CN117925390A - Microorganism gradient concentration sample preparation system and method - Google Patents
Microorganism gradient concentration sample preparation system and method Download PDFInfo
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- CN117925390A CN117925390A CN202410161030.4A CN202410161030A CN117925390A CN 117925390 A CN117925390 A CN 117925390A CN 202410161030 A CN202410161030 A CN 202410161030A CN 117925390 A CN117925390 A CN 117925390A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 244000005700 microbiome Species 0.000 title claims description 43
- 238000000034 method Methods 0.000 title description 13
- 239000007788 liquid Substances 0.000 claims abstract description 73
- 239000000243 solution Substances 0.000 claims abstract description 58
- 235000015097 nutrients Nutrition 0.000 claims abstract description 54
- 239000012452 mother liquor Substances 0.000 claims abstract description 35
- 238000010790 dilution Methods 0.000 claims abstract description 9
- 239000012895 dilution Substances 0.000 claims abstract description 9
- 230000000813 microbial effect Effects 0.000 claims abstract description 8
- 238000005464 sample preparation method Methods 0.000 claims abstract description 4
- 238000007789 sealing Methods 0.000 claims description 31
- 230000001580 bacterial effect Effects 0.000 claims description 29
- 238000003756 stirring Methods 0.000 claims description 29
- 238000005273 aeration Methods 0.000 claims description 25
- 238000005070 sampling Methods 0.000 claims description 9
- 230000005526 G1 to G0 transition Effects 0.000 claims description 8
- 230000005484 gravity Effects 0.000 claims description 8
- 230000012010 growth Effects 0.000 claims description 8
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000002906 microbiologic effect Effects 0.000 claims 3
- 210000001503 joint Anatomy 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000005189 flocculation Methods 0.000 abstract description 5
- 230000016615 flocculation Effects 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 238000012546 transfer Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 52
- 238000012360 testing method Methods 0.000 description 8
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- 241000894006 Bacteria Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 241000195597 Chlamydomonas reinhardtii Species 0.000 description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 2
- 241000192710 Microcystis aeruginosa Species 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
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- 238000012258 culturing Methods 0.000 description 1
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Abstract
The invention relates to the technical field of microbial sample preparation, and particularly discloses a microbial concentration gradient sample preparation system and a microbial concentration gradient sample preparation method, wherein the microbial concentration gradient sample preparation system comprises a plurality of mother liquor tanks, the bottoms of the mother liquor tanks are respectively provided with a mother liquor outlet, and valves are arranged on the mother liquor outlets; each mother liquor outlet is connected in parallel through a liquid outlet pipe, and a first sample pool is arranged below the liquid outlet position of the liquid outlet pipe; the invention can form samples with different gradient concentrations rapidly, improves the working efficiency, and is beneficial to rapid and accurate research of experiments; the invention can realize the addition and transfer of the nutrient solution through simple operation, thereby realizing the dilution of the multiple rate and greatly improving the working efficiency and the sample preparation efficiency; the invention improves the concentration accuracy of the sample and reduces the occurrence of flocculation.
Description
Technical Field
The invention relates to the technical field of microbial sample preparation, in particular to a microbial gradient concentration sample preparation system and a method.
Background
The following methods are often used for measuring the microorganism concentration: dry weight method, blood cell plate count method, PCT plate count method, biosensor assay, metabolite method, optical assay, etc. There are significant advantages to optical assays over the various methods described above. The method characterizes the microorganism concentration in terms of turbidity, can achieve high-accuracy measurement (R 2 > 0.9) within 1min, and automatically measures in a non-contact and non-destructive manner. In general, microorganisms can be regarded as particles having a diameter of about 1. Mu.m, which, upon irradiation with light, reflect, scatter, absorb or transmit. According to lambert's law, the higher the microorganism concentration, the more light is absorbed, reflected and scattered by the solution, and the more significant the attenuation of the light intensity. Therefore, the concentration change of the microorganisms can be indirectly measured by monitoring the transmitted light intensity in real time.
However, when the OD value is measured by an optical measurement method, on one hand, the microorganism culture is separated from the OD measurement, the sampling damages the original environment, and the sample is consumed while the risk of bacterial contamination is increased, and on the other hand, the whole growth cycle time of the microorganism is long, and the sampling workload is large. Taking E.coli as an example, the sample is taken and measured once OD after 10h into the plateau at half an hour intervals.
