Turbid traditional Chinese medicine antibacterial MIC detection method based on TTC indicator and detection kit thereof
By combining TTC indicator with a focusing microplate, the problem of turbidity in the detection of antibacterial MIC of traditional Chinese medicine was solved, achieving accurate MIC value positioning and stable detection results.
Patent Information
- Application Number
- CN202610235212.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-15
AI Technical Summary
In the detection of antibacterial MIC in traditional Chinese medicine, it is difficult to distinguish between turbid medicine solutions and bacterial growth turbidity. Existing technologies cannot achieve simple, accurate and standardized detection.
Using TTC indicator combined with a focusing microplate, bacteria are precipitated and focused at the bottom of the well by gravity. The TTC colorimetric signal is combined with the precipitation focusing effect of the well plate to distinguish between the turbidity of the Chinese medicine solution itself and the turbidity caused by bacterial growth.
The colorimetric identification is significantly improved, the MIC value is accurately located, the colorimetric error is reduced, and the results are highly stable, meeting the detection requirements of traditional Chinese medicine antibacterial experiments.
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Figure CN122038524A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial detection technology, specifically relating to a method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator and its detection kit. Background Technology
[0002] The microbroth dilution method is a classic method for determining the MIC (Minimum Inhibitory Concentration) value of antimicrobial drugs. Its core principle is to determine the bacterial growth status by observing the clarity or turbidity of the wells in a 96-well plate, thereby determining the minimum concentration of the drug that inhibits bacterial growth. Due to its simplicity and low cost, this method is widely used in the detection of antimicrobial activity and drug susceptibility testing of Western medicines.
[0003] However, in antibacterial experiments using traditional Chinese medicine (TCM), many active ingredients or their extracts exhibit significant turbidity, especially at medium to high concentrations (typically ≥4 mg / mL). The turbidity state and the turbidity in the experimental wells caused by bacterial growth and reproduction are difficult to discern visually, making it impossible to accurately determine the critical concentration for bacterial inhibition and severely impacting the reliability and accuracy of MIC (micron emission index) results. Currently, there are few solutions to address the interference of turbidity in TCM, primarily relying on centrifugation to reduce the turbidity of the supernatant and quantitative detection using turbidimeters. However, centrifugation not only increases experimental steps but may also introduce systematic errors due to loss of active ingredients with precipitation or uneven distribution. While turbidimetry can quantify turbidity, it cannot substantially distinguish whether the turbidity originates from the herbal solution itself or bacterial growth, and requires additional specialized equipment, making it difficult to meet the simple, convenient, accurate, and standardized detection requirements in TCM antibacterial experiments.
[0004] 2,3,5-Triphenyltetrazolium chloride (TTC), a commonly used indicator of microbial metabolism, can be reduced to red triphenylformazan (TPF) by dehydrogenases of live bacteria. This causes the system with bacterial growth to appear red, while the system without bacterial growth retains its original color. This reagent is inexpensive and readily available, making it suitable for widespread application in grassroots laboratories. It is worth noting that current technologies generally use flat-bottomed plates for detection, resulting in dispersed bacterial growth signals. Even when combined with TTC color development, the red signal easily blends with the turbid background of traditional Chinese medicine, leading to insufficient identification. Summary of the Invention
[0005] To address the technical shortcomings of existing methods for detecting antibacterial MIC in traditional Chinese medicine (TCM), which make it difficult to distinguish between turbid medicinal solutions and bacterial growth turbidity, this invention aims to provide a TTC-based method for detecting antibacterial MIC in turbid TCM. This method effectively solves the technical problems of distorted TCM MIC detection results and the inability to achieve simple, accurate, and standardized detection.
[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator, comprising the following steps: S1. Screening for the optimal working concentration of TTC culture medium and suitable well plate that has no antibacterial effect on the target bacteria and can make the bacterial sedimentation and color development at the bottom of the well clear. S2. Select the appropriate well plate determined in step S1, and set up a blank control, a drug-TTC reaction control and a positive control. All control groups and experimental groups use the same well plate type and system configuration. Add serially diluted turbid Chinese medicine solution, bacterial solution of the test strain and TTC optimal working concentration culture medium determined in step S1 to each well, and control the final volume of each well to construct the experimental system and control system. S3. Place the experimental system and control system constructed in step S2 in a constant temperature environment for incubation; S4. Based on the precipitation focusing effect of the well plate and combined with the expected results of each control well, the MIC value of the turbid Chinese medicine against the target bacteria was determined by observing the color of the bottom of the experimental well and the precipitation state.
