Combined culture device and culture method thereof

Abstract

This invention belongs to the technical field of culture devices, specifically disclosing a combined culture device and its culture method. In this invention, the first container is a container that can be filled with gas. The bottom of the first container has a protruding end, and the other end of the protruding end is provided with an inoculation ring. The inoculation ring is used to inoculate the bacterial solution to be cultured into a second container. A gas channel is connected between the interior of the protruding end and the first inner cavity. The gas channel is used to introduce gas from the first inner cavity into the second container for the growth and reproduction of the bacterial solution in the second container. By providing a sealing ring, the connection between the protrusion and the groove can be strengthened, preventing carbon dioxide gas leakage from the second container. The culture device of this invention has a simple structure, is small and convenient, and is suitable for the rapid culture of some bacteria in the laboratory. Therefore, it has certain application prospects in the field of culture devices.

Description

Technical Field

[0001] This invention belongs to the field of culture device technology, specifically relating to a combined culture device and its culture method. Background Technology

[0002] Mycobacterium tuberculosis (M. tuberculosis) is an acid-fast, rod-shaped bacillus, mainly including human, bovine, African, and murine types, with the human type being the most pathogenic to humans. The tubercle bacillus is slender and slightly curved, approximately (1–4) × 0.4 μm in size. It lacks flagella and spores, cannot produce toxins or endotoxins, is obligately aerobic, requires high culture standards, and grows slowly. Its cell components mainly include lipids, polysaccharides, and proteins. Numerous studies have shown that Mycobacterium tuberculosis is a facultative parasite because it initially replicates within infected macrophages, hence the term intracellular bacteria; then, as the disease progresses, it also replicates extracellularly in necrotic tissue. The pathogenicity of Mycobacterium tuberculosis is related to the inflammatory response caused by its massive intracellular proliferation, the toxicity of its cell and cell wall components and metabolites, and the body's anti-tuberculosis immune response. Mycobacterium tuberculosis has a thick cell wall with a high lipid content. Its cytoplasmic membrane is surrounded by a rigid peptidoglycan (PG) layer. Between the PG layer and the plasma membrane are various immunogenic proteins, and a large amount of mycolic acid surrounds the PG layer. The main pathogenic strain to humans is Mycobacterium humane (also known as tuberculosis bacillus), the causative agent of tuberculosis in humans. Mycobacterium tuberculosis culture is one of the gold standards commonly used in laboratory testing for tuberculosis, and is frequently used for etiological diagnosis of tuberculosis and susceptibility testing of anti-tuberculosis drugs.

[0003] There are various culture media for Mycobacterium tuberculosis. Initial isolation of Mycobacterium tuberculosis requires a nutrient-rich medium. A commonly used medium is Lowenstein-Jensen solid medium, which contains egg yolk, glycerol, potato starch, inorganic salts, and malachite green. Malachite green inhibits the growth of other bacteria, facilitating isolation and long-term culture. Egg yolk contains lipid growth factors that stimulate growth. Depending on the amount of inoculated bacteria, colony growth is generally visible in 2-4 weeks. The colonies are granular, nodular, or cauliflower-like, milky white or pale yellow, and opaque. Commonly used liquid media include 7H9. Because the surface area of ​​contact with nutrients is larger in liquid media, bacterial growth is more rapid, with noticeable growth generally visible in 1-2 weeks. Clinical practice has also shown that liquid culture has a positive rate several times higher than solid culture.

[0004] Besides the culture medium, temperature and other culture conditions also significantly affect the growth of Mycobacterium tuberculosis. Mycobacterium tuberculosis is generally cultured under conditions of 40-50% oxygen, 5-10% CO2, a temperature of 36℃±5℃, and a suitable pH of 6.8-7.2. Studies have shown that higher CO2 concentrations promote the growth of Mycobacterium tuberculosis. Therefore, the culture of Mycobacterium tuberculosis generally needs to be carried out in a dedicated CO2 incubator or a gas-generating bag with CO2 production capabilities. These operations are cumbersome, and CO2 incubators and gas-generating bags are generally quite expensive.

[0005] To provide optimal culture conditions for Mycobacterium tuberculosis more conveniently, it is necessary to develop a modular culture device. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a combined culture device and its culture method. It solves the problems of existing bacterial culture methods requiring specialized CO2 incubators or gas-generating bags with CO2 production capabilities, which result in cumbersome operation and generally high costs for CO2 incubators and gas-generating bags.

