Device for culturing bacteriophage

Abstract

The utility model discloses a device for culturing bacteriophages, and relates to the technical field of microorganism culture. The device comprises a container cover, a container, a sample inlet pipe and a sample outlet pipe, the container is in airtight connection with the sample inlet pipe and the sample outlet pipe respectively; the container cover is detachably connected with the opening of the container in an airtight manner; a through vent hole is formed in the container cover; the sample inlet pipe and the sample outlet pipe are respectively communicated with the inner side and the outer side of the container, and a sample inlet pipe port is higher than a sample outlet pipe port on the inner side of the container; an air filter membrane is arranged in the air vent, the sample inlet pipe is provided with a bacterial filter, and a sealing plug is detachably mounted at a port, outside the container, of the sample outlet pipe. By adopting the device, when the phage type is replaced, the experiment does not need to be terminated, and the culture can be continued by continually supplementing a fresh culture medium and phage suspension from the sample feeding pipe, so that the continuity of phage culture and screening is realized until the phage capable of splitting target bacteria is screened out.

Description

Technical Field

[0001] This utility model relates to the field of microbial culture technology, and in particular to a device for culturing bacteriophages. Background Technology

[0002] Bacteriophages are viruses capable of infecting bacteria. Based on their life cycle within the host bacteria, they can be classified into lysogenic phages and lytic phages. With the development of viral metagenomic sequencing, it has been discovered that our previous understanding of phages was only the tip of the iceberg, and a vast number of phages remain undiscovered. Different types and quantities of phages are distributed from the human gastrointestinal tract to the oceans worldwide. The cultivation and screening of phages are crucial steps in phage research.

[0003] Currently, the main traditional method for screening bacteriophages involves co-inoculating bacteriophage samples collected from nature or bacteriophage samples of known bacteriophage species collected from a bacteriophage library with the target strain in a bacterial culture flask containing culture medium for a period of time. After a period of time, the culture needs to be terminated, the device opened, and the presence of the target bacteriophage in the culture is detected by the double-layer plate culture method.

[0004] Traditional phage culture methods typically involve culturing bacteriophages in a single bacterial culture flask. This method has several drawbacks: the apparatus must be opened and the experiment stopped before subsequent tests can be performed. This leads to: 1. If the phage sample fails to infect or lyse the target bacteria, the culture process must be restarted from scratch; 2. Since some phages capable of lysing specific pathogens are not abundant in nature, testing too early can also affect the screening results. Therefore, using traditional screening devices is extremely labor-intensive. Utility Model Content

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a device for culturing bacteriophages, which can collect bacteriophages during the culturing process without terminating the experiment for detection, thereby achieving continuous culturing and screening of bacteriophages.

[0006] To achieve the above objectives, this utility model is implemented through the following technical solution:

[0007] This invention provides an apparatus for culturing bacteriophages, including a container cap, a container, an inlet tube, and an outlet tube; the container is airtightly connected to the inlet tube and the outlet tube respectively; the container cap is detachably airtightly connected to the opening of the container.

[0008] The container lid is provided with a through vent; the sample inlet tube and the sample outlet tube are respectively connected to the inside and outside of the container, and on the inside of the container, the port of the sample inlet tube is higher than the port of the sample outlet tube.

[0009] An air filter membrane is provided in the vent, a bacterial filter is provided in the sample inlet tube, and a sealing plug is detachably installed at the port of the sample outlet tube outside the container.

[0010] Optionally, the container lid has internal threads, and the container opening has external threads, with the external threads engaging with the internal threads.

[0011] Optionally, the vent is connected to an air pump, and the vent and the air pump are connected by a flexible hose, which is provided with a sealing clamp.

[0012] Optionally, the vent is provided with a hose connector that protrudes from the surface of the container lid.

[0013] Optionally, the injection tube is connected to a feed pump.

[0014] Optionally, the feed pump is a peristaltic pump.

[0015] Optionally, the container is a conical flask, and the inlet tube and the outlet tube penetrate the wall of the conical flask.

[0016] Optionally, the pore size of the bacterial filter is less than 0.22 micrometers.

[0017] Optionally, the pore size of the air filter membrane is less than 0.22 micrometers.

