CN112029679B - Large-scale culture method of helicobacter pylori - Google Patents
Large-scale culture method of helicobacter pylori Download PDFInfo
- Publication number
- CN112029679B CN112029679B CN202010805872.0A CN202010805872A CN112029679B CN 112029679 B CN112029679 B CN 112029679B CN 202010805872 A CN202010805872 A CN 202010805872A CN 112029679 B CN112029679 B CN 112029679B
- Authority
- CN
- China
- Prior art keywords
- liquid
- culture
- helicobacter pylori
- solid
- solid culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/26—Conditioning fluids entering or exiting the reaction vessel
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/02—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by impregnation, e.g. using swabs or loops
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
- C12M37/04—Seals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Sustainable Development (AREA)
- Clinical Laboratory Science (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Thermal Sciences (AREA)
- Analytical Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to a large-scale culture method of helicobacter pylori, which utilizes a helicobacter pylori liquid-solid culture device and comprises the following steps: (A) coating the stirrer with 1mg/ml fibrinogen; (B) preparing a solid culture medium; (C) helicobacter pylori is prepared into 1 × 109Injecting the bacterium solution per ml into a pylorus screw bacterium solution-solid culture device, coating bacteria, and culturing with a solid culture medium; (D) preparing a liquid culture medium; (E) culturing helicobacter pylori in solid culture medium for 12 hr, injecting liquid culture medium, culturing in liquid culture medium for 12 hr, stirring, culturing for 1-2 days, discharging liquid culture medium, and collecting helicobacter pylori. The invention has short culture period, simple operation and more helicobacter pylori, and provides basic conditions for preparing inactivated whole-bacterium vaccine.
Description
Technical Field
The invention belongs to a microbial culture method, and particularly relates to a large-scale culture method of helicobacter pylori.
Background
Helicobacter pylori is a spiral, micro-anaerobic, very demanding bacterium for growth conditions and is currently the only microorganism species known to be viable in the human stomach. Because the requirement on the culture condition of the helicobacter pylori is high, the number of bacteria obtained by the traditional liquid culture and solid culture is small, the cost is high, the culture period is long, the in-vitro large-scale culture is difficult to realize, and the difficulty is brought to the preparation of the inactivated whole-bacterium vaccine.
Disclosure of Invention
The invention aims to provide a large-scale culture method for obtaining helicobacter pylori with a large number of bacteria, which has short culture period and simple and convenient operation.
The invention adopts the following technical scheme:
a large-scale culture method of helicobacter pylori, which utilizes a helicobacter pylori liquid-solid culture device and comprises the following steps:
(A) coating a stirrer in a pyloric screw bacteria liquid-solid culture device with 1mg/ml fibrinogen;
(B) preparation of solid medium: weighing 3.9g Columbia blood agar, adding 100ml distilled water, placing in a high pressure steam sterilizer for high pressure sterilization, gradually cooling to 60 deg.C, adding 10ml fetal calf serum, immediately injecting into a pylorus screw bacteria liquid-solid culture device, and cooling for solidification;
(C) helicobacter pylori is prepared into 1 × 109Injecting the bacterium solution per ml into a solid culture part of a helicobacter pylori bacterium solution-solid culture device, uniformly coating helicobacter pylori on the surface of a solid culture medium by using a stirrer, and culturing the solid culture medium by using clean mixed gas with the culture temperature of 37 ℃ and the ventilation volume of 1L/min;
(D) preparing a liquid culture medium: weighing 3.7g brain heart leachate broth, placing in 100ml distilled water, placing in a high pressure steam sterilizer for high pressure sterilization, gradually cooling to normal temperature, and adding 10ml fetal calf serum for use;
(E) culturing helicobacter pylori in solid culture for 12 hr, injecting the obtained liquid culture medium into the liquid culture part of the helicobacter pylori liquid-solid culture device, and performing static culture of the liquid culture medium at 37 deg.C with ventilation of 1L/min; after culturing for 12 hours, stirring the liquid culture medium at a rotation speed of 20 rpm, continuing culturing for 1-2 days under stirring, discharging the liquid culture medium, and collecting helicobacter pylori.
