CN112683904B - An in-situ characterization device and characterization method for the interaction between microorganisms and solid surfaces - Google Patents

Abstract

The invention relates to an in-situ characterization device and a characterization method for interaction of microorganisms and a solid surface, and belongs to the cross technical field of microscopy, microbiology and materials science. The in-situ characterization device comprises a temperature control hot table, a microorganism culture tank, a glass substrate, a solid surface material, an inverted microscope, an image sensor and an upper computer. The in-situ characterization device is convenient to build, simple to operate and wide in application range, realizes in-situ, real-time and long-time observation of the interaction of the microorganisms and the solid surface in an inverted microscope bright field under the condition of not marking the microorganisms by combining the in-situ characterization method, and can be effectively used for researching the adhesion kinetics and the proliferation behavior of the microorganisms on the solid surface.

Description

An in-situ characterization device and characterization method for the interaction between microorganisms and solid surfaces

technical field

The invention belongs to the interdisciplinary technical field of microscopy, microbiology and materials science, and particularly relates to an in-situ characterization device and a characterization method for the interaction between microorganisms and solid surfaces.

Background technique

Microorganisms are ubiquitous and closely related to human beings. The existence of beneficial microbial systems not only determines the health of the human body (such as intestinal flora), but also plays an important role that cannot be replaced by other substances in wastewater treatment, metal recovery, and preparation of fine chemicals. However, while bringing convenience to human production and life, the high energy consumption caused by the growth of microorganisms on the surfaces of food packaging, ship transportation, biological materials, implanted devices, etc. has also brought a heavy economic burden to human beings, and even Directly threaten the safety of human life (such as the SARS virus in 2003 and the spread of the new coronavirus at the end of 2019). Both beneficial and harmful microorganisms tend to adhere and proliferate on solid surfaces. Therefore, it is of great theoretical and practical significance to study the interaction between microorganisms and solid surfaces.

In the prior art, the devices used to characterize the interaction between microorganisms and solid surfaces mainly include scanning electron microscopes, atomic force microscopes, fluorescence microscopes, laser scanning confocal microscopes, and quartz crystal microscales. Among them, the scanning electron microscope has high resolution, high magnification, and large depth of field, which can clearly observe bacteria and solid surfaces (Colloids & Surfaces B Biointerfaces [J], 2015, 135: 549-555), but this method requires microbial samples to be analyzed. Dehydration and fixation treatment, and then observe the microbial morphology on the solid surface under an electron microscope, that is to say, this method only characterizes the final result of the interaction between the two, and cannot achieve real-time tracking. Atomic force microscopy is mainly used for the acquisition of ultra-high-resolution surface topography of microorganisms and the quantitative characterization of microbial surface adhesion (Nat. Commun. [J], 2010, 1:26), but cannot observe the movement phenotype of microorganisms on the surface . Fluorescence microscopy (ACS Applied Materials&Interfaces [J], 2018, 10(11): 9225–9234) and laser scanning confocal microscopy (Biofouling [J], 2014, 30(9-10): 1023-1033.) can detect microorganisms For real-time observation, especially the laser scanning confocal microscope, which adds a laser scanning device on the basis of the fluorescence microscope, adopts the principle and device of conjugate focusing, and cooperates with the focus stabilization system to realize the dynamic observation of living cells for several hours. However, both methods require fluorescent protein labeling, so there are problems such as the effect of phototoxicity on the activity of microorganisms, and the inability to observe light quenching for a long time. In addition, in fluorescence mode, microorganisms and surface patterns cannot be observed under the lens at the same time. The sensitivity of the quartz crystal microbalance can reach the nanogram level, which can sensitively detect the adhesion and desorption behavior of microorganisms, but it reflects the overall state and cannot observe the real-time movement behavior of individual microorganisms (Applied & Environmental Microbiology [J] ], 2005, 71(5):2705-2712.).

In view of the shortcomings of the existing technology, it is necessary to develop an in-situ characterization device and characterization method that is easy to operate and can observe the interaction between microorganisms and solid surfaces in real time.

SUMMARY OF THE INVENTION

In order to solve the technical problems in the prior art, the present invention provides an in-situ characterization device and a characterization method for the interaction between microorganisms and solid surfaces.

