Effectiveness of red spinach (Amaranthus tricolor) extract as an alternative to eosin in histological staining

The high usage of eosin as a cytoplasmic stain in histological preparations involves relatively high costs and poses potential environmental and toxicity risks, thereby necessitating a natural dye alternative. Red spinach (Amaranthus tricolor) contains anthocyanins, which have the potential to be used as a substitute for eosin. This study aim to compare the effectiveness of red spinach extract with eosin as a cytoplasmic stain in histological preparations. This study employed a quasi-experimental laboratory design with a descriptive quantitative approach involving 24 myoma tissue preparations divided into four groups: Hematoxylin-Eosin (control), and Hematoxylin combined with red spinach extract at concentrations of 10%, 20%, and 40%. Staining quality was assessed based on cytoplasmic staining intensity, nucleus-cytoplasm contrast, and tissue structure clarity by an anatomical pathologist. Statistical analysis was performed using the Kruskal-Wallis test followed by the Mann-Whitney test. The results showed a significant difference between the control group and all red spinach extract groups (p < 0.001). The Hematoxylin-Eosin group obtained the highest mean rank (21.50), while all red spinach extract groups shared the same mean rank (9.50). The findings indicate that red spinach extract at all tested concentrations has not been able to provide a staining quality equivalent to that of eosin. Unlike red spinach extract, which is a mixture of various natural compounds, eosin possesses more standardized chemical characteristics and usage, resulting in a more consistent dye. Staining histological preparations using hematoxylin combined with red spinach extract at concentrations of 10%, 20%, and 40% has not been able to produce a quality equivalent to eosin. This applies to the conditions of this study using extract concentrations of 10%, 20%, and 40% via the maceration method; therefore, it cannot yet be concluded that red spinach extract is generally unsuitable as an alternative to eosin. Future research is recommended to use higher extract concentrations, more optimal extraction methods, and longer staining durations.