Oral cavity microorganism separation culture method

Abstract

The invention belongs to the technical field of culture methods of microorganisms, and particularly relates to an oral cavity microorganism isolated culture method which comprises the following steps: (I) collecting a sample; (2) sterilizing and disinfecting the chip and the assembly; (3) assembling a chip; (IV) performing in-situ culture; and (V) separation and purification. The oral microorganism isolated culture method provided by the invention is simple to operate, the growth of oral microorganisms fits the growth condition of original ecological microorganisms, the growth speed of the oral microorganisms is not influenced, the problem of nutrient demand does not need to be considered, the operation simplicity is improved, and experimental verification shows that the separation capacity of the oral microorganisms is remarkably improved, and the oral microorganisms can be effectively cultured. The experiment difficulty is reduced, and more possibilities are provided for clinical research and practical application.

Description

Technical Field

[0001] This invention relates to the field of microbial culture methods, and more specifically, to a method for isolating and culturing oral microorganisms. Background Technology

[0002] The oral microbial community is an important component of the human microbial community. The balance of the oral microbial community directly affects various health conditions such as periodontal disease, dental caries, and overall health. In the medical field, microbial isolation and culture devices can be used to diagnose and treat diseases. The oral microorganisms obtained can not only help us understand the pathogenesis and pathological process of oral diseases, but also provide new ideas and methods for the prevention and treatment of oral diseases.

[0003] Currently, most methods for isolating and culturing oral microorganisms rely on traditional culture media and culture devices. These methods often have problems such as complex operation, long culture cycle, and high risk of contamination. Furthermore, the natural growth environment of oral microorganisms is complex, and existing technologies cannot completely replicate the original environment of oral microorganisms.

[0004] In summary, it is very important to propose a method for isolating and culturing oral microorganisms. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for isolating and culturing oral microorganisms.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows: A method for isolating and culturing oral microorganisms, the key of which is the use of a microporous array chip with a length of 5-7 mm, a width of 3-5 mm, and a thickness of 1-2 mm, wherein the microporous array chip is provided with several interconnected culture micropores; Includes the following steps: (I) Sample collection: Collect oral microbial samples for later use; (II) Sterilization and disinfection of chips and components: ① Sterilize the microporous array chip for later use; ② Disinfect the first filter membrane, the second filter membrane and the connecting device for later use; (III) Chip assembly: Fix the first filter membrane to the underside of the microporous array chip, prepare a solid culture medium for microorganisms, heat and sterilize it, and while the solid culture medium is not solidified, immerse the microporous array chip with the first filter membrane fixed in the culture medium. After the culture medium solidifies, remove it and remove the residual culture medium around it; inject the oral microbial sample collected in the sample collection step from the upper surface of the microporous array chip until the saliva no longer seeps into the micropores. After the injection is completed, fix the second filter membrane to the upper side of the microporous array chip. (IV) In situ culture: The assembled chip is adhered to the oral cavity of the subject and cultured for 1-14 days; (V) Separation and purification: Take out the cultured chip, remove the first and second filter membranes of the chip under sterile conditions, place the chip in a microbial liquid culture medium, separate the microorganisms cultured in the micropores, and culture the liquid culture medium containing microorganisms to obtain the isolated oral microorganisms.

[0007] As a further improvement of the present invention, the microporous array chip is a well plate based on polypropylene or polyetheretherketone; the microporous array chip is provided with multiple microporous arrays, and each microporous array contains multiple culture microwells.

[0008] As a further improvement of the present invention, the culture micropores of different micropore arrays have different pore sizes.

[0009] As a further improvement of the present invention, the pore size of the culture micropores ranges from 8μm to 540μm, and the distance between the pores is 0.2 mm.

[0010] As a further improvement of the present invention, the pore size of the culture micropore is 400 μm.

[0011] As a further improvement of the present invention, the oral microbial samples in step (I) are saliva samples, subgingival plaque samples, and gingival crevicular fluid samples.