In order to solve the above-mentioned technical problems, the technical scheme adopted by the present invention is as follows:

The in-situ characterization device for the interaction between microorganisms and a solid surface of the present invention comprises a temperature-controlling heat stage, the temperature-controlling heat stage is a container with an open top, and the top opening is provided with a temperature-controlling stage cover, and the material of the temperature-controlling and heating stage cover is It is a transparent material, and there are through holes on the bottom surface of the inner wall of the temperature control heating table;

The in-situ characterization device further includes a microbial culture tank, a glass substrate, a solid surface material, an inverted microscope, an image sensor and an upper computer;

The microorganism cultivation pool is a container with openings at both ends, the bottom end is fixed on the bottom surface of the inner wall of the temperature control heat table, the top opening is provided with a microorganism cultivation pool cover, and the lower part of the inner wall of the microorganism cultivation pool is provided with an annular baffle plate. The outer edge of the baffle is fixed along the inner wall of the microorganism cultivation pool; the materials of the microorganism cultivation pool and the cover of the microorganism cultivation pool are all transparent materials;

The edge of the glass substrate is sandwiched between the lower surface of the annular baffle and the bottom surface of the inner wall of the temperature control heat table, and the upper surface of the glass substrate and the lower surface of the annular baffle are sealed and fixed to form a glass substrate as the bottom, A container with a microbial culture tank as a side wall for holding microbial solutions;

The solid surface material is arranged in the inner cavity of the microorganism culture tank, and the lower surface of the solid surface material is attached to the middle of the upper surface of the glass substrate, and the outer edge of the lower surface of the solid surface material is sealed and fixed on the upper surface of the glass substrate, The surface of the solid surface material is a plane, a surface with an irregular pattern or a surface with a regular pattern, and the material of the solid surface material is a transparent material; the total thickness of the glass substrate and the solid surface material is <0.36mm;

The light source of the inverted microscope is a halogen lamp, and the light emitted by the light source sequentially passes through the temperature-controlled heating table cover, the microorganism culture tank cover, the solid surface material and the glass substrate, and then is amplified by the objective lens of the inverted microscope and transmitted to the image sensor;

The image sensor collects the optical signal of the inverted microscope, converts the optical signal into an analog current signal, amplifies and converts the analog current signal, and transmits the obtained digital signal to the host computer;

The host computer processes the received digital signal to obtain a real-time picture of the behavior of microorganisms on the solid surface material, and analyzes the picture.

Further, the temperature-controlling heating stage is of a cube structure, and the through hole is a circle.

Further, the microorganism culture tank is of a cylindrical structure, the annular baffle is a circular shape, and the annular baffle and the microorganism culture tank are integrally formed.

Further, the glass substrate is disc-shaped.

Further, the thickness of the glass substrate is 0.05mm-0.30mm, and the thickness of the solid surface material is 0.05mm-0.30mm.

Further, the upper surface of the glass substrate and the lower surface of the annular baffle are pasted and fixed by sealant, and the outer edge of the lower surface of the solid surface material is sealed and fixed to the middle of the upper surface of the glass substrate by sealant.

Further, the bonding method between the lower surface of the solid surface material and the upper surface of the glass substrate is as follows: immerse the solid surface material in sterile water and then take it out, lay it flat on the middle of the upper surface of the glass substrate, and then combine the solid surface material with the upper surface of the glass substrate. The glass substrate is placed in a biochemical incubator at 37°C, and the solid surface material can be attached to the glass substrate after the water evaporates completely.

Further, the solid surface material is a surface with a regular pattern, and the regular pattern is a raised cylindrical shape, a raised conical shape, a raised hemispherical shape, a raised square shape, a raised honeycomb shape, and a raised shape. One of the shark skin shape, the raised ridge shape, the recessed cylindrical shape, the recessed conical shape, the recessed hemispherical shape, the recessed square shape, the recessed honeycomb shape, the recessed shark skin shape, the recessed ridge shape or a mixture of various.

Further, the inverted microscope is equipped with an eyepiece with a magnification of 10 times, and an objective lens with a maximum magnification of 100 times.

Further, the image sensor is a CCD image sensor.

