Anaerobic bacteria co-culture device and application
By designing a simple anaerobic co-culture device, the problems of complex operation and high cost in the existing technology have been solved, realizing low-cost anaerobic co-culture and promoting research on intestinal flora interactions.
Patent Information
- Application Number
- CN202511042092.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-11-07
AI Technical Summary
Existing bacterial co-culture techniques are complex and costly, making it difficult to simulate the anaerobic or microanaerobic environment of gut microbiota in the laboratory, thus affecting research on gut microbiota interactions.
An anaerobic co-culture device consisting of a first-screw syringe, a Luer-patterned adapter, a tubing, a filter membrane clamp, and a filter membrane is used. Through the design of the connection and the filter membrane, the co-culture of two anaerobic bacteria is achieved, simulating an anaerobic environment.
It enables simple and low-cost co-culture of anaerobic bacteria, avoids cross-contamination, is suitable for co-culture of various anaerobic bacteria, and promotes research on intestinal flora interactions.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microorganisms, in particular to an anaerobic bacteria co-culture device and application. BACKGROUND
[0002] Intestinal flora plays an important role in human health. The balance of intestinal flora is closely related to the human digestive, metabolic and immune systems. With the gradual deepening of the study of intestinal flora, more and more studies have shown that there are complex interactions between the microorganisms present in the intestine. Studying the interaction mechanisms between intestinal flora has important scientific value and application prospects. On the one hand, it helps us to better understand the physiological functions and pathological mechanisms of intestinal microecology, and provides a theoretical basis for the prevention and treatment of intestinal-related diseases. For example, certain intestinal flora imbalance is closely related to the occurrence and development of diseases such as obesity, diabetes, and inflammatory bowel disease. On the other hand, by studying the interaction of intestinal flora, potential microbial resources with application value can be tapped, such as the development of new probiotics.
[0003] Pathogenic bacteria are a class of microorganisms that can break through the host immune barrier and cause infectious diseases. Their pathogenicity and transmission characteristics pose a persistent threat to human health. Salmonella is a class of gram-negative, facultative anaerobic bacilli in the Enterobacteriaceae family. As one of the most important foodborne pathogenic bacteria in the world, Salmonella is transmitted through the fecal-oral route, mainly contaminating poultry meat (the contamination rate of retail raw chicken in China is about 40%), eggs (shell and internal contamination risk), dairy products and fresh fruits and vegetables. The clinical manifestations of Salmonella infection are gastroenteritis (6-72 hour incubation period, acute fever, abdominal pain, diarrhea), enteric fever (typhoid / paratyphoid) and septicemia.
[0004] Probiotics are live microorganisms that are designated to be planted in the human body and can produce benefits to the health of the host, and play an important role in maintaining human health. Probiotics composed of a variety of microbial communities can inhibit the growth of harmful bacteria by competing for nutrients and attachment sites, thereby resisting the invasion of pathogens and protecting the body from invasion. Existing studies have shown that probiotics can be effectively applied to the treatment of various gastrointestinal diseases, including acute infectious diarrhea, ulcerative colitis, etc. Notably, probiotics have been shown to have the potential to prevent and treat cancer, and may become an adjunctive drug against cancer in the future. Therefore, it is particularly important to uncover the key mechanisms of the interaction between probiotics and other bacteria.
[0005] Currently, the co-culture techniques of bacteria mainly include compartment culture method, microfluidic chip technology and cell culture plug-in method. Although these existing techniques meet the needs of intestinal flora co-culture to some extent, they all have complex operation, high cost and need anaerobic culture equipment such as anaerobic / microaerophilic workstation and anaerobic tank, which increases the complexity of experiment. With the in-depth research of intestinal flora, in vitro simulation of the interaction of intestinal flora in the intestinal tract becomes an important issue. In the research of intestinal flora interaction, it is crucial to provide intestinal flora with a more natural interaction condition. Therefore, an effective and convenient co-culture device for simulating the interaction of bacteria in the anaerobic or microaerobic environment of the intestinal tract is urgently needed, which facilitates laboratory operation and in-depth research, provides a powerful tool for the research of intestinal flora interaction mechanism and promotes the research progress in microbiology and related fields. SUMMARY
[0006] The purpose of the present application is to overcome the shortcomings of the prior art and provide an anaerobic bacteria co-culture device.
