CN112683904B - In-situ characterization device and characterization method for interaction between microorganisms and solid surface - Google Patents
In-situ characterization device and characterization method for interaction between microorganisms and solid surface Download PDFInfo
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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
Technical Field
The invention belongs to the cross technical field of microscopy, microbiology and materials science, and particularly relates to an in-situ characterization device and a characterization method for interaction of microorganisms and a solid surface.
Background
Microorganisms are ubiquitous and closely related to humans. The existence of beneficial microbial systems not only determines the health condition of human bodies (such as intestinal flora), but also plays an important role which cannot be replaced by other substances in the aspects of wastewater treatment, metal recovery, fine chemical preparation and the like. However, while bringing convenience to human production and life, the problems of high energy consumption caused by the growth of microorganisms on the surfaces of food packaging, shipping, biological materials, implanting instruments and the like also bring heavy economic burden to human beings and even directly threaten the life safety of human beings (such as the spread of SARS virus in 2003 and new coronavirus in 2019). Whether beneficial or harmful, tend to adhere to and proliferate on solid surfaces, and therefore, there is important theoretical and practical significance in studying the interaction of microorganisms with solid surfaces.
In the prior art, devices for characterizing the interaction between microorganisms and a solid surface mainly include a scanning electron microscope, an atomic force microscope, a fluorescence microscope, a laser scanning confocal microscope, a quartz crystal micro-microscope and the like. The scanning electron microscope has high resolution, high magnification and large depth of field, and can clearly observe bacteria and solid Surfaces (Colloids & Surfaces B Biointerfaces [ J ],2015,135:549-555), but the method needs to carry out dehydration and fixation treatment on a microorganism sample, and then observe the form of the microorganism fixed on the solid surface under the electron microscope, that is, the method only characterizes the final result of the interaction of the two, and cannot realize real-time tracking. The atomic force microscope is mainly used for obtaining the ultra-high resolution surface topography of microorganisms and quantitatively characterizing the surface adhesion of the microorganisms (nat. Commun [ J ],2010,1:26), and the movement phenotype of the microorganisms on the surface cannot be observed. The fluorescence microscope (ACS Applied Materials & Interfaces [ J ],2018,10(11): 9225-. However, both methods require fluorescent protein labeling, and thus there are problems that phototoxicity affects microbial activity, that quenching by light cannot be observed for a long time, and the like, and in addition, in the fluorescent mode, microorganisms and surface patterns cannot be observed under the lens at the same time. The sensitivity of the quartz crystal microbalance can reach nanogram level, the adhesion and desorption behaviors of microorganisms can be sensitively detected, but the quartz crystal microbalance reflects the whole state and cannot observe the real-time movement behaviors of individual microorganisms (Applied and Environmental Microbiology [ J ],2005, 71(5): 2705-2712.).
Aiming at the defects of the prior art, it is necessary to develop an in-situ characterization device and a characterization method which are simple and convenient to operate and can observe the interaction between microorganisms and a solid surface in real time.
