Our results demonstrate that ethanol treatment can be used to evaluate large collections of environmental samples for the presence of giant viruses and to provide insight into understanding their ecology. This study should also facilitate the isolation of giant viruses using other species of protozoa in addition to Acanthamoeba spp. Abstract Objective: Giant viruses of the Megavirales order have been neglected in the literature because they are removed from samples during viral purification for

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... viral metagenomic studies. Isolation via amoeba coculture has low efficiency and is extremely time-consuming. Thus, our objective was to improve Megavirales detection and recovery by using a new protocol that will eliminate most bacteria present in environmental samples while preserving giant virus viability. Methods: In this study, we tested the ability of a number of disinfection protocols to kill contaminating bacteria. These treatments were ethanol, UV irradiation, desiccation, glutaraldehyde and thermal shock. Results: Of all the treatments, a brief ethanol treatment did not significantly reduce the titer of viable viral particles of Acanthamoeba polyphaga mimivirus or Marseillevirus , whereas it efficiently killed Escherichia coli . This treatment was applied to environmental samples that previously tested positive for giant viruses and was shown to eliminate contaminating bacteria, whereas it allowed for the isolation of the giant viruses. Conclusion: