AU2021100145A4

AU2021100145A4 - Large-scale culture method of helicobacter pylori

Info

Publication number: AU2021100145A4
Application number: AU2021100145A
Authority: AU
Inventors: Dailun HU; Bao’en SHAN; Lianmei ZHAO
Original assignee: Hebei Medical University; Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
Current assignee: Hebei Medical University; Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
Priority date: 2020-08-12
Filing date: 2021-01-11
Publication date: 2021-04-08
Anticipated expiration: 2029-01-11
Also published as: CN112029679A; CN112029679B

Abstract

The present disclosure relates to a large-scale culture method of Helicobacterpylori. The method utilizes a H. pylori liquid-solid culture device and includes the following steps: (A) coating a stirrer with 1 mg/ml fibrinogen; (B) preparing a solid medium; (C) preparing H. pylori into a 1 x 10' cells/ml bacterial suspension, injecting the bacterial suspension into the H. pylori liquid-solid culture device, spreading H. pylori, and culturing on the solid medium; (D) preparing a liquid medium; and (E) after culturing H. pylori on the solid medium for 12 h, injecting the liquid medium, culturing on the liquid medium for 12 h, continuing the culture for 1-2 days under stirring, releasing the liquid medium, and collecting H. pylori. The present disclosure features short culture period, convenient operation, and a plurality of H. pylori obtained, providing a basic condition for preparing inactivated whole-cell vaccines. DRAWINGS 15 21 17 /19 / 22 10 20/- - 12 14-- 13 6 1 ,7 S 2' 18~ FIG.1I Page Iof1I

Description

DRAWINGS

15 21

17 /19 / 22 10 20/- - 12

14-- 13

6

1 ,7 S 2' 18~ FIG.1I

Page Iof1I

LARGE-SCALE CULTURE METHOD OF HELICOBACTER PYLORI TECHNICAL FIELD

The present disclosure relates to a microbial culture method, and in particular to a large-scale culture method of Helicobacterpylori.

BACKGROUND

H. pylori is a spiral, microanaerobic bacterium that restricts on growth conditions, and is the only microorganism species known to be able to survive in the human stomach. Due to the high requirements for H. pylori culture conditions, conventional liquid culture and solid culture produce a small number of bacteria, with high cost and long culture period. These culture methods are difficult to achieve large-scale culture in vitro, posing a difficulty in preparing inactivated whole-cell vaccines.

SUMMARY

An objective of the present disclosure is to provide a large-scale culture method of H. pylori with a short culture period, convenient operation and a plurality of bacteria obtained.

The present disclosure adopts the following technical solution:

Provided is a large-scale culture method of H. pylori, where the method utilizes a H. pylori liquid solid culture device and includes the following steps:

step (A), coating a stirrer in the H. pylori liquid-solid culture device with1 mg/ml fibrinogen;

step (B), preparation of solid medium: weighing 3.9 g of Columbia Blood Agar Base into 100 ml of distilled water, autoclaving in an autoclave sterilizer, gradually cooling to 60°C, adding and immediately injecting 10 ml of fetal calf serum (FBS) into the H. pylori liquid-solid culture device, cooling and curing;

step (C), preparing H. pylori into a1 x 109 cells/ml bacterial suspension, injecting the bacterial suspension into a solid culture member of the H. pylori liquid-solid culture device, spreading H. pylori on the surface of the solid medium evenly using the stirrer, and culturing on the solid medium at 37°C under an atmosphere of clean mixed gas with an aeration volume of 1 L/min;

step (D), preparation of liquid medium: weighing 3.7 g of Brain Heart Infusion Broth into 100 ml of distilled water, autoclaving in the autoclave sterilizer, and then gradually cooling to room temperature, and adding 10 ml of FBS for use; and

step (E), after culturing H. pylori on the solid medium for 12 h, injecting a resulting liquid medium into a liquid culture member of the H. pylori liquid-solid culture device, and conducting static culture on the liquid medium at 37°C under an atmosphere of clean mixed gas with an aeration volume of 1 L/min; after culturing for 12 h, stirring the liquid medium at 20 r/min, continuing the culture for 1-2 days under stirring, releasing the liquid medium, and collecting H. pylori.

Herein, in steps (C) and (E), a gas introduced may be a clean mixed gas with an oxygen content of 5%, specifically including the following components in volume fraction: 10% C02, 85% N2, and %02.

Herein, the H. pylori liquid-solid culture device may include a culture flask, a sealing cap arranged on the upper part of the culture flask, and a heating platform arranged under the culture flask; the culture flask may include a solid culture member, a liquid culture member, and an indicator ring arranged between the solid culture member and the liquid culture member.

Herein, a fixed hub may be arranged at the center of the bottom of the solid culture member, the bottom of the solid culture member may be laterally in contact with the heating platform, and the side wall of the solid culture member may be provided with a solid medium filling pipe. The height of the fixed hub may be higher than the distance between the indicator ring and the bottom of the solid culture member.

Herein, the bottom side wall of the liquid culture member may be provided with a discharge pipe and an air intake pipe.

Herein, the sealing cap may include a cover body sealed with a top opening of the liquid culture member, a bacterial suspension filling pipe, an exhaust pipe, a liquid medium filling pipe and a spreading arrangement arranged in the cover body.

Herein, the spreading arrangement may include a rotary rod penetrating the cover body, a stirrer arranged on the rotary rod, and a motor that drives the rotary rod to rotate; one end of the rotary rod may be provided with a slot, the other end may be rotatably connected with the fixed hub, a drive shaft of the motor may be inserted into the slot and fixedly connected by a fixing bolt, and a seal sleeve may be arranged between the rotary rod and the cover body.

Herein, a support bar may be further provided on the cover body, one end of the support bar may be fixedly connected with the cover body, and the other end may befixedly connected with the housing of the motor.

Herein, the solid culture member may be a truncated cone-shaped container, the liquid culture member may be a cylinder-shaped container, and a diameter of the cylinder may be equal to a diameter of the top surface of the truncated cone.

Herein, a waste liquid pipe may be provided at the bottom of the solid culture member.

Herein, the support bar may be telescopic.

Herein, the solid medium filling pipe, the discharge pipe, the air intake pipe, the bacterial suspension filling pipe, the exhaust pipe, the liquid medium filling pipe, and the waste liquid pipe may be provided with valves.

Herein, a gas filter may be provided at the outer end of the exhaust pipe.

Herein, the stirrer may be a flat plate arranged along the axis of the rotary rod, the bottom margin of the stirrer may be straight and arranged horizontally, and the lower end of the stirrer may be provided with a reserved slot for receiving the fixed hub.

Herein, the stirrer and the rotary rod maybe integrally formed of stainless steel.

Herein, the solid culture member and the liquid culture member may be integrally formed of glass or stainless steel.

The present disclosure has the following beneficial effects: the method of the present disclosure may realize the large-scale culture of H. pylori; the method not only shortens the culture period (which may be reduced by 2-3 days), but also increases the number of bacteria harvested by 10-20 times, providing a basic condition for preparing inactivated whole-cell vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural schematic view of a H. pylori liquid-solid culture device of the present disclosure.

Herein, 1 represents a heating platform, 2 represents a solid culture member, 3 represents a liquid culture member, 4 represents an indicator ring, 5 represents a fixed hub, 6 represents a solid medium filling pipe, 7 represents a discharge pipe, 8 represents a liquid medium filling pipe, 9 represents a cover body, 10 represents a bacterial suspension filling pipe, 11 represents an exhaust pipe, 12 represents an air intake pipe, 13 represents a rotary rod, 14 represents a stirrer, 15 represents a motor, 16 represents a seal sleeve, 17 represents a support bar, 18 represents a waste liquid pipe, 19 represents a valve, 20 represents a gas filter, 21 represents a slot , 22 represents a fixing bolt, and 23 represents a reserved slot.

DETAILED DESCRIPTION

The technical solution of the present disclosure will be described below clearly and completely with reference to the drawing in the embodiment of the present disclosure. It is clear that the described embodiment is only a part of, not all of, the embodiments of the present disclosure.