Further, there are Infinity Analyze, FastStone Capture and Image J in the host computer. Infinity Analyze converts the digital signal of the image sensor into a picture, FastStone Capture converts the real-time picture screen recording into a video, and Image J analyzes the video.

Further, the microorganism is a mixture of one or more of bacteria, fungi, algae and cells.

The present invention also provides an in-situ characterization device utilizing the interaction of the above-mentioned microorganisms with the solid surface. The in-situ characterization method for the interaction between the microorganisms and the solid surface of the present invention is as follows:

After culturing the microorganisms in the culture medium, the concentration of less than ten microorganisms can be observed in the objective lens field of the inverted microscope by 100 times, and the microorganism solution is obtained, and then the microorganism solution is taken and added to the microorganism culture tank. The surface material does not exceed 4/5 of the capacity of the microorganism culture tank, adjust the temperature, turn on the inverted microscope, adjust the height of the objective lens of the inverted microscope until it focuses on the solid surface material, turn on the image sensor and the host computer, record and analyze the microorganisms in the solid in real time. behavior on surface materials.

Compared with the prior art, the beneficial effects of the present invention are:

The in-situ characterization device for the interaction between microorganisms and solid surfaces provided by the present invention is convenient to build, simple to operate, and has a wide range of applications. Combined with the in-situ characterization method for the interaction of microorganisms and solid surfaces provided by the present invention, it realizes no need to label microorganisms. The in-situ, real-time, and long-term observation of the interaction between microorganisms and solid surfaces in the bright field of an inverted microscope can be effectively used to study the adhesion kinetics and proliferation behavior of microorganisms on solid surfaces.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

1 is a schematic structural diagram of the in-situ characterization device for the interaction between microorganisms and solid surfaces of the present invention;

Fig. 2 is the axonometric exploded view along the A-A direction of Fig. 1;

3 is a schematic diagram of the structure of several common solid surfaces of the in-situ characterization device for the interaction between microorganisms and solid surfaces of the present invention, a-f are respectively a convex square shape, a concave square shape, a convex cylindrical shape, and a concave shape. Cylindrical, Raised Sharkskin, Raised Triangular and Cylindrical Mixed Figures, Raised Ridges.

Figure 4 is a screenshot of the interaction between Staphylococcus aureus and the surface of a 5 μm hemispherical patterned polyurethane film in Example 1 of the present invention. The incubation times a-d are 1h, 2h, 3h and 4h, respectively.

5 is the movement trajectory of Staphylococcus aureus on the surface of the 5 μm hemispherical patterned polyurethane film in Example 1 of the present invention, and marks 1, 2, and 3 represent the trajectory of bacterial movement for 1-2h, 2-3h and 3-4h, respectively.

6 is the movement trajectory of Staphylococcus aureus on the surface of the 2 μm hemispherical patterned polyurethane film in Example 2 of the present invention, and the marks 1, 2, and 3 represent the trajectory of bacterial movement for 1-2h, 2-3h and 3-4h, respectively.

In the figure, 1. Temperature-controlled heating table, 1-1, Temperature-controlled heating table cover, 2. Microbial culture tank, 2-1, Microbial culture tank cover, 3. Glass substrate, 4. Solid surface material, 5. Microbial solution , 6, sealant, 7, inverted microscope, 8, image sensor, 9, host computer.

Detailed ways

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with specific embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention rather than limiting the patent requirements of the present invention.

As shown in Figures 1-2, the in-situ characterization device for the interaction between microorganisms and solid surfaces of the present invention includes a temperature-controlled heating stage 1, a microorganism culture tank 2, a glass substrate 3, a solid surface material 4, an inverted microscope 7, and an image sensor. 8 and the host computer 9.

The temperature control heat table 1 is a container with an open top, and a temperature control heat table cover 1-1 is provided at the top opening, and the temperature control heat table cover 1-1 can seal the top opening of the temperature control heat table 1 . In order not to block the light path, the temperature-controlled heating stage cover 1-1 is made of transparent material. The bottom surface of the inner wall of the temperature control heating table 1 is provided with through holes, which are generally circular. Preferably, the temperature control heating table 1 has a cubic structure, such as a rectangular parallelepiped or a cube. A heating device and a temperature control device are arranged in the shell of the control heat stage 1 to control the temperature. The temperature-controlled heating stage 1 is in the prior art and can be obtained commercially, such as Heating Insert P 2000 from PECON.

