Integrated device and method for mixing bacterial samples based on isodensity adjustment and batch volume ratio mixing

Abstract

The application belongs to the technical field of automatic configuration of biological samples, and particularly relates to a bacteria sample mixing integrated device and method based on equal-concentration adjustment and batch volume ratio mixing. A detection unit measures the optical density value of bacteria liquid in real time and feeds back to a control unit. The control unit controls the injection amount of diluent according to the comparison result of the measured value and the target value, so as to realize equal-concentration closed-loop adjustment. A switching valve group sequentially selects each equal-concentration bacteria liquid according to a preset volume ratio sequence. A quantitative suction unit quantitatively sucks the bacteria liquid and injects it into a mixing cavity for mixing. The control unit adjusts the execution priority of each ratio before entering the mixing, and executes the suction and mixing of each volume ratio according to the adjusted priority until the preparation of all preset volume ratios is completed. The equal-concentration adjustment in a specific manner ensures the consistency of the concentrations of the bacteria liquids, the execution priority adjustment reduces the interference of pipeline residues on subsequent trace samples, and the preparation precision of each volume ratio sample is ensured.

Description

Technical Field

[0001] This invention belongs to the field of automated biological sample preparation technology, specifically relating to an integrated device and method for mixing bacterial samples based on isoconcentration adjustment and batch volume ratio mixing. Background Technology

[0002] Bacterial contamination in water bodies is a significant risk factor affecting drinking water safety, and the synergistic pathogenic effects caused by mixed bacterial contamination make bacterial identification particularly urgent. Multi-wavelength transmission spectroscopy combined with machine learning algorithms has been proven to be able to rapidly identify mixed bacteria in water bodies. This requires establishing a standard spectral database covering different mixing ratios, which necessitates the preparation of a large number of standard bacterial samples with known species and mixing ratios. This preparation process includes two core steps: equal concentration adjustment and mixing at different volume ratios.

[0003] Currently, the above steps are generally performed manually: operators use a spectrophotometer to measure the OD value of each bacterial culture, manually calculate and add diluent, and repeatedly measure and adjust until the target is met; then, for each preset volume ratio, the corresponding volume of bacterial culture is manually pipetted and mixed, and the container must be manually cleaned after each ratio is completed. This manual preparation method is inefficient, taking 1 to 2 hours to prepare a set of mixed samples, which is difficult to meet the needs of large-scale library construction; manual measurement, calculation and pipetting are prone to errors, resulting in limited volume accuracy and poor sample consistency and repeatability; only one ratio can be processed at a time, making batch continuous preparation impossible; manual cleaning is not thorough, posing a risk of cross-contamination. In addition, existing automated sample preparation devices are usually only for single concentration adjustment or fixed ratio mixing, lacking an integrated device that can simultaneously achieve automatic adjustment of multiple bacterial species at equal concentrations and automatic mixing of programmable volume ratio sequences. Summary of the Invention

[0004] The purpose of this invention is to provide an integrated device and method for mixing bacterial samples based on equal concentration adjustment and batch volume ratio mixing, so as to solve the technical problems in the existing preparation of mixed bacterial standard samples, where equal concentration adjustment of each bacterial solution depends on manual step-dilution, and where pipeline residues interfere with trace proportions of samples when mixing at different volume ratios.

[0005] The present invention achieves the above objectives through the following technical solutions: In a first aspect, the present invention proposes an integrated bacterial sample mixing device based on equal concentration adjustment and batch volume ratio mixing, comprising: The liquid storage unit is used to store various original bacterial solutions and diluents; The detection unit is used for online real-time measurement of the optical density value of the bacterial solution; The control unit is used to receive the optical density value fed back by the detection unit, and control the amount of diluent injected according to the comparison result between the optical density value and the target isoconcentration value, so as to perform isoconcentration adjustment until the concentration of each bacterial solution reaches the target isoconcentration. The switching valve assembly has multiple inlets and switchable outlets, and is controlled by the control unit to sequentially select bacterial solutions of equal concentration according to a preset volume ratio sequence. The quantitative aspiration unit is used to quantitatively aspirate a set volume of bacterial solution from the corresponding bacterial solution bottle after the switching valve group selects the corresponding bacterial solution, and inject it into the mixing chamber to achieve mixing of different volume ratios; The control unit is also used to adjust the execution priority of each proportion in the volume ratio sequence before entering the volume ratio mixing, and control the switching valve group and the quantitative aspiration unit to perform the aspiration and mixing operations of each volume ratio according to the adjusted priority, until all preset volume ratios are completed.

[0006] Furthermore, the specific method by which the control unit adjusts the execution priority is as follows: obtain the amount of bacterial solution absorbed for each ratio in the volume ratio sequence, reorder the volume ratio sequence in descending order of the minimum absorbed amount in each ratio, and use the sorted order as the execution priority; for a mixture of two bacteria, the minimum absorbed amount in each ratio is the smaller value of the absorbed amounts of the two bacterial solutions in that ratio; for a mixture of three bacteria, the minimum absorbed amount in each ratio is the minimum value of the absorbed amounts of the three bacterial solutions in that ratio.

[0007] Furthermore, the control unit is also used to read the amount of bacterial solution absorbed corresponding to each ratio in the volume ratio sequence before the isoconcentration adjustment, and calculate the total volume requirement of each bacterial solution. The volume ratio sequence contains n volume ratios. For a mixture of two bacteria, the total volume requirement of bacterial solution A is calculated using the following formula: ; The total volume requirement for bacterial culture B is calculated using the following formula: ; in, Let A be the volume of bacterial culture A drawn in the i-th volume ratio. The volume of bacterial solution B drawn in the i-th volume ratio; Based on the current volume of bacterial solution in each vial and the measured optical density value, the expected volume to be obtained after achieving the target concentration is calculated using the following formula. : ; in, This represents the current volume of bacterial solution in the bacterial solution bottle. This is the measured optical density value. The target isoconcentration value; If the expected obtainable volume is less than the product of the total volume requirement and the preset safety margin coefficient, then the total amount of bacterial solution is insufficient and the equal concentration adjustment process is paused until it is re-verified and passed.

[0008] Furthermore, when the control unit performs isoconcentration adjustment, it acquires the measured optical density value of the bacterial solution obtained by the detection unit and performs interval judgment: The measured optical density value of the bacterial solution obtained by the detection unit is obtained, and concentration processing, dilution adjustment or dilution process is performed according to whether the measured optical density value is lower than the second threshold, between the second threshold and the first threshold or higher than the first threshold, wherein the first threshold is greater than the second threshold; The dilution process includes: the control unit calculates the required volume of diluent to be added according to the following formula and injects it into the bacterial culture bottle: ; in This is the volume of diluent to be added. This represents the current volume of bacterial solution in the bacterial solution bottle; Pump the bacterial solution into the circulation path and return it to the bacterial solution bottle through the switching valve group. After circulating and mixing, measure the optical density value again. Repeat the above steps until the deviation between the measured optical density value and the target concentration value is not greater than the preset deviation threshold. When the measured optical density value is higher than the first threshold, a preset ratio of the total amount of diluent is injected first, and after circulating and mixing, the optical density value is measured again. Based on the measured deviation, the remaining amount of diluent is added or the dilution is terminated.

[0009] Furthermore, it also includes a concentration unit, which is used to introduce the bacterial solution into a micro centrifuge for centrifugation and concentration when the measured optical density value is less than the second threshold, discharge the supernatant, inject diluent to resuspend the precipitate and return it to the storage unit; the control unit calculates the amount of diluent to be injected for resuspension based on the bacterial solution volume before centrifugation and the measured optical density value, so that the optical density value of the bacterial solution after resuspension rises back to between the second threshold and the first threshold.

[0010] Furthermore, the switching valve assembly includes a multi-channel switching valve and a four-way valve. The multi-channel switching valve has multiple inlets and a common outlet, and the common outlet can be selectively connected to the detection unit or the quantitative aspiration unit. The inlet of the four-way valve is connected to the outlet of the detection unit, the first outlet of the four-way valve is connected to the reflux port of each bacterial culture bottle, the second outlet is connected to the waste culture bottle, and the third outlet is connected to the concentration unit.

[0011] Secondly, the present invention proposes a method for mixing bacterial samples, implemented based on the aforementioned integrated device, comprising the following steps: S1: The target isoconcentration value and a volume ratio sequence containing multiple different volume ratios are set through the control unit; S2: The optical density value of each bacterial solution is measured online in real time by the detection unit and fed back to the control unit. The control unit controls the amount of diluent injected based on the comparison between the measured optical density value and the target isoconcentration value until the concentration of each bacterial solution reaches the target isoconcentration. S3: Before entering the volume ratio mixing, obtain the amount of bacterial solution taken for each ratio in the volume ratio sequence, and reorder the volume ratio sequence in descending order of the minimum amount taken for each ratio. S4: In the sorted order, for each volume ratio, quantitatively sample the corresponding volume of bacterial solution of equal concentration and collect it into the mixing chamber for mixing. Repeat this process until all preset volume ratios are completed.

[0012] Furthermore, prior to the concentration adjustment steps such as step S2, a volume budget prediction step is also included: The volume of each bacterial solution is read from the volume ratio sequence, and the total volume requirement of each bacterial solution is calculated. Based on the current volume of bacterial solution in each bottle and the measured optical density value, the expected volume that can be obtained after reaching the target concentration is determined. If the expected volume that can be obtained is less than the product of the total volume requirement and the preset safety margin coefficient, the total amount of bacterial solution is insufficient and the concentration adjustment process is paused until it is re-verified and passed.

[0013] Furthermore, in step S4, the volume ratio mixing step, the following sub-steps are performed for each volume ratio: S4.1: Select the first type of bacterial solution channel, quantitatively draw a set volume of the first type of bacterial solution and inject it into the mixing chamber; S4.2: Select the second type of bacterial solution channel, quantitatively draw a set volume of the second type of bacterial solution and inject it into the same mixing chamber; S4.3: If it is a mixture of three bacteria, continue to select the third bacterial solution channel, quantitatively draw up the set volume of the third bacterial solution and inject it into the same mixing chamber; S4.4: Start the stirrer to mix the ingredients; S4.5: Output the mixed bacterial solution; S4.6: Clean the mixing chamber; S4.7: Load the next volume ratio, return to S4.1, until all preset volume ratios are completed.

[0014] The beneficial effects of this invention are as follows: 1. This invention utilizes a specific adjustment method comprised of an online detection and control unit, eliminating the need for manual measurement of OD values ​​and calculation of water addition. This ensures that bacterial solutions of varying initial concentrations can accurately reach the same target concentration value. For cases of excessively high or low concentrations, segmented dilution strategies and centrifugal concentration are employed, effectively overcoming the problems of excessive water addition in the nonlinear region and inaccurate adjustment below the detection limit, significantly broadening the device's concentration adaptability range. Simultaneously, the control unit adjusts the priority of each ratio before volumetric mixing, ensuring that bacterial solutions of equal concentrations are mixed sequentially according to the adjusted order. This avoids deviations in the preparation of subsequent micro-samples due to pipeline residue, guaranteeing the preparation accuracy of each volumetric sample. Furthermore, automatic cleaning after each mixing eliminates cross-contamination between samples.

