Device capable of rapidly diluting concentration of luminous bacteria in gradient manner

Abstract

The invention discloses a device for gradient dilution of luminous bacteria concentration, which comprises a dilution container device 1 and a power stirring device 7, the dilution container device 1 comprises a circular bottom plate 4 and a plurality of cylindrical partition plates 2 with different radiuses, the circular bottom plate 4 is provided with a plurality of annular tracks 6, the partition plates 2 and the corresponding annular tracks 6 are mutually clamped, and the power stirring device 7 is connected with the power stirring device 7. A closed container is formed, a stirring rod 5 is further arranged on the circular bottom plate 4, and the power stirring device 7 provides power for the stirring rod 3. According to the device, personal errors can be reduced; rapid gradient dilution is realized, and the experiment efficiency is improved; the dilution ratio is accurately controlled, and the accuracy of experimental results is ensured; operation is simple and adaptability is high; the cost is reasonable.

Description

Technical Field

[0001] This invention belongs to the field of bacterial culture technology, and in particular relates to a device for rapidly gradient dilution of luminescent bacteria concentration. Background Technology

[0002] Bioluminescent bacteria are a class of bacteria that can produce luciferase under normal physiological conditions and use it to emit visible fluorescence. Currently, known bioluminescent bacteria mainly involve the genera *Vibrio*, *Shewanella*, *Photobacterium*, and *Photorhabdus*, among which *Photobacterium luminiferum* (bright bioluminescent bacteria) is a prominent example. PRPhosphoreum Vibrio fischeri ( V.fischeri ) and Vibrio qinghaiensis ( V. ginghaiensis These are widely used in environmental toxicity testing (PAULV D) and other methods. et. al. (King JM, 2006). These luminescent bacteria release visible fluorescence with wavelengths of 450-490 nm through a redox reaction catalyzed by bacterial luciferase in the presence of oxygen, involving the oxidation-reduction reaction of long-chain fatty aldehydes (RCHO) and reduced flavin mononucleotides (FMNH2). The luminescent properties of luminescent bacteria have always been a topic of considerable interest, and researchers from various countries have conducted numerous studies on this subject, such as the luminescence mechanism of luminescent bacteria, the Lux luminescent gene system, and its expression product luciferase (King JM, 2006). et. al., A 1990 study showed that any factor that interferes with or damages the normal physiological metabolic processes of bacteria will have varying degrees of impact on the luminescence intensity of luminescent bacteria (Fernandezpinas F). et. al. , 2014).

[0003] Therefore, utilizing luminescent bacteria as indicator organisms to monitor the pollution risk of toxic and harmful substances in the environment has become one of the main applications of luminescent bacteria. The relationship between luminescent bacteria and acutely toxic substances in the environment, as well as research on using marine luminescent bacteria as biosensors, are currently hot topics. As a toxicity monitoring method, luminescent bacteria toxicity monitoring, due to its rapid, accurate, and easily quantifiable characteristics, has been widely used in drinking water and lake water quality comprehensive evaluation, and early warning of major water pollution incidents. However, its application in wastewater treatment plant influent water quality monitoring and early warning is relatively limited. Applying luminescent bacteria toxicity monitoring to water quality early warning is of great significance for wastewater treatment plant influent water quality early warning.

[0004] Current techniques for processing and analyzing luminescent bacteria, such as the traditional manual dilution method, involve cross-dilution in acute cytotoxicity tests, using 2% NaCl solution to obtain test solutions with varying concentration gradients for each concentrated water sample (e.g., ...). Figure 1The volume fractions of the diluted sample test solutions were 4.17%, 6.25%, 8.33%, 16.7%, 25.0%, 33.3%, 50.0%, 66.7%, and 100% (C1-C10) of the concentrated water sample (Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 3: Method using freeze-dried bacteria: ISO 11348—3 [S]. Switzerland: ISO, 2007.). This method is simple to operate, low in cost, and highly accurate, and is used by most laboratories when diluting luminescent bacteria. However, this method is time-consuming and prone to cross-contamination.