Wherein, in the step (C) and the step (E), the introduced gas is clean with oxygen content of 5 percentThe mixed gas specifically comprises the following components in volume fraction: 10% CO2、85% N2、5% O2。
The helicobacter pylori liquid-solid culture device comprises a culture bottle, a sealing cover arranged at the upper part of the culture bottle and a heating platform arranged below the culture bottle; the culture flask includes a solid culture part, a liquid culture part, and an indicator ring disposed between the solid culture part and the liquid culture part.
The bottom center department of solid culture portion is provided with fixed cover, the bottom outside of solid culture portion contacts with heating platform, the lateral wall of solid culture portion is provided with solid medium filler tube. The height of the fixed sleeve is higher than the distance between the indicating ring and the bottom of the solid culture part.
Wherein, the bottom lateral wall of liquid culture portion is provided with bleeder line and intake pipe.
The sealing cover comprises a cover body sealed with an opening at the top end of the liquid culture part, and a bacterial liquid filling pipe, an exhaust pipe, a liquid culture medium filling pipe and a coating device which are arranged on the cover body.
The coating device comprises a rotating rod penetrating through the cover body, a stirrer arranged on the rotating rod and a motor driving the rotating rod to rotate; the one end of dwang is provided with the slot, and the other end rotates with fixed cover to be connected, the drive shaft cartridge of motor is in the slot and through fixing bolt fixed connection, be provided with the seal cover between dwang and the lid.
The motor cover is characterized in that a support rod is further arranged on the cover body, one end of the support rod is fixedly connected with the cover body, and the other end of the support rod is fixedly connected with a shell of the motor.
Wherein the solid culture part is a cone frustum-shaped container, the liquid culture part is a cylindrical container, and the diameter of the cylinder is equal to the diameter of the upper top surface of the cone frustum.
Wherein a waste liquid pipe is arranged at the bottom of the solid culture part.
Wherein, the bracing piece is the telescopic link.
Valves are arranged on the solid culture medium filling pipe, the liquid discharge pipe, the air inlet pipe, the bacterial liquid filling pipe, the exhaust pipe, the liquid culture medium filling pipe and the waste liquid pipe.
Wherein, the outer end of the exhaust pipe is provided with a gas filter.
The stirrer is a flat plate which is axially arranged along the rotating rod, the bottom edge of the stirrer is a straight edge and is horizontally arranged, and the lower end of the stirrer is provided with a yielding groove for accommodating the fixing sleeve.
Wherein, agitator and dwang are stainless steel material integrated into one piece.
Wherein the solid culture part and the liquid culture part are integrally formed by glass or stainless steel materials.
The invention has the beneficial effects that: the method of the invention can realize the large-scale culture of the helicobacter pylori, not only shortens the culture period (can reduce 2-3 days), but also improves the number of the harvested bacteria by 10-20 times, and provides basic conditions for preparing inactivated whole-bacteria vaccine.
Drawings
FIG. 1 is a schematic structural view of a helicobacter pylori liquid-solid culture apparatus according to the present invention.
Wherein, 1 heating platform, 2 solid culture part, 3 liquid culture part, 4 indicating rings, 5 fixing sleeves, 6 solid culture medium filling pipes, 7 liquid discharging pipes, 8 liquid culture medium filling pipes, 9 cover bodies, 10 bacteria liquid filling pipes, 11 exhaust pipes, 12 air inlet pipes, 13 rotating rods, 14 stirrers, 15 motors, 16 sealing sleeves, 17 supporting rods, 18 waste liquid pipes, 19 valves, 20 gas filters, 21 slots, 22 fixing bolts and 23 abdicating grooves.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
As shown in FIG. 1, a helicobacter pylori liquid-solid culture device comprises a culture bottle, a sealing cover arranged at the upper part of the culture bottle and a heating platform 1 arranged below the culture bottle; the culture flask comprises a solid culture part 2, a liquid culture part 3, and an indicator ring 4 disposed between the solid culture part 2 and the liquid culture part 3. The solid culture part 2 is a truncated cone-shaped container, the liquid culture part 3 is a cylindrical container, and the diameter of the cylindrical container is equal to the diameter of the upper top surface of the truncated cone. The solid culture part 2 and the liquid culture part 3 are integrally formed by glass or stainless steel materials. The indicating ring can be a scale or an annular boss and is used for calibrating the addition amount of the solid culture medium. The device can realize solid-liquid culture of helicobacter pylori, greatly reduce intermediate operation process, is convenient to use, and can improve the number of finally obtained bacteria by 10-20 times.
