CN113637613B - Culture medium for mycoplasma pneumoniae of human origin and preparation method and application thereof - Google Patents
Culture medium for mycoplasma pneumoniae of human origin and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a mycoplasma pneumoniae culture medium and a preparation method thereof, wherein the culture medium is prepared from a basic culture medium, a culture medium additive, fetal calf serum and penicillin sodium according to a volume ratio of 800 ml: (0.5 to 1.5) ml: (180 to 220) ml: (2.0 to 3.0) ml; the basic culture medium comprises tryptone, soybean peptone, bacterial peptone, sodium pyruvate, beef extract powder, yeast extract powder, sodium chloride, L-cysteine, glucose and phenol red; the culture medium additive comprises glutathione and vitamin C. The culture medium can meet the growth requirement of the human mycoplasma pneumoniae, greatly accelerates the growth and propagation speed of the mycoplasma pneumoniae, solves the problems of complex culture conditions, slow growth speed of the human mycoplasma pneumoniae, complex culture steps and the like of the conventional mycoplasma pneumoniae culture medium, and has important significance for acquisition of mycoplasma pneumoniae antigens and scientific research thereof.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a mycoplasma culture medium preparation technology, and particularly relates to a human mycoplasma pneumoniae culture medium and a preparation method and application thereof.
Background
The mycoplasma pneumoniae is a pathogen of human mycoplasma pneumonia, mainly causes interstitial pneumonia of human, sometimes causes bronchopneumonia, is primary atypical pneumonia, can occur all the year round, and brings huge challenges to human health. Since the pathogenic microorganism is mycoplasma, the pathogenic culture is extremely difficult.
At present, the original mycoplasma pneumoniae is not provided with a special culture medium temporarily, and other culture mediums are used for culturing the original mycoplasma pneumoniae, so that the defects of complex culture conditions, slow growth and propagation speed of the original mycoplasma pneumoniae, complex culture steps and the like exist, and the corresponding target cannot be achieved.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a culture medium for human mycoplasma pneumoniae and a preparation method and application thereof, the culture medium greatly improves the growth and propagation speed of the human mycoplasma pneumoniae, fully meets the nutritional requirement and the growth requirement of the human mycoplasma pneumoniae, and has important significance for isolated culture, enrichment and amplification, epidemic disease prevention and control and molecular diagnosis of the human mycoplasma pneumoniae; the preparation method does not need the support of large instruments and equipment, is suitable for large-scale production, and has important significance for acquisition of mycoplasma pneumoniae antigens and scientific research thereof.
The technical scheme of the invention is as follows:
a culture medium for human mycoplasma pneumoniae is prepared from basic culture medium, culture medium additives, fetal calf serum and penicillin sodium according to a volume ratio of 800 ml: (0.5 to 1.5) ml: (180 to 220) ml: (2.0 to 3.0) ml; the basic culture medium comprises tryptone, soybean peptone, bacterial peptone, sodium pyruvate, beef extract powder, yeast extract powder, sodium chloride, L-cysteine, glucose and phenol red; the culture medium additive comprises glutathione and vitamin C.
The culture of the human mycoplasma pneumoniae has special requirements on culture conditions, environment and nutrition, and the culture medium has important functions on serum and the external gas environment for the growth of the serum and the like besides organic carbon sources, nitrogen sources and inorganic salts for maintaining the growth, the propagation and the survival of cells. The invention designs the components, contents, preparation processes and culture processes of various nutrients in the culture medium of the mycoplasma pneumoniae according to the nutritional requirements in the processes of growth, propagation, metabolism and the like of the mycoplasma pneumoniae. According to the characteristics of the requirements of the growth of the external gas environment of the mycoplasma pneumoniae and the like, the improved culture medium can be used for eliminating the limitation of the traditional 5% carbon dioxide incubator, and is more suitable for large-scale production and popularization.
In the human mycoplasma pneumoniae culture medium, the tryptone, the soybean peptone and the bactopeptone provide necessary nitrogen sources for the human mycoplasma pneumoniae culture medium, the sodium pyruvate, the glucose and the like provide carbon sources for the human mycoplasma pneumoniae culture medium, the glutathione and the vitamin C provide a required redox environment for the human mycoplasma pneumoniae culture medium, and the phenol red is used as an indicator for indicating the growth and metabolism conditions of the human mycoplasma pneumoniae culture medium.