[0007] To address the aforementioned problems in the prior art, the present invention is achieved through the following technical solution:

[0008] A combined culture device includes a first container and a second container:

[0009] The first container includes a top cover, a first inner cavity, and an extended end. The first inner cavity is located in the middle of the first container. The top cover covers the first inner cavity. The first inner cavity is filled with carbon dioxide gas. The extended end is located at the bottom of the first container. An inoculation ring is provided at the other end of the extended end. The bottom of the first inner cavity is connected to one end of a gas channel. The other end of the gas channel passes through the bottom of the first container and extends into the interior of the extended end.

[0010] The second container includes a second inner cavity located in the middle of the second container, and the second inner cavity contains a culture medium;

[0011] The bottom of the first container and the top of the second container are sealed together.

[0012] Furthermore, the first container also includes protrusions symmetrically arranged on the outer side wall of the first container; the second container also includes grooves symmetrically arranged on the inner side wall of the second container, and the protrusions and the grooves are engaged and connected.

[0013] Furthermore, a sealing ring is provided between the protrusion and the groove.

[0014] Furthermore, the protruding end includes a break opening, which is disposed on a gas passage extending into the interior of the protruding end.

[0015] Furthermore, a heating device is connected to the bottom of the second container.

[0016] Furthermore, the first container is made of polypropylene.

[0017] Furthermore, the second container is made of polypropylene.

[0018] Furthermore, an elastic device is provided on the outer wall of the second container.

[0019] The present invention also provides a cultivation method.

[0020] The cultivation method includes the following steps:

[0021] S1. Inflation: Open the top cover of the first container and fill the first inner cavity of the first container with carbon dioxide gas. After inflation is complete, close the top cover to complete the entire inflation process.

[0022] S2, Injecting Culture Medium: Add culture medium to the second container;

[0023] S3. Sampling: Place the inoculation loop on the bottom protruding end of the first container described in step S1 into the container containing the bacterial culture to be cultured, and gently stir to complete the entire sampling process.

[0024] S4. Break: Lean the first container described in step S3 against the second container described in step S2, apply gentle force, and the protruding end breaks off from the break point. Then the inoculation loop with bacterial solution falls into the bottom of the second container containing the culture medium.

[0025] S5. Sealed culture: Stretch the side wall of the second container described in step S4 outward, and then insert the protrusion on the first container described in step S4 into the groove on the inner side wall of the second container to complete the sealing process; then connect the sealed device to the heating device to culture the bacterial solution.

[0026] Furthermore, all of the S1 to S5 operations are performed in a sterile environment.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1) In this invention, the first container is a container that can be filled with gas. The bottom of the first container is provided with an extended end, and the other end of the extended end is provided with an inoculation ring. The inoculation ring is used to inoculate the bacterial solution to be cultured into the second container. A gas channel is connected between the inside of the extended end and the first inner cavity. The gas channel is used to introduce the gas in the first inner cavity into the second container for the growth and reproduction of the bacterial solution in the second container. By providing a sealing ring, the connection between the protrusion and the groove can be strengthened to prevent carbon dioxide gas leakage in the second container.

[0029] 2) The culture device of the present invention has a simple structure, is small and convenient, and is suitable for the rapid culture of some bacteria in the laboratory. Therefore, it has certain application prospects in the field of culture devices. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the combined culture device structure of the present invention;

[0032] Figure 2 This is a schematic diagram of the culture process of the culture device of the present invention;

[0033] The meanings of the reference numerals in the figure are as follows: 1: First container; 11: Top cover; 12: First inner cavity; 13: Carbon dioxide gas; 14: Protrusion; 15: Sealing ring; 16: Gas channel; 17: Protruding end; 171: Fold-off end; 18: Inoculation loop; 2: Second container; 21: Groove; 22: Second inner cavity; 23: Culture medium; 3: Heating device. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "top / bottom," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0036] Please see Figure 1 A combined culture device includes a first container 1 and a second container 2.

[0037] The first container 1 includes a top cover 11, a first inner cavity 12, and an extension end 17. The first inner cavity 12 is located in the middle of the first container 1. The top cover 11 covers the first inner cavity 12. The first inner cavity 12 is filled with carbon dioxide gas 13. The extension end 17 is located at the bottom of the first container 1. An inoculation ring 18 is provided at the other end of the extension end 17. The bottom of the first inner cavity 12 is connected to one end of a gas channel 16. The other end of the gas channel 16 passes through the bottom of the first container 1 and extends into the interior of the extension end 17.

[0038] The second container 2 includes a second inner cavity 22, which is located in the middle of the second container 2, and the second inner cavity 22 contains a culture medium 23.

[0039] The bottom of the first container 1 and the top of the second container 2 are sealed together.

[0040] Furthermore, the first container 1 also includes a protrusion 14, which is symmetrically arranged on the outer side wall of the first container 1; the second container 2 also includes a groove 21, which is symmetrically arranged on the inner side wall of the second container 2, and the protrusion 14 and the groove 21 are engaged and connected.

