Staged phage culture and detection sampling device

Abstract

A staged phage culture and detection sampling device comprises a base, the upper surface of the base is provided with a bottom frame and a rectangular clamping groove, the bottom frame is fixed to the base, the rectangular clamping groove is located in the center of the bottom frame, and a cell culture assembly is placed in the rectangular clamping groove; in conclusion, the timing exposure of different cell culture unit grooves is realized by utilizing the stepping motor, the transmission belt and the shielding cloth, so that the contact time of the bacteriophage and the host bacteria can be accurately controlled; the sampling rod is pushed to sample by adopting an electric scissor telescopic structure, so that the manual intervention is reduced, and the risk of cross contamination is reduced; the atomizer module is used for uniformly introducing bacteriophage steam fog into the culture medium; the traditional manual sampling process is improved in an automatic and staged mode, the pollution problem possibly caused by frequent opening of a culture device is solved, and the experimental operation efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bacteriophage cultivation, and in particular relates to a staged bacteriophage cultivation and detection sampling device. Background Art

[0003] Bacteriophages are ubiquitous organisms that specifically use bacteria as hosts. They are a mass of genetic material wrapped in a protein shell. Most phages also have a "tail" that is used to inject genetic material into the host.

[0004] The abuse and improper use of antibiotics in medicine and agriculture have promoted the spread of drug-resistant genes, making the global bacterial resistance problem increasingly serious, resulting in more and more bacteria becoming resistant to commonly used antibiotics and forming "super bacteria". Infections caused by these resistant bacteria not only increase the difficulty of treatment and medical costs for patients, but also pose a serious threat to public health. Therefore, there is an urgent need to find antibacterial methods that replace antibiotics. Phage inhibition has become a potential solution because of its strong specificity, ability to accurately attack specific bacteria without affecting beneficial bacteria, and low side effects. In addition, phages can be used in combination with antibiotics to enhance the efficacy, and because they are naturally occurring viruses, they have good biocompatibility and a small immune response, making them suitable for a variety of patients. At the same time, the preparation and preparation of phages are relatively simple, and they have the potential for personalized treatment. Therefore, in order to realize the application of phages in clinical practice, we need to carry out phage cultivation.

[0005] The basic steps for culturing phages usually include the following key links: (1) Selecting host bacteria: Select host bacteria corresponding to the target phage. Ensure that the host bacteria are culturable and are the specific infection targets of the phage; (2) Obtaining phages: Phages can be extracted from natural environments (such as soil, water sources, etc.), or known phage strains can be used; (3) Phage isolation: Phages are isolated by dilution or filtration, the sample is diluted, and mixed with host bacteria to observe whether plaques are formed, thereby determining the presence of phages; (4) Cultivating phages: Cultivate them together with host bacteria in a culture medium. Commonly used culture media include LB liquid culture medium or other culture media suitable for host bacteria. The culture time is generally from a few hours to a few days, and then the host bacteria are filtered using a 0.22um filter membrane to obtain the phage culture solution; (5) Quantification and identification: After gradient dilution, the double-layer plate method is used to determine the plaque forming unit (PFU) method to quantify the phage concentration, and molecular biological identification, such as PCR or sequencing, is performed to confirm its type and characteristics; (6) Storage of phages: The extracted phages are stored under appropriate conditions, usually frozen at -80°C to maintain their activity.

[0006] The current method of sampling bacteriophages is: at the time point when sampling is required, use a pipette to draw 1 ml of sample, filter and detect the titer. In addition, since it is necessary to detect the titer changes during the phage culture process and during the application process, we need to take samples at fixed intervals and detect the growth status of the phage or its host bacteria during the culture process multiple times. When sampling phages now, it is usually considered to be directly sampled on the surface of the culture medium. For example, to detect the growth inhibition of bacteria by phages, we need to directly use the sampler to insert the sample, and send the cotton swab head with the sample for inspection or place it in a constant temperature environment of 4°C for unified inspection. In order to detect the impact of phages on bacteria in different time periods, it is usually necessary to conduct inspections every 10-30 minutes, so multiple sampling is required for inspection. However, it is necessary to record the time and conduct manual sampling at regular intervals. At present, when sampling, it is necessary to open the culture device and then dip the bacterial culture medium, which is a cumbersome operation. Summary of the invention

