CN111024594B - Observation method for metal material corroded by microorganism adhesion - Google Patents
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N17/00—Investigating resistance of materials to the weather, to corrosion, or to light
- G01N17/006—Investigating resistance of materials to the weather, to corrosion, or to light of metals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/32—Polishing; Etching
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract
The invention provides a method for observing a metal material corroded by microorganism adhesion, which sequentially comprises the following steps: polishing the surface of a metal sample; carrying out electrolytic double spraying on a metal sample by taking perchloric acid ethanol solution as electrolyte; cleaning a metal sample with an ethanol solution immediately, drying the metal sample in an oxygen-free manner, and then preserving the metal sample in vacuum; standing the metal sample in a marine microorganism solution; a metal sample is washed by PBS buffer solution and then soaked in glutaraldehyde solution with the mass fraction of 2.5-2.7% for 10-15 min; the liquid adhering to the metal sample was removed and observed by TEM. The observation method can realize the observation and recording of the dynamic process of the microbial corrosion interface on the surface of the metal material corroded by the microbes, and the observation method can be used for observing the microbial corrosion after polishing the metal sample, so that compared with the prior method of polishing the sample after the microbial corrosion, the method avoids the loss of the information of the microbes and obtains more information of the metal material corroded by the microbes.
Description
Technical Field
The invention relates to the field of microscopic detection of materials, in particular to a method for observing a metal material corroded by microorganism adhesion.
Background
The 21 st century is that of the ocean. A large amount of surface protection materials are required in the fields of marine resource development, marine transportation, harbors, defense construction and the like. Statistically, the material damage related to the marine microorganism attachment accounts for 70-80% of the total amount of the marine material, and the energy consumption and corrosion loss caused by the attachment of the microorganism is up to billions of dollars every year, so that the research on the marine surface protective material with the attachment corrosion of the microorganism is particularly important, and a corresponding method is needed for researching the marine surface protective material with the attachment corrosion of the microorganism, but the existing detection method of the marine surface protective material with the attachment corrosion of the microorganism can cause the loss of microorganism information in the sample processing process.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for observing a metal material corroded by microorganism adhesion.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for observing microbial adhesion corrosion of a metal material, the method comprising the steps of:
(1) polishing the metal sample and then performing mechanical surface polishing;
(2) under the conditions that the polishing voltage is 20-25V and the temperature is minus 20-minus 22 ℃, carrying out electrolytic double spraying on the metal sample obtained in the step (1) by taking perchloric acid ethanol solution as electrolyte;
(3) cleaning the metal sample treated in the step (2) by using an ethanol solution immediately and then drying without oxygen;
(4) dissolving bittern in deionized water to obtain solution A with salinity of 2.8-3.5%, and adding marine microorganisms and a culture medium into the sterilized and cooled solution A to obtain a marine microorganism solution;
(5) immersing the metal sample treated in the step (3) in the marine microorganism solution in the step (4) and standing for 24-72 hours;
(6) washing the metal sample treated in the step (5) by using a PBS (phosphate buffer solution) and then soaking the metal sample in a glutaraldehyde solution with the mass fraction of 2.5-2.7% for 10-15 min;
(7) and (4) removing the adhering liquid from the metal sample treated in the step (6) and observing the metal sample by using a TEM.
The observation method can realize the observation and recording of the dynamic process of the microbial corrosion interface on the surface of the metal material corroded by the microbes, and the observation method can be used for observing the microbial corrosion after polishing the metal sample, so that compared with the prior method of polishing the sample after the microbial corrosion, the method avoids the loss of the information of the microbes and obtains more information of the metal material corroded by the microbes.
Preferably, the material of the metal sample is carbon steel, low alloy steel, stainless steel or copper-containing steel.
The inventor finds that the observation method can be suitable for all metal materials applicable to ocean engineering application, and has better effect when the material of the metal sample is carbon steel, low alloy steel, stainless steel or copper-containing steel.
Preferably, the material of the metal sample is EH32 ship plate steel, 316L stainless steel, and 316L stainless steel containing 1.0 wt% of copper.
