CN113789283A - Culture medium for rapidly culturing mycobacterium tuberculosis and preparation method and application thereof - Google Patents
Culture medium for rapidly culturing mycobacterium tuberculosis and preparation method and application thereof Download PDFInfo
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- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 128
- 239000001963 growth medium Substances 0.000 title claims abstract description 78
- 238000012258 culturing Methods 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title abstract description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 72
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 37
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 37
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a culture medium for rapidly culturing mycobacterium tuberculosis and a preparation method and application thereof, wherein bovine serum albumin, glucose, Dubos broth basis, glycerol and tween are used as raw materials and are mixed in a proper weight ratio, the bovine serum albumin can play a role in physiological and mechanical protection for the mycobacterium tuberculosis, the glucose and Dubos broth basis can provide carbon sources and nutrient substances required for rapid growth of the mycobacterium tuberculosis, and the glycerol and the Tween can effectively prevent the mycobacterium tuberculosis from aggregating into clusters in the proliferation process and are matched with the carbon sources and the nutrient substances, so that the contact inhibition of cell growth is avoided, and the rapid and high-density proliferation of the mycobacterium tuberculosis and the research of the application of the mycobacterium tuberculosis are facilitated. The method for rapidly culturing the mycobacterium tuberculosis by using the culture medium can realize rapid and high-density culture of the mycobacterium tuberculosis by activating the mycobacterium tuberculosis by using the Sauton culture medium and then culturing, and the culture period is shortened to 15-16 days.
Description
Technical Field
The invention belongs to the technical field of culture media, and particularly relates to a culture medium for rapidly culturing mycobacterium tuberculosis as well as a preparation method and application thereof.
Background
Mycobacterium tuberculosis, commonly known as Mycobacterium tuberculosis, is the causative agent of tuberculosis. The culture of the tubercle bacillus has decisive significance in the aspects of tuberculosis diagnosis, observation of chemotherapy effect, epidemiological indexes, research of the effect of antituberculosis drugs and the like. For a long time, the prior art adopts a Roche medium for culture, however, the growth of the Mycobacterium tuberculosis is slow, the culture period of the conventional culture method is about 30 days generally, and the detection of tuberculosis and the research of related medicaments are seriously influenced.
The prior art often shortens the growth cycle of Mycobacterium tuberculosis by optimizing the culture medium. Tanking et al found that phytohormone has an obvious proliferation promoting effect on tubercle bacillus, and the culture medium containing phytohormone is used for culturing the tubercle bacillus in a sputum specimen, so that the average time for obtaining a positive culture result of the sputum specimen is 15 days, but the cost is high. Huangqiwen, etc. developed a novel liquid culture medium for rapidly culturing mycobacterium tuberculosis based on fresh coconut juice and horse serum, but the cost is high, and the novel liquid culture medium is mainly used for detecting mycobacterium tuberculosis and is difficult to realize high-density culture.
In conclusion, the culture medium for mycobacterium tuberculosis in the prior art still has the problems of high cost, difficulty in realizing high-density culture, incapability of meeting the requirement of developing mycobacterium tuberculosis vaccines and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a culture medium which is low in price and suitable for rapidly culturing mycobacterium tuberculosis at high density, and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
a culture medium for rapidly culturing mycobacterium tuberculosis comprises raw materials of bovine serum albumin, glucose, Dubos broth base, glycerol and Tween.
The mass ratio of the bovine serum albumin, the glucose and the Dubos broth is (3-7): (5.5-9.5): (1.0-1.6), including but not limited to 4:6:1.2, 5:7:1.4, 5.5:8:1.5, 6:8.5:1.5, or 6.5:9: 1.4.
The glycerol is present in the culture medium at a volume percentage of 0.03% to 0.07%, including but not limited to 0.04%, 0.05% or 0.06%;
the volume percentage of the tween in the culture medium is 0.06% -0.1%, including but not limited to 0.07%, 0.08% or 0.09%;
the tween is tween 80.
The preparation method of the culture medium for rapidly culturing the mycobacterium tuberculosis comprises the following steps:
(1) weighing serum albumin solution and glucose according to the weight, dissolving the serum albumin solution and the glucose in physiological saline, and sterilizing to obtain bovine serum albumin solution;
(2) weighing Dubos broth base according to the weight, dissolving in deionized water, heating to boil, cooling to 50-60 ℃, and sterilizing to obtain Dubos broth base solution;
(3) and (3) adding the bovine serum albumin solution, glycerol and tween into the Dubos broth base solution obtained in the step (2) to obtain the culture medium for rapidly culturing the mycobacterium tuberculosis.
In the step (1), the sterilization is performed by filtering with a sterile filter; preferably, sterile filtration is carried out using a 0.22-0.4 μm (e.g., 0.22 μm, 0.3 μm, or 0.35 μm) sterile filter.
In the step (2), the sterilization is performed by high-pressure steam, preferably by high-pressure steam at the temperature of 115 ℃ and 121 ℃ for 15-30 min; after sterilization, the temperature was reduced to 50-60 ℃ to obtain the Dubos broth base solution.
The culture medium is applied to the rapid culture of the mycobacterium tuberculosis.
The method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps:
(S1) preparing a Sauton culture medium, and dissolving the bacterial colony or freeze-dried powder of the mycobacterium tuberculosis in the Sauton culture medium for activation to obtain activated mycobacterium tuberculosis;
(S2) adding the activated Mycobacterium tuberculosis of the step (S1) into the culture medium for rapidly culturing the Mycobacterium tuberculosis for culturing, thereby realizing the rapid culture of the Mycobacterium tuberculosis.
In the invention, the proliferation speed of the mycobacterium tuberculosis can be further accelerated by activating the mycobacterium tuberculosis by adopting the Sauton culture medium.
In step (S2), the culture temperature is 25-30 deg.C, including but not limited to 26 deg.C, 27 deg.C, 28 deg.C or 29 deg.C; the culture period is 15-16 days.
The invention has the following beneficial effects:
(1) according to the culture medium for rapidly culturing the mycobacterium tuberculosis, bovine serum albumin, glucose, Dubos broth basis, glycerol and Tween are adopted as raw materials and are subjected to proper weight proportion, wherein the bovine serum albumin can play a role in physiological and mechanical protection of the mycobacterium tuberculosis, the glucose and Dubos broth basis provides carbon sources and nutrient substances required by rapid growth of the mycobacterium tuberculosis, and the glycerol and the Tween can effectively prevent the mycobacterium tuberculosis from aggregating into clusters in the proliferation process and are matched with the carbon sources and the nutrient substances, so that the contact inhibition of cell growth is avoided, and the culture medium is beneficial to rapid and high-density proliferation of the mycobacterium tuberculosis and is applied to research on tuberculosis.
(2) According to the method for rapidly culturing the mycobacterium tuberculosis, the mycobacterium tuberculosis is activated by utilizing the Sauton culture medium, and then the activated mycobacterium tuberculosis is added into the culture medium for rapidly culturing the mycobacterium tuberculosis for culturing, so that the mycobacterium tuberculosis can be rapidly cultured at high density, and the culture period is shortened to 15-16 days.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a growth curve of Mycobacterium tuberculosis in example 1;
FIG. 2 is a photograph showing a stained specimen of a culture solution before the culture in example 1;
FIG. 3 is a photograph showing the stained culture broth after the completion of the culture in example 1;
FIG. 4 is a growth curve of Mycobacterium tuberculosis in example 2;
FIG. 5 is a growth curve of Mycobacterium tuberculosis in example 3;
FIG. 6 is a growth curve of Mycobacterium tuberculosis in example 4;
FIG. 7 is a growth curve of Mycobacterium tuberculosis in comparative example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the embodiments described are merely exemplary of the invention, and not of the invention in its entirety. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
In the following examples, reagents and instruments are not specified by manufacturers, and are all conventional products commercially available from normal sources.
Example 1
The embodiment provides a culture medium for rapidly culturing mycobacterium tuberculosis, which is prepared by adopting the following method:
(1) weighing 5g of Bovine Serum Albumin (BSA) powder and 7.5g of anhydrous D-glucose powder, dissolving in 100g of physiological saline, and filtering with a 0.22 μm sterile filter after complete dissolution to obtain a BSA solution;
(2) weighing 1.3g of Dubos broth base powder (Difco base culture medium of BD company, USA), dissolving in 180mL of deionized water, stirring with a glass rod, heating with microwave oven to boil, cooling to 55 deg.C, sterilizing with high pressure steam (121 deg.C, 30min), and cooling to 55 deg.C to obtain Dubos broth base solution;
(3) adding 20mL of the BSA solution into the Dubos broth base solution, and respectively adding glycerol and Tween 80(Tween-80) according to the volume percentage of 0.05% and 0.08%, thus obtaining the culture medium for rapidly culturing the mycobacterium tuberculosis.