[0008] The present invention aims to provide a staged phage culture and detection sampling device to solve the technical problem of the cumbersome operation of manual sampling in multiple time periods of existing phage culture.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions: A staged phage culture and detection sampling device comprises a base, wherein the upper surface of the base is provided with a bottom frame and a rectangular slot, the bottom frame is fixed on the base, the rectangular slot is located at the center of the bottom frame, and a cell culture assembly is placed in the rectangular slot; The cell culture assembly includes a cell culture container, a culture tank is arranged on the top of the cell culture container, two partitions are fixed in the culture tank, the two partitions divide the culture tank into three cell culture unit tanks, two through slits and two guide slits are arranged on the top side of the culture tank, the two through slits are arranged correspondingly, and the two guide slits are arranged correspondingly; A hollow body is arranged at the lower part of the cell culture container, and a multi-time period culture control mechanism is arranged in the hollow body, and the multi-time period culture control mechanism comprises a stepping motor, an active roller, a driven roller, two transmission belts and a plurality of guide rollers, and the active roller, the driven roller and all the guide rollers are respectively installed in the hollow body at the lower part of the cell culture container by a rotational manner, and the active roller and the driven roller are located at two ends of the hollow body, and the stepping motor is connected to the active roller by transmission, and two transmission belts are respectively arranged on the active roller, the driven roller and all the guide rollers, and the two transmission belts are arranged correspondingly, and a shielding cloth is connected between the two transmission belts, and the shielding cloth is used to cover three cell culture unit slots, and the two side edges of the shielding cloth are respectively located in the two guide seams, and the side edges of the shielding cloth are provided with sealing strips, and the sealing strips are used to seal the gap between the shielding cloth and the guide seam; An automatic sampling mechanism is installed on the top of the bottom frame. The automatic sampling mechanism includes a pushing mechanism, a sampler and a middle box. The middle box is a box structure with an open bottom. A sampling box is fixed in the middle box. Three sleeves are fixed on the top of the sampling box. Three sampling holes are opened at the bottom of the sampling box. The three sampling holes and the three sleeves are arranged correspondingly up and down. A round plugging plate is installed at each sampling hole. The round plugging plate is used to block the sampling hole, and the round plugging plate is hingedly installed on the side wall of the sampling hole through a pin shaft. A torsion spring is arranged on the pin shaft, and the torsion spring is used to reset the round plugging plate. The sampler comprises three sampling rods, the upper ends of the three sampling rods are fixedly connected by a cross bar, the three sampling rods are respectively inserted into three sleeves, the pushing mechanism and the cross bar are both located above the sampling box, the pushing mechanism is used to push and pull the cross bar up and down, and a sampling head is arranged at the lower end of each sampling rod; A refrigerant box is installed on the top of the middle box body, and a liquid filling port is arranged on the refrigerant box; a controller and an atomizer module are also arranged in the base, the atomizer module is connected to an atomizer tube through a pipeline, an atomizer liquid port is arranged on the top of the atomizer tube, and the atomizer tube is connected to the side wall of the bottom frame through a hose; The controller is respectively connected to the atomizer module, the stepper motor and the driving mechanism for control. The side wall of the base is provided with an atomization adjustment knob, which is used to control the atomizer module.

[0010] Furthermore, a light strip is provided on the upper surface of the base, the light strip is provided around the outer periphery of the bottom frame, and a lighting switch for controlling the light strip is provided on the side wall of the base.

[0011] Furthermore, the inner wall of the sampling box is provided with a sponge layer, and the sponge layer absorbs physiological saline.

[0012] Furthermore, the pushing mechanism is an electric scissors-type telescopic structure, and the electric scissors-type telescopic structure is controlled by a controller.

