A buffer and its use in improving stability of resazurin

Abstract

The application discloses a buffer for improving stability of resazurin. In view of the problem of resazurin instability under illumination in RPMI-1640 culture medium, the application provides a buffer containing a compound with structure I, the compound with structure I is reasonably combined with other components in the buffer, the illumination stability of the culture system is obviously improved, and the color of the buffer can be maintained unchanged for more than 5 hours under illumination.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a buffer solution and its application in improving the stability of resazurin. Background Technology

[0002] Razor blue, also known as sodium azoride, is a chemical substance with the molecular formula C. 12 H6NNaO4 is a chromogenic solution commonly used in the study of biological materials. It is used as a redox indicator in the analysis of bacterial activity and the proliferation of bacterial, yeast, or mammalian cells. It is commonly used to measure bacterial and eukaryotic cell viability, as a cell staining agent, and also as an acid-base indicator, with a pH range of 3.8 (orange) to 6.5 (deep purple). In the cytoplasm, resazurin can be reduced from its blue state to a pink, strongly fluorescent form called halogenated resazurin under the action of various reductases. Further reactions result in a colorless and non-fluorescent form, dihydrohalogenated resazurin.

[0003] Current techniques often involve adding rezathoate to RPMI-1640 medium for culturing and monitoring the metabolic responses of cells, bacteria, and fungi. After incubation, the presence of microorganisms or their growth and metabolism are detected by color changes (from blue to red or purple). However, this detection system is highly sensitive to light, requiring strict light-protection during use. Otherwise, even a short period of light exposure can cause the blue to turn purple or red, affecting the colorimetric monitoring results. Therefore, there is a need for a compound that can improve the stability of rezathoate, allowing the RPMI-1640 medium containing rezathoate to maintain its blue color for an extended period under light conditions without turning red, thus accurately monitoring and reflecting the true growth and metabolism of microorganisms. Summary of the Invention

[0004] In view of this, the present invention provides a buffer solution and its application in improving the stability of resamaritan. Adding the buffer solution of the present invention to the reaction system of RPMI-1640 culture medium and resamaritan, or using the buffer solution of the present invention to prepare the resamaritan chromogenic solution, can significantly improve the light stability of the reaction system.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] Application of compounds with Formula I structure in improving the light stability of azuran.

[0007]

[0008] R1 is selected from H, -COCH2(OCH2CH2). n OCH3 or -COCH2(OCH2CH2) nOH, where n is any integer between 4 and 12.

[0009] In this invention, the resplenium is sodium resplenium salt, and in specific embodiments, the resplenium and resplenium indicator used are sodium resplenium salt. Studies have shown that adding the buffer solution of this invention to RPMI-1640 culture medium containing resplenium indicator, or using the buffer solution of this invention and resplenium to prepare a resplenium colorimetric solution, both achieve the goal of maintaining the blue color without turning red after 5 hours of light exposure, indicating that the compound of this invention and its buffer solution can significantly improve the photostability of resplenium in the RPMI-1640 system.

[0010] Specifically, the compound of Formula I is selected from one or more of 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 12-ether acetyl-5-benzyl)-benzotriazole, and 2-(2-hydroxy-5-methylphenyl)benzotriazole.

[0011] This invention provides a buffer solution for improving the stability of resazurin, comprising water, an osmotic pressure regulator, a pH regulator, a solubilizer, and a compound of formula I;

[0012]

[0013] R1 is selected from H, -COCH2(OCH2CH2). n OCH3 or -COCH2(OCH2CH2) n OH, where n is any integer between 4 and 12.

[0014] Specifically, the compound of Formula I is selected from one or more of 2-(2-hydroxy-3-monomethyl ether polyoxyethylene (5) ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene (9) ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene (12) ether acetyl-5-benzyl)-benzotriazole, and 2-(2-hydroxy-5-methylphenyl)benzotriazole. Studies have shown that buffer solutions prepared from the above compounds can significantly improve the photostability of the rezakura reaction system.

[0015] This invention has found that some compounds in the structure of Formula I are ineffective. The ineffective compounds that are not effective in improving the stability of azurium under light include: 4-aminobenzotriazole, 1H-benzotriazole-1-acetonitrile, 1-(2-pyrrolecarbonyl)benzotriazole, etc.

