阅读说明：本技术 一种唾液产气微生物的分离方法 (Method for separating salivary aerogenic microorganisms ) 是由 高茜 米其利 罗娜 王雪 许力 管莹 向海英 曾婉俐 杨光宇 李雪梅 于 2020-12-03 设计创作，主要内容包括：本发明公开了一种唾液产气微生物的分离方法,包括如下步骤：(1)稀释采集的唾液；(2)将稀释的唾液涂布于不同类型的培养基上,在厌氧环境中恒温培养；(3)待培养基上生长出菌落后,取无菌薄膜贴于培养基上,判断菌落是否产气；(4)选取产气的菌落进行划线纯化后,接种至同类型的培养基上；(5)重复执行步骤(3)和(4)；(6)对步骤(5)中得到产气菌落进行划线纯化,直到得到单菌落；(7)将单菌落接种至装有同类型的培养基并放置了杜氏小管的离心管中进行恒温培养,出现气泡的菌落即为唾液产气微生物。本公开的唾液产气微生物的分离方法在菌株分离的同时展开产气微生物的筛选,可有效提高唾液产气微生物的分离和筛选效率。(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.)

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.

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.