When a series of tests of microorganism control are carried out on microalgae, the growth period of the microalgae is longer, 5-20 d is generally needed, meanwhile, the growth state of the microorganism is continuously changed, the microorganism is required to be ensured to be in the logarithmic growth phase during the tests, samples with different concentration gradients are generally needed to be arranged in the same series of tests, a great amount of time and energy are consumed if the samples are missed, and the operation is easy to make mistakes; in preparing samples of different concentration gradients, how to ensure the accuracy of the different concentrations is also a matter of concern, as well as flocculation if stirring is inadequate.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a microorganism gradient concentration sample preparation system and a method, which can rapidly form samples with different gradient concentrations, improve the working efficiency and facilitate the rapid and accurate research of experiments; the invention can realize the addition and transfer of the nutrient solution through simple operation, thereby realizing the dilution of the multiple rate and greatly improving the working efficiency and the sample preparation efficiency; the invention improves the concentration accuracy of the sample and reduces the occurrence of flocculation.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The microorganism concentration gradient sample preparation system comprises a plurality of mother liquor tanks, wherein the bottoms of the mother liquor tanks are respectively provided with a mother liquor outlet, and valves are arranged on the mother liquor outlets; each mother liquor outlet is connected in parallel through a liquid outlet pipe, and a first sample pool is arranged below the liquid outlet position of the liquid outlet pipe;
A plurality of second sample cells are sequentially arranged below the first sample cell, and the first sample cell and the plurality of second sample cells have the same capacity and are arranged in series in a stepped shape; a first sample outlet is formed above one side of the first sample pool, and second sample outlets are formed above one side of the plurality of second sample pools; the samples at the first sample outlet and the second sample outlet flow into corresponding sample cells below, and a comparison sample cell is arranged below the second sample outlet of the second sample cell at the lowest end;
The system also comprises a nutrient solution pond, wherein the bottom of the nutrient solution pond is provided with a nutrient solution outlet, and a valve is arranged on the nutrient solution outlet; the nutrient solution outlet is connected with the second sample cell through a shunt structure; and an aeration stirring device is arranged in the second sample tank.
Further, a shunt structure is arranged above each second sample cell, the shunt structure comprises a first shunt tube, a second shunt tube and a third shunt tube which are mutually communicated, the second shunt tube is connected with the top of one side of the second sample cell below, and a plurality of groups of third shunt tubes of the shunt structure are connected with the first shunt tubes end to end; the first shunt tube positioned at the uppermost shunt structure is communicated with the nutrient solution outlet, and the third shunt tube outlet positioned at the lowermost shunt structure is positioned above the comparison sample cell;
the liquid inlet of the second shunt tube is arranged right below the liquid outlet of the first shunt tube of each component of the shunt structure, and the liquid inlet of the third shunt tube is communicated with the side of the first shunt tube, so that nutrient solution of the first shunt tube flows into the second shunt tube through gravity.
Furthermore, a stepping motor is arranged on the side wall of the second sample tank, the stepping motor is positioned between the second shunt pipe and the second sample outlet, the output end of the stepping motor stretches into the second sample tank, the output end of the stepping motor is fixedly connected with a sealing plate, and the stepping motor drives the sealing plate to rotate so that the sealing plate can butt-seal the second sample outlet or the second shunt pipe.
Further, the distance between the first sample outlet and the bottom of the first sample tank is nine tenths of the overall depth of the first sample tank, and the distance between the second sample outlet and the bottom of the second sample tank is nine tenths of the overall depth of the second sample tank.
Further, a valve is arranged on the first sample outlet, and a valve is arranged on the second shunt pipe positioned at the lowest shunt structure.
Further, the aeration stirring device comprises a spiral stirring head positioned in the second sample tank, the spiral stirring head comprises a straight line pipe and a spiral pipe which are communicated, the straight line pipe is rotationally connected with the side wall of the second sample tank through a rotary sealing bearing, a bracket is fixedly connected with the outside of the side wall of the second sample tank through a bolt, and a rotary motor and an air pump are arranged on the bracket; a first gear is fixedly connected to a straight line pipe positioned outside the second sample tank, a second gear is fixedly connected to an output shaft of the rotating motor, and the first gear is meshed with the second gear; the output end of the air pump is in rotary sealing connection with the end part of the straight line pipe.
Furthermore, one end of the spiral tube is closed, a continuous disturbance blade is fixedly connected to the tube wall on the outer side of the spiral tube, a plurality of aeration heads are uniformly arranged on the tube wall on the inner side of the spiral tube, and the adjacent aeration heads are oppositely arranged.
Further, the number of the second sample cells is 4.
Further, an OD value monitor is arranged in the mother liquid pool.
