CN113218928A - Colorimetric method for rapidly determining antibacterial activity based on fluorescent probe - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The invention discloses a colorimetric method for rapidly determining bacteriostatic activity based on a fluorescent probe, which is used for determining the metabolic activity change of bacteria before and after bacteriostatic substance treatment by using a fluorescent probe 5-cyano-2,3-ditolyl tetrazole chloride (CTC) to evaluate the bacteriostatic activity of a test object. The test method can analyze the activity of the antibacterial substance in a very short time (2 h), has high sensitivity, is suitable for quickly measuring the antibacterial activity of antibacterial natural products or chemical synthesis antibacterial substances, and has innovation and application value.
Description
Technical Field
The invention belongs to the field of antibiosis, and relates to a colorimetric method for rapidly determining bacteriostatic activity based on a fluorescent probe.
Background
Microorganisms are widely distributed in nature, play an important role in different ecosystems, are important and key participants of the circulation of substances such as nitrogen, carbon and the like, and are closely related to human life. Microorganisms are in various postures, and most of microorganisms bring great benefits to people, such as lactic acid bacteria, yeast and edible fungi used for food manufacturing, streptomycete and filamentous fungi used for antibiotic production, various anaerobic microorganisms used for environmental management, and the like. But part of the microorganisms bring great harm to the health of human beings, such as food-borne pathogenic bacteria, which cause diseases and even death of human beings, and spoilage microorganisms, which cause food spoilage and loss of edible value or cause food poisoning of eaters. In addition, some of the mold can cause the mildew of chemical materials, plastic products and textiles, and cause serious damage.
At present, the search for natural bacteriostatic substances from plants, animals and microorganisms is an important research content. And the antibacterial substance can be prepared by chemical modification and synthesis based on the chemical structure of the natural antibacterial component. These antimicrobial components and compounds are important sources of antiseptics and antibiotics. The determination of the antibacterial activity is an important prerequisite for evaluating the antibacterial action of natural products and chemical compounds. At present, methods such as a punching method, an oxford cup method, a paper method, a minimum inhibitory concentration method, a minimum bactericidal concentration method and the like are generally adopted to determine and evaluate the bacteriostatic effect and the bacteriostatic spectrum of a test object, but the methods have the defects of long period, complicated steps, low sensitivity and the like. Therefore, the development of a rapid and high-sensitivity antibacterial activity analysis method is of great significance, and the development of antibacterial research is facilitated.
Disclosure of Invention
The invention aims to provide a colorimetric method for rapidly determining bacteriostatic activity based on a fluorescent probe, which is suitable for analyzing the inhibition and bactericidal activity of water-soluble bacteriostatic substances on bacteria, takes 5-cyano-2,3-ditolyl tetrazolium chloride (5-cyano-2, 3-ditolyl tetrazolium chloride, CTC) as the fluorescent probe, is mainly applied to the identification of bacterial death and activity, is a colorless water-soluble azolium salt, can be reduced into a water-insoluble red fluorescent substance accumulated in cells by dehydrogenase in an electron respiration transmission chain on a bacterial cell membrane (see figure 1), and the strong red color indicates high bacterial activity.
In order to achieve the purpose, the invention adopts the following technical scheme:
a colorimetric method for rapidly determining bacteriostatic activity based on a fluorescent probe mainly comprises the following steps (see figure 2):
(1) 5-cyano-2,3-ditolyl tetrazolium chloride CTC is used as a fluorescent probe, sterile physiological saline or PBS buffer solution is used for preparing 50 mM fluorescent staining solution, and the fluorescent staining solution is preserved at 4 ℃ for later use;
(2) preparing bacterial suspension (such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus, etc.) with bacteria concentration of 1 × 10 with sterile normal saline7~1×108 CFU/mL, used after 1 h of preparation;
(3) sucking a proper amount of bacterial suspension, adding a bacteriostatic substance to be tested, wherein the final concentration is 0.05-0.1 mg/mL, acting at normal temperature for 15-30 min, and then adding sterile normal saline with 10 times of the original volume to dilute, so as to stop the action of the bacteriostatic substance on thallus cells;
(4) sucking 1 mL of diluted sample, adding CTC fluorescent staining solution to enable the final concentration to be 5 mM, and staining for 30 min at 37 ℃;
(5) centrifuging the dyed sample, removing the supernatant, adding 2 mL of dimethyl sulfoxide to extract a CTC reduction product in the bacterial cells, and stirring the product when the product is not used;
(6) centrifuging after extraction is finished, taking dimethyl sulfoxide as a blank, and measuring the absorbance value of the supernatant at 470 nm;
(7) and (3) taking the non-bacteriostatic treatment of the bacterial suspension as a reference, and evaluating the bacteriostatic activity of the bacteriostatic substance according to the change of the absorbance value, wherein the smaller the absorbance, the stronger the bacteriostatic activity of the bacteriostatic substance is, and the larger the absorbance value, the weaker the bacteriostatic activity or no bacteriostatic activity of the bacteriostatic substance is.
The invention has the beneficial effects that: 1) the test of the bacteriostatic activity can be completed in a short time (about 2 hours), while the traditional method needs at least more than 12 hours, so that the test time is obviously shortened; 2) The accuracy is obviously improved, and the tiny difference of the bacteriostatic activity among different bacteriostatic components can be measured; 3) the accuracy of the analysis result is high.
Drawings
FIG. 1 is a diagram of the mechanism of bacterial staining;
FIG. 2 is a schematic flow chart of the experimental operation steps of the colorimetric method of the present invention;
FIG. 3 is a graph comparing whether ɛ -polylysine solution was used for treatment in example 1.
Detailed Description
The following examples further illustrate the method of use of the invention, but are intended to be illustrative only and not to limit the scope of the invention in any way. Furthermore, modifications and substitutions in the details and form of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but the modifications and substitutions are within the scope of the present invention.
Example 1 ɛ bacteriostatic Activity of polylysine against Bacillus subtilis
1. Inoculating activated Bacillus subtilis to a medium containing 30 mL of nutrient broth, culturing at 37 deg.C and 150 rpm/min for 18 h, and culturing at a concentration of about 5 × 108 CFU/mL；
2. Centrifuging the bacterial liquid at 8000 rpm for 10 min, discarding supernatant, resuspending with sterile normal saline, and adjusting bacterial concentration to 1 × 108 CFU/mL；
3. Sucking 1 mL of bacterial suspension into a sterile centrifuge tube, adding a sterile ɛ -polylysine solution to a final concentration of 0.1 mg/mL, treating for 30 min, and adding 10 mL of sterile normal saline to dilute so as to stop or weaken the effect of ɛ -polylysine on the somatic cells; a bacterial suspension without ɛ -polylysine solution was used as a control (ɛ -polylysine solution was replaced with sterile physiological saline), and the other procedures were the same;
4. sucking 1 mL of diluted sample, adding CTC fluorescent staining solution (BS-02 CTC rapid staining kit from Japan Co-ordinated chemical) with final concentration of 5 mM, staining at 37 deg.C for 30 min, and the staining effect of different treatment groups is shown in figure 3;
5. centrifuging the dyed sample at 8000 rpm for 10 min, removing the supernatant, adding 2 mL of dimethyl sulfoxide into the precipitated bacterial cells to extract a CTC reduction product in the bacterial cells, stirring at intervals during the extraction process, and extracting for 30 min;
6. centrifuging at 8000 rpm for 10 min after extraction, collecting supernatant, and determining absorbance at 470 nm with dimethyl sulfoxide as blank;
7. the result shows that the absorbance value is 0.21, the absorbance of the control group is 0.57, and the bacterial activity is reduced by 63% after the bacterial cells are treated by ɛ -polylysine, thereby showing that the ɛ -polylysine has strong bacteriostatic activity on the bacillus subtilis.
Comparative example 1 the conventional method for testing the bacteriostatic activity of ɛ -polylysine on bacillus subtilis is as follows:
1. inoculating activated Bacillus subtilis to a medium containing 30 mL of nutrient broth, culturing at 37 deg.C and 150 rpm/min for 18 h, and culturing at a concentration of about 5 × 108 CFU/mL；
2. Centrifuging the bacterial liquid at 8000 rpm for 10 min, discarding supernatant, resuspending with sterile normal saline, and adjusting bacterial concentration to 1 × 108 CFU/mL；
3. Sucking 2 mL of bacterial suspension into a sterile centrifuge tube, adding a sterile ɛ -polylysine solution to a final concentration of 0.1 mg/mL, treating for 30 min, and adding 20 mL of sterile normal saline to dilute so as to stop or weaken the effect of ɛ -polylysine on the somatic cells; a bacterial suspension without ɛ -polylysine solution was used as a control (ɛ -polylysine solution was replaced with sterile physiological saline), and the other procedures were the same;
4. 0.1 mL of the diluted sample was spread on nutrient agar plates, incubated at 37 ℃ for 24 h, and counted. The nutrient agar formula comprises: 10g of peptone, 5g of yeast extract, 5g of sodium chloride, 1g of glucose, 15 g of agar and 1000mL of water.
5. The results showed that the amount of Bacillus subtilis was 9.5 log CFU/mL after treatment with ɛ -polylysine solution and the amount of Bacillus subtilis was 3.7 log CFU/mL for the control, indicating that the bacterial activity decreased by 61% after treatment of the cells with ɛ -polylysine solution. The detection time of the detection method of comparative example 1 was 18 to 24 hours; therefore, the analysis result of the invention can analyze the activity of the bacteriostatic substance within 2 hours, and is more accurate and rapid.
Example 2 bacteriostatic Activity of protamine on E.coli
1. Inoculating activated Escherichia coli into 100 mL triangular flask containing 30 mL LB medium, culturing at 37 deg.C and 220 rpm/min for 12 hr to obtain thallus cell concentration of about 1 × 109 CFU/mL；
2. Centrifuging the cultured bacterial liquid at 8000 rpm for 10 min, discarding supernatant, resuspending with sterile physiological saline, and adjusting cell concentration to 1 × 108 CFU/mL；
3. Sucking 1 mL of bacterial suspension into a sterile centrifuge tube, adding a protamine solution subjected to filtration sterilization to a final concentration of 0.05 mg/mL, treating for 15 min, and adding 10 mL of sterile normal saline for dilution to stop or weaken the effect of the protamine solution on bacterial cells; the bacterial suspension without the protamine solution is used as a control (the protamine solution is replaced by sterile normal saline), and other operation methods are the same;
4. taking 1 mL of diluted sample, adding CTC fluorescent staining solution (BS-02 CTC rapid staining kit from Japan Dojinghu chemical) with a final concentration of 5 mM, and staining at 37 deg.C for 30 min;
5. centrifuging the dyed sample at 8000 rpm for 10 min, removing the supernatant, adding 2 mL of dimethyl sulfoxide into the precipitated thalli cells to extract CTC reduction products accumulated in the thalli cells, stirring at intervals in the extraction process, and extracting for 30 min;
6. centrifuging the extracted sample at 8000 rpm for 10 min, taking the supernatant, and determining the absorbance value at 470 nm by using dimethyl sulfoxide as a blank;
7. the results show that the absorbance value of the protamine treated sample is 0.34, the absorbance of the control group is 0.64, and the bacterial activity of the bacterial cells is reduced by 47% after the protamine treatment, thereby showing that the protamine has stronger inhibition effect on the escherichia coli.
Comparative example 2 the conventional method for testing the bacteriostatic activity of protamine on escherichia coli is as follows:
1. inoculating activated Escherichia coli into 100 mL triangular flask containing 30 mL LB medium, culturing at 37 deg.C and 220 rpm/minCulturing for 12 hr to obtain thallus cell concentration of about 1 × 109 CFU/mL；
2. Centrifuging the cultured bacterial liquid at 8000 rpm for 10 min, discarding supernatant, resuspending with sterile physiological saline, and adjusting cell concentration to 1 × 108 CFU/mL；
3. Sucking 2 mL of bacterial suspension into a sterile centrifuge tube, adding the filtered and sterilized protamine solution with the final concentration of 0.05 mg/mL, processing for 15 min, and adding 20 mL of sterile normal saline for dilution to stop or weaken the effect of the protamine solution on the bacterial cells; the same procedure was followed using a bacterial suspension without protamine as a control (replacing the protamine solution with sterile physiological saline).
4. 0.1 mL of the diluted sample was spread on nutrient agar plates, incubated at 37 ℃ for 24 h, and counted. The nutrient agar formula comprises: 10g of peptone, 5g of yeast extract, 5g of sodium chloride, 1g of glucose, 15 g of agar and 1000mL of water.
5. The results show that the number of the Escherichia coli treated by the protamine solution is 9.7 log CFU/mL, the number of the Escherichia coli of the control is 5.2 log CFU/mL, and the bacterial activity of the bacterial cells is reduced by 46 percent after the protamine solution is treated. The detection time of the detection method of comparative example 1 was 18 to 24 hours; therefore, the analysis result of the invention can analyze the activity of the bacteriostatic substance within 2 hours, and is more accurate and rapid.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (4)
1. A colorimetric method for rapidly determining bacteriostatic activity based on a fluorescent probe is characterized by comprising the following steps:
(1) 5-cyano-2,3-ditolyl tetrazolium chloride CTC is used as a fluorescent probe, sterile physiological saline or PBS buffer solution is used for preparing 50 mM fluorescent staining solution, and the fluorescent staining solution is preserved at 4 ℃ for later use;
(2) preparing bacterial suspension with sterile physiological saline to make the cell concentration of bacterial cell 1 × 107~1×108 CFU/mL, used after 1 h of preparation;
(3) sucking a proper amount of bacterial suspension, adding a bacteriostatic substance to be tested, acting at normal temperature for 15-30 min, adding sterile normal saline with the volume 10 times of the original volume to dilute, and stopping the action of the bacteriostatic substance on thallus cells;
(4) sucking 1 mL of diluted sample, adding CTC fluorescent staining solution to enable the final concentration to be 5 mM, and staining for 30 min at 37 ℃;
(5) centrifuging the dyed sample, removing the supernatant, adding 2 mL of dimethyl sulfoxide to extract a CTC reduction product in the bacterial cells, and stirring the product when the product is not used;
(6) centrifuging after extraction is finished, taking dimethyl sulfoxide as a blank, and measuring the absorbance value of the supernatant at 470 nm;
(7) and (3) taking the non-bacteriostatic treatment of the bacterial suspension as a reference, and evaluating the bacteriostatic activity of the bacteriostatic substance according to the change of the absorbance value, wherein the smaller the absorbance, the stronger the bacteriostatic activity of the bacteriostatic substance is, and the larger the absorbance value, the weaker the bacteriostatic activity or no bacteriostatic activity of the bacteriostatic substance is.
2. The colorimetric method for rapid determination of bacteriostatic activity based on a fluorescent probe as claimed in claim 1, wherein the bacterial suspension in the step (2) comprises one of escherichia coli, bacillus subtilis and staphylococcus aureus.
3. The colorimetric method for rapid determination of bacteriostatic activity based on a fluorescent probe as claimed in claim 1, wherein the bacteriostatic substance is water-soluble.
4. The colorimetric method for rapid determination of bacteriostatic activity based on a fluorescent probe as claimed in claim 1, wherein the final concentration of the bacteriostatic substance added in step (3) is 0.05-0.1 mg/mL.
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