Disclosure of Invention
The invention provides an in-situ characterization device and a characterization method for interaction of microorganisms and a solid surface, aiming at solving the technical problems in the prior art.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the in-situ characterization device for the interaction of microorganisms and a solid surface comprises a temperature control hot table, wherein the temperature control hot table is a container with an opening at the top end, a temperature control hot table cover is arranged at the opening at the top end, the temperature control hot table cover 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;
the in-situ characterization device also comprises a microorganism culture pond, a glass substrate, a solid surface material, an inverted microscope, an image sensor and an upper computer;
the microorganism culture pond 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 heating table, the opening at the top end is provided with a microorganism culture pond cover, the lower part of the inner wall of the microorganism culture pond is provided with an annular baffle, and the outer edge of the annular baffle is fixed along the inner wall of the microorganism culture pond; the microbial culture pool and the microbial culture pool cover are made of transparent materials;
the edge of the glass substrate is clamped between the lower surface of the annular baffle and the bottom surface of the inner wall of the temperature control hot table, and the upper surface of the glass substrate and the lower surface of the annular baffle are fixed in a sealing manner to form a container which takes the glass substrate as the bottom and the microorganism culture pond as the side wall and is used for containing microorganism solution;
the solid surface material is arranged in the inner cavity of the microorganism culture pond, the lower surface of the solid surface material is attached to the middle part of the upper surface of the glass substrate, the outer edge of the lower surface of the solid surface material is fixed on the upper surface of the glass substrate in a sealing mode, the surface of the solid surface material is a plane surface, a surface with irregular patterns or a surface with regular patterns, and the solid surface material is a transparent material; the total thickness of the glass substrate and the solid surface material is <0.36 mm;
the light source of the inverted microscope is a halogen lamp, and light emitted by the light source passes through the temperature control heating platform cover, the microbial culture pool cover, the solid surface material and the glass substrate in sequence, is amplified through the objective lens of the inverted microscope and is transmitted to the image sensor;
the image sensor collects optical signals of the inverted microscope, converts the optical signals into analog current signals, amplifies and performs analog-to-digital conversion on the analog current signals, and transmits obtained digital signals to the upper computer;
and the upper computer processes the received digital signals to obtain a real-time picture of the behavior of the microorganisms on the solid surface material, and analyzes the picture.
Furthermore, the temperature control hot table is of a square structure, and the through hole is circular.
Furthermore, the microbial cultivation pond is of a cylindrical structure, the annular baffle is of a circular ring shape, and the annular baffle and the microbial cultivation pond are integrally formed.
Further, the glass substrate has a disk shape.
Further, the thickness of the glass substrate is 0.05mm-0.30mm, and the thickness of the solid surface material is 0.05mm-0.30 mm.
Furthermore, the upper surface of the glass substrate and the lower surface of the annular baffle plate are fixedly adhered through a sealant, and the outer edge of the lower surface of the solid surface material is fixedly sealed in the middle of the upper surface of the glass substrate through the sealant.
Further, the method for attaching the lower surface of the solid surface material to the upper surface of the glass substrate is as follows: and (3) immersing the solid surface material in sterile water, taking out, flatly paving the solid surface material in the middle of the upper surface of the glass substrate, putting the solid surface material and the glass substrate in a biochemical incubator at 37 ℃, and attaching the solid surface material and the glass substrate after the moisture is completely volatilized.
Further, the solid surface material is a surface having a regular pattern of one or a mixture of raised cylinders, raised cones, raised hemispheres, raised tetragonal shapes, raised honeycombs, raised sharkskin, raised ridges, recessed cylinders, recessed cones, recessed hemispheres, recessed tetragonal shapes, recessed honeycombs, recessed sharkskin, recessed ridges.
Further, the inverted microscope is provided 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, the upper computer comprises definition analysis, FastStone Capture and Image J, wherein the definition analysis converts digital signals of the Image sensor into pictures, the FastStone Capture converts real-time picture recording into videos, and the Image J analyzes the videos.
Further, the microorganism is one or more of bacteria, fungi, algae and cells.
The invention also provides an in-situ characterization device utilizing the interaction between the microorganisms and the solid surface, and the in-situ characterization method for the interaction between the microorganisms and the solid surface comprises the following steps:
culturing microorganisms in a culture solution, diluting the microorganisms to a concentration which is 100 times that of microorganism individuals observed in the field of view of an objective lens of an inverted microscope, obtaining a microorganism solution, adding the microorganism solution into a microorganism culture pool, submerging a solid surface material by the microorganism solution and is not more than 4/5 of the capacity of the microorganism culture pool, adjusting the temperature, opening the inverted microscope, adjusting the height of the objective lens of the inverted microscope until the objective lens is focused on the solid surface material, opening an image sensor and an upper computer, and recording and analyzing the behavior of the microorganisms on the solid surface material in real time.
Compared with the prior art, the invention has the beneficial effects that:
the in-situ characterization device for the interaction between the microorganisms and the solid surface is convenient to build, simple to operate and wide in application range, and by combining the in-situ characterization method for the interaction between the microorganisms and the solid surface, the in-situ, real-time and long-time observation of the interaction between the microorganisms and the solid surface in an inverted microscope bright field is realized without marking the microorganisms, so that the in-situ characterization device can be effectively used for researching the adhesion kinetics and the proliferation behavior of the microorganisms on the solid surface.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic structural diagram of an in situ characterization device for the interaction of microorganisms with a solid surface according to the present invention;
FIG. 2 is an isometric exploded view of FIG. 1 taken along A-A;
FIG. 3 is a schematic diagram of several common solid surfaces of the in situ characterization device of the interaction of microorganisms with the solid surface of the present invention, and a-f are respectively convex tetragonal shape, concave tetragonal shape, convex cylindrical shape, concave cylindrical shape, convex sharkskin shape, convex triangular and cylindrical mixed pattern, and convex ridge shape.
FIG. 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, wherein the incubation times a-d are 1h, 2h, 3h and 4h, respectively.
FIG. 5 is a trace of Staphylococcus aureus on the surface of a 5 μm hemispherical patterned polyurethane film in example 1 of the present invention, where the marks 1, 2, and 3 represent the traces of movement of bacteria for 1-2h, 2-3h, and 3-4h, respectively.
FIG. 6 is a trace of Staphylococcus aureus on the surface of a 2 μm hemispherical patterned polyurethane film in example 2 of the present invention, where the marks 1, 2, and 3 represent the traces of movement of bacteria for 1-2h, 2-3h, and 3-4h, respectively.
In the figure, 1, a temperature control hot table, 1-1, a temperature control hot table cover, 2, a microorganism culture pond, 2-1, a microorganism culture pond cover, 3, a glass substrate, 4, a solid surface material, 5, a microorganism solution, 6, a sealant, 7, an inverted microscope, 8, an image sensor, 9 and an upper computer.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.
As shown in FIGS. 1-2, the device for in-situ characterization of the interaction of microorganisms and a solid surface according to the present invention comprises a temperature-controlled thermal 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 an upper computer 9.
The temperature control heating table 1 is a container with an opening at the top end, a temperature control heating table cover 1-1 is arranged at the opening at the top end, and the opening at the top end of the temperature control heating table 1 can be sealed by the temperature control heating table cover 1-1. In order not to block the light path, the temperature control heating table cover 1-1 is made of transparent material. The bottom surface of the inner wall of the temperature control hot table 1 is provided with a through hole which is usually circular. Preferably, the temperature control heating table 1 is of a cubic structure, such as a cuboid or a cube. And a heating device and a temperature control device are arranged in the shell of the control heating platform 1 and used for controlling the temperature. The temperature-controlled hot plate 1 is known in the art and is commercially available, for example, from Heating Insert P2000 of PECON.
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 heat platform 1, the opening of the top end is provided with a microorganism culture pond cover 2-1, the microorganism culture pond cover 2-1 can seal the opening of the top end of the microorganism culture pond 2, 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. Preferably, the microorganism culture pond 2 is of a cylindrical structure, and the annular baffle 2-2 is of an annular shape. Preferably, the annular baffle 2-2 and the microorganism culture tank 2 are integrally formed.
The edge of the glass substrate 3 is clamped between the lower surface of the annular baffle plate 2-2 and the bottom surface of the inner wall of the temperature control heating table 1, and the upper surface of the glass substrate 3 and the lower surface of the annular baffle plate 2-2 are sealed and fixed 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 sealing and fixing mode is preferably that the sealing and fixing mode is that the sealing glue 6 is pasted and fixed, and the sealing glue 6 is preferably NOA63 and is solidified by ultraviolet light. The glass substrate 3 is preferably in the shape of a disk. The glass substrate 3 can be replaced, and a suitable glass substrate 3 with clear focusing can be selected according to the thickness of the solid surface material 4, wherein the sum of the thicknesses of the glass substrate 3 and the solid surface material 4 is less than 0.36mm, preferably, the thickness of the glass substrate 3 is 0.05mm-0.30mm, and the thickness of the solid surface material 4 is 0.05mm-0.30 mm.
