In-vitro microorganism concentration on-line detection device and method

Abstract

The application discloses a kind of in-vitro microbial concentration online detection device and method, device includes: container seat is equipped with for accommodating transparent circular test tube installation through-hole;Light source module is installed in the side of container seat;Transmitted light sensor is installed in the position opposite light source module on container seat;Multiple scattering light sensors are installed on container seat and are arranged around installation through-hole;Control processing unit is electrically connected with transmitted light sensor and multiple scattering light sensors;Control processing unit is configured to: based on transmitted light signal, according to Lambert-Beer law calculation first OD value;Based on multiple scattering light signals, scattering light intensity distribution information is obtained, according to standard curve between scattering light intensity distribution and OD value, determine second OD value.The device volume micro, compact structure, can realize from low to high full concentration range with wide range, high precision Real-time, online, non-contact, multi-channel detection, avoid the pollution risk and the operational complexity caused by sampling.

Description

Technical Field

[0001] This invention belongs to the field of microbial laboratory research, specifically relating to an online detection device and method for microbial concentration in test tubes. Background Technology

[0002] During microbial culture, it is necessary to periodically monitor the growth status of microorganisms. The absorbance method is commonly used to detect the microbial content in the culture medium. The theoretical basis for concentration detection is Beer-Lambert's law: when a beam of parallel monochromatic light passes perpendicularly through a uniform, non-scattering absorbing substance, its absorbance A or optical density (OD) is directly proportional to the concentration c of the absorbing substance and the thickness d of the absorbing layer. Therefore, concentration can be expressed in terms of absorbance. The mathematical expression is OD = kcd, where k is a proportionality constant. Absorbance is a measure of the degree to which a substance absorbs light, defined as OD = logcd 10 (I0 / I), where I0 is the reference light intensity and I is the transmitted light intensity of the sample.

[0003] Conventional measurement methods typically employ a spectrophotometer to detect the absorbance of the sampled culture medium. However, because the bacterial culture is a suspension, light scattering can cause nonlinear responses in high concentration ranges (e.g., exceeding 0.8 OD). Therefore, it is usually necessary to dilute the sample to a linear range of 0.4 OD to 0.8 OD for measurement, and then calculate the theoretical concentration using the dilution factor. This method is not only cumbersome and prone to contamination, but also cannot meet the needs of real-time online monitoring.

[0004] To achieve real-time online detection, existing technologies also employ absorbance measurement based on transmitted light and establish a standard curve of concentration versus OD value to cover the nonlinear range. However, this method is limited by the linear range of the detection system, making it difficult to achieve accurate detection at high concentrations. Even with modern instruments increasing the measurable upper limit to 5 OD, it still cannot meet the requirements for real-time measurement of higher concentrations.

[0005] In addition, some studies have used scattered light for concentration detection. For example, Chinese patent application 201610341529.9 discloses a detection device that combines transmitted light and backscattered light. By adjusting the distance between the light source and the receiver, the detection range is widened to a high concentration range (such as 15 OD), and a pre-calibrated scattered light intensity-concentration curve is used for conversion. However, the bacterial culture system itself is unstable, and the backscattered light signal is weak and easily fluctuates, resulting in poor measurement repeatability and insufficient accuracy. Furthermore, it is difficult to effectively connect the signal with the transmitted light detection range, making it difficult to form a complete and reliable full-concentration calibration curve.

[0006] Figure 1 A schematic diagram of transmitted and astigmatic light on the cross-section of a circular test tube containing bacterial solution when a collimated beam passes through it. Figure 1In the diagram, A is a circular test tube, B is the incident light, C is the transmitted light, D is the forward scattered light, E is the 90-degree scattered light, and F is the backscattered light. Figure 2 This diagram illustrates the changes in the intensity distribution of transmitted and scattered light across the cross-section of a circular test tube containing bacterial solutions of varying concentrations, as a collimated light beam passes through it. Figure 1 and Figure 2 It is known that the response characteristics of scattered light at different angles to concentration changes vary across different concentration ranges: forward scattered light is more sensitive at low concentrations, 90-degree scattered light still has a good response at medium to high concentrations, while backscattered light signals increase with increasing concentration at high concentrations, covering a wider range. Therefore, relying solely on scattered light measurements from a single angle often results in limited detection limits and low accuracy.