[0018] The beneficial effects of this utility model are as follows:

[0019] This invention features a sample inlet tube equipped with a bacterial filter to prevent contaminating bacteria from entering the container through the tube. During operation, the target bacterial strain is placed in the container, and the container opening is sealed. Then, sterile fresh culture medium and phage suspension are added separately through the sample inlet tube. Upon reaching the set culture stage, the sample inlet is closed, and sterile air is pumped in through the vent, allowing the culture mixture in the container to be pumped out through the outlet tube. If the target phage is not obtained, fresh culture medium and phage suspension can be added again through the sample inlet tube to continue the culture process without terminating the experiment. The phage species can be changed for screening, achieving continuity in phage culture and screening. During the process, only a single target bacterium exists inside the container, greatly simplifying the culture process. Simultaneously, operators can flexibly adjust culture conditions and time to facilitate effective phage replication, easily increasing phage abundance and achieving effective detection. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of this utility model. The illustrative embodiments of this utility model and their descriptions are used to explain this utility model and do not constitute an improper limitation of this utility model.

[0021] The diagram exaggerates the spacing or dimensions between parts to show their positions; the diagram is for illustrative purposes only.

[0022] Figure 1 This is a schematic diagram of the device for culturing bacteriophages in Example 1.

[0023] Figure 2 This is a schematic diagram of the container lid in Example 1.

[0024] Figure 3 This is a schematic diagram of the sample inlet tube in Example 1.

[0025] Figure 4 This is a schematic diagram of the sample tube in Example 1.

[0026] Among them, 1. Container cap; 2. Container; 21. External thread; 3. Vent; 31. Hose connector; 32. Air filter membrane; 33. Internal thread; 34. Air-sealing cap; 4. Sample inlet tube; 41. Bacterial filter; 5. Sample outlet tube; 51. Sealing plug. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] A device for culturing bacteriophages, such as Figure 1 As shown, it includes a container cap 1, a container 2, an inlet tube 4, and an outlet tube 5; the container 2 is airtightly connected to the inlet tube 4 and the outlet tube 5 respectively; the container cap 1 is detachably airtightly connected to the opening of the container 2.

[0029] The container lid 1 is provided with a through vent 3; the sample inlet tube 4 and the sample outlet tube 5 are respectively connected to the inside and outside of the container 2, and the port of the sample inlet tube 4 is higher than the port of the sample outlet tube 5 inside the container 2.

[0030] like Figure 2 As shown, an air filter membrane 32 is installed in the vent 3, such as Figure 3 As shown, the sample inlet tube 4 is equipped with a bacterial filter 41, such as... Figure 4 As shown, the port of the sample tube 5 outside the container 2 is detachably fitted with a sealing plug 51.

[0031] With this setup, during phage culture, closing the inlet tube 4 and simultaneously pumping sterile air through the vent 3 allows the culture mixture in container 2 to be pumped out through the outlet tube 5 without terminating the experiment. Fresh culture medium or phage suspension can then be added from the inlet tube 4 to continue culturing the target bacteria or screening the phage. Because the entire phage screening process revolves around a specific target strain, ensuring that only a single target strain exists in container 2 allows for the addition of phages of different types or sources until a specific phage capable of infecting and lysing the target bacteria is selected.

[0032] like Figure 2 As shown, the container lid 1 has an internal thread 33, and the opening of the container 2 has an external thread 21. The external thread 21 and the internal thread 33 cooperate with each other, which facilitates the installation, disassembly and cleaning of the device.

[0033] Vent 3 is connected to an air pump, and vent 3 and air pump are connected by a hose. The hose is equipped with a sealing clamp, which can allow air to pass through the inside of container 2 or prevent gas exchange between the inside and outside of container 2 according to the culture conditions of the target bacteria.

[0034] like Figure 2 As shown, the vent 3 is provided with a hose connector 31 that protrudes from the surface of the container cover 1.

[0035] Alternatively, an air-sealing cover 34 is provided outside the vent to seal the vent and provide an anaerobic bacterial culture environment. When it is necessary to pump out the culture mixture in container 2 for testing, the air-sealing cover 34 is opened, the vent 3 is connected to the air pump, and air is pumped into container 2 to expel the culture mixture.

