CN110846247A - Culture medium for culturing and separating microorganisms in cutting fluid and preparation method and application thereof - Google Patents
Culture medium for culturing and separating microorganisms in cutting fluid and preparation method and application thereof Download PDFInfo
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- CN110846247A CN110846247A CN201911103562.8A CN201911103562A CN110846247A CN 110846247 A CN110846247 A CN 110846247A CN 201911103562 A CN201911103562 A CN 201911103562A CN 110846247 A CN110846247 A CN 110846247A
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- culture medium
- cutting fluid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a culture medium for culturing and separating microorganisms in cutting fluid, and a preparation method and application thereof, wherein the culture medium comprises the following components: beef extract, peptone, triethanolamine borate and derivatives thereof, oleic acid, methanol, organic phosphate and derivatives thereof and distilled water; when the solid culture medium is prepared, a certain amount of agar is also required to be added. The cutting fluid microorganism separation culture medium disclosed by the invention solves the problems that the existing culture medium cannot culture and separate microorganisms in the cutting fluid or has poor culture and separation effects; the culture medium has low cost, convenient and quick preparation and use methods and good separation culture effect, is particularly suitable for quickly culturing and separating microorganisms in the cutting fluid, and lays a foundation for further developing a bacteriostatic agent with good effect and inhibiting the degradation of the cutting fluid and the corrosion of the microorganisms.
Description
Technical Field
The invention belongs to the field of microorganisms, relates to a culture medium, and particularly relates to a culture medium for culturing and separating microorganisms in a cutting fluid, and a preparation method and application thereof.
Background
The water-soluble cutting fluid is an oil-in-water emulsified phase and is easy to breed microorganisms and fungi. The corrosion loss caused by microorganisms in the fields of ocean, petroleum pipelines, nuclear power and the like has attracted much attention, and in the cutting processing industry, the corrosion of a workpiece, a cutter and a machine tool caused by microorganisms bred in a cutting emulsion has a non-trivial influence. When an aluminum alloy part is processed in an alkaline cutting emulsion, microbial corrosion phenomena such as white spots, black spots and the like are easily caused, the lubricity and cooling performance of the cutting fluid are reduced due to microorganisms, the cutting fluid is degraded, and the corrosion of the aluminum alloy is accelerated. In addition, the aluminum alloy is expensive, the turnover period of the aluminum alloy parts is long, the precision requirement is high, and particularly in the aerospace industry, once the aluminum alloy parts are corroded due to the degradation of microorganisms and cutting emulsion, the aluminum alloy parts can only be scrapped, so that huge economic loss is caused to enterprises. In addition, the metabolic activity of microorganisms can lead to a reduction in the performance of the cutting emulsion, and once the metalworking fluid fails, these additives can never reconstitute the original working fluid. Finally, the cutting emulsion deteriorates, degrades, decreases in performance, and becomes a waste liquid prematurely, increasing the cost of the enterprise, and the direct discharge of the waste cutting emulsion liquid poses a hazard to the environment and human health.
The current research generally considers that the aluminum alloy corrosion phenomenon is caused by the degradation of the cutting fluid and the microbial corrosion, and the degradation of the cutting fluid is caused by the reproduction and growth of microbes. The research on the root cause and mechanism of corrosion is the basis for solving the corrosion problem in the aluminum alloy cutting process. In order to deeply study the root cause and corrosion mechanism of corrosion, find out which microorganisms cause corrosion and destroy the cutting fluid, develop a bacteriostatic agent with good effect, and inhibit the degradation of the cutting fluid and the corrosion of the microorganisms, the culture and separation of the microorganisms are required. The existing culture medium cannot meet the growth of special microorganisms in the cutting fluid, so that the culture medium is used for culture and separation; therefore, a special culture medium which takes the main components of the cutting fluid as nutrient substances and is supplemented with common nutrient elements and can provide a good nutrient substance basis for common and special microorganisms in the cutting fluid needs to be developed.
Disclosure of Invention
In view of the above problems, the present invention is to provide a culture medium for culturing and separating microorganisms in a cutting fluid, which has the advantages of convenience, rapidness, low price, and the like, and can simultaneously culture and separate most microorganisms in the cutting fluid.