[0015] 2. Through the above-mentioned scheme, the present invention enables the preparation of mixed samples of multiple bacterial species and multiple volume ratios to be completed continuously in the same device. The volume ratio sequence can be freely programmed, supporting the mixing of two or three bacteria. Furthermore, by adding a storage unit, it can be expanded to include more bacterial species. It is suitable for the batch sample preparation needs when constructing a mixed bacterial standard spectral database based on transmission spectroscopy technology. Attached Figure Description

[0016] Figure 1 This is a system block diagram of an integrated bacterial sample mixing device according to an embodiment of the present invention; Figure 2 This is a schematic diagram of a bacterial sample mixing integrated device according to an embodiment of the present invention; Figure 3 This is a schematic flowchart of a bacterial sample mixing method according to an embodiment of the present invention; Figure 4 This is another schematic diagram of a bacterial sample mixing method proposed in one embodiment of the present invention. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0018] Example 1

[0019] Please see Figure 1 This embodiment provides an integrated bacterial sample mixing device based on equal concentration adjustment and batch volume ratio mixing. The device includes a liquid storage unit, a detection unit, a control unit, a switching valve group, a quantitative aspiration unit, and a mixing chamber.

[0020] Please see Figure 2The storage unit includes three bacterial culture bottles, one deionized water bottle, and one waste liquid bottle. The three bacterial culture bottles are used to store different types of original bacterial cultures, such as culture A, culture B, and culture C; the deionized water bottle is used to store sterile deionized water as a source of diluent and cleaning solution; and the waste liquid bottle is used to collect waste liquid generated during dilution, cleaning, and concentration processes.

[0021] Understandably, the number of bacterial culture bottles can be configured according to the actual number of bacterial species to be treated. For example, two bacterial culture bottles can be configured when treating two types of bacteria, and this can be further expanded to include more species. Correspondingly, the system also includes a liquid pump unit connected to the outlet of each bacterial culture bottle for the delivery of the bacterial culture.

[0022] The detection unit, located in the circulation path of the device, consists of an LED light source, a miniature flow cell, and a photodetector. The LED and photodetector are positioned on opposite sides of the flow cell. The flow cell is an optical detection chamber through which the bacterial solution flows. Driven by the flow path, the bacterial solution flows through the internal channels of the flow cell. The measuring light emitted by the LED passes through the bacterial solution in the flow cell. The photodetector receives the transmitted light and outputs a voltage signal proportional to the light intensity. This signal is transmitted to the control unit via a data line. The control unit pre-stores the voltage value for a deionized water blank, which is used for subsequent OD value calculation. This detection unit is used to measure the optical density value of the flowing bacterial solution online in real time and feeds the measurement results back to the control unit.

[0023] Control unit ( Figure 2 The control system can be implemented using a PLC controller or a microcontroller system. The control unit is connected via signal lines to each pump, valve, the photodetector output of the detection unit, the stirrer motor, and the concentration unit within the device. It is used to collect signals and issue control commands. The control unit receives the optical density value fed back from the detection unit and, based on the comparison between the optical density value and the target isoconcentration value, controls the amount of diluent injected to perform isoconcentration adjustment until the concentration of each bacterial culture reaches the target isoconcentration.

[0024] In addition, the control unit is also used to adjust the execution priority of each proportion in the volume ratio sequence before entering the volume ratio mixing, and control the switching valve group and the quantitative aspiration unit to perform the aspiration and mixing operations of each volume ratio according to the adjusted priority, until all preset volume ratios are completed.

[0025] The switching valve assembly includes a multi-channel switching valve and a four-way valve. The multi-channel switching valve is an electrically operated rotary valve with multiple inlets and a common outlet. Each inlet is connected to the outlet of a bacterial culture pump and the outlet of a dilution pump via piping. The common outlet can be selectively connected to either the detection unit or the quantitative aspiration unit. The four-way valve is an electrically operated directional valve with one inlet and three outlets. Its inlet connects to the outlet of the detection unit. The first outlet is connected to a four-way connector via a return main line. The other three outlets of this four-way connector are connected to the return ports of three bacterial culture bottles for circulating mixing and resuspension return. The second outlet connects to a waste bottle for emptying and discharging supernatant. The third outlet connects to the concentration unit via a concentration inlet pipe for introducing over-diluted bacterial culture into the concentration unit for concentration. The switching valve assembly is controlled by a control unit to sequentially select bacterial cultures of equal concentration according to a preset volume ratio sequence.

[0026] The quantitative aspiration unit is installed between the common outlet of the multi-channel switching valve and the inlet of the mixing chamber. After the switching valve group selects the corresponding bacterial solution, the quantitative aspiration unit quantitatively aspirates a set volume of bacterial solution from the corresponding bacterial solution bottle and injects it into the mixing chamber to achieve mixing at different volume ratios. In a preferred embodiment, the quantitative aspiration unit can be implemented using a syringe pump or a peristaltic pump. A syringe pump can achieve higher volumetric accuracy and is suitable for scenarios requiring extremely high accuracy for small-volume samples; a peristaltic pump has the advantages of simple structure and easy maintenance, and can be selected according to actual needs.