The inventor finds that the fetal calf serum has important significance on the growth and the propagation of the human mycoplasma pneumoniae. The mycoplasma pneumoniae of human origin hardly grows in the culture medium without serum, and the culture effect is not ideal; the cost is increased due to excessive addition of fetal calf serum in the culture medium, and the expected growth and propagation effects cannot be achieved due to too little addition of fetal calf serum, so that high requirements are provided for the proportion of the culture medium. The addition amount of the fetal calf serum is gradually adjusted in an experiment, and the growth and reproduction effects of the human mycoplasma pneumoniae are remarkably improved after the proportion of the fetal calf serum is gradually increased to 20% of the fetal calf serum, and then the growth and reproduction speed is increased continuously until 30% of the fetal calf serum is added, so that the growth and reproduction speed is not greatly improved compared with that of the 20% of the fetal calf serum. Therefore, based on the experimental results, the addition ratio of fetal bovine serum was determined as: basic culture medium: fetal bovine serum 800 ml: (180 to 220) ml; preferably, the fetal calf serum is added in the proportion of the basic culture medium: fetal bovine serum 800 ml:200 And (3) ml.
Further, the fetal bovine serum is inactivated serum; fetal bovine serum was inactivated at 56 ℃ for 30 min, and filter sterilized with a 0.45 μm filter.
Further, the penicillin sodium is 120 ten thousand units of penicillin sodium which is diluted to 120 ten thousand units by sterile deionized water, and the penicillin sodium is filtered and sterilized by a 0.45-micron filter.
Further, sterile deionized water is adopted in the culture medium additive to dissolve glutathione and vitamin C, and a 0.45-micron filter is used for filtering and sterilizing.
Further, the pH value of the culture medium of the mycoplasma pneumoniae is 7.2 to 7.6; the mycoplasma pneumoniae culture medium can also be added with agar to prepare a solid culture medium for use.
Preferably, the volume ratio of the basal medium, the medium additive, the fetal calf serum and the 120 ten thousand units of penicillin sodium is 800 ml: (0.7 to 1.3) ml: (190 to 210) ml: (2.3 to 2.5) ml;
every 800 mL basal medium contains: 4-6 g of tryptone, 4-6 g of soybean peptone, 4-6 g of bacterial peptone, 8-12 g of sodium pyruvate, 4-6 g of beef extract, 4-6 g of yeast extract, 2-3 g of sodium chloride, 0.1-0.5 g of L-cysteine, 4-6 g of glucose and 0.02 g phenol red; the additive comprises the following components in every 50 mL culture medium: 0.8-1.2 g of glutathione and 5.5-6.5 g of vitamin C; adjusting the pH value to 7.2-7.6.
Preferably, the culture medium is prepared from basic culture medium, culture medium additives, fetal bovine serum and 120 ten thousand units of penicillin sodium according to a volume ratio of 800 mL:1 mL:200 mL:2.4 mL is prepared;
every 800 mL basal medium contains: tryptone 5.0 g, soy peptone 5.0 g, bactopeptone 5.0 g, sodium pyruvate 10.0 g, beef extract powder 5.0 g, yeast extract powder 5.0 g, sodium chloride 2.5 g, L-cysteine 0.3 g, glucose 5.0 g, phenol red 0.02 g; the additive comprises the following components in every 50 mL culture medium: glutathione 1 g, vitamin C6 g.
The preparation method of the culture medium of the human mycoplasma pneumoniae comprises the following steps:
(1) Respectively weighing tryptone, soybean peptone, bacterial peptone, sodium pyruvate, beef extract powder, yeast extract powder, sodium chloride, L-cysteine, glucose and phenol red, adding distilled water, fully mixing and dissolving, adjusting the pH to 7.2-7.6, and sterilizing at 121 ℃ for 30 min under high pressure to prepare a basic culture medium; wherein the adopted distilled water is double distilled water;
(2) Respectively weighing glutathione and vitamin C, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, and preparing to obtain a culture medium additive;
(3) And cooling the basic culture medium to room temperature, and adding a culture medium additive, inactivated fetal calf serum and 120 ten thousand units of penicillin sodium into the basic culture medium according to the volume ratio to prepare the human mycoplasma pneumoniae culture medium.