Further, the method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps
(S1) preparing a Sauton culture medium: mixing 2g citric acid, 0.05g ferric ammonium citrate, 0.5g MgSO4、0.5g K2HPO44.0g L-aspartic acid, 60mL of glycerol and 800mL of deionized water are fully mixed, after the components are fully dissolved, 6mol/L of NaOH is used for adjusting the pH value of the solution to 7.2, the deionized water is continuously added to supplement the solution to 1000mL, and the solution is sterilized at 121 ℃ for 20min under high pressure to obtain a Sauton culture medium;
adding the bacterial colony of the mycobacterium tuberculosis into a centrifugal tube (Eppendorf, Ep tube) filled with the Sauton culture medium for activation, and storing at low temperature after the bacterial colony is dissolved into uniform and stable liquid to obtain activated mycobacterium tuberculosis;
(S2) adding 500. mu.L of the activated Mycobacterium tuberculosis of the step (S1) to 200mL of the rapid Mycobacterium tuberculosis culture medium at 25 ℃ for culturing; and detecting the absorbance value of the culture solution under 600nm, recording the data result, and dyeing and observing the culture solution before and after the culture.
The growth curve of Mycobacterium tuberculosis prepared according to the absorbance value is shown in FIG. 1, the Mycobacterium tuberculosis rapidly proliferates, and the culture period is shortened to 15 days; the staining results are shown in fig. 2 and 3, where fig. 2 is a staining microscopic image of the culture solution before the culture and shows that the cell density is low, and fig. 3 is a staining microscopic image of the culture solution after the culture is completed and shows that the cell density is significantly increased.
Example 2
The embodiment provides a culture medium for rapidly culturing mycobacterium tuberculosis, which is prepared by adopting the following method:
(1) weighing 3g of Bovine Serum Albumin (BSA) powder and 5.5g of anhydrous D-glucose powder, dissolving in 100g of physiological saline, and filtering by using a sterile filter of 0.22 μm after complete dissolution to obtain a BSA solution;
(2) weighing 1.0g of Dubos broth base powder (Difco base culture medium of BD company, USA), dissolving in 180mL of deionized water, stirring with a glass rod, heating with microwave oven to boil, cooling to 55 deg.C, sterilizing with high pressure steam (121 deg.C, 30min), and cooling to 55 deg.C to obtain Dubos broth base solution;
(3) adding 20mL of the BSA solution into the Dubos broth base solution, and respectively adding glycerol and Tween 80(Tween-80) according to the volume percentage of 0.03% and 0.1%, thus obtaining the culture medium for rapidly culturing the mycobacterium tuberculosis.
Further, the method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps
(S1) preparing a Sauton culture medium: mixing 2g citric acid, 0.05g ferric ammonium citrate, 0.5g MgSO4、0.5g K2HPO44.0g L-aspartic acid, 60mL of glycerol and 800mL of deionized water are fully mixed, after the components are fully dissolved, 6N NaOH is used for adjusting the pH value of the solution to 7.2, deionized water is continuously added to supplement the solution to 1000mL, and autoclaving is carried out at 121 ℃ for 20min to obtain a Sauton culture medium;
adding the bacterial colony of the mycobacterium tuberculosis into a centrifugal tube (Eppendorf, Ep tube) filled with the Sauton culture medium for activation, and storing at low temperature after the bacterial colony is dissolved into uniform and stable liquid to obtain activated mycobacterium tuberculosis;
(S2) adding 500. mu.L of the activated Mycobacterium tuberculosis of the step (S1) to 200mL of the rapid Mycobacterium tuberculosis culture medium at 30 ℃ for culturing; the absorbance value of the culture solution under 600nm is detected, the data result is recorded, the growth curve of the prepared mycobacterium tuberculosis is shown in figure 4, the rapid proliferation of the mycobacterium tuberculosis can be seen, and the culture period is shortened to 16 days.