[0013] Furthermore, two baffles are fixed to the bottom surface of the sampling box, and the middle sampling hole is located between the two baffles.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention realizes timed exposure of different cell culture unit slots by utilizing a stepper motor, a transmission belt and a shielding cloth, which helps to accurately control the contact time between the bacteriophage and the host bacteria; an electric scissor-type telescopic structure is used to push the sampling rod for sampling, which reduces manual intervention and reduces the risk of cross-contamination; the low-temperature environment provided by the freezing liquid box helps to maintain sample activity; the nebulizer module is used to evenly introduce bacteriophage vapor into the culture medium; the present invention improves the traditional manual sampling process in an automated and staged manner, solves the contamination problem that may be caused by frequently opening the culture device, and improves the efficiency of experimental operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 It is a structural schematic diagram of a staged phage culture and detection sampling device of the present invention; Figure 2 This is a schematic diagram of the installation of the bottom frame; Figure 3 is a schematic diagram of the structure of the cell culture assembly; Figure 4 is a schematic diagram of the structure of a cell culture container; Figure 5 A schematic diagram of the structure of the control mechanism for multi-time period cultivation; Figure 6 This is a schematic diagram of the state where the sampling box is installed in the middle box; Figure 7 It is a connection diagram of the sampling box and the sampler; Figure 8 It is a structural schematic diagram of the automatic sampling mechanism. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The present invention is further described in detail below in conjunction with embodiments.

[0020] like Figure 1-8 As shown, a specific embodiment of a staged phage culture and detection sampling device provided by the present invention is as follows: A staged phage culture and detection sampling device comprises a base 1, the upper surface of the base 1 is provided with a bottom frame 2 and a rectangular card slot 3, the bottom frame 2 is fixed on the base 1, the rectangular card slot 3 is located at the center of the bottom frame 2, and a cell culture assembly 4 is placed in the rectangular card slot 3; The cell culture assembly 4 includes a cell culture container 5, such as Figure 4 As shown, a culture tank 6 is arranged on the top of the cell culture container 5, and two partitions 7 are fixed in the culture tank 6. The two partitions 7 divide the culture tank 6 into three cell culture unit tanks 8. Two through slits 9 and two guide slits 10 are arranged on the top side of the culture tank 6. The two through slits 9 are arranged correspondingly, and the two guide slits 10 are arranged correspondingly. The lower part of the cell culture container 5 is provided with a cavity 11, and a multi-time period culture control mechanism is provided in the cavity 11, such as Figure 5 As shown, the multi-time period culture control mechanism includes a stepper motor, an active roller 12, a driven roller 13, two transmission belts 14 and a plurality of guide rollers 15. The active roller 12, the driven roller 13 and all the guide rollers 15 are respectively installed in the hollow body 11 at the lower part of the cell culture container 5 in a rotating manner. The active roller 12 and the driven roller 13 are located at both ends of the hollow body 11. The stepper motor is connected to the active roller 12 in transmission. The two transmission belts 14 are respectively arranged on the active roller 12, the driven roller 13 and all the guide rollers 15. The two transmission belts 14 are arranged correspondingly. A shielding cloth 16 is connected between the two transmission belts 14. The shielding cloth 16 is used to cover the three cell culture unit slots 8 (at this time, the bottom surface of the shielding cloth 16 is in contact with the top of the partition 7). The two side edges of the shielding cloth 16 are respectively located in the two guide seams 10, and the side edges of the shielding cloth 16 are provided with sealing strips. The sealing strips are used to seal the gap between the shielding cloth 16 and the guide seams 10. An automatic sampling mechanism is installed on the top of the bottom frame 2. The automatic sampling mechanism includes a pushing mechanism 17, a sampler and a middle box 18. The middle box 18 is a box structure with an open bottom. A sampling box 19 is fixed in the middle box 18. Three sleeves 20 are fixed on the top of the sampling box 19. Three sampling holes 21 are opened at the bottom of the sampling box 19. The three sampling holes 21 are arranged correspondingly to the three sleeves 20. A round plugging plate 22 is installed at each sampling hole 21. The round plugging plate 22 is used to block the sampling hole 21, and the round plugging plate 22 is hingedly installed on the side wall of the sampling hole 21 through a pin shaft. A torsion spring is arranged on the pin shaft, and the torsion spring is used to reset the round plugging plate 22. The sampler includes three sampling rods 23, the upper ends of the three sampling rods 23 are fixedly connected by a cross bar 24, the three sampling rods 23 are respectively inserted into three sleeves 20, the pushing mechanism 17 and the cross bar 24 are both located above the sampling box 19, the pushing mechanism 17 is used to push and pull the cross bar 24 up and down, and a sampling head 25 is provided at the lower end of each sampling rod 23; A refrigerant box 26 is installed on the top of the middle box 18, and a liquid filling port 27 is provided on the refrigerant box 26; a controller and an atomizer module are also provided in the base 1, and the atomizer module is connected to an atomizer tube 28 through a pipeline, and an atomizer liquid port 29 is provided on the top of the atomizer tube 28, and the atomizer tube 28 is connected to the side wall of the bottom frame 2 through a hose 30; The controller is respectively connected to the atomizer module, the stepper motor and the driving mechanism 17 . The side wall of the base 1 is provided with an atomization adjustment knob 31 , which is used to control the atomizer module.

