Acanthamoeba is a free-living ameba that is found throughout the world and that causes encephalitis, keratitis, and cutaneous infections in humans. It has two stages in its life cycle: a trophic stage and a resistant cyst stage. We describe here the ability of Acanthamoeba cysts to survive desiccation for more than 20 years.Acanthamoeba, a free-living ameba, is an opportunistic pathogen of humans and other animals, including gorillas, monkeys, dogs, ovines, bovines, horses, and kangaroos, as well as birds, reptiles, amphibians, and fishes. In humans, Acanthamoeba causes a spectrum of diseases, including infections of the central nervous system, namely, granulomatous amebic encephalitis (GAE); infection of the skin; and Acanthamoeba keratitis (AK), an infection of the eye. GAE and cutaneous infections have often occurred in patients with human immunodeficiency virus infection and AIDS, as well as immunodeficient patients, including transplant recipients. Acanthamoeba keratitis, however, has occurred in immunocompetent persons wearing soft contact lenses and those with trauma to the eye. Acanthamoeba feeds on bacteria and occurs worldwide. It has been isolated from a number of habitats, including soil; freshwater ponds; pools; lakes; brackish water; seawater; heating, ventilating, and air-conditioning filters; and medical equipment, such as gastric wash tubing, dental irrigation units, contact lens paraphernalia, as well as vegetables, cell cultures, and even human and other animal tissues (13,21,26).Acanthamoeba has two stages in its life cycle: a trophozoite stage and a cyst stage. Both the trophozoite and the cyst are uninucleate, although binucleate trophozoites are occasionally seen. The nucleus is characterized by a large densely staining nucleolus. The trophozoite feeds on bacteria and reproduces by binary fission. The cyst stage is a dormant and resistant stage. The cyst has double walls. The outer ectocyst is wrinkled and is proteinaceous, whereas the inner cyst wall, the endocyst, is either stellate, polygonal, round, or oval and contains cellulose (15, 16). According to a few previous studies the cyst stage of Acanthamoeba spp. is resistant to extreme physical and chemical conditions, including pH 2.0, freezing, ␥ irradiation (250 rads), and UV irradiation (800 mJ/cm 2 ) (2, 8); moist heat (60°C) with a contact time of 60 min (11); prolonged storage at room temperature for 24 months (4) or 24 years at 4°C in water (14); and heavy metals and polychlorinated biphenyls (PCBs) (18).Over the past 30 years, we have established in culture 45isolates obtained from diverse human specimens, including cerebrospinal fluid (CSF), brain, skin, and nasal and corneal tissues, as well as contact lens paraphernalia and water. The isolates were grown on nonnutrient agar plates coated with live Escherichia coli cells. After the amebae differentiated into cysts, the agar plates were tightly wrapped with Parafilm and stored at room temperature in laboratory cabinets (Table 1). The agar plates that were retrieved from storage...