Method for determining total number of viable bacteria based on WST-8 chromogenic reaction

A WST-8, color reaction technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, color/spectral characteristic measurement, etc., can solve problems such as low sensitivity, difficulty in color reaction, lack of applied research, etc. , to achieve high sensitivity, wide range of bacterial detection, and improved sensitivity

Pending Publication Date: 2021-11-19
WUHAN TEXTILE UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, this method has not been used for bacterial detection, which may be due to the fact that dehydrogenase is mainly distributed on the inner mitochondrial membrane, while bacteria do not have a mitochondrial structure, and the content of dehydrogenase in bacteria is less than that in cells; on the other hand The existence of the bacterial wall makes it difficult for the chromogenic reagent WST-8 and the electron carrier to enter the bacterium, the chromogenic reaction is difficult to occur, and the sensitivity is low, so there is a lack of research on its application in bacteria

Method used

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  • Method for determining total number of viable bacteria based on WST-8 chromogenic reaction
  • Method for determining total number of viable bacteria based on WST-8 chromogenic reaction
  • Method for determining total number of viable bacteria based on WST-8 chromogenic reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0051] A method for measuring the total number of live bacteria based on WST-8 color reaction, comprising the following steps:

[0052] (1) Make a standard curve

[0053] (11) Resuspend the Escherichia coli bacterial liquid with PBS buffer (pH=7.4) and carry out gradient dilution to prepare standard sample suspensions with different concentration gradients (for example, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL);

[0054] (12) Prepare a chromogenic substrate solution comprising WST-8, electron carriers and nutrients, the concentration of WST-8 is 0.05mM, the concentration of electron carriers is 2μM, and the concentration of nutrients sodium chloride is 1mM;

[0055] Mix the standard sample suspensions with different concentration gradients with the chromogenic substrate solution respectively, and react for 2 hours to obtain the standard reaction solution to be tested;

[0056] (13) measure the absorbance value at 450nm of the standard...

Embodiment 2

[0065] A method for measuring the total number of live bacteria based on WST-8 color reaction, comprising the following steps:

[0066] (1) Make a standard curve

[0067] (11) Resuspend the Staphylococcus aureus bacterial liquid with PBS buffer (pH=7.4) and carry out gradient dilution to prepare standard sample suspensions with different concentration gradients (for example, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL);

[0068] (12) Prepare a chromogenic substrate solution comprising WST-8, electron carriers and nutrients, the concentration of WST-8 is 0.05mM, the concentration of electron carriers is 2μM, and the concentration of nutrient peptone is 0.2mM;

[0069] Mix the standard sample suspensions with different concentration gradients with the chromogenic substrate solution respectively, and react for 2 hours to obtain the standard reaction solution to be tested;

[0070] (13) measure the absorbance value at 450nm of the standard r...

Embodiment 3

[0078] A method for determining the total number of viable bacteria based on the WST-8 color reaction, compared with Example 1, the difference is that the reaction time between Escherichia coli and the color substrate is 6h. Others are substantially the same as in Embodiment 1, and will not be repeated here.

[0079] The standard curve is Y=0.11718log 10 X-0.34481 (R 2 =0.87324), the linear concentration range of detectable bacteria is 10 3 -10 7 CFU / mL.

[0080] Adopt step (2) similar method to test unknown escherichia coli concentration, obtain escherichia coli concentration and be 8.9 * 10 7 CFU / mL; the concentration of Escherichia coli measured by plate counting method was 6.79×10 7 CFU / mL, the relative standard deviation of the bacterial concentration obtained by the two test methods is 31.08%. It can be seen that when the reaction time is too long, the bacterial growth enters the logarithmic phase and increases rapidly, which makes the linear relationship poor. The...

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Abstract

The invention provides a method for determining the total number of viable bacteria based on a WST-8 chromogenic reaction. The method comprises the following steps: mixing a resuspended to-be-detected bacterial suspension with a chromogenic substrate solution to react for 0.5-4 hours to obtain a to-be-detected reaction solution; and determining the absorbance value of the reaction liquid to obtain the total number of viable bacteria. The chromogenic substrate solution is prepared from 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfo phenyl)-2H-tetrazolium salt, an electron carrier, namely, 1-methoxy-5-methylphenazine methyl sulfate, and a bacterial growth nutrient substance. According to the method, the chromogenic reaction of WST-8 is used for detecting the viable bacteria for the first time, and the content of dehydrogenase in bacterial cells can be effectively increased due to existence of bacterial growth nutrient substances in the chromogenic substrate solution, so that the sensitivity of bacterial detection is improved. The method has the characteristics of convenience in operation, rapidness, high sensitivity and wide bacterial detection range.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for measuring the total number of living bacteria based on WST-8 color reaction. Background technique [0002] Bacteria are ubiquitous in the environment, and humans are exposed to a variety of bacteria every day, most of which are harmless to humans, and many even form beneficial relationships with their hosts. However, pathogenic bacteria can cause human infection, the number of bacteria required for infection is usually small, and the resistance of these bacteria is also increasing, posing a serious threat to public health. Therefore, the total number of bacteria is a recognized indicator of international drinking water, food and other hygienic bacteriology. In order to ensure the life and health of human beings and improve the living environment of human beings, it is very necessary to measure the total number of bacteria in water quality an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N21/31C12Q1/06
CPCG01N21/78G01N21/31C12Q1/06
Inventor 钟亚平王栋鲁振坦杨显红
Owner WUHAN TEXTILE UNIV
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