Example 3
The embodiment provides a culture medium for rapidly culturing mycobacterium tuberculosis, which is prepared by adopting the following method:
(1) weighing 7g of Bovine Serum Albumin (BSA) powder and 9.5g of anhydrous D-glucose powder, dissolving in 100g of physiological saline, and filtering by using a sterile filter of 0.22 μm after complete dissolution to obtain a BSA solution;
(2) weighing 1.6g of Dubos broth base powder (Difco base culture medium of BD company, USA), dissolving in 180mL of deionized water, stirring with a glass rod, heating with microwave oven to boil, cooling to 55 deg.C, sterilizing with high pressure steam (121 deg.C, 30min), and cooling to 55 deg.C to obtain Dubos broth base solution;
(3) adding 20mL of BSA solution into the Dubos broth base solution, and respectively adding glycerol and Tween 80(Tween-80) according to the volume percentage of 0.07 percent and 0.06 percent to obtain the culture medium for rapidly culturing the mycobacterium tuberculosis.
Further, the method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps
(S1) preparing a Sauton culture medium: mixing 2g citric acid, 0.05g ferric ammonium citrate, 0.5g MgSO4、0.5g K2HPO44.0g L-aspartic acid, 60mL of glycerol and 800mL of deionized water are fully mixed, after the components are fully dissolved, 6N NaOH is used for adjusting the pH value of the solution to 7.2, deionized water is continuously added to supplement the solution to 1000mL, and autoclaving is carried out at 121 ℃ for 20min to obtain a Sauton culture medium;
adding the bacterial colony of the mycobacterium tuberculosis into a centrifugal tube (Eppendorf, Ep tube) filled with the Sauton culture medium for activation, and storing at low temperature after the bacterial colony is dissolved into uniform and stable liquid to obtain activated mycobacterium tuberculosis;
(S2) adding 500. mu.L of the activated Mycobacterium tuberculosis of the step (S1) to 200mL of the rapid Mycobacterium tuberculosis culture medium at 25 ℃ for culturing; detecting the absorbance value of the culture solution under 600nm, recording the data result, and obtaining the growth curve of the mycobacterium tuberculosis as shown in figure 5, wherein the rapid proliferation of the mycobacterium tuberculosis can be seen, and the culture period is shortened to 15-16 days.
Example 4
The embodiment provides a culture medium for rapidly culturing mycobacterium tuberculosis, which is prepared by adopting the following method:
(1) weighing 5g of Bovine Serum Albumin (BSA) powder and 7.5g of anhydrous D-glucose powder, dissolving in 100g of physiological saline, and filtering with a 0.22 μm sterile filter after complete dissolution to obtain a BSA solution;
(2) weighing 1.3g of Dubos broth base powder (Difco base culture medium of BD company, USA), dissolving in 180mL of deionized water, stirring with a glass rod, heating with microwave oven to boil, cooling to 55 deg.C, sterilizing with high pressure steam (121 deg.C, 30min), and cooling to 55 deg.C to obtain Dubos broth base solution;
(3) adding 20mL of BSA solution into the Dubos broth base solution, and respectively adding glycerol and Tween 80(Tween-80) according to the volume percentage of 0.07 percent and 0.06 percent to obtain the culture medium for rapidly culturing the mycobacterium tuberculosis.
Further, the method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps
Adding 500 mu LSauton culture medium dissolved mycobacterium tuberculosis into 200mL of the rapid mycobacterium tuberculosis culture medium for culture at 25 ℃; the absorbance value of the culture solution under 600nm is detected, the data result is recorded, the growth curve of the prepared mycobacterium tuberculosis is shown in figure 6, the rapid proliferation of the mycobacterium tuberculosis can be seen, and the culture period is shortened to 18-20 days.
Example 5
The embodiment provides a culture medium for rapidly culturing mycobacterium tuberculosis, which is prepared by adopting the following method:
(1) weighing 2g of Bovine Serum Albumin (BSA) powder and 4g of anhydrous D-glucose powder, dissolving in 100g of physiological saline, and filtering by using a sterile filter of 0.22 mu m after complete dissolution to obtain a BSA solution;
(2) weighing 0.8g of Dubos broth base powder (Difco base culture medium of BD company, USA), dissolving in 180mL of deionized water, stirring with a glass rod, heating with microwave oven to boil, cooling to 55 deg.C, sterilizing with high pressure steam (121 deg.C, 30min), and cooling to 55 deg.C to obtain Dubos broth base solution;
(3) adding 20mL of the BSA solution into the Dubos broth base solution, and respectively adding glycerol and Tween 80(Tween-80) according to the volume percentage of 0.05% and 0.08%, thus obtaining the culture medium for rapidly culturing the mycobacterium tuberculosis.
