CN112129797A - Bacterium sample preparation method and kit for scanning electron microscope - Google Patents
Bacterium sample preparation method and kit for scanning electron microscope Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
- G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/60—Specific applications or type of materials
- G01N2223/612—Specific applications or type of materials biological material
Abstract
The invention discloses a bacteria sample preparation method and a kit for a scanning electron microscope, belonging to the field of microorganism detection methods. The sample preparation method comprises the following steps: 1) covering the glass sheet with chitosan solution, and naturally drying to cover a layer of chitosan film on the surface of the glass sheet; 2) placing the bacterial suspension on the surface of a glass sheet coated with a chitosan membrane, and standing to enable thalli to be adsorbed on the chitosan membrane; 3) fixing aldehyde solution, and washing with phosphate buffer solution; 4) dehydrating the ethanol solution with gradient concentration; 5) drying at a carbon dioxide critical point; 6) and (6) gold plating. The kit comprises the chitosan solution in the step 1). The method can firmly fix the bacteria on the glass sheet and is convenient for observation by a scanning electron microscope.
Description
Technical Field
The invention belongs to the field of microbial detection methods.
Background
Most biological samples including bacteria have the characteristics of high water content, soft texture, poor conductivity and the like, and before scanning electron microscope observation, the samples are correspondingly treated to contain no moisture, keep the appearance of the samples to the maximum extent, and have good conductivity and higher secondary electron emissivity. Fixing the sample by using neutral formalin, glutaraldehyde or other aldehyde solutions to keep the appearance of the sample and harden the sample; then, dehydrating by using ethanol with concentration gradient, and gradually replacing the water contained in the sample by using the ethanol; removing ethanol in the sample by using a carbon dioxide critical point drying method to completely dry the sample; and finally, plating a layer of gold on the surface of the sample by an ion sputtering method to ensure that the sample is conductive and is convenient for observation by a scanning electron microscope.
The existing fixing, cleaning and dehydrating of bacteria samples are carried out in a centrifugal tube, after each step of operation, thalli and a fixing solution, a phosphate buffer solution and ethanol with various concentrations need to be separated through centrifugation, a supernatant is discarded, then a solution of the next step of operation is added to resuspend the thalli, and the operation is repeated. The dried thallus powder is picked out and stuck on a carbon adhesive tape and then plated with gold by ion sputtering. Study on the influence of the Wangwei and the like on the morphology and surface ultrastructure of escherichia coli in simulated microgravity environment [ J ]. chemical and biological engineering, 2011, 28: 14-17 discloses a method for preparing a sample of escherichia coli for a scanning electron microscope, wherein in the steps of fixing, cleaning, gradient dehydration and the like, each step needs to be centrifuged at 4000rpm for 3-5 min, the supernatant is discarded, the next reagent is added, a dropper is used for sucking back for several times, and the bacterium blocks are scattered. Zhouyi et al in observing the influence of an unplated conductive film on escherichia coli damage and images using a scanning electron microscope [ J ]. agricutural science, 2009, 37: 15692-. A method for quickly preparing a staphylococcus aureus sample for a scanning electron microscope (application number 201610481585.2) by Songming pearl and the like discloses a method for preparing the staphylococcus aureus sample for the scanning electron microscope, wherein in the steps of fixing, cleaning, gradient dehydration and the like, the centrifugation is carried out for 3-5 min at 8000rpm in each step, the supernatant is discarded, the next reagent is added, and the reagent is blown and beaten for 3-5 times by a liquid transfer gun each time to break up bacterium blocks. In the above published papers and patents, the whole sample preparation process requires centrifugation and re-suspension of the cells many times, which not only consumes a lot of time, but also easily causes shedding of fine structures such as flagella and the like in the frequent operation process; on the other hand, a plurality of centrifuge tubes are simultaneously put into a carbon dioxide critical point dryer for drying, once the air pressure changes slightly, the thalli are easily sprayed out of the centrifuge tubes, and the cross contamination of samples is caused.