Furthermore, the invention provides the application of the culture medium of the mycoplasma pneumoniae in the rapid enrichment culture of the mycoplasma pneumoniae.
Furthermore, the invention provides an application of the culture medium of the mycoplasma pneumoniae of human origin in preparing the molecular diagnostic antigen of the mycoplasma pneumoniae of human origin.
The invention also provides a culture method of the human mycoplasma pneumoniae, which comprises the following steps: the mycoplasma pneumoniae is inoculated into the prepared mycoplasma pneumoniae culture medium, sealing and static culture are carried out at 37 ℃, 5% carbon dioxide culture is not needed, and therefore a carbon dioxide incubator is not needed for culture.
The invention also provides application of the cultured human mycoplasma pneumoniae in preparing mycoplasma pneumoniae vaccines and antigens of molecular diagnostic kits.
The invention has the beneficial effects that:
(1) The human mycoplasma pneumoniae culture medium is designed according to the physiological metabolism and the favorite characteristics of the human mycoplasma pneumoniae, so that the growth and the propagation speed of the mycoplasma pneumoniae are greatly increased, the culture density of the mycoplasma pneumoniae is greatly improved, the culture time can be shortened, and the human mycoplasma pneumoniae culture medium has important significance in acquisition of mycoplasma pneumoniae antigens and scientific research on the acquisition of the mycoplasma pneumoniae antigens.
(2) The culture medium fully meets the nutritional requirements and growth requirements of the human mycoplasma pneumoniae, is suitable for multiple fields of enrichment culture, separation diagnosis, academic research, molecular diagnosis antigen preparation, vaccine preparation and the like of the human mycoplasma pneumoniae, and has important significance for disease prevention and control, molecular diagnosis and the like.
(3) The culture medium for the human mycoplasma pneumoniae has the advantages of easily obtained components, clear dosage and simple preparation method, simplifies the culture method of the mycoplasma pneumoniae, does not need 5% carbon dioxide culture in use, gets rid of the inconvenience of using a carbon dioxide incubator for culture, improves the feasibility of large-scale production and use of the human mycoplasma pneumoniae, and has strong popularization.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the embodiments described below are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. For further understanding of the present invention, the present invention will be further described with reference to examples.
Unless otherwise specified, the technical means employed in the examples and comparative examples are conventional means well known to those skilled in the art, and the test materials used are conventional test materials in the art and commercially available.
Example 1
The culture medium of the human mycoplasma pneumoniae is prepared from a basic culture medium, a culture medium additive, fetal calf serum and 120 ten thousand units of penicillin sodium according to a volume ratio of 800 mL:1 mL:200 mL:2.4 mL is prepared;
every 800 mL basal medium contains: tryptone 5.0 g, soybean peptone 5.0 g, bacterial peptone 5.0 g, sodium pyruvate 10.0 g, beef extract 5.0 g, yeast extract 5.0 g, sodium chloride 2.5 g, L-cysteine 0.3 g, glucose 5.0 g, phenol red 0.02 g; the additive for every 50 mL culture medium comprises: glutathione 1 g, vitamin C6 g;
the preparation method comprises the following steps:
(1) Respectively weighing tryptone 5.0 g, soybean peptone 5.0 g, bacterial peptone 5.0 g, sodium pyruvate 10.0 g, beef extract powder 5.0 g, yeast extract powder 5.0 g, sodium chloride 2.5 g, L-cysteine 0.3 g, glucose 5.0 g, phenol red 0.02 g, adding double distilled water, fully mixing and dissolving, adjusting pH to 7.4, adjusting the double distilled water to 800 mL, carrying out autoclaving at 121 ℃ for 30 min, and preparing a basic culture medium;
(2) Respectively weighing glutathione 1 g and vitamin C6 g, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, and metering the volume to 50 mL to prepare a culture medium additive;
(3) Inactivating fetal calf serum at 56 deg.C for 30 min, filtering with 0.45 μm filter, and sterilizing; diluting the penicillin sodium into 120 ten thousand units of penicillin sodium by adopting sterile deionized water, and filtering and sterilizing by using a 0.45-micrometer filter;
(4) Cooling the basic culture medium to room temperature, adding 1 mL culture medium additive, 200 mL inactivated fetal calf serum and 2.4 mL 120 ten thousand units of penicillin sodium into 800 mL basic culture medium according to volume ratio, and fully and uniformly mixing to prepare the human mycoplasma pneumoniae culture medium.
