M. Tech. Water care / Free-living amoebae are important predators that feed on bacteria, fungi and algae by phagocytosis. However, a number of bacteria have become resistant to, and are able to survive, grow and exit Free-living amoebae. Free-living amoebae are ubiquitous organisms that have been isolated from various man-made and environmental water systems. Some of these free-living amoebae host amoeba-resistant bacteria such as Legionella spp. Vibrio cholerae and Mycobacterium species which are regarded as human pathogens. The objective of this study was to establish amoebal enrichment and co-culture techniques in order to isolate and identify amoebae and amoeba-resistant bacteria from seeded and environmental water samples. The aim of the study was to introduce and establish amoebal co-culture and amoebal enrichment techniques in South Africa, to provides South African laboratories with baseline information on the efficiency of culture-based methods as well as analytical requirements like incubation temperature, media preparation and storage conditions for amoebal enrichment and co-culture techniques for the detection and isolation of amoebae and amoeba-resistant bacteria from environmental water systems.