Background: Methylene blue was used as a vital stain for the assessment of viability of protoscolices from hydatid cysts taking advantage of the chemical nature of the dye as a redox indicator and the kinetically distinct molecular transfer systems of Echinococcus protoscolex for uptake of materials across the tegument. Aim: The present study attempts to validate the application of methylene blue staining for assessment of viability of protoscolices. Methods: To validate the criteria by which viability is assessed, control tests were performed using normal protoscolices and protoscolices previously treated with distilled water at 60°C for 5 minutes. Performance of methylene blue was further studied at intervals over a period of 50 minutes after protoscolex exposure using 1% dye concentration. Results: Normal protoscolices were able to adsorb and reduce the dye and have, therefore, lost the blue color. Protoscolices previously treated with warm water on the other hand, being functionally dead, failed to reduce the adsorbed dye and permanently retained the blue color. Results also indicated that a clear distinction between dead and alive protoscolices can be made within 1 minute. Reading of the test after 10 minutes would be misleading giving a false result. Conclusion: These findings suggest that viability of protoscolices can be assessed on the basis of acquisition and loss/retaining of the dye blue color. Increasing the concentration of methylene blue to 1% was noticed to be associated with remarkable enhancement of contractility, sucker movement, and evagination. Such an excitatory action of the dye may be exploited in viability tests which adopt these criteria.

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Sodium hypochlorite (NaOCI) is used extensively as an artificial exsheathment medium of nematode larvae in a diversity of studies such as comparing the efficacy of drugs, assessment of resistance to anthelmintics or evaluation of plant extracts as anthelmintics by virtue of its unique capacity for tissue dissolution. Tests with NaOCI indicate that the compound, although highly effective as exsheathing agent, the infectivity of the exsheathed larvae produced was significantly low suggesting reduced viability. We used Strongyloides papillosus larvae, a nematode that naturally lacks a protective sheath and a potentially highly motile organism, as a model to exclude or confirm possible negative effects on the viability of the parasite. Motility was taken as a viability assay. Larvae were designated as actively motile, sluggish, or immotile. Results were presented as supplementary movie files. The viability of larvae is dependent on both the concentration and the time of exposure to the compound. There are certain concentration (C) and time (T) limits beyond which viability is impaired (0.3% ˃ C ˃ 0.2%; 10 min ˃ T ˃ 5 min). The results support the conclusions from infectivity tests that where NaOCI is used as an exsheathment medium, results should be interpreted with caution as the compound is potentially capable of reducing the viability of larvae. It may even induce structural damage.