Example 2
The culture medium of the human mycoplasma pneumoniae is prepared from a basic culture medium, a culture medium additive, fetal calf serum and 120 ten thousand units of penicillin sodium according to a volume ratio of 800 mL:0.5 mL:180 mL:2.0 mL is prepared;
every 800 mL basal medium contains: tryptone 4.0 g, soybean peptone 4.0 g, bacterial peptone 4.0 g, sodium pyruvate 8.0 g, beef extract powder 4.0 g, yeast extract powder 4.0 g, sodium chloride 2.0 g, L-cysteine 0.1 g, glucose 4.0 g, phenol red 0.02 g; the additive comprises the following components in every 50 mL culture medium: 0.8 g of glutathione, 5.5 g of vitamin C;
the preparation method comprises the following steps:
(1) Respectively weighing tryptone 4.0 g, soybean peptone 4.0 g, bactopeptone 4.0 g, sodium pyruvate 8.0 g, beef extract powder 4.0 g, yeast extract powder 4.0 g, sodium chloride 2.0 g, L-cysteine 0.1 g, glucose 4.0 g, phenol red 0.02 g, adding double distilled water, fully mixing and dissolving, adjusting the pH to 7.2, adjusting the pH to 800 mL, sterilizing at 121 ℃ for 30 min under high pressure, and preparing a constant volume basal medium;
(2) Respectively weighing glutathione 0.8 g and vitamin C5.5 g, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, and metering to 50 mL to obtain a culture medium additive;
(3) Inactivating fetal calf serum at 56 deg.C for 30 min, filtering with 0.45 μm filter, and sterilizing; diluting the penicillin sodium into 120 ten thousand units of penicillin sodium by adopting sterile deionized water, and filtering and sterilizing by adopting a 0.45-micrometer filter;
(4) Cooling the basal culture medium to room temperature, adding 0.5 mL culture medium additive, 180 mL inactivated fetal calf serum and 2.0 mL 120 ten thousand unit penicillin sodium into 800 mL basal culture medium according to volume ratio, and fully and uniformly mixing to prepare the human mycoplasma pneumoniae culture medium.
Example 3
The culture medium of the human mycoplasma pneumoniae is prepared from a basic culture medium, a culture medium additive, fetal calf serum and 120 ten thousand units of penicillin sodium according to a volume ratio of 800 mL:1.5 mL:220 mL:2.6 mL is prepared;
every 800 mL basal medium contains: tryptone 6.0 g, soybean peptone 6.0 g, bacterial peptone 6.0 g, sodium pyruvate 12.0 g, beef extract powder 6.0 g, yeast extract powder 6.0 g, sodium chloride 3.0 g, L-cysteine 0.5 g, glucose 6.0 g, phenol red 0.02 g; the additive comprises the following components in every 50 mL culture medium: glutathione 1.2 g, vitamin C6.5 g;
the preparation method comprises the following steps:
(1) Respectively weighing tryptone 6.0 g, soybean peptone 6.0 g, bactopeptone 6.0 g, sodium pyruvate 12.0 g, beef extract powder 6.0 g, yeast extract powder 6.0 g, sodium chloride 3.0 g, L-cysteine 0.5 g, glucose 6.0 g, phenol red 0.02 g, adding double distilled water, fully mixing and dissolving, adjusting the pH to 7.6, adjusting the double distilled water to 800 mL, and carrying out autoclaving at 121 ℃ for 30 min to prepare a constant volume basal medium;
(2) Respectively weighing glutathione 1.2 g and vitamin C6.5 g, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, metering the volume to 50 mL, and preparing to obtain a culture medium additive;
(3) Inactivating fetal calf serum at 56 deg.C for 30 min, filtering with 0.45 μm filter, and sterilizing; diluting the penicillin sodium into 120 ten thousand units of penicillin sodium by adopting sterile deionized water, and filtering and sterilizing by adopting a 0.45-micrometer filter;
(4) Cooling the basic culture medium to room temperature, adding 1.5 mL culture medium additive, 220 mL inactivated fetal calf serum and 2.6 mL 120 ten thousand units of penicillin sodium into 800 mL basic culture medium according to volume ratio, and fully and uniformly mixing to prepare the human mycoplasma pneumoniae culture medium.