[0041] In this invention, the first container 1 is a container that can be filled with gas. The bottom of the first container 1 is provided with an extended end 17, and the other end of the extended end 17 is provided with an inoculation ring 18. The inoculation ring 18 is used to inoculate the bacterial solution to be cultured into the second container 2. A gas channel 16 is connected between the inside of the extended end 17 and the first inner cavity 12. The gas channel 16 is used to introduce the gas in the first inner cavity 12 into the second container 2 for the growth and reproduction of the bacterial solution in the second container 2. It should be noted that the volumes of the first container 1 and the second container 2 can be adjusted as needed to achieve the gas concentration required for cultivation. The inoculation ring 18 can be designed as a ring with different diameters or a porous shape according to the volume of bacterial solution to be taken, so that the bacterial solution of 1 to 100 μL can be adsorbed by surface tension.

[0042] Furthermore, a sealing ring 15 is provided between the protrusion 14 and the groove 21. By providing the sealing ring 15, the connection between the protrusion 14 and the groove 21 can be strengthened, preventing the leakage of carbon dioxide gas 13 in the second container 2.

[0043] Furthermore, the protruding end 17 includes a break opening 171, which is disposed on the gas channel 16 extending into the interior of the protruding end 17. By providing the break opening 171, it can be further ensured that the protruding end 17 breaks at the break opening 171, facilitating the diffusion of carbon dioxide gas 13 in the first container 1 into the second container 2.

[0044] Furthermore, a heating device 3 is connected to the bottom of the second container 2. The heating device 3 can be adjusted to the appropriate temperature according to the optimal growth temperature of the bacterial culture. Optionally, the heating device 3 is a constant temperature water bath, or any other device with temperature control.

[0045] Furthermore, both the first container 1 and the second container 2 are made of polypropylene. Polypropylene is a polymer material that is non-toxic to bacteria.

[0046] Furthermore, an elastic device 24 is provided on the outer wall of the second container 2. For example, the elastic device 24 can be a rubber ring fixedly fitted on the outer wall, or other existing fixing methods are acceptable. When the first container 1 and the second container 2 are sealed together, the side wall of the second container 2 is first stretched outward. At this time, the elastic device 24 is in a stretched state. Then, the protrusion on the first container is engaged in the groove on the inner side wall of the second container 2. The elastic device 24 retracts, causing the side wall of the second container 2 to return to its original position, further ensuring the reliability of the connection between the protrusion 14 and the groove 21. At the same time, it also facilitates the installation of the first container 1 and the second container 2.

[0047] It should be noted that the gas channel 16 is a narrow and elongated channel. By making the gas channel 16 a narrow and elongated channel, excessive gas outflow and large gas loss can be avoided when the break point 171 breaks.

[0048] Please see Figure 2 A cultivation method comprising the following steps:

[0049] S1. Inflation: Open the top cover 11 on the first container 1 and fill the first inner cavity 12 of the first container 1 with carbon dioxide gas 13. After inflation is complete, close the top cover 11 to complete the entire inflation process.

[0050] S2, Inject culture medium: Add culture medium 23 to the second container 2;

[0051] S3. Sampling: Place the inoculation loop 18 on the bottom protruding end 17 of the first container 1 mentioned in step S1 into the container containing the bacterial solution to be cultured, and gently stir to complete the entire sampling process.

[0052] S4. Breaking: Lean the first container 1 described in step S3 against the second container 2 described in step S2, and apply gentle force. The protruding end 17 breaks off from the break 171, and then the inoculation loop 18 with bacterial solution falls into the bottom of the second container 2 containing the culture medium 23.

[0053] S5. Sealed culture: Stretch the side wall of the second container 2 described in step S4 outward, and then insert the protrusion 14 on the first container 1 described in step S4 into the groove 22 on the inner side wall of the second container 2 to complete the sealing process; then connect the sealed device to the heating device 3 to culture the bacterial solution.

[0054] Furthermore, all of the S1 to S5 operations are performed in a sterile environment.

[0055] To verify the cultivation effect of the combined cultivation device of the present invention, the effects of culturing Mycobacterium bovis (BCG) using the combined cultivation device of the present invention and the traditional CO2 incubator were compared in the laboratory. The first inner cavity 12 of the first container 1 has a volume of approximately 25 μL and is filled with 100% CO2. The second inner cavity 22 of the second container 2 has a volume of approximately 200 μL. 50 μL of 7H9 culture medium was added to the second inner cavity 22. Approximately 10 μL of BCG bacterial solution was taken using an inoculation loop 18 and... Figure 2After the inoculation and gas introduction are completed, the CO2 gas concentration in the culture device is about 10%. The temperature of the heating device 3 is set to 37°C and cultured for 4 days (96 hours). Then, the bacterial solution is inactivated at 95°C for 30 minutes. Finally, the solution is mixed by vortexing and then briefly separated. 5 μL of the supernatant is taken as a template and the nucleic acid changes are measured using a quantitative PCR kit (provided by Wuhan Saisrui Microbial Technology Co., Ltd.) to verify whether Mycobacterium bovis has grown.