[0012] As a further improvement of the present invention, the first and second filter membranes are polycarbonate filter membranes with a pore size of 0.03 μm and a thickness of 15-20 μm; the connecting device is food-grade silicone; the filter membranes are fixed to the microporous array chip by food-grade silicone, so that each pore is separated from the other pores and used as a separate microbial culture chamber for culturing microorganisms.

[0013] As a further improvement of the present invention, the sterilization method in steps (II) and (III) is high-pressure steam sterilization at 121°C; The disinfection method in step (II) is ultraviolet light irradiation. In step (III), the microbial solid culture medium is LB solid culture medium; The microbial liquid culture medium in step (V) is LB liquid culture medium.

[0014] As a further improvement of the present invention, in step (IV), the lower side of the assembled chip first filter membrane is adhered to the buccal side of the subject's maxillary first molar using dental adhesive.

[0015] As a further improvement of the present invention, the specific operation for separating the microorganisms cultured in the microwells in step (V) is as follows: use a vortex mixer at 1000-1500 rpm for 10-20 seconds to wash out the microbial cells cultured in the microwells.

[0016] As a further improvement of the present invention, in step (V), the liquid culture medium containing microorganisms is isolated and cultured by means of streak plate separation, spread plate separation, or / and pour plate separation.

[0017] The beneficial effects of adopting the above technical solution are as follows: This invention provides a method for isolating and culturing oral microorganisms, comprising (I) sample collection; (II) sterilization and disinfection of the chip and components; (III) chip assembly; (IV) in situ culture; and (V) isolation and purification. The method for isolating and culturing oral microorganisms provided in this application is simple to operate, allowing the growth of oral microorganisms to closely resemble the growth conditions of native microorganisms, without affecting the growth rate of oral microorganisms, eliminating the need to consider nutrient requirements, and improving operational simplicity. Experimental verification has shown that it significantly improves the isolation capacity of oral microorganisms, reduces experimental difficulty, and provides more possibilities for clinical research and practical applications. Attached Figure Description

[0018] Figure 1 This is a flowchart of the isolation and culture method of the present invention; Figure 2 This is a schematic diagram of the assembled micro-hole array chip of the present invention; Figure 3 yes Figure 2 An explosion diagram; Figure 4 This is a top view of a micro-hole array chip provided by the present invention; Figure 5 This is a top view of another micro-hole array chip provided by the present invention; Figure 6 This is a graph showing the results of microbial abundance in the test samples of this application. In the graph, Group I is the group cultured using the method in Example 1 of this application, Group C is the group cultured using the traditional culture method, and Group M is the saliva sample detected by high-throughput sequencing.

[0019] Wherein: 1 is the first filter membrane, 2 is the micropore array chip, 3 is the second filter membrane, 201 is the culture micropore, and 202 is the micropore array. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0021] The flowchart of the isolation and culture method in this application is as follows: Figure 1 As shown, the specific steps include sample collection, sterilization and disinfection of the chip and components, chip assembly, in-situ culture, and separation and purification.

[0022] like Figures 2-5As shown, it comprises, from bottom to top: a first filter membrane 1, a microporous array chip 2, and a second filter membrane 3; the first filter membrane 1 is used to be specifically bonded to the subject's teeth. The micropore array chip 2 is provided with a plurality of through-hole culture micropores 201. The first filter membrane 1 covers the bottom of all culture micropores 201, and the second filter membrane 3 covers the top of all culture micropores 201. The culture micropores 201 are used for microbial growth and reproduction.

[0023] This invention does not specifically limit the material and size of the aforementioned first filter membrane 1, microporous array chip 2, and second filter membrane 3, as long as they are suitable for microbial culture. For example, the microporous array chip 2 is made of polypropylene, which has good mechanical properties and biocompatibility and will not produce toxicity when cultured in the oral cavity. For example, the microporous array chip 2 is made of polyetheretherketone. The first filter membrane 1 and the second filter membrane 3 are both made of polycarbonate, and the pore size of the first filter membrane 1 and the second filter membrane 3 is 0.03 μm, ensuring that the first filter membrane 1 and the second filter membrane 3 can effectively isolate the culture micropores 201 for cultivating oral microorganisms, avoiding the influence of the external environment on the microorganisms.

