A plastic bag air-extraction microbial culture method and device

Abstract

This invention discloses a method for microbial culture using a plastic bag with vacuum extraction, comprising: placing a culture medium inoculated with the microorganisms to be cultured inside a sealed plastic bag with excellent thermal conductivity and permeability; using a vacuum extraction device to extract the air from the plastic bag to reduce its internal oxygen content, forming a microaerobic or anaerobic environment with low redox potential; and subsequently heat-sealing the vacuum port of the plastic bag. The device mainly includes the plastic bag and a vacuum sealing machine. This invention replaces the traditional chemical oxygen absorption method with physical vacuum extraction, solving the problems of high cost, cumbersome operation, chemical pollution and safety hazards, difficulty in observation, inaccurate temperature control, and inability to conduct long-term culture by existing methods. It has the advantages of low cost, simple operation, safety and environmental protection, easy observation, and the ability to maintain a stable culture environment for a long time, and is particularly suitable for the isolation, culture, and research of microorganisms such as bacteria, fungi, mycoplasma, and chlamydia.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and more specifically to a method and apparatus for culturing microaerophilic or anaerobic microorganisms. Background Technology

[0002] Microorganisms can be classified into aerobic bacteria, facultative anaerobes, and obligate anaerobes / microaerobics based on their oxygen requirements. Microaerobic bacteria and strict anaerobes require a specific low-oxygen environment (low redox potential) for cultivation. Currently, commonly used methods for creating microaerobic environments in laboratories include: ① Candle method: A candle is lit inside a sealed desiccator to consume oxygen. This method is simple, but temperature control is inaccurate (due to poor thermal conductivity of the desiccator), observation is inconvenient, environmental stability is poor (failure is easily caused by operation or candle extinguishing), and there is a risk of spillage. ② Chemical reagent method: Such as the pyrogallol-alkali oxygen absorption method. This method absorbs oxygen relatively thoroughly, but the reagents used are toxic, easily deteriorate (due to oxidation), and produce corrosive waste liquid, posing safety hazards and environmental pollution, and are also costly. ③ Anaerobic tank / bag method: An anaerobic environment is created inside a sealed tank / bag using commercially available anaerobic gas-generating bags and oxygen-absorbing bags. Operation is simple, but consumables (especially imported products) are expensive, cannot be reused, and generate solid waste. ④ Anaerobic incubator method: The air inside the chamber is replaced by an inert gas (such as nitrogen). Environmental control is precise, but the equipment is expensive (tens of thousands to hundreds of thousands of yuan), operating costs are high (continuous gas consumption), maintenance is complex, and airflow inside the chamber can easily cause moisture evaporation from the culture medium. ⑤ Hengette drum method: Used for strictly anaerobic bacterial culture; the equipment is expensive and relies on imports, operation is complex, and consumables are specialized.

[0003] The methods described above all have drawbacks to varying degrees, such as high cost, cumbersome operation, risk of contamination, difficulty in real-time observation, or unsuitability for long-term cultivation. Therefore, developing a low-cost, high-efficiency, safe, environmentally friendly, and easily observable microaerobic culture method has significant application value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and apparatus for microbial culture by air extraction from plastic bags. This method creates and maintains a stable low redox potential environment at low cost through physical air extraction.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for microbial culture by vacuuming plastic bags, comprising the following steps: S0. Modify the vacuum sealing machine by adding a digital pressure gauge with a pressure range of -0.1 to 0.1 MPa (with 1 standard atmosphere as the zero standard). S1. Prepare a sealed plastic bag with a viewing window and at least one air vent; S2. Place the culture medium inoculated with the microorganisms to be cultured into the plastic bag; S3. Use an air extraction device to extract the air from the plastic bag through the air extraction port to create a low oxidation-reduction potential environment inside the bag. S4. After the pressure is reduced to the specified pressure, the air extraction port is sealed to isolate the inside of the plastic bag from the external environment; S5. Place the sealed plastic bag in a constant temperature incubator for incubation.

