• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.118-123
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• 2008

A new method for antimicrobial susceptibility testing of vitro-cultured bacteria on an ordinary fluorescence spectrometer was developed. The viable bacteria reduced 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles that displayed intense resonance scattering light. The assay showed a linear relationship between the number of viable bacteria and the intensity of resonance scattering light. Dead bacteria were unable to reduce MTT. Methicillin-resistant Staphylococcus aureus exposed to flavonoids from Marchantia convoluta showed a flavonoids concentration-dependent inhibition of the ability to reduce MTT. In the assay, less than 12 h was required to attain susceptibility results and fewer bacteria were utilized than in traditional methods. The RLS technique could, in combination with the MTT assay, be a rapid and sensitive measuring method to determine the in vitro activity of new antimicrobials.

• BMB Reports
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• v.38 no.3
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• pp.366-369
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• 2005

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome $b_5$, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of $A_{610}$. MTT reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;20\;{\mu}M$, $k_{cat}\;=\;1,910\;min^{-1}$). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.494-498
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• 2008

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored water-soluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to $3.0{\times}10^7$ sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at $17^{\circ}C$ using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory.

• Biomedical Science Letters
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• v.22 no.4
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• pp.167-173
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• 2016

The broth microdilution technique used to measure the minimal inhibitory concentration (MIC) of natural compounds against bacteria is problematic: it is difficult to visualize bacterial growth due to the color of the natural compound. Therefore, the use of a colorimetric method with a redox indicator by broth microdilution can simplify it and increase its objectivity. This study evaluated the usefulness of the colorimetric method in measuring the MIC of Phellinus baumii against methicillin-resistant Staphylococcus aureus (MRSA). The inhibition in disc diffusion method was observed from $8,192{\mu}g/mL$ P. baumii in all 10 MRSA isolates examined; however, the MIC ranges of the 10 MRSA isolates was $512{\sim}2,048{\mu}g/mL$ by broth microdilution using a colorimetric method; with the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) indicator. In addition, the MIC of P. baumii by broth microdilution using MTT as indicator yielded excellent results. However, the 2, 3, 5-triphenyltetrazolium chloride (TTC) results could not be determined due to the color of the TTC indicator. The MICs of four antibiotics against MRSA using MTT or TTC were equal to those determined by visual interpretation. In conclusion, to evaluate the antibacterial effects of a natural compound, the broth microdilution technique is considered to be better than the disc diffusion method. Moreover, to resolve the problems caused by the colors of natural compounds, a colorimetric method such as that using MTT may be very valuable.

• Environmental Analysis Health and Toxicology
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• v.9 no.1_2
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• pp.19-23
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• 1994

The tetrazolium dye, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cell, and this reaction is used as the end point in a rapid drug screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantative relationship is established between cell number and MTT-formazan production. Several conditions were examined to devise an in vitro assay method in primary cultured hepatocytes, such as optimum wavelength, optimal MTT concentration, optimal incubation time, and cell density.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.4
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• pp.1169-1172
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• 2004

The cytotoxic activity of 13(E)-labd-13-ene-8α, 15-diol (1) was evaluated against tumor cell lines. A comparison of IC/sub 50/ values of this compound in cancer cell lines showed that their susceptibility to this compound decreased in the following order: P388>B16{F10>MDA-MB-231>A549>KB>SNU-C4 by the MTT method. 13(E}-Labd-13-ene-8α, 15-diol (1) was the most effective growth inhibitor of P388 murine leukaemia cell lines, producing approximately 8.3㎍/mL of IC/sub 50/ in the MTT method.

• Bulletin of the Korean Chemical Society
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• v.35 no.7
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• pp.1933-1938
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• 2014

Despite increasing importance of in vitro cell-based assays for the assessment of nanoparticles (NPs) cytotoxicity, their suitability for the assessment of NPs toxicity is still in doubt. Here, limitations of widely used cell viability assay protocol (i.e., MTT asssay) for the cytotoxicity assessment of P25 $TiO_2$ NPs were carefully examined and an alternative toxicity assessment method to overcome these limitations was proposed, where the artifacts caused by extracellularly formed formazan and light scattered by agglomerated NPs were minimized by measuring only the intracellular formazan via image cytometric methods.

• Microbiology and Biotechnology Letters
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• v.25 no.3
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• pp.335-337
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• 1997

In this study, we show a convenient MTT assay for detect the susceptibility of yeast-like form of Trichosporon beigelii against antifungal agents. This assay was developed based on mitocondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan. Cells of T beigelii are seeded into 96-well microtiter plates, and antifungal agents, amphotericin B, magainin and CA-ME hybrid peptide were added with various concentrations. After 24 hr incubation, MTT was added, then incubations were continued for 4 hr. Formazan formation was quantified photometrically after extraction of the formazan with acid sodium dodesyl sulfate (SDS). From this assay, we could obtained MICs of antifungal agents against T. beigelii. The presented method can easily be used as an effective methods to assess the antiftingal action of various agents on yeasts with minimal amounts of antifungal agents.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.15 no.1
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• pp.37-50
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• 1989

The in Vitro chemosensitivity of fibroblast cell strains was determined using a semiautomated tetrazolium-based colorimetric assay(MTT assay) to 16 cosmetic materials. This assay is useful method to evaluate toxic effects of the chemicals. From assay results, we determined that the preservatives are more toxic than moisteurizers. The chemicals in the same group have a different toxicity. That is, in preservatives, Germall -115 is more toxic than Danisol -M, -p, and in surfactant sodium laurel sulfate than Myrj 52, and in moisteurizers, 1, 3-butylene glycol is more safe than the others. When the results from this assay for preservatives were compared with patch test results, good correlation was observed. Forthemore, this assay method can be used together with Patch test for the evaluation of the chemical toxicity, particularly in cosmetic field.