Search

CN-121975665-A - Culture medium and culture method for culturing mycoplasma pneumoniae

CN121975665ACN 121975665 ACN121975665 ACN 121975665ACN-121975665-A

Abstract

The invention relates to a culture medium for culturing mycoplasma pneumoniae and a culture method, belonging to the technical field of microorganisms, wherein the culture medium for culturing mycoplasma pneumoniae comprises a mycoplasma culture medium and a growth promoting additive, and the growth promoting additive comprises horse serum or fetal bovine serum, cholesterol, penicillin sodium and HEPES buffer solution; the method for culturing the mycoplasma pneumoniae changes the culturing mode from static to dynamic, improves the culturing efficiency of the mycoplasma pneumoniae, improves the thallus yield of the mycoplasma pneumoniae, has short period of preparing the mycoplasma pneumoniae, low cost, high viable bacteria density of the culture and high yield, and is suitable for culturing the mycoplasma pneumoniae on a large scale.

Inventors

  • YANG FAN
  • GUO HUI
  • MENG DEZHI
  • WANG TING
  • LIU WANJIAN

Assignees

  • 山东硕景生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260407

Claims (6)

  1. 1. A culture medium for culturing mycoplasma pneumoniae in human is characterized by comprising a mycoplasma culture medium and a growth promoting additive, wherein the growth promoting additive comprises 10-20% horse serum or fetal bovine serum, 0.1mg/mL cholesterol, 100 ten thousand units of penicillin sodium and 1mM HEPES buffer solution, and the mycoplasma culture medium comprises 10.0g/L of pig stomach digests, 5.0g/L of beef extract powder, 5.0g/L of yeast extract powder, 2.5g/L of sodium chloride, 5.0g/L of glucose and 0.02g/L of phenol red.
  2. 2. A preparation method of a culture medium for culturing human mycoplasma pneumoniae is characterized by comprising the following steps: (1) Preparing a basic culture medium, namely weighing 27.52g of mycoplasma culture medium, adding pure water, fully dissolving and uniformly mixing, adjusting the pH value to 7.2-7.6, fixing the volume to 1000mL by using the pure water, and sterilizing at 121 ℃ under high pressure to 20min to obtain the basic culture medium; (2) Preparation of the medium additive: S1, taking horse serum or fetal bovine serum, and inactivating for 30min at 56 ℃; S2, weighing 1g of cholesterol, adding sterile pure water, dissolving to a constant volume of 10mL, and filtering and sterilizing by a 0.22 mu m filter under the sterile condition, wherein the concentration is 100 mg/mL; S3, weighing 2.38g of HEPES powder, adding sterile pure water, dissolving to a constant volume of 10mL, and filtering and sterilizing by a 0.22 mu M filter under the sterile condition, wherein the concentration is 1M; s4, adding 1.6mL of sterile pure water into 160 ten thousand units of penicillin sodium powder for dissolution, wherein the concentration is 100 ten thousand units/mL; (3) The basal medium is cooled to room temperature, and under the aseptic condition, culture medium additives are added according to the volume ratio of the basal medium, namely serum, cholesterol, penicillin sodium and HEPES buffer solution of 1000:111-250:1.1-1.25:1.1-1.25:1.1-1.25, so as to obtain the culture medium for culturing the mycoplasma pneumoniae of human beings.
  3. 3. A method for culturing human mycoplasma pneumoniae is characterized by comprising the following steps: Activating the standard strain ATCC 15531 strain of the mycoplasma pneumoniae, namely inoculating the standard strain ATCC 15531 strain of the mycoplasma pneumoniae to the culture medium for culturing the mycoplasma pneumoniae obtained in the claim 2 according to the volume ratio of 1:10 by taking 1X 10 9 CCU/mL as an initial inoculation concentration, and culturing for 2-3 days; S02, inoculating the activated strain to the culture medium for culturing the mycoplasma pneumoniae of the human being, which is obtained in the claim 2, according to the volume ratio of 1:10, placing the strain into a 5% carbon dioxide cell bottle-rotating machine for culturing, collecting the culture solution for centrifugation when the color of the mycoplasma pneumoniae culture solution is changed from red to clear yellow and the pH value is reduced to 5.0-5.5, and obtaining the precipitate, namely the mycoplasma pneumoniae of the human being, wherein the rotation speed is 12000rpm and the time is 60 min.
  4. 4. The method of claim 3, wherein in step S02, the temperature of the culture in the carbon dioxide cell flask-transferring machine is in the range of 36℃to 37 ℃.
  5. 5. The method of claim 3, wherein the rotational speed of the culture in the carbon dioxide cell flask-rotating machine in step S02 is in the range of 60-70rpm.
  6. 6. The method of claim 3, wherein in step S02, the culture is performed in a carbon dioxide cell flask-rotating machine for 3 to 4 days.