[0016] In this invention, the solvent of the buffer solution is water. Specifically, the buffer solution comprises the following components at the following concentrations: 0.1M-0.15M of the compound of Formula I, 2.5-5 g / L of osmotic pressure regulator, 0.05-0.2M of pH buffer, and 0.03wt%-0.06wt% of solubilizer. The concentration of each component refers to its final concentration in the buffer solution.

[0017] In some embodiments, the osmotic pressure regulator includes at least one of sodium citrate, sucrose, and bovine serum albumin; the pH buffer includes at least one pair of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and sodium carbonate-sodium bicarbonate buffer; and the solubilizer includes at least one of polysorbate 20, polysorbate 60, and polysorbate 80.

[0018] In some embodiments, the buffer solution further includes growth factors; the growth factors include at least one selected from para-aminobenzoic acid, riboflavin, biotin, and phenylalanine; the concentration of the growth factors is 0.02-0.15 g / L, specifically 0.02 g / L, 0.025 g / L, 0.05 g / L, 0.08 g / L, 0.12 g / L, and 0.15 g / L.

[0019] In some specific embodiments, the buffer solution of the present invention comprises water and components of the following concentrations:

[0020] Sodium citrate 2.5 g / L, sodium carbonate-sodium bicarbonate 0.08 M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole 0.1 M, p-aminobenzoic acid 0.025 g / L, polysorbate 20 0.05 wt%.

[0021] Alternatively, it may include: sucrose 5 g / L, sodium carbonate-sodium bicarbonate 0.08 M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole 0.12 M, polysorbate 60 0.05 wt%, and phenylalanine 0.025 g / L.

[0022] Alternatively, it may include: sodium citrate 5 g / L, disodium hydrogen phosphate (sodium dihydrogen phosphate) 0.1 M, 2-(2-hydroxy-5-methylphenyl)benzotriazole 0.13 M, phenylalanine 0.025 g / L, and polysorbate 20 g / L.

[0023] 0.05 wt%.

[0024] This invention provides a method for improving the light stability of azuran in a culture medium, comprising: adding the buffer solution described in this invention to RPMI-1640 culture medium containing azuran;

[0025] Alternatively, it may include: dissolving resplenium in the buffer solution described in the present invention as a solvent to prepare a resplenium chromogenic solution, and then adding the resplenium chromogenic solution to RPMI-1640 medium.

[0026] In some embodiments, the amount of buffer added is 1-2.5 mL of the buffer described in this invention per liter of RPMI-1640 medium. In some specific embodiments, 1 mL, 1.2 mL, 1.5 mL, 1.8 mL, 2 mL, 2.2 mL, or 2.5 mL of the buffer described in this invention may be added per liter of RPMI-1640 medium.

[0027] By adding resamaritan and the buffer solution described in this invention using the two methods described above, the final concentration of resamaritan is 1.4-100 mg / L and 1-2.5 mL of the buffer solution described in this invention per liter of culture system. In some specific embodiments of this invention, each 500 mL culture system contains 6 mg of resamaritan and 1.2 mL of the buffer solution described in this invention, i.e., each liter of culture system contains 12 mg of resamaritan and 2.4 mL of the buffer solution described in this invention.

[0028] RPMI-1640 culture medium has a complex nutrient composition. When the indicator resazurin is added, it easily changes from blue to red under light, affecting the colorimetric monitoring results of microorganisms. Therefore, this system must be protected from light. This invention addresses the light instability of resazurin during use by providing a buffer solution. Adding this buffer solution to RPMI-1640 culture medium containing resazurin significantly improves the light stability of the culture system, maintaining its color for more than 5 hours under light. Attached Figure Description

[0029] Figures 1-1 to 1-5 The following are the comparison results of the OD values ​​of the buffer solutions of formulations 1-1 to 1-5 in Example 4 and the control group under different light exposure times;

[0030] Figures 2-1 to 2-5 The following are the comparison results of the OD values ​​of the buffer solutions of formulations 2-1 to 2-5 in Example 5 and the control group under different light exposure times;

[0031] Figures 3-1 to 3-5 The results show the comparison of OD values ​​of the buffer solutions of formulations 3-1 to 3-5 in Example 6 and the control group under different light exposure times. Detailed Implementation

[0032] This invention provides a buffer solution and its application in improving the stability of rezakura. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0033] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0034] The present invention will be further illustrated below with reference to the embodiments:

[0035] Example 1: Buffer formulation of the present invention

[0036] Formula 1-1: 0.1M 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole, with water as the solvent.

[0037] Formula 1-2: Sodium citrate 2.5g / L, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole 0.1M, solvent is water.