[0027] The mixing chamber is a cylindrical, sealed container equipped with an electric stirrer. It has multiple ports connecting to the outlet of the quantitative aspiration unit, the inlet of the cleaning solution, the outlet of the cleaning waste solution, and the output port. The bottom of the mixing chamber is conical for easy evacuation. The mixing chamber receives and mixes the bacterial solution aspirated in each quantitative aspiration. An output pump is installed in the outlet pipeline of the mixing chamber to deliver the mixed bacterial solution to an external spectrometer.

[0028] Furthermore, the device also includes a liquid pump unit. The liquid pump unit includes a bacterial culture pump, a dilution pump, a cleaning pump, a metering pump, and an output pump. The bacterial culture pump is installed on the outlet line of each bacterial culture bottle to pump the bacterial culture from each bottle. The dilution pump is installed on the outlet line of the deionized water bottle, and its outlet is connected to one inlet of a multi-channel switching valve for injecting deionized water into the bacterial culture bottle for dilution. The cleaning pump is also installed on the outlet line of the deionized water bottle, and its outlet is divided into two paths: one path connects to the cleaning solution inlet of the mixing chamber for cleaning the mixing chamber; the other path connects to the resuspension / cleaning inlet of the concentration unit for resuspension of precipitates after centrifugation and cleaning of the concentration unit. The function of the metering pump has been described in the description of the metering unit, and the function of the output pump has been explained in the description of the mixing chamber, and will not be repeated here.

[0029] Furthermore, the device also includes a concentration unit. The concentration unit comprises a miniature centrifuge, whose concentration inlet is connected to the third outlet of a four-way valve via a concentration inlet pipe, and its waste outlet is connected to a waste bottle for discharging the supernatant after centrifugation. A resuspension / washing inlet is connected to the outlet of a washing pump via piping for injecting deionized water into the centrifuge to resuspend the precipitate or wash the centrifuge. The concentrated resuspension outlet is connected to the reflux ports of three bacterial culture bottles via reflux pipes. The speed and running time of the miniature centrifuge are controlled by a control unit.

[0030] Through the specific structural design of the aforementioned switching valve group, the device can flexibly switch between different working modes, including measurement mode, dilution mode, circulation mixing mode, venting mode, concentration mode, and suction mixing mode.

[0031] Based on the aforementioned device, the control unit achieves isoconcentration regulation through online detection and closed-loop control: the detection unit measures the optical density value of each bacterial solution online in real time and feeds it back to the control unit. The control unit determines the amount of diluent to be added based on the comparison between the measured value and the target value, and precisely injects it into the corresponding bacterial solution bottle via a dilution pump. After injection, the bacterial solution is pumped into the circulation path by the bacterial solution pump and returned to the bacterial solution bottle via a switching valve assembly. The circulating flow agitates the liquid in the bottle, ensuring uniform concentration. The optical density value is then measured again, and the above operation is repeated until the deviation between the measured value and the target value is within the allowable range. Thus, without manual measurement of the OD value or calculation of the water addition, bacterial solutions with different initial concentrations can accurately reach the same target isoconcentration value, ensuring the consistency of the concentration of each bacterial solution.

[0032] In this embodiment, the optical density value (OD value) is calculated based on the Lambert-Beer law:

[0033] in, The transmitted light intensity of sterile deionized water. The intensity of transmitted light from the bacterial culture. (OD) 600 Within the range of 0.2-0.8, the OD value showed a good linear relationship with the bacterial concentration; when the OD value was within this range... 600 When the OD value is greater than 0.8, light scattering deviates from the Lambert-Beer law, resulting in a non-linear relationship between the measured value and the actual concentration; when the OD value is greater than 0.8, light scattering deviates from the Lambert-Beer law, causing a non-linear relationship between the measured value and the actual concentration; 600 When the OD value is less than 0.2, it exceeds the accurate detection limit of most spectrophotometers. Therefore, during the isoconcentration adjustment process, the OD value of each bacterial solution needs to be controlled within the linear range of 0.2-0.8 to ensure the accuracy of concentration adjustment. Based on this, this embodiment sets the first threshold for OD value judgment to 0.8 and the second threshold to 0.2, as the basis for subsequent range judgment and dilution / concentration processing.

[0034] The control unit adjusts the execution priority of each ratio before mixing by volume. After each mixing is completed, the control unit automatically controls the cleaning solution to rinse the mixing chamber, eliminating cross-contamination between samples.

[0035] As a further preferred embodiment, the control unit adjusts the execution priority in the following way: It obtains the amount of bacterial solution absorbed for each proportion in the volume ratio sequence, reorders the volume ratio sequence in descending order of the minimum absorbed amount in each proportion, and uses the reordered order as the execution priority. For a mixture of two bacteria, the minimum absorbed amount in each proportion is the smaller value of the absorbed amounts of the two bacterial solutions in that proportion; for a mixture of three bacteria, the minimum absorbed amount in each proportion is the smallest value of the absorbed amounts of the three bacterial solutions in that proportion.

[0036] It is understandable that during volume-ratio mixing, a small amount of bacterial solution from the previous sample will inevitably remain on the inner wall of the tubing. When the aspirated volume for a certain proportion is small, the residual amount accounts for a relatively large proportion of the total volume of that proportion, thus significantly affecting the preparation accuracy of that proportion. By adjusting the priority method described above, the micro-absorption proportion is moved to a later position in the sequence. Utilizing the flushing effect of the preceding large-volume aspiration operation on the tubing, the interference of tubing residue on subsequent micro-samples is effectively reduced, further ensuring the preparation accuracy of samples at each volume ratio. This method is particularly suitable for batch mixing preparation scenarios with large volume ratio ranges.