[0007] Preferably, the optimal working concentration of TTC is determined through a dual screening process: First, the non-specific reduction interference of the traditional Chinese medicine solution to TTC is eliminated by pre-validation of the drug-TTC reaction for the target bacteria. Then, the optimal working concentration of TTC is determined by a dual method of "verifying antibacterial safety by colony counting on broth agar plates" and "observing the clarity of color development on round / pointed bottom 96-well plates". The optimal working concentration of TTC that "has no antibacterial effect on the target bacteria (no statistical difference in colony count compared with the blank control, P>0.05), clear and distinguishable color development, and does not react non-specifically with the traditional Chinese medicine solution" is obtained. More preferably, in step S1, TTC is mixed with culture medium to prepare a series of TTC-culture medium systems, and a well plate with a better focusing effect than a flat plate is selected; the antibacterial safety of the TTC concentration is verified by the coating method, and the system is incubated in a constant temperature environment to screen out the optimal working concentration of TTC that has no antibacterial effect on the target bacteria and can make the bacterial precipitate at the bottom of the well clearly visible to the naked eye.
[0008] More preferably, in step S1, the TTC concentration in the TTC-culture medium system is 2.5~80 μg / mL, prepared using a 2-fold serial dilution method; the culture medium is MH broth or a target bacteria-specific culture medium; the appropriate well plate is a sterile round-bottom 96-well plate or a sterile pointed-bottom 96-well plate (using gravity to cause bacterial precipitation and focusing at the bottom of the well), and 100 μL of culture medium containing the optimal working concentration of TTC, 50 μL of serially diluted turbid traditional Chinese medicine solution, and a final concentration of 5×10⁻⁶ are added to each well sequentially. 5 A target bacterial culture of CFU / mL was prepared in a volume of 50 μL, and an experimental system with a final volume of 200 μL was constructed.
[0009] Preferably, the optimal working concentration of TTC should simultaneously meet the following conditions: 1) there is no statistically significant difference in the number of colonies on the culture medium compared to the blank plate without TTC; 2) the red signal at the bottom of the well in the adapter plate is clearly visible to the naked eye; 3) there is no non-specific red color development in the drug-TTC reaction control well.
[0010] Preferably, in steps S1 and S3, the temperature for cultivation in the constant temperature environment is 35±1℃, and the cultivation time is 16~24h.
[0011] Preferably, in step S2, the control group is configured as follows: Blank control: Culture medium containing the optimal working concentration of TTC, without drugs or bacterial suspension; Drug-TTC reaction control: Culture medium containing traditional Chinese medicine solutions of various concentrations and the optimal working concentration of TTC, without bacterial culture; Positive control: Culture medium containing the optimal working concentration of TTC and bacterial suspension of the test strain, without drugs.
[0012] Preferably, in step 2, the final concentration range of the turbid Chinese medicine solution is 0.5~64 mg / mL, which can be flexibly adjusted according to the preliminary experimental MIC value to ensure coverage of the expected antibacterial concentration range.
[0013] Preferably, the detection system can incorporate antibacterial Western medicines, and is suitable for determining the antibacterial effect in multi-drug susceptibility tests involving traditional Chinese medicine-Western medicine and traditional Chinese medicine-traditional Chinese medicine combinations.
[0014] Preferably, in step S4, the expected results for the control wells are: no specific red precipitate at the bottom of the wells for the blank control and the drug-TTC reaction control, only the original turbid color of the drug solution or a small amount of precipitate produced by the drug itself; and a clear specific red precipitate at the bottom of the wells for the positive control.
[0015] Preferably, in step S4, the criteria for judging the experimental wells are as follows: the appearance of red precipitate at the bottom of the well indicates that the concentration of the drug solution in the well has not reached the antibacterial threshold and has no obvious antibacterial effect; as the concentration gradient of the traditional Chinese medicine increases, the first occurrence of no obvious red color at the bottom of the well and only maintaining the original turbid color of the drug solution is taken as the antibacterial critical point, and the lowest concentration of traditional Chinese medicine corresponding to this is the MIC value of the turbid traditional Chinese medicine against the target bacteria.