The bottom center department of solid culture portion 2 is provided with fixed cover 5, the bottom outside of solid culture portion 2 contacts with heating platform 1, the lateral wall of solid culture portion 2 is provided with solid medium filler pipe 6. The solid culture part is used for culturing helicobacter pylori by using a solid culture medium, the heating platform is used for heating the whole device, the proper temperature for culture is maintained, and the solid culture medium can be melted after the culture is finished, so that the device is beneficial to discharging the solid culture medium and killing microorganisms in waste liquid. The height of the fixed sleeve 5 is higher than the distance between the indicating ring 4 and the bottom of the solid culture part 2; namely, the upper end opening of the fixing sleeve is higher than the plane of the indicating ring, and when the solid culture medium is filled, the solid culture medium cannot be filled into the fixing sleeve.
The liquid culture part 3 has a liquid discharge pipe 7 and an air inlet pipe 12 on the bottom side wall thereof. The liquid culture section is used for culturing helicobacter pylori using a liquid culture medium. Because the helicobacter pylori is a micro-anaerobic bacterium, the air inlet pipe can introduce a trace amount of oxygen into the culture medium in the culture process, and the proper culture condition is ensured. After the culture is finished, the liquid culture medium and the helicobacter pylori therein are discharged from the liquid discharging pipe at the bottom of the liquid culture part, and the bacterial liquid is collected.
The seal cap comprises a cap body 9 for sealing the top opening of the liquid culture part 3, a bacterial liquid filling pipe 10, an exhaust pipe 11, a liquid medium filling pipe 8 and a coating device provided on the cap body 9. The coating device comprises a rotating rod 13 penetrating through the cover body 9, a stirrer 14 arranged on the rotating rod 13 and a motor 15 driving the rotating rod 13 to rotate; the one end of dwang 13 is provided with slot 21, and the other end rotates with fixed cover 5 to be connected, the drive shaft cartridge of motor 15 is in slot 21 and through fixing bolt 22 fixed connection, be provided with seal cover 16 between dwang 13 and the lid 9.
The sealing cover seals the whole device, the exhaust pipe exhausts outwards, clean mixed gas (the oxygen content is 5%) containing trace oxygen enters the device through the air inlet pipe and then is exhausted through the exhaust pipe, a positive pressure environment is formed, external air is prevented from entering the device to cause bacteria contamination, the outlet end of the exhaust pipe is further provided with a gas filter 20, and external microorganisms are further prevented from entering the device. The slot is arranged at the upper end of the rotating rod, the driving shaft of the motor is inserted into the slot and fixed through the fixing bolt, and the change of the vertical height of the stirrer can be realized through the change of the relative position of the driving shaft of the motor and the rotating rod. In the lower extreme of dwang inserted fixed cover, the motor drove the dwang rotatory and then drive the agitator and rotate, and the groove of stepping down can give out the rotatory position of confession agitator, guarantees that the agitator all can rotate at any height. Through the relative position of adjustment motor drive shaft and dwang for the lower terminal surface of agitator contacts with the upper surface of solid medium, when carrying out solid culture, the fungus liquid coating on solid medium surface is come with to the agitator. When liquid culture is performed, the agitator may function to agitate the liquid culture medium.
Still be provided with bracing piece 17 on the lid 9, bracing piece 17 one end and lid 9 fixed connection, the other end and the casing fixed connection of motor 15. Preferably, the supporting rod is a telescopic rod. Although the final upper plane of the solid culture medium can be kept on the plane of the indicating ring by controlling the filling amount of the solid culture medium, the position of the upper plane of the solid culture medium can be changed to a certain extent in the experimental process, and the supporting rod can also drive the motor to be finely adjusted up and down, so that the lower end face of the stirrer is in contact with the upper surface of the solid culture medium, and the coating function is realized.
The method for culturing the helicobacter pylori by using the device comprises the following steps:
(1) the stirrer was coated with 1mg/ml fibrinogen.
