrogen in the range of 0.14 to 1.25 P.P.. Such variations provided neither consistent nor marked differences in the results obtained, probably because of the high degree of cyst destruction under all conditions employed.
In general, it is concluded that ozone in aqueous solution is highly cysticidal for E. histolytica and that the cysticidal action does not appear to be influenced to any great extent by pH, temperature, or organic nitrogen. It is concluded, further, that the evaluation of cysticidal agents by cultural means should employ a method of planting treated cysts in decreasing numbers whereby cyst destruction can be estimated in instances in which it is not complete.
1 Laboratory of Tropical Diseases, Microbiological Institute, National Institutes of Health, Bethesda, Maryland.
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