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TK Beattie, A Tomlinson, AK McFayden, DV Seal; Most Probable Number Method of Amoebal Enumeration in the Determination of the Amoebacidal Activity of Multi-purpose Solutions . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3099.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A Most Probable Number (MPN) method was employed to enumerate amoebae in evaluating the efficacy of six multi-purpose solutions (MPS) against Acanthamoeba castellanii cysts and trophozoites. Methods: Cysts or trophozoites were inoculated into ReNu MultiPlus (RM), Opti-Free Express (OFE) Complete (C), Solo-care (S), All-in-One (AO), and All-in-One Light (AOL). The active ingredient in all solutions was 0.0001% polyhexamethylene biguanide (PHMB), with the exception of AO which contained 0.0005% PHMB and OFE which contained 0.001% Polyquad & 0.0005% Aldox. After 0, 2, 4, 8 and 24h exposure, samples were neutralised in DE broth prior to dilution in amoebal saline. Five x 1ml, 5 x 0.1ml and 5 x 0.01ml aliquots of each amoebal saline dilution were inoculated onto non-nutrient agar plates seeded with heat killed bacteria. Plates were incubated at 30ºC for 7-14 days and examined daily for viable amoebae. Results were entered into a MPN statistical table to determine the MPN of amoebae. Experiments were run in triplicate for cysts and trophozoites. Results: After the minimum recommended disinfection time (S 10min, OFE 6h, all others 4h), AO gave a log reduction of ≷3 against cysts, however, all other MPS failed to achieve a log reduction of 1. By 24h AO achieved total kill of cysts (log reduction of 3.78), RM gave a log reduction of 2.62, and all other MPS reached a log reduction of ∼1. Against trophozoites, AO, RM and OE achieved a total kill (log reduction of ≷3) after the minimum recomended disinfection time, however, the other MPS failed to reach a log reduction of 1. After 24h all MPS proved trophoziticidal, achieving, with the exception of Complete (log reduction 2.95), total kill (log reduction ≥3). Conclusion: MPN provides a simple method of enumeration that relies on establishing the presence or absence of amoebal growth on culture plates, and determining the MPN of amoebae present from statistical tables. Utilising this method RM and OFE demonstrated effective trophoziticidal activity after the minimum recommended disinfection time, however only AO proved effective against both cysts and trophozoites after the minimum recommended disinfection time.
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