All other embodiments obtained by those of ordinary skill in the art based on the embodiment of the present disclosure without creative efforts shall fall within the scope of the present disclosure.

As shown in FIG. 1, a H. pylori liquid-solid culture device includes a culture flask, a sealing cap arranged on the upper part of the culture flask, and a heating platform 1 arranged under the culture flask; the culture flask includes a solid culture member 2, a liquid culture member 3, and an indicator ring 4 arranged between the solid culture member 2 and the liquid culture member 3. The solid culture member 2 is a truncated cone-shaped container, the liquid culture member 3 is a cylinder-shaped container, and a diameter of the cylinder is equal to a diameter of the top surface of the truncated cone. The solid culture member 2 and the liquid culture member 3 is integrally formed of glass or stainless steel. The indicator ring may be a scale or a ring-shaped boss, used to calibrate the amount of solid medium added. The device may not only realize the solid-liquid culture of H. pylori and substantially reduce the intermediate operation process in a convenient manner, but also increase the number of bacteria finally obtained by 10-20 times.

A fixed hub 5 is arranged at the center of the bottom of the solid culture member 2, the bottom of the solid culture member 2 is laterally in contact with the heating platform 1, and the side wall of the solid culture member 2 is provided with a solid medium filling pipe 6. The solid culture member is used to culture H. pylori with the solid medium. The heating platform not only heats the whole device to maintain an appropriate temperature for the culture, but also melts the solid medium after the culture is completed, being conducive to the discharge of the solid medium from the device and the elimination of microorganisms in waste liquid. The height of the fixed hub 5 is higher than the distance between the indicator ring 4 and the bottom of the solid culture member 2; that is, the top opening of the fixed hub is higher than the plane where the indicator ring is located. When the solid medium is injected, the solid medium will not be poured into the fixed hub.

The bottom side wall of the liquid culture member 3 is provided with a discharge pipe 7 and an air intake pipe 12. The liquid culture member is used to culture H. pylori with the liquid medium. Since H. pylori is a microanaerobe, the air intake pipe may inject minute amount of oxygen into the culture medium during the culture, so as to ensure appropriate culture conditions. When the culture is over, the liquid medium and the H. pylori therein are discharged from the discharge pipe at the bottom of the liquid culture member, and a bacterial suspension is collected.

The sealing cap includes a cover body 9 sealed with a top opening of the liquid culture member 3, a bacterial suspension filling pipe 10, an exhaust pipe 11, a liquid medium filling pipe 8 and a spreading arrangement arranged in the cover body 9. The spreading arrangement includes a rotary rod 13 penetrating the cover body 9, a stirrer 14 arranged on the rotary rod 13, and a motor 15 that drives the rotary rod 13 to rotate; one end of the rotary rod 13 is provided with a slot 21, the other end is rotatably connected with the fixed hub 5, a drive shaft of the motor 15 is inserted into the slot 21 and fixedly connected by a fixing bolt 22, and a seal sleeve 16 is arranged between the rotary rod 13 and the cover body 9.

The stirrer 14 is a flat plate arranged along the axis of the rotary rod 13, the bottom margin of the stirrer is straight and arranged horizontally, and the stirrer 14 and the rotary rod 13 are integrally formed of stainless steel; to further improve spreading quality, a glass rod may be arranged (bonded) at the lower end of the stirrer to prevent scratches on the surface of the solid medium during spreading. The lower end of the stirrer 14 is provided with a reserved slot 23 for receiving the fixed hub 5. The number of stirrers is not limited, as long as a plurality of stirrers may be uniformly arranged on the rotary rod along the circumferential direction, and the specific shape may be a circle and a polygon, and preferably a rectangle.

