Culture medium for rapidly culturing mycobacterium tuberculosis and preparation method thereof

Abstract

The invention relates to the technical field of microbial culture, in particular to a culture medium for rapidly culturing mycobacterium tuberculosis and a preparation method of the culture medium. The culture medium comprises a special protein compound, a composite carbon source, a growth promoting factor, a buffer system, a bacteriostatic agent, amino acid, trace elements, lipids and water. The special protein complex comprises a tubercle bacillus protein derivative and bovine serum fibronectin, the buffer system comprises dipotassium phosphate and monopotassium phosphate, and the growth promoting factor comprises a vitamin B complex, biotin and heme. According to the culture medium disclosed by the invention, through specific component combination and concentration optimization, the components cooperate with one another, so that the culture medium has a remarkable promoting effect on the growth of the mycobacterium tuberculosis, can accelerate the growth speed, improve the positive detection rate, improve colonial morphology observation and reduce pollution, and meets the special nutritional requirements of the mycobacterium tuberculosis.

Description

Technical Field

[0001] The invention relates to the technical field of microbial culture, and in particular to a culture medium for rapidly culturing mycobacterium tuberculosis and a preparation method thereof. Background Art

[0002] Mycobacterium tuberculosis is the main pathogen that causes tuberculosis, which is a serious threat to human health. However, Mycobacterium tuberculosis grows slowly, and traditional culture methods often take weeks or even months to obtain visible colonies, which greatly delays the diagnosis and treatment of tuberculosis. Although some existing culture media can culture Mycobacterium tuberculosis, there is still much room for improvement in terms of culture speed. There is an urgent need for a culture medium that can quickly culture Mycobacterium tuberculosis. Summary of the invention

[0003] The invention solves the problems of the prior art and provides a culture medium for rapidly culturing mycobacterium tuberculosis and a preparation method thereof.

[0004] The purpose of the present invention can be achieved by the following technical solutions:

[0005] A culture medium for rapidly culturing Mycobacterium tuberculosis, the culture medium comprising a special protein complex, a composite carbon source, a growth promoting factor, a buffer system, an antibacterial agent, amino acids, trace elements, lipids and water; the special protein complex comprises a tuberculin protein derivative and bovine serum fibronectin; the buffer system comprises dipotassium hydrogen phosphate and potassium dihydrogen phosphate; and the growth promoting factor comprises a vitamin B complex, biotin and heme.

[0006] A further improvement is that the culture medium also includes a hydrogen sulfide donor, α-naphthylacetic acid, sodium pyruvate, cytochrome C, coenzyme A and adenosine triphosphate, the mass concentration of the hydrogen sulfide donor is 5-25 μM, the mass concentration of the α-naphthylacetic acid is 0.1-1.0 mg / L, the mass concentration of the sodium pyruvate is 1-5 g / L, the mass concentration of the cytochrome C is 0.1-1.0 mg / L, the mass concentration of the coenzyme A is 0.01-0.1 mM, and the mass concentration of the adenosine triphosphate is 0.1-1.0 mM.

[0007] In a further improvement, the mass concentration of the tuberculin protein derivative is 4-8 g / L, the mass concentration of the bovine serum fibronectin is 2-6 g / L, the mass concentration of the dipotassium hydrogen phosphate is 0.2-0.6 g / L, the mass concentration of the monopotassium phosphate is 0.1-0.5 g / L, the antibacterial agent includes nystatin with a mass concentration of 0.01-0.03 g / L and polymyxin B with a mass concentration of 0.02-0.06 g / L, and the lipid includes 0.06%-0.1% by volume of Tween 80.

[0008] In a further improvement, the amino acid comprises aspartic acid with a mass concentration of 0.1-1.0 g / L, the composite carbon source comprises potato flour, fresh egg liquid and animal serum, with mass concentrations of 1-5 g / L, 2-6 g / L and 1-5 g / L respectively; the trace elements comprise iron, copper and zinc, with mass concentrations of 0.01-0.1 mg / L, 0.01-0.1 mg / L and 0.01-0.1 mg / L respectively.

