CN108753596B

CN108753596B - Microchamber microfluidic system for detecting microorganism growth images

Info

Publication number: CN108753596B
Application number: CN201810312529.5A
Authority: CN
Inventors: 孙雪松; 杜高飞
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-04-09
Filing date: 2018-04-09
Publication date: 2022-05-17
Anticipated expiration: 2038-04-09
Also published as: CN108753596A

Abstract

The invention discloses a micro-chamber micro-fluidic system for detecting a microorganism growth image, which comprises a microscope, a constant-current injection pump, an injector, a constant-temperature objective table, a micro-chamber micro-fluidic chip, a collector and a CMOS video detection component, wherein the constant-current injection pump is connected with the microscope; the constant-temperature objective table is arranged on an objective of the microscope; the micro-chamber micro-fluidic chip is arranged on the constant-temperature objective table; the independently designed micro-chamber structure can be used for the real-time culture of microorganisms, the CMOS video detection component is connected and fixed above the microscope, and the CMOS video detection component is used for measuring the morphological change of bacteria in each micro-chamber in the micro-chamber micro-fluidic chip in a matched manner; the CMOS video detection device also comprises an operation unit which is based on the principle of realizing linear spatial filtering and a corresponding segmentation formula and coefficient and is used for obtaining clear gray image information of microorganisms. The method effectively overcomes the technical defects that the traditional microorganism image detection method is time-consuming, labor-consuming, expensive, single in function and incapable of acquiring real-time and real microorganism growth image information for a long time, and has a wide application prospect.

Description

Microchamber microfluidic system for detecting microorganism growth images

Technical Field

The invention belongs to the technical field of microorganism detection, and particularly relates to a micro-chamber micro-fluidic system for detecting a microorganism growth image.

Background

The culture of microorganisms is a basic technology in the fields of life science and medical research, industrial fermentation and food detection, but in the current bacterial culture detection field, only one detection index of a bacterial growth curve is usually obtained, namely, a turbidity detection device, such as a spectrophotometer, an enzyme-labeling instrument and the like, is used for obtaining a variation curve of the density and the number of bacterial thalli. However, with the development of science and technology and economy, the detection technology cannot meet the requirements of the current scientific research and the detection field of the food fermentation industry. Therefore, a detection device with high throughput, less sample consumption, high speed, miniaturization, automation, visualization, integration and portability is urgently needed.

The micro-fluidic technology is a technology for controlling micro fluid (with volume generally from picoliter to nanoliter) to flow, transfer heat and transfer mass by utilizing a micro-scale pipeline, and relates to multiple subjects such as chemistry, biology, biomedical engineering, materials, microelectronics and the like. Microfluidic technology began in the 90's of the 20 th century, and was first introduced in 1992 by professor Andreas Manz of switzerland, through more than twenty years of development, it has evolved from the first capillary electrophoresis technology, a one-dimensional continuous flow chip, to a chip with three-dimensional, high-throughput, and multifunctional integration. Today, microfluidic chips are widely used in analysis, synthesis, separation and detection of biochemical samples (nucleic acids, proteins, etc.). Compared with other analysis technologies, the microfluidic chip has obvious technical advantages and is characterized in that various unit technologies with different functions can be combined and integrated on a micro platform, so that the scientific research process is more efficient and convenient.

The micro-fluidic chip is used as a core platform of the micro-fluidic technology, the processing technology of the micro-fluidic chip originates from the micro-processing technology of a semiconductor integrated circuit chip, and the micro-processing technology is developed for a long time, so far, common micro-fluidic chip processing methods comprise an etching method, a hot pressing method, a molding method, an injection molding method, a laser ablation method, a soft lithography technology, a LIGA technology and the like.

However, with the development of microfluidic technology, the application field of the microfluidic technology is also expanded to the research of cells and bacteria at present, Torisawa et al (2007) designs and prepares a three-dimensional chip in which a micro-pipeline made of PDMS material and a micro-porous plate made of silicon are connected together, cultures multi-cell spheroids on the chip, and researches the formation of the spheroids through continuous observation for 14 days. Lanning et al designed a T-shaped microfluidic chip in 2008, and utilized two liquid flows to mix in a main pipe to form laminar flow, and generated a concentration gradient, and observed the migration of bacteria in such an environment. It was observed that the bacteria, despite the presence of the liquid flow, could migrate in the laminar flow, moving them in a direction that favours their own survival. In 2010, Kim and the like design a cell co-culture model based on a microfluidic chip technology, simulate the infection process of enterohemorrhagic escherichia coli on Hala cells, observe the colonization and propagation of bacteria on the cells, and form a bacterial envelope.