[0024] Regarding the size, in order to avoid causing discomfort to the subject when the device is placed on the buccal side of the subject's maxillary first molar, the microporous array chip 2 has a length of 5-7mm, a width of 3-5mm, and a thickness of 1-2mm, with its four sides transitioned by arcs; the thickness of the first filter membrane 1 and the second filter membrane 3 is 15-20μm.

[0025] The dimensions of the culture micropores 201 in the micropore array chip 2 are described below.

[0026] In one possible implementation, several culture microwells 201 are arranged with different pore sizes, ranging from tens of micrometers to hundreds of micrometers. This range of pore sizes better meets the needs of oral microbial cultivation. Of course, the culture microwells 201 are arranged in an array and are independent of each other. Specifically, the microwell array chip 2 has multiple microwell arrays 202, each containing multiple culture wells. The culture wells within the same microwell array 202 have the same pore size, but the pore sizes of the culture wells in different microwell arrays 202 are different. For example, as... Figure 5 As shown, the number of micropore arrays 202 is set to five, and the pore sizes of the culture wells contained in the five micropore arrays 202 are 8 micrometers, 20 micrometers, 60 micrometers, 180 micrometers and 540 micrometers respectively.

[0027] In another possible implementation, the size of the culture micropores 201 can be set to a single size, i.e., forming a micropore array. In this case, as... Figure 4 As shown, the inner diameter of the micropores can be set to 400 μm, and the distance between any two culture micropores 201 is 200 μm.

[0028] In one possible implementation, the collected oral microbial sample is a saliva sample. The collection method is as follows: a disposable saliva collector is used, and the patient must not eat, chew gum, or smoke for 30 minutes prior to collection. After rinsing the mouth with purified water, the saliva is spat into a test tube containing a funnel until the saliva volume reaches 3 ml. The funnel is then rotated off the test tube, the cap is closed and tightened, and the tube is labeled and transported to the laboratory via cold chain, where it is stored at -80°C.

[0029] In one possible implementation, the collected oral microbial samples are subgingival plaque samples. The collection method is as follows: the sampling operator uses a supragingival curette to remove supragingival plaque and soft deposits, dries a cotton ball to isolate it from moisture, and gently scrapes subgingival plaque around the tooth root using a Gracy curette, placing it in a 1.5 mL EP tube. When the amount of subgingival plaque is small, PBS is used to flush the plaque off the sampler and place it in the tube, which is then sent to the laboratory for storage at -80°C within 4 hours. After collection, the sample is placed in a sampling tube containing preservation solution, stirred for 30-40 seconds, and then rapidly shaken for at least 30 seconds before being transferred to a -80°C freezer on dry ice.

[0030] In one possible implementation, the collected oral microbial sample is a gingival crevicular fluid sample. The collection method is as follows: probing removes gingival plaque and tartar, the tooth surface is dried, and a 2mm × 10mm Whatman filter paper is deeply inserted into the bottom of the gingival sulcus for 30 seconds. The filter paper is then removed and placed in an EP tube. The EP tube is pre-filled with 0.2 mL of phosphate-buffered saline (PBS), vortexed for 1 hour, centrifuged at 3000 rpm for 10 minutes (centrifugation radius 12 cm), and the supernatant is stored at -80°C for later use.

[0031] The LB solid culture medium used in this application consists of: 1.0 g tryptone, 0.5 g yeast extract, 1.0 g sodium chloride, and 1.5 g agar powder, with distilled water added to 100 ml, and autoclaved at 121°C for 30 minutes.

[0032] The LB liquid medium is made of 1.0g tryptone, 0.5g yeast extract and 1.0g sodium chloride, with distilled water added to 100ml, and autoclaved at 121℃ for 30 minutes.