[0021] The specific culture detection sampling process of the present invention is as follows: the three cell culture unit slots 8 of the present invention are all used to contain cell culture medium. In the initial state, the three cell culture unit slots 8 are covered by the shielding cloth 16 on the top, and the three cell culture unit slots 8 are independent of each other. Then, the liquid containing bacteriophage is added to the atomizer tube 28 through the atomization liquid port 29, and the atomizer module is started. The vapor mist containing bacteriophage is slowly and continuously passed into the bottom frame 2 through atomization. In addition, the working power of the atomizer module can be controlled by the atomization adjustment knob 31 to adjust the atomization speed; then the stepper motor is started, and the stepper motor drives the active roller 12 to rotate. Through the pulling of the active roller 12, the two transmission belts 14 are driven, so that part of the shielding cloth 16 enters the hollow body 1 at the lower part of the cell culture container 5 through one of the slits 9. 1. After part of the shielding cloth 16 is moved, the first cell culture unit slot 8 is exposed, and the vapor mist containing bacteriophages contacts the cell culture medium in the cell culture unit slot 8; after 10 minutes, the stepper motor is started again, and the transmission belt 14 pulls the shielding cloth 16 to move a certain distance again, so that the second cell culture unit slot 8 is exposed. After another 10 minutes of exposure time, the stepper motor is started again, and the transmission belt 14 pulls the shielding cloth 16 into the cavity 11, and the third cell culture unit slot 8 is exposed. After another 10 minutes, the exposure of the third cell culture unit slot 8 is completed. Finally, the exposure time of the first cell culture unit slot 8 is 30 minutes, the exposure time of the second cell culture unit slot 8 is 20 minutes, and the exposure time of the third cell culture unit slot 8 is 10 minutes.

[0022] Next, the controller starts the pushing mechanism 17, which is preferably an electric scissor-type telescopic structure (existing conventional technology, the specific structure is not repeated here), the electric scissor-type telescopic mechanism pushes the cross bar 24 to move downward, the three sampling rods 23 push open the three circular plugging plates 22, and are inserted downward into the cell culture medium in the three cell culture unit slots 8, and the sampling head 25 at the end is dipped for sampling. After sampling, the electric scissor-type telescopic mechanism pulls the cross bar 24 upward to reset, and the three sampling rods 23 are retracted from the three sampling holes 21 into the sampling box 19, and the circular plugging plate 22 blocks the sampling hole 21 again under the action of the torsion spring; in order to avoid cross contamination during the sampling process, two baffles 32 can be set on the bottom surface of the sampling box 19, and the middle sampling hole 21 is located between the two baffles 32, and the baffle 32 isolates the three sampling holes 21 from each other.

[0023] Preferably, a sponge layer 33 may be provided on the inner wall of the sampling box 19 . The sponge layer 33 is adsorbed with physiological saline, and the sponge layer can ensure a relatively moist space in the sampling box 19 .

[0024] In addition, the freezing liquid box 26 is used to contain freezing liquid. The freezing liquid box 26 can provide a relatively low temperature environment for the sample after sampling to keep the sample active.