[0006] Furthermore, the plastic bag is made of food-grade or medical-grade plastic film such as polyethylene (PE) or polypropylene (PP), which has excellent thermal conductivity (thermal conductivity preferably ≥0.2 W / (m·K)) and high transparency (light transmittance preferably ≥80%), and can withstand a certain negative pressure (with atmospheric pressure as the reference), to ensure uniform heat transfer and facilitate direct observation.

[0007] Furthermore, after evacuation, the redox potential inside the bag drops to below 5% of oxygen, which meets the cultivation requirements of most microaerophilic bacteria. By controlling the evacuation time and observing the digital pressure gauge readings, it can be further adjusted to below 1% to meet more stringent anaerobic requirements.

[0008] Furthermore, using an integrated vacuum sealing machine for vacuuming and heat sealing operations is highly efficient and provides reliable sealing.

[0009] The present invention also provides an apparatus for implementing the above method, comprising a plastic bag unit, a support placed inside the plastic bag unit, the support including at least one horizontal tray.

[0010] Compared with the prior art, the present invention has the following significant advantages: 1. Extremely low cost: The core consumable is inexpensive plastic bags (priced at about 0.1-0.3 yuan each, which can be reused), and the equipment only requires a low-cost vacuum sealing machine (priced at around 100 yuan). The overall cost is only one-hundredth to one-tenth of that of traditional methods.

[0011] 2. Safe and environmentally friendly: No toxic chemical reagents are involved in the entire process, and no harmful waste gas, waste liquid or solid waste is generated, avoiding chemical hazards and environmental pollution.

[0012] 3. Simple and quick operation: The steps are simple, requiring no complicated pretreatment or gas control, and can be easily completed by one person.

[0013] 4. Easy to observe: The transparent bag can be placed directly in the incubator to observe the growth of microorganisms without opening and disturbing the environment, achieving non-destructive monitoring.

[0014] 5. Stable and independent culture environment: Each sample is individually packaged, without interference, avoiding cross-contamination and mutual influence of harmful metabolites (mainly gases such as hydrogen sulfide).

[0015] 6. Suitable for long-term culture: The completely sealed environment effectively prevents the evaporation of moisture from the culture medium, and can maintain culture for several weeks without drying out. It can be used for slow-growing microorganisms such as tuberculosis bacilli and mycoplasma.

[0016] 7. Precise temperature control: The plastic bag has good thermal conductivity and can quickly balance with the temperature of the incubator, ensuring accurate and stable incubation temperature.

[0017] This invention differs fundamentally from commercially available anaerobic bag methods: This invention employs a "physical air extraction and oxygen removal" principle, directly extracting air from the bag to create a low oxidation-reduction potential environment without relying on chemical reagents. In contrast, commercially available anaerobic bags rely on chemical reagents reacting with oxygen to absorb oxygen, which not only involves the consumption and deterioration of chemical reagents but also generates solid waste and is costly. This invention has fundamental advantages in cost control, safety, and environmental friendliness. Attached Figure Description

[0018] Figure 1 This is a front view of a first embodiment of the apparatus for the microbial culture method described in this invention.

[0019] Figure 2 This is a top view of a first embodiment of the apparatus for the microbial culture method described in this invention.

[0020] Figure 3 This is a front view of a second embodiment of the apparatus for the microbial culture method described in this invention.

[0021] Figure 4 This is a top view of a second embodiment of the apparatus for the microbial culture method described in this invention.

[0022] Attached labels: 1-bag body, 2-bag body seal, 3-support, 4-culture dish, 5-exhaust port. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] Example 1: Isolation and culture of Bifidobacterium Prepare a Figure 1 and Figure 2 The polyethylene plastic bag 1 shown has an opening and closing point 2, which is transparent and has a pre-installed support 3 inside.

[0025] Place the MRS agar plate containing the intestinal sample into culture dish 4, and then place culture dish 4 into the tray groove of support 3.