Preferably, the marine microorganism is vibrio natriegens or pseudomonas aeruginosa.
The inventor finds that the observation method can be suitable for all culturable marine microorganisms, and the observation effect is better when the method is suitable for vibrio natriegens or pseudomonas aeruginosa.
Preferably, in the step (2), the mass fraction of the perchloric acid ethanol solution is 5-10%.
The inventor finds that, when the electrolyte for electrolytic double spraying in the step (2) of the method is a perchloric acid ethanol solution with the mass fraction of 5% -10%, the information obtained by the observation method is larger and clearer.
Preferably, in the step (2), the flow rate of the electrolyte is 9ml/cm2～11ml/cm2。
Preferably, in the step (1), the metal sample is ground by using sand paper with the mesh number gradually increased to 2000 meshes, the mechanical surface is polished until no scratch is observed through a metallographic microscope, the mechanical surface is cleaned and dried in an oxygen-free manner after being polished, the cleaning agent for cleaning the mechanical surface after being polished is an ethanol solution, and the nitrogen blow-drying method for drying the mechanical surface without oxygen after being polished is adopted.
According to the observation method, the mechanical surface of the metal sample is polished to be free of scratches when being observed by a metallographic microscope, so that the influence of the surface condition of the metal sample on an observation result is avoided, and the information obtained by the observation method is larger and clearer.
Preferably, in the step (3), the metal sample is stored under vacuum after oxygen-free drying, and the nitrogen blow drying is adopted as the oxygen-free drying method.
The observation method avoids the surface of the metal sample from being oxidized after the sample treated in the step (3) is stored in vacuum.
Preferably, in the step (4), the OD value of the marine microorganism solution is 2.0-4.8, and the culture medium is 2216E liquid culture medium.
Preferably, in the step (4), the sterilization temperature is 120-121 ℃, the sterilization time is 15-20 min, and the temperature after sterilization and cooling is 20-25 ℃.
Preferably, in the step (1), the thickness of the metal sample before treatment is 0.5mm to 0.7 mm.
The observation method described above enables more and clearer information to be obtained using metal samples having a thickness of 0.5mm to 0.7 mm.
Preferably, in the step (7), the method for removing the adhering liquid from the metal sample treated in the step (6) comprises the following steps: and (4) sequentially placing the metal samples treated in the step (6) into ethanol solutions with the volume fractions gradually increasing from 50% to 100%.
Preferably, in the step (7), the method for removing the adhering liquid from the metal sample treated in the step (6) comprises the following steps: and (3) dehydrating the metal sample treated in the step (6) in ethanol solutions with the volume fractions of 50%, 60%, 70%, 80%, 90% and 100% in sequence.
The invention has the beneficial effects that: the invention provides an observation method of a microorganism attached corrosion metal material, which can realize observation and recording of a dynamic process of a microorganism corrosion interface on the surface of the microorganism attached corrosion metal material.
Drawings
FIG. 1 is a diagram showing the effect of the observation method according to the embodiment of the present invention, in which (A) is a control diagram and (B) is a sample diagram.
FIG. 2 is a diagram showing the effect of the observation method according to the embodiment of the present invention, wherein (A) is a control diagram and (B) is a sample diagram.
FIG. 3 is a diagram showing the effect of the observation method according to the embodiment of the present invention, wherein (A) is a control diagram and (B) is a sample diagram.
FIG. 4 is a diagram showing the effect of the observation method according to the embodiment of the present invention, wherein (A) is a control diagram and (B) is a sample diagram.