A sample preparation method utilizing the microorganism concentration gradient sample preparation system comprises the following steps:
S1, determining the number of mother liquor pools according to the growth cycle of target microorganisms, and inoculating the target microorganisms in the mother liquor pools in batches; monitoring the OD value of the bacterial liquid by an OD value monitor, when the OD value of the bacterial liquid reaches the intersection point of the logarithmic phase and the stationary phase, opening a valve corresponding to a mother liquid outlet to enable the bacterial liquid to enter a first sample pool, when the OD value of the bacterial liquid is lower than a target range, continuing to culture, and when the OD value of the bacterial liquid is higher than the target range, adding a culture medium to dilute to a lower concentration to continue to culture;
S2, when the bacterial liquid in the first sample tank is full, opening a valve of a first sample outlet to enable one tenth of the bacterial liquid in the first sample tank to flow into the second sample tank below, opening a valve of a nutrient solution outlet to enable the nutrient solution to flow into the second shunt pipe from the first shunt pipe through gravity and further enter the second sample tank below to be mixed with the bacterial liquid, opening a rotating motor and an air pump, simultaneously performing nutrient solution adding and stirring aeration operation, and completing multiplying power dilution when the nutrient solution is filled in the second sample tank;
s3, after the second sample tank is filled with the nutrient solution, starting a stepping motor to enable a sealing plate to rotate 180 degrees to seal the second shunt pipe, enabling the nutrient solution of the first shunt pipe to change the flow direction and flow into a third shunt pipe, enabling the nutrient solution to enter the sample tank below through a next shunt structure, enabling a second sample outlet of the second sample tank to be opened, enabling one tenth of bacteria in the second sample tank to flow into the sample tank below, and then performing the next sample preparation cycle;
s4, after the sample preparation is carried out on the plurality of second sample tanks through the cyclic operation, the valve on the second shunt pipe of the lowest shunt structure is closed, so that the nutrient solution finally flows into the comparison sample tank to be used as a comparison sample, and the corresponding concentration in the comparison sample tank is 0 at the moment, and finally the sample preparation operation is finished.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, through the cooperation monitoring of the mother liquid pool and the OD value monitor which are arranged in parallel, the bacterial liquid with the OD value at the intersection point of the logarithmic phase and the stationary phase can be efficiently and quickly obtained, the continuous sample adding of the first sample pool is realized, the test period is effectively reduced, the test efficiency is accelerated, and the microorganism physiological uniformity is high; meanwhile, through the first sample outlet and the plurality of second sample tanks which are arranged in series, samples can be quickly circulated, so that samples with different gradient concentrations can be quickly formed, the working efficiency is improved, and the rapid and accurate research of experiments is facilitated.
(2) According to the invention, through the communication design of the multi-component flow distribution structures, the liquid inlet of the second flow distribution pipe is arranged right below the liquid outlet of the first flow distribution pipe of each component flow distribution structure, and the liquid inlet of the third flow distribution pipe is communicated with the side of the first flow distribution pipe, so that the nutrient solution of the first flow distribution pipe flows into the second flow distribution pipe through gravity at first, then flows into different sample tanks in an accurate and stepwise manner by matching with the stepping motor and the sealing plate, meanwhile, the circulation of samples can be synchronously promoted, and the addition and the transfer of the nutrient solution can be realized through simple operation, thereby realizing the dilution of the doubling rate, and greatly improving the working efficiency and the sample preparation efficiency.
(3) According to the invention, the spiral stirring head is driven to rotate by the rotating motor, and the structural design of the spiral pipe and the disturbance blade can fully stir the sample, so that the sample is uniformly mixed, the generation of spray can be reduced, the splash loss of the sample caused by large liquid level fluctuation is prevented, and the concentration accuracy of the sample is improved; meanwhile, the aeration heads are oppositely arranged on the pipe wall at the inner side of the spiral pipe, so that the concentration precision is influenced by the loss caused by great fluctuation of the liquid sample is further reduced through convection impact while the aeration is realized, and the oppositely arranged aeration heads can increase the bottom liquid circulation of the second sample tank, so that the flocculation is reduced under the convection impact.
Drawings
FIG. 1 is a schematic diagram showing the overall structure of a microorganism concentration gradient sample preparation system according to the present invention;
FIG. 2 is a schematic diagram of a second sample cell and an aeration stirring device of the microorganism concentration gradient sample preparation system of the invention;
FIG. 3 is a schematic view of a second sample cell and an aeration stirring device of the microorganism concentration gradient sample preparation system of the invention;
FIG. 4 is a schematic diagram of the structure of a spiral stirring head of the microorganism concentration gradient sample preparation system of the invention;
FIG. 5 is an OD-time curve of Chlamydomonas reinhardtii in an example of a microorganism concentration gradient sampling system according to the present invention;
FIG. 6 is an OD-time curve of Chlorella in an embodiment of a microorganism concentration gradient sample system of the present invention;
FIG. 7 is an OD-time curve of Microcystis aeruginosa in an embodiment of a microorganism concentration gradient sampling system of the present invention;
FIG. 8 is an OD-time curve of a Tetrastigmata obliqua in an example of a microorganism concentration gradient sampling system according to the present invention.