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 solid surface material 4 is guaranteed not to drift during the whole characterization period, microorganism solution does not penetrate between the solid surface material 4 and the glass substrate 3, the sealing and fixing mode is preferably fixed through a sealing glue 6, the sealing glue 6 is preferably NOA63, and ultraviolet light curing is adopted. The solid surface material 4 should be a transparent material, and is not limited to a polymer material, and a polyurethane film, a polystyrene film, a polydimethylsiloxane film, or the like is commonly used. The lower surface of the solid surface material 4 and the upper surface of the glass substrate 3 are bonded in the following manner: 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, then placing 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. The surface of the solid surface material 4 may be planar, have an irregular pattern or have a regular pattern, preferably a regular pattern of one or more 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, as shown in fig. 3, the size of the regular pattern is 0.5 μm to 50 μm, and the area of the solid surface material 4 is typically 1cm x 1 cm. The solid surface material 4 is typically prepared using a two-stage replication process (Advanced Materials,1997,9(2): 147-.
The inverted microscope 7 is used for observing the solid surface material 4, an objective lens of the inverted microscope 7 is arranged right below the solid surface material 4, a 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 through the objective lens of the inverted microscope 7 and is transmitted to the image sensor 8. Preferably, the inverted microscope 7 is equipped with an eyepiece with a magnification of 10, an objective with a maximum magnification of 100, preferably an objective with a magnification of 100.
The image sensor 8 collects an optical signal of the inverted microscope 7, converts the optical signal into an analog current signal, amplifies and analog-to-digital 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 upper computer 9 processes the received digital signals to obtain real-time pictures of the behaviors of the microorganisms on the solid surface material 4, and analyzes the pictures, such as the behaviors of microorganism adhesion, migration, aggregation, proliferation and the like. Preferably, the upper computer 9 includes Infinity Analyze, FastStone Capture and Image J, the Infinity Analyze converts the digital signal of the Image sensor 8 into a picture, the FastStone Capture converts the picture screen into a video, the Image J analyzes the video, the adhesion kinetics and proliferation behavior research is completed, and the position of a pixel point where the microorganism passes is recorded manually through a TrackMate plug-in to obtain the movement track of the microorganism. The software is commercially available.
In the technical scheme, the microorganism is one or a mixture of bacteria, fungi, algae and cells. The size of each microorganism is 0.5-10 μm.
By utilizing the in-situ characterization device for the interaction between the microorganisms and the solid surface, the in-situ characterization method for the interaction between the microorganisms and the solid surface comprises the following steps:
culturing microorganisms in a culture solution, diluting the culture solution to a concentration which is 100 times that of microorganism individuals observed in a field of view of an objective lens of an inverted microscope 7, obtaining a microorganism solution 5, adding the microorganism solution 5 into a microorganism culture pond 2, submerging a solid surface material 4 by the microorganism solution 5 and not exceeding 4/5 of the capacity of the microorganism culture pond 2, adjusting the temperature, opening the inverted microscope 7, adjusting the height of the objective lens of the inverted microscope 7 until the objective lens is focused on the solid surface material 4, opening an image sensor 8 and an upper computer 9, and recording and analyzing the behavior of the microorganisms on the solid surface material 4 in real time.