(2) Preparing a solid culture medium. 3.9g of Columbia blood agar is weighed and put into 100ml of distilled water, and the mixture is placed into a high-pressure steam sterilizer for high-pressure sterilization under the conditions of high pressure of 103.4kPa, temperature of 121.3 ℃ and 15-20 minutes. After gradually cooling to 60 ℃, 10ml of fetal calf serum is added, the mixture is immediately injected into the fixed culture part from a solid culture medium filling pipe, the upper surface of the liquid level is flush with the indicating ring, and the mixture is cooled and solidified.
(3) Helicobacter pylori is prepared into 1 × 109Injecting bacterial liquid per ml from a bacterial liquid filling pipe, adjusting a fixing bolt or a supporting rod to enable the lower end of the stirrer to be in contact with the upper surface of the solid culture medium, and rotating the stirrer to coat bacteria. Culturing at 37 deg.C with 1L/min of clean mixed gas (10% CO)2，85% N2，5% O2）。
(4) Preparing a liquid culture medium. Weighing 3.7g brain heart leachate broth, placing in 100ml distilled water, placing in a high pressure steam sterilizer, and autoclaving under the conditions of pressure 103.4kPa, temperature 121.3 deg.C, and maintaining for 15-20 min. After gradually cooling to normal temperature, 10ml fetal calf serum is added for standby.
(5) Quiet and beautifulCulturing helicobacter pylori in solid culture for 12 hr, injecting liquid culture medium from liquid culture medium filler pipe, culturing at 37 deg.C with 1L/min of clean mixed gas (10% CO)2，85% N2，5% O2). The culture is carried out for 12 hours in a standing way, the helicobacter pylori is adhered to a stirrer coated with fibrinogen, and the rapid growth is carried out by utilizing the nutrition of a liquid culture medium. Adjusting the fixing bolt or the supporting rod to separate the lower end of the stirrer from the upper surface of the solid culture medium, starting the motor to drive the stirrer to rotate at the rotating speed of 20 revolutions per minute, continuing culturing for 1-2 days, discharging the bacterial liquid from the liquid discharge pipe, and collecting bacteria.
(6) After the bacteria are collected, the temperature of the heating platform is increased to melt the solid culture medium, and the melted solid culture medium is discharged from the waste liquid pipe.
After the device and the method are adopted, the intermediate operation process in the helicobacter pylori solid-liquid culture process is greatly reduced, the use is convenient, the culture period can be at least shortened by 2-3 days, and basic conditions are provided for preparing inactivated whole-bacterium vaccines.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A large-scale culture method of helicobacter pylori is characterized in that the method utilizes a helicobacter pylori liquid-solid culture device and comprises the following steps:
(A) coating a stirrer in a pyloric screw bacteria liquid-solid culture device with 1mg/ml fibrinogen;
(B) preparation of solid medium: weighing 3.9g of Columbia blood agar, putting the Columbia blood agar into 100ml of distilled water, placing the Columbia blood agar into a high-pressure steam sterilizer for high-pressure sterilization, gradually cooling to about 60 ℃, adding 10ml of fetal calf serum, immediately injecting a pyloric screw bacteria liquid-fixed culture part of a solid culture device from a solid culture medium filling pipe, flushing the upper surface of the liquid surface with an indicating ring, and cooling and solidifying;
(C) helicobacter pylori is prepared into 1 × 109Injecting bacterial liquid per ml from a bacterial liquid filling pipe, adjusting a fixed bolt or a support rod to enable the lower end of the stirrer to be in contact with the upper surface of the solid culture medium, rotating the stirrer to coat helicobacter pylori, and culturing the solid culture medium at the culture temperature of 37 ℃ with clean mixed gas with the ventilation volume of 1L/min;
(D) preparing a liquid culture medium: weighing 3.7g brain heart leachate broth, placing in 100ml distilled water, placing in a high pressure steam sterilizer for high pressure sterilization, gradually cooling to normal temperature, and adding 10ml fetal calf serum for use;