In addition, wang xiao dong et al disclosed a method for preparing a sticky sheet for a scanning electron microscope observation sheet and a preparation method thereof (application No. 201610965957.9), which mainly prepared sticky sheet agent from glutinous rice flour and egg white, mixed with thalli fixed with glutaraldehyde, adhered the mixed solution on a glass slide, and dried naturally and then subjected to conventional gradient dehydration. The preparation process of the sticky tablet comprises grinding and drying glutinous rice flour (the temperature must reach 130 ℃ and the duration is not more than 2 hours) to achieve a better gelatinization effect, the egg white is preferably selected from native eggs, and the filtration sterilization is performed according to the technical requirements of aseptic operation of microbiological experiments. The preparation of the sticking agent in the invention is complex, the requirement on raw materials is high, and the sticking agent and the thalli are mixed together, so that part of the thalli is possibly wrapped by the sticking agent, and the complete appearance can not be observed under a scanning electron microscope.
Disclosure of Invention
The invention aims to solve the problems that: provides a novel bacterial sample preparation method for scanning electron microscope observation.
The technical scheme of the invention is as follows:
the invention provides a method for fixing bacteria on a glass sheet, which comprises the following steps:
1) covering the glass sheet with chitosan solution, and naturally drying to cover a layer of chitosan film on the surface of the glass sheet;
2) and (3) placing the bacterial suspension on the surface of the glass sheet coated with the chitosan membrane, and standing to enable the bacteria to be adsorbed on the chitosan membrane.
The method as described above, the concentration of the chitosan solution in step 1) is 0.1-3% (m/v), preferably 2.5% (m/v).
The method comprises the steps of 1) taking an acetic acid solution as a solvent for the chitosan solution;
preferably, the concentration of the acetic acid solution is 0.5-2% (v/v); further preferably, the acetic acid solution concentration is 1% (v/v).
The method is as described above, the standing in the step 2) lasts for more than 5 min; preferably, the standing duration is 30 min.
As in the previous method, the bacterium is Escherichia coli or Staphylococcus aureus.
The invention also provides a bacteria sample preparation method for the scanning electron microscope, which comprises the following steps:
1) immobilizing the bacterial suspension to the cell slide using the method described above;
2) adding aldehyde solution to fix bacteria, and washing with phosphate buffer solution;
3) adding ethanol solution with gradient concentration to dehydrate bacteria;
4) drying the bacteria at the critical point of carbon dioxide;
5) plating gold on the surface of the bacteria.
In the sample preparation method, the bacteria are Escherichia coli or Staphylococcus aureus.
According to the sample preparation method, the aldehyde solution in the step 2) is glutaraldehyde, formaldehyde or paraformaldehyde solution.
The sample preparation method as described above, the OD of the bacterial suspension in the step 1)6000.2 to 0.8; preferably, the concentration is 0.3 to 0.6.
The invention also provides a bacterial mucoadhesive tablet for scanning electron microscope samples, which is a chitosan solution with the concentration of 0.1-3% (m/v) prepared by taking 0.5-2% (v/v) acetic acid solution as a solvent.
The adhesive tablet as described above, wherein the acetic acid solution has an acetic acid content of 1% (v/v);
and/or the concentration of chitosan in the chitosan solution is 0.1-3% (m/v); preferably, it is 2.5%.
The present invention also provides a bacterial sampling kit for scanning electron microscopy, comprising the adhesive sheet according to claim 8 or 9.
The invention has the beneficial effects that:
compared with the conventional sample preparation method for the scanning electron microscope, the method reduces the operation steps of centrifugation and thallus re-suspension, reduces the probability of damage to fine structures such as bacterial flagella and the like on one hand, greatly shortens the sample preparation time on the other hand, and reduces the workload. In addition, bacteria are adsorbed on the chitosan membrane in the early stage of sample preparation, so that the operation of picking out the bacteria and adhering the bacteria to a carbon tape after the bacteria are dried in the conventional method is omitted. Finally, in the sample prepared by the invention, the thalli are basically and uniformly distributed on the cell slide, the stacking phenomenon of large thalli and thick thalli which influence the image acquisition does not occur, and the efficiency of acquiring the image by an electron microscope is greatly improved.