The reference numerals are as follows:
A mother liquor tank 100; an OD value monitor 110; a mother liquor outlet 120; a liquid outlet pipe 130; a first sample cell 200; a first sample outlet 210; a second sample cell 300; a second sample outlet 310; a stepper motor 320; a sealing plate 330; a rotary seal bearing 340; aeration stirring device 400; helical stirring head 410; a straight line pipe 411; a spiral tube 412; a perturbing vane 413; an aeration head 414; a first gear 420; a second gear 430; a rotation motor 440; an air pump 450; a bracket 460; a nutrient solution bath 500; a nutrient solution outlet 510; a first shunt 610; a second shunt tube 620; a third shunt 630; the sample cell 700 is compared.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. Of course, the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Although the steps of the present invention are arranged by reference numerals, the order of the steps is not limited, and the relative order of the steps may be adjusted unless the order of the steps is explicitly stated or the execution of a step requires other steps as a basis. It is to be understood that the term "and/or" as used herein relates to and encompasses any and all possible combinations of one or more of the associated listed items.
Examples
1-8, A microorganism concentration gradient sample preparation system comprises a mother liquor pond 100, wherein the mother liquor pond 100 is used for culturing microorganisms such as Chlamydomonas reinhardtii, chlorella, microcystis aeruginosa or Alternaria obliqua, and the culture temperature of target microorganisms is 25-40 ℃; the plurality of mother liquor tanks 100 are arranged, the bottom of each mother liquor tank 100 is provided with a mother liquor outlet 120, and valves are arranged on the mother liquor outlets 120; each mother liquor outlet 120 is connected in parallel through a liquid outlet pipe 130, a first sample pool 200 is arranged below the liquid outlet position of the liquid outlet pipe 130, and bacteria liquid flowing out of the liquid outlet pipe 130 can fall into the first sample pool 200;
A plurality of second sample cells 300 are sequentially arranged below the first sample cell 200, and the first sample cell 200 and the plurality of second sample cells 300 have the same capacity and are arranged in series in a ladder shape; a first sample outlet 210 is formed above one side of the first sample cell 200, and second sample outlets 310 are formed above one side of the plurality of second sample cells 300; the samples of the first sample outlet 210 and the second sample outlet 310 flow into the corresponding sample cells below, and a comparison sample cell 700 is arranged below the second sample outlet 310 of the second sample cell 300 positioned at the lowest end;
through a pre-experiment, determining an OD-time curve of the target microorganism, as shown in fig. 5-8;
Microorganisms are grown dynamically, with growth rates varying, typically including a slow phase, a log phase, a stationary phase, and a decay phase.
Determining the required concentration of the sample cell according to the concentration gradient required by the test, wherein the concentration is 1X 10 8CFU/mL、1×107CFU/mL、1×106CFU/mL、1×105 CFU/mL and 0 CFU/mL;
further, an OD monitor 110 is provided in the mother liquor tank 100. The OD value monitor 110 can monitor whether the microorganism in the mother liquor tank 100 reaches the end of the logarithmic phase of growth, and then the composite target bacterial solution is put into the first sample tank 200 by opening the valve of the mother liquor outlet 120.
It should be noted that the OD monitor 110 is a well-known technology, and the OD may be measured by optical measurement, which is not described in detail herein.
According to the invention, through the matched monitoring of the mother liquid pool 100 and the OD value monitor 110 which are arranged in parallel, the bacterial liquid with the OD value at the intersection point of the logarithmic phase and the stationary phase can be efficiently and quickly obtained, the continuous sample adding of the first sample pool 200 is realized, the test period is effectively reduced, the test efficiency is accelerated, and the physiological uniformity of microorganisms is high; meanwhile, through the first sample outlet 210 and the plurality of second sample tanks 300 which are arranged in series, samples can be quickly circulated, so that samples with different gradient concentrations can be quickly formed, the working efficiency is improved, and the rapid and accurate research of experiments is facilitated.
The invention can quickly lead the first sample cell 200 to acquire the bacterial liquid which is enough to meet the OD value at the intersection point of the logarithmic phase and the stationary phase.
The system also comprises a nutrient solution pond 500, wherein a nutrient solution outlet 510 is arranged at the bottom of the nutrient solution pond 500, and a valve is arranged on the nutrient solution outlet 510; the nutrient solution outlet 510 is connected with the second sample cell 300 through a shunt structure; an aeration stirring device 400 is installed inside the second sample cell 300.
Further, a shunt structure is disposed above each second sample cell 300, and the shunt structure includes a first shunt tube 610, a second shunt tube 620 and a third shunt tube 630 that are mutually communicated, where the second shunt tube 620 is connected to the top of one side of the second sample cell 300 below, and multiple groups of third shunt tubes 630 of the shunt structure are connected end to end with the first shunt tube 610; the first shunt tube 610 positioned at the uppermost shunt structure is communicated with the nutrient solution outlet 510, and the outlet of the third shunt tube 630 positioned at the lowermost shunt structure is positioned above the comparison sample cell 700;
Directly below the liquid outlet of the first shunt tube 610 of each component of the flow structure is the liquid inlet of the second shunt tube 620, and the liquid inlet of the third shunt tube 630 is communicated with the side of the first shunt tube 610, so that the nutrient solution of the first shunt tube 610 flows into the second shunt tube 620 first by gravity.
Further, a stepper motor 320 is mounted on the sidewall of the second sample cell 300, the stepper motor 320 is located between the second shunt tube 620 and the second sample outlet 310, an output end of the stepper motor 320 extends into the second sample cell 300, and an output end of the stepper motor 320 is fixedly connected with a sealing plate 330, and the stepper motor 320 drives the sealing plate 330 to rotate, so that the sealing plate 330 performs butt-joint sealing on the second sample outlet 310 or the second shunt tube 620.
It should be noted that, the stepper motor 320 is a well-known technique, and may be fixed to rotate at a certain angle, which is not described in detail herein.
Further, in order to improve the tightness of the sealing plate 330, the second shunt tube 620 and the second sample outlet 310 may be provided with a rubber sealing ring or other structures or a magnetic fixing device at the corresponding interfaces, so as to achieve a sealing connection, which will not be further described herein.
Further, the distance from the first sample outlet 210 to the bottom of the first sample cell 200 is nine tenths of the overall depth of the first sample cell 200, and the distance from the second sample outlet 310 to the bottom of the second sample cell 300 is nine tenths of the overall depth of the second sample cell 300. By setting the height of the first 210 and second 310 sample outlets, the volume of liquid flowing out of the outlets in a filled condition of the sample cell is fixed.
According to the invention, through the communication design of the multi-component flow distribution structures, meanwhile, the liquid inlet of the second flow distribution pipe 620 is arranged right below the liquid outlet of the first flow distribution pipe 610 of each component flow distribution structure, and the liquid inlet of the third flow distribution pipe 630 is communicated with the side of the first flow distribution pipe 610, so that the nutrient solution of the first flow distribution pipe 610 flows into the second flow distribution pipe 620 firstly through gravity, then flows into different sample pools accurately and stepwise by matching with the stepping motor 320 and the sealing plate 330, meanwhile, the circulation of samples can be synchronously promoted, the addition and the transfer of the nutrient solution can be realized through simple operation, the dilution of the multiple rate is realized, and the working efficiency and the sample preparation efficiency of the samples are greatly improved.
Specifically, the principle of the dilution with the multiple ratio is that, for example, when the concentration of the first sample cell 200 is 1×10 8 CFU/mL, one tenth of the concentration is placed in the second sample cell 300 through the first sample outlet 210, then nine times of nutrient solution is added to the second sample cell 300 to fill the second sample cell 300 for dilution, and finally the concentration in the second sample cell 300 becomes 1×10 7 CFU/mL, so that the cycle can be continuously diluted with multiple times.
Further, a valve is installed at the first sample outlet 210, and a valve is installed at the second shunt tube 620 positioned at the lowermost shunt structure.
Further, the aeration stirring device 400 includes a spiral stirring head 410 located inside the second sample tank 300, the spiral stirring head 410 includes a straight pipe 411 and a spiral pipe 412 that are communicated, the straight pipe 411 is rotationally connected with the side wall of the second sample tank 300 through a rotary sealing bearing 340, a bracket 460 is fixedly connected with the outside of the side wall of the second sample tank 300 through a bolt, and a rotary motor 440 and an air pump 450 are installed on the bracket 460; a first gear 420 is fixedly connected to the linear tube 411 positioned outside the second sample cell 300, a second gear 430 is fixedly connected to the output shaft of the rotating motor 440, and the first gear 420 and the second gear 430 are meshed; the output end of the air pump 450 is in rotary sealing connection with the end of the straight tube 411.
When the rotation motor 440 rotates, the second gear 430 is driven to rotate, the second gear 430 rotates the first gear 420 through engagement, and the rotation of the first gear 420 drives the screw stirring head 410 to rotate.
It should be noted that the rotary sealing structure is a mature technology in the prior art, for example, a rotary sealing ring and the like, only needs to realize rotary sealing connection, and is not described in detail herein, and other positions where the rotary sealing connection is needed are the same as those of the rotary sealing connection, for example, the structure of a motor output shaft and the like.
Further, one end of the spiral tube 412 is closed, a continuous turbulence blade 413 is fixedly connected to the wall of the outer side of the spiral tube 412, a plurality of aeration heads 414 are uniformly arranged on the wall of the inner side of the spiral tube 412, and the adjacent aeration heads 414 are oppositely arranged.
The spiral stirring head 410 is driven to rotate by the rotating motor 440, and the structural design of the spiral pipe 412 and the disturbance blade 413 can fully stir the sample, so that the sample is uniformly mixed, the generation of spray can be reduced, the splash loss of the sample caused by large liquid level fluctuation is prevented, and the concentration accuracy of the sample is improved; meanwhile, the aeration is realized through the aeration heads 414 which are oppositely arranged on the pipe wall at the inner side of the spiral pipe 412, the fluctuation of the liquid sample is further reduced through convection impact so as to cause loss and influence on concentration precision, and the oppositely arranged aeration heads 414 can increase the liquid circulation at the bottom of the second sample tank 300, so that flocculation is reduced under the convection impact.
The stirring effect can be improved by matching the stirring blades 413 with the spiral pipe 412, and meanwhile, the stability of liquid in the stirring process can be ensured.
Further, the number of the second sample cells 300 is 4.
A sample preparation method utilizing the microorganism concentration gradient sample preparation system comprises the following steps:
S1, determining the number of the mother liquid tanks 100 according to the growth cycle of target microorganisms, and inoculating the target microorganisms in the mother liquid tanks 100 in batches; monitoring the OD value of the bacterial liquid by an OD value monitor 110, when the OD value of the bacterial liquid reaches the intersection point of the logarithmic phase and the stationary phase, opening a valve corresponding to a mother liquid outlet 120 to enable the bacterial liquid to enter a first sample pool 200, when the OD value of the bacterial liquid is lower than a target range, continuing to culture, and when the OD value of the bacterial liquid is higher than the target range, adding a culture medium to dilute to a lower concentration, and continuing to culture; when the intersection point of the logarithmic phase and the stationary phase is reached, the mother liquor is led out from the mother liquor pool.
S2, when the bacterial liquid in the first sample tank 200 is full, opening a valve of the first sample outlet 210 to enable one tenth of the bacterial liquid in the first sample tank 200 to flow into the second sample tank 300 below, opening a valve of the nutrient solution outlet 510 to enable the nutrient solution to flow into the second shunt pipe 620 from the first shunt pipe 610 through gravity and further enter the second sample tank 300 below to be mixed with the bacterial liquid, opening the rotating motor 440 and the air pump 450, and simultaneously performing nutrient solution adding and stirring aeration operation, and completing multiplying power dilution when the nutrient solution is filled in the second sample tank 300;
S3, after the second sample cell 300 is filled with the nutrient solution, starting the stepper motor 320, rotating the sealing plate 330 by 180 degrees to seal the second shunt tube 620, changing the flow direction of the nutrient solution of the first shunt tube 610 into the third shunt tube 630, and then entering the sample cell below through the next shunt structure, simultaneously starting the second sample outlet 310 of the second sample cell 300, enabling one tenth of the bacteria solution of the second sample cell 300 to flow into the sample cell below, and then performing the next sample preparation cycle;
And S4, after the sample preparation is performed on the plurality of second sample tanks 300 through the cyclic operation, the valve on the second shunt pipe 620 positioned at the lowest end of the shunt structure is closed, so that the nutrient solution finally flows into the comparison sample tank 700 to be used as a comparison sample, and the corresponding concentration in the comparison sample tank 700 is 0 at the moment, and finally the sample preparation operation is completed.
It should be noted that the valve structure of the present application may be a manual valve or an automatic structure such as an electric valve, which will not be further described herein.
The structure of the present application having a certain height is supported by an external support structure, which is not described in detail herein; meanwhile, the application relates to electric equipment such as a motor and the like which are powered and supported by an external power supply and controlled, and the details are not described herein.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (10)
1. The microorganism concentration gradient sample preparation system comprises a plurality of mother liquor tanks (100), and is characterized in that the mother liquor tanks (100) are provided with a plurality of mother liquor outlets (120) at the bottom of each mother liquor tank (100), and valves are arranged on the mother liquor outlets (120); each mother liquor outlet (120) is connected in parallel through a liquid outlet pipe (130), and a first sample pool (200) is arranged below a liquid outlet position of the liquid outlet pipe (130);
A plurality of second sample cells (300) are sequentially arranged below the first sample cell (200), and the first sample cell (200) and the plurality of second sample cells (300) have the same capacity and are arranged in series in a stepped shape; a first sample outlet (210) is formed above one side of the first sample cell (200), and second sample outlets (310) are formed above one side of the plurality of second sample cells (300); the samples at the first sample outlet (210) and the second sample outlet (310) flow into corresponding sample cells below, and a comparison sample cell (700) is arranged below the second sample outlet (310) of the second sample cell (300) at the lowest end;
The system also comprises a nutrient solution pond (500), wherein a nutrient solution outlet (510) is arranged at the bottom of the nutrient solution pond (500), and a valve is arranged on the nutrient solution outlet (510); the nutrient solution outlet (510) is connected with the second sample cell (300) through a shunt structure; an aeration stirring device (400) is arranged in the second sample tank (300).