[0005] In addition, there are now automated diluents specifically designed for diluting microbial and cell suspensions. These instruments can accurately execute dilution steps according to preset programs, reducing human error and improving efficiency. For luminescent bacteria, these devices are particularly helpful for rapidly preparing large numbers of samples for analysis. In recent years, microfluidics technology has been increasingly widely used in biological research. Through microchannel networks, fluid flow and mixing processes can be precisely controlled at a very small scale, thereby achieving high-precision dilution of luminescent bacteria. This technology has advantages such as high efficiency and reagent savings, and is of great value in high-throughput screening and precision experiments. However, the instruments used in the above two methods are expensive and have high maintenance costs, making them unsuitable for all environments. Summary of the Invention

[0006] Given the limitations of existing methods, it is particularly important to develop a device for rapidly gradient dilution of luminescent bacteria concentrations. Such a device should possess the following characteristics: reduced human error; rapid gradient dilution to improve experimental efficiency; precise control of dilution ratios to ensure the accuracy of experimental results; ease of operation and strong adaptability; and reasonable cost.

[0007] Based on the above background, the present invention aims to provide a device for gradient dilution of luminescent bacteria concentration, which can effectively overcome the limitations of existing methods and improve the accuracy and efficiency of luminescent bacteria concentration determination.

[0008] The present invention adopts the following technical solution: A device for gradient dilution of luminescent bacteria concentration includes a dilution container device (1) and a power stirring device (7). The dilution container device (1) includes a circular base plate (4) and several cylindrical partitions (2) with different radii. Several annular tracks (6) are provided on the circular base plate (4). The partitions (2) and the corresponding annular tracks (6) are interlocked to form a sealed container. A stirring rod (5) is also provided on the circular base plate (4). The power stirring device (7) provides power to the stirring rod (5).

[0009] In a preferred embodiment of the present invention, four annular tracks (6) are provided on the circular base plate (4), and four cylindrical partitions (2) are correspondingly provided. By changing the circumference of the tracks (6), i.e., the bottom radius of the cylindrical partitions (2), the volume of the container is changed, thereby achieving dilution. In a preferred embodiment, the ratio of the radii of the annular tracks (6) is 1.12:1.60:2.26:3.19.

[0010] In a preferred embodiment of the present invention, the cylindrical partition (2) is provided with scale lines, which can adjust the sample addition height according to experimental needs, for example, it can be 5cm.

[0011] In a preferred embodiment of the present invention, the power stirring device (7) is provided with a timing knob (8) and a speed adjustment knob (9) to adjust the speed of the stirring rod while controlling the switch. The stirring rod (5) is a pointer stirring rod.

[0012] In a preferred embodiment of the present invention, the power stirring device (7) is provided with a buckle for connecting with the stirring rod (5).

[0013] In a preferred embodiment of the present invention, the cylindrical partition (2) is made of polypropylene (PP).

[0014] In a preferred embodiment of the present invention, the track (6) is fitted with an O-ring made of fluororubber (FKM) for sealing with the cylindrical partition (2).

[0015] In a preferred embodiment of the present invention, the cylindrical partition (2) is provided with a handle (3) on the upper side, which can facilitate the loading and unloading of the partition.

[0016] In a preferred embodiment of the present invention, the power stirring device (7) is made of aluminum alloy and has a cylindrical appearance.

[0017] The main reasons for using rapid serial dilutions of luminescent bacteria include the following: 1. Accurate determination of bacterial concentration: In microbiological research, accurate determination of bacterial concentration is crucial for evaluating experimental results and comparing bacterial growth dynamics under different treatment conditions. Rapid serial dilutions ensure that a series of dilutions are obtained in a short time, allowing for accurate counting within a low concentration range.

[0018] 2. Avoid over-dilution: In traditional dilution methods, excessively high dilution factors can lead to bacterial counts falling below the detection limit, making accurate counting impossible. Rapid gradient dilution avoids over-dilution, ensuring that bacterial counts in all samples remain within a countable range.