Compared with the sticking agent (prepared from glutinous rice flour and egg white) of the Wang Xiaodong and the like, the sticking agent disclosed by the invention is simple to prepare and operate, has lower material requirement and is more beneficial to industrial production.
Research reports that chitosan is grafted on the surface of medical terylene to reduce the adhesion of bacteria (chemical grafting method preparation of medical terylene surface chitosan and bacterial adhesion research, functional material, volume 35 of 2004, page 2443). The invention increases the adhesion of bacteria through chitosan, and a large amount of bacteria still do not fall off after the chitosan is cleaned by phosphate buffer solution, ethanol and other solutions, thereby obtaining unexpected technical effects.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a scanning electron microscope image (5 μm scale) of Pseudomonas aeruginosa according to example 1 of the present invention.
FIG. 2 is a scanning electron microscope image (scale of 1 μm) of Pseudomonas aeruginosa according to example 1 of the present invention.
FIG. 3 is a scanning electron microscope image (5 μm scale) of Staphylococcus aureus in example 2 of the present invention.
FIG. 4 is a graph showing the effect of Staphylococcus aureus in example 2 of the present invention on scanning electron microscopy (scale of 1 μm).
FIG. 5 is a graph showing the effect of different concentrations of chitosan on the ratio of Staphylococcus aureus adhesion.
Detailed Description
EXAMPLE 1 scanning Electron microscopy of Pseudomonas aeruginosa
1. Method of producing a composite material
(1) Taking 100mL of 1% acetic acid solution, slowly adding 0.5g of chitosan, stirring, heating and dissolving to obtain 0.5% chitosan solution;
(2) taking a cell slide (a round glass sheet) with the diameter of 8mm, cleaning with absolute ethyl alcohol, and naturally drying;
(3) putting the clean cell slide into the chitosan solution, enabling the surface of the cell slide to be stained with the chitosan solution, taking out the cell slide and naturally drying the cell slide to enable the surface of the cell slide to be covered with a layer of chitosan film;
(4) collecting Pseudomonas aeruginosa in logarithmic phase, centrifuging at 4 deg.C for 5min at 3000g, removing supernatant, adding 0.1M phosphate buffer solution with pH of 7.3, resuspending thallus, and adjusting bacterial suspension concentration to OD600＝0.3～0.6；
(5) Sucking 15 mu L of bacterial suspension, dripping the bacterial suspension on the surface of a cell climbing sheet coated with chitosan, standing for 1h at room temperature to enable thalli to be adsorbed on the surface of a chitosan membrane;
(6) placing the cell slide into a 12-hole plate, adding 1mL of glutaraldehyde solution with the concentration of 2.5% (v/v) into each hole, and fixing at 4 ℃ overnight;
(7) discarding glutaraldehyde solution, adding 1mL of 0.1M phosphate buffer solution with pH7.3 into each well, and slowly shaking on a decoloring shaking table for 5min to clean cell slide;
(8) discarding the phosphate buffer solution, sequentially adding 1mL of 50%, 60%, 70%, 80%, 90% and 100% (v/v) ethanol solution with concentration gradient to each hole for dehydration, adding the ethanol solution, slowly shaking on a decoloring shaking table for 5min, then discarding the ethanol solution, and then adding the ethanol solution with the next concentration to repeat the operation;
(9) drying the cell slide in a carbon dioxide critical point dryer;
(10) adhering the dried cell slide to a sample table of a scanning electron microscope through a carbon adhesive tape, and plating gold in an ion sputtering instrument to finish the preparation of the sample;
(11) and (3) placing the prepared sample into a scanning electron microscope to observe the form of the pseudomonas aeruginosa.
2. Results
As shown in the figures 1 and 2, under a scanning electron microscope, the pseudomonas aeruginosa sample prepared by the method has complete thallus form and relatively uniform distribution, is independently scattered and is stacked in multiple layers, but does not have large thallus and thallus stack with large fluctuation difference, and a target area for picking the image is easy to find.
EXAMPLE 2 scanning Electron microscopy of Staphylococcus aureus
1. Method of producing a composite material
(1) Taking 100mL of 1% acetic acid solution, slowly adding 1.5g of chitosan, stirring, heating and dissolving to obtain 1.5% chitosan solution;
(2) taking a cell slide with the diameter of 6mm, cleaning with absolute ethyl alcohol, and naturally drying;
(3) putting the clean cell slide into the chitosan solution, enabling the surface of the cell slide to be stained with the chitosan solution, taking out the cell slide and naturally drying the cell slide to enable the surface of the cell slide to be covered with a layer of chitosan film;
(4) collecting Staphylococcus aureus in logarithmic phase, centrifuging at 4 deg.C for 5min at 3000g, removing supernatant, adding 0.1M phosphate buffer solution with pH of 7.3, resuspending thallus, and adjusting suspension concentration to OD600＝0.2～0.5；
(5) Sucking 10 μ L of bacterial suspension, dripping onto the surface of cell slide coated with chitosan, standing at 4 deg.C for 30min to make thallus adsorbed onto the chitosan membrane surface;
(6) placing the cell slide into a 24-well plate, adding 0.7mL of glutaraldehyde solution with the concentration of 2.5% (v/v) into each well, and fixing at 4 ℃ overnight;
(7) discarding glutaraldehyde solution, adding 0.1M phosphate buffer solution (0.7 mL) with pH of 7.3 into each well, and slowly shaking on a decoloring shaking table for 5min to clean cell slide;
(8) discarding the phosphate buffer solution, sequentially adding 0.7mL of 50%, 60%, 70%, 80%, 90% and 100% (v/v) ethanol solution with concentration gradient to each well for dehydration, adding the ethanol solution, slowly shaking on a decoloring shaking table for 5min, then discarding the ethanol solution, and then adding the ethanol solution with the next concentration to repeat the operation;
(9) drying the cell slide in a carbon dioxide critical point dryer;
(10) adhering the dried cell slide to a sample table of a scanning electron microscope through a carbon adhesive tape, and plating gold in an ion sputtering instrument to finish the preparation of the sample;
(11) and (3) putting the prepared sample into a scanning electron microscope to observe the form of the staphylococcus aureus.
2. Results
Under a scanning electron microscope, the staphylococcus aureus sample prepared by the method has complete bacteria shape and relatively uniform distribution, is independently scattered and stacked in multiple layers, does not have large bacteria and bacteria with large fluctuation difference, and is easy to find a target area (fig. 3 and fig. 4) for image collection.
EXAMPLE 3 Effect of different concentrations of Chitosan on bacterial sample preparation and scanning Electron microscopy
1. Method of producing a composite material
(1) Slowly adding chitosan with different masses into acetic acid solution with the concentration of 1%, stirring, heating and dissolving to prepare chitosan solution with the concentration of 0.1%, 1.5%, 2.5% and 3% (w/v);
(2) placing the cell slide with diameter of 8mm cleaned with anhydrous ethanol and naturally air dried into chitosan solutions with different concentrations, allowing the surface of the cell slide to be stained with the chitosan solution, taking out and naturally drying, and covering a layer of chitosan membrane on the surface of the cell slide to obtain chitosan cell slide with concentration of 4 chitosan
(3) Collecting Staphylococcus aureus in logarithmic growth phaseCentrifuging at 4 deg.C for 5min at 3000g, discarding supernatant, adding 0.1M phosphate buffer solution with pH7.3 to resuspend thallus, and adjusting OD of suspension600To about 0.2
(4) Sucking 10 μ L of bacterial suspension, respectively dripping onto the surface of 4 chitosan cell slide, standing at 4 deg.C for 30min to make thallus adsorbed onto the chitosan membrane surface;
(5) placing the cell slide into a 12-hole plate, adding 1mL of glutaraldehyde solution with the concentration of 2.5% (v/v) into each hole, and fixing at 4 ℃ overnight;
(6) discarding glutaraldehyde solution, adding 1mL of 0.1M phosphate buffer solution with pH7.3 into each well, and slowly shaking on a decoloring shaking table for 5min to clean cell slide;