Although the microfluidic chip has been developed in the field of bacteria and cells, the culture of microorganisms still has many technical defects, for example, a channel culture mode is adopted, and the growth and movement of microorganisms are limited due to the culture of a single channel; in addition, a double-layer limited culture mode is adopted, namely, microorganisms are inoculated on the bottom layer, then a polymer film is covered on the upper layer, and the permeation culture is carried out by a method of flowing a culture medium on the upper layer.

Disclosure of Invention

In order to solve the defects and shortcomings of the prior art, the invention provides a micro-chamber micro-fluidic chip for real-time culture and observation of microorganisms. The micro-fluidic chip has the characteristics of light transmission, oxygen permeation, high flux, less sample consumption, miniaturization, automation, visualization, portability and the like, and can be used for carrying out real-time online detection on the shape and growth of bacteria.

It is still another object of the present invention to provide a micro-chamber microfluidic system for detecting the growth of microorganisms.

The above object of the present invention is achieved by the following technical solutions:

a microchamber micro-fluidic chip for real-time culture and observation of microorganisms comprises a chip main body, wherein the chip main body comprises at least one micro-fluidic channel, and the micro-fluidic channel consists of a liquid inlet 1, a main channel 2 and a liquid outlet 3 which are sequentially communicated; the middle section of the main channel 1 is of a broken line type, the broken line type main channel extends outwards at the corner along the liquid flow direction to form an annular channel and form a microchamber 4 of a cofferdam structure, and a T-shaped angle 5 is arranged at one end, close to the main channel, of the microchamber 4; a micro-column 6 is arranged on a channel between the micro-chamber 4 and the main channel 1; the cofferdam of the microchamber 4 is semi-closed, incompletely closed or fully closed, and the chip main body is provided with at least three different types of microchambers (4).

The microfluidic chip is provided with a micro chamber for independent culture, and the micro chamber adopts three different designs according to different functions; the semi-closed microchamber is used as an observation microchamber for culture observation of bacterial growth, and due to the adoption of the semi-closed design, a culture medium can be fully mixed with bacteria in the microchamber when passing through the annular channel, so that the culture microchamber is suitable for culture observation of bacterial growth; the incompletely-closed microchamber is used as a chemotaxis tracking microchamber and is suitable for the growth observation and the chemotaxis tracking of bacteria, when different culture mediums pass through the annular channel, because the microchamber and the annular channel are incompletely closed, the bacteria in the microchamber can sense the culture mediums flowing through the annular channel, thereby generating chemotactic motion; the completely closed microchamber is used as a biofilm measuring microchamber, and because no opening is arranged between the annular channel and the microchamber, bacteria grow in the peripheral channel of the microchamber, and the whole microchamber plays a role in adhering a microcolumn. In addition, the microcolumn is a multi-column transmission structure, the microcolumn and the T-shaped angle play a role in a valve, the function of transmitting bacteria liquid and culture medium to the annular channel and the microchamber is realized by utilizing the adhesion effect of the microcolumn, and the T-shaped angle simultaneously plays a shunting role, so that liquid flow has directionality and can flow in from one end of the annular channel and flow out from the other end of the annular channel; meanwhile, the cofferdam of each microchamber also has the function of a valve, so that bacteria and culture medium can flow through the annular channel and the microchamber.

Preferably, the depth of the main channel is 35 μm, and the depth of the micro chamber is 8 μm; if the depth is too deep, the bacteria culture chamber is easy to be laminated, thus being unfavorable for observation, and if the depth is too shallow, the flux is insufficient when the bacteria are cultured, thus affecting the observation result.

Preferably, the diameter of the micro chamber is 150-160 [ mu ] m or 250-260 [ mu ] m, 150-160 [ mu ] m is suitable for 40 times of observation under an objective lens, and 250-260 [ mu ] m is suitable for 20 times of observation of the objective lens.

Preferably, the semi-closed microchamber has an opening of about one third of the circumference of the microchamber.

Preferably, the size of the opening of the incompletely-closed microchamber is slightly larger than the diameter of the bacteria and is 5-10 mu m.