2. The microbial concentration gradient sample preparation system according to claim 1, wherein a shunt structure is arranged above each second sample cell (300), the shunt structure comprises a first shunt tube (610), a second shunt tube (620) and a third shunt tube (630) which are communicated with each other, the second shunt tube (620) is connected with the top of one side of the second sample cell (300) below, and a plurality of groups of third shunt tubes (630) of the shunt structures are connected with the first shunt tube (610) end to end; the first shunt pipe (610) positioned at the uppermost shunt structure is communicated with the nutrient solution outlet (510), and the outlet of the third shunt pipe (630) positioned at the lowermost shunt structure is positioned above the comparison sample cell (700);
The liquid inlet of the second shunt pipe (620) is arranged right below the liquid outlet of the first shunt pipe (610) of each component of the shunt structure, and the liquid inlet of the third shunt pipe (630) is communicated with the side of the first shunt pipe (610), so that nutrient solution of the first shunt pipe (610) flows into the second shunt pipe (620) through gravity at first.
3. The microorganism concentration gradient sample preparation system according to claim 2, wherein a stepper motor (320) is mounted on the side wall of the second sample cell (300), the stepper motor (320) is located between the second shunt tube (620) and the second sample outlet (310), the output end of the stepper motor (320) extends into the second sample cell (300) and the output end of the stepper motor (320) is fixedly connected with a sealing plate (330), and the stepper motor (320) drives the sealing plate (330) to rotate so that the sealing plate (330) performs butt joint sealing on the second sample outlet (310) or the second shunt tube (620).
4. The microbial concentration gradient sampling system according to claim 1, wherein the first sample outlet (210) is located at a distance of nine tenths of the overall depth of the first sample cell (200) from the bottom of the first sample cell (200), and the second sample outlet (310) is located at a distance of nine tenths of the overall depth of the second sample cell (300) from the bottom of the second sample cell (300).
5. A microbiological concentration gradient sampling system according to claim 1, wherein the first sample outlet (210) is provided with a valve and the second shunt tube (620) at the lowermost shunt structure is provided with a valve.
6. The microorganism concentration gradient sample preparation system according to claim 1, wherein the aeration stirring device (400) comprises a spiral stirring head (410) positioned inside the second sample tank (300), the spiral stirring head (410) comprises a straight line pipe (411) and a spiral pipe (412) which are communicated, the straight line pipe (411) is rotationally connected with the side wall of the second sample tank (300) through a rotary sealing bearing (340), a bracket (460) is fixedly connected outside the side wall of the second sample tank (300) through a bolt, and a rotary motor (440) and a gas pump (450) are installed on the bracket (460); a first gear (420) is fixedly connected to a linear pipe (411) positioned outside the second sample tank (300), a second gear (430) is fixedly connected to an output shaft of the rotating motor (440), and the first gear (420) is meshed with the second gear (430); the output end of the air pump (450) is in rotary sealing connection with the end part of the straight pipe (411).
7. The microorganism concentration gradient sample preparation system according to claim 6, wherein one end of the spiral tube (412) is closed, a continuous disturbance blade (413) is fixedly connected to a tube wall outside the spiral tube (412), a plurality of aeration heads (414) are uniformly arranged on the tube wall inside the spiral tube (412), and adjacent aeration heads (414) are oppositely arranged.
8. A microbiological concentration gradient sampling system according to claim 1, wherein the number of second sample wells (300) is 4.
9. A microbiological concentration gradient sampling system according to claim 1, wherein an OD monitor (110) is provided in the mother liquor cell (100).
10. A sample preparation method using the microorganism concentration gradient sample preparation system according to any one of claims 1 to 9, comprising the steps of:
s1, determining the number of mother liquor pools (100) according to the growth cycle of target microorganisms, and inoculating the target microorganisms in the mother liquor pools (100) in batches; monitoring the OD value of the bacterial liquid by an OD value monitor (110), when the OD value of the bacterial liquid reaches the intersection point of the logarithmic phase and the stationary phase, opening a valve corresponding to a mother liquid outlet (120) to enable the bacterial liquid to enter a first sample pool (200), when the OD value of the bacterial liquid is lower than a target range, continuing to culture, and when the OD value of the bacterial liquid is higher than the target range, adding a culture medium to dilute to a lower concentration and continuing to culture;
S2, when the bacterial liquid in the first sample tank (200) is full, opening a valve of a first sample outlet (210), enabling one tenth of the bacterial liquid in the first sample tank (200) to flow into a second sample tank (300) below, opening a valve of a nutrient solution outlet (510), enabling the nutrient solution to flow into a second shunt pipe (620) from a first shunt pipe (610) by gravity, further entering the second sample tank (300) below to be mixed with the bacterial liquid, opening a rotating motor (440) and an air pump (450), simultaneously performing nutrient solution adding and stirring aeration operation, and completing multiplying power dilution when the nutrient solution is full of the second sample tank (300);
S3, after the second sample tank (300) is filled with the nutrient solution, starting a stepping motor (320), enabling a sealing plate (330) to rotate 180 degrees to seal a second shunt pipe (620), enabling the nutrient solution of the first shunt pipe (610) to flow into a third shunt pipe (630) in a flow direction, enabling the nutrient solution to enter the sample tank below through a next shunt structure, enabling a second sample outlet (310) of the second sample tank (300) to be opened, enabling one tenth of the nutrient solution of the second sample tank (300) to flow into the sample tank below, and then performing the next sample preparation cycle;
S4, after the sample preparation is carried out on the plurality of second sample tanks (300) in a circulating mode, the valve on the second shunt pipe (620) of the lowest shunt structure is closed, so that nutrient solution finally flows into the comparison sample tank (700) to serve as a comparison sample, and at the moment, the corresponding concentration in the comparison sample tank (700) is 0, and finally the sample preparation operation is finished.