(7) discarding the phosphate buffer solution, sequentially adding 1mL of 50%, 60%, 70%, 80%, 90% and 100% (v/v) ethanol solution with concentration gradient to each hole for dehydration, adding the ethanol solution, slowly shaking on a decoloring shaking table for 5min, then discarding the ethanol solution, and then adding the ethanol solution with the next concentration to repeat the operation;
(8) removing the ethanol solution, adding 1mL of 0.1M phosphate buffer solution with pH7.3 into each well, and slowly shaking on a decoloring shaking table for 5min to clean the cell slide;
(9) discarding the phosphate buffer solution, adding 0.5mL of DAPI dye solution with the concentration of 20 mu g/mL into each hole, and processing for 5min in a dark place;
(10) discarding DAPI staining solution, adding 1mL of 0.1M phosphate buffer solution with pH7.3 into each well, and slowly shaking on a decoloring shaking table for 5min to clean cell slide, wherein the operation is carried out for 3 times, which is an experimental group;
(11) sucking 10 mu L of bacterial suspension, respectively dripping the bacterial suspension on the surface of the 4 chitosan cell slide, and naturally drying;
(12) placing the cell slide into a 12-well plate, adding 0.5mL of DAPI staining solution with the concentration of 20 mu g/mL into each well, and processing for 5min in a dark place;
(13) repeating the step (10), wherein the step is a control group;
(14) respectively observing the experimental group and the control group by using an inverted fluorescence microscope, and counting fluorescence signals;
(15) calculating the percentage of bacterial adhesion by the formula: percent adhesion ═ (number of experimental/control fluorescence signals) × 100%;
(16) the above operation was repeated 3 times.
2. Results
With the increase of the chitosan concentration, the adhesion percentage of the staphylococcus aureus on the cell slide is gradually increased, and the number of the thalli observed by a scanning electron microscope is increased (figure 5). If the adhesion percentage is low, the thallus observed under a scanning electron microscope is sparse, image acquisition is not facilitated, and the concentration of the bacterial suspension needs to be improved to achieve the effect of high-concentration chitosan cell slide. The adhesion performance of the cell climbing sheets with the chitosan concentration of 2.5 percent and 3 percent to staphylococcus aureus has no obvious difference, and because the chitosan film of the cell climbing sheets with the chitosan concentration of 3 percent is thicker, in practical application, after the cell climbing sheets are dried by carbon dioxide critical points, partial chitosan film of partial cell climbing sheets can deform and break, but the observation of a scanning electron microscope on bacteria is not influenced.
Considering comprehensively, the cell slide with 2.5% chitosan concentration is the best scheme.
In conclusion, compared with the conventional operation, the method reduces the operation steps of centrifugation and thallus re-suspension of the bacterial sample in the sample preparation process of the scanning electron microscope, reduces the probability of damage to fine structures such as bacterial flagella and the like on one hand, greatly shortens the sample preparation time on the other hand, and reduces the workload. In addition, bacteria are adsorbed on the chitosan membrane in the early stage of sample preparation, so that the operation of picking out the bacteria and adhering the bacteria to a carbon tape after the bacteria are dried in the conventional method is omitted. Finally, in the sample prepared by the invention, thalli are complete in shape and basically and uniformly distributed on the cell slide, the phenomenon of stacking of large thalli and thick thalli which influence image acquisition does not occur, and the efficiency of acquiring images by an electron microscope is greatly improved.
Claims (10)
1. A method of immobilizing bacteria on a glass sheet, comprising: the method comprises the following steps:
1) covering the glass sheet with chitosan solution, and naturally drying to cover a layer of chitosan film on the surface of the glass sheet;
2) and (3) placing the bacterial suspension on the surface of the glass sheet coated with the chitosan membrane, and standing to enable the bacteria to be adsorbed on the chitosan membrane.