Preferably, the microfluidic chip is made of PDMS material. The PDMS material can provide a stable space structure and a ventilation environment for the culture of the microorganisms and the chemostatic culture of the microorganisms, the microchamber is highly adapted to the growth and movement of various microorganisms such as cell bacteria and the like, and a good light-transmitting medium is provided for the later observation.

The invention also provides application of the micro-chamber micro-fluidic chip in preparation of a microorganism growth image detection system.

A micro-chamber micro-fluidic system for detecting a microorganism growth image comprises a microscope, a constant-flow injection pump, an injector, a constant-temperature objective table, any one of the micro-chamber micro-fluidic chips, a collector and a CMOS video detection assembly; the constant-temperature objective table is arranged on an objective of the microscope; the microchamber microfluidic chip is arranged on a constant-temperature objective table, provides constant culture temperature for the chip and is adaptive to a microscope and the chip in size; the CMOS video detection component is fixed above the microscope through connection, and is used for measuring the morphological change of bacteria in each microchamber in the microchamber microfluidic chip in a matched manner; a liquid inlet of the microchamber microfluidic chip is connected with a constant-current injection pump, and an injector is arranged on the constant-current injection pump; and a liquid outlet of the micro-chamber micro-fluidic chip is connected with a collector.

Preferably, the CMOS video detection assembly includes an arithmetic unit, which is based on a median filtering principle and corresponding segmentation formulas and coefficients, for obtaining clear grayscale image information of microorganisms.

Preferably, the operation unit performs image filtering based on a mat2gray function and an imfilter function, and the image segmentation adopts a bwaeopen function and a bwaeclean function and a circular segmentation formula S = (R)²-r²）。

Specifically, an imfilter function (implementing a linear spatial filter function) is adopted, parameters are set to be 'corr', namely correlation, and 'reproduction', namely the size of an image is expanded by copying the value of the outer boundary, the size of an average filter window is 30, and a minimum filter pixel threshold value is 0.035.

A bweareaopen function (MATLAB) and a bwearclear function, with parameter thresholds P of 0.3 and 5000, respectively, and a neighborhood of 8.

Preferably, the CMOS video detection component is a digital camera; the Nikon D7100 camera is preferable, a high-speed recording unit with a transmission pixel of 1080P and a frequency of 10 frames/second can be realized, microorganisms moving at high speed can be recorded clearly and accurately, more reliable image information can be obtained, and the price is lower than that of an industrial camera with the same performance. Matched image filtering and segmentation algorithms, the mat2gray function and the imfilter function can filter the image, remove image noise and smooth the backgroundThe uniform function and the bweraopen function can divide and remove the graphic structures of the non-microorganisms with too small and too large areas in the image, thereby obtaining accurate and real graphic data, and the annular division formula S = (R)²-r²) The segmentation of the biomembrane region can be realized.

Preferably, the microscope is a fluorescence microscope, and the fluorescence module is a CCD fluorescent lamp and can emit blue light, red light and green light.

Preferably, the system also comprises a video recorder, a mobile hard disk and a display, wherein the video recorder is connected with the display and is respectively connected with the COMS video detection component, the mobile hard disk and the display.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a micro-chamber micro-fluidic chip for real-time culture and observation of microorganisms, which has the characteristics of light transmission, oxygen permeation, high flux, less sample consumption, miniaturization, automation, visualization, portability and the like, a series of visual data such as morphological information, motion information, group response information, division model information, interaction information with other species cells and the like of bacteria can be obtained by utilizing the visual online real-time detection and culture of the chip, and the data can be used for judging a series of important functional indexes such as the species, the activity, the drug resistance, the toxicity, the field planting, the infection capability and the like of the bacteria to carry out real-time online detection on the morphology and the growth of the bacteria.

(2) The invention provides a micro-chamber micro-fluidic system for detecting images of microorganism growth, which can obtain image information of microorganism growth, division, movement, death, formation of a biological membrane and the like in real time through the real-time constant culture of a micro-fluidic chip and the recording function of a CMOS (complementary metal oxide semiconductor), a matched arithmetic unit can remove noise points and miscellaneous points, clearly segment the morphological characteristics of the microorganism, and provide reliable image information and data information for establishing models of growth, movement, division, death, formation of the biological membrane and the like of the microorganism at the later stage.

Drawings

Fig. 1 is a top view of a microchamber microfluidic chip of example 1.

Fig. 2 is a front view of a micro-chamber microfluidic chip of example 1.

Fig. 3 is a top view of a microchamber microfluidic chip of example 1.