[0007] In summary, existing online methods for detecting microbial concentrations have significant limitations: methods based on transmitted light offer high accuracy but have a low detection limit; methods based on single-angle scattered light are susceptible to signal fluctuations and struggle to seamlessly integrate with the transmitted light range, making it difficult to achieve stable and accurate real-time monitoring across the entire concentration range. How to achieve real-time online monitoring with both wide range and high accuracy across the entire concentration range, from low to high, remains a pressing technical challenge in this field. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide an online detection device and method for microbial concentration in test tubes, addressing the shortcomings of existing technologies. The detection device of this invention is miniature and compact, and can be directly placed in conventional microbial culture shakers and other equipment. Furthermore, the device can be mass-produced, achieving real-time, online, non-contact, multi-channel detection with a wide range of concentrations from low to high, while avoiding the contamination risks and operational complexities associated with sampling.

[0009] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: an online detection device for microbial concentration in test tubes, comprising:

[0010] The container holder has mounting holes for accommodating transparent circular test tubes;

[0011] A light source module, installed on one side of the container base, is used to emit a collimated beam whose optical axis passes through the center of the mounting through hole;

[0012] A transmitted light sensor is installed on the container base at a position opposite to the light source module, and is used to receive the transmitted light signal after the collimated beam passes through the culture medium in the test tube.

[0013] Multiple scattered light sensors are mounted on the container base and arranged around the mounting through hole to receive scattered light signals from different angles from the culture medium in the test tube;

[0014] A control processing unit is electrically connected to the transmitted light sensor and the plurality of scattered light sensors; the control processing unit is configured to: calculate a first OD value based on the transmitted light signal according to the Lambert-Beer law, and obtain scattered light intensity distribution information based on the plurality of scattered light signals, and determine a second OD value according to a pre-stored standard curve between the scattered light intensity distribution and the OD value.

[0015] The principle of this invention is as follows:

[0016] First, based on the Lambert-Beer law, the absorbance (OD value) of the bacterial solution can be calculated by detecting the intensity of transmitted light. This OD value shows a good linear relationship with the bacterial solution concentration in the low concentration range. However, as the bacterial solution concentration increases, the particle scattering effect intensifies, and the OD value no longer exhibits a linear relationship with concentration, rendering the Lambert-Beer law inapplicable. Simultaneously, the transmitted light signal attenuates sharply, making it difficult for the sensor to reliably detect, thus placing extremely high demands on the system's dynamic range and signal-to-noise ratio.

[0017] Secondly, as the bacterial concentration changes, the spatial distribution of its scattered light exhibits a regular evolution: at lower concentrations, the intensity of forward small-angle scattered light increases with increasing concentration and is highly sensitive to minute concentration changes, which is beneficial for achieving high-precision detection; as the concentration further increases, the forward scattered light gradually weakens and its sensitivity decreases, while the lateral scattered light signal at approximately 90° to the incident light direction strengthens and maintains high sensitivity to concentration changes; when the concentration continues to increase to a higher range, both forward and lateral scattered light weaken, while backscattered light significantly strengthens, becoming the main optical signal reflecting concentration changes.

[0018] Based on the aforementioned variation pattern between concentration and scattered light distribution, this invention acquires the spatial distribution characteristics of scattered light signals from multiple angles and establishes a standard curve between the scattered light distribution and the OD value. Using this standard curve, high-precision, non-contact online measurement of bacterial suspension OD values ​​can be achieved over a wide concentration range, thus simultaneously ensuring high accuracy in the low-concentration region and a wide detection upper limit in the high-concentration region.

