Use of 5-Cyano-2,3-Ditolyl-Tetrazolium Chloride Staining as an Indicator of Biocidal Activity in a Rapid Assay for Anti-Acanthamoeba Agents

Abstract

The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguanide (PHMB) and commercial soft contact lens (SCL) disinfectant solutions. The treated Acanthamoeba organisms were stained with CTC, and their respiratory activity was determined by the intensity of fluorescence in a fluorescence microplate reader. The survival rates of the same samples were determined by a culture-dependent biocidal assay using the Spearman-Karber method. Our results showed that the respiratory activities determined by the CTC biocidal assay and the survival rates determined by the culture-dependent biocidal assay for Acanthamoeba trophozoites and cysts decreased in a dose-dependent way after PHMB treatments, and the results were significantly correlated (r ‫؍‬ 0.83 and P < 0.01 for trophozoites; r ‫؍‬ 0.60 and P < 0.01 for cysts; Spearman rank correlation test). The respiratory activities in the trophozoites and cysts treated with SCL disinfectant solutions were significantly correlated with the survival rate (r ‫؍‬ 0.70 and P < 0.01 for trophozoites; r ‫؍‬ 0.64 and P < 0.01 for cysts; Spearman rank correlation test). The significant correlation of the results indicated that the CTC biocidal assay can be used as an alternative method to a culture-dependent biocidal assay. The CTC biocidal assay is a rapid and simple method to test the effectiveness of disinfectant solutions against Acanthamoeba trophozoites and cysts.

Effect of Photodynamic Therapy with Methylene Blue onAcanthamoebaIn Vitro

“…In contrast, the CTC biocidal assay can be carried out in only a few hours for trophozoites (30 minutes for staining and 30 minutes for fixation after the treatment), and in a day for cysts, which require preincubation for 16 hours before CTC staining. In addition, in both our MB-PDT experiments and a previous study that evaluated treatment of Acanthamoeba with PHMB and SCL disinfectants, 23 the respiratory activity determined by the CTC biocidal assay was confirmed to be significantly correlated with the survival rate determined by the culturedependent biocidal assay.…”

“…To estimate the efficacy of treatment, the 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) biocidal assay was performed as described previously 23 to assess the respiratory activity of Acanthamoeba. Briefly, reagents from the bacterial staining/CTC staining kit (Bacstain-CTC Rapid Staining Kit; Dojindo Laboratories, Kumamoto, Japan) were added to samples, and cells were incubated for 30 minutes at 258C according to the manufacturer's recommendations.…”

“…The CTC biocidal assay was also performed on cysts as described. 23 After treatment with PDT, cysts were preincubated for 16 hours in PYG medium to facilitate the CTC biocidal assay.…”

Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway

“…Acanthamoeba castellanii (strain ATCC 30011) was obtained from the American Type Culture Collection, and cells were axenically cultured in peptone-yeast-glucose (PYG) medium at 26°C (23). Artemether (purity of Ͼ98.0%, provided by Qing Xu from Guilin Medical College, China) was dissolved in 98% ethanol at a final concentration of 8 mg/ml.…”

Effects of Multipurpose Solutions on the Viability and Encystment of Acanthamoeba Determined by Flow Cytometry

“…Recently, Kobayashi et al 23 introduced usage of 5-cyano-2,3ditolyl-tetrazolium chloride staining for rapid determination of the respiratory activity of Acanthamoeba. This culture-dependent assay including 30-minute incubation is also useful for evaluating the disinfecting efficacy of MPS; however, our present method has advantage in the simplicity of the procedure without the use of physiological cultivation in the assay.…”