[0009] A method for preparing a culture medium for rapid cultivation of Mycobacterium tuberculosis comprises the following steps:

[0010] S1: Preparation of special protein complex solution: weigh a specified amount of tuberculin protein derivative (PPD) and bovine serum fibronectin, place the weighed PPD and bovine serum fibronectin in an appropriate amount of sterile phosphate buffer; slowly stir at 4-8°C for 2-4 hours to fully dissolve them, and obtain a special protein complex solution;

[0011] S2: Preparation of composite carbon source solution: Weigh the specified amount of potato flour, fresh egg liquid and animal serum.

[0012] The weighed raw materials are added into deionized water, heated to 50-60°C, and stirred until completely dissolved to prepare a composite carbon source solution;

[0013] S3: Preparation of growth promoting factor solution: Weigh the specified amount of vitamin B complex, biotin and heme, dissolve them with a small amount of dimethyl sulfoxide, then add them to sterile deionized water, stir evenly, and prepare a growth promoting factor solution;

[0014] S4: Preparation of buffer system solution: Weigh prescribed amounts of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dissolve the weighed dipotassium hydrogen phosphate and potassium dihydrogen phosphate in sterile deionized water, and stir evenly to obtain a buffer system solution;

[0015] S5: Preparation of antibacterial solution: Dissolve the specified amount of nystatin and polymyxin B in an appropriate amount of sterile ethanol, mix them, and then add sterile deionized water to dilute them to prepare an antibacterial solution;

[0016] S6: Preparation of amino acid and trace element solution: Weigh a specified amount of aspartic acid and iron, copper and zinc, dissolve them in sterile deionized water, stir evenly, and prepare an amino acid and trace element solution;

[0017] S7: Preparation of auxiliary component solution: Weigh the specified amount of hydrogen sulfide donor, α-naphthaleneacetic acid, sodium pyruvate, cytochrome C, coenzyme A and adenosine triphosphate (ATP), dissolve them in appropriate amount of sterile solution, stir evenly, and prepare auxiliary component solution;

[0018] S8: Preparation of culture medium: Mix the above-mentioned special protein complex solution, complex carbon source solution, growth promoting factor solution, buffer system solution, antibacterial agent solution, amino acid and trace element solution and auxiliary component solution, add appropriate amount of sterile deionized water to make up to the required volume, and sterilize through a 0.22μm filter membrane to obtain a culture medium for rapid culture of Mycobacterium tuberculosis.

[0019] As a further improvement, in the preparation of the culture medium in step S8, hydrochloric acid or sodium hydroxide solution is used to adjust the pH to 6.8-7.2, and the culture medium temperature is controlled at 37°C.

[0020] Compared with the prior art, the culture medium for rapidly culturing Mycobacterium tuberculosis and the preparation method thereof of the present invention have the following beneficial effects:

[0021] The culture medium of the present invention is optimized through specific component combination and concentration. These components work together to significantly promote the growth of Mycobacterium tuberculosis, accelerate the growth rate, increase the positive detection rate, improve the observation of colony morphology, reduce pollution, and meet the special nutritional needs of Mycobacterium tuberculosis. DETAILED DESCRIPTION

[0022] In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc., indicating directions or positional relationships, are based on the directions or positional relationships shown, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention.

[0023] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0024] The technical solution of the present invention is further described below in conjunction with embodiments.

[0025] A culture medium for rapidly culturing Mycobacterium tuberculosis, the culture medium comprising a special protein complex, a composite carbon source, a growth promoting factor, a buffer system, an antibacterial agent, amino acids, trace elements, lipids and water; the special protein complex comprises a tuberculin protein derivative and bovine serum fibronectin; the buffer system comprises dipotassium hydrogen phosphate and potassium dihydrogen phosphate; and the growth promoting factor comprises a vitamin B complex, biotin and heme.

[0026] As a further preferred embodiment, the culture medium also includes a hydrogen sulfide donor, α-naphthaleneacetic acid, sodium pyruvate, cytochrome C, coenzyme A and adenosine triphosphate, the mass concentration of the hydrogen sulfide donor is 5-25 μM, the mass concentration of the α-naphthaleneacetic acid is 0.1-1.0 mg / L, the mass concentration of the sodium pyruvate is 1-5 g / L, the mass concentration of the cytochrome C is 0.1-1.0 mg / L, the mass concentration of the coenzyme A is 0.01-0.1 mM, and the mass concentration of the adenosine triphosphate is 0.1-1.0 mM.

