Amoebae graze on bacteria from the environment. In some cases, bacteria acquire resistance to the destructive action of amoebae and survive inside the host. The resistance of microorganisms to amoebae is often accompanied by resistance to the physiologically related human macrophage and displays increased pathogenic potential to humans. The association of bacteria with amoebae, that are ubiquitous in the environment, protects the bacteria from common disinfection measures. This leads to the creation of reservoirs of pathogenic microorganisms that are difficult to eradicate. This process occurs widely in the environment, but is of special concern in hospitals, where many established and emerging pathogens involved in nosocomial disease, such as Legionella pneumophila, are increasingly being identified in association with amoebae. One particularly important amoeba-associated microorganism (AAM) is the newly recognized giant mimivirus, a member of the Nucleo-Cytoplasmic Large Deoxyribovirus (NCLDV) group. The virus has been isolated from hospital sources in France and, together with other members of the NCLDV group, is known from metagenomic studies to be widespread in the Oceans, representing one of the main groups of marine viruses. Our project aims to detect amoeba and potentially pathogenic AAMs with particular relevance to the NCLDVs in the environment and in hospital facilities. In the present work we have developed methods for detection, isolation and in vitro culture of amoebae from several environmental sources including hospital dust, sea water and estuarine sediments.