[0019] 3. Improve experimental efficiency: Time is a vital resource in scientific research and production. Rapid gradient dilution can significantly shorten experimental preparation time and improve experimental efficiency, especially when a large number of samples need to be counted.

[0020] 4. Preventing contamination: Contamination is a common problem during bacterial culture. Rapid gradient dilution can be completed in a short time, reducing the time samples are exposed to air and to the operator's hands, thereby reducing the risk of contamination.

[0021] 5. Adaptable to diverse experimental needs: In fields such as microbiology research, environmental monitoring, and biotechnology, experiments may require bacterial samples of varying concentrations. Rapid gradient dilution can quickly generate a series of samples with different concentrations to meet the needs of different experiments.

[0022] 6. Studying bacterial growth kinetics: When studying the growth curves, growth rates and other kinetic parameters of bacteria, rapid gradient dilution can help researchers quickly obtain a series of bacterial samples at different times to facilitate the analysis of bacterial growth patterns.

[0023] On the other hand, the present invention provides a method for gradient dilution of luminescent bacteria concentration, comprising inserting each partition into a corresponding track to form a multi-layer cylindrical container, adding the original luminescent bacteria solution to the innermost cylinder, and then adding dilution solvent to each partition to the scale line, and removing the partitions sequentially from the inside to the outside by means of the handle, thereby diluting the luminescent bacteria solution by 2 times sequentially.

[0024] The device of the present invention can dilute the concentration of luminescent bacteria solution by 4 gradients at a time, that is, by 16 times.

[0025] In a preferred embodiment of the present invention, the diluting solvent is selected from NaCl solution, for example, 2% NaCl solution.

[0026] Technical effect

[0027] This invention features graduated lines on a cylindrical partition, allowing the diluent to be added only until the lines are filled. By adjusting the volume of the cylinder, the volume of solution added is determined, making dilution more efficient. No additional solution volume calculations are needed during dilution, making it more convenient to add larger volumes of solution and avoiding forgetting the dosage when using a pipette.

[0028] This invention applies shear force and eddy current effects to the surrounding liquid as the stirring rod rotates rapidly within it, causing the liquid to flow and ultimately achieve uniform mixing. Apart from the pointer stirring rod, the entire system has no moving parts that directly contact the liquid, reducing the potential risk of sample contamination. Due to the absence of complex mechanical structures, the containers used are easy to clean and sterilize. Users can adjust the stirring speed and time as needed to adapt to different experimental requirements.

[0029] This invention features a handle on a cylindrical partition, such as a tilted metal handle inserted in the middle for easy gripping. The tilted handle utilizes leverage; by applying force to pry out one end, the entire partition can be easily removed. Directly handling the partition with hands is risky due to the low friction between gloves and the smooth surface, making it difficult to apply force and potentially contaminating the liquid inside the container. Using the external handle to press down and pry out one end of the partition facilitates its removal and ensures a sterile environment within the partition.

[0030] The device for gradient dilution of luminescent bacteria concentration in this invention utilizes high-temperature resistant materials, facilitating sterilization and allowing for multiple cycles of use. The central stirring rod can be perfectly secured with the clips in the power stirring device. Stirring can be started by turning on the speed adjustment knob, and the stirring speed can also be adjusted. The stirring time can be adjusted for different liquid dilution volumes by adjusting the timer knob.

[0031] The luminescent bacteria gradient dilution device of this invention has good sealing performance to avoid external contamination and ensure the stability of dilution conditions. It also has the following characteristics: it can reduce human error; it can achieve rapid gradient dilution and improve experimental efficiency; it can accurately control the dilution ratio and ensure the accuracy of experimental results; it is easy to operate and highly adaptable; and it is reasonably priced. Attached Figure Description

[0032] Figure 1 A schematic diagram of a traditional manual dilution method is shown; Figure 2 This is a schematic diagram of a device for gradient dilution of luminescent bacteria concentration according to a preferred embodiment of the present invention.

