*4.1. Methanol Was Determined to Be the Optimal Extraction Solvent*

The results demonstrated that methanol was a more suitable solvent when compared against ethanol (Figure 1a). The molecule analysis produced six reproducible results: four of the molecules had one outlier and the other two had three reproducible results with one, which suggested ethanol was a better extraction solvent. All of the outliers and results that suggested ethanol was better came from a single sample (sample A). This could have been caused by an error in aliquot division when mixing a culture of cells, or homogeneity of the sample may not have been successfully achieved. The number of molecules extracted were consistent with metabolite concentration analysis, with sample set A being the only triplicate in which ethanol demonstrated better metabolite extraction than methanol. Overall, these results indicate that for *Blastocystis* ST7, methanol is a better extraction solvent. This is in contradiction to one past publication, in which a comparison between methanol and ethanol both produced similar results [29].