[0036] The sample inlet tube 4 is connected to the feed pump, which is used to replenish sterile fresh culture medium and phage suspension into container 2. (The last part, "due to...", appears to be incomplete and unrelated to the preceding sentence.) Figure 3 As shown, the sample inlet tube 4 is equipped with a bacterial filter 41, which can prevent other bacteria from entering the container 2 through the sample inlet tube 4, so that only the target bacteria exist inside the container 2.

[0037] The feed pump is a peristaltic pump to accurately control the amount of fresh culture medium and phage suspension added, and to prevent the substances in the injection tube 4 from flowing backward. A valve is installed between the peristaltic pump and the injection tube 4.

[0038] Container 2 is a conical flask. The inlet tube 4 and outlet tube 5 are respectively through the wall of the conical flask. The device for obtaining the cultured bacteriophage can be installed using common laboratory equipment. The conical flask has a specification of 250mL. The inner diameter of the inlet tube 4 is matched with the size of the cone of a medical syringe to be compatible with commonly used laboratory instruments. The inner diameter of the outlet tube 5 is the same as the inner diameter of the inlet tube 4.

[0039] Among them, the medical syringe cone conforms to the requirements of GB / T 1962.1 or GB / T 1962.2 and is a commonly used device in this field.

[0040] like Figure 4 As shown, the sealing plug 51 is made of elastic rubber and can seal the sample tube 5 when testing is not required.

[0041] The pore size of the bacterial filter 41 is less than 0.22 micrometers. This size is smaller than the size of bacteria but larger than the general size of bacteriophages, which can prevent the entry of contaminating bacteria without blocking the entry of bacteriophages into container 2.

[0042] The air filter membrane 32 has a pore size of less than 0.22 micrometers, which is also used to prevent the entry of miscellaneous bacteria; in addition, by processing the incoming raw material of the air pump, contamination inside container 2 can also be avoided in terms of raw materials.

[0043] The method of using the device for culturing bacteriophages includes the following steps:

[0044] S1. Add the target bacteria into container 2;

[0045] S2. Seal the sealing plug 51, connect the container cap 1 and the container 2 together in an airtight manner through the thread, remove the air pump connected to the vent 3, open the valve between the sample inlet tube 4 and the peristaltic pump, and replenish the container 2 with fresh culture medium and phage suspension through the peristaltic pump. During the process, the gas in the container 2 escapes through the vent 3.

[0046] S3. Seal the valve between the sample inlet tube 4 and the peristaltic pump. By adjusting the air-sealing cap 34 or the sealing clamp on the air pump hose, keep the air inlet 3 open or closed to create a suitable culture environment for the target bacteria in the culture medium for culture.

[0047] S4. At the appropriate detection time point, remove the sealing plug 51, close the sample inlet tube 4, and connect the air vent 3 to the air pump. Turn on the air pump to fill the container 2 with sterile air, so that the culture mixture is pumped out from the sample outlet tube 5 under air pressure for detection. Since the liquid level of the culture mixture needs to be above the port of the sample outlet tube 5 during the pumping process, the volume of the residue in the container 2 can be adjusted by tilting the container 2 to adjust the positional relationship between the liquid level and the port of the sample outlet tube 5.

[0048] S5. After pumping is complete, check whether there is a sufficient abundance of phages in the pumped material. By repeating S2~S3, continuous culture and screening can be achieved.

[0049] In this scenario, if researchers find that the detection time is too early, they can use the remaining culture material in container 2 to add fresh culture medium and continue culturing. Under continuous culturing conditions, it is easier to screen for bacteriophages that can lyse the target bacteria.

[0050] After each pumping process, a portion of the culture mixture remains in the sample tube. This portion is expelled during the next pumping of a new culture mixture. The residual culture mixture from the previous pumping is only a small amount and generally does not affect the phage screening results. The influence of the residue on the test results can be avoided by discarding the initial pumped sample and collecting the samples from subsequent pumped samples.

[0051] Example 2

[0052] The practical applications of the bacteriophage culturing device in Example 1 include the following aspects.

[0053] I. Advantages of continuous culture and screening devices.