Example 4
The culture medium of the human mycoplasma pneumoniae is prepared from a basic culture medium, a culture medium additive, fetal calf serum and 120 ten thousand units of penicillin sodium according to a volume ratio of 800 mL:1 mL:210 mL:2.4 mL is prepared;
every 800 mL basal medium contains: tryptone 5.0 g, soybean peptone 5.0 g, bacterial peptone 5.0 g, sodium pyruvate 10.0 g, beef extract powder 5.0 g, yeast extract powder 5.0 g, sodium chloride 2.5 g, L-cysteine 0.3 g, glucose 5.0 g,20.0g agar powder, phenol red 0.02 g; the additive for every 50 mL culture medium comprises: glutathione 1 g, vitamin C6 g;
the preparation method comprises the following steps:
(1) Respectively weighing tryptone 5.0 g, soybean peptone 5.0 g, bacterial peptone 5.0 g, sodium pyruvate 10.0 g, beef extract powder 5.0 g, yeast extract powder 5.0 g, sodium chloride 2.5 g, L-cysteine 0.3 g, glucose 5.0 g,20.0g agar powder and phenol red 0.02 g, adding double distilled water, fully mixing and dissolving, adjusting the pH to 7.4, fixing the volume of the double distilled water to 800 mL, and autoclaving at 121 ℃ for 30 min to prepare a basal medium;
(2) Respectively weighing glutathione 1 g and vitamin C6 g, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, and metering the volume to 50 mL to prepare a culture medium additive;
(3) Inactivating fetal calf serum at 56 deg.C for 30 min, filtering with 0.45 μm filter, and sterilizing; diluting the penicillin sodium into 120 ten thousand units of penicillin sodium by adopting sterile deionized water, and filtering and sterilizing by using a 0.45-micrometer filter;
(4) Cooling the basic culture medium to 40 ℃, adding 1 mL culture medium additive, 210 mL inactivated fetal calf serum and 120 million units of penicillin sodium of 2.4 mL into 800 mL basic culture medium according to volume ratio, fully and uniformly mixing, and pouring plates to obtain the human mycoplasma pneumoniae culture medium.
Example 5
The culture medium of the human mycoplasma pneumoniae is prepared from a basic culture medium, a culture medium additive, fetal calf serum and 120 ten thousand units of penicillin sodium according to a volume ratio of 800 mL:0.7 mL:190 mL:2.2 mL is prepared;
every 800 mL basal medium contains: tryptone 6.0 g, soybean peptone 6.0 g, bacterial peptone 4.0 g, sodium pyruvate 12.0 g, beef extract powder 4.0 g, yeast extract powder 4.0 g, sodium chloride 3.0 g, L-cysteine 0.5 g, glucose 6.0 g, phenol red 0.02 g; the additive comprises the following components in every 50 mL culture medium: glutathione 0.8 g, vitamin C5.5 g;
the preparation method comprises the following steps:
(1) Weighing tryptone 6.0 g, soybean peptone 6.0 g, bacterial peptone 4.0 g, sodium pyruvate 12.0 g, beef extract powder 4.0 g, yeast extract powder 4.0 g, sodium chloride 3.0 g, L-cysteine 0.5 g, glucose 6.0 g, phenol red 0.02 g, adding double distilled water, fully mixing and dissolving, adjusting pH to 7.2, adjusting the double distilled water to 800 mL, carrying out autoclaving at 121 ℃ for 30 min, and preparing a basal medium;
(2) Respectively weighing glutathione 0.8 g and vitamin C5.5 g, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, and metering to 50 mL to obtain a culture medium additive;
(3) Inactivating fetal calf serum at 56 deg.C for 30 min, filtering with 0.45 μm filter, and sterilizing; diluting the penicillin sodium into 120 ten thousand units of penicillin sodium by adopting sterile deionized water, and filtering and sterilizing by adopting a 0.45-micrometer filter;
(4) Cooling the basic culture medium to room temperature, adding 0.7 mL culture medium additive, 190 mL inactivated fetal calf serum and 2.2 mL 120 ten thousand units of penicillin sodium into 800 mL basic culture medium according to volume ratio, and fully and uniformly mixing to prepare the human mycoplasma pneumoniae culture medium.
