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Prototheca Isolation Medium (PIM) was proven to be effective in selectively isolating Prototheca, even from heavily contaminated natural sources. Measurements of the autospores of 125 isolates of Prototheca indicated that there might exist two sub-genera, one with small autospores and one with large autospores. The addition of one percent sodium acetate to PIM resulted in a medium that inhibited the growth of isolates of the small spored sub-genus while allowing isolates of the large spored sub-genus to grow normally. A survey of various environmental habitats showed that Prototheca was common in aquatic habitats such as streams and sewage. The largest populations were found in sewage, in effluent from a slaughterhouse, and in slime flux of trees. In streams, the largest populations occurred in the bottom sediment. A study of the relation of Prototheca populations at six stream sites to chemical and physical characteristics of these sites revealed that the total population and the population of the small spored sub-genus correlated positively with pH, BOD, and rainfall and negatively with water temperature and organic carbon. The population of the large spored sub-genus correlated positively with pH and BOD and negatively with water temperature. There was some indication that increases in populations were due to the addition of cells to the stream from an external source, especially during times of heavy rainfall. A more detailed survey of some of the sampling sites revealed pipes carrying raw sewage and large numbers of Prototheca cells into the streams. Survival studies in the laboratory and in the field using membrane filter chambers showed that at some of the sites the Prototheca cells died off rapidly while at others some of the cells survived for up to eleven days. The results of this investigation of the occurrence of Prototheca indicate that it is a common aquatic microorganism. It enters streams and rivers with raw sewage with more entering when sewage flow is increased by rainfall. In some streams the cells are able to survive for long periods of time but probably do not reproduce to any significant extent due to low nutrient levels.