Fig. 4 is a view of the microchamber region of the microchamber microfluidic chip of example 1.

Fig. 5 shows the specific preparation parameters of the micro-chamber microfluidic chip in example 1.

Fig. 6 is a schematic view of a micro-chamber microfluidic system for detecting a microorganism growth image in example 2.

Fig. 7 shows the image recording result of application example 1.

Fig. 8 shows the partitioning result of application example 1.

Fig. 9 shows the statistical results of the gray scale values of application example 1.

Fig. 10 shows the image recording results of application example 2.

FIG. 11 shows the results of the separation in application example 2.

FIG. 12 is a graph showing the movement locus of some of the bacteria in application example 2.

Fig. 13 shows the image recording and segmentation results of application example 3.

FIG. 14 shows the results of the biofilm measurement in application example 3.

Drawing notes: 1-liquid inlet; 2-a main channel; 3-a liquid outlet; 4-a microchamber; 5-T type angle; 6-microcolumn.

Detailed Description

The invention provides a microorganism detection device and a detection method, which can effectively overcome the technical defects of time consumption, labor consumption, low accuracy, incapability of recording image information for a long time and difficulty in image segmentation in the traditional microorganism morphology detection method.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Materials:

the strain is as follows: escherichia coli MC4100 Escherichia coli BW25113

Culture medium:

LB culture medium: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), and 10g/L sodium chloride (NaCl).

M9 medium:

(1) firstly, 1M MgSO (MgSO) is prepared₄：MgSO₄·7H₂Dissolving O2.46 g in 10ML double distilled water, and autoclaving for use;

(2) preparation of 1M CaCl₂：CaCl₂·6H₂Dissolving O2.191 g in 10ML double distilled water, and autoclaving for standby;

(3) 5 XM 9 salt solution was prepared: na (Na)₂PO₄·7H₂O 12.8g，KH₂PO₄ 3.0g，NaCl 0.5g，NH₄Cl 1.0g is dissolved by adding 200mL of double distilled water, sterilized for 15 minutes at 121 ℃, and then the solution is prepared by the steps of: the three samples are prepared respectively, bottled respectively, and can be sent to high pressure together.

(4) Preparing a 20% glucose solution: dissolving 4g of glucose in 20mL of double distilled water, and filtering and sterilizing by using a 0.22 micron filter;

(5) sterile protocol M9 Medium 5 XM 9 salt solution 200mL 1M MgSO₄2mL of 20% glucose solution 20mL of 1M CaCl₂Sterile double distilled water was added to 0.1mL to 1000 mL.

CMOS imaging system:

equipment: guangzhou Mingmei microscope ML32, Nikon camera D7100, Philips high-definition display screen, KDS-100-CE constant-current injection pump, Changzhou Xiangtian constant-temperature objective table, Seagate mobile hard disk, Zhicheng constant-temperature incubator ZDP-A2080A, hard disk 2T, hard disk box and Houji TBOX PRO recording box.

Software: nikon camera control pro, Adobe Illustrator, MATLAB.

An injector: 2.5mL (several), 1mL (several), 500 μ l (2), 50 μ l (1).

And (3) filter membrane: 0.22 μm.

The raw materials used in the present invention are all commercially available.

Example 1

As shown in fig. 1 to 4, the present embodiment discloses a microchamber microfluidic chip for real-time cultivation and observation of microorganisms, the microfluidic chip includes a chip main body and a PDMS film integrally molded therewith, the chip main body includes 4 microfluidic channels, and the microfluidic channels are composed of a liquid inlet 1, a main channel 2 and a liquid outlet 3 which are sequentially communicated; the middle part of the main channel 1 is a micro-chamber area of the micro-fluidic chip, the middle sections of the main channels 1 are broken line type, the included angles between the broken line type main channels are 90 degrees, the broken line type main channels extend outwards along the liquid flow direction at the corners to form annular channels and form micro-chambers 4 of cofferdam structures, and one ends of the micro-chambers 4 close to the main channels are provided with T-shaped angles 5; a micro-column 6 is arranged on a channel between the micro-chamber 4 and the main channel 1; the cofferdam of the microchamber 4 is semi-closed, incompletely closed or fully closed, the openings of the cofferdam are all arranged at one end of the microchamber far away from the main channel, and at least three microchambers 4 of different types are arranged on the chip main body.