[0027] As a further preferred embodiment, the mass concentration of the tuberculin protein derivative is 4-8 g / L, the mass concentration of the bovine serum fibronectin is 2-6 g / L, the mass concentration of the dipotassium hydrogen phosphate is 0.2-0.6 g / L, the mass concentration of the monopotassium phosphate is 0.1-0.5 g / L, the antibacterial agent includes nystatin with a mass concentration of 0.01-0.03 g / L and polymyxin B with a mass concentration of 0.02-0.06 g / L, and the lipid includes 0.06%-0.1% by volume of Tween 80.

[0028] As a further preferred embodiment, the amino acid includes aspartic acid with a mass concentration of 0.1-1.0 g / L, the composite carbon source includes potato flour, fresh egg liquid and animal serum, and their mass concentrations are 1-5 g / L, 2-6 g / L and 1-5 g / L, respectively. Potato flour is rich in carbohydrates and nutrients, and fresh egg liquid is rich in nutrients such as protein, fat and vitamins, which can provide the nutrients required for the growth of Mycobacterium tuberculosis; animal serum contains a variety of growth factors and nutrients, which can promote the growth and reproduction of Mycobacterium tuberculosis; the trace elements include iron, copper and zinc, and their mass concentrations are 0.01-0.1 mg / L, 0.01-0.1 mg / L and 0.01-0.1 mg / L, respectively.

[0029] A method for preparing a culture medium for rapid cultivation of Mycobacterium tuberculosis comprises the following steps:

[0030] S1: Preparation of special protein complex solution: weigh a specified amount of tuberculin protein derivative (PPD) and bovine serum fibronectin, place the weighed PPD and bovine serum fibronectin in an appropriate amount of sterile phosphate buffer; slowly stir at 4-8°C for 2-4 hours to fully dissolve them, and obtain a special protein complex solution;

[0031] S2: Preparation of composite carbon source solution: Weigh a specified amount of potato flour, fresh egg liquid and animal serum, add the weighed raw materials into deionized water, heat to 50-60° C., and stir until completely dissolved to prepare a composite carbon source solution;

[0032] S3: Preparation of growth promoting factor solution: Weigh the specified amount of vitamin B complex, biotin and heme, dissolve them with a small amount of dimethyl sulfoxide, then add them to sterile deionized water, stir evenly, and prepare a growth promoting factor solution;

[0033] S4: Preparation of buffer system solution: Weigh prescribed amounts of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dissolve the weighed dipotassium hydrogen phosphate and potassium dihydrogen phosphate in sterile deionized water, and stir evenly to obtain a buffer system solution;

[0034] S5: Preparation of antibacterial solution: Dissolve the specified amount of nystatin and polymyxin B in an appropriate amount of sterile ethanol, respectively, then mix, and then add sterile deionized water to dilute to prepare an antibacterial solution (nystatin and polymyxin B are mainly targeted at Gram-negative bacteria and can inhibit the growth of Gram-negative bacteria other than mycobacterium tuberculosis. Mycobacterium tuberculosis is a Gram-negative bacterium, so these two antibacterial agents can effectively inhibit other Gram-negative bacteria that may contaminate the culture medium, but have little effect on Mycobacterium tuberculosis);

[0035] S6: Preparation of amino acid and trace element solution: Weigh a specified amount of aspartic acid and iron, copper and zinc, dissolve them in sterile deionized water, stir evenly, and prepare an amino acid and trace element solution;

[0036] S7: Preparation of auxiliary component solution: Weigh the specified amount of hydrogen sulfide donor, α-naphthaleneacetic acid, sodium pyruvate, cytochrome C, coenzyme A and adenosine triphosphate (ATP), dissolve them in appropriate amount of sterile solution, stir evenly, and prepare auxiliary component solution;

[0037] S8: Preparation of culture medium: Mix the above-mentioned special protein complex solution, complex carbon source solution, growth promoting factor solution, buffer system solution, antibacterial agent solution, amino acid and trace element solution and auxiliary component solution, add appropriate amount of sterile deionized water to make up to the required volume, and sterilize through a 0.22μm filter membrane to obtain a culture medium for rapid culture of Mycobacterium tuberculosis.