[0054] Three groups of bacteriophages were artificially set up: bacteriophages infected with Escherichia coli, bacteriophages infected with Klebsiella pneumoniae, and bacteriophages infected with Pseudomonas aeruginosa, which were numbered bacteriophage ①, bacteriophage ②, and bacteriophage ③, respectively, along with a Pseudomonas aeruginosa bacterial suspension. The process of isolating bacteriophages infected with and lysing Pseudomonas aeruginosa in nature was simulated. Bacteriophages ①, ②, and ③ simulated different samples collected in nature, respectively, and the screening effects of traditional bacteriophage screening methods and the continuous culture screening device of Example 1 on bacteriophages infected with Pseudomonas aeruginosa were compared.

[0055] 1) According to the method used in Example 1, the screening process of Pseudomonas aeruginosa phages using the continuous culture screening device of Example 1 includes: loading Pseudomonas aeruginosa bacterial suspension into a container, installing the container cap, and sequentially adding phage ①, phage ②, and phage ③ into the container through the injection tube in chronological order. Each incubation time is 24 hours. During this period, the co-culture is pumped out, and the double-layer plate culture method is used to determine whether the culture medium contains phages infected with lysed Pseudomonas aeruginosa.

[0056] The screening schedule for the continuous screening device was as follows: *Pseudomonas aeruginosa* and phage ① samples were pumped into a continuous screening culture bottle and incubated for 24 hours without opening the culture device. The pumped-out culture medium was then used to determine whether it contained phages capable of infecting and lysing *P. aeruginosa*. Next, phage ② samples were pumped in and incubated for another 24 hours without opening the culture device. The pumped-out culture medium was then used to determine whether it contained phages capable of infecting and lysing *P. aeruginosa*. Then, phage ③ samples were pumped in and incubated for another 24 hours without opening the culture device. The pumped-out culture medium was then used to determine whether it contained phages capable of infecting and lysing *P. aeruginosa*. The experiment concluded after phage ③, which could infect and lyse *P. aeruginosa*, was identified. The total screening time was 72 hours.

[0057] 2) The screening process for Pseudomonas aeruginosa phages using traditional phage screening methods includes: incubating Pseudomonas aeruginosa sequentially with phage ①, phage ②, and phage ③; after 24 hours of incubation with phage ①, the apparatus is opened, and a double-layer plate culture method is used to determine whether the culture medium contains phages infecting lysed Pseudomonas aeruginosa. Subsequently, the bacteria are incubated sequentially with phage ② and phage ③ for another 24 hours until phages infecting lysed Pseudomonas aeruginosa are screened out.

[0058] The traditional screening method timeline is as follows: *Pseudomonas aeruginosa* and phage ① sample co-incubate (24h); open the culture device and remove the culture medium; use a double-layer plate culture method to determine if the culture medium contains phages that infect and lyse *P. aeruginosa*; wash the culture bottle, autoclave the culture medium and culture bottle (12h); inoculate *P. aeruginosa* and phage ② sample co-incubate (24h); open the culture device and remove the culture medium; use a double-layer plate culture method to determine if the culture medium contains phages that infect and lyse *P. aeruginosa*; wash the culture bottle again, autoclave the culture medium and culture bottle (12h); inoculate *P. aeruginosa* and phage ③ sample co-incubate (24h); open the culture device and remove the culture medium; use a double-layer plate culture method to determine if the culture medium contains phages that infect and lyse *P. aeruginosa*; the experiment ends after screening out phage ③ that can infect and lyse *P. aeruginosa*, taking a total of 96h.

[0059] It can be seen that both the continuous culture screening device and the traditional screening method can effectively screen for bacteriophages that infect and lyse *Pseudomonas aeruginosa*. However, because the traditional screening method requires opening the experimental apparatus, *Pseudomonas aeruginosa* needs to be recultured before mixing with the next bacteriophage sample. Furthermore, once the culture bottle is opened, it needs to be autoclaved to avoid contamination, thus taking longer. In contrast, the continuous culture device does not require opening the culture bottle; the culture medium is simply pumped out to replace the next bacteriophage sample. Therefore, the time required to screen the three bacteriophage samples is significantly shortened.

[0060] II. To investigate whether the residual previous mixed culture in the sample tube has any impact on the experimental results.