As a specific embodiment, the microchamber area comprises 8 microchambers, wherein 4-1 and 4-4 are all microchambers in a semi-closed design, and the diameters of the microchambers can be divided into four types, namely 250 mu m, 260 mu m, 150 mu m and 160 mu m; 4-2, 4-5 are incompletely closed microchambers with diameters of 260 μm and 160 μm respectively; 4-3, 4-6 are completely closed micro chambers with the diameter of 250 μm and 150 μm. The microchamber with the diameter of 250 to 260 mu m is suitable for observation under a 20-time objective lens, and the microchamber with the diameter of 150 to 160 mu m is suitable for observation under a 40-time objective lens.

In one embodiment, the semi-enclosed microchamber has an opening sized to be one third of the circumference of the microchamber.

As a specific embodiment, the size of the opening of the incompletely closed microchamber is slightly larger than the diameter of the bacteria and is 8 mu m.

As a specific implementation mode, the width of the main channel 1 is 100 mu m, the diameter of the annular channel is 360 mu m, and the distance between the annular channel and the adjacent main channel is 300 mu m.

In a specific embodiment, the depth of the main channel is 35 mu m, and the depth of the microchamber is 8 mu m.

The microfluidic chip of the embodiment is provided with a micro chamber for independent culture, and the micro chamber adopts three different designs according to different functions; the semi-closed microchamber is used as an observation microchamber for culture observation of bacterial growth, and due to the adoption of the semi-closed design, a culture medium can be fully mixed with bacteria in the microchamber when passing through the annular channel, so that the culture microchamber is suitable for culture observation of bacterial growth; the incompletely-closed microchamber is used as a chemotaxis tracking microchamber and is suitable for the growth observation and the chemotaxis tracking of bacteria, when different culture mediums pass through the annular channel, because the microchamber and the annular channel are incompletely closed, the bacteria in the microchamber can sense the culture mediums flowing through the annular channel, thereby generating chemotactic motion; the completely closed microchamber is used as a biofilm measuring microchamber, and because no opening is arranged between the annular channel and the microchamber, bacteria grow in the peripheral channel of the microchamber, and the whole microchamber plays a role in adhering a microcolumn. In addition, the microcolumn is a multi-column transmission structure, the microcolumn and the T-shaped angle play a role in a valve, the function of transmitting bacteria liquid and culture medium to the annular channel and the microchamber is realized by utilizing the adhesion effect of the microcolumn, and the T-shaped angle simultaneously plays a shunting role, so that liquid flow has directionality and can flow in from one end of the annular channel and flow out from the other end of the annular channel; meanwhile, the cofferdam of each microchamber also has the function of a valve, so that bacteria and culture medium can flow through the annular channel and the microchamber. The micro-chamber micro-fluidic chip can be used for culturing bacteria in three different micro-chambers and observing the bacteria simultaneously according to different requirements.

The specific preparation parameters of the micro-chamber micro-fluidic chip are shown in detail in fig. 5, and the preparation process is carried out according to the conventional technology in the field.

Example 2

As shown in fig. 6, the present embodiment discloses a micro-chamber microfluidic system for detecting a microorganism growth image, which comprises a microscope, a constant-flow injection pump, an injector, a constant-temperature stage, the micro-chamber microfluidic chip described in embodiment 1, a collector, a CMOS video detection assembly, a video recorder, a mobile hard disk and a display; the constant-temperature objective table is arranged on an objective of the microscope; the microchamber microfluidic chip is arranged on a constant-temperature objective table, provides constant culture temperature for the chip and is adaptive to a microscope and the chip in size; the CMOS video detection component is fixed above the microscope through connection, and is used for measuring morphological change of bacteria in each microchamber in the microchamber microfluidic chip in a matched manner; a liquid inlet of the microchamber microfluidic chip is connected with a constant-current injection pump, and an injector is arranged on the constant-current injection pump; a liquid outlet of the micro-chamber micro-fluidic chip is connected with a collector; the video recorder is connected with the display and is respectively connected with the COMS video detection component, the mobile hard disk and the display.

As a specific implementation mode, the CMOS video detection component comprises an operation unit, and the operation unit is based on a median filtering principle and a corresponding segmentation formula and coefficients and is used for obtaining clear grayscale image information of microorganisms.