[0038] Formulas 1-3: Sodium citrate 2.5g / L, sodium carbonate-sodium bicarbonate 0.08M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole 0.1M, solvent is water.

[0039] Formulas 1-4: Sodium citrate 2.5 g / L, sodium carbonate-sodium bicarbonate 0.08 M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole 0.1 M, p-aminobenzoic acid 0.025 g / L, solvent is water.

[0040] Formulas 1-5: Sodium citrate 2.5 g / L, sodium carbonate-sodium bicarbonate 0.08 M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole 0.1 M, p-aminobenzoic acid 0.025 g / L, polysorbate 200.05 wt%, solvent is water.

[0041] Example 2: Buffer formulation of the present invention

[0042] Formula 2-1: 0.12M 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole. Solvent is water.

[0043] Formula 2-2: 5 g / L sucrose, 0.12 M 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole, with water as the solvent.

[0044] Formulas 2-3: 5 g / L sucrose, 0.08 M sodium carbonate-sodium bicarbonate, 0.12 M 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole, with water as the solvent.

[0045] Formulas 2-4: sucrose 5g / L, sodium carbonate-sodium bicarbonate 0.08M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole 0.12M, phenylalanine 0.025g, solvent is water.

[0046] Formulas 2-5: sucrose 5g / L, sodium carbonate-sodium bicarbonate 0.08M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole 0.12M, polysorbate 60 0.05wt%, phenylalanine 0.025g / L, solvent is water.

[0047] Example 3: Buffer formulation of the present invention

[0048] Formula 3-1: 0.13M 2-(2-hydroxy-5-methylphenyl)benzotriazole, in water.

[0049] Formula 3-2: Sodium citrate 5g / L, 2-(2-hydroxy-5-methylphenyl)benzotriazole 0.13M, solvent is water.

[0050] Formula 3-3: Sodium citrate 5g / L, disodium hydrogen phosphate (sodium dihydrogen phosphate) 0.1M, 2-(2-hydroxy-5-methylphenyl)benzotriazole 0.13M, solvent is water.

[0051] Formulas 3-4: Sodium citrate 5g / L, disodium hydrogen phosphate (sodium dihydrogen phosphate) 0.1M, 2-(2-hydroxy-5-methylphenyl)benzotriazole 0.13M, phenylalanine 0.025g / L, solvent is water.

[0052] Formulas 3-5: Sodium citrate 5g / L, disodium hydrogen phosphate (sodium dihydrogen phosphate) 0.1M, 2-(2-hydroxy-5-methylphenyl)benzotriazole 0.13M, phenylalanine 0.025g / L, polysorbate 20 0.05wt%, solvent is water.

[0053] Example 4

[0054] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 3 mL of rezinex chromogenic reagent (the concentration of rezinex in the rezinex chromogenic reagent is 2 g / L), and then add 1.2 ml of any of the buffer solutions from Example 1; or prepare a rezinex indicator using 1.2 mL of the buffer solution from Example 1 and solid rezinex sodium salt (the concentration of rezinex in the buffer solution is 5 g / L), and add 1.2 mL of the rezinex indicator to 500 ml of culture medium. This ensures that each 500 ml of RPMI-1640 culture medium contains 1.2 mL of the buffer solution from Example 1. Finally, illuminate the entire system with an LED (not less than 20W), and record the color of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of illumination. Detect the blue OD of the system using a UV spectrophotometer. 600 The color retention rate was calculated, and the results are shown in Tables 1-1 to 1-5. The control group did not add the buffer solution of the present invention, while the experimental group added the buffer solution of Example 1 of the present invention. Figures 1-1 to 1-5 The OD values ​​of the control group and experimental group were measured under different light exposure times in Tables 1-1 to 1-5.

[0055] Table 1-1 Detection results of the control group and experimental group (Formula 1-1) under different light exposure times.

[0056]

[0057] Table 1-2 Detection results of control group and experimental group (formula 1-2) under different light exposure times

[0058]

[0059] Table 1-3 Detection results of control group and experimental group (formulas 1-3) under different light exposure times.

[0060]

[0061] Table 1-4 Detection results of control group and experimental group (formulas 1-4) under different light exposure times

[0062]

[0063] Table 1-5 Detection results of control group and experimental group (formulas 1-5) under different light exposure times.