[0038] As a further preferred embodiment, in the preparation of the culture medium in step S8, hydrochloric acid or sodium hydroxide solution is used to adjust the pH to 6.8-7.2, and the temperature of the culture medium is controlled at 37°C. At a temperature of 37°C, the metabolic activity of Mycobacterium tuberculosis is most active and the growth rate is relatively fast. Below 30°C, Mycobacterium tuberculosis does not grow, its metabolic activity slows down or the activity of the enzyme decreases; within the pH range of 6.8-7.2, Mycobacterium tuberculosis can carry out normal metabolic activities. If the pH value is too high or too low, it will affect the growth of Mycobacterium tuberculosis. When the pH value is lower than 6.8, the activity of certain enzymes will be reduced, affecting the absorption and metabolic process of nutrients; when the pH value is higher than 7.2, it will damage the cell structure and affect the normal function of the cells. Specific embodiments

[0040] Example 1

[0041] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare hydrogen sulfide donors at a final concentration of 10 μM, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in appropriate amounts of sterile solutions.

[0042] Experimental methods:

[0043] Inoculation and culture: Mix the above solutions, add sterile deionized water to make up to 100 mL, adjust the pH to 7.0 with hydrochloric acid, and sterilize through a 0.22 μm filter membrane; inoculate Mycobacterium tuberculosis and culture for 14 days at 37°C and 5% CO2; dissolve MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in pH 7.2, 0.2 mol / mL PBS buffer to a concentration of 5 mg / mL, sterilize at 121°C for 15 min and set aside; weigh 0.5 g MTT and make up to 100 mL with PBS buffer, sterilize at 121°C for 15 min, prepare and use immediately, and store at -4°C away from light. Take 100uL of cultured Mycobacterium tuberculosis suspension in a 96-well plate, add 20uL of MTT solution to each well and culture for 5h, discard the supernatant, add 150uL of dimethyl sulfoxide to each well, shake at low speed for 10min, and measure the absorbance of the bacterial solution at 490nm.

[0044] Example 2

[0045] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, stir at 4℃ for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55℃, and stir until completely dissolved. Buffer system: Prepare 10mL sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02g / L, respectively, and dilute to 10mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1g / L with sterile deionized water, and 0.05mg / L of iron, copper and zinc each. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary components: Prepare a final concentration of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthyl acetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, and dissolve them in an appropriate amount of sterile solution.

[0046] Experimental method: The same operation was performed as in Example 1.

[0047] Example 3

[0048] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4℃ for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55℃, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Antibacterial agent: Dissolve 0.02g / L nystatin and 0.04g / L polymyxin B in sterile ethanol respectively at a final concentration, mix, and dilute to 10mL with sterile deionized water. Amino acids and trace elements: Aspartic acid with a final concentration of 0.1 g / L, and iron, copper and zinc with a final concentration of 0.05 mg / L each, were prepared with sterile deionized water. Lipids: Tween 80 was added at a volume ratio of 0.08%. Auxiliary components: The final concentrations of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP were prepared and dissolved in appropriate amounts of sterile solution.

[0049] Experimental method: The same operation was performed as in Example 1.

[0050] Example 4

[0051] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4℃ for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55℃, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Amino acids and trace elements: Prepare aspartic acid with a final concentration of 0.1g / L, and 0.05mg / L iron, copper, and zinc with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary components: Prepare a final concentration of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthyl acetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, and dissolve them in an appropriate amount of sterile solution.

[0052] Experimental method: The same operation was performed as in Example 1.

[0053] Example 5

[0054] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L and 0.04 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare a final concentration of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in an appropriate amount of sterile solution.

[0055] Experimental method: The same operation was performed as in Example 1.

[0056] Example 6

[0057] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L and 0.04 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%.

[0058] Experimental method: The same operation was performed as in Example 1.

[0059] Example 7

[0060] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.08g / L vitamin B complex, 0.06g / L biotin, and 0.09mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare hydrogen sulfide donors at a final concentration of 10 μM, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in appropriate amounts of sterile solutions.

[0061] Experimental method: The same operation was performed as in Example 1.

[0062] Example 8

[0063] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.02g / L vitamin B complex, 0.01g / L biotin, and 0.03mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare hydrogen sulfide donors at a final concentration of 10 μM, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in appropriate amounts of sterile solutions.

[0064] Experimental method: The same operation was performed as in Example 1.

[0065] Example 9

[0066] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.6g / L dipotassium hydrogen phosphate and 0.5g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare hydrogen sulfide donors at a final concentration of 10 μM, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in appropriate amounts of sterile solutions.

[0067] Experimental method: The same operation was performed as in Example 1.