In order to achieve the purpose, the invention adopts the technical scheme that: a medium for culturing and isolating microorganisms in cutting fluids, the medium comprising the following components: beef extract, peptone, triethanolamine borate and derivatives thereof, oleic acid, methanol, organic phosphate and derivatives thereof and distilled water.
Beef extract and peptone are nutrient substances cultured by common microorganisms, the beef extract provides necessary carbon source, phosphate and vitamin for the microorganisms, and the peptone provides necessary carbon source, nitrogen source and growth factor for the microorganisms; the triethanolamine borate and the derivatives thereof are nitrogen-containing aliphatic compounds which can provide necessary carbon source and nitrogen source for microorganisms and maintain the pH value of the culture medium to be stable; oleic acid is an unsaturated fatty acid, is commonly found in plants, can provide a nitrogen source for microorganisms, is beneficial to long-term storage of a culture medium and prevents the culture medium from being oxidized; methanol is saturated monohydric alcohol which can be utilized by microorganisms to provide carbon source for the microorganisms to synthesize protein; the organic phosphate and the derivative thereof are aliphatic compounds containing nitrogen and phosphorus, and can provide a carbon source, a nitrogen source and a phosphorus source for microorganisms.
The components of the existing culture medium and the cutting fluid are greatly different, the culture medium suitable for culturing and separating microorganisms in the cutting fluid is explored and finally designed according to the components of the cutting fluid, triethanolamine borate, oleic acid, methanol and organic phosphate are used as main nutrient substances, common nutrient substances such as beef extract and peptone are used as auxiliary nutrient substances, and a good nutrient environment can be provided for the microorganisms in the cutting fluid.
In a preferred embodiment of the present invention, the pH of the medium is 8.3 to 8.5.
The pH value is another main factor influencing the culture of the microorganisms, and the pH value is adjusted to be 8.3-8.5 according to the properties of the cutting fluid, so that a good acid-base environment can be further provided for the target microorganisms, and the generation of mixed bacteria is reduced.
As a preferred embodiment of the present invention, the medium comprises the following components: 2-8 g/L of beef extract, 2-8 g/L of peptone, 40-120 g/L of triethanolamine borate and derivatives thereof, 3-8 g/L of oleic acid, 1-5 g/L of methanol, 1-5 g/L of organic phosphate and derivatives thereof and distilled water.
The invention optimizes each component of the culture medium, can rapidly separate the microorganism of the cutting fluid under the proportioning condition, and has better effect and low price.
As a preferred embodiment of the present invention, the medium consists of the following components: 5g of beef extract, 5g of peptone, 80g of triethanolamine borate and derivatives thereof, 5.5g of oleic acid, 3g of methanol, 3g of organic phosphate and derivatives thereof and 1000mL of distilled water.
As a preferred embodiment of the present invention, the triethanolamine borate and the derivative thereof are selected from at least one of monoethanolamine borate, diethanolamine borate and triethanolamine borate.
Monoethanolamine borate, diethanolamine borate and triethanolamine borate are easy to be decomposed and utilized by microorganisms.
As a preferable embodiment of the invention, the organic phosphate and the derivative thereof are selected from at least one of organic triethyl phosphate, organic tripropyl phosphate and organic diethyl phosphate.
The organic triethyl phosphate, the organic tripropyl phosphate and the organic diethyl phosphate are easy to decompose and utilize by microorganisms.
Further, the culture medium further comprises agar.
The culture medium of the present invention can be prepared into a solid culture medium by adding a certain amount of agar.
Preferably, the culture medium contains 10-15 g/L agar.
The invention also provides a preparation method of the culture medium, which is characterized by comprising the following steps: adding triethanolamine borate, oleic acid, methanol, organic phosphate, beef extract, peptone and agar into distilled water in sequence, mixing well, adjusting pH, and heating for sterilization.
The preparation method can fully dissolve all the components in water, is not easy to generate water-insoluble substances, and is beneficial to full utilization of all the components.
When the liquid culture medium is prepared, agar does not need to be added, and the liquid culture medium can be used for culturing and separating microorganisms after being sterilized and cooled to room temperature; when preparing the solid culture medium, agar is required to be added, the agar is subpackaged into plates after sterilization, and the plates are cooled to form solid plates which are further used for culturing and separating microorganisms.
Further, the sterilization process is heating at 121 ℃ for 15 min.
The culture medium usually contains rich nutrition, various microorganisms can be introduced in the preparation process, and the rapid high-temperature sterilization can kill the mixed bacteria to reduce the damage to the nutrient components.
The culture medium can be used for culturing and separating microorganisms in cutting fluid.
The culture medium is specially proportioned according to the components of the cutting fluid, and is particularly suitable for culturing and separating microorganisms in the cutting fluid.
The invention also provides a method for culturing and separating microorganisms in cutting fluid by using the culture medium, which comprises the following steps: and (3) adding the cutting fluid sample into the culture medium, and culturing for 24-48 h at 30-37 ℃.
The cutting fluid sample may be a cutting fluid which is directly obtained from a processing place and generates certain corruption (fresh cutting fluid is milk white, and the cutting fluid is generally creamy yellow), or a corrupted cutting fluid which is diluted by a certain concentration. When the microorganism is cultured and separated, the operation can be carried out according to the general culture and separation steps of the microorganism.
Further, when the culture medium is a liquid culture medium, shaking culture needs to be carried out in a shaking table, and the rotating speed is 80-120 r/min.
When liquid culture medium is used for culture, a shaking table is used for shaking to improve the culture effect.
The cutting fluid microorganism separation culture medium disclosed by the invention solves the problems that the existing culture medium cannot culture and separate microorganisms in the cutting fluid or has poor culture and separation effects; the preparation and use methods of the culture medium are convenient and quick, the culture medium is low in cost and good in separation culture effect, and the culture medium is particularly suitable for quickly culturing and separating microorganisms in the cutting fluid, and lays a foundation for further developing a bacteriostatic agent with good effect and inhibiting the degradation of the cutting fluid and the corrosion of the microorganisms.
Drawings
FIG. 1 the density of bacteria in solution was observed under an inverted fluorescence microscope.
FIG. 2 shows the growth curve of the strain in the purified cutting fluid.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
An embodiment of the present invention relates to a medium for isolating and culturing microorganisms in a cutting fluid, the medium comprising the following components: 2g of beef extract, 2g of peptone, 40g of triethanolamine borate and derivatives thereof, 3g of oleic acid, 1g of methanol, 1g of organic phosphate and derivatives thereof and 1000mL of distilled water; the triethanolamine borate and the derivative thereof are triethanolamine borate; the organic phosphate and the derivatives thereof are organic triethyl phosphate.
Example 2
An embodiment of the present invention relates to a medium for isolating and culturing microorganisms in a cutting fluid, the medium comprising the following components: 8g of beef extract, 8g of peptone, 120g of triethanolamine borate and derivatives thereof, 8g of oleic acid, 5g of methanol, 5g of organic phosphate and derivatives thereof and 1000mL of distilled water; the triethanolamine borate and the derivative thereof are monoethanolamine borate; the organic phosphate and the derivative thereof are organic diethyl phosphate.
Example 3
An embodiment of the present invention relates to a medium for isolating and culturing microorganisms in a cutting fluid, the medium comprising the following components: 3.5g of beef extract, 3.5g of peptone, 60g of triethanolamine borate and derivatives thereof, 4g of oleic acid, 2g of methanol, 2g of organic phosphate and derivatives thereof and 1000mL of distilled water; the triethanolamine borate and the derivative thereof are diethanolamine borate; the organic phosphate and the derivative thereof are organic phosphoric acid tripropyl ester.
Example 4
An embodiment of the present invention relates to a medium for isolating and culturing microorganisms in a cutting fluid, the medium comprising the following components: 6.5g of beef extract, 6.5g of peptone, 100g of triethanolamine borate and derivatives thereof, 7g of oleic acid, 4g of methanol, 4g of organic phosphate and derivatives thereof and 1000mL of distilled water; the triethanolamine borate and the derivative thereof are triethanolamine borate; the organic phosphate and the derivatives thereof are organic triethyl phosphate.
Example 5
An embodiment of the present invention relates to a medium for isolating and culturing microorganisms in a cutting fluid, the medium comprising the following components: 5g of beef extract, 5g of peptone, 80g of triethanolamine borate and derivatives thereof, 5.5g of oleic acid, 3g of methanol, 3g of organic phosphate and derivatives thereof and 1000mL of distilled water; the triethanolamine borate and the derivative thereof are triethanolamine borate; the organic phosphate and the derivatives thereof are organic triethyl phosphate.