[0016] This invention also discloses a detection kit for implementing the above-described method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator, comprising: Focused microplates, TTC reagent, and culture medium; The focusing microplate is used to concentrate bacteria at the bottom of the well; the TTC reagent, when mixed with the culture medium, can be configured into a TTC-culture medium system for screening the optimal working concentration; the optimal working concentration is the concentration that has no inhibitory effect on the target bacteria, provides clear and distinguishable color development, and does not react non-specifically with the turbid Chinese medicine solution.
[0017] Preferably, the focusing microplate is a round-bottomed 96-well plate or a pointed-bottomed 96-well plate.
[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator. It innovatively combines the TTC colorimetric signal with the precipitation focusing effect of a well plate. The well plate can utilize gravity to cause bacteria to concentrate and precipitate at the bottom of the well, forming an aggregation area. This allows the red signal generated by TTC reduction to be focused locally, significantly improving the colorimetric recognition against a turbid background (the red precipitate forms a strong color contrast with the upper turbid medicine solution, and the accuracy of visual identification is significantly improved compared to a flat-bottom plate). This method thoroughly distinguishes between the turbidity of the traditional Chinese medicine solution itself and the turbidity caused by bacterial growth, achieving precise positioning of the MIC value and solving the core technical problem that traditional methods are difficult to determine.
[0019] Furthermore, well plates with better focusing effect than flat-bottomed plates were selected, namely, sterile round-bottomed 96-well plates or sterile pointed-bottomed 96-well plates. The focusing precipitation effect fully reduced the color development error, and the MIC determination consistency rate of three independent parallel experiments reached 100%, with high result stability.
[0020] Furthermore, the optimal working concentration of TTC has been validated for strain compatibility to ensure that it does not inhibit bacterial growth and avoids introducing additional interference due to the indicator. Attached Figure Description
[0021] Figure 1 This is a flowchart of the TTC indicator-based method for detecting the antibacterial MIC of turbid traditional Chinese medicine according to the present invention. Figure 2 The results of the screening for the optimal working concentration of TTC; Figure 3 The results are from the MIC detection experiment of acetofumaric acid. Detailed Implementation
[0022] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
[0023] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.
[0024] The present invention will now be described in further detail with reference to the accompanying drawings: See Figure 1 A method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator, comprising the following steps: Step 1: Screening for the optimal working concentration of TTC and the appropriate plate type for the target bacteria. Preparation of TTC-medium system: Mix TTC with MH broth or target bacteria-specific medium to prepare a 2-fold serial dilution system with a concentration of 2.5~80 μg / mL; Use sterile 96-well plates with round or pointed bottoms (bacterial sedimentation is better focused than flat-bottomed plates). Add 100 μL of the above TTC-medium system to each well, then add to a final concentration of 5 × 10⁻⁶. 5 100 μL of the test strain culture with a CFU / mL concentration was prepared, with a final volume of 200 μL per well. Three parallel wells and a blank control (containing only TTC-medium medium) were also set up. Parallel verification of the antibacterial safety of TTC concentrations: The test strains were inoculated in parallel on broth agar plates containing corresponding gradient concentrations of TTC using the spread method (the inoculation concentration was based on a plate count of 30-300 CFU / plate without TTC) to determine the highest TTC concentration that had no antibacterial effect on bacteria after culture. The optimal working concentration of TTC is determined by the highest TTC concentration that, after incubation at 35±1℃ for 16~24 h, exhibits no inhibitory effect on the target bacteria while allowing for clear and visually identifiable bacterial precipitation at the bottom of the well.
[0025] Step 2: Construction of the antibacterial experimental system of turbid traditional Chinese medicine solution and TTC indicator Select the round-bottom or pointed-bottom 96-well plate determined in the above steps, and add to each well sequentially: 50 μL of serially diluted turbid traditional Chinese medicine solution (final concentration covering 0.5~64 mg / mL, the concentration range can be flexibly adjusted according to the preliminary experimental MIC value), with a final concentration of 5×10⁻⁶. 5 50 μL of the bacterial culture of the test strain with CFU / mL, 100 μL of the TTC optimal working concentration culture medium determined in step 3.2.1, and a final volume of 200 μL per well; Three sets of control wells were set up to ensure the accuracy of the experimental system: Blank control: 200 μL of MH broth containing the optimal working concentration of TTC (without drugs and bacterial culture) was used to eliminate non-specific reduction interference of TTC by the culture medium itself. Drug-TTC reaction control: 100 μL of each grade concentration of traditional Chinese medicine solution + 100 μL of TTC optimal working concentration culture medium (without bacterial solution) to eliminate non-specific colorimetric interference between the drug and TTC; Positive control: 100 μL of MH broth containing the optimal working concentration of TTC + 100 μL of bacterial suspension of the test strain (without drug) was used to verify the colorimetric effect under normal bacterial growth conditions.
