Purpose:
The most commonly reported methods for outgrowth and enumeration of surviving Acanthamoebae following treatment with disinfecting solutions require use of bacterial cells and time-consuming individual microscopic observations of plated cells over a 14 day period to determine log reduction values. Here we compare performance of a simple colorimetric assay in axenic culture medium plus a TTZ (tetrazolium) cellular respiration based dye to that of outgrowth plus microscopy with live E. coli.
Methods:
Trophozoites of Acanthamoeba castellanii ATCC 50370 were cultured in axenic chemically defined AC6 medium without any antibiotics, treated with PBS (Phosphate Buffered Saline) or 10 ppm alexidine control solutions for 1, 4, or 24 hours, then aliquots were neutralized and serially diluted into 96-well plates containing either AC6 medium plus TTZ or E. coli in Ringer’s solution and monitored for 14 days. Axenic plates were scored by visual observation of TTZ reduction to a dark purple whereas E. coli plates were scored by microscopic inspection of each well for trophozoite growth. Log reduction values were determined with the most probable number method using Spearman-Karber computations.
Results:
See table for comparison of results obtained by both methods.
Conclusions:
A simplified, colorimetric outgrowth enumeration method can yield quantitative log reduction values and kill kinetics similar to those obtained with more labor-intensive microscopic methods that require co-culture with live bacteria. Moreover, the colorimetric method uses axenic culture of Acanthamoebae throughout, and is thus consistent with ISO/FDA test standards for disinfection efficacy testing against bacteria and fungi which all specify use of axenic culture methods.
Keywords: Acanthamoeba • keratitis • contact lens