Comparative example 1
The present invention is a comparative example of a medium for isolating and culturing microorganisms in a cutting fluid, which is the same as in example 5 except for the following components: beef extract 0.5 g.
Comparative example 2
The present invention is a comparative example of a medium for isolating and culturing microorganisms in a cutting fluid, which is the same as in example 5 except for the following components: peptone 0.5 g.
Comparative example 3
The present invention is a comparative example of a medium for isolating and culturing microorganisms in a cutting fluid, which is the same as in example 5 except for the following components: triethanolamine borate and its derivatives 10 g.
Comparative example 4
The present invention is a comparative example of a medium for isolating and culturing microorganisms in a cutting fluid, which is the same as in example 5 except for the following components: oleic acid 0.5 g.
Comparative example 5
The present invention is a comparative example of a medium for isolating and culturing microorganisms in a cutting fluid, which is the same as in example 5 except for the following components: methanol 0.3 g.
Comparative example 6
The present invention is a comparative example of a medium for isolating and culturing microorganisms in a cutting fluid, which is the same as in example 5 except for the following components: organic phosphate and its derivative 0.3 g.
First, separation effect
The cutting fluid is obtained from a Shanghai automobile part processing workshop, the temperature is 30 ℃, the pH value is 8.3, and the cutting fluid is milky yellow and is obviously different from milky white of fresh cutting fluid.
In the preparation process of the culture medium of the embodiment and the comparative example, the solvents are added with triethanolamine borate and derivatives thereof, oleic acid, methanol, organic phosphate and derivatives thereof, beef extract and peptone in sequence, and after being uniformly mixed, the pH is adjusted to 8.3; 2216E is adjusted to pH 8.3 with LB medium, heated at 121 deg.C for 15min for sterilization, and then cooled to room temperature for use.
In each example, comparative example and 2216E, 10-15 g/L of agar is added to a solid culture medium corresponding to the LB culture medium, and after sterilization treatment, a solid culture medium plate is poured for standby.
1mL of the waste cutting fluid was taken and subjected to 1: dilution was performed with a 10 fold dilution, 5 dilution gradient. And (3) respectively coating 100 mu L of the final dilution gradient solution on the solid culture medium, the comparative liquid culture medium, the 2216E solid culture medium and the LB solid culture medium of each embodiment, finally putting the final dilution gradient solution into a biochemical incubator to culture for 24-48 h at the temperature of 30-37 ℃, then carrying out bacterial purification for three times, finally carrying out 16S rDNA identification on the obtained bacterial strain, and counting the number of the obtained bacterial strain.
Meanwhile, 100 mu L of taken cutting fluid samples are added into the liquid culture medium 2216E and LB liquid culture medium of each example and each comparative example, are placed on a shaker to be cultured for 24-48 h under the conditions of 30-37 ℃ and 80-120 r/min, and are sent to a biological company together with waste cutting fluid for carrying out microbial diversity test to determine the total number of the strains contained in each sample, and the ratio of the number of the microbial strains cultured by the liquid culture medium to the total number of the strains of the cutting fluid is calculated. The results are shown in Table 1.
TABLE 1 Effect of separation of the respective media
The culture medium for culturing and separating the microorganisms in the cutting fluid disclosed by the invention has a good bacterial effect, the number of the bacterial genera which can be cultured in the embodiment 5 accounts for 76.5 percent of the total number of the microbial genera, and the number of the strains identified by separation is 16, which is far higher than that of other common culture media.
Second, culture Effect
Adding 100 mu L of the taken cutting fluid sample into the liquid culture medium, the 2216E liquid culture medium and the LB liquid culture medium in the embodiment 5, putting the cutting fluid sample on a shaking bed, culturing for 24-48 h under the conditions of 30-37 ℃ and 80-120 r/min, adding acridine orange for dyeing for 10-15 min, taking 100-200 mu L of the cutting fluid sample, dropwise adding the cutting fluid sample on an aluminum alloy plate, and observing the concentration/density of bacteria in the solution under an inverted fluorescence microscope; the results of the experiment are shown in FIG. 1(a. cutting fluid; b. example 5 medium; c.2216E medium; d.LB medium).
In comparison with the cutting fluid collected on site, the concentration of microorganisms in the liquid culture medium of example 5 is similar to that of the cutting fluid, and the concentration of bacteria in the common 2216E, LB culture medium is lower. The culture medium is suitable for culturing microorganisms in the cutting fluid and has basically the same total concentration of bacteria as the actual cutting fluid.
