Figure 2. Direct and indirect cytopathic effects of Acanthamoeba on corneal fibroblasts. A: Acanthamoebae were placed on corneal fibroblasts at the center of 6-cm dishes and incubated at 25 °C for 2 days. The fibroblasts are uniformly stained with Giemsa solution in a control dish (left). The central area where Acanthamoebae were placed shows no staining (indicating loss of corneal fibroblasts) in a treated dish (right). B: MTT assay showed there was no significant difference of optical density value in the outer dishes with corneal fibroblasts with or without insert culture dishes bearing Acanthamoebae. Significant low optical density value is detected in Acanthamoebae direct adhesion group, compared with insert culture dishes bearing Acanthamoebae. (n=6) Amoeba; Acanthamoeba, Inserter; insert culture dish. C: Phase contrast microscopy shows many corneal fibroblasts are detached and Acanthamoebae adhere to corneal fibroblasts and the dish surface. Arrowheads show active Acanthamoebae co-cultured with corneal fibroblasts. D: Confluent human corneal fibroblasts are seen. Acanthamoebae in the insert culture dishes with 0.4 µm pores are not observed. Similar findings were obtained with repeated two sets of experiments. Representative data are shown. Scale bar=10 µm.