CN112501241A - Method for separating salivary aerogenic microorganisms - Google Patents
Method for separating salivary aerogenic microorganisms Download PDFInfo
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- CN112501241A CN112501241A CN202011410962.6A CN202011410962A CN112501241A CN 112501241 A CN112501241 A CN 112501241A CN 202011410962 A CN202011410962 A CN 202011410962A CN 112501241 A CN112501241 A CN 112501241A
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- saliva
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- 244000005700 microbiome Species 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 25
- 210000003296 saliva Anatomy 0.000 claims abstract description 40
- 239000001963 growth medium Substances 0.000 claims abstract description 29
- 230000001580 bacterial effect Effects 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 7
- 210000005239 tubule Anatomy 0.000 claims abstract description 7
- 239000011248 coating agent Substances 0.000 claims abstract description 5
- 238000000576 coating method Methods 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract description 3
- 239000002609 medium Substances 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 239000007621 bhi medium Substances 0.000 claims description 4
- 239000006161 blood agar Substances 0.000 claims description 4
- 239000003086 colorant Substances 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000000926 separation method Methods 0.000 abstract description 13
- 238000012216 screening Methods 0.000 abstract description 12
- 239000010408 film Substances 0.000 description 15
- 238000004519 manufacturing process Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000025157 Oral disease Diseases 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 208000030194 mouth disease Diseases 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940092559 enterobacter aerogenes Drugs 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 208000028169 periodontal disease Diseases 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- 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
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a method for separating salivary aerogenic microorganisms, which comprises the following steps: (1) diluting the collected saliva; (2) coating the diluted saliva on different types of culture media, and culturing at constant temperature in an anaerobic environment; (3) after the bacteria grow out of the culture medium, taking a sterile film to be pasted on the culture medium, and judging whether the bacterial colony generates gas or not; (4) selecting a colony generating gas, streaking and purifying the colony, and then inoculating the colony to a culture medium of the same type; (5) repeatedly executing the steps (3) and (4); (6) streaking and purifying the aerogenic colonies obtained in the step (5) until single colonies are obtained; (7) inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed in a Du's tubule for constant temperature culture, wherein the colony with bubbles is the salivary aerogenic microorganism. The method for separating the salivary aerogenic microorganisms can be used for screening the aerogenic microorganisms while separating the strains, and can effectively improve the separation and screening efficiency of the salivary aerogenic microorganisms.
Description
Technical Field
The invention relates to the field of microorganism separation, in particular to a method for separating salivary aerogenic microorganisms.
Background
The microorganisms in saliva are abundant and diverse, and have various physiological functions. The community structure characteristics and the steady state of salivary microorganisms are closely related to the oral cavity and the whole body health of a host, and the pathological changes of the salivary microorganisms can induce oral infectious diseases such as caries, periodontal disease and the like and are also related to systemic diseases such as diabetes, rheumatoid arthritis, cardiovascular diseases and the like.
The salivary microorganisms include aerogenic bacteria (also called aerogenic microorganisms), such as perfringens and enterobacter aerogenes. The aerogenic microorganisms in the saliva have potential close relationship with oral diseases and other systemic diseases, so the aerogenic microorganisms are often used as clinical disease monitoring bacteria, and the research on the aerogenic microorganisms is beneficial to preventing and treating the oral diseases.
At present, the isolation of the salivary aerogenic microorganism needs to obtain a pure culture strain by coating and separation, and then the isolated salivary aerogenic microorganism can be obtained only by testing the aerogenic condition. Because the physiological function of the microorganism producing the gas by the saliva is complex and the living environment is difficult to simulate, only a few strains can be obtained by pure culture, so that the gas producing strains obtained by further gas producing screening experiments are very few.
Therefore, how to provide a method capable of effectively improving the efficiency of isolating and screening the aerogenesis microorganisms becomes a technical problem which needs to be solved urgently in the field.
