A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay

Abstract

Significance and Impact of the Study: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on reduction in a tetrazolium salt (WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi.Keywords electron mediator, fungi, naphthoquinone, proliferation assay, susceptibility testing, tetrazolium salt. Abstract A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations (MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi.

A colorimetric assay using tetrazolium salts with an electron mediator to evaluate yeast growth in opaque dispersed systems

“…These prepared detection reagents contained 10.0 mM of TTC with C1 (TTC1) and C2 (TTC2) mediator levels or 2.5 mM of INT with C1 (INT1) and C2 (INT2) mediator levels. Detection reagents concentrations were selected taking into account those proposed by [1,6,10].…”

“…For example, the monotetrazolium salt, 2-(4iodophenyl)-3-(4nitrophenyl)-5-phenyl tetrazolium chloride (INT), has been used effectively to study mitochondrial electron transport [9]. Furthermore, to increase the sensitivity of these indicators, intermediate electron acceptors are used since they facilitate reduction reactions [10]. The 2-methoxy-1, 4naphthoquinone (MNQ) was successfully used as an electron mediator to evaluate effectiveness of antimicrobials [11].…”

Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays

“…Moreover, the WST-8 assay is more sensitive than WST-1, especially at neutral pH [21]. Therefore, the WST-8 colorimetric method is useful for the rapid determination of NAD(P)H and could be a valuable tool for screening cell viability and cytotoxicity [26–28]. WST-8 was used in the qualitative measurement of glucose-6-phosphate dehydrogenase (G6PD) activity.…”

Evaluation of Antibiotic Penicillin G Activities Based on Electrochemical Measurement of a Tetrazolium Salt

“…11) The reduced coenzymes nicotinamide adenine dinucleotide (NADH) and FADH 2 exist in living cells and their function is to transfer electrons through redox reactions. [12][13][14][15] The tetrazolium salt [16][17][18][19][20][21] 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), which is soluble and has excellent cell membrane permeability, permeates the cell membrane and forms a reduced complement in living cells. [22][23][24][25][26][27][28][29] MTT receives electrons from the enzyme and is reduced to insoluble formazan, which is then deposited (Fig.…”