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.

Fig 1

Acanthamoeba castellanii trophozoites stained with CTC. Merged fluorescence images of red-fluorescing CTC formazan and differential interference contrast images of trophozoites stained with CTC are shown. (A) CTC formazan accumulates within trophozoites, as shown by red fluorescence. (B) Trophozoite from panel A at higher magnification. (C) Inhibition of respiration of trophozoites by addition of sodium azide. (D) Respiration-inhibited trophozoite from panel C at higher magnification.

Fig 2

Acanthamoeba castellanii cysts stained with CTC. Merged fluorescence images of red-fluorescing CTC formazan and differential interference contrast images of cysts stained with CTC are shown. (A) CTC formazan accumulates within cysts, as shown by red fluorescence. (B) Cyst from panel A at higher magnification. (C) Inhibition of respiration of cysts by addition of sodium azide. (D) Respiration-inhibited cyst from panel C at higher magnification.

Fig 3

CTC staining of PHMB-treated Acanthamoeba trophozoites. Merged fluorescence images of red-fluorescing CTC formazan and differential interference contrast images of trophozoites stained with CTC after PHMB treatments are shown. CTC staining was performed on trophozoites treated with PHMB (0.2, 1, or 10 ppm) for 4 h at 25°C.

Fig 4

Respiratory activity and survival rate of Acanthamoeba trophozoites after PHMB treatment. The respiratory activity (CTC biocidal assay) was compared with the survival rate (culture-dependent biocidal assay) for trophozoites after treatment with PHMB (25°C, 4 h). Error bars represent the standard errors of the means for four experiments. ND, not detected (<1%).

Fig 5

Respiratory activity and survival rate of Acanthamoeba trophozoites after treatment with SCL disinfectant solutions. The respiratory activity (CTC biocidal assay) was compared with the survival rate (culture-dependent biocidal assay) for trophozoites after treatment with SCL disinfectant solutions (25°C, 4 h). Error bars represent the standard errors of the means for four experiments. ND, not detected (<1%).

Fig 6

CTC staining of PHMB-treated Acanthamoeba cysts. Merged fluorescence images of red-fluorescing CTC formazan and differential interference contrast images of cysts stained with CTC after PHMB treatments are shown. CTC staining was performed on cysts treated with PHMB (1, 10, or 100 ppm) for 4 h at 25°C.

Fig 7

Respiratory activity and survival rate of Acanthamoeba cysts after PHMB treatment. The respiratory activity (CTC biocidal assay) was compared with the survival rate (culture-dependent biocidal assay) for cysts after treatment with PHMB (25°C, 4 h). Error bars represent the standard errors of the means for four experiments. ND, not detected (<1%).

Fig 8

Respiratory activity and survival rate of Acanthamoeba cysts after treatment with SCL disinfectant solutions. The respiratory activity (CTC biocidal assay) was compared with the survival rate (culture-dependent biocidal assay) for cysts after treatment with SCL disinfectant solutions (25°C, 4 h). Error bars represent the standard errors of the means for four experiments.