Comparative example 1
Weighing 5.0 g tryptone, 5.0 g soybean peptone, 5.0 g bacterial peptone, 5.0 g beef extract powder, 5.0 g yeast extract powder, 2.5 g sodium chloride, 0.3 g L-cysteine, 5.0 g glucose and 0.02 g phenol red respectively, fully mixing and dissolving by using distilled water, fixing the volume to 800 mL, and autoclaving at 121 ℃ for 15 min to prepare a basal medium; inactivating the fetal calf serum at 56 deg.C for 30 min, and filtering with 0.45 μm filter for sterilization;
adding 200 mL fetal calf serum and 120 kilomega units of penicillin sodium of 2.4 mL into 800 mL basal medium cooled to room temperature, mixing uniformly, and adjusting the pH to 7.2-7.6 to obtain the medium A.
Comparative example 2
Weighing 5.0 g tryptone, 5.0 g soybean peptone, 5.0 g bacterial peptone, 10.0 g sodium pyruvate, 5.0 g beef extract powder, 5.0 g yeast extract powder, 2.5 g sodium chloride, 0.3 g L-cysteine, 5.0 g glucose, 0.02 g phenol red respectively, fully mixing and dissolving by using distilled water, fixing the volume to 800 mL, and autoclaving at 121 ℃ for 15 min to prepare a basal medium; inactivating fetal calf serum at 56 deg.C for 30 min, and filtering with 0.45 μm filter for sterilization;
adding 200 mL fetal calf serum and 120 kilomega units of penicillin sodium of 2.4 mL into 800 mL basal medium cooled to room temperature, mixing uniformly, and adjusting the pH to 7.2-7.6 to obtain the medium B.
Comparative example 3
Weighing 5.0 g tryptone, 5.0 g soybean peptone, 5.0 g bacterial peptone, 10.0 g sodium pyruvate, 5.0 g beef extract powder, 5.0 g yeast extract powder, 2.5 g sodium chloride, 0.3 g L-cysteine, 5.0 g glucose, 0.02 g phenol red respectively, fully mixing and dissolving by using distilled water, fixing the volume to 800 mL, and autoclaving at 121 ℃ for 15 min to prepare a basal medium; inactivating fetal calf serum at 56 deg.C for 30 min, and filtering with 0.45 μm filter for sterilization;
adding 100 mL fetal calf serum and 120 kilomega units of penicillin sodium of 2.4 mL into a 900 mL basic culture medium cooled to room temperature, uniformly mixing, and adjusting the pH to 7.2-7.6 to obtain a culture medium C.
Test example 1
Culture of a human-derived mycoplasma pneumoniae:
1. the test method comprises the following steps:
a human-derived Mycoplasma pneumoniae was inoculated into each of the media prepared in examples 1 to 5, and placed in a 37 ℃ chamber and left to stand.
Culture was performed under the same conditions using SP4 Medium (Remel Co.), ATCC Medium 2611, mycoplasma broth Medium (HB 7025-2), mycoplasma pneumoniae liquid isolation Medium (Chanda Co.), mycoplasma broth base CM403 (OXOID Co.), mycoplasma pneumoniae rapid culture kit (Daiichi Bioengineering Co., ltd.), MP rapid culture solution, mycoplasma pneumoniae broth base (Qingdao Nippon Japan), and PPLO Medium (BD Co.) as controls.
The cultures were left to stand, the change in color of the medium was observed, and the time required for the medium color to change from red to yellow was recorded.