[0026] All control wells and experimental wells used the same plate type and system configuration.
[0027] Step 3: Experimental culture The experimental system and control system constructed above were placed in a constant temperature incubator at 35±1℃ and incubated for 16~24 h. The color and precipitation state at the bottom of the well were then observed.
[0028] Step 4: Result Judgment Criteria Based on the precipitation focusing effect of round-bottom / pointed-bottom orifice plates, the red signal contrasts sharply with the turbid upper layer of drug solution, and is determined by the following criteria: 1) The results of the control wells must meet expectations: there should be no specific red precipitate at the bottom of the blank control and drug-TTC reaction control wells, only the original turbidity of the drug solution or a small amount of precipitate produced by the drug itself (no bacterial growth, TTC has not been reduced); the bottom of the positive control wells should show a clear specific red precipitate (normal bacterial growth, TTC has been reduced). 2) Well determination: If a red precipitate appears at the bottom of the well, it indicates that the bacteria are growing normally and reducing TTC. The concentration of the drug solution in the well has not reached the inhibition threshold and has no obvious antibacterial effect. As the concentration of the Chinese medicine increases, the lowest concentration of the Chinese medicine that first appears without obvious red color at the bottom of the well and only maintains the original turbidity of the drug solution is the MIC value of the turbid Chinese medicine against the target bacteria.
[0029] The specific implementation methods and operational details of the present invention are described in detail below with reference to specific embodiments. These embodiments are only for explaining the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods in the field of microbiological experiments; reagents and materials without specified specifications are all conventional consumables and reagents that can be purchased through commercial channels.
[0030] 1. Experimental materials Strains: Staphylococcus aureus ATCC29213 (standard quality control strain); Drug: Acetic acid fumarate (purity ≥98%); Reagents: MH broth, TTC powder, sterile water, 0.22 μm filter membrane; Instruments: Sterile polystyrene 96-well plates with flat / round / pointed bottoms, 35±1℃ constant temperature incubator, ultra-clean workbench, adjustable pipettes (10 μL~1000 μL).
[0031] 2. Screening experiment for optimal working concentration of TTC (1) Preparation of TTC stock solution: Accurately weigh TTC powder, dissolve and dilute with sterile water to prepare a stock solution with a concentration of 800 μg / mL, and store at 4℃ protected from light for later use; (2) Concentration gradient setting: A round-bottom 96-well plate was selected. The TTC stock solution was serially diluted 2 times with MH broth so that the final concentrations of each well were 2.5, 5, 10, 20, 40 and 80 μg / mL, respectively. Three parallel wells were set for each concentration, and 100 μL of MH broth containing TTC was added to each well. (3) System construction: Add a final concentration of 5×10 to each of the above wells. 5 100 μL of ATCC29213 bacterial culture (CFU / mL) was added to each well, with a final volume of 200 μL. A blank control well was also set up (containing only 200 μL of TTC-MH broth at the corresponding concentration). (4) Safety verification of TTC concentration for antibacterial activity: Broth agar plates containing the above gradient concentrations of TTC were prepared, and ATCC29213 bacterial suspension was inoculated by the spread method (the inoculation concentration was based on the number of colonies of TTC-free plates being 30~300 CFU / plate), and 3 replicate plates were set for each concentration; (5) Incubation and observation: 96-well plates and agar plates were incubated in a constant temperature incubator at 35±1℃ for 16-20 h. The results showed that when the TTC concentration was ≤20 μg / mL, there was no statistically significant difference in the number of colonies on the agar plates compared with the blank plates without TTC (P>0.05), indicating that this concentration range had no inhibitory effect on ATCC29213. In the 96-well plates, red precipitates were observed at the bottom of the wells in the concentration range of 2.5-80 μg / mL, but the red signal was weak or absent and the identification was insufficient at concentrations of 2.5 μg / mL and below. 20 μg / mL, 40 μg / mL, and 80 μg / mL all showed obvious color development. Considering both the antibacterial safety and color development effect, 20 μg / mL was determined to be the optimal working concentration of TTC for ATCC29213.
