Axenic Cultivation and Pathogenic Assays of Acanthamoeba Strains Using Physical Parameters

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays. Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cul­tured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotoler­ance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fiftyµl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture me­diums. Cultures were incubated at 37oC and 42 oC. All plates were monitored for 24 h, 48 h and 72 h. Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axeni­cally after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 oC) and osmolarity (1.5 M) and thus they were classified as weak pathogens. Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.