2. And (3) test results:
the color change time of the culture medium under the same conditions in each group of culture medium is shown in Table 1.
TABLE 1 time to color change of each medium under the same conditions
| Culture medium | Time at which color change occurs |
| Mycoplasma pneumoniae culture media of examples 1 to 5 | 40h |
| SP4 Medium (Remel Co.) | 72h |
| ATCC®Medium 2611 | 96h |
| Mycoplasma broth culture medium (HB 7025-2) | 144h |
| Mycoplasma pneumoniae liquid isolation medium (Lechangda company) | 144h |
| Mycoplasma broth base CM403 (OXOID Corp.) | 120h |
| Mycoplasma pneumoniae rapid culture kit (Zhuhai Dier bioengineering Co., ltd.) | 168h |
| MP rapid culture solution | 144h |
| Mycoplasma pneumoniae broth foundation (Qingdao Nishui) | 168h |
| PPLO Medium (BD Co.) | 120h |
| Medium A (comparative example 1) | 120h |
| Medium B (comparative example 2) | 96h |
| Medium C (comparative example 3) | 144h |
1000 mL of the culture medium groups in Table 1 are respectively measured, strains are inoculated, the strains are cultured for 7 days under the same culture conditions, the strains are centrifuged at 12000 rpm for 60 min to collect the strains, and the wet weight of the strains is weighed, and the results are shown in Table 2.
TABLE 2 Wet weight of bacteria cultured in each medium under the same conditions
| Culture medium | Dampness and gravity of fungi |
| Mycoplasma pneumoniae of human origin culture media of examples 1 to 5 | 0.2000g |
| SP4 Medium (Remel Co.) | 0.1630g |
| ATCC®Medium 2611 | 0.1501g |
| Mycoplasma broth culture medium (HB 7025-2) | 0.1423g |
| Mycoplasma pneumoniae liquid isolation medium (Lechangda company) | 0.1389g |
| Mycoplasma broth base CM403 (OXOID Corp.) | 0.1702g |
| Mycoplasma pneumoniae rapid culture kit (Zhuhai Dier bioengineering Co., ltd.) | 0.1401g |
| MP rapid culture solution | 0.1008g |
| Mycoplasma pneumoniae broth foundation (Qingdao Nishui) | 0.1521g |
| PPLO Medium (BD Co.) | 0.1432g |
| Medium A (comparative example 1) | 0.1721g |
| Medium B (comparative example 2) | 0.1323g |
| Medium C (comparative example 3) | 0.1056g |
As can be seen from tables 1 and 2, compared with the SP4 Medium (Remel Corp.), ATCC Medium 2611, mycoplasma broth Medium (HB 7025-2), mycoplasma pneumoniae liquid isolation Medium (Chanda Corp.), mycoplasma pneumoniae broth base CM403 (OXOID Corp.), mycoplasma pneumoniae rapid culture kit (Du Zhuhai Diel bioengineering Co., ltd.), MP rapid culture solution, mycoplasma pneumoniae broth base (Qingdao Nippon water) and PPLO Medium (BD Corp.), the Mycoplasma pneumoniae culture Medium prepared by the present invention is more suitable for rapid culture and mass fermentation of Mycoplasma pneumoniae of human origin, and compared with the culture mediums prepared by comparative examples 1 to 3, it can be found that the growth and propagation rates of Mycoplasma pneumoniae of human origin are reduced by adjusting the raw material composition or the ratio of the Mycoplasma pneumoniae culture Medium of the present invention.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or modification made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The mycoplasma pneumoniae culture medium is characterized by comprising a basic culture medium, a culture medium additive, fetal bovine serum and penicillin sodium according to a volume ratio of 800 ml: (0.5 to 1.5) ml: (180 to 220) ml: (2.0 to 3.0) ml; the basic culture medium comprises tryptone, soybean peptone, bacterial peptone, sodium pyruvate, beef extract powder, yeast extract powder, sodium chloride, L-cysteine, glucose and phenol red; the culture medium additive comprises glutathione and vitamin C; every 800 ml basal medium contains: 4-6 g of tryptone, 4-6 g of soybean peptone, 4-6 g of bacterial peptone, 8-12 g of sodium pyruvate, 4-6 g of beef extract, 4-6 g of yeast extract, 2-3 g of sodium chloride, 0.1-0.5 g of L-cysteine, 4-6 g of glucose and 0.02 g phenol red; the additive comprises the following components in every 50 ml culture medium: 0.8 to 1.2 g of glutathione and 5.5 to 6.5 g of vitamin C; adjusting the pH value to 7.2-7.6.
