Imaging Acanthamoeba Disinfection with Contact Lens Solutions

Similar to our previous description [8 (link)], sterile aluminum transmission flow cells (Biosurface Technologies Corporation, Bozeman, MT, USA) were assembled with glass coverslips (Figure 1). Acanthamoeba suspensions (3.0 × 10⁴ cells/mL) in one-quarter Ringer’s solution were added through the ports of the flow cells. Each flow cell was placed on the microscope stage and kept there for the duration of the experiment, to reduce physical interference with Acanthamoeba behavior. The flow cell ports were clamped shut to stop the flow of the Acanthamoeba suspension, and Acanthamoeba trophozoites were allowed to adhere to glass coverslips for one hour. Following this, flow cell solutions were either maintained as one-quarter Ringer’s solution, or changed to one of the four contact lens care solutions (CLCs). To exchange fluids, 4 mL of a solution was slowly added through the ports of the flow cell, taking care not to disturb the adhered amoebae. The flow cell ports were again clamped to prevent fluidic movement, and amoebae were imaged for 6 h (6 h was used to create identical testing conditions, as the majority of the solutions used specify a 6-h disinfection time, although PAPB/PQ requires only 4 h). After 6 h, all solutions were exchanged for axenic culture media (AC6) to determine the ability of Acanthamoebae to recover following disinfection. The AC6 condition was imaged for a further 12 h, for a total experimental imaging time of 18 h. Images were taken at 4× magnification using a Nikon Eclipse Ti-U Microscope (Nikon, Tokyo, Japan), every 24 s. The images were subsequently combined into a video format using NIS Elements AR 3.2.