(E) culturing helicobacter pylori in solid culture for 12 hr, injecting the obtained liquid culture medium into the liquid culture part of the helicobacter pylori liquid-solid culture device, and performing static culture of the liquid culture medium at 37 deg.C with ventilation of 1L/min; after culturing for 12 hours, adjusting a fixing bolt or a supporting rod to separate the lower end of the stirrer from the upper surface of the solid culture medium, then starting a motor to drive the stirrer to rotate, stirring the liquid culture medium at 20 revolutions per minute, continuously culturing for 1-2 days, discharging the liquid culture medium, and collecting helicobacter pylori;
(F) after bacteria are collected, the temperature of the heating table is increased to melt the solid culture medium, and the melted solid culture medium is discharged from the waste liquid pipe;
the helicobacter pylori liquid-solid culture device comprises a culture bottle, a sealing cover arranged at the upper part of the culture bottle and a heating platform (1) arranged below the culture bottle; the culture bottle comprises a solid culture part (2), a liquid culture part (3) and an indicating ring (4) arranged between the solid culture part (2) and the liquid culture part (3);
a fixed sleeve (5) is arranged at the center of the bottom of the solid culture part (2), the outer side of the bottom of the solid culture part (2) is in contact with the heating platform (1), and a solid culture medium filling pipe (6) is arranged on the side wall of the solid culture part (2); the height of the fixed sleeve (5) is higher than the distance between the indicating ring (4) and the bottom of the solid culture part (2);
a liquid discharging pipe (7) and an air inlet pipe (12) are arranged on the side wall of the bottom of the liquid culture part (3);
the sealing cover comprises a cover body (9) sealed with the top end opening of the liquid culture part (3), a bacterial liquid filling pipe (10), an exhaust pipe (11), a liquid culture medium filling pipe (8) and a coating device, wherein the bacterial liquid filling pipe, the exhaust pipe (11), the liquid culture medium filling pipe and the coating device are arranged on the cover body (9);
the coating device comprises a rotating rod (13) penetrating through the cover body (9), a stirrer (14) arranged on the rotating rod (13) and a motor (15) driving the rotating rod (13) to rotate; the one end of dwang (13) is provided with slot (21), and the other end rotates with fixed cover (5) to be connected, the drive shaft cartridge of motor (15) is in slot (21) and through fixing bolt (22) fixed connection, be provided with seal cover (16) between dwang (13) and lid (9).
2. The method for the mass culture of helicobacter pylori according to claim 1, wherein the gas introduced in the steps (C) and (E) is a clean mixed gas containing 5% oxygen.
3. The mass culture method of helicobacter pylori according to claim 1, wherein the cover (9) is further provided with a support rod (17), one end of the support rod (17) is fixedly connected with the cover (9), and the other end is fixedly connected with the housing of the motor (15).
4. The method for the mass culture of helicobacter pylori according to claim 1, wherein the solid culture part (2) is a container having a truncated cone shape, and the liquid culture part (3) is a container having a cylindrical shape with a diameter equal to the diameter of the upper top surface of the truncated cone.
5. The method for the large-scale culture of helicobacter pylori according to claim 1, wherein a waste liquid tube (18) is provided at the bottom of the solid culture part (2).
6. The method for the mass culture of helicobacter pylori according to claim 1, wherein the support rods (17) are telescopic rods.
7. The method for the large-scale culture of helicobacter pylori according to claim 5, wherein valves (19) are provided on the solid medium filling pipe (6), the liquid discharging pipe (7), the air inlet pipe (12), the bacterial liquid filling pipe (10), the air outlet pipe (11), the liquid medium filling pipe (8) and the waste liquid pipe (18).
8. The method for the mass culture of helicobacter pylori according to claim 1, wherein a gas filter (20) is provided at the outer end of the gas exhaust tube (11).
9. The method for mass culture of helicobacter pylori according to claim 1, wherein the stirrer (14) is a flat plate disposed axially along the rotating shaft (13), and the lower end of the stirrer (14) is provided with a relief groove (23) for receiving the fixing sheath (5).