[0064]

[0065]

[0066] The results showed that the reaction system of resamaritan and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED), the experimental group did not change color, with a color retention rate of 99.70%, while the control group without buffer turned purplish-red, with a color retention rate of 63.25%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.47%, while the control group without buffer turned red, with a color retention rate of 46.46%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.77%, while the control group without buffer turned red, with a color retention rate of 22.73%. After 40 minutes, the system with buffer did not change color, remaining blue, with a color retention rate of 99.62%, while the system without buffer turned red and became brighter, with a color retention rate of 16.72%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.39%, while the system without buffer turned red, and the color retention rate decreased to 11.94%.

[0067] The above results show that the reaction system with the buffer solution of the present invention (formulations 1-1 to 1-5) remained unchanged in red for 5 hours after the light reaction time was extended from 10 minutes, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0068] Example 5

[0069] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 3 mL of rezinex chromogenic reagent (the concentration of rezinex in the rezinex chromogenic reagent is 2 g / L), and then add 1.2 ml of any of the buffer solutions from Example 2; or prepare a rezinex indicator using 1.2 mL of any of the buffer solutions from Example 2 and solid rezinex sodium salt (the concentration of rezinex in the buffer solution is 5 g / L), and add 1.2 mL of the rezinex indicator to the culture medium. Finally, ensure that each 500 ml of RPMI-1640 culture medium contains 1.2 mL of the buffer solution from Example 2. Illuminate the entire system with an LED, and record the color of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of illumination. Detect the blue OD of the system using a UV spectrophotometer. 600 The color retention rate was calculated, and the results are shown in Tables 2-1 to 2-5. The control group did not add the buffer solution of the present invention, while the experimental groups were each added with one of the buffer solutions of Example 2 of the present invention. Figures 2-1 to 2-5 The OD values ​​of the control group and experimental group were measured under different light exposure times in Tables 2-1 to 2-5.

[0070] Table 2-1 Detection results of the control group and experimental group (Formula 2-1) under different light exposure times.

[0071]

[0072] Table 2-2 Detection results of control group and experimental group (formula 2-2) under different light exposure times

[0073]

[0074] Table 2-3 Detection results of control group and experimental group (formula 2-3) under different light exposure times

[0075]

[0076] Table 2-4 Detection results of control group and experimental group (formula 2-4) under different light exposure times

[0077]

[0078] Table 2-5 Detection results of control group and experimental group (formula 2-5) under different light exposure times

[0079]

[0080] The results showed that the reaction system of resamaritan and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED), the experimental group did not change color, with a color retention rate of 99.41%, while the control group without buffer turned purplish-red, with a color retention rate of 65.41%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.55%, while the control group without buffer turned red, with a color retention rate of 43.95%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 98.89%, while the control group without buffer turned red, with a color retention rate of 22.27%. After 40 minutes, the system with buffer did not change color, remaining blue, with a color retention rate of 99.85%, while the system without buffer turned red and became brighter, with a color retention rate of 15.78%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.41%, while the system without buffer turned red, and the color retention rate decreased to 12.91%.

[0081] The reaction system with the added buffer solution of the present invention (formulations 2-1 to 2-5) remained red for 5 hours after the light reaction time was extended from 10 minutes to 5 hours, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0082] Example 6

[0083] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 3 mL of rezinex chromogenic reagent (2 g / L), and then add 1.2 ml of any of the buffer solutions from Example 3; or prepare rezinex indicator (5 g / L) using the buffer solution from Example 3, and add 1.2 mL of the rezinex indicator to the culture medium. The final concentration should be 1.2 mL of buffer solution from Example 3 per 500 ml of RPMI-1640 culture medium. Illuminate the entire system with an LED and record the color of the culture medium at 0, 10, 20, 40, and 5 hours of illumination. Detect the blue OD value using a UV spectrophotometer. 600 The color retention rate was calculated, and the results are shown in Tables 3-1 to 3-5. The control group did not add the buffer solution of the present invention, while the experimental group added any of the buffer solutions in Example 3 of the present invention. Figures 3-1 to 3-5 The OD values ​​of the control group and experimental group were measured under different light exposure times in Tables 3-1 to 3-5.

[0084] Table 3-1 Detection results of the control group and experimental group (formula 3-1) under different light exposure times.

[0085]

[0086] Table 3-2 Detection results of control group and experimental group (formula 3-2) under different light exposure times

[0087]

[0088] Table 3-3 Detection results of control group and experimental group (formula 3-3) under different light exposure times.