[0026] Insert one end of the bag seal 2 into the integrated vacuum sealing machine that combines the vacuuming and sealing equipment. Start the machine and vacuum for about 30 seconds. Observe the pressure value through the digital pressure gauge. When the value shows -0.01, it is equivalent to 5% oxygen at normal pressure. If a lower oxidation-reduction potential is required, you can continue to vacuum, such as when the digital display shows -0.02.

[0027] After the air extraction is complete, the sealing function is activated and the machine automatically seals the seal.

[0028] Place the sealed plastic bag 1 into a 37℃ constant temperature incubator and incubate for 48-72 hours.

[0029] During the incubation period, colony growth can be observed directly through the viewing window area 6 of the bag. After the incubation is complete, cut open the sealed area of ​​the plastic bag by 2-3 cm and remove the petri dish for identification. The colonies grow vigorously and have typical morphology. The remaining part of the bag can be reused.

[0030] Example 2: Long-term culture of mycoplasma Using the same plastic bag 1 and support 3 as in Example 1, the mycoplasma-specific solid culture medium plate inoculated with clinical samples was placed into the culture dish 4, and then the culture dish 4 was placed in the tray groove of the support 3.

[0031] Connect the integrated vacuum sealing machine according to the steps in Example 1, start the machine to vacuum for 35 seconds, and observe the pressure value through the digital pressure gauge. When the value shows -0.01, it is equivalent to 5% oxygen at normal pressure. If a lower oxidation-reduction potential is required, you can continue to vacuum, such as when the digital display shows -0.02.

[0032] After evacuation, heat seal the bag and place it in a 37°C incubator for incubation.

[0033] Because the environment inside the bag is stable and the humidity is well maintained (the surface of the culture medium is still moist after 10 days of cultivation, without any cracking), and because the support 3 avoids the problem of breakage caused by direct contact between the culture dish and the bag, it can be continuously cultured for 5-7 days or even longer, making it convenient to observe the slowly growing mycoplasma colonies ("fried egg-like" colonies).

[0034] Example 3: Culture of Clostridium perfringens By modifying the petri dish, such as increasing its thickness and material toughness (referred to as an improved petri dish), the pressure that the petri dish can withstand (withstanding -0.1 MPa with atmospheric pressure as a zero reference) can be increased, enabling scaffold-free vacuum culture, as described below: After the improved petri dishes are manufactured, they are sterilized by the manufacturer using ethylene oxide or ultraviolet light according to standard procedures. The culture medium (such as TSC tryptone sulfite cycloserine agar base) is poured in the same way as ordinary disposable petri dishes. Liver tissue fluid can be added at a volume ratio of 5% to prepare plate culture medium.

[0035] Using the same plastic bag 1 as in Example 1, the modified culture dish inoculated with the clinical sample was placed in the plastic bag 1, and then the plastic bag 1 was connected to the integrated vacuum sealing machine. The machine was started to vacuum for 35 seconds, and the pressure value was observed through the digital pressure gauge. When the value showed -0.01, it was equivalent to 5% oxygen at normal pressure (if a lower oxidation-reduction potential is required, vacuuming can continue, such as when the digital display shows -0.02).

[0036] After evacuation, heat seal the bag and place it in a 37°C incubator for incubation.

[0037] During the incubation period, colony growth can be observed directly through the viewing window area 6 of the bag. After the incubation is complete, cut open the sealed area of ​​the plastic bag by 2-3 cm and remove the petri dish for bacterial identification. The colonies grow vigorously and have typical morphology. The remaining part of the bag can be reused.

[0038] Example 4: Culture of Helicobacter pylori Prepare a Figure 3 and Figure 4 The polyethylene plastic bag 1 shown has an opening and closing point 2, which is the bag's seal. It is completely transparent and has a pre-installed support 3 inside. The polyethylene plastic bag 1 has a thermal conductivity ≥0.2 W / (m·K) and a light transmittance ≥85%.

[0039] Place the Columbia blood agar plate inoculated with Helicobacter pylori (standard strain ATCC 43504) into petri dish 4, and then place petri dish 4 into the tray groove of support 3, ensuring that the surface of the plate is unobstructed.