[0007] The second purpose of the present application is to provide the application of the anaerobic bacteria co-culture device in anaerobic bacteria co-culture.
[0008] The technical solution of the present application is as follows:
[0009] The anaerobic bacteria co-culture device comprises a first screw injection syringe 1 and a second screw injection syringe 8, the first screw injection syringe 1 is connected with a first luer outer thread adapter 2, a first hose 3, a filter membrane clamp 4, a second hose 5, a second luer outer thread adapter 6 and the second screw injection syringe 8 in sequence, and the filter membrane clamp 4 is provided with a filter membrane 7.
[0010] The specification of the filter membrane 7 is preferably 0.22 μm filter membrane.
[0011] The application of the above anaerobic bacteria co-culture device in anaerobic bacteria co-culture preferably comprises the following steps:
[0012] 1) In a biological safety cabinet, the above anaerobic bacteria co-culture device is used;
[0013] 2) The plungers of the first screw injection syringe 1 and the second screw injection syringe 8 are pulled out respectively; the culture medium is added into the barrel of the first screw injection syringe 1, the plunger of the first screw injection syringe 1 is put into the barrel of the first screw injection syringe 1, the plunger of the first screw injection syringe 1 is pushed to push and fill the culture medium into the second screw injection syringe 8; the culture medium is filled into the second screw injection syringe 8, and the plunger of the first screw injection syringe 1 is pulled out;
[0014] 3) The plunger of the second screw injection syringe 8 is put into the barrel of the second screw injection syringe 8, the plunger of the second screw injection syringe 8 is pushed to push and fill the culture medium into the barrel of the first screw injection syringe;
[0015] 4) Add the first anaerobic bacteria into the barrel of the first screw syringe 1, and fill the barrel of the first screw syringe 1 with the culture medium; put the piston of the first screw syringe 1 into the barrel of the first screw syringe 1;
[0016] 5) pull out the piston of the second screw syringe 8, add the second anaerobic bacteria into the barrel of the second screw syringe 8, and supplement the culture medium, and put the piston of the second screw syringe 8 into the barrel of the second screw syringe 8;
[0017] 6) anaerobic culture, remove the bacterial liquid in the barrel of the first screw syringe 1 and the barrel of the second screw syringe 8 to obtain the first anaerobic bacteria liquid and the second anaerobic bacteria liquid.
[0018] Preferably, the first anaerobic bacteria is Salmonella typhimurium 14028s 3297:gfp with a fluorescent protein gene.
[0019] Preferably, the second anaerobic bacteria is Bifidobacterium breve, Faecalibacterium prausnitzii, Akkermansia or Prevotella.
[0020] Advantages of the present application:
[0021] The anaerobic bacteria co-culture device of the present application has simple structure, low cost and convenient use, can easily simulate an anaerobic environment and co-culture anaerobic bacteria under the condition of no anaerobic culture equipment, well cultures two different bacteria in the same liquid environment without cross contamination, is suitable for co-culturing of various common anaerobic bacteria such as Salmonella typhimurium, Bifidobacterium breve, Faecalibacterium prausnitzii, Akkermansia or Prevotella, and is of great benefit to the initial research of bacterial interaction related research projects. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 Figure 1 is a schematic diagram of an anaerobic bacteria co-culture device; wherein A is an exploded view of an anaerobic bacteria co-culture device; B is a schematic diagram of a filter membrane clamp 4; C is a schematic diagram of assembly of the filter membrane clamp 4 and the filter membrane 7.
[0023] Figure 2 Figure 2 is a co-culture picture of S.Tm and Bi.Breve;
[0024] Figure 3 Figure 3 is a fluorescence observation after co-culture of S.Tm and Bi.Breve;
[0025] Figure 4 Figure 4 is a co-culture picture of S.Tm and F.p.;
[0026] Figure 5 Figure 5 is a fluorescence observation after co-culture of S.Tm and F.p.;
[0027] Figure 6 Picture of co-culture of S.Tm and AKK;
[0028] Figure 7 Fluorescence observation after co-culture of S.Tm and AKK;
[0029] Figure 8 Picture of co-culture of S.Tm and P.copri;
[0030] Figure 9 Fluorescence observation after co-culture of S.Tm and P.copri. DETAILED DESCRIPTION
[0031] The application will be further described below in conjunction with specific examples.