The microbial culture tank 2 is a container with openings at both ends, the bottom end is fixed on the bottom surface of the inner wall of the temperature control heat table 1, and the top opening is provided with a microbial culture pond cover 2-1, which is capable of culturing microorganisms. The top opening of the pool 2 is sealed, the lower part of the inner wall of the microorganism culture pool 2 is provided with an annular baffle 2-2, and the outer edge of the annular baffle 2-2 is fixed along the inner wall of the microorganism culture pool 2. Preferably, the microorganism culture tank 2 has a cylindrical structure, and the annular baffle 2-2 is a circular shape. Preferably, the annular baffle 2-2 and the microorganism culture tank 2 are integrally formed.

The edge of the glass substrate 3 is sandwiched between the lower surface of the annular baffle 2-2 and the bottom surface of the inner wall of the temperature control heat table 1, and the upper surface of the glass substrate 3 is sealed and fixed with the lower surface of the annular baffle 2-2 to form A container with the glass substrate 3 as the bottom and the microorganism culture tank 2 as the side wall is used for holding the microorganism solution 5 . The preferred method of sealing and fixing is to stick and fix through the sealant 6, and the sealant 6 is preferably NOA63, which is cured by ultraviolet light. The glass substrate 3 is preferably disk-shaped. The glass substrate 3 can be replaced. Specifically, a suitable and clearly focused glass substrate 3 is selected according to the thickness of the solid surface material 4. The sum of the thicknesses of the glass substrate 3 and the solid surface material 4 is <0.36 mm. The thickness is 0.05mm-0.30mm, and the thickness of the solid surface material 4 is 0.05mm-0.30mm.

The solid surface material 4 is arranged in the inner cavity of the microorganism culture tank 2, and the lower surface of the solid surface material 4 is attached to the middle of the upper surface of the glass substrate 3, and the outer edge of the lower surface of the solid surface material 4 is sealed and fixed on the glass substrate 3. On the upper surface, it is ensured that the solid surface material 4 does not drift during the entire characterization period, and that the microbial solution does not penetrate between the solid surface material 4 and the glass substrate 3. The sealing and fixing method is preferably pasted and fixed by the sealant 6, and the sealant 6 is preferably NOA63, which adopts ultraviolet light. light curing. The solid surface material 4 should be a transparent material, not limited to polymer materials, commonly used are polyurethane films, polystyrene films, polydimethylsiloxane films, and the like. The bonding method between the lower surface of the solid surface material 4 and the upper surface of the glass substrate 3 is: immerse the solid surface material 4 in sterile water and then take it out, lay it flat on the middle of the upper surface of the glass substrate 3, and then place the solid surface material 4 Put the glass substrate 3 in a biochemical incubator at 37°C, and the solid surface material 4 and the glass substrate 3 can be attached after the water evaporates completely. The surface of the solid surface material 4 can be a flat surface, a surface with an irregular pattern, or a surface with a regular pattern, preferably a surface with a regular pattern, and the regular pattern is a raised cylindrical shape, a raised conical shape, a raised hemisphere shape, raised quadrangle, raised honeycomb, raised sharkskin, raised ridge, recessed cylinder, recessed cone, recessed hemisphere, recessed tetrahedron, recessed honeycomb As shown in FIG. 3 , the size of the regular pattern is 0.5 μm-50 μm, and the area of the solid surface material 4 is usually 1 cm×1 cm. The solid surface material 4 is usually prepared by a two-stage replication molding process (Advanced Materials, 1997, 9(2): 147-149.), and then stored in a desiccator.

The inverted microscope 7 is used to observe the solid surface material 4, the objective lens of the inverted microscope 7 is placed directly below the solid surface material 4, the light source of the inverted microscope 7 is a halogen lamp, and the light emitted by the light source passes through the temperature-controlled heating stage cover 1-1, The microorganism culture tank cover 2 - 1 , the solid surface material 4 and the glass substrate 3 are then magnified by the objective lens of the inverted microscope 7 and transmitted to the image sensor 8 . Preferably, the inverted microscope 7 is equipped with an eyepiece with a magnification of 10 times, an objective lens with a maximum magnification of 100 times, and the objective lens is preferably an oil lens with a magnification of 100 times.