[0019] Preferably, the plurality of scattered light sensors are at the same height as the light source module and the transmitted light sensor to avoid measurement errors caused by uneven concentration distribution in the upper and lower layers of the liquid or misalignment of the sensors, thus ensuring the efficiency of transmitted light reception and the accuracy of scattered light acquisition at various angles.

[0020] Preferably, with the line connecting the light source module and the transmitted light sensor as the boundary, the plurality of scattered light sensors are distributed on one side of the line, and the angle between any two adjacent scattered light sensors is 15 degrees to 30 degrees. In this technical solution, multiple scattered light sensors are concentrated on one side of the line, reducing the number of mounting reference surfaces and lowering the difficulty of precision machining and calibration.

[0021] Alternatively, as a preferred embodiment, the multiple scattered light sensors are distributed on both sides of the line connecting the light source module and the transmitted light sensor. The angular position distribution of all the scattered light sensors on the horizontal plane satisfies the following: Mirroring the angular position of a scattered light sensor on one side of the line to the other side, using the line as an axis of symmetry, together with the original angular position of the scattered light sensor on the other side, forms an equivalent sensor group. In this equivalent sensor group, after sorting by angular position from smallest to largest, the angle between any two adjacent angular positions is between 15 and 30 degrees. This design, with multiple scattered light sensors distributed on both sides of the line, offers flexible layout, stronger measurement stability and robustness. When establishing standard curves and processing real-time data, the mirror principle can be used to simplify calculations, while providing more comprehensive spatial sampling. This allows for more accurate capture of the complete spatial distribution characteristics of scattered light in the culture medium on the horizontal plane, providing more reliable data for establishing more accurate concentration-distribution relationship curves.

[0022] Smaller angles (e.g., 15 degrees) offer high sensitivity in low-concentration regions but have a narrow effective range; larger angles (e.g., 30 degrees) provide a wider range and better bridge different concentration ranges. By employing multiple scattered light sensors distributed on one or both sides of the line, within a preferred angle range of 15 to 30 degrees, it ensures that a sensor signal at a suitable angle maintains high sensitivity to concentration changes across a wide concentration range from low to high, thus achieving wide-range, high-precision detection. The number of scattered light sensors should be at least three. At least three scattered light sensors provide the minimum requirement for constructing one-dimensional characteristic information of the scattered light spatial distribution, while more sensors allow for a finer scattered light intensity distribution curve, resulting in a more accurate standard curve. The number of scattered light sensors can be determined according to the specific application requirements.

[0023] Preferably, the container base is a polyhedral cylindrical structure, with the mounting through-hole located at its center; the light source module, the transmitted light sensor, and multiple scattered light sensors are respectively mounted in radial through-holes opened on different sides of the container base. The polyhedral cylindrical container base provides a precise and fixed mounting reference surface for the light source module, the transmitted light sensor, and the multiple scattered light sensors, ensuring that all optical axes are strictly aligned with the center of the test tube, thereby ensuring high accuracy and repeatability of the detection.

[0024] Preferably, the light source module includes a laser diode and a collimating lens. The laser light source module composed of a laser diode and a collimating lens is small in size and has low power consumption, which helps to ensure the clarity and stability of the transmitted light and scattered light signals.

[0025] Preferably, a positioning mechanism is provided within the mounting through hole, which is used to coaxially position the test tube within the mounting through hole. The positioning mechanism can specifically be a rubber ring, an elastic positioning element, a spring, etc.

[0026] A method for online detection of microbial concentration in test tubes using the above-mentioned device includes the following steps:

[0027] S1. Provide a reference tube containing liquid culture medium and a sample tube containing a bacterial culture medium sample;

[0028] S2. Place the reference tube in the mounting through hole of the container seat to obtain the transmitted light reference intensity I0;

[0029] S3. Place the sample tube into the mounting through hole of the container seat and obtain the transmitted light sample intensity I;

[0030] S4. According to the formula OD = log 10 (I0 / I) Calculate the first OD value of the bacterial culture sample;

[0031] S5. When the first OD value is lower than a preset threshold, the first OD value is used as the final OD output;

[0032] When the first OD value reaches or exceeds the preset threshold, the scattered light intensity signal collected by the plurality of scattered light sensors is acquired, the scattered light intensity distribution information is calculated based on the scattered light intensity signal, and the pre-stored standard curve is queried to determine the corresponding second OD value as the final OD output.

[0033] The above detection method is simple to operate. It organically combines the high precision advantage of the transmission light method in the low concentration range with the wide range advantage of the multi-angle scattering light method in the high concentration range, and finally realizes real-time, accurate and online monitoring of microbial concentration in a wide range from close to 0 OD to more than 30 OD.

[0034] Preferably, the preset threshold is 0.6 OD to 1.0 OD. This preset threshold range falls within the region where the linearity of the traditional transmission method is still good (generally considered to be below 0.8 OD). Using transmission method results below this threshold can fully utilize its recognized high accuracy. When the concentration exceeds this threshold and the Lambert-Beer law gradually fails, timely switching avoids significant errors caused by continuing to use the transmission method in the nonlinear region, ensuring the reliability of measurement results across the entire concentration range.

[0035] Preferably, the method for establishing the standard curve includes the following steps: preparing a series of standard bacterial culture samples with known OD values; measuring the intensity of scattered light from each standard bacterial culture sample at different angles using the device; constructing scattered light intensity distribution information based on the scattered light intensity at each angle; and establishing a relationship curve between the scattered light intensity distribution information and the known OD values. The above method for establishing the standard curve is highly operable, enabling standardized operation for establishing standard curves for different batches and different users, thereby ensuring the repeatability and consistency of the method when applied at different times and locations.

[0036] Compared with existing technologies, this invention has the following advantages: The online microbial concentration detection device in test tubes is miniature in size and simultaneously possesses a transmitted light detection channel and multiple scattered light detection channels. At low concentrations, it performs high-precision measurements based on transmitted light signals to determine conventional OD values, meeting the real-time culture detection needs of some bacterial cultures with low growth concentrations. At higher concentrations, based on multi-angle scattered light distribution information and comprehensively considering the variation patterns of scattered light intensity at various angles, it can achieve high-precision high-concentration OD value measurement even when the bacterial culture concentration is too high, and greatly improves the detection upper limit of OD values. This invention overcomes the shortcomings of traditional transmitted light methods, which cannot accurately measure in high-concentration areas due to insufficient light intensity and severe nonlinearity, and the large signal fluctuations and insufficient accuracy of single-angle scattered light methods. The device of this invention has a compact structure and can be directly placed in conventional microbial culture shakers and other equipment to provide an external growth environment for microbial growth. Furthermore, this detection device can be mass-produced to achieve wide-range, high-precision real-time, online, non-contact, multi-channel detection across the entire concentration range from low to high, avoiding the contamination risks and operational complexities associated with sampling. Attached Figure Description

[0037] Figure 1 A schematic diagram of transmitted and scattered light on the cross-section of a circular test tube containing bacterial solution when a collimated light beam passes through it.

[0038] Figure 2 A schematic diagram showing the changes in the intensity distribution of transmitted and scattered light on the cross-section of a circular test tube containing bacterial solutions of different concentrations when a collimated light beam passes through it.

[0039] Figure 3 This is a schematic diagram of the longitudinal section of the detection device in Example 1;

[0040] Figure 4 This is a schematic diagram showing the distribution of the light source module, the transmitted light sensor, and multiple scattered light sensors on the cross-section of the detection device in Example 1.

[0041] Figure 5 This is a schematic diagram showing the distribution of the light source module, the transmitted light sensor, and multiple scattered light sensors on the cross-section of the detection device in Example 2;

[0042] Figures 3-5 The specific reference numerals in the attached figures are as follows:

[0043] 1-Container base, 11-Mounting through hole, 12-Rubber ring, 13-Pressure ring, 14-Radial through hole, 15-Threaded hole, 21-Laser diode, 22-Collimating lens, 3-Transmitted light sensor, 4-Scattered light sensor, 5-Test tube, 51-Cultivation medium, 6-Connection wire. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0045] Example 1: An online detection device for microbial concentration in test tubes, such as... Figure 3 and Figure 4 As shown, the device includes a container base 1, a light source module, a transmitted light sensor 3, five scattered light sensors 4, and a control and processing unit (not shown in the figure). This online detection device is fixed in a conventional microbial culture shaker (not shown in the figure). This online detection device is compact and miniaturized, and multiple units can be replicated as needed to form a multi-channel parallel detection system on the shaker.

[0046] In Example 1, the container base 1 adopts a regular dodecahedral prism structure with a total height of 100 mm. A mounting through-hole 11 with an inner diameter of 19 mm is opened at its geometric center for placing a standard circular test tube 5 with an outer diameter of 18 mm and a height of 150 mm. Three rubber rings 12 are installed vertically at intervals within the mounting through-hole 11 for frictional contact with the outer wall of the standard circular test tube 5, coaxially positioning the test tube 5 within the mounting through-hole 11. The lowermost rubber ring 12 is pressed tightly by a pressure ring 13. Several radial through-holes 14 are opened at the same height on different sides of the container base 1, each radial through-hole 14 being 30 mm above the bottom of the container base 1. A threaded hole 15 is pre-drilled at the bottom of the container base 1, allowing it to be securely fixed to a suitable position on a microbial culture shaker using screws.

[0047] In Example 1, the light source module is installed in a radial through-hole 14 on one side of the container base 1. The light source module consists of a single-wavelength laser diode 21 with an emission wavelength of 660 nm and a collimating lens 22. The light emitted by the laser diode 21 passes through the collimating lens 22 to form a collimated beam whose optical axis passes through the center of the mounting through-hole 11 (i.e., the center of the test tube 5).

[0048] In Example 1, the transmitted light sensor 3 is installed in another radial through-hole 14 on the container base 1, opposite to the light source module. The receiving surface of the transmitted light sensor 3 is aligned with the center of the mounting through-hole 11 to receive the transmitted light signal after the collimated beam penetrates the culture medium 51 in the test tube 5.

[0049] In Example 1, five scattered light sensors 4 are installed in five additional radial through-holes 14 within a continuous circumferential range of the surface containing the light source module and the transmitted light sensor 3. Each scattered light sensor 4 is installed in one radial through-hole 14. These five scattered light sensors 4 are arranged around the mounting through-hole 11, and the normal of the receiving surface of each scattered light sensor 4 points precisely to the center of the mounting through-hole 11. Figure 4 As shown, with the line 6 connecting the light source module and the transmitted light sensor 3 as the boundary, these 5 scattered light sensors 4 are distributed on one side of the line 6, and the included angle between any two adjacent scattered light sensors 4 is 30 degrees.

[0050] In Example 1, the control processing unit is placed outside the microbial culture shaker. The laser diode 21, the transmitted light sensor 3, and the five scattered light sensors 4 are all electrically connected to the control processing unit via cables. This control processing unit (typically a circuit board containing a microprocessor and accompanying software) is configured to perform the following core functions: control the light source to emit light and synchronously or sequentially acquire signals from the transmitted light sensor 3 and each of the scattered light sensors 4; calculate the first OD value based on the transmitted light signal according to the Lambert-Beer law; acquire scattered light intensity distribution information (such as the intensity vector or normalized distribution of signals at various angles) based on multiple scattered light signals, and determine the second OD value according to a pre-stored standard curve between the scattered light intensity distribution and the OD value.