[0040] First, seal the bag opening 2. Then, open the sealing cover of the suction opening 5. Connect the suction device to the suction opening 5 and start suctioning for 25 seconds. Observe the pressure value through the digital pressure gauge. When the value shows -0.01, it is equivalent to 5% oxygen at normal pressure. If a lower oxidation-reduction potential is required, you can continue suctioning, such as when the digital display shows -0.02.

[0041] Stop the vacuuming process, remove the vacuuming equipment, cover it with a sealed cap, and place the plastic bag in a 37°C constant temperature incubator for 72 hours.

[0042] During the culture, the colony morphology can be observed in real time through the transparent window. After the culture is completed, the plate is taken out for biochemical identification. The results show that Helicobacter pylori is growing well and has typical colony characteristics.

[0043] Experimental example: Comparative verification with existing technologies To verify the technical advantages of this invention, the candle jar method and the commercially available anaerobic bag method (imported anaerobic gas-generating bags), which are widely used in the prior art, were selected as controls. Helicobacter pylori, Bifidobacterium, and Mycoplasma were used as test subjects in parallel comparative experiments. The key results are summarized in the table below:

[0044] Results Analysis: This invention is significantly superior to the candle jar method in terms of cost, ease of operation, environmental stability (especially humidity maintenance), culture effect, and safety and environmental protection, and is comparable to or even better than the expensive commercial anaerobic bag method (especially in terms of cost and environmental protection). This proves that this invention, through the technical solution of "physical air extraction and oxygen removal + transparent thermally conductive plastic bag + compartmentalized support", achieves a high-quality microaerophilic culture environment at extremely low cost.

[0045] The above embodiments and experimental examples fully illustrate the specific implementation methods and beneficial effects of the present invention. The scope of protection of the present invention is not limited to the above embodiments. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for microbial culture by vacuuming plastic bags, characterized in that, Includes the following steps: S1. Prepare a sealed plastic bag with a viewing window and at least one air vent; S2. Place the culture medium inoculated with the microorganisms to be cultured into the plastic bag; S3. Use an air extraction device to extract the air from the plastic bag through the air extraction port to reduce the pressure inside the bag and create a low oxidation-reduction potential environment. S4. After the pressure is reduced to the specified pressure, the air extraction port is sealed to isolate the inside of the plastic bag from the external environment; S5. Place the sealed plastic bag in a constant temperature incubator for incubation.

2. The method for microbial culture by vacuuming plastic bags according to claim 1, characterized in that, In step S1, the plastic bag is made of polyethylene, polypropylene, or composite film material, with a thermal conductivity of not less than 0.2 W / (m·K) and a light transmittance of not less than 80%.

3. The method for microbial culture by vacuuming plastic bags according to claim 1, characterized in that, In step S2, the plastic bag also contains a support for supporting and separating the petri dishes, the support comprising at least one horizontal tray.

4. The method for microbial culture by vacuuming plastic bags according to claim 1, characterized in that, In step S3, the low-oxygen environment refers to an environment where the oxidation-reduction potential inside the bag is equivalent to that of 2-5% oxygen.

5. The method for microbial culture by vacuuming plastic bags according to claim 1, characterized in that, In steps S3 and S4, an integrated vacuum sealing machine is used to complete the vacuuming and heat sealing operations.

6. The method for microbial culture by vacuuming plastic bags according to claim 1, characterized in that, The microorganisms to be cultured are microaerophilic or anaerobic bacteria, including Bifidobacterium, lactic acid bacteria, mycoplasma, chlamydia, Campylobacter jejuni, or Helicobacter pylori.

7. A microbial culture apparatus for implementing the method according to any one of claims 1-6, characterized in that, include: A plastic bag unit, which is a sealable bag body made of transparent thermally conductive material, with at least one heat-sealable air extraction / sealing port on the bag body; A support frame placed inside the plastic bag unit, the support frame comprising At least one horizontal tray.

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