Disclosure of Invention
An object of the present invention is to provide a novel technical solution for a method for separating a microorganism producing saliva, which can effectively improve the efficiency of screening and separation of the microorganism producing saliva.
According to a first aspect of the present invention, there is provided a method of isolating a microorganism that produces gas from saliva.
The method for separating the aerogenesis microorganisms comprises the following steps:
step (1): diluting the collected saliva;
step (2): coating the diluted saliva on different types of culture media, and culturing at constant temperature in an anaerobic environment;
and (3): after the bacteria grow out of the culture medium, taking the sterile film to be pasted on the culture medium, and judging whether the bacterial colony generates gas or not according to whether the air bubble is formed under the sterile film;
and (4): selecting a colony generating gas, streaking and purifying the colony, and then inoculating the colony to a culture medium of the same type;
and (5): repeatedly performing the step (3) and the step (4);
and (6): streaking and purifying the aerogenic colonies obtained in the step (5) until single colonies are obtained;
and (7): inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed in a Du's tubule for constant temperature culture, wherein the colony with bubbles is the salivary aerogenic microorganism.
Optionally, the step (1) is specifically as follows:
the collected saliva was diluted with sterile water over 0.5h to a volume percentage of one thousandth of the saliva.
Optionally, the culture medium in step (2) includes BHI medium, TSA medium, anaerobic agar medium, EG medium and blood agar medium.
Optionally, the step (2) is specifically as follows:
the diluted saliva was spread on various types of media, placed in an anaerobic jar and incubated in a 37 ℃ incubator for 48 h.
Optionally, the step (2) is specifically as follows:
150 μ L of diluted saliva was spread on different types of media and placed in an anaerobic jar and incubated in a 37 ℃ incubator for 48 h.
Optionally, the sterile film in the step (3) is an upper film of a coliform group test piece.
Optionally, the step (4) is specifically as follows:
colonies of different morphologies, colors and sizes and with air bubbles were picked and streaked for purification and inoculated onto the same type of medium.
Optionally, the number of times of repeatedly performing the step (3) and the step (4) in the step (5) is 1.
Optionally, the step (7) is specifically as follows:
inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed with a Duchenne tubule, placing the centrifugal tube in an incubator at 37 ℃ for culture, wherein the colony with bubbles is the salivary aerogenic microorganism.
Optionally, the method further comprises the step (8):
carrying out enrichment culture on the salivary aerogenic microorganisms.
The method for separating the salivary aerogenic microorganisms can be used for screening the aerogenic microorganisms while separating the strains, and can effectively improve the separation and screening efficiency of the salivary aerogenic microorganisms. And different types of culture mediums are adopted for carrying out separation culture simultaneously, which is beneficial to obtaining more aerogenic strains to the maximum extent.
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.
The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.
Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.
In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.
The present disclosure provides a method for separating a microorganism producing saliva, comprising the steps of:
step (1): the collected saliva was diluted.
In order to improve the separation and screening efficiency, the step (1) can be embodied as follows:
the collected saliva was diluted with sterile water over 0.5h to a volume percentage of one thousandth of the saliva.
Step (2): the diluted saliva was spread on different types of media and incubated under anaerobic conditions at constant temperature.
The culture medium in step (2) may include BHI medium, TSA medium, anaerobic agar medium, EG medium and blood agar medium in order to obtain more aerogenesis strains to the maximum.
In order to improve the separation and screening efficiency, the step (2) can be embodied as follows:
the diluted saliva was spread on various types of media, placed in an anaerobic jar and incubated in a 37 ℃ incubator for 48 h.
Further, the step (2) is specifically as follows:
150 μ L of diluted saliva was spread on different types of media and placed in an anaerobic jar and incubated in a 37 ℃ incubator for 48 h.
And (3): after the bacteria grow out of the culture medium, the sterile film is taken to be pasted on the culture medium, and whether the bacterial colony generates gas is judged according to whether the air bubble is formed under the sterile film.
The sterile film in the step (3) can be an upper film of a coliform group test piece.