[0037] As a further preferred embodiment, the control unit is also used to perform a volume budget prediction before the concentration adjustment. Specifically, the control unit reads the amount of bacterial solution absorbed for each ratio in the volume ratio sequence and calculates the total volume requirement for each bacterial solution. The volume ratio sequence contains n volume ratios. For a mixture of two bacteria, the total volume requirement for bacterial solution A is calculated using the following formula:

[0038] The total volume requirement for bacterial culture B is calculated using the following formula:

[0039] in, Let A be the volume of bacterial culture A drawn in the i-th volume ratio. The volume of bacterial solution B drawn in the i-th volume ratio; Based on the current volume of bacterial solution in each vial and the measured optical density value, the expected volume to be obtained after achieving the target concentration is calculated using the following formula. :

[0040] in, This represents the current volume of bacterial solution in the bacterial solution bottle. This is the measured optical density value. The target isoconcentration value; If the expected volume is less than the product of the total required volume and the preset safety margin factor, an indication will be given that the total bacterial volume is insufficient, and the isoconcentration adjustment process will be paused until re-verification is successful. By using this volume estimation prediction, it is possible to determine in advance whether the available volume of each bacterial culture is sufficient to meet the mixing requirements of all preset volume ratios before the isoconcentration adjustment begins. This effectively avoids situations where insufficient bacterial culture is discovered during the adjustment process, leading to operation interruption or sample rejection. As an example, the preset safety margin factor can be set according to the specific experimental accuracy requirements; for example, a 20% margin can be reserved.

[0041] As a further preferred embodiment, when the control unit performs isostatic adjustment, it acquires the measured optical density value of the bacterial solution obtained by the detection unit and performs interval judgment. Specifically, depending on whether the measured optical density value is lower than a second threshold, between the second threshold and the first threshold, or higher than the first threshold, it performs concentration treatment, dilution adjustment, or dilution process respectively, wherein the first threshold is greater than the second threshold.

[0042] As an example, the second threshold can be 0.2, and the first threshold can be 0.8. When the measured OD value is between 0.2 and 0.8, the bacterial concentration is within the usable linear range, and dilution adjustment can be directly initiated; when the measured OD value is greater than 0.8, the dilution procedure is executed; when the measured OD value is less than 0.2, the concentration process is executed.

[0043] The dilution process includes: the control unit calculates the required volume of diluent to be added using the following formula and injects it into the bacterial culture bottle:

[0044] in This is the volume of diluent to be added. The current volume of bacterial solution in the bacterial solution bottle is given. After injecting the calculated amount of diluent into the bacterial solution bottle, the bacterial solution is pumped into the circulation path and returned to the bacterial solution bottle through the switching valve group. After circulation and mixing, the optical density value is measured again. The above steps are repeated until the deviation between the measured optical density value and the target isoconcentration value is not greater than the preset deviation threshold.

[0045] In cases where the measured optical density value is higher than the first threshold, a preset ratio of the total amount of diluent is injected first, the mixture is circulated and mixed, and the optical density value is measured again. Based on the measured deviation, the remaining amount of diluent is added or the dilution is terminated.

[0046] Understandably, at high concentrations, the OD value may deviate from a linear relationship with the concentration. Adding the entire calculated amount of diluent at once could lead to excessive water addition due to nonlinear errors. The segmented dilution strategy described above, which involves adding a portion of the calculated water volume first, mixing thoroughly, and then determining whether to add the remaining water based on actual measurements, effectively avoids the problem of excessive water addition due to nonlinear errors and improves the accuracy of dilution adjustments.

[0047] As a further preferred embodiment, the device also includes a concentration unit, used to introduce the bacterial solution into a microcentrifuge for centrifugal concentration when the measured optical density value is less than a second threshold. Specifically, the four-way valve is switched to the third outlet, and the bacterial solution is pumped into the microcentrifuge through the concentration inlet pipe for centrifugation. After centrifugation, the waste discharge valve is opened to discharge the supernatant into a waste bottle, and then a diluent is injected to resuspend the precipitate before returning it to the storage unit. The control unit calculates the amount of diluent injected for resuspension based on the bacterial solution volume before centrifugation and the measured optical density value, so that the optical density value of the bacterial solution after resuspension rises back to between the second threshold and the first threshold.

[0048] Through the aforementioned concentration unit, the device can process excessively dilute bacterial solutions with concentrations below the detection limit, concentrating them to a linear range that can be accurately measured, thus expanding the device's adaptability to bacterial solutions with different initial concentrations.

[0049] As a further preferred embodiment, the switching valve assembly includes a multi-channel switching valve and a four-way valve. The multi-channel switching valve has multiple inlets and a common outlet, which can be selectively connected to the detection unit or the quantitative aspiration unit. The inlet of the four-way valve is connected to the outlet of the detection unit, the first outlet of the four-way valve is connected to the reflux port of each bacterial culture bottle for circulation mixing and resuspension reflux; the second outlet is connected to the waste bottle for emptying and discharging the supernatant; and the third outlet is connected to the concentration unit for introducing the diluted bacterial culture into a centrifuge for concentration. Through the specific structural arrangement of the switching valve assembly described above, flexible switching between different operating modes (measurement, dilution, circulation mixing, emptying, concentration, and aspiration mixing) of the device is achieved.

[0050] As a further preferred embodiment, the apparatus also includes a waste liquid bottle for collecting waste liquid generated during the dilution, washing, and concentration processes. The waste liquid bottle ensures that all waste liquid generated during the entire preparation process has a unified collection container, avoiding pollution caused by indiscriminate discharge of waste liquid.