In the technical scheme, the culture solution and culture of the microorganismThe conditions and the temperature regulation vary according to the microorganism, and can be determined according to the prior art, the microorganism of the present embodiment is, for example, staphylococcus aureus, preferably, the culture solution is TSB culture solution, and the culture conditions of the microorganism are: culturing with shake bacteria at 37 deg.C and 180rpm for 12h, and adjusting temperature to 37 deg.C; if Escherichia coli is used, the culture solution is preferably LB culture solution, and the culture conditions of the microorganism are as follows: culturing at 37 deg.C and 180rpm for 12h, and adjusting temperature to 37 deg.C. The microorganism of the present embodiment is preferably Staphylococcus aureus at a concentration of 105CFU/ml, e.g.E.coli, preferably at a concentration of 106CFU/ml。
The terms used in the present invention generally have 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 solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The in-situ characterization device for the interaction of microorganisms and a solid surface comprises a temperature-controlled hot table 1, 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, wherein the temperature-controlled hot table 1 is a cuboid container with an opening at the top end, a temperature-controlled hot table cover 1-1 is arranged at the opening at the top end, and a circular through hole is formed in the bottom surface of the inner wall of the temperature-controlled hot table 1; the microorganism culture pond 2 is a cylindrical 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 a circular 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 glass substrate 3 is disc-shaped, the edge of the glass substrate is clamped between the lower surface of the annular baffle plate 2-2 and the bottom surface of the inner wall of the temperature control heating table 1, and the upper surface of the glass substrate 3 and the lower surface of the annular baffle plate 2-2 are fixedly adhered through sealant 6NOA63 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 microorganism solution 5. The lower surface of the solid surface material 4 was fitted 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 was fixed to the upper surface of the glass substrate 3 by sealing with sealant 6NOA 63. The inverted microscope 7 is used for observing the solid surface material 4, an objective lens of the inverted microscope 7 is arranged under the solid surface material 4, a light source of the inverted microscope 7 is a halogen lamp, 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 through the objective lens of the inverted microscope 7 and is transmitted to the image sensor 8, the inverted microscope 7 is provided with an eyepiece with the magnification of 10 times, and the highest magnification is an oil lens with the magnification of 100 times. The image sensor 8 is a CCD image sensor, and the image sensor 8 collects an optical signal of the inverted microscope 7, converts the optical signal into an analog current signal, and amplifies and analog-to-digital converts the analog current signal to obtain a digital signal which is transmitted to the upper computer 9. The upper computer 9 comprises definition Analyze, FastStone Capture and Image J, wherein the definition Analyze converts the digital signals of the Image sensor 8 into pictures, the FastStone Capture converts picture recording into videos, the frame number is 25, and the Image J analyzes the videos. Wherein the solid surface material 4 is a polyurethane film having an area of 1cm × 1cm and a thickness of 0.10mm, and the surface pattern is a hexagonal close-packed hemispherical projection having a diameter of 5 μm. The thickness of the glass substrate 3 was 0.10 mm.
The in situ characterization method of the interaction of microorganisms with solid surfaces is as follows:
placing Staphylococcus aureus in TSB culture solution, shake culturing at 180rpm and 37 deg.C for 12h, diluting to 10%5CFU/ml, taking 2ml, adding into the microorganism culture pond 2, adjusting to 37 ℃, opening the inverted microscope 7, adjusting the height of an objective lens of the inverted microscope 7 until the objective lens is focused on the solid surface 4, opening the image sensor 8 and the upper computer 9, processing the received digital signals by the upper computer 9 to obtain the microorganism on the solid surface material (4)Real-time pictures of behaviors, and analyzing the pictures.
In FIG. 4, a-d are the results of bacterial adhesion and proliferation of Staphylococcus aureus of this example at time points of 1h, 2h, 3h and 4h, respectively. Fig. 5 shows the movement locus of staphylococcus aureus in the embodiment, wherein the marks 1, 2 and 3 respectively represent the movement locus of bacteria in different time periods of 1-2h, 2-3h and 3-4h, and meanwhile, the adhesion kinetic parameters of 175 pixels in the average movement path, 70s in average time and the like are obtained.
