Quantitative measurement of bacterial growth by the reduction of tetrazolium salts.

Tetrazolium salts can be used for measuring the reducing activity of microbial cultures colorimetrically, because the reduced form of these redox indicators is conspicuously colored (B. Jambor, Tetrazolium Salze in der Biologie, G. Fischer, Jena, 1960; B. J. Liska et al., J. Dairy Sci. 41:776, 1958). In an actively growing culture, the reducing activity is proportional with the number of bacteria in the culture, assuming that all bacteria are in a similar metabolic state. We correlated the reducing activity of nutrient broth cultures of Escherichia coli and Staphylococcus aureus with their viable count. We have found that under controlled conditions the color intensity of the reduced tetrazolium salt, formed by the reducing activity of bacteria, was proportional with the number of actively growing bacteria in the culture. Although tetrazolium salts inhibit the growth of bacteria to some extent (E. D. Weinberg, J. Bacteriol. 66:240, 1953; P. S. May et al., Proc. Soc. Exptl. Biol. Med. 105:364, 1960), we used tetrazolium salts in a concentration that had very little growth inhibition, or we used them under conditions which did not affect the quantitative determination of bacterial growth. Triphenyl tetrazolium chloride (TTC; Nutritional Biochemicals Corp., Cleveland, Ohio) and blue tetrazolium (BT; DAJAC Labs, Philadelphia, Pa.) were used in the experiments. The growth inhibitory effect of TTC and BT for the two test organisms is shown in Table 1. Since BT was more toxic than TTC for these bacteria, we carried out most experiments with TTC, although similar results were also obtained with BT. The TTC concentration used in the tests was 0.1 the 50% inhibition concentration. The 1TC reduction of E. coli and S. aureus cultures during a 30hr incubation period with TTC is shown in Fig. 1. The red formazan (the reduced form of TTC) was extracted from the culture broth with an equal amount of n-butanol, according to Barnes (E. M. Barnes, J. Gen. Microbiol. 14:57, 1956). The nbutanol-culture broth mixture was kept frozen overnight before the two layers were separated by centrifugation. The color intensity of the clear upper layer (the n-butanol solution of formazan) was measured in a Spectronic-20 colorimeter at 490 mm. An n-butanol extract of a control culture was used for a blank. The TTC reducing activity of the culture was proportional with the number of newly formed cells, i.e., the actively growing portion of the bacterial population. This point is clear especially in Fig. 2. The TTC reduction was carried out with different inoculum levels. The log A/Ao ratio represents the increment in the population of the