[0051] In a preferred embodiment, the control unit also incorporates optical density linear range parameters for various bacterial species, calling up the corresponding upper and lower limits of the linear range based on the bacterial species selected by the user. Specifically, the system includes a table of OD linear range parameters for common bacteria such as Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. After the user selects a bacterial species, the system automatically calls up the corresponding upper and lower limits of the linear range, making the control strategy more targeted.

[0052] In a preferred embodiment, the control unit employs a short-duration, multiple-stirring mode for highly viscous bacterial strains. This mode involves alternating stirring and stopping operations several times. Specifically, certain bacteria, such as those producing extracellular polysaccharides, experience increased viscosity in the bacterial solution under prolonged continuous stirring, affecting the stability of OD measurements and the uniformity of mixing. For such strains, the system automatically adopts a short-duration, multiple-stirring mode, for example, stirring for 10 seconds, stopping for 5 seconds, and repeating 2-3 times, to avoid the increased viscosity caused by prolonged continuous stirring.

[0053] As an alternative, the peristaltic pump in the above embodiments can be replaced by a syringe pump. Syringe pumps achieve higher volumetric accuracy and are suitable for scenarios requiring extremely high precision for small-volume samples. The 600nm LED + photodetector in the OD detection module can be replaced by a miniature fiber optic spectrometer. The spectrometer can simultaneously acquire transmission spectra at multiple wavelengths, allowing for the measurement of not only OD... 600 It can also monitor other wavelength information for more complex concentration determination. The mechanical stirrer in the mixing chamber can be replaced by a magnetic stir bar. A magnetic stir bar is placed at the bottom of the mixing chamber, and a rotating magnet is set below the chamber to drive it. This avoids the sealing problems caused by the stirring shaft passing through the chamber and makes it easier to achieve aseptic operation.

[0054] Example 2

[0055] This embodiment provides a method for mixing bacterial samples, which is implemented based on the integrated device in Embodiment 1. Please refer to... Figure 3 and Figure 4 The method includes the following steps.

[0056] S1: The target isoconcentration value and a sequence of volume ratios containing multiple different volume ratios are set through the control unit. Specifically, the operator inputs the following parameters through the human-machine interface of the control unit: target isoconcentration OD value, volume ratio mixing list, stirring time for each ratio, and washing parameters. The control unit stores the above parameters in memory and generates a task queue to be executed.

[0057] S2: The optical density value of each bacterial solution is measured online in real time by the detection unit and fed back to the control unit. The control unit controls the amount of diluent injected based on the comparison between the measured optical density value and the target isoconcentration value until the concentration of each bacterial solution reaches the target isoconcentration.

[0058] In a preferred embodiment, step S2 specifically includes: measuring the measured optical density value of the bacterial suspension; and performing concentration treatment, dilution adjustment, or dilution process according to whether the measured optical density value is lower than a second threshold, between the second threshold and a first threshold, or higher than the first threshold, wherein the first threshold is greater than the second threshold. For example, the second threshold can be 0.2, and the first threshold can be 0.8.

[0059] The dilution process includes: the control unit calculates the required volume of diluent to be added using the following formula and injects it into the bacterial culture bottle:

[0060] in This is the volume of diluent to be added. This represents the current volume of bacterial solution in the bacterial solution bottle.

[0061] The bacterial culture is pumped into the circulation path and returned to the bacterial culture bottle via a switching valve assembly. After thorough mixing, the optical density value is measured again. This process is repeated until the deviation between the measured optical density value and the target concentration value is no greater than a preset deviation threshold. For example, the preset deviation threshold can be 0.02, and typically 2-3 cycles are sufficient to achieve the target.

[0062] For high-concentration cases where the measured optical density value is higher than the first threshold, first inject a preset proportion of the total amount of diluent, circulate and mix thoroughly, then remeasure the optical density value. Based on the measured deviation, add the remaining amount of diluent or terminate the dilution process. As an example, the preset proportion can be 80%.

[0063] Through the above steps, by utilizing a specific adjustment method composed of online detection and control units, it is possible to achieve the same target concentration value for each bacterial solution with different initial concentrations without the need for manual measurement of OD value and calculation of water addition.

[0064] S3: Before entering the volume ratio mixing, obtain the amount of bacterial solution taken for each ratio in the volume ratio sequence, and reorder the volume ratio sequence in descending order of the minimum amount taken for each ratio.

[0065] In a preferred embodiment, for a mixture of two bacteria, the minimum amount to be taken in each proportion is the smaller of the amounts of the two bacterial solutions taken in that proportion; for a mixture of three bacteria, the minimum amount to be taken in each proportion is the minimum of the amounts of the three bacterial solutions taken in that proportion.

[0066] It is understandable that during the volume-ratio mixing process, a small amount of bacterial solution from the previous sample will inevitably remain on the inner wall of the tubing. Adjusting the micro-absorption ratio to a later position in the sequence utilizes the flushing effect of the preceding large-volume aspiration operation on the tubing, effectively reducing the interference of tubing residue on subsequent micro-samples, thereby ensuring the preparation accuracy of samples at each volume ratio.

[0067] S4: In the sorted order, for each volume ratio, quantitatively sample the corresponding volume of bacterial solution of equal concentration and collect it into the mixing chamber for mixing. Repeat this process until all preset volume ratios are completed.

[0068] In a preferred embodiment, step S4 performs the following sub-steps for each volume ratio: S4.1: Select the first bacterial solution channel, quantitatively draw a set volume of the first bacterial solution and inject it into the mixing chamber.

