CN110577897A - A method for rapidly isolating Lactobacillus reuteri from mouse intestine - Google Patents

Abstract

The invention discloses a method for rapidly separating lactobacillus reuteri in intestinal tracts of mice, which comprises the step of taking colon contents of the mice to perform selective primary culture of lactobacillus in MRS broth culture medium. Anaerobic culture is carried out for 72h for gram staining microscopic examination and catalase test, and the lactobacillus is confirmed to be bacillus by microscopic examination, wherein the bacillus is rod-shaped, gram staining positive and catalase negative; inoculating the mixed flora to a LAMVAB plate culture medium for subculture, and separating lactobacillus with different levels by color and form observation after 72 hours; and selecting a colony which is completely light green in morphology, round and relatively large, inoculating the colony to an improved BHI agar culture medium for purification culture, and culturing for 18h to obtain a biochemical identification experiment, wherein the lactobacillus reuteri (Lactobacillus reuteri) can be identified by only metabolizing maltose, raffinose and sucrose. The improved BHI agarThe lipid culture medium comprises brain heart extract powder, tryptone, sodium chloride, disodium hydrogen phosphate dodecahydrate, glucose, agar, Tween 80, and H₂And O. The method is efficient and simple.

Description

A method for rapidly isolating Lactobacillus reuteri from mouse intestine

technical field

The invention relates to a method for isolating Lactobacillus reuteri, in particular to a method for rapidly isolating Lactobacillus reuteri in mouse intestines.

Background technique

Gut microbiome is a large and complex biological community that symbiotically lives in the intestine, and is widely involved in digestion and absorption, immune metabolism, neurodevelopment and other functions, and is closely related to health and disease. At present, intestinal microbes have been considered as an independent "organ", but the mechanism of their physiological and pathological effects is still unclear. The efficient isolation and culture of intestinal bacteria is a strong support and an important basis for comprehensively studying their biochemical characteristics, clarifying their biological functions and mechanisms, and promoting clinical transformation. However, due to differences in growth factor requirements and culture conditions, there is still a lack of targeted isolation and culture methods for most bacteria, and the establishment of individual culture conditions for different strains still requires a lot of research work.

Lactobacillus reuteri (Lactobacillus reuteri) is currently known as a type of lactobacillus that can exist in the intestinal tract of almost all vertebrates and mammals. Studies have shown that Lactobacillus reuteri has the functions of regulating intestinal microecology, improving allergic constitution, alleviating immune dysfunction, etc. It has become a new type of probiotic lactic acid bacteria recognized internationally, and has high theoretical research and production application value. Most of the current isolation and culture methods for Lactobacillus reuteri are based on the MRS broth medium of conventional Lactobacillus. The cultivation methods for Lactobacillus reuteri on the market use cornmeal or a series of crops such as cornflour to cultivate jointly. Bacteria in the MRS medium are similar in shape, all are white opaque, round or slightly irregular, difficult to observe with the naked eye, and the manual screening procedure is complicated; at the same time, the growth time of Lactobacillus reuteri on MRS is shorter than that of other lactobacilli. Delayed, the growth state particles are small and flat, the culture time needs 48-72h, and the growth takes a long time. Therefore, conventional Lactobacillus isolation medium (MRS medium) cannot meet the requirements for efficient isolation and cultivation of Lactobacillus reuteri, and improving related methods is of great value for in-depth research on its function and application transformation.

Contents of the invention

Purpose of the invention: The purpose of the invention is to provide a method for rapidly isolating Lactobacillus reuteri from mouse intestinal contents through efficient separation and obvious visual observation.

Technical solution: The present invention provides a method for rapidly isolating Lactobacillus reuteri from mouse intestines, comprising the following steps:

(1) Take the mouse cecal contents and carry out the selective primary culture of Lactobacillus reuteri in MRS broth medium to obtain mixed flora, anaerobic culture for 72 hours to do Gram staining microscopic examination and catalase test, and pass Microscopic examination results showed that rod-shaped, Gram-positive and catalase-negative bacteria were confirmed as Lactobacillus;

(2) Inoculate the mixed flora on the LAMVAB plate medium for subculture, and isolate different levels of Lactobacillus through color and shape observation;

(3) Pick the colonies whose colonies are all light green, round and relatively large, and inoculate them on the improved BHI agar medium for purification and culture.