The image sensor 8 collects the optical signal of the inverted microscope 7 , converts the optical signal into an analog current signal, amplifies and converts the analog current signal, and transmits the obtained digital signal to the upper computer 9 . The image sensor 8 is preferably a CCD image sensor.

The host computer 9 processes the received digital signal to obtain a real-time picture of the behavior of microorganisms on the solid surface material 4, and analyzes the picture, such as analyzing the behaviors of microorganisms such as adhesion, migration, aggregation, and proliferation. Preferably, there are Infinity Analyze, FastStone Capture and Image J in the host computer 9, Infinity Analyze converts the digital signal of the image sensor 8 into a picture, FastStone Capture converts the picture recording screen into a video, and Image J analyzes the video to complete the adhesion For the study of kinetics and proliferation behavior, the trackmate is usually used to manually record the position of the pixel points that the microorganisms pass through to obtain the microbial movement trajectory. The above software is available through commercial purchase.

In the above technical solution, the microorganism is a mixture of one or more of bacteria, fungi, algae and cells. The size of individual microorganisms is 0.5 μm-10 μm.

Utilizing the in-situ characterization device for the interaction of the microorganisms with the solid surface, the in-situ characterization method for the interaction between the microorganisms and the solid surface of the present invention is as follows:

The microorganisms are cultivated in the culture solution, diluted to a concentration of less than ten microorganisms in the field of view of the objective lens of the inverted microscope 7, and the microorganism solution 5 is obtained, and the microorganism solution 5 is added to the microorganism culture tank 2. 5 Submerge the solid surface material 4 and do not exceed 4/5 of the capacity of the microbial culture tank 2, adjust the temperature, turn on the inverted microscope 7, adjust the height of the objective lens of the inverted microscope 7 until it focuses on the solid surface material 4, turn on the image sensor 8 and the host computer 9. Record and analyze the behavior of microorganisms on the solid surface material 4 in real time.

In the above-mentioned technical scheme, the nutrient solution, culturing conditions and adjustment temperature of the microorganism are different according to the difference of the microorganism, and the specific can be determined according to the prior art. As the microorganism of the present embodiment adopts Staphylococcus aureus, as preferably, the nutrient solution is TSB. Culture medium, the culture conditions of microorganisms are: 180rpm, 37°C under the conditions of shaking bacteria for 12h, and the temperature is adjusted to 37°C; if Escherichia coli is used, preferably, the culture medium is LB culture medium, and the culture conditions of microorganisms are: 180rpm, 37°C The bacteria were shaken for 12 h under the condition of ℃, and the temperature was adjusted to 37 ℃. As the microorganism of this embodiment, if Staphylococcus aureus is used, preferably, the concentration is 10 ⁵ CFU/ml, and if Escherichia coli is used, preferably, the concentration is 10 ⁶ CFU/ml.

Terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The materials, reagents, devices, instruments, equipment, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