[0025] A light strip 34 is also provided on the upper surface of the base 1 , and the light strip is arranged around the outer periphery of the bottom frame 2 . A lighting switch 35 for controlling the light strip is provided on the side wall of the base 1 .

[0026] It should be noted that, in this article, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0027] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A staged phage culture and detection sampling device, characterized in that: It comprises a base, wherein the upper surface of the base is provided with a bottom frame and a rectangular slot, the bottom frame is fixed on the base, the rectangular slot is located at the center of the bottom frame, and a cell culture assembly is placed in the rectangular slot; The cell culture assembly includes a cell culture container, a culture tank is arranged on the top of the cell culture container, two partitions are fixed in the culture tank, the two partitions divide the culture tank into three cell culture unit tanks, two through slits and two guide slits are arranged on the top side of the culture tank, the two through slits are arranged correspondingly, and the two guide slits are arranged correspondingly; A hollow body is arranged at the lower part of the cell culture container, and a multi-time period culture control mechanism is arranged in the hollow body, and the multi-time period culture control mechanism comprises a stepping motor, an active roller, a driven roller, two transmission belts and a plurality of guide rollers, and the active roller, the driven roller and all the guide rollers are respectively installed in the hollow body at the lower part of the cell culture container by a rotational manner, and the active roller and the driven roller are located at two ends of the hollow body, and the stepping motor is connected to the active roller by transmission, and two transmission belts are respectively arranged on the active roller, the driven roller and all the guide rollers, and the two transmission belts are arranged correspondingly, and a shielding cloth is connected between the two transmission belts, and the shielding cloth is used to cover three cell culture unit slots, and the two side edges of the shielding cloth are respectively located in the two guide seams, and the side edges of the shielding cloth are provided with sealing strips, and the sealing strips are used to seal the gap between the shielding cloth and the guide seam; An automatic sampling mechanism is installed on the top of the bottom frame. The automatic sampling mechanism includes a pushing mechanism, a sampler and a middle box. The middle box is a box structure with an open bottom. A sampling box is fixed in the middle box. Three sleeves are fixed on the top of the sampling box. Three sampling holes are opened at the bottom of the sampling box. The three sampling holes and the three sleeves are arranged correspondingly up and down. A round plugging plate is installed at each sampling hole. The round plugging plate is used to block the sampling hole, and the round plugging plate is hingedly installed on the side wall of the sampling hole through a pin shaft. A torsion spring is arranged on the pin shaft, and the torsion spring is used to reset the round plugging plate. The sampler comprises three sampling rods, the upper ends of the three sampling rods are fixedly connected by a cross bar, the three sampling rods are respectively inserted into three sleeves, the pushing mechanism and the cross bar are both located above the sampling box, the pushing mechanism is used to push and pull the cross bar up and down, and a sampling head is arranged at the lower end of each sampling rod; A refrigerant box is installed on the top of the middle box body, and a liquid filling port is arranged on the refrigerant box; a controller and an atomizer module are also arranged in the base, the atomizer module is connected to an atomizer tube through a pipeline, an atomizer liquid port is arranged on the top of the atomizer tube, and the atomizer tube is connected to the side wall of the bottom frame through a hose; The controller is respectively connected to the atomizer module, the stepper motor and the driving mechanism for control. The side wall of the base is provided with an atomization adjustment knob, which is used to control the atomizer module.

2. A staged phage culture and detection sampling device according to claim 1, characterized in that: A light strip is also arranged on the upper surface of the base, and the light strip is arranged around the outer periphery of the bottom frame. A lighting switch for controlling the light strip is arranged on the side wall of the base.

3. A staged phage culture and detection sampling device according to claim 1, characterized in that: The inner wall of the sampling box is provided with a sponge layer, and physiological saline is absorbed by the sponge layer.

4. A staged phage culture and detection sampling device according to claim 1, characterized in that: The pushing mechanism is an electric scissor-type telescopic structure, and the electric scissor-type telescopic structure is controlled by a controller.

5. The staged phage culture and detection sampling device according to claim 1, characterized in that: Two baffles are fixed on the bottom surface of the sampling box, and the central sampling hole is located between the two baffles.

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