[0051] Example 2: An online detection device for microbial concentration in test tubes, differing from Example 1 in that, in Example 2, five scattered light sensors 4 are distributed on both sides of the line 6 connecting the light source module and the transmitted light sensor 3, as shown below. Figure 5 As shown, there are 3 scattered light sensors 4 on one side of line 6 and 2 scattered light sensors 4 on the other side of line 6. The angular distribution of all scattered light sensors 4 on the horizontal plane satisfies the following: With line 6 as the axis of symmetry, mirroring the angular positions of the scattered light sensors 4 located on one side of line 6 to the other side of line 6, together with the original angular positions of the scattered light sensors 4 located on the other side, forms an equivalent sensor group; in the equivalent sensor group, after sorting by angular position from smallest to largest, the angle between any two adjacent angular positions is 30 degrees. In Example 2, after mirroring the two scattered light sensors 4 on the upper side of line 6 to the lower side of line 6, their positional distribution is similar to... Figure 4 The five scattered light sensors are positioned identically. The mirror principle can be used to simplify calculations when establishing standard curves and processing real-time data.

[0052] Example 3: An online detection method for in vitro microbial concentration using the device of Example 1, comprising the following steps:

[0053] S1. Prepare two sterilized test tubes. One test tube is used as a reference tube and liquid culture medium is added. The other test tube is used as a sample tube and bacterial culture medium sample is added. Ensure that the liquid level in both the reference tube and the sample tube exceeds the height of the radial through hole 14 by 5 mm, and seal the openings of both the reference tube and the sample tube with silicone plugs or breathable membranes to prevent contamination.

[0054] S2. Place the reference tube in the mounting through hole 11 of the container seat 1, turn on the light source through the control processing unit, and record the intensity value measured by the transmission light sensor 3 at this time as the transmission light reference intensity I0. After completion, remove the reference tube.

[0055] S3. Place the sample tube into the mounting through hole 11 of the container seat 1, set the shaker equipped with the device to the culture conditions, and start the control processing unit to automatically execute the measurement cycle at the set time interval (e.g., every 5 minutes). In each measurement cycle, the control processing unit first acquires the signal of the transmitted light sensor 3 to obtain the transmitted light sample intensity I.

[0056] S4. According to the formula OD = log 10 (I0 / I) Calculate the first OD value of the bacterial culture sample;

[0057] S5. When the first OD value is lower than the preset threshold of 0.8 OD, it is determined that the bacterial concentration is low and the Lambert-Beer law applies. At this time, the control processing unit directly outputs and records the first OD value as the final OD value of the current culture medium.

[0058] When the first OD value is ≥0.8 OD, it is determined that the bacterial culture has entered the high-concentration nonlinear region. At this time, the control processing unit immediately and synchronously acquires the signals from the five scattered light sensors 4 to obtain a set of scattered light intensity values ​​at five angles. Then, the control processing unit calculates the scattered light intensity distribution information based on this set of intensity values ​​(for example, by forming a feature vector from the five intensity values, or by performing normalization processing), and queries the pre-stored standard curve database. By comparing the real-time measured distribution information with the standard curve, the corresponding second OD value is determined, and this value is output and recorded as the final OD value of the current culture medium.

[0059] The method for establishing the above standard curve includes the following steps:

[0060] 1) Use a shaker to culture the bacterial culture to the plateau phase to obtain a high-concentration stock solution;

[0061] 2) Using the dilution method and spectrophotometer measurement, the OD values ​​of the stock solution and a series of gradient dilutions were accurately calibrated to obtain a series of standard bacterial culture samples with known OD values;

[0062] 3) Using the apparatus of Example 1, the intensity of scattered light at five angles was measured for each standard bacterial culture sample;

[0063] 4) For each standard bacterial culture sample, construct the scattered light intensity distribution information (such as a normalized intensity vector) from the scattered light intensity at its five angles.

[0064] 5) Using the scattered light intensity distribution information of all standard bacterial culture samples and their corresponding known OD values, establish the relationship curve between the two through a fitting algorithm (such as multiple linear regression or neural network), and store this relationship curve as a standard curve in the control processing unit.