FIG. 5 is a diagram showing the effect of the observation method according to the embodiment of the present invention, wherein (A) is a control diagram and (B) is a sample diagram.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The method for observing the metal material adhered and corroded by the microorganisms, which is an embodiment of the invention, comprises the following steps:
(1) cutting a stainless steel metal sample to be 0.5-0.7 mm in thickness, sequentially polishing the stainless steel metal sample with the thickness of 0.5-0.7 mm by using 150-mesh, 400-mesh, 800-mesh, 1500-mesh and 2000-mesh abrasive paper, then polishing the surface by using a metal test polishing machine at the rotating speed of 120-150 rmp until no scratch is observed by a metallographic microscope, and cleaning by using an ethanol solution and drying by using nitrogen after polishing the surface;
(2) under the conditions that the polishing voltage is 20-25V and the temperature is 20-22 ℃ below zero, the metal sample obtained in the step (1) takes 5-10% of perchloric acid ethanol solution as electrolyte for electrolytic double spraying, and the flow rate of the electrolyte is 9ml/cm2～11ml/cm2；
(3) Cleaning the metal sample treated in the step (2) with an ethanol solution within 2-6 s, drying the metal sample with nitrogen, and performing vacuum storage on the metal sample after drying with nitrogen;
(4) dissolving bittern in deionized water to obtain a solution A with the salinity of 2.8-3.5%, and adding 2216E bacterial liquid of vibrio natriegens into the sterilized and cooled solution A to obtain a marine microorganism solution, wherein the OD value of the marine microorganism solution is 2.0-4.8;
(5) immersing the metal sample treated in the step (3) in the marine microorganism solution in the step (4) and standing for 24 hours;
(6) washing the metal sample treated in the step (5) with PBS buffer solution for 3-4 times, and soaking in glutaraldehyde solution with the mass fraction of 2.5% -2.7% for 10-15 min;
(7) and (5) placing the metal sample treated in the step (6) in ethanol solutions with volume fractions of 50%, 60%, 70%, 80%, 90% and 100% in sequence, dehydrating, and observing by using a TEM (transmission electron microscope).
A control experiment was set up, which differs from this example in that: in the step (4), the 2216E bacterial liquid of vibrio natriegens is not added into the solution A, and in the step (5), the metal sample treated in the step (3) is immersed into the sterilized and cooled solution A.
As shown in FIG. 1(B), Vibrio natriegens have firmly attached to the surface of the stainless steel sample without causing destructive influence on the structure (dislocations, grain boundaries, etc.) of the stainless steel sample; as shown in FIG. 1(A), the corrosion of the control sample is mainly uniform corrosion, and the microstructure of the matrix has no obvious change, which illustrates that the method of the embodiment can be used for observing the influence of microorganisms on the corrosion of the metal sample.
Example 2
As a method for observing a metal material corroded by microorganisms attached thereto according to an embodiment of the present invention, the only differences between this embodiment and embodiment 1 are: the metal sample is a carbon steel metal sample.
As shown in FIG. 2(B), Vibrio natriegens have firmly attached to the surface of the carbon steel sample, and have had a destructive effect on the internal structure (dislocations, grain boundaries, etc.) of the carbon steel sample, resulting in perforation. As shown in fig. 2(a), the control sample has no obvious perforation, the corrosion is mainly uniform corrosion, and the microstructure of the matrix has no obvious change, which illustrates that the method of this embodiment can observe the influence of the microorganisms on the corrosion of the metal sample.
Example 3
As an observation method of a metal material corroded by microorganism adhesion, the only difference between this embodiment and embodiment 1 is: the metal samples were low alloy steel metal samples.
As shown in fig. 3(B), vibrio natriei is required to be firmly attached to the surface of the low alloy steel metal sample, and destructive influence is caused on the internal structure (dislocation, grain boundary and the like) of the low alloy steel metal sample, so that corrosion gaps are caused. As shown in FIG. 3(A), the low alloy steel metal sample has no obvious perforation, mainly uniform corrosion and no obvious change of the microstructure of the matrix.
Example 4
As a method for observing a metal material corroded by microorganisms attached thereto according to an embodiment of the present invention, the only differences between this embodiment and embodiment 1 are: in the step (4), the marine microorganism is pseudomonas aeruginosa.
As shown in fig. 4(B), pseudomonas aeruginosa had firmly attached to the surface of the stainless steel metal sample, and had a destructive effect on the internal structure (dislocations, grain boundaries, etc.) of the stainless steel metal sample. As shown in FIG. 4(A), the stainless steel metal sample has no obvious perforation, the corrosion is mainly uniform corrosion, and the microstructure of the matrix has no obvious change.