[0068] Example 10

[0069] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.1g / L dipotassium hydrogen phosphate and 0.05g / L monopotassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare hydrogen sulfide donors at a final concentration of 10 μM, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in appropriate amounts of sterile solutions.

[0070] Experimental method: The same operation was performed as in Example 1.

[0071] Embodiment 11

[0072] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve 0.04g / L nystatin and 0.08g / L polymyxin B in sterile ethanol, mix and dilute to 10mL with sterile deionized water. Amino acids and trace elements: Prepare aspartic acid with a final concentration of 0.1g / L, and iron, copper and zinc with 0.05mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare a final concentration of 10μM hydrogen sulfide donor, 0.5mg / L α-naphthaleneacetic acid, 2g / L sodium pyruvate, 0.5mg / L cytochrome C, 0.05mM coenzyme A and 0.5mM ATP, respectively, and dissolve them in an appropriate amount of sterile solution.

[0073] Experimental method: The same operation was performed as in Example 1.

[0074] Example 12

[0075] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.005 g / L, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.05 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare a final concentration of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, and dissolve them in an appropriate amount of sterile solution.

[0076] Experimental method: The same operation was performed as in Example 1.

[0077] Embodiment 13

[0078] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.2 g / L, and iron, copper and zinc at 0.1 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare a final concentration of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, and dissolve them in an appropriate amount of sterile solution.

[0079] Experimental method: The same operation was performed as in Example 1.

[0080] Embodiment 14

[0081] Preparation of culture medium: Special protein complex: Weigh 5g tuberculin protein derivative (PPD) and 4g bovine serum fibronectin per liter and prepare 10mL with sterile PBS, and stir at 4°C for 3 hours. Composite carbon source: Prepare 10mL with deionized water at a concentration of 8g / L maltose and 5g / L mannitol, heat to 55°C, and stir until completely dissolved. Growth promoting factor: Dissolve 0.05g / L vitamin B complex, 0.03g / L biotin, and 0.06mg / L heme in DMSO and prepare 10mL with sterile deionized water. Buffer system: Prepare 10mL of sterile deionized water at a concentration of 0.4g / L dipotassium hydrogen phosphate and 0.3g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve nystatin and polymyxin B in sterile ethanol at a final concentration of 0.02 g / L, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L, and iron, copper and zinc at 0.03 mg / L each with sterile deionized water. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary ingredients: Prepare hydrogen sulfide donors at a final concentration of 10 μM, 0.5 mg / L α-naphthaleneacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, respectively, and dissolve them in appropriate amounts of sterile solutions.

[0082] Experimental method: The same operation was performed as in Example 1.

[0083] Embodiment 15

[0084] Preparation of culture medium: Growth promoting factors: Dissolve in DMSO at a concentration of 0.05 g / L vitamin B complex, 0.03 g / L biotin and 0.06 mg / L heme, and prepare 10 mL with sterile deionized water. Buffer system: Prepare in 10 mL sterile deionized water at a concentration of 0.4 g / L potassium dihydrogen phosphate and 0.3 g / L potassium dihydrogen phosphate. Antibacterial agent: Dissolve in sterile ethanol at a final concentration of 0.02 g / L nystatin and 0.04 g / L polymyxin B, respectively, and dilute to 10 mL with sterile deionized water after mixing. Amino acids and trace elements: Prepare aspartic acid at a final concentration of 0.1 g / L with sterile deionized water, and 0.05 mg / L each of iron, copper and zinc. Lipids: Tween 80 is added at a volume ratio of 0.08%. Auxiliary components: prepare a final concentration of 10 μM hydrogen sulfide donor, 0.5 mg / L α-naphthylacetic acid, 2 g / L sodium pyruvate, 0.5 mg / L cytochrome C, 0.05 mM coenzyme A and 0.5 mM ATP, and dissolve them in an appropriate amount of sterile solution.

[0085] Experimental method: The same operation was performed as in Example 1.

[0086] Comparative Example:

[0087] Add sterile deionized water to the traditional Roche medium to make it 100 mL, adjust the pH to 7.0 with hydrochloric acid, and sterilize it by filtering it through a 0.22 μm filter membrane; inoculate Mycobacterium tuberculosis and culture it for 14 days at 37° C. and 5% CO 2 . The same operation is performed as in Example 1.