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010805872.0A CN112029679B (en) | 2020-08-12 | 2020-08-12 | Large-scale culture method of helicobacter pylori |
| AU2021100145A AU2021100145A4 (en) | 2020-08-12 | 2021-01-11 | Large-scale culture method of helicobacter pylori |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010805872.0A CN112029679B (en) | 2020-08-12 | 2020-08-12 | Large-scale culture method of helicobacter pylori |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112029679A CN112029679A (en) | 2020-12-04 |
| CN112029679B true CN112029679B (en) | 2022-01-11 |
Family
ID=73577838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010805872.0A Active CN112029679B (en) | 2020-08-12 | 2020-08-12 | Large-scale culture method of helicobacter pylori |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112029679B (en) |
| AU (1) | AU2021100145A4 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322155B (en) * | 2021-05-31 | 2022-07-22 | 济南千麦医学检验有限公司 | Incubator for helicobacter pylori |
| CN114854528A (en) * | 2022-05-11 | 2022-08-05 | 江苏省家禽科学研究所 | Culture device and culture method suitable for campylobacter microaerophilic |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403986A (en) * | 2014-11-07 | 2015-03-11 | 德州创迪微生物资源有限责任公司 | Microbe spore culture method and equipment |
| CN107674826A (en) * | 2017-09-04 | 2018-02-09 | 陕西众兴高科生物科技有限公司 | A kind of liquid bacterial culture device |
| CN109810927A (en) * | 2019-03-21 | 2019-05-28 | 贵州医科大学 | A kind of helicobacter pylori liquid cultivating method |
| CN209602512U (en) * | 2018-12-24 | 2019-11-08 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Muscardine solid culture inoculating facility |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2213730Y (en) * | 1994-10-12 | 1995-11-29 | 叶大林 | Three-phase substratum |
| EP2233577A1 (en) * | 2009-03-20 | 2010-09-29 | F. Hoffmann-La Roche AG | Process and apparatus for testing substances for potential carcinogenicity |
-
2020
- 2020-08-12 CN CN202010805872.0A patent/CN112029679B/en active Active
-
2021
- 2021-01-11 AU AU2021100145A patent/AU2021100145A4/en not_active Ceased
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403986A (en) * | 2014-11-07 | 2015-03-11 | 德州创迪微生物资源有限责任公司 | Microbe spore culture method and equipment |
| CN107674826A (en) * | 2017-09-04 | 2018-02-09 | 陕西众兴高科生物科技有限公司 | A kind of liquid bacterial culture device |
| CN209602512U (en) * | 2018-12-24 | 2019-11-08 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Muscardine solid culture inoculating facility |
| CN109810927A (en) * | 2019-03-21 | 2019-05-28 | 贵州医科大学 | A kind of helicobacter pylori liquid cultivating method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112029679A (en) | 2020-12-04 |
| AU2021100145A4 (en) | 2021-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112029679B (en) | Large-scale culture method of helicobacter pylori | |
| US7628528B2 (en) | Pneumatic bioreactor | |
| US8822208B2 (en) | Bioreactor and method | |
| CN213447122U (en) | Device for solid culture of helicobacter pylori bacterial liquid | |
| CN208038422U (en) | A kind of Yoghourt fermentation system | |
| CN208395183U (en) | A kind of vertical type solid fermentor | |
| JPH0286762A (en) | Air scattering system | |
| CN210163462U (en) | Vertical rotary tank bioreactor for large-scale cell culture | |
| JP2004249271A (en) | Stirring method for double tank and stirrer | |
| CN213172363U (en) | Compound bacterial fermentation cylinder of benefit fungus | |
| CN207904278U (en) | A kind of poly-nuclear algae element centrifugation fermentation extraction element | |
| CN219326756U (en) | Microbial agent fermenting installation | |
| JPH01124379A (en) | Culture apparatus | |
| CN208346149U (en) | Stirring reactor in process of high-density fermentation | |
| JPH0659203B2 (en) | Incubator | |
| GB2163453A (en) | Vessel for culturing cells on microcarriers or in capsules | |
| CN213977648U (en) | Beauty product raw materials for production bioreactor | |
| CN110791431A (en) | Online ultrasonic stripping cell disruption bioreactor and virus harvesting method | |
| JPS6214778A (en) | Deep fermentation apparatus | |
| CN211814387U (en) | Microbial reaction device for producing D-amino acid | |
| CN211999740U (en) | Pig feed fermenting installation | |
| CN220376695U (en) | Constant-temperature fermentation culture device for microbial flocculant producing bacteria | |
| CN210237641U (en) | Basket type bioreactor | |
| CN117511713B (en) | Full-automatic fermentation tank for microbial culture medium and fermentation method thereof | |
| CN219228884U (en) | Be applied to raw materials fermenting installation of milk product jar production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |