Method for quickly measuring total number of live bacteria of luminous bacteria by using MTT method

A luminescent bacteria, rapid determination technology, applied in the determination/inspection of microorganisms, chemiluminescence/bioluminescence, biochemical equipment and methods, etc. The detection method is simple, rapid and reproducible

Inactive Publication Date: 2010-01-06
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no relevant report on the application of MTT method

Method used

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  • Method for quickly measuring total number of live bacteria of luminous bacteria by using MTT method
  • Method for quickly measuring total number of live bacteria of luminous bacteria by using MTT method
  • Method for quickly measuring total number of live bacteria of luminous bacteria by using MTT method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Add 0.3mL MTT solution to 3mL of properly diluted fermentation broth, mix well, place at 25°C for 2h, centrifuge at 10,000r / min for 10min, discard the supernatant, add 3mL DMSO to fully dissolve the precipitate, and use UV-Vis spectroscopy The photometer scans the maximum absorption wavelength, and measures the light absorption value in the range of 400nm-800nm. Depend on figure 1 It can be seen that its maximum absorption peak is at 500nm.

Embodiment 2

[0015] Using DMSO as a reference, sterile saline, sterile blank medium, fermentation supernatant (after centrifugation at 10,000r / min, absorb the supernatant) and dead bacteria (boiling water bath for 30min) as samples to be tested, use MTT The light absorption values ​​measured by the method are listed in Table 1. It can be seen from the table that these substances have little interference with the measurement results. It can be seen that the MTT method is related to the amount of viable bacteria, but has nothing to do with the properties of the solids and metabolites contained in the medium, which largely solves the problem of using the turbidity method due to the solids contained in the medium. Serious interference with the measurement of live bacteria by the dry weight method of the bacteria.

[0016] Table 1 Absorbance value when different substances are blank controls

[0017]

Embodiment 3

[0019] Add 0.3mL of MTT solution to 3mL of properly diluted fermentation broth, mix well, place at 25°C for 2h, centrifuge at 10,000r / min for 10min, discard the supernatant, add 3mL of DMSO to fully dissolve the precipitate, and place at 500nm Measure the absorbance. At the same time, the centrifuged bacteria were redissolved with physiological saline, and after gradient dilution, they were spread on a plate and counted colonies.

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Abstract

The invention aims at establishing a method for quickly measuring total number of live bacteria of luminous bacteria, in particular to a method for counting the live bacteria based on the MTT coloration principle. The invention adopts MTT colorimetric method to measure the viable count of the luminous bacteria and carries out optimizing selection on the detection condition of the MTT colorimetric method. Therefore, the optimal method for measuring total number of the live bacteria of the luminous bacteria is determined. In the invention, first, the product of formazan generated from the reaction of succinate dehydrogenase in MTT and live thalli is dissolved in DMSO, light absorption value is determined within the range of 400nm-800nm, and the largest detected absorption wavelength is determined to be 500nm. By optimizing time and influencing the light absorption value by temperature, the optimal measurement condition is detected: the reaction time is 2h, and the reaction temperature is 25 DEG C. The invention has novel detection principle, simple and fast detection method, good repeatability, can be used for the quantitative detection of the number of the live bacteria of the luminous bacteria in the fields of food hygiene and safety, environmental monitoring and the like. The invention can also be applied to the tests of microbiology, immunology and other disciplines and used for measurement of the activity and number of the live bacteria materials. The invention can also develop the method for measuring the total number of the bacteria which is used for replacing the traditional method for measuring the total number of the live bacteria.

Description

technical field [0001] The invention relates to a method for rapidly measuring the total number of living bacteria of luminescent bacteria, in particular to a method for counting living bacteria based on the principle of MTT color development. Background technique [0002] As far as the inventor knows by consulting data and document retrieval: [0003] At present, in the experiments of microbiology, immunology and other disciplines, a certain concentration of living bacteria is often used as experimental materials, which involves a problem of counting live bacteria. Several bacterial counting methods commonly used in microbiology are turbidimetric method, plate counting method and so on. The turbidimetric method is simple to operate, but it can only measure the number of bacteria, and cannot judge the activity of bacteria; although the plate count method measures the number of viable bacteria, the method is cumbersome, the repeatability is poor, and the time required is lon...

Claims

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Application Information

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IPC IPC(8): G01N21/76C12Q1/02
Inventor 王静雪林洪王亚群
Owner OCEAN UNIV OF CHINA
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