Reassessment of tetrazolium bromide as a viability stain for spores of vesicular‐arbuscular mycorrhizal fungi

Abstract

Techniques for the determination of viability of field‐collected vesicular‐arbuscular mycorrhizal spores must be valid for spores of different ages and of varying levels of peridium (outer wall of unispored sporocarp) development. The use of the dehydrogenase‐activated stain 3‐(4,5‐dimethylthiazol‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) for determining viability of endogonaceous spores was reassessed. Freshly isolated and stored spores of three isolates of Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe were stained using two different protocols: Miller, Torres, and McClean (Mycological Research, in press) and An and Hendrix (Mycologia 80: 259–261). Time course results from selected experiments were analyzed graphically, and pooled results from all experiments were analyzed by factorial analysis of variance to determine statistically significant differences among the protocols, storage regime of spores, control killing method, and the effect of added cobalt ions. Significant nondehydrogenase‐specific staining occurred in both types of controls of freshly isolated spores using Miller, Torres, and McClean's method, rendering it inadequate for viability determination. With An and Hendrix's protocol, fresh nonkilled spores stained significantly more than ethanol‐treated spores, although these results underestimated viability as determined by two other assessment procedures, and autoclaving of fresh spores was not a satisfactory control treatment. Addition of cobalt to the incubation solution effectively drove all formazan production to the blue color but did not change the total percentage of colored spores produced, regardless of protocol used. Results corroborate the use of An and Hendrix's protocol with stored spores if color interpretation was corrected to include both blue and red colors as indicators of viability, results were read before nonspecific staining starts, and timing was extended to 75 hours. Both staining methods work with uniformly stored spores using specific incubation times, but not with spores of mixed ages, which could include young spores, making MTT not useful as a viability stain for field‐isolated spores.