Effectiveness of tetrazolium salts in microbial ecological studies in submerged soil

Several trials to obtain information on the living microorganisms at micro-sites of the submerged soil by the novel utilization of the tetrazolium salts were described. Results are summarized as follows:

(1) The air-dried soil was incubated with TIC (triphenyl tetrazolium chloride) under submerged condition according to the modified double layer plate method. The red formazan (the reduced product of TIC) was precipitated at the biologically active sites by the dehy. drogenase activity. Using a binocular microscope, most of the biologically active sites were found to be localized on and around the organic debris.

(2) The amount of nitrogen mineralized by incubating the soil under submerged condi. tion correlated well not only with the amount of formazan formed biologically in the submerged soil but also with the amount of formazan formed non-biologically by heating the soil with TIC in Ba(OH). solution. In addition, the pattern of precipitation of the nonbiologically formed formazan was very similar to that of the biologically formed formazan. Accordingly, the substances which reduced TIC non-biologically were considered to be closely associated with the substrates for the microorganisms.

(3) The plant debris stained with the red formazan could be mounted on a slide glass and observed at high magnification for the detailed study on the process of decomposition. Addition of lactophenol cotton blue to the plant debris could visualize the microorganisms responsible for the decomposition of the plant debris.

(4) NTB (nitro tetrazolium blue) was the most suitable for staining individual microorganisms growing on cellulosic materials or plant debris. The double staining with NTB and rosebengal made the discrimination between dead or resting cells and viable ones possible when this technique was applied to the decomposed filter paper.