[0056] Meanwhile, using a traditional culture method, 10 μL of the same BCG bacterial suspension was pipetted into a reagent tube containing 50 μL of 7H9 medium and then placed in a CO2 incubator (10% CO2) at 37°C. During the culture, the tube was left loose for one day to ensure sufficient CO2 exchange to the top of the medium to promote tuberculosis growth, after which it was sealed tightly. After 4 days of culture, the bacterial suspension was inactivated at 95°C for 30 min. Finally, the mixture was vortexed, briefly centrifuged, and 5 μL of the supernatant was used as a template. A quantitative PCR kit was used to detect changes in nucleic acid to verify whether Mycobacterium bovis had grown.

[0057] The specific results are shown in Table 1:

[0058] Table 1. Results of Mycobacterium bovis culture

[0059]

[0060]

[0061] As shown in Table 1, the growth of BCG strains cultured using the combined culture device of this invention is consistent with that of traditional CO2 incubator culture, with no difference in Ct values ​​after 4 days of growth. This result indicates that the combined culture device of this invention can culture and grow some bacteria. Furthermore, the device is simple in structure, small and convenient, and suitable for the rapid culture of some bacteria in the laboratory.

[0062] The above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the scope of the technology disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention.

Claims

1. A combined culture device, comprising a first container (1) and a second container (2), characterized in that: The first container (1) includes a top cover (11), a first inner cavity (12) and an extension end (17). The first inner cavity (12) is located in the middle of the first container (1). The top cover (11) covers the first inner cavity (12). The first inner cavity (12) is filled with carbon dioxide gas (13). The extension end (17) is located at the bottom of the first container (1). The other end of the extension end (17) is provided with an inoculation ring (18). The bottom of the first inner cavity (12) is connected to one end of a gas channel (16). The other end of the gas channel (16) passes through the bottom of the first container (1) and extends into the interior of the extension end (17). The second container (2) includes a second inner cavity (22), which is located in the middle of the second container (2), and the second inner cavity (22) contains a culture medium (23). The bottom of the first container (1) and the top of the second container (2) are sealed together; The protruding end (17) includes a break (171) disposed on a gas passage (16) extending into the interior of the protruding end (17).

2. The combined culture device as described in claim 1, characterized in that, The first container (1) further includes a protrusion (14), which is symmetrically arranged on the outer side wall of the first container (1); the second container (2) further includes a groove (21), which is symmetrically arranged on the inner side wall of the second container (2), and the protrusion (14) and the groove (21) are engaged and connected.

3. The combined culture device as described in claim 2, characterized in that, A sealing ring (15) is also provided between the protrusion (14) and the groove (21).

4. The combined culture device as described in claim 1 or 2, characterized in that, The bottom of the second container (2) is connected to a heating device (3).

5. The combined culture device as described in claim 1 or 2, characterized in that, The first container (1) is made of polypropylene.

6. The combined culture device as described in claim 1 or 2, characterized in that, The second container (2) is made of polypropylene.

7. The combined culture device as described in claim 6, characterized in that, An elastic device (24) is provided on the outer wall of the second container (2).

8. A cultivation method using the cultivation apparatus of claim 1, characterized in that, Includes the following steps: S1. Inflation: Open the top cover (11) on the first container (1), and fill the first inner cavity (12) in the first container (1) with carbon dioxide gas (13). After inflation is completed, close the top cover (11) to complete the entire inflation process. S2, Inject culture medium: Add culture medium (23) into the second container (2); S3. Sampling: Place the inoculation loop (18) on the bottom protruding end (17) of the first container (1) mentioned in step S1 into the container containing the bacterial solution to be cultured, stir gently, and complete the entire sampling process; S4, break: lean the first container (1) described in step S3 against the second container (2) described in step S2, apply gentle force, and the protruding end (17) breaks from the break point (171), and then the inoculation loop (18) with bacterial solution falls into the bottom of the second container (2) containing the culture medium (23); S5. Sealed culture: Stretch the side wall of the second container (2) described in step S4 outward, and then insert the protrusion (14) on the first container (1) described in step S4 into the groove (22) on the inner side wall of the second container (2) to complete the sealing process; then connect the sealed device to the heating device (3) to carry out bacterial culture.

9. The cultivation method as described in claim 8, characterized in that, All operations S1 to S5 are performed in a sterile environment.

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