Staining of Parasitic Helminths by Extracts of Allium cepa, Juglans regia, and Rubia tinctorum: An Approach to Herbal Dyes

Background: Although carmine, as a synthetic dye, is the major substance for staining helminths, it may impose some adverse effects on human health. In the present study, we evaluated the aqueous extracts of onion (Allium cepa) skin, walnut (Juglans regia) husk, and madder (Rubia tinctorum) roots as potential herbal dyes for staining parasitic helminths. Methods: Aqueous solutions (5%, 10% and 20%, w/v) of each herbal prepared from dried and powdered husk of walnut, skins of red onion, and madder roots in distilled water. Parasitic helminths including Fasciola spp., Dicrocoelium denderiticum, Echinococcus gronulosus protoscolices, Moniezia spp., and Haemonchus contortus were stained by different concentrations of herbal dyes according to carmine staining method. The structural clarity and quality of stained internal organs of the helminths such as suckers, intestine, and reproductive systems were scored by semiquantitative evaluation in comparison with carmine stained samples. Results: The optimum concentrations of extracts for helminths staining were 10% (w/v) of A. cepa and J. regia, and 20% (w/v) of R. tinctorum with final scores of 3.1, 3 and 2.8, respectively. In general, A. cepa and J. regia extracts showed higher quality in staining Platyhelminthes, while R. tinctorum extract presented relatively higher quality in staining Nematoda. Conclusion: Considering proper quality of A. cepa, J. regia and R. tinctorum extracts in staining the helminths, they may be safe, eco-friendly, and inexpensive alternatives to carmine dye.