Tetranitro-blue Tetrazolium Reduction in Bacillus Subtilis

In order to decide whether in the bacterial cell the enzymes of the respiratory chain are located in "particulate elements" (1) or in the plasma membrane (2) we investigated the reduction of potassium tellurite in Gram-positive and Gram-negative bacteria. For the first time in 1962 (3), and in more detail in 1964 (4), we reported that with Bacillus subtilis we had found that reduction products of tellurite are deposited in or on the membranes of the particular organelles previously described by one of us (5, 6), and called mesosomes by Fitz-James (7). This localization of the reduced tellurite seemed to indicate that in this Gram-positive bacterium the membranous organelles represent the bacterial equivalents of tbe mito-chondria of higher cells. For this reason we now use the term chondrioids (8) to indicate discrete elements possessing reductive activity. VanderWinkel and Murray in 1962 (9), using 2,3,5-triphenyl tetrazolium, found in Bacillus subtilis "white spaces" near mesosomes, probably caused by dissolution of the formazan formed, and concluded that the mesosomes would be the sites of the oxidative-reductive system of the cell. Because it is unsatisfactory that the formazan itself was not visualized, it was deemed desirable to reexamine, with the help of one of the newer tetrazolium compounds, the contention of the mitochondrial nature of the membranous structure in the cell. At present few attempts have been made to define precisely the step in the respiratory chain that leads directly or indirectly to tellurite reduction. Of old, the reduction of tetrazolium has been considered specific for mitochondrial function. The newer nitro-blue tetrazolium and tetranitro-blue tetrazolium compounds appear to accept electrons directly from the respiratory chain: according to Novikoff (10), possibly at a step before coenzyme Q and cytochrome b. The use of tetranitro-blue tetrazolium (TNBT) would have certain advantages over that of nitro-blue tetra-zolium (NBT); it would suffer, for instance, less of the non-specific affinity of the diformazan for lipid (1 l, 12). With the TNBT method, deposits of diformazan under the electron microscope were seen to occur in or on the cristae mitochondriales of the cells of various tissues (13-15). It seemed important to make use of the new compounds in a renewed search for the sites of enzymes of the respiratory chain in bacteria (16). MATERIALS AND METHODS Bacillus subtilis strain Marburg was cultivated under vigorous agitation in Difco heart infusion broth. In the early logarithmic phase the ceils were spun down …