[0033] In the attached diagram, 1: dilution container device, 2: partition, 3: handle, 4: base plate, 5: stirring rod, 6: track, 7: power stirring device, 8: timer knob, 9: speed adjustment knob, 10: buckle. Detailed Implementation

[0034] Example 1

[0035] See Figure 1 This embodiment provides a device for gradient dilution of luminescent bacteria concentration, including a dilution container device (1) and a power stirring device (7).

[0036] Dilution container device (1): The partition (2) of this device is made of polypropylene (PP) and has four partitions of different diameters. The partition radius, scale height and sample volume are shown in Table 1. Each partition has a handle (3) for easy handling to avoid contaminating the inside of the container. The circular base plate (4) has four circular tracks (6), and each track (6) has an O-ring for fixing and sealing. The bottom plate (4) also has a stirring rod in the middle to help the liquid mix thoroughly and the dilution gradient be accurate.

[0037] Table 1

[0038] Power stirring device (7): The stirring rod (5) in the middle of the circular base plate (4) can be perfectly fixed with the buckle (10) in the power stirring device (7). The stirring can be started after the speed adjustment knob (9) is turned on, and the stirring speed can also be adjusted. The stirring time can be adjusted according to different liquid dilution volumes by adjusting the timer knob (8).

[0039] In this embodiment, there are four partitions. In other embodiments, there may be other numbers of partitions, and the track of the base plate may be changed accordingly.

[0040] Before use, insert the partitions of each size into the track, add 5 mL of luminescent bacteria solution into the innermost cylinder, and then add the corresponding dilution solution to each partition until the 5 cm mark. Squeeze the handles to remove the partitions in sequence to achieve a gradient dilution of 2 times. This invention can dilute the luminescent bacteria solution concentration by 4 gradients at once, that is, a dilution of 16 times.

[0041] Each partition is a cylindrical tubular structure. In this embodiment, the partition and the bottom plate are made of transparent polypropylene sheet. This material is easy to disassemble, lightweight, and inexpensive. If there are special requirements, it can be immediately replaced with other materials.

[0042] The base plate contains O-rings of the appropriate size in its track. The partition plate can be inserted into the track to completely seal it and prevent the liquid from flowing between each other.

[0043] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A device for gradient dilution of luminescent bacteria concentration, comprising a dilution container device 1 and a power stirring device 7, wherein the dilution container device 1 comprises a circular base plate 4 and a plurality of cylindrical partitions 2 with different radii, the circular base plate 4 is provided with a plurality of annular tracks 6, the partitions 2 and the corresponding annular tracks 6 are interlocked to form a sealed container, the circular base plate 4 is also provided with a stirring rod 5, and the power stirring device 7 provides power to the stirring rod 5.

2. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The circular base plate 4 is provided with four annular tracks 6, and four cylindrical partitions 2 are provided accordingly.

3. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 2, wherein, The ratio of the radii of the circular track 6 is 1.12:1.60:2.26:3.

19.

4. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The power stirring device 7 is equipped with a timer knob 8 and a speed adjustment knob 9, which can adjust the speed of the stirring rod while controlling the switch.

5. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The power stirring device 7 is equipped with a buckle for connecting to the stirring rod 5.

6. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The cylindrical partition 2 is made of polypropylene.

7. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The track 6 is equipped with an O-ring made of fluororubber.

8. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The cylindrical partition 2 is provided with a handle 3.

9. The apparatus for gradient dilution of luminescent bacteria concentration according to claim 1, wherein, The power stirring device 7 is made of aluminum alloy and has a cylindrical appearance.

10. A method for gradient dilution of luminescent bacteria concentration, comprising inserting each partition into a corresponding track to form a multi-layer cylindrical container, adding the original luminescent bacteria solution to the innermost cylinder, adding dilution solvent to each partition to the mark, and removing the partitions sequentially from the inside to the outside via the handle, thereby diluting the luminescent bacteria solution by 2 times sequentially.

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