An in-situ characterization device for the interaction between microorganisms and solid surfaces, including a temperature-controlled heating stage 1, a microorganism culture tank 2, a glass substrate 3, a solid surface material 4, an inverted microscope 7, an image sensor 8 and a host computer 9, and a temperature-controlled heating stage 1 It is a rectangular parallelepiped container with an open top, and the top opening is provided with a temperature control heat table cover 1-1, and the bottom surface of the inner wall of the temperature control heat table 1 is provided with a circular through hole; the microorganism culture tank 2 is a cylindrical container with openings at both ends. The bottom end is fixed on the bottom surface of the inner wall of the temperature control heat table 1, the top opening is provided with a microorganism cultivation pool cover 2-1, and the lower part of the inner wall of the microorganism cultivation pool 2 is provided with a circular ring baffle 2-2 , the outer edge of the annular baffle 2-2 is fixed along the inner wall of the microbial culture tank 2; the glass substrate 3 is disc-shaped, and the edge is sandwiched between the lower surface of the annular baffle 2-2 and the bottom surface of the inner wall of the temperature control heat table 1 and the upper surface of the glass substrate 3 and the lower surface of the annular baffle 2-2 are pasted and fixed by the sealant 6NOA63 to form a container with the glass substrate 3 as the bottom and the microorganism culture pool 2 as the side wall, for carrying Microbial solution 5. The lower surface of the solid surface material 4 is attached to the center of the upper surface of the glass substrate 3 , and the lower surface of the outer edge of the solid surface material 4 is sealed and fixed on the upper surface of the glass substrate 3 with sealant 6NOA63. The inverted microscope 7 is used to observe the solid surface material 4, the objective lens of the inverted microscope 7 is placed directly below the solid surface material 4, the light source of the inverted microscope 7 is a halogen lamp, and the light emitted by the light source passes through the temperature-controlled heating stage cover 1-1, The microbial culture tank cover 2-1, the solid surface material 4 and the glass substrate 3 are magnified by the objective lens of the inverted microscope 7 and transmitted to the image sensor 8. The inverted microscope 7 is equipped with an eyepiece with a magnification of 10 times, and the maximum magnification is 100. times the oil mirror. The image sensor 8 is a CCD image sensor. The image sensor 8 collects the optical signal of the inverted microscope 7, converts the optical signal into an analog current signal, and amplifies and converts the analog current signal, and the obtained digital signal is transmitted to the host computer 9. . There are Infinity Analyze, FastStone Capture and Image J in the host computer 9. Infinity Analyze converts the digital signal of the image sensor 8 into a picture, FastStone Capture converts the picture recording screen into a video, the frame number is 25 frames, and Image J analyzes the video. The solid surface material 4 is a polyurethane film with an area of 1 cm×1 cm, a thickness of 0.10 mm, and a surface pattern of hexagonal close-packed hemispherical protrusions with a diameter of 5 μm. The thickness of the glass substrate 3 was 0.10 mm.

In situ characterization methods for the interaction of microorganisms with solid surfaces are as follows:

Put Staphylococcus aureus in the TSB medium, shake the bacteria at 180rpm and 37°C for 12h, dilute to 10 ⁵ CFU/ml, add 2ml to the microbial culture pool 2, adjust to 37°C, open and invert Microscope 7, adjust the height of the objective lens of the inverted microscope 7 until it is focused on the solid surface 4, turn on the image sensor 8 and the upper computer 9, and the upper computer 9 processes the received digital signal to obtain the behavior of microorganisms on the solid surface material (4). Real-time picture, and analyze the picture.

In FIG. 4 , a-d are respectively the bacterial adhesion and proliferation results of Staphylococcus aureus of the present embodiment at time points of 1 h, 2 h, 3 h and 4 h. Fig. 5 is the locus of Staphylococcus aureus of the embodiment, the marks 1, 2, 3 represent the trajectories of bacteria in different time periods of 1-2h, 2-3h, and 3-4h respectively, and the average motion distance obtained simultaneously is 175 pixels, average Adhesion kinetic parameters such as 70 s were used.

Example 2

The in-situ characterization device for the interaction between microorganisms and solid surfaces is the same as in Example 1, except that the solid surface material 4 is replaced by a polyurethane film, with an area of 1 cm × 1 cm, a thickness of 0.05 mm, and a surface pattern of hexagonal close-packed hemispheres with a diameter of 2 μm bulge. The thickness of the glass substrate 3 is 0.17 mm.

In situ characterization methods for the interaction of microorganisms with solid surfaces are as follows:

Put Staphylococcus aureus in the TSB medium, shake the bacteria at 180rpm and 37°C for 12h, dilute to 10 ⁵ CFU/ml, add 2ml to the microbial culture pool 2, adjust to 37°C, open and invert Microscope 7, adjust the height of the objective lens of the inverted microscope 7 until it is focused on the solid surface 4, turn on the image sensor 8 and the upper computer 9, and the upper computer 9 processes the received digital signal to obtain the behavior of microorganisms on the solid surface material (4). Real-time picture, and analyze the picture.

Fig. 6 is the movement trajectory of Staphylococcus aureus on the surface of this embodiment, and the marks 1, 2, and 3 represent the bacterial movement trajectory in different time periods of 1-2h, 2-3h, and 3-4h, respectively, and the average movement distance is obtained at the same time. Adhesion kinetic parameters such as 195 pixels and an average time of 52 s.