The sealing cap seals the entire device, and the exhaust pipe exhausts outward. A clean mixed gas containing trace oxygen (5% oxygen) enters the device through the air intake pipe and exits the device through the exhaust pipe to form a positive pressure environment, so as to prevent outside air from entering the device to cause bacterial contamination; the outlet end of the exhaust pipe is further provided with a gas filter 20 to further prevent external microorganisms from entering the device. The upper end of the rotary rod is provided with a slot, and a drive shaft of the motor is inserted into the slot and fixed by a fixing bolt. The change of the relative position between the drive shaft of the motor and the rotary rod may realize the change of the vertical height of the stirrer. The lower end of the rotary rod is inserted into the fixed hub, and the motor drives the rotary rod to rotate, so as to drive the stirrer to rotate. The reserved slot may make room for the stirrer to rotate, ensuring that the stirrer may rotate at any height. By adjusting the relative position between the drive shaft of the motor and the rotary rod, the lower end surface of the stirrer is in contact with the upper surface of the solid medium. When solid culture is performed, the stirrer spreads the bacterial suspension on the surface of the solid medium. When liquid culture is performed, the stirrer may play a role in stirring the liquid medium.

A support bar 17 is further provided on the cover body 9, one end of the support bar 17 is fixedly connected with the cover body 9, and the other end is fixedly connected with the housing of the motor 15. Preferably, the support bar may be telescopic. Although controlling the filling amount of the solid medium may control the upper plane of a final solid medium to remain on the plane where the indicator ring is located, the position of the upper plane of the solid medium may still change during the experiment. The support bar may further drive the motor to trim up and down to ensure that the lower end surface of the stirrer is in contact with the upper surface of the solid medium, realizing the spreading function.

The solid medium filling pipe 6, the discharge pipe 7, the air intake pipe 12, the bacterial suspension filling pipe 10, the exhaust pipe 11, the liquid medium filling pipe 8, and the waste liquid pipe 18 are provided with valves 19. A waste liquid pipe 18 is provided at the bottom of the solid culture member 2.

A method for culturing H. pylori using the device is as follows:

(1) Coat the stirrer with 1 mg/ml fibrinogen.

(2) Preparation of solid medium. Weigh 3.9 g of Columbia Blood Agar Base into 100 ml of distilled water, autoclave the Columbia Blood Agar Base in an autoclave sterilizer for 15-20 min at 103.4 kPa and 121.3°C. After gradually cooling to 60°C, add and immediately inject 10 ml of FBS into the solid culture member from the solid medium filling pipe. The upper surface of the liquid level is flush with the indicator ring. Cool and cure the solid medium.

(3) Prepare H. pylori into a1 x 109 cells/ml bacterial suspension, inject the bacterial suspension into the bacterial suspension filling pipe, adjust the fixing bolt or the support bar so that the lower end of the stirrer is in contact with the upper surface of the solid medium, and rotate the stirrer to spread the bacteria. The culture is conducted at 37°C under an atmosphere of clean mixed gas (10% C02, 85% N2, and 5% 02) with an aeration volume of 1 L/min.

(4) Preparation of liquid medium. Weigh 3.7 g of Brain Heart Infusion Broth into 100 ml of distilled water, and autoclave the Brain Heart Infusion Broth in the autoclave sterilizer for 15-20 min at 103.4 kPa and 121.3°C. After gradually cooling to room temperature, add 10 ml of FBS for use.

(5) After culturing H. pylori on the solid medium for 12 h, inject the liquid medium from the liquid medium filling pipe, and culture at 37°C under an atmosphere of clean mixed gas (10% C02, % N2, and 5% 02) with an aeration volume of 1 L/min. After static culture for 12 h, H. pylori adheres to the fibrinogen-coated stirrer and grows quickly by means of nutrients of the liquid medium. Adjust the fixing bolt or the support bar to separate the lower end of the stirrer from the upper surface of the solid medium, and start the motor to drive the stirrer to rotate at 20 r/min; continue the culture for 1-2 days, release the liquid medium from the discharge pipe, and collect the bacteria.

(6) After collecting the bacteria, increase the temperature of the heating platform to melt the solid medium, and release the melted solid medium from the waste liquid pipe.

After adopting the device and the method, the intermediate operation process during the solid liquid culture of H. pylori is substantially reduced in a convenient manner, and the culture period may be shortened by at least 2-3 days, providing a basic condition for preparing inactivated whole-cell vaccines.