Further, the method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps
(S1) preparing a Sauton' S medium in the same manner as in example 1;
adding the bacterial colony of the mycobacterium tuberculosis into a centrifugal tube (Eppendorf, Ep tube) filled with the Sauton culture medium for activation, and storing at low temperature after the bacterial colony is dissolved into uniform and stable liquid to obtain activated mycobacterium tuberculosis;
(S2) adding 500. mu.L of the activated Mycobacterium tuberculosis of the step (S1) to 200mL of the rapid Mycobacterium tuberculosis culture medium at 25 ℃ for culturing, wherein the culture period is shortened to 15-16 days.
Example 6
The embodiment provides a culture medium for rapidly culturing mycobacterium tuberculosis, which is prepared by adopting the following method:
(1) weighing 9g of Bovine Serum Albumin (BSA) powder and 12g of anhydrous D-glucose powder, dissolving in 100g of physiological saline, and filtering by using a sterile filter of 0.22 mu m after complete dissolution to obtain a BSA solution;
(2) weighing 2g of Dubos broth base powder (Difco basic culture medium of BD company, USA), dissolving in 180mL of deionized water, stirring with a glass rod, heating with microwave oven to boil, cooling to 55 deg.C, sterilizing with high pressure steam (121 deg.C, 30min), and cooling to 55 deg.C to obtain Dubos broth base solution;
(3) adding 20mL of the BSA solution into the Dubos broth base solution, and respectively adding glycerol and Tween 80(Tween-80) according to the volume percentage of 0.05% and 0.08%, thus obtaining the culture medium for rapidly culturing the mycobacterium tuberculosis.
Further, the method for rapidly culturing the mycobacterium tuberculosis by applying the culture medium comprises the following steps
(S1) preparing a Sauton' S medium in the same manner as in example 1;
adding the bacterial colony of the mycobacterium tuberculosis into a centrifugal tube (Eppendorf, Ep tube) filled with the Sauton culture medium for activation, and storing at low temperature after the bacterial colony is dissolved into uniform and stable liquid to obtain activated mycobacterium tuberculosis;
(S2) adding 500. mu.L of the activated Mycobacterium tuberculosis of the step (S1) to 200mL of the rapid Mycobacterium tuberculosis culture medium at 25 ℃ for culturing, wherein the culture period is shortened to 15-16 days.
Comparative example 1
This comparative example differs from example 1 only in that: when the medium for rapidly culturing mycobacterium tuberculosis was prepared, the glycerol and tween 80 added in step (3) were replaced with the same amount of sterile water, and the rest were the same as in example 1.
The growth curve of the cultured Mycobacterium tuberculosis in this comparative example is shown in FIG. 7, the proliferation of Mycobacterium tuberculosis is slow, and the culture period is more than 30 days.
Comparative example 2
This comparative example differs from example 1 only in that: when the medium for rapidly culturing Mycobacterium tuberculosis was prepared, the glycerol added in step (3) was replaced with an equal amount of sterile water, and the rest was the same as in example 1. The culture period is more than 30 days.
Comparative example 3
This comparative example differs from example 1 only in that: when the medium for rapidly culturing mycobacterium tuberculosis is prepared, the tween 80 added in the step (3) is replaced by the same amount of sterile water, and the rest is the same as that in the example 1. The culture period is more than 30 days.
Comparative example 4
This comparative example differs from example 1 only in that: when the medium for rapidly culturing mycobacterium tuberculosis is prepared, the tween 80 added in the step (3) is replaced by the same amount of tween 20, and the rest is the same as that in the example 1. The culture period is more than 20 days.
Comparative example 5
This comparative example differs from example 1 only in that: when the medium for rapidly culturing mycobacterium tuberculosis is prepared, the tween 80 added in the step (3) is replaced by the same amount of ethylene glycol, and the rest is the same as that in the example 1. The culture period is more than 30 days.
