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

Abstract

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method’s applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.

Microbial respiration in contrasting ocean provinces via high-frequency optode assays

“…A more extensive study by Garcıá-Martıń et al (2019b) validated the INT-based respiration assay using Winkler-derived O 2 measurements for marine plankton, reporting reasonable agreement with incubations times tailored to each method (2 h for INT and 20-48 h for O 2 incubation). However, given evidence of potential underestimation by INT methods despite their validation against O 2 techniques (Villegas- Mendoza et al, 2015;Garcıá-Martıń et al, 2019c;Baños et al, 2020), future research should further explore how INT, O 2 optode, and BP-derived respiration rates compare. Additionally, the potential of emerging techniques, such as new single-cell respiration measurements (Munson-McGee et al, 2022), should be investigated.…”

“…Alternatives to the O 2 -based assays, including respiratory stains such as in vivo 2-para-(iodophenyl)-3 (nitrophenyl)-5(phenyl) tetrazolium chloride (INT) measure activity of the electron transport system which can yield highly precise and sensitive measures of respiration without a long incubation period (Griffith, 1988), but are still time and wet-lab resource intensive with limited sample throughput. While environmental and culture studies find the INT method to be representative of marine plankton respiration (Garcıá-Martıń et al, 2019b andGarcıá-Martıń et al, 2019c), other studies caution that INT may underestimate respiration due to toxicity effects (Villegas-Mendoza et al, 2015;Baños et al, 2020).…”

Current Perspectives on Viable but Non-Culturable Foodborne Pathogenic Bacteria: A Review

“…The fluorescence can be observed and quantified by fluorescence microscopy or flow cytometry. The typically used dyes include CTC or INT [ 57 , 58 , 59 ]. For instance, Zhu et al [ 60 ] successfully quantified the number of E. coli in the VBNC state after UV treatment using CTC flow cytometry.…”