[0065] Through the above process, this method achieves fully automated, seamless, and high-precision real-time online OD value monitoring of microbial culture processes from low to high concentrations, without the need for manual sampling or dilution.

Claims

1. An online detection device for microbial concentration in test tubes, characterized in that, include: The container holder has mounting holes for accommodating transparent circular test tubes; A light source module, installed on one side of the container base, is used to emit a collimated beam whose optical axis passes through the center of the mounting through hole; A transmitted light sensor is installed on the container base at a position opposite to the light source module, and is used to receive the transmitted light signal after the collimated beam passes through the culture medium in the test tube. Multiple scattered light sensors are mounted on the container base and arranged around the mounting through hole to receive scattered light signals from the culture medium in the test tube at different angles, including forward scattered light, side scattered light, and back scattered light; the multiple scattered light sensors are at the same height as the light source module and the transmitted light sensor; A control processing unit is electrically connected to the transmitted light sensor and the plurality of scattered light sensors; the control processing unit is configured to: calculate a first OD value based on the transmitted light signal according to the Lambert-Beer law, and obtain scattered light intensity distribution information based on the plurality of scattered light signals, and determine a second OD value according to a pre-stored standard curve between the scattered light intensity distribution and the OD value; in: With the line connecting the light source module and the transmitted light sensor as the boundary, the plurality of scattered light sensors are distributed on one side of the line, and the included angle between any two adjacent scattered light sensors is 15 degrees to 30 degrees. Alternatively, taking the line connecting the light source module and the transmitted light sensor as the boundary, the plurality of scattered light sensors are distributed on both sides of the line. The angular position distribution of all the scattered light sensors on the horizontal plane satisfies the following: with the line as the axis of symmetry, after mirroring the angular position of the scattered light sensor located on one side of the line to the other side of the line, it together with the angular position of the original scattered light sensor located on the other side forms an equivalent sensor group; in the equivalent sensor group, after sorting by angular position from smallest to largest, the included angle between any two adjacent angular positions is 15 degrees to 30 degrees.

2. The online detection device for microbial concentration in test tubes according to claim 1, characterized in that, The container base is a polyhedral cylindrical structure, with the mounting through hole located at its center; the light source module, the transmitted light sensor, and multiple scattered light sensors are respectively installed in radial through holes opened on different sides of the container base.

3. The online detection device for microbial concentration in test tubes according to claim 1, characterized in that, The light source module includes a laser diode and a collimating lens.

4. The online detection device for microbial concentration in test tubes according to claim 1, characterized in that, The mounting through hole is equipped with a positioning mechanism, which is used to coaxially position the test tube within the mounting through hole.

5. A method for online detection of microbial concentration in test tubes using the apparatus according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Provide a reference tube containing liquid culture medium and a sample tube containing a bacterial culture medium sample; S2. Place the reference tube in the mounting through hole of the container seat to obtain the transmitted light reference intensity I0; S3. Place the sample tube into the mounting through hole of the container seat and obtain the transmitted light sample intensity I; S4. According to the formula OD = log 10 (I0 / I) Calculate the first OD value of the bacterial culture sample; S5. When the first OD value is lower than a preset threshold, the first OD value is used as the final OD output; When the first OD value reaches or exceeds the preset threshold, the scattered light intensity signal collected by the plurality of scattered light sensors is acquired, the scattered light intensity distribution information is calculated based on the scattered light intensity signal, and the pre-stored standard curve is queried to determine the corresponding second OD value as the final OD output.

6. The method for online detection of microbial concentration in test tubes according to claim 5, characterized in that, The preset threshold is 0.6 OD to 1.0 OD.

7. The method for online detection of microbial concentration in test tubes according to claim 5, characterized in that, The method for establishing the standard curve includes the following steps: preparing a series of standard bacterial culture samples with known OD values; measuring the intensity of scattered light at different angles for each standard bacterial culture sample using the device; constructing scattered light intensity distribution information based on the scattered light intensity at each angle; and establishing a relationship curve between the scattered light intensity distribution information and the known OD values.

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