As a specific implementation manner, the arithmetic unit performs image filtering based on a mat2gray function and an imfilter function, and image segmentation adopts a bweraopen function and a bweraclean function and a circular segmentation formula S = (R)²-r²）。

As a specific implementation, the CMOS video detection component is a digital camera; the Nikon D7100 camera is preferable, a high-speed recording unit with a transmission pixel of 1080P and a frequency of 10 frames/second can be realized, microorganisms moving at high speed can be recorded clearly and accurately, more reliable image information can be obtained, and the price is lower than that of an industrial camera with the same performance. Matched image filtering and segmentation algorithm, mat2gray function and imfilter function can be used for carrying out image filtering and segmentation on imagesFiltering to remove noise of the image and smooth the nonuniformity of the background, wherein the bweraopen function and the bweraclean function can be used for segmenting and removing the graphic structures of the microorganisms with too small and too large areas in the image so as to obtain accurate and real graphic data, and an annular segmentation formula S = (R) is used for segmenting²-r²) The segmentation of the biomembrane region can be realized.

In a specific embodiment, the microscope is a fluorescence microscope, and the fluorescence module is a CCD fluorescent lamp and can emit blue light, red light and green light.

Application example 1 Observation of bacterial growth culture

1. Coli MC4100 was activated overnight.

2. And opening an injection pump to set parameters, wherein the parameters of the injection pump are 2.5mL in specification, the flow rate is 4mL/h, the volume of the culture medium is 2.5mL, and sucking the bacterial liquid by using a 2.5mL disposable needle tube for later use.

3. The power switch is turned on, camera parameters (live view, light sensitivity H1 and visual field DX), microscope parameters (an objective lens 40X, matching 40, light intensity is maximum, an interface is 0.6), video recorder parameters (HDMI and 1080P) constant-temperature object stage parameters (42 ℃) and constant-temperature incubator parameters (37 ℃), the chip is communicated with an in-out sample tube, the chip is fixed by a special chip fixing clamp, manual focusing is carried out, and focusing is carried out after focusing.

4. The syringe needle is connected to the orifice of the sampling tube, the injection pump is opened, and simultaneously the video recorder is opened to start to record videos.

5. Observing the entering situation of the bacterial liquid, setting the parameters of the injection pump as the flow rate of 2mL/h after the bacterial liquid is concentrated in the microchamber, and replacing with new LB (adding a ciprofloxacin culture solution with a certain concentration into the micro-chamber to wash the residual bacterial liquid in the pipeline for 1 hour.

6. The parameters of the injection pump are reset to be 1mL/h of flow rate, and the stable culture stage is started.

7. And after the video recording is finished, taking out the mobile hard disk, turning off the power supply, and flushing the chip at a high speed by using 30% hydrogen peroxide, wherein the parameter of the injection pump is 5 mL/h.

8. Processing image information

It can be seen from the image record (fig. 7) and the segmentation result (fig. 8) that the heterotypic growth process of the escherichia coli MC4100 after ciprofloxacin is added is successfully recorded, the change of the shape is clear, the bacteria are grown into long rods, the result shown in fig. 9 is the average gray value statistics of fig. 8, and the result is a smooth logarithmic growth model curve, so the system can accurately obtain the morphological information and the growth curve of the bacteria.

Application example 2 growth observation and chemotaxis tracking of bacteria

1. Coli BW25113 was activated overnight with M9 medium.

2. And opening the setting parameters of the injection pump, wherein the parameters of the injection pump are 2.5 mL. The flow rate is 4mL/h, the volume of the LB medium is 2.5mL, and a 2.5mL disposable needle tube is used for sucking the bacteria liquid for standby.

5. And (4) observing the entering condition of the bacterial liquid, setting the parameters of the injection pump to be the flow rate of 2mL/h after the bacterial liquid is concentrated in the microchamber, and replacing with a new LB culture solution to wash the residual bacterial liquid in the pipeline for 1 hour.

6. The parameters of the injection pump are reset to be the flow rate of 0ul/h, and the stable culture stage is started.

8. Processing image information

As can be seen from the image record (FIG. 10) and the segmentation result (FIG. 11), the invention successfully records the chemotactic movement process of Escherichia coli BW25113 in the movement from the nutrient-deficient M9 culture medium to the nutrient-rich LB culture medium, and the movement track of part of bacteria in FIG. 12 truly reflects the movement process.