[0032] Screw-in syringe (flute / L108977, also known as disposable plastic syringe for laboratory; 3 mL)
[0033] Luer adapter (4.8 luer PC: Guangzhou Sanglong)
[0034] Hose (silicone hose)
[0035] Filter membrane clamp (polytetrafluoroethylene filter membrane clamp: GLASS / HONPZSX0753)
[0036] Filter membrane (specification 0.22 μm filter membrane, diameter 25 mm: JinTeng / TJMF0424)
[0037] The disclosure of the above components is to enable those skilled in the art to better implement it, but does not limit the application.
[0038] The bacterial sources used in the following examples are as follows:
[0039] The original strain of Salmonella typhimurium 14028s 3297:gfp with fluorescent protein gene: Salmonella typhimurium was purchased from the American Type Culture Collection (ATCC) with the number ATCC14028, and reference was made to Xiao Hong, He Zhen, Tian Qi, et al. Construction of fluorescent reporter strain of Salmonella typhimurium and its application in tracing of porcine alveolar macrophages [J]. North China Journal of Agricultural Sciences, 2023, 38(02): 232-238. The gfp fluorescent protein gene was inserted to obtain Salmonella typhimurium 14028s 3297:gfp with fluorescent protein gene, which is referred to as S.Tm.
[0040] The source of the second anaerobic bacteria used in the examples: Bifidobacterium breve, Faecalibacterium prausnitzii, Akkermansia or Prevotella was purchased from Beina Biology (Suzhou Beina Chuilian Biotechnology Co., Ltd.) in March 2024.
[0041] Bifidobacterium breve / BNCC186529, abbreviated as Bi.Breve;
[0042] Faecalibacterium prausnitzii / BNCC363610, abbreviated as F.p.
[0043] Akkermansia muciniphila / BNCC341917, abbreviated as Akk
[0044] Prevotella copri / BNCC337399, abbreviated as P.copri
[0045] Example 1
[0046] The anaerobic bacteria co-culture device (see Figure 1 ), comprising a first screw syringe 1 and a second screw syringe 8, the first screw syringe 1 is connected with a first luer external swab adapter 2, a first hose 3, a filter membrane clamp 4, a second hose 5, a second luer external swab adapter 6 and the second screw syringe 8 in sequence, and the filter membrane clamp 4 is provided with a filter membrane 7.
[0047] The specification of the filter membrane 7 is preferably 0.22 μm filter membrane.
[0048] Example 2
[0049] Pre-treatment of the first anaerobic bacteria and the second anaerobic bacteria:
[0050] (1) Resuscitation of the first anaerobic bacteria and the second anaerobic bacteria:
[0051] The first anaerobic bacteria (Salmonella typhimurium 14028s 3297:gfp with a fluorescent protein gene) was taken out at -80°C, thawed at room temperature, inoculated on a Chl LB plate by plate streaking method, and cultured overnight at 37°C. A single colony was inoculated into 10 mL BHI medium and cultured overnight at 37°C under anaerobic conditions (sealed).
[0052] The second anaerobic bacteria (Bifidobacterium breve, Faecalibacterium prausnitzii, Akkermansia muciniphila and Prevotella copri were resuscitated by the same method as follows)
[0053] The second anaerobic bacteria (Bifidobacterium breve, Faecalibacterium prausnitzii, Akkermansia muciniphila and Prevotella copri were resuscitated by the same method as follows)
[0054] (2) Preparation of anaerobic co-culture device: Wash the other parts of the anaerobic co-culture device (excluding the 0.22μm filter membrane) with water, immerse them in a 75% ethanol aqueous solution overnight, take them out, sterilize them under ultraviolet light in a biosafety cabinet, dry them, and assemble them together; sterilize the 0.22μm filter membrane under ultraviolet light and put it into the filter membrane holder.
[0055] Example 3
[0056] The application of anaerobic co-culture devices in anaerobic co-culture includes the following steps:
[0057] 1) Use a sterilized anaerobic co-culture device in a biosafety cabinet;
[0058] 2) Pull out the pistons of the first screw-type syringe 1 and the second screw-type syringe 8 respectively; add BHI culture medium into the barrel of the first screw-type syringe 1, put the piston of the first screw-type syringe 1 into the barrel of the first screw-type syringe 1, push the piston of the first screw-type syringe 1 to push the BHI culture medium into the second screw-type syringe 8; fill the second screw-type syringe 8 with BHI culture medium, and pull out the piston of the first screw-type syringe 1.
[0059] 3) Insert the piston of the second screw syringe 8 into the barrel of the second screw syringe 8, push the piston of the second screw syringe 8 to push and fill the BHI culture medium into the barrel of the first screw syringe;
[0060] 4) Add the first type of anaerobic bacteria (take 2 μL of overnight cultured Salmonella Typhimurium suspension carrying fluorescent protein gene, measure OD600~1) into the barrel of the first screw syringe 1, and fill the entire barrel of the first screw syringe 1 with BHI medium; put the piston of the first screw syringe 1 into the barrel of the first screw syringe 1.
[0061] 5) Pull out the piston of the second screw-type syringe 8, add the second type of anaerobic bacteria into the barrel of the second screw-type syringe 8 (take 2 mL of overnight cultured Bifidobacterium breve suspension, measure OD600-1, and supplement with BHI medium, and put the piston of the second screw-type syringe 8 into the barrel of the second screw-type syringe 8).
[0062] 6) After anaerobic incubation at 37℃ for 18 hours, the bacterial culture was removed from the barrels of the first screw-type syringe 1 and the second screw-type syringe 8 to obtain the first and second anaerobic bacterial cultures. The OD values were measured at incubation times of 0 hours and 18 hours, respectively. 600 .
[0063] After 18 hours, both bacterial cultures were observed to be turbid. The co-culture device for one type of anaerobic bacteria was well-sealed with no leakage. The OD values of the two bacterial cultures were... 600 ~1, see Figure 2 .
[0064] Example 4
[0065] Fluorescence observation of Salmonella typhimurium with fluorescent protein gene and Bifidobacterium breve co-cultured bacteria suspension
[0066] (1) Salmonella typhimurium with fluorescent protein gene sample preparation:
[0067] ① Take 5 μL of the co-cultured Salmonella typhimurium with fluorescent protein gene suspension in Example 3 and spread it on a clean slide, and dry it at 37°C for 5 min.
[0068] ② Add 4% paraformaldehyde and fix it at room temperature for 5 min, and gently rinse the paraformaldehyde with PBS.
[0069] ③ Add 20 μL of anti-fluorescence quenching mounting medium (Company: beyotime, Product number: P0126-25mL), cover the cover glass, and observe under a fluorescence microscope.
[0070] (2) Bifidobacterium breve sample preparation:
[0071] ④ Take 5 μL of the co-cultured Bifidobacterium breve suspension in Example 3 and spread it on a clean slide, and dry it at 37°C for 5 min.
[0072] ⑤ Add 4% paraformaldehyde and fix it at room temperature for 5 min, and gently rinse the paraformaldehyde with PBS.
[0073] ⑥ Add 20 μL of anti-fluorescence quenching mounting medium, cover the cover glass, and observe under a fluorescence microscope.
[0074] It is observed that the Bifidobacterium breve liquid has no fluorescence, and the Salmonella typhimurium with fluorescent protein gene liquid has significant fluorescence, indicating that the co-cultivation device can ensure that the two bacteria are not contaminated, as shown in Figure 3 .
[0075] Example 5
[0076] Application of anaerobic bacteria co-cultivation device in anaerobic bacteria (Salmonella typhimurium with fluorescent protein gene and Prevotella xylaniphila) co-cultivation, including the following steps:
[0077] Use Prevotella xylaniphila to replace Bifidobacterium breve in Example 3, and the rest is the same as Example 3. The results are shown in Figure 4 .