In specific implementation, a bag of unopened coliform group test piece is taken, the upper thin film is taken off, the upper thin film is cut into a proper shape and is attached to the upper part of the culture medium, and the gas production condition is observed. If gas-producing colonies are present, gas bubbles will appear under the film. If no gas-producing bacterial colony exists, no bubble appears under the film.
In addition, positive control can be performed by using Escherichia coli with specific gas production capacity to verify whether the test system is normal.
And (4): and selecting the aerogenic colonies, streaking and purifying the aerogenic colonies, and then inoculating the aerogenic colonies to the culture medium of the same type.
In order to improve the separation and screening efficiency, the step (4) can be embodied as follows:
colonies of different morphologies, colors and sizes and with air bubbles were picked and streaked for purification and inoculated onto the same type of medium.
The medium of the same type in the step (4) is the same type of medium as the medium in the step (2) in which the aerogenic strain is cultured.
And (5): and (5) repeatedly executing the step (3) and the step (4).
In specific implementation, in order to accelerate the speed of separation and screening on the basis of ensuring the separation effect, the times of repeatedly executing the step (3) and the step (4) are 1 time. That is, step (4) and step (5) are performed twice.
And (6): and (4) streaking and purifying the aerogenic colonies obtained in the step (5) until single colonies are obtained.
The number of repetitions of step (6) can be determined by one skilled in the art based on whether a single colony is obtained. In specific implementation, the step (6) can be repeatedly executed for 2-3 times.
And (7): inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed in a Du's tubule for constant temperature culture, wherein the colony with bubbles is the salivary aerogenic microorganism.
In order to improve the separation and screening efficiency, the step (7) is specifically as follows:
inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed with a Duchenne tubule, placing the centrifugal tube in an incubator at 37 ℃ for culture, wherein the colony with bubbles is the salivary aerogenic microorganism.
The medium of the same type in the step (7) is the same type of medium as the medium in the step (2) in which the aerogenic strain is cultured.
In order to obtain more aerogenesis microorganisms, the method for isolating aerogenesis microorganisms of the present disclosure further comprises the step (8):
carrying out enrichment culture on the salivary aerogenic microorganisms.
The experimental procedures used in the examples below are conventional unless otherwise specified, the materials and reagents used therein are commercially available, and the equipment used in the experiments are well known to those skilled in the art without otherwise specified.
Example 1
(1) Saliva sample processing
The collected saliva was diluted with sterile water over 0.5h to a volume percentage of one thousandth of the saliva.
(2) Flat coating
150 μ L of diluted saliva was pipetted onto plates in BHI medium, TSA medium, anaerobic agar medium, EG medium and blood agar medium, respectively.
(3) Gas production observation
Placing the culture medium in an anaerobic jar, placing in an incubator at 37 ℃ for culturing for 48h, taking an unopened coliform group test piece after bacterial colony grows out, removing the upper layer film, cutting the upper layer film into a proper shape, sticking the upper layer film on the culture medium, and observing the gas production condition. If gas-producing colonies are present, gas bubbles will appear under the film. If no gas-producing bacterial colony exists, no bubble appears under the film. Positive control can be performed by using Escherichia coli with definite gas production capacity to verify whether the test system is normal.
(4) Strain isolation
Selecting colonies with different shapes, colors and sizes and generated bubbles, continuously streaking and purifying, inoculating to the same type of culture medium, and performing pure culture.
(5) Obtaining a single colony
And (3) repeatedly performing the operations of gas production condition observation and strain separation to obtain gas production colonies. And (4) streaking and purifying the aerogenic colonies until single colonies are obtained.
(6) Verification of gas production situation
And (4) carrying out classification and numbering according to the shape, size and color of single colonies in the culture medium after purification. Single colonies of different numbers were inoculated into centrifuge tubes containing the same type of media and Duchenne tubes placed. And placing the centrifugal tube in an incubator at 37 ℃, and culturing until bubbles appear in the tube, wherein bacterial colonies generated by the bubbles in the tube are the salivary aerogenic microorganisms with aerogenic capability.
