S16: Methods For The Evaluation Of The Effects Contact Lens Cleaning Solutions On Acanthamoeba Killing and Cyst production.

Acanthamoeba keratitis is an infection that has received considerable attention during the past year. The infection is caused by several species of Acanthamoeba. The organism is ubiquitous and has been reported from house dust and residential water supplies. As part of our ongoing efforts in applied microbiology we have developed methods to rapidly evaluate killing of both trophozoites and cysts. The complexity of the life cycle and other factors contribute to variations in the results obtained.  Axenically grown trophozoites(1x10⁶ /ml) of Acanthamoeba sp were exposed to common MPS solutions for 6h, following the neutralization with Letheen broth for 1hr.  The cells were concentrated by centrifugation and serial dilutions were plated onto dilute PYG agar with heat killed Enterobacter aerogenes.  Following incubation for 48 -72 hours plaques in the enterobacter lawn were enumerated. Similar analyses with Acanthamoeba cysts induced by several methods are also reported.  Cyst induction by MPS solutions were evaluated by introducing 0.5 ml trophozoite suspensions containing 5x10⁶trophozoites/ml into  1.5ml of MPS. Following incubation for 24 hours at room temperature aliquots were neutralized in Letheen broth and incubated for 1h at room temperature. The entire volume of the suspension was then filtered through an 0.45 µm pore sized cellulose acetate membrane. The membrane was stained with Calcifluor white and observed for cyst. Ten microscope fields were counted and the total number of cysts produced are calculated. Results are reported relative to saline or other commercial solutions.  Data for several commercial solutions will be discussed.