Example 3

The in-situ characterization device for the interaction between microorganisms and solid surfaces is the same as in Example 1, except that the solid surface material 4 is replaced by a polystyrene film, with an area of 1 cm × 1 cm and a thickness of about 0.05 mm; the surface pattern is hexagonal close-packed and the diameter is 5μm hemispherical protrusions. The thickness of the glass substrate 3 is 0.17 mm.

In situ characterization methods for the interaction of microorganisms with solid surfaces are as follows:

Put Staphylococcus aureus in the TSB medium, shake the bacteria at 180rpm and 37°C for 12h, dilute to 10 ⁵ CFU/ml, add 2ml to the microbial culture pool 2, adjust to 37°C, open and invert Microscope 7, adjust the height of the objective lens of the inverted microscope 7 until it is focused on the solid surface 4, turn on the image sensor 8 and the upper computer 9, and the upper computer 9 processes the received digital signal to obtain the behavior of microorganisms on the solid surface material (4). Real-time picture, and analyze the picture.

Example 4

The in-situ characterization device for the interaction between microorganisms and solid surfaces is the same as in Example 1, except that the solid surface material 4 is replaced by a polyurethane film, with an area of 1 cm × 1 cm, a thickness of 0.05 mm, and a surface pattern of hexagonal close-packed hemispheres with a diameter of 5 μm bulge. The thickness of the glass substrate 3 is 0.17 mm.

In situ characterization methods for the interaction of microorganisms with solid surfaces are as follows:

Put E. coli in the LB medium, shake the bacteria at 180rpm and 37°C for 12h, then dilute to 10 ⁶ CFU/ml, add 2ml to the microbial culture pool 2, adjust to 37°C, and turn on the inverted microscope 7 , adjust the height of the objective lens of the inverted microscope 7 until it focuses on the solid surface 4, turn on the image sensor 8 and the upper computer 9, and the upper computer 9 processes the received digital signal to obtain a real-time picture of the behavior of microorganisms on the solid surface material (4). , and analyze the screen.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. However, the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (10)

1. The in-situ characterization device for the interaction of microorganisms and a solid surface comprises a temperature control hot table (1), wherein the temperature control hot table (1) is a container with an opening at the top end, a temperature control hot table cover (1-1) is arranged at the opening at the top end, the temperature control hot table cover (1-1) is made of a transparent material, and a through hole is formed in the bottom surface of the inner wall of the temperature control hot table (1);

the in-situ characterization device is characterized by further comprising a microorganism culture pond (2), a glass substrate (3), a solid surface material (4), an inverted microscope (7), an image sensor (8) and an upper computer (9);

the microorganism culture pond (2) is a container with two open ends, the bottom end of the microorganism culture pond is fixed on the bottom surface of the inner wall of the temperature control hot table (1), the opening at the top end is provided with a microorganism culture pond cover (2-1), the lower part of the inner wall of the microorganism culture pond (2) is provided with an annular baffle (2-2), and the outer edge of the annular baffle (2-2) is fixed along the inner wall of the microorganism culture pond (2); the microbial culture tank (2) and the microbial culture tank cover (2-1) are made of transparent materials;

the edge of the glass substrate (3) is clamped between the lower surface of the annular baffle (2-2) and the bottom surface of the inner wall of the temperature control hot table (1), and the upper surface of the glass substrate (3) is hermetically fixed with the lower surface of the annular baffle (2-2) to form a container which takes the glass substrate (3) as the bottom and the microorganism culture pond (2) as the side wall and is used for containing a microorganism solution (5);

the solid surface material (4) is arranged in the inner cavity of the microorganism culture pond (2), the lower surface of the solid surface material (4) is attached to the middle of the upper surface of the glass substrate (3), the outer edge of the lower surface of the solid surface material (4) is sealed and fixed on the upper surface of the glass substrate (3), the surface of the solid surface material (4) is a plane, a surface with an irregular pattern or a surface with a regular pattern, and the solid surface material (4) is a transparent material; the total thickness of the glass substrate (3) and the solid surface material (4) is less than 0.36 mm;

