Estimation of Microbial Count in Raw Milk Based on a Colorimetric Reaction with Vitamin K3 and Tetrazolium Salt.

Ukeda, Hiroyuki; Goto, Yukihiko; Sawamura, Masayoshi; Kusunose, Hirozo; Kamei, Toshiro

doi:10.3136/nskkk.42.627

Cited by 3 publications

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“…The authors reported the microbial counting method of raw milk based on the reduction reaction of NBT mediated with menadione at neutral pH (Ukeda et al, 1995). We observed that, when the method was applied to commercially available UHT-treated milks, a similar reduction reaction occurred despite the fact that the milks contained no microorganisms.…”

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confidence: 76%

“…Menadione has been known as an effective substrate of ETS of microorganisms and applied to the determination of ETS activity using a combination of tetrazolium salts (Ukeda et a/., 1995;Roslev & King, 1993). We observed that the addition of menadione at the saturation level increased the reaction rate to about two-fold (Ukeda et a/., 1995). Also, its effectiveness has been shown as a redox mediator between dihydropyridine H. UKEDA et al .…”

Section: Methodsmentioning

confidence: 99%

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Reduction of Tetrazolium Salt XTT with UHT-Treated Milk: Its Relationship with the Extent of Heat-Treatment and Storage Conditions.

Ukeda

Goto

Sawamura

et al. 1995

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Agricu/ture, Kochi University, Monobe B-200, Nankoku Co., Ltd., 1-2l-3, Sakae-cho, Higashimurayama, Tokyo 783, Japa n 189, Japan UHT-treated milk reduced 3'-{1-L(phenylamino)-carbonylJ-3,1~tetrazolium}-bis(4hmethoxy-6-nitro)benzenesulfonic acid hydrate (XTT). The reducibility was caused by the Maillard reaction intermediate between milk protein and lactose. First, the assay procedure of the XTT reduction was optimized using the heated model solution containing casein and lactose and UHT-treated milk. When the XTT reduction assay was applied to two kinds of UHT-treated milks under optimum conditions, the ability of each milk sample to reduce XTT significantly reflected the extent of the heat treatment as well as the evaluation method using the hydroxymethylfurfural (HMF) value. In contrast to the HMF value, the ability of UHT-treated milk gradually decreased depending upon the storage period and temperature. These results suggest not only that the present XTT assay is applicable to estimate the extent of heat treatment but also that the reducibility of the milk sample heat-treated under a given condition can serve to estimate the storage period if the storage temperature is known or vice versa. The method is much simpler (only mixing of the XTT solution containing menadione with milk sample) and quicker (about 20 min) with a satisfactory reproducibility than the conventional methods to estimate the extent of the Maillard reaction such as the lactulose or HMF determination.

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confidence: 76%

Section: Methodsmentioning

confidence: 99%

Reduction of Tetrazolium Salt XTT with UHT-Treated Milk: Its Relationship with the Extent of Heat-Treatment and Storage Conditions.

Ukeda

Goto

Sawamura

et al. 1995

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“…Cell proliferation and viability assays are of particular importance for routine applications in bacterial biology, so the detection of viable microbes is of major concern for many applications such as testing antibiotic effects against microbes. ,,, If this technique were to be used as an alternative to colony counting, it could also be employed for viability and activity detection.…”

Section: Resultsmentioning

confidence: 99%

“…As a different metabolism-based system, tetrazolium salts have become one of the most widely used tools in cell biology. ,− These salts, such as MTT and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (Scheme ), are redox dyes that can rapidly penetrate into intact biological cells, where they are enzymatically reduced to colored formazan derivatives, which can be assessed by colorimetry or fluorometry . The salts do not actually measure the number of viable cells, but rather respond to an integrated set of enzymatic activities that are related to cell metabolism .…”

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Electrochemical Detection of Viable Bacterial Cells Using a Tetrazolium Salt

et al. 2018

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In this study, electrochemical detection of viable bacterial cells was performed using a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which was converted to an insoluble and redox active formazan compound in viable microbial cells. The insolubility of this formazan was effectively exploited as a surface-confined redox event. An indium-tin-oxide electrode was applied to a microbial suspension that had been incubated with MTT and was heated to dry for the extraction and adsorption of formazan. Drying led to the appearance of a distinctive voltammetric oxidation peak at +0.1 V vs Ag|AgCl, the magnitude of which was successfully correlated to the number of viable microbes in the suspension. Thus, the electrochemical detection of formazan was effectively coupled with the thermal lysis of microbes. It is also noteworthy that this lysis-adsorption technique was highly selective to the hydrophobic formazan molecule due to the removal of hydrophilic cell components during equilibration in a phosphate buffer before voltammetric measurement. This technique was capable of detecting microbes above 2.8 × 10 CFU mL and required only a 1 h incubation. The results of this study indicate that the sensitivity of the present technique is up to 10 000-fold higher than that of MTT colorimetry. The higher sensitivity was mainly ascribed to the concentration of the microbially produced formazan on the electrode by thorough desiccation of the bacterial suspension.

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