The above descriptions are only preferred embodiments of the present disclosure and are not intended to limit the present disclosure. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure shall be included in the scope of the present disclosure.

Claims

What is claimed is:

1. A large-scale culture method of Helicobacter pylori, wherein the method utilizes a Helicobacterpyloriliquid-solid culture device and comprises the following steps:

step (A), coating a stirrer in the Helicobacterpylori liquid-solid culture device with 1 mg/ml fibrinogen;

step (B), preparation of solid medium: weighing 3.9 g of Columbia Blood Agar Base into 100 ml of distilled water, autoclaving in an autoclave sterilizer, gradually cooling to 60°C, adding and immediately injecting 10 ml of fetal calf serum (FBS) into the Helicobacterpylori liquid-solid culture device, cooling and curing;

step (C), preparing Helicobacter pylori into a 1 x 109 cells/ml bacterial suspension, injecting the bacterial suspension into a solid culture member of the Helicobacterpyloriliquid solid culture device, spreading Helicobacterpylori,and culturing on the solid medium at 37°C under an atmosphere of clean mixed gas with an aeration volume of 1 L/min;

step (E), after culturing Helicobacterpylori on the solid medium for 12 h, injecting a resulting liquid medium into a liquid culture member of the Helicobacterpylori liquid-solid culture device, and conducting static culture on the liquid medium at 37°C under an atmosphere of clean mixed gas with an aeration volume of 1 L/min; after culturing for 12 h, stirring the liquid medium at 20 r/min, continuing the culture for 1-2 days, releasing the liquid medium, and collecting Helicobacterpylori.

2. The large-scale culture method of Helicobacterpyloriaccording to claim 1, wherein in steps (C) and (E), a gas introduced is a clean mixed gas with an oxygen content of 5%.

3. The large-scale culture method of Helicobacterpyloriaccording to claim 1, wherein the Helicobacterpyloriliquid-solid culture device comprises a culture flask, a sealing cap arranged on the upper part of the culture flask, and a heating platform arranged under the culture flask; the culture flask comprises a solid culture member, a liquid culture member, and an indicator rin arranged between the solid culture member and the liquid culture member;

a fixed hub is arranged at the center of the bottom of the solid culture member, the bottom of the solid culture member is laterally in contact with the heating platform, and the side wall of the solid culture member is provided with a solid medium filling pipe; the height of the fixed hub is higher than the distance between the indicator ring and the bottom of the solid culture member; the bottom side wall of the liquid culture member is provided with a discharge pipe and an air intake pipe; the sealing cap comprises a cover body sealed with a top opening of the liquid culture member, a bacterial suspension filling pipe, an exhaust pipe, a liquid medium filling pipe and a spreading arrangement arranged in the cover body; and the spreading arrangement comprises a rotary rod penetrating the cover body, a stirrer arranged on the rotary rod, and a motor that drives the rotary rod to rotate; one end of the rotary rod is provided with a slot, the other end is rotatably connected with the fixed hub, a drive shaft of the motor is inserted into the slot and fixedly connected by a fixing bolt, and a seal sleeve is arranged between the rotary rod and the cover body.

4. The large-scale culture method of Helicobacterpyloriaccording to claim 3, wherein a support bar iis further provided on the cover body, one end of the support bar is fixedly connected with the cover body, and the other end is fixedly connected with the housing of the motor;

wherein the solid culture member is a truncated cone-shaped container, the liquid culture member is a cylinder-shaped container, and a diameter of the cylinder is equal to a diameter of the top surface of the truncated cone;

wherein the support bar is telescopic;

wherein a gas filter is provided at the outer end of the exhaust pipe;

wherein the stirrer is a flat plate arranged along the axis of the rotary rod, and the lower end of the stirrer is provided with a reserved slot for receiving the fixed hub.

5. The large-scale culture method of Helicobacterpyloriaccording to claim 3, wherein a waste liquid pipe is provided at the bottom of the solid culture member;

wherein the solid medium filling pipe, the discharge pipe, the air intake pipe, the bacterial suspension filling pipe, the exhaust pipe, the liquid medium filling pipe, and the waste liquid pipe are provided with valves.