the light source of the inverted microscope (7) is a halogen lamp, and light emitted by the light source sequentially passes through the temperature control heating platform cover (1-1), the microorganism culture pool cover (2-1), the solid surface material (4) and the glass substrate (3), is amplified by an objective lens of the inverted microscope (7), and is transmitted to the image sensor (8);

the image sensor (8) collects optical signals of the inverted microscope (7), converts the optical signals into analog current signals, amplifies and performs analog-to-digital conversion on the analog current signals, and transmits the obtained digital signals to the upper computer (9);

and the upper computer (9) processes the received digital signals to obtain a real-time picture of the behavior of the microorganisms on the solid surface material (4), and analyzes the picture.

2. The in-situ characterization device of microorganism and solid surface interaction according to claim 1, wherein the temperature controlled thermal stage (1) is a cube structure with a circular through hole; the microorganism culture pond (2) is of a cylindrical structure, the annular baffle (2-2) is of a circular ring shape, and the annular baffle (2-2) and the microorganism culture pond (2) are integrally formed; the glass substrate (3) is disc-shaped.

3. An in situ characterization device of microorganism interaction with a solid surface according to claim 1, characterized in that the glass substrate (3) has a thickness of 0.05mm-0.30mm and the solid surface material (4) has a thickness of 0.05mm-0.30 mm.

4. An in-situ characterization device for microorganism interaction with a solid surface according to claim 2, wherein the upper surface of the glass substrate (3) is adhesively fixed with the lower surface of the ring-shaped baffle plate by a sealant (6), and the outer edge of the lower surface of the solid surface material (4) is fixed in the middle of the upper surface of the glass substrate (3) by the sealant (6).

5. The apparatus for in situ characterization of microorganism and solid surface interaction according to claim 4, characterized in that the lower surface of the solid surface material (4) is attached to the upper surface of the glass substrate (3) in a way that: and (3) immersing the solid surface material (4) in sterile water, taking out, flatly spreading the solid surface material (4) in the middle of the upper surface of the glass substrate (3), putting the solid surface material (4) and the glass substrate (3) in a biochemical incubator at 37 ℃, and attaching the solid surface material (4) and the glass substrate (3) after the moisture is completely volatilized.

6. The apparatus for in situ characterization of microorganism-solid surface interaction according to claim 1, characterized in that the solid surface material (4) is a surface having a regular pattern of one or a mixture of raised cylindrical, raised conical, raised hemispherical, raised tetragonal, raised honeycomb, raised sharkskin, raised ridge, depressed cylindrical, depressed conical, depressed hemispherical, depressed tetragonal, depressed honeycomb, depressed sharkskin, depressed ridge.

7. The apparatus for in situ characterization of microorganism interaction with solid surfaces according to claim 1, characterized in that the inverted microscope (7) is equipped with an eyepiece at 10 x magnification, an objective at the highest magnification of 100 x; the image sensor (8) is a CCD image sensor.

8. An in situ characterization device of microorganism and solid surface interaction according to claim 1, characterized in that there are Infinity Analyze, FastStone Capture and Image J in the upper computer (9), the Infinity Analyze converts the digital signal of the Image sensor (8) into pictures, the FastStone Capture converts the picture screen into video, and the Image J analyzes the video.

9. The apparatus of claim 1, wherein the microorganism is a mixture of one or more of bacteria, fungi, algae, and cells.

10. A method for in situ characterization using an in situ characterization device for the interaction of microorganisms with a solid surface according to any one of claims 1 to 9,

after the microorganisms are cultured in the culture solution, the microorganisms are diluted to a concentration which is 100 times that of the individual microorganisms can be observed in the field of view of an objective lens of an inverted microscope (7), a microorganism solution is obtained, then the microorganism solution is added into a microorganism culture pond (2), the microorganism solution (5) submerges a solid surface material (4) and does not exceed 4/5 of the capacity of the microorganism culture pond (2), the temperature is adjusted, the inverted microscope is opened, the height of the objective lens of the inverted microscope is adjusted until the objective lens is focused on the solid surface material (4), an image sensor (8) and an upper computer (9) are opened, and the behaviors of the microorganisms on the solid surface material (4) are recorded and analyzed in real time.

Images