[0061] After co-culturing Klebsiella pneumoniae phage ② with Pseudomonas aeruginosa for 24 hours, the mixed culture was pumped out. Then, 100 mL of fresh culture medium was pumped into the mixed culture of phage ③, and the culture was incubated again for 24 hours. Subsequently, five 15 mL centrifuge tubes were used to collect the pumped mixed cultures. Since the first centrifuge tube contained residual mixed culture of phage ② from the previous culture, only the first centrifuge tube contained mixed culture of phage ②. After the first centrifuge tube was collected, the remaining mixed culture in the exit tube contained mixed culture of phage ③. By evaluating the number of phages in the first centrifuge tube and the other four centrifuge tubes, it was possible to determine whether the residue in the exit tubes would affect the screening results. Then, the number of phages infected and lysed with Pseudomonas aeruginosa in each pumped-out sample was determined using a double-layer plate culture method with a double-layer Pseudomonas aeruginosa suspension.

[0062] The phage count results of the mixed cultures collected from each segment (five centrifuge tubes) are shown in Table 1: There was no significant difference in the number of phage ③ in the mixed cultures of each segment. The first centrifuge tube contained both the mixed culture of phage ② remaining from the sample tube and the mixed culture of phage ③. The other four centrifuge tubes did not contain the mixed culture of phage ②, but only the mixed culture of phage ③. The results of all five centrifuge tubes were the same. In other words, this device can screen for the target phage in the presence of multiple phage mixtures, and the residual mixed culture from the previous sample tube has no effect on the experimental results.

[0063] Table 1. Phage Count Results

[0064]

[0065] Because this device can continuously replenish fresh culture medium, if the target phage is not detected in the culture sample taken last time, the culture in the culture bottle can be cultured again to extend the culture time, allowing the phage to replicate effectively and increase its abundance. This avoids having to open the culture device for detection, which would terminate the experiment.

[0066] In recent years, the widespread use of antibiotics has led to the emergence and spread of numerous drug-resistant bacteria, posing a serious threat to human health. With continued antibiotic use and the evolution and spread of drug-resistant bacteria, there may be no effective antibiotics available to treat pathogenic infections in the future. Bacteriophages are viruses capable of infecting bacteria, and based on their life cycle within the host bacteria, they can be classified into lysogenic phages and lytic phages. Combating drug-resistant bacteria through phage infection is an effective means of treating pathogenic infections in the future. The phage culturing device in this embodiment can be used for the continuous culture and screening of lysed specific pathogens, and has wide applications in phage screening.

[0067] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An apparatus for culturing bacteriophages, characterized in that, It includes a container cap, a container, an inlet tube, and an outlet tube; the container is airtightly connected to the inlet tube and the outlet tube respectively; the container cap is detachably airtightly connected to the opening of the container. The container lid is provided with a through vent; the sample inlet tube and the sample outlet tube are respectively connected to the inside and outside of the container, and on the inside of the container, the port of the sample inlet tube is higher than the port of the sample outlet tube. An air filter membrane is provided in the vent, a bacterial filter is provided in the sample inlet tube, and a sealing plug is detachably installed at the port of the sample outlet tube outside the container.

2. The apparatus for culturing bacteriophages according to claim 1, characterized in that... The container lid has internal threads, and the container opening has external threads, with the external threads engaging with the internal threads.

3. The apparatus for culturing bacteriophages according to claim 1, characterized in that, The vent is connected to the air pump, and the vent and the air pump are connected by a flexible hose, which is equipped with a sealing clamp.

4. The apparatus for culturing bacteriophages according to claim 3, characterized in that, The vent is equipped with a flexible hose connector that protrudes from the surface of the container lid.

5. The apparatus for culturing bacteriophages according to claim 1, characterized in that, An airtight cover is installed outside the vent.

6. The apparatus for culturing bacteriophages according to claim 1, characterized in that, The injection tube is connected to the feed pump.

7. The apparatus for culturing bacteriophages according to claim 6, characterized in that, The feed pump is a peristaltic pump.

8. The apparatus for culturing bacteriophages according to claim 1, characterized in that, The container is a conical flask, and the inlet tube and the outlet tube penetrate the wall of the conical flask.

9. The apparatus for culturing bacteriophages according to claim 1, characterized in that, The air filter membrane has a pore size of less than 0.22 micrometers.

10. The apparatus for culturing bacteriophages according to claim 1, characterized in that, The pore size of the bacterial filter is less than 0.22 micrometers.

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