Fluorescence detection method of total number of living bacteria

A technology for fluorescence detection and viable count, which is applied in fluorescence/phosphorescence, biochemical equipment and methods, and microbial determination/inspection. The effect of fast detection, simple operation and high sensitivity

Pending Publication Date: 2020-05-15
西安润嬴生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods have their own characteristics. Compared with the traditional plate counting method, the detection accuracy, sensitivity, and detection speed have been greatly improved. Strict conditions, not applicable to all institutions and units and on-site testing

Method used

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  • Fluorescence detection method of total number of living bacteria
  • Fluorescence detection method of total number of living bacteria
  • Fluorescence detection method of total number of living bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1 (sample to be tested is the cultured bacterium liquid)

[0078] A fluorescent detection method for the total number of living bacteria, comprising the following steps:

[0079] Step S01, centrifuge at 8000r / min at 4°C for 15min, pour off the supernatant, add an equal volume of Tris-HCl (pH=8) solution to resuspend, pipette evenly, and centrifuge at 8000r / min at 4°C After 15 minutes, the supernatant was poured off, and the above-mentioned resuspension steps were repeated three times to obtain the test sample liquid of the liquid bacterial suspension;

[0080] Step S02, establishing the linear relationship equation between the relative fluorescence intensity value and the number of viable bacteria as follows:

[0081] The sample solution to be tested and the fluorescent dye Calcein UltraGreen TM AM mixed reaction, carry out gradient dilution (wherein, described gradient dilution factor is 5 * 10 -2 , 2.5×10 -2 , 2×10 -2 , 1.25×10 -2 、10 -2 , 5×10 -3 ...

Embodiment 2

[0090] Embodiment 2 (the sample to be tested is the cultured bacterium liquid)

[0091] A fluorescent detection method for the total number of living bacteria, comprising the following steps:

[0092] Step S01, centrifuge at 8000r / min at 4°C for 15min, pour off the supernatant, add an equal volume of Tris-HCl (pH=8) solution to resuspend, pipette evenly, and centrifuge at 8000r / min at 4°C After 15 minutes, the supernatant was poured off, and the above-mentioned resuspension steps were repeated three times to obtain the test sample liquid of the liquid bacterial suspension;

[0093] Step S02, establishing the linear relationship equation between the relative fluorescence intensity value and the number of viable bacteria as follows:

[0094] The sample solution to be tested and the fluorescent dye Calcein UltraGreen TM AM mixed reaction, carry out gradient dilution (wherein, described gradient dilution factor is 5 * 10 -2 , 2.5×10 -2 , 2×10 -2 , 1.25×10 -2 、10 -2 , 5×10 ...

Embodiment 3

[0103] Embodiment 3 (sample to be tested is a certain water supply plant water sample)

[0104] A fluorescent detection method for the total number of living bacteria, comprising the following steps:

[0105] Step S01, take 50 mL of water sample, and filter it through a 0.22 μm mixed cellulose filter membrane sterilized by high-pressure steam at 121°C with a vacuum filtration device. Sterilized tweezers grip the edge of the filter membrane to remove the filter membrane, add 50mL of sterile water, and vortex shaker for 30min, so that the bacteria enriched on the filter membrane can be eluted from the filter membrane to obtain the sample solution to be tested ;

[0106] Step S02, establishing the linear relationship equation between the relative fluorescence intensity value and the number of viable bacteria as follows:

[0107] Mix the sample solution to be tested with the fluorescent dye, react at 37°C for 20min, and carry out gradient dilution to the stained reaction solutio...

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Abstract

The invention belongs to the technical field of compound materials, and particularly relates to a fluorescence detection method of a total number of living bacteria. The fluorescence detection methodof the total number of the living bacteria comprises the following steps: conducting a mixing reaction on a to-be-detected sample, with an unknown number of the living bacteria, and fluorescent dye toobtain to-be-detected reaction liquid subjected to fluorescence staining; and detecting relative fluorescence intensity of the to-be-detected reaction liquid to obtain the number of the living bacteria in the to-be-detected reaction liquid. By means of the fluorescence detection method of the total number of the living bacteria, the number of the living bacteria which actually exist in an environment can be reflected, and the fluorescence detection method of the total number of the living bacteria is high in detection speed, convenient, simple, easy to operate, high in sensibility, accurate and good in dynamic range, can detect multiple kinds of microorganisms, and can achieve field monitoring.

Description

technical field [0001] The invention belongs to the technical field of detecting the total number of live bacteria, and in particular relates to a fluorescence detection method for the total number of live bacteria. Background technique [0002] The total number of bacteria is recognized as a hygienic bacteriological indicator of drinking water and food at home and abroad. Its measurement technology is widely used in food hygiene and safety, disease prevention and environmental monitoring. Detection of the total number of bacteria, and detection of pathogenic bacteria in the air in public places such as hospitals and stations. Such detection of the total number of bacteria can help improve the quality of human living environment and ensure the health of the population. In addition, the detection of the total number of bacteria can also be applied to the cultivation of microorganisms, such as reflecting the growth of microorganisms in the fermentation process of microorganis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06G01N21/64
CPCC12Q1/06G01N21/6428
Inventor 聂麦茜第五振军王磊张宇婷尹秋月聂红云
Owner 西安润嬴生物科技有限公司
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