[0032] See results Figure 2 , Figure 2 The TTC concentrations in the seven wells from left to right are 1.25, 2.5, 5, 10, 20, 40, and 80 μg / mL, respectively. The rightmost well (the eighth one) is a blank control well without TTC.
[0033] 3. MIC detection experiment of acetofumaric acid (1) Drug dilution: Prepare acetofumaric acid stock solution (concentration 80 mg / mL), sterilize it by aseptic filtration through a 0.22 μm filter membrane, and then perform a 2-fold serial dilution with MH broth to achieve a final concentration ranging from 0.5 to 64 mg / mL. (2) Experimental system construction: A round-bottom 96-well plate was used. 50 μL of TTC-MH broth (final concentration 20 μg / mL), 100 μL of acetic acid solution with gradient concentrations, and 50 μL of ATCC29213 bacterial suspension (final concentration 5×10⁻⁶) were added to each well sequentially. 5 (CFU / mL), gently tap the plate wall to mix, final volume of 200 μL per well; (3) Setting of reference holes: Blank control: 200 μL of MH broth containing 20 μg / mL TTC; Drug-TTC reaction control: 100 μL of gradient concentration acetofumaric acid solution + 100 μL of MH broth containing a final concentration of 20 μg / mL TTC; Positive control: 100 μL of MH broth containing TTC + 100 μL of ATCC29213 bacterial suspension (final concentrations: 20 μg / mL and 5×10⁻⁶, respectively). 5 (CFU / mL) (4) Culture and Result Observation: After incubation at 35±1℃ for 16~20 h, the results of the control wells were as expected: no specific red signal was observed at the bottom of the blank control and drug-TTC reaction control wells, and the positive control wells showed clear color. In the experimental group, when the concentration of acetofumaric acid was <4 mg / mL, a clear red precipitate was visible at the bottom of each well, and the upper layer maintained the turbid color of the drug solution. When the concentration reached 4 mg / mL, no red precipitate was generated at the bottom of the wells, and only the yellow turbid color of the traditional Chinese medicine solution was maintained. Furthermore, no red color was observed in each well with a concentration ≥4 mg / mL (as the concentration increased, the turbidity of the drug solution increased, but no red precipitate was observed at the bottom of the wells). Overall judgment: The MIC value of acetofumaric acid against ATCC29213 was 4 mg / mL.
[0034] See results Figure 3 , Figure 3 In the first row, wells 1-3 are blank controls, and wells 4-6 are positive controls. In the second row, the leftmost well is a positive growth well without TTC and drug solution, followed by drug-TTC reaction control wells with gradient concentrations. In the third row, from left to right, the drug solution concentrations are: positive growth wells without TTC and drug solution, and positive growth wells with TTC and drug solution, with drug solution concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 mg / mL, respectively.
[0035] The MIC detection experiment for acetofumaric acid was repeated using 96-well plates with flat bottoms, round bottoms, and pointed bottoms, respectively. The visual judgment time and judgment consistency were recorded. The results showed that with the flat-bottomed plate, the bacterial growth signal was dispersed, and the red color blended with the turbid background, resulting in a 73% consistency rate among the three testers. With the round / pointed bottom plate, the red signal was concentrated through the precipitation focusing effect, and the consistency rate among the three testers reached 100%, significantly improving the detection efficiency and the reliability of the results.
[0036] In summary, this invention innovatively combines TTC indicator screening with well plate structure optimization to form a synergistic solution, which is key to overcoming the bottlenecks of existing technologies. The detection method of this invention uses well plates that are routine laboratory consumables, requiring no additional specialized equipment. The operation procedure conforms to standard microbiological experimental practices, and the reagent cost is low, making it easy to promote and apply in grassroots laboratories. This method can be widely applied to the MIC detection of various turbid traditional Chinese medicine extracts and compound preparations of traditional Chinese medicine, and can also be extended to the effect determination of drug sensitivity tests between traditional Chinese medicine and Western medicine, and between traditional Chinese medicines, making it applicable to flexible scenarios.
[0037] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.