Example 2
In situ characterization device of the interaction of microorganisms with solid surface, as in example 1, only the solid surface material 4 was replaced with a polyurethane film having an area of 1cm × 1cm and a thickness of 0.05mm, and the surface pattern was hexagonal close-packed hemispherical protrusions with a diameter of 2 μm. The thickness of the glass substrate 3 was 0.17 mm.
The in situ characterization method of the interaction of microorganisms with solid surfaces is as follows:
placing Staphylococcus aureus in TSB culture solution, shake culturing at 180rpm and 37 deg.C for 12h, diluting to 10%5And (3) taking 2ml of the CFU/ml, adding the 2ml of the CFU/ml into the microorganism culture pond 2, adjusting the temperature to 37 ℃, opening the inverted microscope 7, adjusting the height of an objective lens of the inverted microscope 7 until the objective lens is focused on the solid surface 4, opening the image sensor 8 and the upper computer 9, processing the received digital signals by the upper computer 9, obtaining a real-time picture of the behavior of the microorganism on the solid surface material (4), and analyzing the picture.
Fig. 6 shows the movement locus of staphylococcus aureus on the surface, wherein the marks 1, 2 and 3 represent the movement locus of bacteria in different time periods of 1-2h, 2-3h and 3-4h respectively, and meanwhile, the adhesion kinetic parameters of 195 pixels in the average movement path and 52s in the average time are obtained.
Example 3
In the in-situ characterization device for the interaction between the microorganisms and the solid surface, as in example 1, only the solid surface material 4 is replaced by a polystyrene film, the area is 1cm × 1cm, and the thickness is about 0.05 mm; the surface pattern was hexagonal close-packed hemispherical protrusions with a diameter of 5 μm. The thickness of the glass substrate 3 was 0.17 mm.
The in situ characterization method of the interaction of microorganisms with solid surfaces is as follows:
placing Staphylococcus aureus in TSB culture solution, shake culturing at 180rpm and 37 deg.C for 12h, diluting to 10%5And (3) taking 2ml of the CFU/ml, adding the 2ml of the CFU/ml into the microorganism culture pond 2, adjusting the temperature to 37 ℃, opening the inverted microscope 7, adjusting the height of an objective lens of the inverted microscope 7 until the objective lens is focused on the solid surface 4, opening the image sensor 8 and the upper computer 9, processing the received digital signals by the upper computer 9, obtaining a real-time picture of the behavior of the microorganism on the solid surface material (4), and analyzing the picture.
Example 4
In situ characterization device of the interaction of microorganisms with solid surface, as in example 1, only the solid surface material 4 was replaced with a polyurethane film having an area of 1cm × 1cm and a thickness of 0.05mm, and the surface pattern was hexagonal close-packed hemispherical protrusions with a diameter of 5 μm. The thickness of the glass substrate 3 was 0.17 mm.
The in situ characterization method of the interaction of microorganisms with solid surfaces is as follows:
placing Escherichia coli in LB culture solution, shake culturing at 180rpm and 37 deg.C for 12h, and diluting to 106And (3) taking 2ml of the CFU/ml, adding the 2ml of the CFU/ml into the microorganism culture pond 2, adjusting the temperature to 37 ℃, opening the inverted microscope 7, adjusting the height of an objective lens of the inverted microscope 7 until the objective lens is focused on the solid surface 4, opening the image sensor 8 and the upper computer 9, processing the received digital signals by the upper computer 9, obtaining a real-time picture of the behavior of the microorganism on the solid surface material (4), and analyzing the picture.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the 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.
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| CN114112920A (en) * | 2021-12-10 | 2022-03-01 | 中国科学院长春应用化学研究所 | Fluorescence detection sample pool capable of intelligently and accurately regulating and controlling solution concentration on line and control method |
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| CN1208739C (en) * | 2003-04-16 | 2005-06-29 | 浙江大学 | Computer controlled low temperature biology microscopic system |
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