(4) After culturing for 18 hours, the biochemical identification test can be done, and it can be confirmed as Lactobacillus reuteri only by metabolizing maltose, raffinose, and sucrose. ;

Further, the formula of the improved BHI agar medium in the step (3) is 3.5g of brain heart extract powder, 2g of tryptone, 1g of sodium chloride, 1.26g of disodium hydrogen phosphate dodecahydrate, 1g of glucose (1% ), agar 2g (2%), Tween 80 100μL, 100mL ddH ₂ O.

Further, the formula of the MRS broth medium is 5.224g of MRS powder and 100ml of LddH ₂ O.

Further, the formulation of the LAMVAB plate medium is 7.25 g of LAMVAB agar, 2 g of agar (2%), 100 mL of dd H2O, and 2 mg of vancomycin.

Further, the preliminary culture method in the step (1) is to dissect a healthy mouse in a biological safety cabinet, draw the contents of the cecum with a sterile syringe, inject it into the liquid MRS medium, and selectively enrich the bacteria in the anaerobic operation box , to obtain a mixed flora.

Further, in the step (2), the method of subculturing and isolating different levels of Lactobacillus reuteri is to use a sterile inoculation loop to inoculate the mixed flora on the LAMVAB plate medium, streak culture, and during the culture process Different metabolites reacted with bromocresol green to obtain bacterial colonies with different colors and sizes.

The present invention adopts a three-step culture method, and uses three kinds of solid or liquid culture medium with different components to carry out selective isolation and subculture of lactobacillus respectively: the MRS medium has the function of isolating lactobacillus, and the subordination level narrows the screening range; LAMVAB medium utilizes the color reaction between bacteria and bromocresol green in its components, and the growth law of the bacteria group can be clearly observed with the naked eye, and the number of bacteria species can be summarized. In general, while improving the specificity of each step, the procedure of biochemical identification in culture omics is simplified, and the repeatability and convenience of bacterial screening are improved;

Beneficial effects: the improved BHI culture medium of the present invention is convenient to obtain, simple to prepare, takes a short time to grow bacteria, and is suitable for laboratory use. The method of the invention establishes a rapid and convenient culture condition for the Lactobacillus reuteri, and can obtain a single colony with good growth state on the improved BHI medium. The method of the invention creates a process for the separation and growth of difficult-to-culture lactobacilli such as Lactobacillus reuteri and other difficult-to-cultivate lactobacilli with less time consumption and simple raw materials.

Description of drawings

Figure 1: Biochemical identification diagram of Lactobacillus, Lactobacillus reuteri DSM20016 (Latin name: Lactobacillus reuteri DSM20016) biochemical identification characteristics: in escin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose Among the 8 carbohydrates, Lactobacillus reuteri only metabolizes maltose, sucrose and raffinose, and was positive in these 3 biochemical identification solutions, and the solution changed from blue purple to yellow within 12-24 hours. In the figure: "√" indicates that the metabolic characteristics conform to Lactobacillus reuteri;

Figure 2: Lactobacillus colonies grown on LAMVAB blank plate (left) and LAMVAB plate inoculated for 72 hours (right) Lactobacillus was inoculated from MRS broth to LAMVAB agar medium. After 72 hours, the medium changed from light green to light yellow;

Figure 3: Colony diagrams of Lactobacillus reuteri DSM20016 grown on the improved BHI solid medium (left) and MRS medium (right) for 18 hours respectively;

Figure 4: Colony diagrams of Lactobacillus reuteri DSM20016 grown on the improved BHI solid medium (left) and MRS medium (right) for 24 hours respectively;

At the same time, under the same culture conditions, inoculate Lactobacillus reuteri DSM20016 into the improved BHI and MRS plate medium, observe the growth status of 18h (see Figure 3) and 24h (see Figure 4), and compare in parallel the improved BHI as Reuteri's The advantages of Lactobacillus fermentation medium, the results show that: Lactobacillus reuteri DSM20016 grows fast on the improved BHI medium, and can obtain colonies in a short period of time for subsequent experiments. The colonies are distinct and scattered, milky white, round (or slightly irregular) raised, and smell like yogurt. On the MRS medium, the growth is slow, and the colony particles are small and flat, which are difficult to observe with the naked eye.

