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Interferences in the Optimization of the MTT Assay for Viability Estimation of Proteus mirabilis
Ewa Grela1, Adam Ząbek1, Agnieszka Grabowiecka1
1Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wroclaw, Poland.
Background:
The chromogenic assay based on MTT bioreduction was adapted to Proteus mirabilis viability estimations. We primarily intended to use the assay for the evaluation of novel antimicrobial compounds, including structures with possible permeabilizing activity. Therefore, the influence of basic permeabilizing agents like Triton X-100 and EDTA upon the MTT assay was studied.
Methods:
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was used as a substrate for the whole-cell dehydrogenase activity estimations. The amount of formazan product was evaluated in the end-point reactions terminated with acidic isopropanol or in the continuous reactions run in the presence of low detergent concentrations.
Results:
The generally established procedure of the end product dissolution with acidic isopropanol caused absorbance instability which strongly affected the results accuracy. The disadvantage was especially pronounced when the assay was conducted in Mueller-Hinton Broth. PBS with 0.01% Triton X-100 used as the reaction medium allowed to omit the formazan dissolution step and follow the microbial MTT reduction in a continuous mode. It was observed that in Proteus mirabilis with a compromised outer membrane the assay score was artificially increased above the untreated control.
Conclusion:
The dependence of the assay results on the cell integrity might be a major drawback of the MTT assay application for the evaluation of novel antimicrobials against Gram-negative microorganisms. On the other hand, the MTT reduction could be conveniently used to assay the permeabilization degree in biotechnological protocols.
Insights
The MTT assay for Proteus mirabilis viability can be affected by cell integrity, potentially skewing antimicrobial compound evaluations. However, continuous MTT reduction monitoring offers a reliable method for assessing cell permeabilization.
Area of Science:
- Microbiology
- Biotechnology
- Assay Development
Background:
- The MTT assay, based on bioreduction, was adapted for Proteus mirabilis viability.
- The study investigated the impact of permeabilizing agents on the MTT assay for antimicrobial compound evaluation.
Purpose of the Study:
- To assess the suitability of the MTT assay for Proteus mirabilis viability.
- To evaluate the influence of Triton X-100 and EDTA on MTT assay results.
- To explore continuous monitoring of MTT reduction.
Main Methods:
- Utilized 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for whole-cell dehydrogenase activity.
- Compared end-point reactions with acidic isopropanol dissolution to continuous reactions with low detergent concentrations.
Main Results:
- Acidic isopropanol dissolution led to absorbance instability, affecting accuracy, especially in Mueller-Hinton Broth.
- Using PBS with 0.01% Triton X-100 enabled continuous MTT reduction monitoring without a dissolution step.
- Compromised outer membranes in Proteus mirabilis artificially increased assay scores.
Conclusions:
- Cell integrity dependence is a significant limitation for MTT assay use with Gram-negative antimicrobials.
- MTT reduction can be effectively utilized to quantify permeabilization in biotechnological applications.
