CN116333859A - Continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle - Google Patents

Continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle Download PDF

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CN116333859A
CN116333859A CN202310275492.4A CN202310275492A CN116333859A CN 116333859 A CN116333859 A CN 116333859A CN 202310275492 A CN202310275492 A CN 202310275492A CN 116333859 A CN116333859 A CN 116333859A
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turbidimeter
bacterial
bacteria
continuous culture
tank
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傅雄飞
程松涛
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention relates to a continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle, belonging to the technical field of microbiology. The invention relates to a continuous culture turbidimeter, which comprises a tank structure, a bacteria concentration detection device and a control system which are connected with each other; the tank structure comprises a tank container, a stirrer arranged in the tank container, a feed pipe, a bacteria outlet pipe and a vent pipe which connect the tank container with the outside; the bacteria concentration detection device comprises a bacteria concentration detector connected with the current-voltage conversion module; the control system comprises a singlechip development board connected with the current-voltage conversion module, and one end of the singlechip development board is connected with the feeding pipe through a second peristaltic pump. The turbidimeter of the invention can realize the growth of bacteria in a stable state so as to carry out the study on bacterial cell cycle. The accurate detection and control of the bacterial concentration are realized, so that the stability of the bacterial growth environment and the accuracy of experimental data are ensured.