2. The method of claim 1, wherein: step 1), the concentration of the chitosan solution is 0.1-3% (m/v); preferably, it is 2.5% (m/v).
3. The method of claim 1 or 2, wherein: step 1), taking an acetic acid solution as a solvent for the chitosan solution;
preferably, the concentration of the acetic acid solution is 0.5-2% (v/v); further preferably, the acetic acid solution concentration is 1% (v/v);
further preferably, said standing of step 2) lasts for more than 5 min; preferably, the standing duration is 30 min.
4. The method of claim 1, wherein: the bacteria is Escherichia coli or Staphylococcus aureus.
5. A bacteria sample preparation method for a scanning electron microscope is characterized by comprising the following steps: the sample preparation method comprises the following steps:
1) fixing the bacterial suspension to a cell slide using the method of any one of claims 1 to 4;
2) adding aldehyde solution to fix bacteria, and washing with phosphate buffer solution;
3) adding ethanol solution with gradient concentration to dehydrate bacteria;
4) drying the bacteria at the critical point of carbon dioxide;
5) plating gold on the surface of the bacteria.
6. The sample preparation method of claim 5, wherein: the bacteria is Escherichia coli or Staphylococcus aureus.
7. The sample preparation method of claim 5, wherein: OD of the bacterial suspension in the step 1)6000.2 to 0.8; preferably, the content is 0.3-0.6;
preferably, the aldehyde solution of step 2) is glutaraldehyde, formaldehyde or paraformaldehyde solution.
8. A bacterial muco-tablet for scanning electron microscope specimens, characterized by: the sticky tablet is a chitosan solution with the concentration of 0.1-3% (m/v) prepared by taking 0.5-2% (v/v) acetic acid solution as a solvent.
9. The sticking tablet according to claim 8, wherein:
the acetic acid content of the acetic acid solution is 1% (v/v);
and/or the concentration of chitosan in the chitosan solution is 0.1-3% (m/v); preferably, it is 2.5% (m/v).
10. A bacterial sampling kit for a scanning electron microscope, characterized in that: the kit comprising the adhesive sheet according to claim 8 or 9.
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Citations (5)
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|---|---|---|---|---|
| JPH1114909A (en) * | 1997-06-20 | 1999-01-22 | Bio Quest:Kk | Polymer coating slide glass |
| US20070020649A1 (en) * | 2005-03-11 | 2007-01-25 | E.I. Du Pont De Nemours And Company | Chitosan capture of microorganisms for detection |
| CN106198135A (en) * | 2016-06-24 | 2016-12-07 | 成都大学 | The method that staphylococcus aureus scanning electron microscope sample is quickly prepared |
| CN106596608A (en) * | 2016-11-01 | 2017-04-26 | 河南科技大学 | Sticking agent for making bacterial scanning electron microscope observation slice and preparation method thereof |
| WO2019229198A1 (en) * | 2018-05-30 | 2019-12-05 | Centre National De La Recherche Scientifique | A film of chitosan and a device comprising the same deposited on a substrate and uses thereof |
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| JPH1114909A (en) * | 1997-06-20 | 1999-01-22 | Bio Quest:Kk | Polymer coating slide glass |
| US20070020649A1 (en) * | 2005-03-11 | 2007-01-25 | E.I. Du Pont De Nemours And Company | Chitosan capture of microorganisms for detection |
| CN106198135A (en) * | 2016-06-24 | 2016-12-07 | 成都大学 | The method that staphylococcus aureus scanning electron microscope sample is quickly prepared |
| CN106596608A (en) * | 2016-11-01 | 2017-04-26 | 河南科技大学 | Sticking agent for making bacterial scanning electron microscope observation slice and preparation method thereof |
| WO2019229198A1 (en) * | 2018-05-30 | 2019-12-05 | Centre National De La Recherche Scientifique | A film of chitosan and a device comprising the same deposited on a substrate and uses thereof |
| US20210215694A1 (en) * | 2018-05-30 | 2021-07-15 | Centre National De La Recherche Scientifique | Film of chitosan and a device comprising the same deposited on a substrate and uses thereof |
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