2. The mycoplasma pneumoniae culture medium of claim 1, wherein the fetal bovine serum is inactivated serum; inactivating fetal calf serum at 56 deg.C for 30 min, filtering with 0.45 μm filter, and sterilizing; the penicillin sodium is 120 ten thousand units of penicillin sodium diluted to 120 ten thousand units by adopting sterile deionized water, and a 0.45 mu m filter is used for filtering and sterilizing; the glutathione and the vitamin C are dissolved in the culture medium additive by adopting sterile deionized water, and the culture medium additive is filtered and sterilized by a 0.45 mu m filter.
3. The mycoplasma pneumoniae culture medium according to claim 1, wherein agar is further added to the mycoplasma pneumoniae culture medium to prepare a solid culture medium.
4. The mycoplasma pneumoniae culture medium of any one of claims 1-3, wherein the basal medium, the culture medium additives, the fetal bovine serum, and the 120-kilo-unit sodium penicillin are in a volume ratio of 800 ml: (0.7 to 1.3) ml: (190 to 210) ml: (2.3 to 2.5) ml.
5. The mycoplasma pneumoniae culture medium according to any one of claims 1-3, wherein the culture medium is prepared from a basal medium, culture medium additives, fetal bovine serum and 120 million units of penicillin sodium in a volume ratio of 800 ml:1 ml:200 ml:2.4 ml is prepared;
every 800 ml basal medium contains: tryptone 5.0 g, soybean peptone 5.0 g, bacterial peptone 5.0 g, sodium pyruvate 10.0 g, beef extract 5.0 g, yeast extract 5.0 g, sodium chloride 2.5 g, L-cysteine 0.3 g, glucose 5.0 g, phenol red 0.02 g; the additive comprises the following components in every 50 ml culture medium: glutathione 1 g, vitamin C6 g.
6. The method for producing the culture medium for mycoplasma pneumoniae as claimed in any one of claims 1 to 5, wherein the method comprises the following steps:
(1) Respectively weighing tryptone, soybean peptone, bacterial peptone, sodium pyruvate, beef extract powder, yeast extract powder, sodium chloride, L-cysteine, glucose and phenol red, adding distilled water, fully mixing and dissolving, adjusting the pH to 7.2-7.6, and sterilizing at 121 ℃ for 30 min under high pressure to prepare a basic culture medium;
(2) Respectively weighing glutathione and vitamin C, adding sterile deionized water for dissolving, filtering and sterilizing by a 0.45 mu m filter, and preparing to obtain a culture medium additive;
(3) And cooling the basic culture medium to room temperature, and adding a culture medium additive, inactivated fetal calf serum and 120 ten thousand units of penicillin sodium into the basic culture medium according to the volume ratio to prepare the human mycoplasma pneumoniae culture medium.
7. Use of the mycoplasma pneumoniae culture medium of any one of claims 1-5 in rapid enrichment culture of mycoplasma pneumoniae.
8. Use of a mycoplasma pneumoniae culture medium according to any one of claims 1 to 5 in the preparation of a mycoplasma pneumoniae molecular diagnostic antigen.
9. A method for culturing a human-derived Mycoplasma pneumoniae comprising inoculating a human-derived Mycoplasma pneumoniae into the human-derived Mycoplasma pneumoniae culture medium according to any one of claims 1 to 5, and culturing the Mycoplasma pneumoniae by a closed-end static culture at 37 ℃ without culturing the Mycoplasma pneumoniae in 5% carbon dioxide.
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| EP3277794A1 (en) * | 2015-04-01 | 2018-02-07 | C.P.M. Di Claudio Piermattei E C. S.A.S. | Culture medium for the isolation of clinical samples of species such mycoplasma including which are not culturable in traditional media |
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