Third, growth conditions
Three microorganisms isolated from the cutting fluid were randomly selected, inoculated into the liquid medium of example 5, the OD value of the corresponding medium was measured by an ultraviolet spectrophotometer at 600nm, and compared with the blank patent medium to draw a growth curve, and the results are shown in fig. 2(a. pseudomonas; b. bacillus; c. providencia).
The results show that the three strains of bacteria have good growth conditions, can be maintained for a long time, and have good culture effects, and further show that the culture medium is suitable for culturing and separating microorganisms in cutting fluid.
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, 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 (10)
1. A medium for culturing and isolating microorganisms in cutting fluids, the medium comprising the following components: beef extract, peptone, triethanolamine borate and derivatives thereof, oleic acid, methanol, organic phosphate and derivatives thereof and distilled water.
2. The culture medium according to claim 1, wherein the pH of the culture medium is 8.3 to 8.5.
3. The culture medium of claim 1, wherein the culture medium comprises the following components: 2-8 g/L of beef extract, 2-8 g/L of peptone, 40-120 g/L of triethanolamine borate and derivatives thereof, 3-8 g/L of oleic acid, 1-5 g/L of methanol, 1-5 g/L of organic phosphate and derivatives thereof and distilled water.
4. The culture medium according to claim 1, wherein the triethanolamine borate and the derivative thereof are at least one selected from the group consisting of monoethanolamine borate, diethanolamine borate, and triethanolamine borate.
5. The culture medium of claim 1, wherein the organic phosphate and its derivatives are at least one selected from the group consisting of triethyl organophosphate, tripropyl organophosphate, and diethyl organophosphate.
6. The culture medium of claim 1, wherein the culture medium further comprises agar.
7. The culture medium of claim 6, further comprising 10-15 g/L agar.
8. A method for preparing a culture medium according to any one of claims 1 to 7, comprising the steps of: adding triethanolamine borate, oleic acid, methanol, organic phosphate, beef extract, peptone and agar into distilled water in sequence, mixing well, adjusting pH, and heating for sterilization.
9. Use of a medium according to any one of claims 1 to 7 for culturing and isolating cutting fluid microorganisms.
10. A method for culturing and separating microorganisms in cutting fluid is characterized by comprising the following steps: adding a cutting fluid sample into the culture medium of any one of claims 1 to 7, and culturing at 30-37 ℃ for 24-48 h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444748A (en) * | 2021-07-22 | 2021-09-28 | 上海绿晟环保科技有限公司 | Cutting fluid anti-deterioration process based on microorganisms |
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| US5132046A (en) * | 1988-03-30 | 1992-07-21 | Berol Nobel Stenungsund Ab | Water-based metal working fluid containing at least one alkanolamine compound as antimicrobial agent and a metal working process performed in the presence of said fluid |
| JPH0695952B2 (en) * | 1989-03-23 | 1994-11-30 | ユシロ化学工業株式会社 | Method for evaluating microbial deterioration resistance of water-soluble oil agents for metal processing |
| CN107815344A (en) * | 2017-11-16 | 2018-03-20 | 广州市联诺化工科技有限公司 | Semi-synthetic cutting fluid of long-life environment-friendly type and preparation method thereof |
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| US5132046A (en) * | 1988-03-30 | 1992-07-21 | Berol Nobel Stenungsund Ab | Water-based metal working fluid containing at least one alkanolamine compound as antimicrobial agent and a metal working process performed in the presence of said fluid |
| JPH0695952B2 (en) * | 1989-03-23 | 1994-11-30 | ユシロ化学工業株式会社 | Method for evaluating microbial deterioration resistance of water-soluble oil agents for metal processing |
| CN107815344A (en) * | 2017-11-16 | 2018-03-20 | 广州市联诺化工科技有限公司 | Semi-synthetic cutting fluid of long-life environment-friendly type and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444748A (en) * | 2021-07-22 | 2021-09-28 | 上海绿晟环保科技有限公司 | Cutting fluid anti-deterioration process based on microorganisms |
| CN113444748B (en) * | 2021-07-22 | 2023-08-15 | 上海绿晟环保科技有限公司 | Cutting fluid degradation-resistant technology based on microorganisms |
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