[0069] S4.2: Select the second bacterial solution channel, quantitatively draw a set volume of the second bacterial solution and inject it into the same mixing chamber.

[0070] S4.3: If it is a mixture of three types of bacteria, continue to select the third bacterial solution channel, quantitatively draw up the set volume of the third bacterial solution and inject it into the same mixing chamber.

[0071] S4.4: Start the stirrer to mix. As an example, the stirrer can be used to stir at a speed of 100~200 r / min for 20~60 seconds.

[0072] S4.5: Discharge the mixed bacterial solution. Specifically, turn on the discharge pump to deliver the mixed bacterial solution through the tubing to the flow cell or cuvette of an external spectrometer for transmission spectroscopy measurement. The discharge pump flow rate is 30 mL / min, and the control unit sets the discharge time according to the total volume of the mixed bacterial solution to ensure complete emptying.

[0073] S4.6: Clean the mixing chamber. Specifically, inject diluent into the mixing chamber, start the agitator to rotate, and then drain the cleaning waste liquid. Repeat this process several times.

[0074] S4.7: Load the next volume ratio, return to S4.1, until all preset volume ratios are completed.

[0075] Through the above-described cyclic process, the device can continuously prepare mixed samples with dozens of volume ratios without human intervention.

[0076] As a further preferred embodiment, before the concentration adjustment step S2, a volume budget prediction step is also included: reading the amount of bacterial solution taken from each ratio in the volume ratio sequence, calculating the total volume requirement of each bacterial solution, and determining the expected volume that can be obtained after reaching the target concentration based on the current volume of bacterial solution in each bacterial solution bottle and the measured optical density value. If the expected volume that can be obtained is less than the product of the total volume requirement and the preset safety margin coefficient, the total amount of bacterial solution is insufficient and the concentration adjustment process is paused until it is re-verified and passed.

[0077] As a further preferred embodiment, the cleaning steps after each mixing are as follows: inject diluent into the mixing chamber, start the stirrer to rotate at low speed for 10 seconds, then open the cleaning waste liquid outlet of the mixing chamber and discharge the cleaning waste liquid into the waste liquid bottle. Repeat this cleaning process 1 to 3 times. After cleaning is completed, turn off the cleaning pump and stirrer.

[0078] As a further preferred embodiment, after all proportions are completed, the system performs a comprehensive cleaning: Diluent is injected into the mixing chamber, the stirrer rotates at high speed for 15-30 seconds, and the mixture is drained into a waste bottle; simultaneously, the multi-channel switching valve is switched sequentially to each bacterial culture channel, the pipeline is flushed with diluent, and the waste liquid is discharged into a waste bottle; the cleaning pump is turned on to inject diluent into the micro centrifuge, which runs at low speed for 10-20 seconds and then drains into a waste bottle, completing the centrifuge cleaning. The system then enters standby mode.

[0079] In a preferred embodiment, for highly viscous bacterial strains, the mixing in step S4.4 employs a short-duration, multiple-stage mixing mode. This mode involves alternating between mixing and stopping operations, repeated several times. Specifically, for example, mixing for 10 seconds, stopping for 5 seconds, and repeating 2-3 times, to avoid increased viscosity caused by prolonged continuous mixing.

[0080] In a preferred embodiment, the control unit has built-in optical density linear range parameters corresponding to various bacterial species. The upper and lower limits of the corresponding linear range are called according to the bacterial species selected by the user, making the control strategy more targeted.

[0081] It is understandable that the parameters involved in the above methods and steps can be set and adjusted by the operator through the human-machine interface of the control unit. The parameters include stirring speed, stirring time, number of cleaning cycles, flow rate, etc., to adapt to different bacterial strains and different experimental needs.

[0082] Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0083] In addition, the functional modules in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit.

[0084] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such 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 this application.

Claims

1. An integrated bacterial sample mixing device based on isoconcentration adjustment and batch volume ratio mixing, characterized in that, include: The liquid storage unit is used to store various original bacterial solutions and diluents; The detection unit is used for online real-time measurement of the optical density value of the bacterial solution; The control unit is used to receive the optical density value fed back by the detection unit, and control the amount of diluent injected according to the comparison result between the optical density value and the target isoconcentration value, so as to perform isoconcentration adjustment until the concentration of each bacterial solution reaches the target isoconcentration. The switching valve assembly has multiple inlets and switchable outlets, and is controlled by the control unit to sequentially select bacterial solutions of equal concentration according to a preset volume ratio sequence. The quantitative aspiration unit is used to quantitatively aspirate a set volume of bacterial solution from the corresponding bacterial solution bottle after the switching valve group selects the corresponding bacterial solution, and inject it into the mixing chamber to achieve mixing of different volume ratios; The control unit is also used to adjust the execution priority of each proportion in the volume ratio sequence before entering the volume ratio mixing, and control the switching valve group and the quantitative aspiration unit to perform the aspiration and mixing operations of each volume ratio according to the adjusted priority, until all preset volume ratios are completed.

2. The integrated bacterial sample mixing device according to claim 1, characterized in that, The specific method by which the control unit adjusts the execution priority is as follows: obtain the amount of bacterial solution taken from each ratio in the volume ratio sequence, reorder the volume ratio sequence in descending order of the minimum amount taken from each ratio, and use the sorted order as the execution priority; for a mixture of two bacteria, the minimum amount taken from each ratio is the smaller value of the amount of bacterial solution taken from the two bacteria in that ratio; for a mixture of three bacteria, the minimum amount taken from each ratio is the minimum value of the amount of bacterial solution taken from the three bacteria in that ratio.

