This report describes tests with Evan's Blue and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) for the assessment of the viability of rhinosporidial endospores. MTT stained a proportion of the spherical bodies that we regard as the Electron Dense Bodies (EDBs), and the cytoplasm of freshly prepared endospores or ones that were stored at 4 °. Slow-freezing at −20 °C, exposure to 10% formalin, or 0.1% sodium azide of the endospores abolished MTT-staining in both sites. Evan's Blue stained the EDBs and cytoplasm of fresh endospores or those stored at 4 °, and of sodium azide-treated or frozen (−20 °)-thawed endospores. TMRE (tetramethyl-rhodamine ethyl ester) specifically labelled the spherical bodies, supporting the conclusion that these spherical bodies have a mitochondrial-like structure. TMRE-staining was however retained in endospores after their treatment with formalin, sodium azide and slow-freezing while MTT-staining was abolished in all these treated endospores.

These results indicate that EvB and TMRE were capable of revealing the morphological integrity of endospores but failed to identify the metabolic inactivation of the endospores after treatment with formalin, sodium azide or slow-freezing. Only MTT was capable of identifying metabolically active endospores and hence their viability and could prove to be of value in standardizing models of infection.