The Chemical Transformation of the Cellular Toxin INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) as an Indicator of Prior Respiratory Activity in Aquatic Bacteria

Abstract

In the ocean, the prokaryote respiration rates dominate the oxidation of organics, but the measurements may be biased due to pre-incubation size filtration and long incubation times. To overcome these difficulties, proxies for microbial respiration rates have been proposed, such as the in vitro and in vivo estimation of electron transport system rates (ETS) based on the reduction of tetrazolium salts. INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) is the most commonly applied tetrazolium salt, although it is toxic on time scales of less than 1 h for prokaryotes. This toxicity invalidates the interpretation of the rate of in vivo INT reduction to formazan as a proxy for oxygen consumption rates. We found that with aquatic bacteria, the amount of reduced INT (F; µmol/L formazan) showed excellent relation with the respiration rates prior to INT addition (R; O2 µmol/L/hr), using samples of natural marine microbial communities and cultures of bacteria (V. harveyi) in batch and continuous cultures. We are here relating a physiological rate with the reductive potential of the poisoned cell with units of concentration. The respiration rate in cultures is well related to the cellular potential of microbial cells to reduce INT, despite the state of intoxication.

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure

“…Therefore, reinforcing previous work [6], the results show that INT reduction can be used as a proxy for plankton respiration when incubated for specifically determined short time periods. However, we cannot recommend a particular maximum incubation time and do not support the idea of a constant incubation time for the INT reduction method with natural plankton or eukaryotic cultures as other authors have suggested for prokaryotes [11]. The maximum incubation time (determined as the time after which INT had a negative effect on oxygen consumption) was not related to cell abundance or by implication to biomass alone.…”

“…The increased sensitivity of this measurement, allowing less disruption of the sample and shorter incubation times (from minutes to hours) compared to the traditional Winkler titration method (usually 24 hours), has enabled improved understanding of the spatial and temporal variability of plankton respiration [8] as well as the apportionment of total plankton respiration to different size classes [8–10]. However, despite validation of INT reduction (INT R ) as a proxy for plankton respiration [6], the method remains controversial [11, 12]. Several researchers have shown that the reduction of soluble tetrazolium salts can have a toxic effect on cells, potentially due to formazan production inhibiting the electron transport system [13], leading to a reduction in metabolic activity [5, 12].…”

A Review of Methods to Determine Viability, Vitality, and Metabolic Rates in Microbiology

“…This can be used to determine the respective proportion of active and inactive cells in various samples by comparison to microscopy count, for example (Dufour and Colon, 1992;Haglund et al, 2002;Bartosch et al, 2003). Similarly, studies have shown that the production of formazan per cell increases following an increase in growth rate (Villegas-Mendoza et al, 2019). On the contrary, at a later stage of batch culture (late stationary phase), where most cells are not growing anymore, staining of such cells by tetrazolium was poor (Fukui and Takii, 1989).…”

High variability in organic carbon sources and microbial activities in the hadopelagic waters

“…The enzymatic electron transport system drives oxidative phosphorylation. It has frequently been used as a proxy for respiration (Minutoli and Guglielmo 2009; Villegas‐Mendoza et al 2019). In vivo electron transport system activity was measured at in situ temperature based on tetrazolium salt 2‐para(iodo‐phenyl)‐3(nitrophenyl)‐5(phenyl)tetrazolium chloride (INT) reduction rate following the protocol of Martinez‐Garcia et al (2009).…”