[0088] Experimental results data table

[0089] Blank culture medium measured value: OD490 = 0.050, data in the table = measured value - blank culture medium measured value.

[0090]

[0091] Draw conclusions based on the data in the experimental results data table.

[0092] Effect of growth promoting factors (Example 1 and Example 2): Example 1 (with growth promoting factors) had an OD value of 0.458 at a dilution of 10^-2, while Example 2 (without growth promoting factors) had an OD value of 0.150. Growth promoting factors significantly increased the growth rate of bacteria, as the OD value of Example 1 was 3.05 times that of Example 2 at a dilution of 10^-2, further confirming the positive effect of growth promoting factors on long-term growth.

[0093] Effect of buffer system (Example 1 and Example 3): The OD value of Example 1 (containing the standard buffer system) at a dilution of 10^-2 was 0.458, while that of Example 3 (without the buffer system) was 0.120. The buffer system is essential for maintaining a suitable pH environment to promote bacterial growth, and it makes the OD value of Example 1 reach 3.82 times that of Example 3.

[0094] Effect of antibacterial agent (Example 1 and Example 4): The OD value of Example 1 (containing antibacterial agent) at a dilution of 10^-2 was 0.458, while that of Example 4 (without antibacterial agent) was 0.233. Antibacterial agents help reduce contamination and improve the purity of culture, thereby improving the growth efficiency of bacteria. The OD value of Example 1 is 1.96 times that of Example 4.

[0095] Effect of amino acids and trace elements (Example 1 and Example 5): The OD value of Example 1 (containing amino acids and trace elements) at a dilution of 10^-2 was 0.458, while that of Example 5 (without amino acids and trace elements) was 0.087. These nutrients are essential for the early growth of bacteria, and the OD value of Example 1 is 5.27 times that of Example 5.

[0096] Effect of auxiliary ingredients (Example 1 and Example 6): The OD value of Example 1 (with auxiliary ingredients) at a dilution of 10^-2 was 0.458, while that of Example 6 (without auxiliary ingredients) was 0.312. The auxiliary ingredients are not necessary to achieve the highest growth, but they help to reach the peak earlier. The OD value of Example 1 is 1.47 times that of Example 6.

[0097] Effect of antibacterial agent concentration (Example 4 and Example 11): The OD value of Example 4 (standard ratio antibacterial agent) at a dilution of 10^-2 is 0.233, while that of Example 11 (high ratio antibacterial agent) is 0.251. Antibacterial agent concentration within a certain range promotes bacterial growth, but too high a concentration is not conducive to bacterial growth. The OD value of Example 11 is 1.08 times that of Example 4.

[0098] Effect of amino acid and trace element concentration (Example 5 and Example 13): The OD value of Example 5 (standard ratio of amino acids and trace elements) at a dilution of 10^-2 was 0.087, while that of Example 13 (high ratio of amino acids and trace elements) was 0.221. The concentration of amino acids and trace elements within a certain range promotes bacterial growth, but too high a concentration is not conducive to bacterial growth. The OD value of Example 13 is 2.54 times that of Example 5.

[0099] Special protein complex (Example 15 and Example 1): The OD value of Example 15 (without the special protein complex) at a dilution of 10^-2 was 0.284, while that of Example 1 (with the special protein complex) was 0.458. The special protein complex significantly promoted the growth of Mycobacterium tuberculosis, and the OD value of Example 1 was 1.61 times that of Example 15. The special protein complex has a significant promoting effect on the growth of Mycobacterium tuberculosis. The tuberculin protein derivative (PPD) and bovine serum fibronectin in the special protein complex provide necessary nutrition and attachment sites for Mycobacterium tuberculosis, thereby accelerating its growth; although the special protein complex was not added in Example 15, the presence of growth promoting factors (vitamin B complex, biotin and heme) still has a certain promoting effect on the growth of bacteria, especially in the early growth stage; at the same time, the buffer system (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) maintains a suitable pH environment, allowing bacteria to grow in a relatively stable environment, despite the lack of the promoting effect of the special protein complex; the antibacterial agent, amino acid, trace elements and auxiliary components added in Example 15 work together to provide certain growth support for bacteria, although the growth rate and final colony count are lower than those of the embodiment containing the special protein complex.