[0089]

[0090] Table 3-4 Detection results of control group and experimental group (formula 3-4) under different light exposure times

[0091]

[0092] Table 3-5 Detection results of control group and experimental group (formula 3-5) under different light exposure times

[0093]

[0094] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED), the experimental group did not change color, with a color retention rate of 99.57%, while the control group without buffer turned purplish-red, with a color retention rate of 58.79%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.21%, while the control group without buffer turned red, with a color retention rate of 39.34%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.42%, while the control group without buffer turned red, with a color retention rate of 21.83%. After 40 minutes, the system with buffer did not change color, remaining blue, with a color retention rate of 99.71%, while the system without buffer turned red and became a brighter red, with a color retention rate of 15.27%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.49%, while the system without buffer turned red, and the color retention rate decreased to 11.31%.

[0095] The above results show that the reaction system with the buffer solution of the present invention (formulations 3-1 to 3-5) remained unchanged in red for 10 minutes and 5 hours, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0096] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of compounds with the structure of Formula I in improving the photostability of resazurite indicators. in, R1 is selected from H, -COCH2(OCH2CH2) n OCH3 or -COCH2(OCH2CH2) n OH, where n is any integer between 4 and 12.

2. The application according to claim 1, characterized in that, The compound of Formula I is selected from one or more of 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 12-ether acetyl-5-benzyl)-benzotriazole, and 2-(2-hydroxy-5-methylphenyl)benzotriazole.

3. A buffer solution for improving the stability of rezuril indicator, characterized in that, Including water, osmotic pressure regulators, pH regulators, solubilizers, and compounds with the structure of formula I; R1 is selected from H, -COCH2(OCH2CH2). n OCH3 or -COCH2(OCH2CH2) n OH, where n is any integer between 4 and 12.

4. The buffer solution according to claim 3, characterized in that, The compound of Formula I is selected from one or more of 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 12-ether acetyl-5-benzyl)-benzotriazole, and 2-(2-hydroxy-5-methylphenyl)benzotriazole.

5. The buffer solution according to claim 3, characterized in that, Includes water as a solvent and components at the following concentrations: Compounds of Formula I, 0.1M-0.15M; osmotic pressure regulator, 2.5-5 g / L; pH buffer, 0.05-0.2M; solubilizer, 0.03wt%-0.06wt%.

6. The buffer solution according to any one of claims 3 to 5, characterized in that, The osmotic pressure regulator includes at least one of sodium citrate, sucrose, and bovine serum albumin; the pH buffer includes at least one pair of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and sodium carbonate-sodium bicarbonate buffer; the solubilizer includes at least one of polysorbate 20, polysorbate 60, and polysorbate 80.

7. The buffer solution according to any one of claims 3 to 6, characterized in that, It also includes growth factors; the growth factors include at least one of para-aminobenzoic acid, riboflavin, biotin, and phenylalanine; the concentration of the growth factors is 0.02-0.15 g / L.

8. The buffer solution according to claim 7, characterized in that, Includes water and components at the following concentrations: Sodium citrate 2.5 g / L, sodium carbonate-sodium bicarbonate 0.08 M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 5-ether acetyl-5-benzyl)-benzotriazole 0.1 M, p-aminobenzoic acid 0.025 g / L, polysorbate 20 0.05 wt%; Alternatively, it may include: sucrose 5 g / L, sodium carbonate-bicarbonate 0.08 M, 2-(2-hydroxy-3-monomethyl ether polyoxyethylene 9-ether acetyl-5-benzyl)-benzotriazole 0.12 M, polysorbate 60 0.05 wt%, and phenylalanine 0.025 g / L; Alternatively, it may include: sodium citrate 5 g / L, disodium hydrogen phosphate (sodium dihydrogen phosphate) 0.1 M, 2-(2-hydroxy-5-methylphenyl)benzotriazole 0.13 M, phenylalanine 0.025 g / L, and polysorbate 20 0.05 wt%.

9. A method for improving the stability of azurlan indicator in a culture medium, characterized in that, include: Add the buffer solution according to any one of claims 3 to 8 to RPMI-1640 medium containing rezamidoide indicator; Alternatively, it may include: mixing the buffer solution according to any one of claims 3 to 8 with the resazu indicator to prepare a resazu colorimetric solution, and then adding the resazu colorimetric solution to RPMI-1640 medium.

10. The method according to claim 9, characterized in that, The buffer solution is added in the following amount: 1-2.5 mL of the buffer solution according to any one of claims 3 to 8 is added per liter of RPMI-1640 medium.

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