Description

Culture medium and culture method for culturing mycoplasma pneumoniae Technical Field The invention belongs to the technical field of microorganisms, and particularly relates to a culture medium for culturing mycoplasma pneumoniae and a culture method. Background Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP), a common pathogen of community-acquired pneumonia (Community acquired pneumonia, CAP), mainly causes upper and lower respiratory tract infections in children and adolescents, and causes laryngitis, pharyngitis, bronchitis and atypical pneumonia, and severe cases can cause extrapulmonary complications. The clinical treatment is mainly carried out by macrolide antibiotics. Mycoplasma pneumoniae infection generally has no specific clinical symptoms, so that accurate diagnosis of early infection is an important basis for treating mycoplasma pneumoniae infection. At present, mycoplasma pneumoniae infection is diagnosed in vitro, natural antigens prepared by mycoplasma pneumoniae cultures are generally utilized to detect IgM and IgG antibodies in serum, and the accurate diagnosis can be realized only by the natural antigens with good specificity and strong antigenicity. Mycoplasma pneumoniae is 0.2-0.3 μm in size, is between virus and bacteria, has no cell wall, has only cell membrane, small genome, simple metabolism, and cannot synthesize cell components such as amino acids, fatty acids, nucleotides, cholesterol and the like, and needs to be obtained from host cells. Therefore, when the mycoplasma pneumoniae is cultured in vitro, the mycoplasma pneumoniae has extremely harsh nutrition requirements, complex culture conditions, long growth period and low cell titer, and can not completely meet the current clinical application requirements of low cost and high yield. At present, mycoplasma culture mediums are various in the market, and no special culture medium special for mycoplasma pneumoniae is available, so that the mycoplasma pneumoniae natural antigens are uneven in quality, and the detection results are inaccurate. At present, the culture of the mycoplasma pneumoniae mainly adopts a method of adding a plurality of growth promoting additives into a basic culture medium, has complex components and difficult preparation, and adopts a culture mode mainly of static culture or suspension culture, has long culture time and low viable bacteria titer, is unfavorable for large-scale culture and antigen preparation, so that a culture medium and a culture mode with simple formula, high growth speed and high yield are necessary to be explored to make up the defects of the existing mycoplasma pneumoniae culture technology. Disclosure of Invention Aiming at the defects, the invention provides the culture medium for culturing the mycoplasma pneumoniae, which has the advantages that various components are simple and easy to obtain, the preparation method of the culture medium is greatly simplified, and the efficiency is improved; the invention also provides a method for culturing the mycoplasma pneumoniae, which changes the culturing mode from static state to dynamic state, improves the culturing efficiency of the mycoplasma pneumoniae, improves the thallus yield of the mycoplasma pneumoniae, has short period for preparing the mycoplasma pneumoniae, low cost, high viable bacteria density of the culture and high yield, and is suitable for culturing the mycoplasma pneumoniae on a large scale. In order to solve the technical problems, the culture medium for the mycoplasma pneumoniae culture comprises a mycoplasma culture medium and a growth promoting additive, wherein the growth promoting additive comprises 10-20% horse serum or fetal calf serum, 0.1mg/mL cholesterol, 100 ten thousand units of penicillin sodium and 1mM HEPES buffer solution, and the mycoplasma culture medium comprises 10.0g/L of pig stomach digests, 5.0g/L of beef extract powder, 5.0g/L of yeast extract powder, 2.5g/L of sodium chloride, 5.0g/L of glucose and 0.02g/L of phenol red. The mycoplasma culture medium and the growth promoting additive provide various nutrients required by mycoplasma pneumoniae growth, promote mycoplasma cell membrane to grow fast, inhibit mixed bacteria proliferation, regulate the pH value of the culture solution and prevent the reduction of acid-producing pH of thalli in the culture process to cause the inactivation of thalli. A preparation method of a culture medium for culturing mycoplasma pneumoniae, comprising the following steps: (1) Preparing a basic culture medium, namely weighing 27.52g of mycoplasma culture medium, adding pure water, fully dissolving and uniformly mixing, adjusting the pH value to 7.2-7.6, fixing the volume to 1000mL by using the pure water, and sterilizing at 121 ℃ under high pressure to 20min to obtain the basic culture medium; (2) Preparation of the medium additive: S1, taking horse serum or fetal bovine serum, and inactivating for 30min at 56 ℃; S2, weighing 1g of cholesterol, adding sterile pur