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Shawn Lynch, Joseph Carr, Christopher Kovacs, Matthew Dehmler, Todd Bassage, Wolfgang Haas, Timothy Morris; A Plaque Method for Direct and Sensitive Enumeration of Surviving Acanthamoeba During Biocidal Efficacy and Regimen Testing of Contact Lens Disinfection Solutions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5449.
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© ARVO (1962-2015); The Authors (2016-present)
Currently, there are no established methods or performance criteria for testing disinfection efficacy of contact lens solutions against amoeba that can cause sight-threatening keratitis. Here we sought to develop robust, reproducible procedures for conducting standardized biocidal efficacy and regimen testing against Acanthamoeba castellanii using a direct plaque enumeration method.
A. castellanii ATCC 50370 trophozoites were cultured in chemically defined antibiotic-free medium (AC6) prior to testing against polyaminopropyl biguanide/polyquaternium or alexidine/ polyquaternium based solutions. For biocidal assays test solutions were inoculated and held for 4 or 24 hr at which time aliquots were removed, neutralized, and serially diluted in AC6 medium containing neutralizers (AC6N). For regimen testing, amoeba in organic soil were inoculated on both sides of contact lens for 5 minutes, followed by treatments with test and control solutions (10 sec. rub, 5 sec. rinse per side, 4 hr soak in lens cases). For both assays, test solutions, amoeba recovered from lenses, and control suspensions of amoeba were neutralized with AC6N, added to standardized suspensions of E. coli plus molten top agar, and overlaid onto nutrient-limited agar plates. Plates were incubated at 28°C and monitored daily for up to 7 days to enumerate plaque forming units (pfu) within the E. coli lawn.
Biocidal activity showed similar results for polyaminopropyl biguanide/polyquaternium and alexidine/polyquaternium based solutions with log reduction values ranging from 1.3 - 2.1 at 4 hr to 3.8 - 4.5 at 24 hr. In comparison, the negative control solution exhibited only <0.5 log reductions at both time points. In regimen testing of lenses inoculated with 4.0 - 6.0 x 104 pfu, total recovery from solutions and test lenses showed that both disinfecting solutions consistently eliminated amoeba, resulting in <1 mean pfu recovered after a 4 hr soak time.
The method outlined here reproducibly yielded quantitative log reduction recovery values for biocidal and regimen testing against Acanthamoeba. The plaque enumeration method is simple, direct, sensitive, and yields test results with 4 - 6 days, which is substantially faster than the 14 - 28 days often reported for indirect (i.e. Most Probable Number) enumeration methods in liquid culture.
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