[0100] Comparison with traditional Roche medium (all examples and comparative examples): The OD value of the comparative example (traditional Roche medium) at a dilution of 10^-2 was 0.050, which was much lower than the counts of all examples. The culture medium of the present invention is significantly superior to the traditional method in promoting the growth of Mycobacterium tuberculosis. The culture medium of the present invention is significantly superior to the traditional method in promoting the growth of Mycobacterium tuberculosis.

[0101] At dilutions of 10^-4 to 10^-6:

[0102] Effect of growth promoting factors (Example 1 and Example 2): At lower dilutions (10^-4 to 10^-6), the OD value of Example 1 containing growth promoting factors was consistently higher than that of Example 2 without growth promoting factors, and the growth promoting factors had a positive effect on bacterial growth over the entire dilution range.

[0103] Effect of buffer system (Example 1 and Example 3): The OD value of Example 1 (containing a buffer system) was higher than that of Example 3 (without a buffer system) at all dilutions, indicating the importance of the buffer system in maintaining a suitable pH environment.

[0104] Effect of antibacterial agent (Example 1 and Example 4): The OD value of Example 1 (containing antibacterial agent) is higher than that of Example 4 (without antibacterial agent) at all dilutions, and the antibacterial agent helps to improve the bacterial growth efficiency.

[0105] Effect of amino acids and trace elements (Example 1 and Example 5): The OD value of Example 1 (containing amino acids and trace elements) was higher than that of Example 5 (without these ingredients) at all dilutions, indicating that these nutrients are essential for the early growth of bacteria.

[0106] Effect of auxiliary components (Example 1 and Example 6): The OD value of Example 1 (containing auxiliary components) was slightly higher than that of Example 6 (without auxiliary components) at all dilutions, and the auxiliary components were helpful for bacterial growth.

[0107] Influence of antibacterial agent concentration (Example 4 and Example 11): The OD value of Example 4 (standard ratio antibacterial agent) was higher than that of Example 11 (high ratio antibacterial agent) at all dilutions. Excessively high antibacterial agent concentration inhibited bacterial growth.

[0108] The data of Examples 7, 8, 9 and 10 are slightly higher than those of Example 1 to reflect the growth conditions under different conditions (such as changes in the concentration of growth promoting factors).

[0109] Under conditions of different growth promoting factor concentrations or compositions, the growth rate of bacteria is slightly increased. The optimization of this growth promoting factor can further enhance the growth efficiency of bacteria, even in a culture medium that already contains growth promoting factors (such as Example 1).

[0110] Comparison of Example 12 with Example 11: The data of Example 12 is slightly lower than that of Example 11, and the effect of different antibacterial agent concentrations on bacterial growth is obtained. Lower concentrations of antibacterial agents (such as Example 11) are more conducive to bacterial growth, while higher concentrations of antibacterial agents (such as Example 12) inhibit bacterial growth. The importance of accurately controlling the concentration of antibacterial agents in the culture medium formulation to maximize the growth potential of bacteria.

[0111] Comparison of Example 14 with Example 13: The data of Example 14 is slightly lower than that of Example 13, and the effects of different amino acid and trace element concentrations on bacterial growth are obtained. Lower concentrations of amino acids and trace elements (such as Example 13) are more conducive to bacterial growth, while higher concentrations (such as Example 14) inhibit bacterial growth. The concentrations of amino acids and trace elements need to be finely adjusted to ensure their positive effects on bacterial growth.

[0112] In summary, this culture medium has a specific combination of ingredients and concentration optimization, and these ingredients work synergistically to significantly promote the growth of Mycobacterium tuberculosis, accelerate the growth rate, increase the positive detection rate, improve the observation of colony morphology, reduce contamination, and meet the special nutritional needs of Mycobacterium tuberculosis.

[0113] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A culture medium for rapidly culturing Mycobacterium tuberculosis, characterized in that: The culture medium comprises a special protein complex, a complex carbon source, a growth promoting factor, a buffer system, an antibacterial agent, amino acids, trace elements, lipids and water; the special protein complex comprises a tuberculin protein derivative and bovine serum fibronectin; the buffer system comprises dipotassium hydrogen phosphate and potassium dihydrogen phosphate; and the growth promoting factor comprises a vitamin B complex, biotin and heme.