Detailed ways

Example 1: Preliminary isolation of Lactobacillus from fresh intestinal contents of mice

Prepare sterile equipment in advance, dissect normal healthy mice in a biosafety cabinet, draw fresh cecal contents with a 1ml sterile syringe, put them into freshly prepared and pre-reduced liquid MRS medium for 48 hours, and store them in an anaerobic operation box at 37°C Internal selective enrichment for the preliminary isolation of Lactobacillus. (Note: the bacteria in the liquid medium are easily contaminated, just ferment once with fresh feces. Microscopic examination shows that there is pollution for the second time, and spherical negative bacteria appear.) After 72 hours, do Gram staining and observe under the microscope to basically rod-shaped Gram-positive bacteria.

The formula of MRS broth medium (100mL) is MRS powder 5.224g, add 100mL ddH ₂ O.

Example 2: After isolating Lactobacillus reuteri from the LAMVAB solid medium for 72 hours, use a sterile inoculation loop to inoculate the mixed flora from the enrichment solution to the LAMVAB plate medium for separation, and 3-5 plates can be streaked at the same time , increase the chance of sieving bacteria. The colony undergoes metabolism during the culture process, and the color of the LAMVAB medium changes from green to light yellow (see Figure 2 left). After 72 hours, a series of green bacterial colonies with different sizes and different coloring states were obtained through the different coloring conditions of the reaction between the substances produced by the bacteria and bromocresol green, and the colony morphology was screened and summarized. Classify the bacteria with the same shape into one type, and record the numbers as 1/2/3...According to the experimental results, the colony shape on the LAMVAB agar medium is all light green, round and relatively large. The identification results were in line with the biochemical indicators of Lactobacillus reuteri.

LAMVAB agar medium (100 mL): LAMVAB agar 7.25 g, agar 2 g (2%), add 100 mL ddH2O. Autoclave at 121°C for 15 minutes, cool to about 50°C, add filter-sterilized vancomycin 2 mg, shake slightly from side to side, and do not introduce air bubbles.

The colony morphology and biochemical identification results are shown in Table 1. There are mainly 7 types of bacteria in the LAMVAB medium:

Table 1 Colony morphology and biochemical identification results

Color (percentage indicates area) size (comparison between) form 90% middle green, covered with light white circle Large particles Round 50% middle green small particles Round 20% dark green in the middle, slightly whitish very small particles Round 5% green + milky white Large particles irregular all light green Large particles Round all light green Large particles irregular dark green, dark green small particles Round

Embodiment 3: Utilize the improved BHI solid medium to rapidly cultivate Lactobacillus reuteri

Classify the colonies with consistent growth status into one category, purify and pass on to the BHI modified agar medium (see Figure 2, right), and record the numbers 1.1/1.2/1.3/2.1/2.2... (the first number represents the number of the plate , the last number represents several different bacteria in the same plate). The above bacteria were cultured in vitro in an anaerobic (H ₂ : 10%; CO ₂ : 5%; N ₂ : 85%) constant temperature incubator at 37°C.

The formula of improved brain-heart infusion (BHI) agar medium (100 mL) is 3.5 g of brain-heart infusion powder (manufacturer: American BD Company), 2 g of tryptone, 1 g of sodium chloride, and 1.26 g of disodium hydrogen phosphate dodecahydrate. g, glucose 1g (1%), agar 2g (2%), Tween 80 (0.1%) 100μL, add 100mL ddH ₂ O. Autoclave at 121°C for 15 minutes.

Example 4: Biochemical identification and sequencing for Lactobacillus reuteri DSM20016

