Validation of the methylene blue test for assessment of viability of protoscolices from hydatid cysts

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.