[0033] The RTV 108 food-grade silicone used in this application was purchased from Momentive, USA.

[0034] The polycarbonate filter membrane was purchased from GVS, USA, item number 1239558.

[0035] The dental adhesive is 3M Dental 8th Generation Adhesive, manufactured by 3M Deutschland GmbH, with product number B5005096099.

[0036] Example 1 Sample collection: Saliva samples were collected from five subjects using a disposable saliva collector. (Inclusion criteria: age 20-50 years, total number of teeth ≥ 20, intact maxillary first molars on both sides, without fillings or restorations. Exclusion criteria: (1) chronic diseases, infectious diseases, genetic diseases and autoimmune diseases; (2) patients with severe oral diseases (such as mucosal diseases, tumors, etc.); (3) acute inflammation, infection or trauma; (4) those who have used antibiotics, probiotics or orthodontic treatment in the past three months.) Avoid eating, chewing gum and smoking within 30 minutes before sample collection. After rinsing the mouth with purified water, saliva was spat into a test tube with a funnel until the saliva volume reached 3ml. Then the funnel was rotated off the test tube, the test tube cap was put on and tightened, and the tube was labeled and transported to the laboratory by cold chain and stored in a -80℃ freezer.

[0037] Sterilization and disinfection of chips and components: Sterilization: The microwell array chip 2 is sterilized by high-pressure steam at 121°C for 30 minutes before use. The chip is a well plate based on polypropylene or polyether ether ketone (PEEK). The well plate contains 32 interconnected culture microwells 201 (pore diameter 400μm, distance between pores 200μm), with dimensions of 5×3×1mm. In this embodiment, a polypropylene well plate is used.

[0038] ② Disinfection: Irradiate the polycarbonate film and RTV 108 food-grade silicone with ultraviolet light for 30 minutes in a biosafety cabinet, and set aside for later use.

[0039] Chip assembly: The first filter membrane 1 (polycarbonate membrane, 15 μm thick, 0.03 μm pore size) was adhered to the underside of the microporous array chip 2 using RTV 108 food-grade silicone, ensuring complete coverage of the underside of the microporous array chip 2. LB solid culture medium was prepared in advance and autoclaved at 121°C for 30 minutes. After the temperature dropped to approximately 55°C, the chip was immersed in the LB solid culture medium. Once the medium solidified, the chip was removed, and any remaining medium was cleaned off. Using a disposable sterile syringe, saliva samples were injected into the upper surface of each micropore 201 of the microporous array chip 2 until no more saliva was observed to seep into the micropores. After the chip was left to stand and no obvious saliva was observed on its surface, the second filter membrane 3 (polycarbonate membrane, 15 μm thick, 0.03 μm pore size) was adhered to the upper side of the microporous array chip 2 using RTV 108 food-grade silicone, ensuring complete coverage of the upper side of the microporous array chip 2. The oral microbial isolation and culture device is obtained after the RTV108 food-grade silicone has completely cured.

[0040] In situ culture: The lower side of the assembled chip first filter membrane 1 was adhered to the buccal side of the subject's maxillary first molar using dental adhesive.

[0041] Separation and purification: ① After 5 days of culture, remove the chip and remove the upper and lower filter membranes in a biosafety cabinet. Place the microwell array chip 2 into a sterile test tube containing 1 ml of LB liquid medium and vortex at 1500 rpm for 20 seconds to wash out the microbial cells in the culture microwells. Use a sterile inoculating loop (10 μl size) to pick up the LB liquid after vortexing and use the streak plate method to incubate at 37°C on blood agar plates (HuanKai Microbial, China, 024070) and brain and heart infusion plates (HuanKai Microbial, China, C28361B1). Observe every 24 hours. When a single colony is observed to form in the terminal zone, use a sterile inoculating loop to pick up single colonies of different sizes and shapes and streak them on the corresponding fresh medium. Repeat the process once more to obtain a medium containing single colonies of oral microorganisms.

[0042] Example 2 The culture medium containing single colonies of oral microorganisms obtained in Example 1 was sent to Shanghai Paisennong Biotechnology Co., Ltd. for 16S rRNA sequencing. The composition and diversity of the oral microbial community were explored through gene sequencing and bioinformatics analysis of single strains.

[0043] Saliva samples from five subjects collected in Example 1 were cultured using a conventional method. The conventional method involved directly dipping a sterile inoculating loop (10 μl size) into the saliva sample and streaking it onto blood agar plates (HuanKai Microbial, China, 024070) and brain and heart immersion agar plates (HuanKai Microbial, China, C28361B1) using the streak plate method. The samples were observed every 24 hours at 37°C. When a single colony was observed to form in the terminal zone, a sterile inoculating loop was used to pick up single colonies of different sizes and shapes and streak them onto the corresponding fresh culture medium. This process was repeated once more to obtain a culture medium containing single colonies of oral microorganisms obtained by the conventional method.

[0044] Culture media containing single colonies of oral microorganisms obtained by traditional culture methods were sent to Shanghai Paisenuo Biotechnology Co., Ltd. for 16S rRNA sequencing, and saliva samples were sent to Shanghai Paisenuo Biotechnology Co., Ltd. for high-throughput sequencing.

[0045] Example of effect 1 High-throughput sequencing results ( Figure 6 The analysis method involved merging sequences obtained from high-throughput sequencing based on 100% sequence similarity to generate characteristic ASV sequences and abundance data tables. ASV classification and taxonomic identification were performed using the HOMD_16S database (Human Oral Microbiome Database). ASV characteristic sequences were compared with reference sequences in the database to obtain taxonomic information for each ASV. The abundance matrix, after removing rare ASVs, was used for subsequent analyses. The samples contained at least 20 genera of bacteria. Haemophilus accounted for 19.7%, Streptococcus for 15.7%, followed by Neisseria for 13.0%, Prevotella for 10.8%, Veillonella for 8.8%, Porphyromonas for 6.4%, Prevotella for 3.4%, Fusobacterium for 3.3%, Salrella for 2.4%, and others for 6.1%.

[0046] Traditional culture methods yielded 62 bacterial strains belonging to 7 genera. 16S rRNA gene sequencing and comparison identified 44 strains as *Streptococcus*, 6 as *Rothia*, 8 as *Neisseria*, 1 as *Staphylococcus*, 1 as *Capnocytophaga*, 1 as *Corynebacterium*, and 1 as *Cutibacterium*.

[0047] Following in situ culture in the oral cavity using the method described in Example 1 of this application, bacteria from the microarray were isolated and purified on blood agar plates and brain-heart infusion plates. Identification using 16S rRNA genes yielded 126 strains belonging to 14 genera. The 14 genera are as follows: Streptococcus, Neisseria, Rothia, Staphylococcus, Veillonella, Gemella, Enterobacte, Granulicatella, Actinomyces, Kingella, Corynebacterium, Moraxella, Eikenella, Schaalia. The results showed that among the 14 genera, *Streptococcus*, *Neisseria*, *Rochetomyces*, *Gymnococcus*, *Veillonella*, *Enterobacter cloacae*, *Streptococcus granulosus*, and *Corynebacterium* were the most numerous. Three of these strains showed less than 98.7% similarity to the full-length 16S rRNA sequence in the NCBI database, confirming them as novel species.

[0048] The statistical analysis of all detected microorganisms is shown in Table 1.

[0049] Table 1. Summary of all genera of microorganisms detected by the method, conventional culture method and high-throughput sequencing method in Example 1 of this application ("-" indicates not detected). The results show that the method in Example 1 of this application can significantly improve the isolation ability of oral microorganisms, which is reflected in two aspects: First, for genera that can be isolated by traditional culture methods, the method in Example 1 of this application can obtain more strains. For example, compared with traditional culture methods, more streptococci, Neisseria, Roche, and Corynebacterium were isolated from the improved chip. Second, the method in Example 1 of this application can culture microbial species that cannot be isolated by traditional culture methods. For example, Veillonella, Gestationococcus, Enterobacter cloacae, Granulococcus, Actinomyces, Chlorella, Moraxella, Ekenella, and Salmonella can only be isolated from the method in Example 1 of this application, and these microorganisms cannot be isolated by traditional culture methods. In terms of comparing the microbial isolation efficiency of the chip, if the total number of microorganisms of 28 genera detected by the three methods is regarded as 100%, high-throughput sequencing can detect 71.43%, the microorganisms obtained by the method in Example 1 of this application account for 50%, while traditional culture only obtains 25% of the microorganisms. Therefore, the method in Example 1 of this application can improve the efficiency of traditional culture by about 2 times.

[0050] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications 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.

Claims

1. A method for isolating and culturing oral microorganisms, characterized in that, A microporous array chip is required, with a length of 5-7 mm, a width of 3-5 mm, and a thickness of 1-2 mm. The microporous array chip has several interconnected culture micropores. Includes the following steps: (I) Sample collection: Collect oral microbial samples for later use; (II) Sterilization and disinfection of chips and components: ① Sterilize the microporous array chip for later use; ② Disinfect the first filter membrane, the second filter membrane and the connecting device for later use; (III) Chip assembly: Fix the first filter membrane to the underside of the microporous array chip, prepare a solid culture medium for microorganisms, heat and sterilize it, and while the solid culture medium is not solidified, immerse the microporous array chip with the first filter membrane fixed in the culture medium. After the culture medium solidifies, remove it and remove the residual culture medium around it; inject the oral microbial sample collected in the sample collection step from the upper surface of the microporous array chip until the saliva no longer seeps into the micropores. After the injection is completed, fix the second filter membrane to the upper side of the microporous array chip. (IV) In situ culture: The assembled chip is adhered to the oral cavity of the subject and cultured for 1-14 days; (V) Separation and purification: Take out the cultured chip, remove the first and second filter membranes of the chip under sterile conditions, place the chip in a microbial liquid culture medium, separate the microorganisms cultured in the micropores, and culture the liquid culture medium containing microorganisms to obtain the isolated oral microorganisms.

2. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, The microwell array chip is a well plate based on polypropylene or polyetheretherketone; the microwell array chip is provided with multiple microwell arrays, and each microwell array contains multiple culture microwells.

3. The method for isolating and culturing oral microorganisms according to claim 2, characterized in that, The culture micropores of different micropore arrays have different pore sizes.

4. The method for isolating and culturing oral microorganisms according to claim 2 or 3, characterized in that, The pore size of the culture micropores ranges from 8 μm to 540 μm, and the distance between the pores is 0.2 mm.

5. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, In step (I), the oral microbial samples are saliva samples, subgingival plaque samples, and gingival crevicular fluid samples.

6. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, The first and second filter membranes are polycarbonate filter membranes with a pore size of 0.03 μm and a thickness of 15-20 μm; the connecting device is food-grade silicone; the filter membranes are fixed to the microporous array chip with food-grade silicone, so that each pore is separated from the other pores, and is used as a separate microbial culture chamber for culturing microorganisms.

7. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, The sterilization method in steps (II) and (III) is high-pressure steam sterilization at 121°C; The disinfection method in step (II) is ultraviolet light irradiation. In step (III), the microbial solid culture medium is LB solid culture medium; The microbial liquid culture medium in step (V) is LB liquid culture medium.

8. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, In step (IV), the lower side of the assembled chip first filter membrane is adhered to the buccal side of the subject's maxillary first molar using dental adhesive.

9. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, The specific operation for separating the microorganisms cultured in the microwells in step (V) is as follows: use a vortex mixer at 1000-1500 rpm for 10-20 seconds to wash out the microbial cells cultured in the microwells.

10. The method for isolating and culturing oral microorganisms according to claim 1, characterized in that, In step (V), the liquid culture medium containing microorganisms is isolated and cultured by means of streak plate separation, spread plate separation, or / and pour plate separation.

Images