[0078] Example 6
[0079] Fluorescence observation of Salmonella typhimurium with fluorescent protein gene and Prevotella xylaniphila co-cultured bacteria suspension
[0080] Use Prevotella xylaniphila to replace Bifidobacterium breve in Example 4, and the rest is the same as Example 4, and the results are shown inFigure 5 .
[0081] Example 7
[0082] Application of anaerobic bacteria co-culture device in co-culture of anaerobic bacteria (Salmonella typhimurium with fluorescent protein gene and Akkermansia muciniphila), including the following steps:
[0083] Replace Akkermansia muciniphila with Bifidobacterium breve in Example 3, and the rest is the same as Example 3. See Figure 6 .
[0084] Example 8
[0085] Fluorescence observation of Salmonella typhimurium with fluorescent protein gene and Akkermansia muciniphila co-cultured bacteria suspension
[0086] Replace Akkermansia muciniphila with Bifidobacterium breve in Example 4, and the rest is the same as Example 4. See Figure 7 .
[0087] Example 9
[0088] Application of anaerobic bacteria co-culture device in co-culture of anaerobic bacteria (Salmonella typhimurium with fluorescent protein gene and Prevotella), including the following steps:
[0089] Replace Akkermansia muciniphila with Bifidobacterium breve in Example 3, and the rest is the same as Example 3. See Figure 8 .
[0090] Example 10
[0091] Fluorescence observation of Salmonella typhimurium with fluorescent protein gene and Prevotella co-cultured bacteria suspension
[0092] Replace Akkermansia muciniphila with Bifidobacterium breve in Example 4, and the rest is the same as Example 4. See Figure 9 .
Claims
1. Anaerobic bacteria co-cultivation device comprising a first screw syringe (1) and a second screw syringe (8), characterized in that The first screw port injector (1) is sequentially connected with a first luer external thread adapter (2), a first hose (3), a filter membrane clamp (4), a second hose (5), a second luer external thread adapter (6) and the second screw port injector (8), and the filter membrane clamp (4) is provided with a filter membrane (7).
2. The apparatus for co-culturing anaerobic bacteria according to claim 1, wherein The filter membrane (7) is a 0.22 mu m filter membrane.
3. Use of the anaerobic bacteria co-cultivation device of claim 1 or 2 in anaerobic bacteria co-cultivation.
4. Use according to claim 3, characterized in that The method comprises the following steps: 1) using the anaerobic bacteria co-cultivation device of claim 1 or 2 in a biological safety cabinet; 2) respectively pulling out the plungers of the first screw port injector (1) and the second screw port injector (8); adding a culture medium into the barrel of the first screw port injector (1), putting the plunger of the first screw port injector (1) into the barrel of the first screw port injector (1), pushing the plunger of the first screw port injector (1), pushing the culture medium and filling the second screw port injector (8); filling the culture medium into the second screw port injector (8) and pulling out the plunger of the first screw port injector (1); 3) putting the plunger of the second screw port injector (8) into the barrel of the second screw port injector (8), pushing the plunger of the second screw port injector (8), pushing the culture medium and filling the barrel of the first screw port injector; 4) adding the first anaerobic bacteria into the barrel of the first screw port injector (1) and filling the barrel of the first screw port injector (1) with the culture medium; putting the plunger of the first screw port injector (1) into the barrel of the first screw port injector (1); 5) pulling out the plunger of the second screw port injector (8), adding the second anaerobic bacteria into the barrel of the second screw port injector (8) and supplementing the culture medium, and putting the plunger of the second screw port injector (8) into the barrel of the second screw port injector (8); 6) anaerobic culture, removing the bacterial liquid in the barrel of the first screw port injector (1) and the barrel of the second screw port injector (8) to obtain the first anaerobic bacteria liquid and the second anaerobic bacteria liquid.
5. Use according to claim 4, characterized in that The first anaerobic bacteria is Salmonella typhimurium 14028s 3297:gfp with a fluorescent protein gene.
6. Use according to claim 4, characterized in that The second anaerobic bacteria is Bifidobacterium breve, Faecalibacterium prausnitzii, Akkermansia or Prevotella.