After 18 hours, Gram staining and catalase test (reaction with 15% hydrogen peroxide) were carried out respectively, and the test sequence was carried out in sequence, and the next one was carried out after each index was met, otherwise such strains were excluded. The catalase test was negative, Gram-positive, and rod-shaped bacteria were screened, and biochemical identification was carried out, that is, with 8 kinds of carbohydrates (escin, salicin, maltose, sucrose, raffinose, mannitol, Sorbitol and cellobiose) were reacted to obtain strains that met the biochemical indicators of Lactobacillus reuteri (see Figure 1). A large number of biochemical identifications are required here. Take 100 μL of biochemical reagents in a 96-well plate, add 20-50 μL of bacterial suspension resuspended in sterile water or directly pick colonies with an inoculation loop, and culture them under anaerobic conditions for 18-24 hours. Carbohydrate results showed that only sucrose, maltose, and raffinose were positive (purple turned yellow), so it can be preliminarily determined as Lactobacillus reuteri from the perspective of biochemical characteristics, metabolic characteristics, and genetic characteristics. Subsequently, the DNA of the purified strain is extracted from the genome level, and the pollution degree of organic matter such as protein in the nucleic acid is detected. The OD260/OD280 ratio is between 1.8-2.2, indicating that the purity meets the standard. In the quantitative experiment of q-PCR amplification, observe the melting curve, determine the specificity of primer and bacterial DNA binding, and distinguish and verify the species level of the strain. In the qualitative experiment of agarose gel electrophoresis, the integrity of the strain level is detected according to the color development of a single band presented by the DNA molecular weight, that is, there is no tailing phenomenon. In order to determine the strain level of the bacteria, full-length gene sequencing (3730 sequencing) was carried out for bacterial species identification. According to the blast results, the sample was Lactobacillus reuteri DSM 20016, complete genome, and the nucleic acid sequencing sequence of the isolated bacteria showed that the results were all Roy Lactobacillus subtilis DSM20016, see table 2.

Table 2 Lactobacillus reuteri DSM20016 (Latin name: Lactobacillus reuteri DSM20016) colony characteristics

Claims (6)

1. a method for rapidly separating lactobacillus reuteri in intestinal tracts of mice continuously adopts three culture media to separate and screen lactobacillus reuteri, and is characterized in that: the method comprises the following steps:

(1) Carrying out selective primary culture on lactobacillus reuteri in MRS broth culture medium by taking colon contents of mice to obtain a mixed flora, carrying out anaerobic culture for 72h for gram-stain microscopic examination and catalase test, and confirming that the lactobacillus is bacilliform, gram-stain positive and catalase negative bacteria according to microscopic examination results;

(2) Inoculating the mixed flora to a LAMVAB plate culture medium for subculture, and observing and separating lactobacillus at different levels through color and form;

(3) Selecting a colony which is completely light green in shape, round and relatively large, inoculating the colony to an improved BHI agar culture medium for purification culture, and performing biochemical identification experiment within 18-24 hours, wherein only metabolizing maltose, raffinose and sucrose confirms that the colony is lactobacillus reuteri (Lactobacillus reuteri), and the formula components of the improved BHI agar culture medium comprise brain heart extract powder, tryptone, sodium chloride, disodium hydrogen phosphate dodecahydrate, glucose, agar, Tween 80, ddH₂O。

2. The method for rapidly isolating lactobacillus reuteri in mouse intestinal tract according to claim 1, wherein the formula of the improved fermentation medium is characterized in that: the formula of the BHI agar medium modified in the step (3) comprises 3.5g of brain heart infusion powder, 2g of tryptone, 1g of sodium chloride, 1.26g of disodium hydrogen phosphate dodecahydrate, 1g (1%) of glucose, 2g (2%) of agar, 100 mu L of Tween 80 (0.1%), and 100mL of ddH₂O。

3. The method for rapidly isolating lactobacillus reuteri in mouse intestinal tract according to claim 1, wherein: the formulation of the MRS broth culture medium in the step (1) is 5.224g of MRS powder and 100ml of ddH₂O。

4. The method for rapidly isolating lactobacillus reuteri in mouse intestinal tract according to claim 1, wherein: the formula of the LAMVAB plate culture medium in the step (2) comprises 7.25g of LAMVAB agar, 2g (2%) of agar and 100ml of ddH₂O, vancomycin 2mg。

5. The method for rapidly isolating lactobacillus reuteri in mouse intestinal tract according to claim 1, wherein: the preliminary culture method in the step (1) comprises the steps of dissecting a healthy mouse in a biological safety cabinet, sucking the cecum content by using a sterile syringe, injecting the cecum content into a liquid MRS culture medium, and selectively enriching bacteria in an anaerobic operation box to obtain a mixed flora.

6. The method for rapidly isolating lactobacillus reuteri in mouse intestinal tract according to claim 1, wherein: and (3) inoculating the mixed flora on an LAMVAB plate culture medium by using an aseptic inoculating loop, performing streak culture, and reacting different metabolic product substances with bromocresol green in the culture process to obtain floras with different colors and sizes.