Description

Continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle
Technical Field
The invention belongs to the technical field of microbiology, and particularly relates to a continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle.
Background
Traditional methods of measuring bacterial growth include sampling from culture and measuring cell density using spectrophotometry or counting cells using a microscope. However, these methods are very time consuming, disrupt the growth environment, and may not accurately reflect the true cell density in the culture.
The development of the turbidimeter is to solve the above problems. A turbidimeter is a continuous culture system that maintains a constant cell density by constantly adjusting the flow rate of fresh medium into the culture vessel and removing an equal amount of spent medium. This allows for stable bacterial growth conditions therein, cell density to be monitored in real time using a turbidimeter, which can measure the optical density of the culture.
However, conventional magnetic stirring bars used in turbidimeters can cause disruption of cell culture, especially when cells are sensitive to shear stress. This has led to the development of new technologies to avoid damaging cells, such as airlift fermenters and Rushton (lashton) impellers, which use different types of agitation to avoid damaging cells. For example, the Cellstat system, which uses a rocking motion to provide agitation and avoid interfering with cell culture, has a separate chamber for measuring the optical density of the culture. However, the agitation method of the Cellstat system may not be as efficient as other methods and may result in inconsistent growth rates throughout the culture vessel. Furthermore, the Cellstat system cannot provide fresh air to the culture, which may limit its ability to maintain optimal growth conditions for certain bacterial species.
On the other hand, existing turbidimeter techniques employ complex optical measurement techniques to measure the optical density of a culture, such as the microscopic observation of bacterial concentration of bacterial fluid under a microfluidic chip, which may be more prone to error and require more calibration than simple nephelometry.
PID full scale (pro-port) -integral (integral) -derivative (derivative) controller. PID stands for "proportional-integral-derivative," which is a control algorithm used in a process control system to regulate and stabilize the process output. It is one of the most widely used control algorithms for various applications such as temperature control, speed control, level control, etc.
The PID algorithm consists of three parts: proportional control, integral control, and differential control. Proportional control involves adjusting the output based on the difference between the desired set point and the current process value multiplied by a proportional gain factor. The integration control integrates the error over time to help reduce steady state error. It involves summing the errors over time and multiplying them by an integral gain factor. Differential control calculates the rate of change of the error and adjusts the output accordingly to help reduce overshoot and ringing. It involves deriving the error and multiplying it by a differential gain factor.
By combining these three components, the PID algorithm can adjust the control signal based on the error between the desired set point and the actual process value, thereby effectively adjusting and stabilizing the output of the process.
The present invention aims to address these limitations and drawbacks of the prior art described above by providing a unique and simple tank structure that allows for efficient agitation without interfering with cell culture and that has a hollow agitator shaft to provide fresh air to the culture. The invention also provides a more accurate and reliable method for monitoring the cell density in real time. Overall, the present invention aims to provide a more efficient and effective method to maintain stable bacterial growth and research topics related to bacterial cell cycle.
Disclosure of Invention
In view of the above problems in the prior art, an object of the present invention is to design and provide a continuous culture turbidimeter and its use for measuring bacterial growth and bacterial cell cycle. The invention successfully solves the technical problems by designing a unique tank structure and a bacteria concentration detection device. The tank structure avoids the influence of friction on bacteria, and fresh air is provided at the same time, so that the bacteria grow in a constant environment. The bacterial concentration detection device ensures the accurate detection and control of the bacterial concentration, thereby ensuring the accuracy of experimental data. The combination of these functions provides a more efficient method to maintain stable bacterial growth and to study topics related to bacterial cell cycle, to provide optimal growth conditions for bacteria and to accurately measure bacterial concentration, so that more accurate experiments and studies can be performed in the field of microbiology.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in one aspect, the invention provides a continuous culture turbidimeter, comprising a tank structure, a bacteria concentration detection device and a control system which are connected with each other;
the tank structure comprises a tank container, a stirrer arranged in the tank container, a feed pipe, a bacteria outlet pipe and a vent pipe which connect the tank container with the outside; the tank structure can be effectively stirred without disturbing the cell culture.
The bacteria concentration detection device comprises a bacteria concentration detector connected with the current-voltage conversion module; bacterial growth can be accurately monitored.
The control system comprises a singlechip development board connected with the current-voltage conversion module, and one end of the singlechip development board is connected with the feeding pipe through a second peristaltic pump, so that the growth conditions of bacteria can be accurately regulated. The single chip development board is programmed to regulate the supply of bacterial nutrition based on the sensor input, and the peristaltic pump is used to supply fresh nutrient solution to the culture and to provide flowing bacterial solution to the cuvette as necessary.
The material of the tank container is transparent, so that the bacterial growth condition can be observed conveniently. The tank structure includes a sealing cover that can be conveniently contacted with the bacterial culture without introducing contaminants. The stirrer is suspended in the tank body container, so that bacteria at the bottom of the tank can be prevented from being rubbed like a magnetic stirring rod. The suspension structure design enables the stirring rod to suspend in the air for stirring, but not at the bottom of the tank, so that damage caused by bacteria friction can be avoided.
The stirrer comprises a magnetic stirrer, a hollow pipe and a stainless steel shaft, wherein the stainless steel shaft is used for fixedly connecting the magnetic stirrer with the hollow pipe, and the hollow pipe is connected with an air compressor and a precise pressure controller. The hollow tube is arranged, so that the fixed shaft of the stirrer is hollow, and the stirrer can simultaneously have the functions of stirring and providing fresh air for bacteria. This design helps to maintain optimal growth conditions for the bacteria. The air flow is controlled by an air compressor and a precision pressure controller, which can precisely regulate the air supply.
The bacteria concentration detector comprises an LED coaxial light source, a flow cuvette with a thin pore canal connected with the LED coaxial light source and a silicon photodiode. The bacteria concentration detector withdraws the bacteria liquid from the culture vessel through the capillary tube and into a microcuvette with a fine channel. And the concentration of bacteria can be accurately measured in real time through the LED coaxial light source and the silicon photodiode.
When the light source voltage is constant, if the bacteria concentration reaches a certain value, the current generated by the silicon photodiode reaches a certain value. And then the current-voltage conversion module generates corresponding voltage and transmits the voltage to the singlechip development board so as to determine whether the bacterial concentration is higher than or lower than a target value. In addition, the singlechip development board can also transmit data to a computer through software matched with the singlechip development board, and a user can check a voltage value on a computer interface. The software interface displays real-time data about bacterial growth and allows the user to adjust growth conditions and alter program parameters as desired.
According to the continuous culture turbidimeter, the flowing cell is controlled by the first peristaltic pump to provide flowing bacterial liquid.
The continuous cultivation turbidimeter is characterized in that the singlechip development board is connected with a computer, and the singlechip development board transmits data to the computer through matched software.
The control system controls the concentration of bacteria in the tank body container according to a PID algorithm.
In a second aspect, the invention provides a method for determining bacterial cell cycle using a continuous culture turbidimeter as defined in any one of the preceding claims.
In a third aspect, the invention provides the use of a continuous culture turbidimeter of any one of the above in a bacterial cell cycle assay;
preferably, the bacterium is E.coli.
In a fourth aspect, the invention provides the use of a continuous culture turbidimeter of any one of the above for detecting bacterial sensitivity to antibiotics, for producing microbial products, for monitoring bacterial contamination in an environment.
Compared with the prior art, the invention has the following beneficial effects:
the present invention provides a more accurate and reliable and cheaper method for studying the relevant topics of bacterial cell cycles. The growth of bacteria in a stable state can be achieved for bacterial cell cycle related studies. The accurate detection and control of the bacterial concentration are realized, so that the stability of the bacterial growth environment and the accuracy of experimental data are ensured. These advantages may lead to more consistent, more reliable results, facilitating research and other applications. Specifically:
1. the unique suspension stirring rod tank structure avoids friction bacteria during stirring at the tank bottom, which is beneficial to stable growth of bacteria, prevents damage to bacterial cells during stirring, and ensures more consistent and reliable results. Compared with the tank body of the traditional bioreactor, the cost is lower, and all parts required by the reaction tank can be obtained only by using a common wide-mouth reagent bottle and performing simple machine tool processing on the tetrafluoro ethylene.
2. The fixed shaft of the stirring shaft is hollow, so that the stirring rod simultaneously realizes the double functions of stirring and providing fresh air for bacteria, and the growth of the bacteria is facilitated by fully utilizing the nutrient components in the culture solution.
3. The bacteria concentration detection device designs a unique bacteria concentration detection device, comprises components such as a micro cuvette, an LED light source, a silicon photodiode and the like, and can monitor the bacteria concentration in real time and accurately, so that the culture condition is better controlled, and the growth and the life cycle of bacteria are favorably researched.
4. The use of micro cuvettes, LED light sources, and silicon photodiodes in the detection device is a more accurate and efficient method than other methods used in the prior art.
Drawings
FIG. 1 is a schematic diagram of a turbidimeter structure;
FIG. 2 is a light source-cuvette-photosensor bacterial concentration detection device;
FIG. 3 shows voltage values corresponding to the bacterial concentration displayed in the SCM development board matching software;
FIG. 4 is a schematic flow chart of a PID algorithm;
FIG. 5 is a picture of the operation of the turbidimeter;
FIG. 6 is a chart showing the change of bacterial concentration with time obtained by sampling the bacterial liquid of the turbidimeter a plurality of times and measuring the optical density value of the bacteria in the spectrophotometer;
the device comprises a 1-tank structure, a 101-tank container, a 102-stirrer, 1021-magnetic stirring rod, 1022-hollow pipe, 1023-stainless steel shaft, 103-feeding pipe, 104-bacteria outlet pipe, 105-ventilating pipe, 106-sealing cover, 107-air compressor, 108-precision pressure controller, 2-bacteria concentration detection device, 201-bacteria concentration detector, 2011-LED coaxial light source, 2012-flow cuvette, 2013-silicon photodiode, 202-current-voltage conversion module, 203-first peristaltic pump, 3-control system, 301-singlechip development board, 302-second peristaltic pump and 303-computer.
Detailed Description
The invention will be further illustrated by the following figures and examples.
Example 1:
a continuous culture turbidimeter comprises a tank body structure 1, a bacteria concentration detection device 2 and a control system 3 which are connected with each other;
the tank structure 1 comprises a tank container 101, a stirrer 102 arranged in the tank container 101, a feed pipe 103 connecting the tank container 101 with the outside, a bacteria outlet pipe 104 and a vent pipe 105; the material of the tank container 101 is transparent; the tank structure 1 comprises a sealing cover 106; the stirrer 102 is suspended in the tank 101. The stirrer 102 includes a magnetic stirrer 1021, a hollow tube 1022, and a stainless steel shaft 1023 for fixedly connecting the magnetic stirrer 1021 and the hollow tube 1022, and the hollow tube 1022 is connected to an air compressor 107 and a precision pressure controller 108. Has the functions of simultaneously stirring and providing fresh air for bacteria, and is helpful for maintaining the optimal growth conditions of the bacteria.
The bacteria concentration detection device 2 comprises a bacteria concentration detector 201 connected with a current-voltage conversion module 202; the bacteria concentration detector 201 includes an LED coaxial light source 2011, a flow cuvette 2012 with a thin bore connected to the LED coaxial light source 2011, a silicon photodiode 2013. The flow cell 2012 is controlled by the first peristaltic pump 203 to provide a flow of bacterial fluid. Bacterial liquid is drawn from the canister 101 through the vent tube 105 and into the flow cuvette 2012 with a fine channel for accurate measurement of bacterial concentration in real time.
The control system 3 comprises a singlechip development board 301 connected with the current-voltage conversion module 202, and one end of the singlechip development board 301 is connected with the feeding pipe 103 through a second peristaltic pump 302. The single-chip microcomputer development board 301 is connected with the computer 303, and the single-chip microcomputer development board 301 transmits data to the computer 303 through matched software. The control system controls the concentration of bacteria in the tank container according to a PID algorithm.
As shown in FIG. 1, the structure of the turbidimeter of the present invention is schematically shown, and the turbidimeter of the present invention can provide optimal growth conditions for bacteria and accurately measure the concentration of bacteria.
In actual use, the tank structure 1 is designed to suspend the stirrer 102 for stirring, so as to avoid bacteria rubbing the tank bottom like the magnetic stirring rod in the prior art. The can 101 is made of transparent material, so that the growth of bacteria can be observed. A sealing cap 106 is also provided to allow easy access to the bacterial culture without introducing contaminants. The stirrer 102 of the turbidimeter is designed as a fixed hollow shaft with the dual function of stirring and providing fresh air for bacteria. The stirrer 102 is composed of a magnetic stirrer 1021, a stainless steel shaft 1023, and a hollow tube 1022 that supplies air to bacteria. The air flow is controlled by an air compressor 107 and a fine pressure controller 108, and the air supply can be precisely regulated.
The bacteria concentration detection device 201 provided in the turbidimeter can accurately monitor the bacteria growth. As shown in fig. 2, the detection device is composed of a flow cuvette 2012 with a thin channel, an LED coaxial light source 2011, and a silicon photodiode 2013. When the light source voltage is constant, if the bacteria concentration reaches a certain value, the current generated by the silicon photodiode 2013 reaches a certain value. The current-to-voltage conversion module 202 then generates a corresponding voltage that is transmitted to the single chip development board 301 to determine whether the bacterial concentration is above or below a target value. In addition, the singlechip development board 301 can also transmit data to the computer 303 through software matched with the singlechip development board, and a user can check the voltage value on a computer interface. As shown in fig. 3, the voltage displayed in the interface is divided by 2058 and multiplied by 3.3 to obtain the actual voltage value. The software interface displays real-time data about bacterial growth and allows the user to adjust growth conditions and alter program parameters as desired.
The turbidimeter is equipped with a control system 3 which can accurately regulate the growth conditions of bacteria. The control system 3 includes a single-chip development board 301 and a plurality of peristaltic pumps. The single chip development board 301 is programmed to regulate the supply of bacterial nutrition based on sensor inputs, a first peristaltic pump 203 for providing the cuvette with flowing bacterial fluid and a second peristaltic pump 302 for supplying fresh nutrient fluid to the culture when necessary.
The PID principle utilized by the present invention is shown in FIG. 4, as shown in the following formula (1), wherein the formula is defined (at time t); the input quantity is r (t); the output quantity is c (t); the deviation amount is e (t) =rin (t) -rout (t);
Figure BDA0004136040930000061
the algorithm is used for regulating the speed of a stepping motor of the peristaltic pump, and the motor adopts PWM to regulate the speed and the rotating speed is expressed in unit rotation/min. rin (t) is a predetermined value of rotational speed; the output quantity rout (t) is an actual value of the motor rotation speed; the actuator is a stepper motor. This causes the input amount rin (t) of the control system to be a rotation speed predetermined value (rotations per minute); the output rout (t) is the actual value of the rotational speed (revolutions per minute); the deviation amount is the difference (revolutions per minute) between the predetermined value and the actual value. Fig. 5 is a picture showing the operation of the turbidimeter.
Example 2:
1. sterilization treatment of the device:
two Beckmann brand sterilization bags are taken, two holes are cut by scissors, and then the cut openings are sealed by sealing films and sterilization adhesive tapes for ventilation during later drying. The tank 101, the feed pipe 103, the bacteria outlet pipe 104 and the vent pipe 105 are respectively put into a sterilizing bag and sealed. The two sterilization bags and the reagent bottle filled with the culture medium are put into an autoclave for sterilization, and the equipment is sterilized for 15-20 minutes at the temperature of 121 ℃. And after sterilization, putting the mixture into an oven for drying, and removing water in the device.
2. Culturing and inoculating bacteria:
bacteria were inoculated overnight in the medium overnight in advance, and the bacteria were diluted 100-fold for activation the next day. The medium was filtered using a reagent bottle and a filter membrane.
After the bacterial concentration reached about 0.3, the bacteria were centrifuged in a high-speed centrifuge at 4 degrees celsius at 3000 rpm, and then resuspended in medium. The lines of the reaction tank were then connected, and the bacterial liquid was poured into the reaction tank, which was performed in an ultra clean bench.
3. The device starts:
the reaction tank is placed in a water bath magnetic stirring pot, a pipeline is connected with a peristaltic pump, a reagent bottle and an air compressor, then a power supply is connected, and the state after the equipment is started is shown in fig. 5.
4. Adjusting parameters of a turbidimeter:
parameters of the turbidimeter, such as media flow rate and air flow rate, can be adjusted according to specific experimental needs and requirements.
Example 3:
1. data collection and analysis
During the course of the experiment, the turbidity of the bacterial culture can be monitored in real time using a spectrophotometer, and data can be collected and analyzed using a computer software program. The program can plot the growth curve of bacteria and provide information about the lag, exponential and stationary phases of bacterial growth. The bacteria were taken out a plurality of times, and the optical density of the bacteria at 600 nm wavelength light was measured each time with a spectrophotometer, and the result is shown in fig. 6, which shows that the turbidimeter can stably control the bacteria concentration.
2. Device maintenance
After each use, the apparatus should be thoroughly cleaned with 70% ethanol and allowed to dry thoroughly before storage. Periodic maintenance of peristaltic pumps and air compressors is also necessary to ensure proper operation of the device.
3. Troubleshooting
If the device is not functioning properly, several troubleshooting steps may be taken. First, it is checked whether all connections are firm and the power supply is operating normally. If the concentration of the bacterial liquid is unstable, the culture medium is checked to ensure that the bacterial liquid is uniformly mixed before being poured into the reaction tank.
If the bacteria concentration fluctuates, it is checked whether the peristaltic pump and the air compressor are operating properly.
If contamination occurs, the apparatus is again sterilized and the experiment repeated using appropriate aseptic techniques.
If the optical density readings are not consistent, please check the spectrophotometer calibration and clean the cuvette prior to each measurement.
Experiments, simulations and use prove that the invention is feasible, and feasibility tests show that the turbidimeter can provide stable bacterial growth and maintain constant bacterial concentration, and the bacterial concentration detection device can provide accurate and precise bacterial concentration measurement. The suspension stirring rod and the stirring shaft have dual functions, and the bacteria concentration detection device is designed, so that bacteria can maintain activity for a long time and grow and monitor the bacteria concentration in real time.

Claims (10)

1. The continuous culture turbidimeter is characterized by comprising a tank body structure, a bacteria concentration detection device and a control system which are connected with each other;
the tank structure comprises a tank container, a stirrer arranged in the tank container, a feed pipe, a bacteria outlet pipe and a vent pipe which connect the tank container with the outside;
the bacteria concentration detection device comprises a bacteria concentration detector connected with the current-voltage conversion module;
the control system comprises a singlechip development board connected with the current-voltage conversion module, and one end of the singlechip development board is connected with the feeding pipe through a second peristaltic pump.
2. The continuous culture turbidimeter of claim 1, wherein the material of the tank container is transparent; the tank structure comprises a sealing cover; the stirrer is suspended in the tank body container.
3. The continuous culture turbidimeter of claim 2, wherein the agitator includes magnetic stirring rod, hollow tube and the stainless steel axle that is used for fixed connection magnetic stirring rod and hollow tube, and hollow tube links to each other with air compressor and accurate pressure controller.
4. The continuous culture turbidimeter of claim 1, wherein the bacterial concentration detector comprises an LED coaxial light source, a flow cuvette having a thin tunnel connected to the LED coaxial light source, and a silicon photodiode.
5. The continuous culture turbidimeter of claim 4, wherein the flow cuvette is controlled by a first peristaltic pump to provide a flow of bacterial fluid.
6. The continuous culture turbidimeter of claim 1, wherein the single-chip development board is connected to a computer, and the single-chip development board transmits data to the computer via a software.
7. The continuous culture turbidimeter of claim 1, wherein the control system controls bacterial concentration in the tank vessel according to a PID algorithm.
8. A method for determining bacterial cell cycle, wherein the determination of bacterial cell cycle is performed using the continuous culture turbidimeter of any one of claims 1-7.
9. Use of the continuous culture turbidimeter of any one of claims 1-7 to determine bacterial cell cycle, providing a stable bacterial growth environment;
preferably, the bacterium is E.coli.
10. Use of the continuous culture turbidimeter of any one of claims 1-7 for detecting bacterial sensitivity to antibiotics, for producing microbial products, for monitoring bacterial contamination in an environment.
CN202310275492.4A 2023-03-21 2023-03-21 Continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle Pending CN116333859A (en)

Priority Applications (1)

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CN202310275492.4A CN116333859A (en) 2023-03-21 2023-03-21 Continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310275492.4A CN116333859A (en) 2023-03-21 2023-03-21 Continuous culture turbidimeter and application thereof in measuring bacterial growth and bacterial cell cycle

Publications (1)

Publication Number Publication Date
CN116333859A true CN116333859A (en) 2023-06-27

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