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1065158A (en) * | 1976-03-23 | 1979-10-30 | Technicon Instruments Corporation | Apparatus and method of fluid sample analysis |
| JPH05296974A (en) * | 1992-04-24 | 1993-11-12 | Toshiba Corp | Ph analyzing system for nuclear power station |
| CN104084245A (en) * | 2014-06-13 | 2014-10-08 | 华中科技大学 | Apparatus and system used for forming concentration gradient |
| KR101619170B1 (en) * | 2015-07-10 | 2016-05-10 | 충남대학교산학협력단 | Microfluidic Chip for Sequencial Monitoring of Cell Susceptiblity to Samples |
| CN205665198U (en) * | 2016-06-03 | 2016-10-26 | 黄春连 | Nutrient solution composition on -line measuring system |
| CN206502830U (en) * | 2017-03-01 | 2017-09-19 | 苏州汶颢微流控技术股份有限公司 | Solution gradient is produced and cell culture microflow control chip |
| CN112881729A (en) * | 2021-01-15 | 2021-06-01 | 中山大学 | Drug concentration gradient generation and sample adding device and application thereof |
| CN213416414U (en) * | 2020-09-22 | 2021-06-11 | 云南超越建设工程有限公司 | Aeration equipment for sewage treatment |
| CN214529079U (en) * | 2021-03-10 | 2021-10-29 | 大连医科大学附属第一医院 | Micro-fluidic bionic model |
| CN115970781A (en) * | 2023-03-21 | 2023-04-18 | 杭州霆科生物科技有限公司 | Quantitative sample adding structure, concentration gradient micro-fluidic chip thereof and control method |
| CN116973182A (en) * | 2022-04-23 | 2023-10-31 | 中国科学院青岛生物能源与过程研究所 | Sample separation method, sample separation device, application and cell activity detection method |
| CN117451919A (en) * | 2023-09-20 | 2024-01-26 | 厦门市吉龙德环境工程有限公司 | Method and device for rapidly titration determination of sample concentration by gradient standard solution |
-
2024
- 2024-02-05 CN CN202410161030.4A patent/CN117925390A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1065158A (en) * | 1976-03-23 | 1979-10-30 | Technicon Instruments Corporation | Apparatus and method of fluid sample analysis |
| JPH05296974A (en) * | 1992-04-24 | 1993-11-12 | Toshiba Corp | Ph analyzing system for nuclear power station |
| CN104084245A (en) * | 2014-06-13 | 2014-10-08 | 华中科技大学 | Apparatus and system used for forming concentration gradient |
| KR101619170B1 (en) * | 2015-07-10 | 2016-05-10 | 충남대학교산학협력단 | Microfluidic Chip for Sequencial Monitoring of Cell Susceptiblity to Samples |
| CN205665198U (en) * | 2016-06-03 | 2016-10-26 | 黄春连 | Nutrient solution composition on -line measuring system |
| CN206502830U (en) * | 2017-03-01 | 2017-09-19 | 苏州汶颢微流控技术股份有限公司 | Solution gradient is produced and cell culture microflow control chip |
| CN213416414U (en) * | 2020-09-22 | 2021-06-11 | 云南超越建设工程有限公司 | Aeration equipment for sewage treatment |
| CN112881729A (en) * | 2021-01-15 | 2021-06-01 | 中山大学 | Drug concentration gradient generation and sample adding device and application thereof |
| CN214529079U (en) * | 2021-03-10 | 2021-10-29 | 大连医科大学附属第一医院 | Micro-fluidic bionic model |
| CN116973182A (en) * | 2022-04-23 | 2023-10-31 | 中国科学院青岛生物能源与过程研究所 | Sample separation method, sample separation device, application and cell activity detection method |
| CN115970781A (en) * | 2023-03-21 | 2023-04-18 | 杭州霆科生物科技有限公司 | Quantitative sample adding structure, concentration gradient micro-fluidic chip thereof and control method |
| CN117451919A (en) * | 2023-09-20 | 2024-01-26 | 厦门市吉龙德环境工程有限公司 | Method and device for rapidly titration determination of sample concentration by gradient standard solution |
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