Example 5
As an observation method of a metal material corroded by microorganism adhesion, the only difference between this embodiment and embodiment 1 is: the metal sample was stainless steel containing 1.0 wt% copper.
As shown in fig. 5(B), pseudomonas aeruginosa failed to adhere to the surface of stainless steel having a copper content of 1.0 wt%, and did not exert a destructive influence on the internal structure (dislocations, grain boundaries, etc.) of stainless steel having a copper content of 1.0 wt%. As shown in FIG. 5(A), the stainless steel containing 1.0 wt% of copper has no significant perforation, the corrosion is mainly uniform corrosion, and the microstructure of the matrix has no significant change.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for observing the adhesion and corrosion of a metal material by microorganisms, which is characterized by comprising the following steps:
(1) polishing a metal sample with the thickness of 0.5 mm-0.7 mm, and then carrying out mechanical surface polishing; the metal sample is made of carbon steel, low alloy steel or copper-containing steel;
(2) carrying out electrolytic double spraying on the metal sample obtained in the step (1) by taking perchloric acid ethanol solution as electrolyte under the conditions that the polishing voltage is 20-25V and the temperature is-20-22 ℃;
(3) cleaning the metal sample treated in the step (2) by using an ethanol solution immediately and then drying without oxygen;
(4) dissolving bittern in deionized water to obtain solution A with salinity of 2.8-3.5%, and adding marine microorganisms and culture medium into the sterilized and cooled solution A to obtain marine microorganism solution;
(5) immersing the metal sample treated in the step (3) in the marine microorganism solution in the step (4) and standing for 24-72 hours;
(6) washing the metal sample treated in the step (5) by using a PBS (phosphate buffer solution) and then soaking the metal sample in a glutaraldehyde solution with the mass fraction of 2.5-2.7% for 10-15 min;
(7) and (4) removing the adhering liquid from the metal sample treated in the step (6) and observing the metal sample by using a TEM.
2. The method of claim 1, wherein the marine microorganism is vibrio natriegens or pseudomonas aeruginosa.
3. The method according to claim 1, wherein in the step (2), the mass fraction of the perchloric acid ethanol solution is 5% to 10%.
4. The method as claimed in claim 1, wherein in the step (1), the metal sample is ground by using sand paper with gradually increasing mesh number to 2000 meshes, the mechanical surface is polished until no scratch is observed by a metallographic microscope, the mechanical surface is cleaned and dried without oxygen after being polished, the cleaning agent for cleaning after the mechanical surface is ethanol solution, and the nitrogen blow drying is adopted for drying without oxygen after the mechanical surface is polished.
5. The method of claim 1, wherein in step (3), the metal sample is stored under vacuum after oxygen-free drying, and the oxygen-free drying method is nitrogen blow drying.
6. The method according to claim 1, wherein in the step (4), the OD value of the marine microorganism solution is 2.0-4.8, and the culture medium is 2216E liquid culture medium.
7. The method according to claim 1, wherein in the step (4), the sterilization temperature is 120-121 ℃, the sterilization time is 15-20 min, and the temperature after sterilization and cooling is 20-25 ℃.
8. The method according to claim 1, wherein in the step (7), the method for removing the adhering liquid from the metal sample treated in the step (6) comprises the following steps: and (4) placing the metal sample treated in the step (6) in ethanol solution with the volume fraction gradually increasing from 50% to 100% for dehydration in sequence.
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| CN101142473A (en) * | 2005-03-17 | 2008-03-12 | 亚什兰许可和知识产权有限公司 | Method for determining and controlling the formation of deposits in a water system |
| CN104404520A (en) * | 2014-11-18 | 2015-03-11 | 河北钢铁股份有限公司 | Metallographic structure etching solution of high-nitrogen stainless steel and metallographic etching method |
| CN109406556A (en) * | 2018-10-30 | 2019-03-01 | 成都先进金属材料产业技术研究院有限公司 | The method for preparing GH4169 high temperature alloy transmission electron microscope sample |
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