Fast alive bacteria amount measurement by fluorescence method
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A technology for rapid determination of the total number of bacteria, applied in the direction of fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of determining the total number of Escherichia coli by NADH fluorescence method
Inactive Publication Date: 2009-08-05
OCEAN UNIV OF CHINA
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[0005] At present, there is no report on the direct use of NADH fluorescence method to determine the total number of Escherichia coli
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Embodiment 1
[0016] The scanning conditions were selected for NADH with a concentration of 0.1 mM, and the excitation and emission wavelengths were determined.
[0017] like figure 1 It is shown that under different excitation wavelengths in the range of 310nm to 390nm, scanning the range of 220 to 800nm, it is found that there is a single emission peak. With the change of excitation wavelength, the peak position in the emission spectrum has almost no shift, and its wavelength range is from 460 to 462nm, and when the excitation wavelength is 340nm, the maximum peak fluorescence intensity is reached.
[0018] Use the obtained three emission wavelengths of 460nm, 461nm and 462nm to re-scan to obtain the excitation peak, scan the excitation wavelength in the range of 220-550nm, and the fluorescence peaks appear at 232nm, 273nm and 342nm respectively ( figure 2 ).
[0019] With 232nm as the excitation wavelength, there is no fluorescence peak at 460-462nm. Among them, 273nm and 342nm are t...
Embodiment 2
[0022] Accurately weigh 0.0355 g of pure NADH, dilute to 50 mL with sterile PBS (pH 8.0), and prepare 1 mM NADH stock solution. Dilute the stock solution, dilute NADH to between 0.1nM and 10mM, measure according to the fluorescence method, take the NADH concentration as the abscissa, and the fluorescence intensity as the ordinate, draw the standard curve of NADH ( image 3 ).
Embodiment 3
[0024] Add 3mL of 0.02M, pH 8.0 sterile Tris-HCl to the collected bacterial cells, and treat at 80°C for 30min. After the sample was cooled to room temperature and shaken thoroughly, it was centrifuged at 10,000 rpm for 10 min at 4°C, and 1 mL of the supernatant was taken for NADH detection immediately.
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Abstract
The invention relates to a method for detecting the total number of bacteria based on the fluorescent method of NADH, in particular to a novel method for rapidly detecting the total number of colon bacteria based on the fluorescent characteristic of the NADH in a colon bacteria cell and the stable-content characteristic of the NADH in the colon bacteria cell, which comprises the processes of extracting NADH in the colon bacteria cell, detecting the content of the NADH in the colon bacteria cell, detecting the fluorescent intensity of the NADH in the colon bacteria cell with a fluorescent spectrophotometer and rapidly detecting the total number of bacteria. The fluorescent detection limit of the NADH is 1 nM, and the content of the NADH ranges from 10 nM to 80 microns and keeps a favorable liner relationship (R = 0.9905) with the fluorescent intensity. The colon bacterial cell is obtained in a decentering way, the NADH in the colon bacteria cell is extracted in a Tris-HCl method, the fluorescent intensity of extracting solution of the NADH is extracted with the excitation wavelength of 342 nm and the emission wavelength of 461 nm, and the colon bacteria of 10 cfu/mL can be detected in one hour. The method adopts novel detection theory, has rapid detection speed and good repeatability, is sensitive and easy, is suitable for quantitatively detecting the number of colon bacteria in the fields of food sanitation and safety, environment detection, and the like, and can replace the traditional method for detecting the number of living cells.
Description
technical field [0001] The invention relates to a method for quickly measuring the total number of live bacteria, in particular to a method for quickly measuring the total number of live bacteria based on the principle of NADH fluorescence. Background technique [0002] Known according to the inventor by consulting data, document retrieval, the method for measuring the total number of bacteria generally adopted at present is mainly plate colony counting method and turbidimetric method, plate colony counting method needs the time of colony cultivation, so measuring time is more than 1 day , and the steps are cumbersome, easy to cause pollution, and manual counting will reduce the accuracy of the measurement due to subjective errors. Nephelometric method is simple and less time-consuming, but this method cannot distinguish between living and dead cells, which limits its application. In recent years, new detection methods have been studied at home and abroad to shorten the det...
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