Nitroblue Tetrazolium (NBT) - Succinate Vital Staining of AM Fungi In Roots (some notes by C. Rivetta and R.M. Miller; comments on procedural details by L. GadesEnvironmental Research Division, Argonne National Laboratory)
BACKGROUND
A procedure for the combination of vital staining and non-vital counterstaining of arbuscular mycorrhizal (AM) hyphae in fibrous roots is described. Vital staining with succinate-NBT reveals potential activity of AM fungal succinate dehydrogenase enzyme (SDH) in roots as purple drops of formazan (Mac Donald and Lewis, 1978; Kough et. al., 1987; Sylvia, 1988). Succinate dehydrogenase is a mitochondria enzyme that acts as a carrier for hydrogen removed in the aerobic oxidation of carbohydrate in the Krebs cycle. Pearse (1972) observed an increase in succinic oxidase activity for tissue in which the increased respiratory rate is part of general increase in activity. The presence of succinate dehydrogenase enzyme has been used to indicate the fungus metabolic activity (Kough et al., 1987; Hamel et al., 1990; Smith and Gianinazzi-Pearson, 1990; Smith and Dickson, 1991; Saito et al., 1993; Schaffer and Peterson, 1993; Tisserant et al., 1993; Smith et al., 1994). When root tissue is counterstained with non-vital acid fuchsin which stains the whole fungal structure pink by binding the acid fuchsin to cell wall material the SDH activity stands out particularly well.
The use of enzymes for accurate histochemical analysis requires control of all the variables capable of modifying the reaction, affecting the process of hydrolysis and subsequent staining. The most important factors are: enzyme concentration; time and temperature of incubation; pH at which the reaction is carried out; the presence of electrolytes, activators and inhibitors; and enzyme stability under the above conditions (Pearse, 1980).
The succinate dehydrogenase enzyme has the ability to remove hydrogen from one substrate (succinate) and transfer it to another (nitro blue tetrazolium salt). The dark coloration obtained in the fungus during the vital staining is due to the precipitation of reduced nitroblue tetrazolium salts as colored formazan in the form of purple drops.
Nitro-BT salts are colorless and act as electron acceptors in enzyme-catalyzed oxidation. Kuhn and Jerchel (1941) found that a number of colorless tetrazolium salts were reduced to colored compounds by plant tissues. The intact membrane of a mitochondrion constitutes a barrier to the penetration of the tetrazolium salt. The molecular size of all tetrazolium salts so far employed is sufficiently small for the diffusion constant to be high enough theoretically to allow rapid penetration into the tissues, but the penetration of Nitro-BT into the mitochondrion is reduced to a low level and the amount of formazan is correspondingly small. This characteristic can be compensated for by incubating roots at high concentration of NBT (4 mg/ml).
It was also found that the effect of pH is not confined to altered kinetics of the enzyme reaction. There is also an effect on the mitochondria, at pH 7.4 and above very considerable swelling take place. When this occurs the velocity of oxidation of succinate is much faster (Pearse, 1972).
Another factor that affects the stability of the mitochondria is the presence or absence of Mg+2 (Pearse, 1968). The mechanism by which magnesium ions exert their protective effect is unexplained although it has been hypothesized that they bind ATP to the mitochondrion, thus preventing its loss. This protection is afforded by the use of five mM-magnesium ions in the incubating medium.
Nitro-BT is not lipid soluble, which is a disadvantage with regard to penetration of lipoprotein barriers, but it is offset by the fact that progressive formazans production in lipids droplets does not occur. The formazans from Nitro-BT appear to be insoluble in those lipids normally found in fungal tissues.
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