Selecting salivary aerogenic microorganism for enrichment culture, and storing the cultured colony in a strain for preservation.
Although some specific embodiments of the present invention have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustrative purposes only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.
Claims (10)
1. A method for separating a microorganism producing saliva, which is characterized by comprising the following steps:
step (1): diluting the collected saliva;
step (2): coating the diluted saliva on different types of culture media, and culturing at constant temperature in an anaerobic environment;
and (3): after the bacteria grow out of the culture medium, taking the sterile film to be pasted on the culture medium, and judging whether the bacterial colony generates gas or not according to whether the air bubble is formed under the sterile film;
and (4): selecting a colony generating gas, streaking and purifying the colony, and then inoculating the colony to a culture medium of the same type;
and (5): repeatedly performing the step (3) and the step (4);
and (6): streaking and purifying the aerogenic colonies obtained in the step (5) until single colonies are obtained;
and (7): inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed in a Du's tubule for constant temperature culture, wherein the colony with bubbles is the salivary aerogenic microorganism.
2. The method for isolating a microorganism producing saliva gas according to claim 1, wherein step (1) is specifically as follows:
the collected saliva was diluted with sterile water over 0.5h to a volume percentage of one thousandth of the saliva.
3. The method for isolating a microorganism producing saliva gas according to claim 1, wherein the medium in the step (2) comprises BHI medium, TSA medium, anaerobic agar medium, EG medium and blood agar medium.
4. The method for isolating aerogenesis microorganisms of saliva according to claim 1, wherein the step (2) is specifically as follows:
the diluted saliva was spread on various types of media, placed in an anaerobic jar and incubated in a 37 ℃ incubator for 48 h.
5. The method for isolating aerogenesis microorganisms of saliva according to claim 4, wherein the step (2) is specifically as follows:
150 μ L of diluted saliva was spread on different types of media and placed in an anaerobic jar and incubated in a 37 ℃ incubator for 48 h.
6. The method for isolating microorganisms producing saliva according to claim 1, wherein the sterile film of step (3) is an upper film of a test strip for coliform group bacteria.
7. The method for isolating aerogenesis microorganisms of saliva according to claim 1, wherein the step (4) is as follows:
colonies of different morphologies, colors and sizes and with air bubbles were picked and streaked for purification and inoculated onto the same type of medium.
8. The method for isolating aerogenesis microorganisms of claim 1, wherein the step (5) is repeated the steps (3) and (4) 1 time.
9. Method for the isolation of microorganisms producing gas by saliva according to claim 1, characterized in that step (7) is in particular as follows:
inoculating the single colony to a centrifugal tube filled with the same type of culture medium and placed with a Duchenne tubule, placing the centrifugal tube in an incubator at 37 ℃ for culture, wherein the colony with bubbles is the salivary aerogenic microorganism.
10. Method for the isolation of microorganisms producing saliva according to any of claims 1 to 9, characterized in that it further comprises a step (8):
carrying out enrichment culture on the salivary aerogenic microorganisms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011410962.6A CN112501241A (en) | 2020-12-03 | 2020-12-03 | Method for separating salivary aerogenic microorganisms |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011410962.6A CN112501241A (en) | 2020-12-03 | 2020-12-03 | Method for separating salivary aerogenic microorganisms |
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| CN112501241A true CN112501241A (en) | 2021-03-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113229524A (en) * | 2021-05-19 | 2021-08-10 | 云南中烟工业有限责任公司 | Extraction method of buccal tobacco leaching liquor, bacteriostasis evaluation and gas production inhibition detection method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113229524A (en) * | 2021-05-19 | 2021-08-10 | 云南中烟工业有限责任公司 | Extraction method of buccal tobacco leaching liquor, bacteriostasis evaluation and gas production inhibition detection method |
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