From the above results, it can be seen that examples 1 to 6, using the medium for rapid culture of Mycobacterium tuberculosis of the present invention, can rapidly and highly densely culture Mycobacterium tuberculosis; compared with the examples 1-3, the Mycobacterium tuberculosis is not activated by the Sauton culture medium in the example 4, the culture period is slightly longer and is 30 days; the mass ratios of bovine serum albumin, glucose and Dubos broth bases were not controlled in examples 5 and 6 at (3-7): (5.5-9.5): (1.0-1.6) the culture period of Mycobacterium tuberculosis is slightly longer, 25 days; compared with example 1, in comparative example 1, glycerol and tween are not added in the medium for rapidly culturing mycobacterium tuberculosis, so that the mycobacterium tuberculosis cannot be rapidly proliferated, the culture period is more than 30 days, in comparative examples 2-3, glycerol and tween 80 are not simultaneously adopted, the growth of the mycobacterium tuberculosis is slow, and the culture period is up to 30 days. In comparative examples 4-5, Tween 80 was replaced with Tween 20 and ethylene glycol, and the growth of Mycobacterium tuberculosis was slow, and the culture period was 20-30 days, thus demonstrating that the effect of rapidly culturing Mycobacterium tuberculosis could only be achieved by using glycerol and Tween 80 simultaneously.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A culture medium for rapidly culturing mycobacterium tuberculosis is characterized in that raw materials of the culture medium comprise bovine serum albumin, glucose, Dubos broth base, glycerol and Tween.
2. The medium for rapidly culturing Mycobacterium tuberculosis according to claim 1, wherein the mass ratio of bovine serum albumin, glucose and Dubos broth base is (3-7): (5.5-9.5): (1.0-1.6).
3. The medium for rapidly culturing Mycobacterium tuberculosis according to claim 1, wherein the glycerol is present in the medium in an amount of 0.03 to 0.07% by volume.
4. The medium for rapidly culturing mycobacterium tuberculosis according to claim 1, wherein the volume percentage of the tween in the medium is 0.06% -0.1%.
5. The medium for rapidly culturing Mycobacterium tuberculosis according to claim 1, wherein the tween is tween 80.
6. The method for preparing a medium for rapid culture of Mycobacterium tuberculosis as set forth in claims 1 to 5, comprising the steps of:
(1) weighing serum albumin solution and glucose according to the weight, dissolving the serum albumin solution and the glucose in physiological saline, and sterilizing to obtain bovine serum albumin solution;
(2) weighing Dubos broth base according to the weight, dissolving in deionized water, heating to boil, cooling to 50-60 ℃, and sterilizing to obtain Dubos broth base solution;
(3) and (3) adding the bovine serum albumin solution, glycerol and tween into the Dubos broth base solution obtained in the step (2) to obtain the culture medium for rapidly culturing the mycobacterium tuberculosis.
7. The method for preparing a medium for rapid culture of Mycobacterium tuberculosis as claimed in claim 6, wherein in the step (1), the sterilization is performed by filtration using a sterile filter;
in the step (2), the sterilization is performed by adopting high-pressure steam, and after the sterilization, the temperature is reduced to 50-60 ℃ to obtain the Dubos broth base solution.
8. Use of the medium of claims 1-5 for rapid culture of mycobacterium tuberculosis.
9. A method for rapidly culturing Mycobacterium tuberculosis by using the culture medium of claims 1-5, which is characterized by comprising the following steps:
(S1) preparing a Sauton culture medium, and dissolving the bacterial colony or freeze-dried powder of the mycobacterium tuberculosis in the Sauton culture medium for activation to obtain activated mycobacterium tuberculosis;
(S2) adding the activated Mycobacterium tuberculosis of the step (S1) into the culture medium for rapidly culturing the Mycobacterium tuberculosis for culturing, thereby realizing the rapid culture of the Mycobacterium tuberculosis.
10. The method for rapid culture of Mycobacterium tuberculosis as claimed in claim 9, wherein the culturing temperature is 25-30 ℃ and the culturing period is 15-16 days in the step (S2).
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Citations (2)
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| CN101925819A (en) * | 2007-12-28 | 2010-12-22 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| US20190137492A1 (en) * | 2016-03-11 | 2019-05-09 | S&R Pharmaceuticals, Llc | Assays And Methods For Detecting Mycobacterial Infections |
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| CN101925819A (en) * | 2007-12-28 | 2010-12-22 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| US20190137492A1 (en) * | 2016-03-11 | 2019-05-09 | S&R Pharmaceuticals, Llc | Assays And Methods For Detecting Mycobacterial Infections |
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