Application example 3 bacterial biofilm measurement

1. Activating Escherichia coli MC4100-EGFP (WT) and MC4100-EGFP drug-resistant bacteria (RCIP) overnight.

3. The power switch is turned on, camera parameters (live view, light sensitivity H1 and visual field DX), microscope parameters (an objective lens 20X, matching 20, light intensity is maximum, an interface is 0.6), video recorder parameters (HDMI and 1080P) constant-temperature objective table parameters (42 ℃) and constant-temperature incubator parameters (37 ℃), the chip is communicated with an in-out sample tube, the chip is fixed by a special chip fixing clamp, manual focusing is carried out, and focusing is carried out after focusing.

8. The writing program processes the image information.

As can be seen from the image recording and segmentation results (FIG. 13), the system successfully records the biofilm formation of Escherichia coli, and after statistical examination, the results of FIG. 14 show that the measured biofilm of RCIP strains with known strong biofilm formation is obviously higher than that of WT strains, which indicates that the invention can be applied to the formation detection of bacterial biofilms.

In summary, the micro-chamber micro-fluidic system for detecting the microbial growth image can successfully achieve the acquisition of the microbial morphology student growth information, can be used for neighborhood of growth tracking, chemotaxis research, biofilm formation research and the like of microbes, can achieve real-time, accurate, fast, high-frequency and high-resolution recording of the image information of the microbes, can accurately segment real microbial morphology information and data information by the corresponding CMOS operation module, provides a good foundation for personalized research of the subsequent research, and provides a high-efficiency and low-cost system in the field of microbial image detection.

Claims

1. A microchamber micro-fluidic chip for real-time culture and observation of microorganisms is characterized by comprising a chip main body, wherein the chip main body comprises at least one micro-fluidic channel, and the micro-fluidic channel consists of a liquid inlet (1), a main channel (2) and a liquid outlet (3) which are sequentially communicated; the middle section of the main channel (2) is of a broken line type, the broken line type main channel extends outwards at the corner along the liquid flow direction to form an annular channel and form a microchamber (4) of a cofferdam structure, and a T-shaped angle (5) is arranged at one end, close to the main channel, of the microchamber (4); a micro-column (6) is arranged on a channel between the micro-chamber (4) and the main channel (2); the cofferdam of the microchamber (4) is semi-closed, incompletely closed or totally closed, and the chip main body is provided with at least three different types of microchambers (4);

the size of the opening of the semi-closed microchamber is one third of the circumference of the microchamber;

the size of the opening of the incompletely closed microchamber is slightly larger than the diameter of the bacteria and is 5-10 mu m.

2. The micro-chamber micro-fluidic chip for real-time culture and observation of microorganisms according to claim 1, wherein the depth of the main channel is 35 μm, and the depth of the micro-chamber is 8 μm.

3. The micro-chamber micro-fluidic chip for real-time culture and observation of microorganisms according to claim 1, wherein the diameter of the micro-chamber is 150-160 μm or 250-260 μm.

4. The micro-chamber micro-fluidic chip for real-time culture and observation of microorganisms according to claim 1, wherein the micro-fluidic chip is made of PDMS material.

5. Use of the microchamber microfluidic chip of any one of claims 1 to 4 in the preparation of a microbial growth image detection system.

6. A micro-chamber micro-fluidic system for detecting a microorganism growth image is characterized by comprising a microscope, a constant-current injection pump, an injector, a constant-temperature objective table, a micro-chamber micro-fluidic chip according to any one of claims 1 to 4, a collector and a CMOS video detection assembly; the constant-temperature objective table is arranged on an objective of the microscope; the micro-chamber micro-fluidic chip is arranged on a constant temperature objective table; the CMOS video detection component is fixed above the microscope through connection, and is used for measuring the morphological change of bacteria in each microchamber in the microchamber microfluidic chip in a matched manner; a liquid inlet of the microchamber microfluidic chip is connected with a constant-current injection pump, and an injector is arranged on the constant-current injection pump; and a liquid outlet of the micro-chamber micro-fluidic chip is connected with a collector.

7. The micro-chamber microfluidic system for detecting images of microorganism growth according to claim 6, wherein the CMOS video detection assembly comprises an arithmetic unit, and the arithmetic unit is based on a median filtering principle and corresponding segmentation formulas and coefficients and is used for obtaining clear gray image information of microorganisms.

8. The microorganism growth image detection microchamber microfluidic system according to claim 7, wherein the arithmetic unit performs image filtering based on a mat2gray function and an imfilter function, and the image segmentation adopts a bweareaopen function and a bwearearclear function and a ring segmentation formula S ═ R2-R2.