Claims
1. A method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator, characterized in that, Includes the following steps: S1. Screening for the optimal working concentration of TTC culture medium and suitable well plate that has no antibacterial effect on the target bacteria and can make the bacterial sedimentation and color development at the bottom of the well clear. S2. Select the appropriate well plate determined in step S1, and set up a blank control, a drug-TTC reaction control and a positive control. All control groups and experimental groups use the same well plate type and system configuration. Add serially diluted turbid Chinese medicine solution, bacterial solution of the test strain and TTC optimal working concentration culture medium determined in step S1 to each well, and control the final volume of each well to construct the experimental system and control system. S3. Place the experimental system and control system constructed in step S2 in a constant temperature environment for incubation; S4. Based on the precipitation focusing effect of the well plate and combined with the expected results of each control well, the MIC value of the turbid Chinese medicine against the target bacteria was determined by observing the color of the bottom of the experimental well and the precipitation state.
2. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 1, characterized in that, In step S1, TTC is mixed with culture medium to prepare a series of TTC-culture medium systems. Well plates with better focusing effect than flat bottom plates are selected. The antibacterial safety of TTC concentration is verified by the coating method. The system is incubated in a constant temperature environment to screen out the optimal working concentration of TTC that has no antibacterial effect on the target bacteria and can make the bacterial precipitate at the bottom of the well clearly visible.
3. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 2, characterized in that, In step S1, the TTC concentration of the TTC-medium system is 2.5~80 μg / mL, and it is prepared by a 2-fold serial dilution method; the culture medium is MH broth or target bacteria-specific culture medium; the appropriate well plate is a sterile round-bottom 96-well plate or a sterile pointed-bottom 96-well plate.
4. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 2, characterized in that, The optimal working concentration of TTC must simultaneously meet the following conditions: 1) there is no statistically significant difference in the number of colonies on the culture medium compared to the blank plate without TTC; 2) the red signal at the bottom of the well in the adapter plate is clearly visible to the naked eye; 3) there is no non-specific red color development in the drug-TTC reaction control well.
5. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 2, characterized in that, In steps S1 and S3, the temperature for cultivation in a constant temperature environment is 35±1℃, and the cultivation time is 16~24 h.
6. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 2, characterized in that, In step S2, the control group is configured as follows: Blank control: Culture medium containing the optimal working concentration of TTC, without drugs or bacterial suspension; Drug-TTC reaction control: Culture medium containing traditional Chinese medicine solutions of various concentrations and the optimal working concentration of TTC, without bacterial culture; Positive control: Culture medium containing the optimal working concentration of TTC and bacterial suspension of the test strain, without drugs.
7. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 2, characterized in that, In step S4, the expected results for the control wells are as follows: the bottom of the blank control and the drug-TTC reaction control wells will not show any specific red precipitate, but will only show the original turbid color of the drug solution or a small amount of precipitate produced by the drug itself; the bottom of the positive control wells will show a clear specific red precipitate.
8. The method for detecting the antibacterial MIC of turbid traditional Chinese medicine based on TTC indicator according to claim 1, characterized in that, In step S4, the criterion for judging the experimental well is: the appearance of red precipitate at the bottom of the well indicates that the concentration of the drug solution in the well has not reached the antibacterial threshold and has no obvious antibacterial effect. As the concentration gradient of the traditional Chinese medicine increases, the first occurrence of no obvious red color at the bottom of the well, with only the original turbid color of the medicine solution remaining, serves as the critical point for antibacterial activity. The lowest concentration of traditional Chinese medicine corresponding to this point is the MIC value of the turbid traditional Chinese medicine against the target bacteria.
9. A detection kit for implementing the TTC indicator-based method for detecting the antibacterial MIC of turbid traditional Chinese medicine as described in any one of claims 1-8, characterized in that, include: Focused microplates, TTC reagent, and culture medium; The focusing microplate is used to concentrate bacteria at the bottom of the well; The TTC reagent, when mixed with the culture medium, can be configured into a TTC-culture medium system for screening the optimal working concentration; the optimal working concentration is the concentration that has no inhibitory effect on the target bacteria, produces a clear and distinguishable color, and does not react non-specifically with the turbid Chinese medicine solution.
10. The detection kit according to claim 9, characterized in that, The focusing microplate is a round-bottomed 96-well plate or a pointed-bottomed 96-well plate.