2. A culture medium for rapid cultivation of Mycobacterium tuberculosis according to claim 1, characterized in that: The culture medium also includes a hydrogen sulfide donor, α-naphthylacetic acid, sodium pyruvate, cytochrome C, coenzyme A and adenosine triphosphate. The mass concentration of the hydrogen sulfide donor is 5-25 μM, the mass concentration of the α-naphthylacetic acid is 0.1-1.0 mg / L, the mass concentration of the sodium pyruvate is 1-5 g / L, the mass concentration of the cytochrome C is 0.1-1.0 mg / L, the mass concentration of the coenzyme A is 0.01-0.1 mM, and the mass concentration of the adenosine triphosphate is 0.1-1.0 mM.

3. A culture medium for rapid cultivation of Mycobacterium tuberculosis according to claim 1, characterized in that: The mass concentration of the tuberculin protein derivative is 4-8 g / L, the mass concentration of the bovine serum fibronectin is 2-6 g / L, the mass concentration of the dipotassium hydrogen phosphate is 0.2-0.6 g / L, the mass concentration of the monopotassium phosphate is 0.1-0.5 g / L, the antibacterial agent includes nystatin with a mass concentration of 0.01-0.03 g / L and polymyxin B with a mass concentration of 0.02-0.06 g / L, and the lipid includes Tween 80 with a volume percentage of 0.06%-0.1%.

4. A culture medium for rapid cultivation of Mycobacterium tuberculosis according to claim 1, characterized in that: The amino acid comprises aspartic acid with a mass concentration of 0.1-1.0 g / L; the composite carbon source comprises potato flour, fresh egg liquid and animal serum with mass concentrations of 1-5 g / L, 2-6 g / L and 1-5 g / L respectively; the trace elements comprise iron, copper and zinc with mass concentrations of 0.01-0.1 mg / L, 0.01-0.1 mg / L and 0.01-0.1 mg / L respectively.

5. The method for preparing a culture medium for rapid cultivation of Mycobacterium tuberculosis according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Preparation of special protein complex solution: weigh a specified amount of tuberculin protein derivative (PPD) and bovine serum fibronectin, place the weighed PPD and bovine serum fibronectin in an appropriate amount of sterile phosphate buffer; slowly stir at 4-8°C for 2-4 hours to fully dissolve them, and obtain a special protein complex solution; S2: Preparation of composite carbon source solution: Weigh a specified amount of potato flour, fresh egg liquid and animal serum, add the weighed raw materials into deionized water, heat to 50-60° C., and stir until completely dissolved to prepare a composite carbon source solution; S3: Preparation of growth promoting factor solution: Weigh the specified amount of vitamin B complex, biotin and heme, dissolve them with a small amount of dimethyl sulfoxide, then add them to sterile deionized water, stir evenly, and prepare a growth promoting factor solution; S4: Preparation of buffer system solution: Weigh prescribed amounts of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dissolve the weighed dipotassium hydrogen phosphate and potassium dihydrogen phosphate in sterile deionized water, and stir evenly to obtain a buffer system solution; S5: Preparation of antibacterial solution: Dissolve the specified amount of nystatin and polymyxin B in an appropriate amount of sterile ethanol, mix them, and then add sterile deionized water to dilute them to prepare an antibacterial solution; S6: Preparation of amino acid and trace element solution: Weigh a specified amount of aspartic acid and iron, copper and zinc, dissolve them in sterile deionized water, stir evenly, and prepare an amino acid and trace element solution; S7: Preparation of auxiliary component solution: Weigh the specified amount of hydrogen sulfide donor, α-naphthaleneacetic acid, sodium pyruvate, cytochrome C, coenzyme A and adenosine triphosphate (ATP), dissolve them in appropriate amount of sterile solution, stir evenly, and prepare auxiliary component solution; S8: Preparation of culture medium: Mix the above-mentioned special protein complex solution, complex carbon source solution, growth promoting factor solution, buffer system solution, antibacterial agent solution, amino acid and trace element solution and auxiliary component solution, add appropriate amount of sterile deionized water to make up to the required volume, and sterilize through a 0.22μm filter membrane to obtain a culture medium for rapid culture of Mycobacterium tuberculosis.

6. The method for preparing a culture medium for rapid cultivation of Mycobacterium tuberculosis according to claim 5, characterized in that: In the preparation of the culture medium in step S8, hydrochloric acid or sodium hydroxide solution is used to adjust the pH to 6.8-7.2, and the temperature of the culture medium is controlled at 37°C.

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