3. The integrated bacterial sample mixing device according to claim 1, characterized in that, The control unit is also used to read the volume of each bacterial solution drawn corresponding to each ratio in the volume ratio sequence before the isoconcentration adjustment, and to calculate the total volume requirement of each bacterial solution. The volume ratio sequence contains n volume ratios. For a mixture of two bacteria, the total volume requirement of bacterial solution A is calculated using the following formula: ； The total volume requirement for bacterial culture B is calculated using the following formula: ； in, Let A be the volume of bacterial culture A drawn in the i-th volume ratio. The volume of bacterial solution B drawn in the i-th volume ratio; Based on the current volume of bacterial solution in each vial and the measured optical density value, the expected volume to be obtained after achieving the target concentration is calculated using the following formula. : ； in, This represents the current volume of bacterial solution in the bacterial solution bottle. This is the measured optical density value. The target isoconcentration value; If the expected obtainable volume is less than the product of the total volume requirement and the preset safety margin coefficient, then the total amount of bacterial solution is insufficient and the equal concentration adjustment process is paused until it is re-verified and passed.

4. The integrated bacterial sample mixing device according to claim 3, characterized in that, When the control unit performs isoconcentration adjustment, it acquires the measured optical density value of the bacterial solution obtained by the detection unit and performs interval judgment: The measured optical density value of the bacterial solution obtained by the detection unit is obtained, and concentration processing, dilution adjustment or dilution process is performed according to whether the measured optical density value is lower than the second threshold, between the second threshold and the first threshold or higher than the first threshold, wherein the first threshold is greater than the second threshold; The dilution process includes: the control unit calculates the required volume of diluent to be added according to the following formula and injects it into the bacterial culture bottle: ； in This is the volume of diluent to be added. This represents the current volume of bacterial solution in the bacterial solution bottle; Pump the bacterial solution into the circulation path and return it to the bacterial solution bottle through the switching valve group. After circulating and mixing, measure the optical density value again. Repeat the above steps until the deviation between the measured optical density value and the target concentration value is not greater than the preset deviation threshold. When the measured optical density value is higher than the first threshold, a preset ratio of the total amount of diluent is injected first, and after circulating and mixing, the optical density value is measured again. Based on the measured deviation, the remaining amount of diluent is added or the dilution is terminated.

5. The integrated bacterial sample mixing device according to claim 4, characterized in that, It also includes a concentration unit, which is used to introduce the bacterial solution into a micro centrifuge for centrifugation and concentration when the measured optical density value is less than the second threshold, discharge the supernatant, inject diluent to resuspend the precipitate and return it to the storage unit; the control unit calculates the amount of diluent to be injected for resuspension based on the bacterial solution volume before centrifugation and the measured optical density value, so that the optical density value of the bacterial solution after resuspension rises back to between the second threshold and the first threshold.

6. The integrated bacterial sample mixing device according to claim 5, characterized in that, The switching valve assembly includes a multi-channel switching valve and a four-way valve. The multi-channel switching valve has multiple inlets and a common outlet, which can be selectively connected to the detection unit or the quantitative aspiration unit. The inlet of the four-way valve is connected to the outlet of the detection unit, the first outlet of the four-way valve is connected to the reflux port of each bacterial culture bottle, the second outlet is connected to the waste bottle, and the third outlet is connected to the concentration unit.

7. A method for mixing bacterial samples, implemented based on the integrated device according to any one of claims 1-6, characterized in that, Includes the following steps: S1: Set the target isoconcentration value and a volume ratio sequence containing multiple different volume ratios through the control unit; S2: The optical density value of each bacterial solution is measured online in real time by the detection unit and fed back to the control unit. The control unit controls the amount of diluent injected based on the comparison between the measured optical density value and the target isoconcentration value until the concentration of each bacterial solution reaches the target isoconcentration. S3: Before entering the volume ratio mixing, obtain the amount of bacterial solution taken for each ratio in the volume ratio sequence, and reorder the volume ratio sequence in descending order of the minimum amount taken for each ratio. S4: In the sorted order, for each volume ratio, quantitatively sample the corresponding volume of bacterial solution of equal concentration and collect it into the mixing chamber for mixing. Repeat this process until all preset volume ratios are completed.

8. A method for mixing bacterial samples according to claim 7, characterized in that, Before the concentration adjustment step S2, a volume budget prediction step is also included: The volume of each bacterial solution is read from the volume ratio sequence, and the total volume requirement of each bacterial solution is calculated. Based on the current volume of bacterial solution in each bottle and the measured optical density value, the expected volume that can be obtained after reaching the target concentration is determined. If the expected volume that can be obtained is less than the product of the total volume requirement and the preset safety margin coefficient, the total amount of bacterial solution is insufficient and the concentration adjustment process is paused until it is re-verified and passed.

9. A method for mixing bacterial samples according to claim 7, characterized in that, In step S4, the volume ratio mixing step, the following sub-steps are performed for each volume ratio: S4.1: Select the first type of bacterial solution channel, quantitatively draw a set volume of the first type of bacterial solution and inject it into the mixing chamber; S4.2: Select the second type of bacterial solution channel, quantitatively draw a set volume of the second type of bacterial solution and inject it into the same mixing chamber; S4.3: If it is a mixture of three bacteria, continue to select the third bacterial solution channel, quantitatively draw up the set volume of the third bacterial solution and inject it into the same mixing chamber; S4.4: Start the stirrer to mix the ingredients; S4.5: Output the mixed bacterial solution; S4.6: Clean the mixing chamber; S4.7: Load the next volume ratio, return to S4.1, until all preset volume ratios are completed.

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