CN110468067B - Bacillus circulans separation and identification method - Google Patents
Bacillus circulans separation and identification method Download PDFInfo
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- 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
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- 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
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- 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
Abstract
The invention provides a bacillus circulans separation and identification method, which comprises the following steps: (1) Selectively culturing environmental bacteria containing bacillus circulans by using a selective enrichment culture medium, reducing the proliferation of non-bacillus circulans and efficiently proliferating the bacillus circulans, wherein the selective enrichment culture medium is an improved tryptone soybean broth culture medium containing cephalosporin antibiotics and penicillin potassium; (2) And (2) culturing the bacterial liquid obtained in the step (1) on a Columbia blood agar plate containing antibiotics, separating strains with hemolytic rings, and identifying according to colony morphology. The method can further use a molecular biological method to identify the strain separated in the step (2). The method can simply and quickly separate and identify the bacillus circulans from the environmental bacteria, and has higher screening precision.
Description
Technical Field
The invention belongs to the field of microbial detection, and particularly relates to a bacillus circulans separation and identification method.
Background
Bacillus (Bacillus) is a gram-positive, aerobic or facultative anaerobic, endospore rod-shaped bacterium, and because of the production of stress-resistant endospores, the Bacillus microorganism is resistant to various extreme environments, such as high temperature, extreme acid, extreme salt, and is resistant to various bactericides. The bacillus includes about 50 kinds of bacillus subtilis, bacillus licheniformis, bacillus pumilus, bacillus circulans, bacillus mycoides, bacillus polymyxa, bacillus anthracis, bacillus cereus and the like.
The conventional detection method for bacillus is to judge according to the characteristics of bacterial colonies and thalli and combines with a strain identification material, but when a sample with unobvious phenotypic characteristics or containing various microorganisms is subjected to misjudgment, the conventional microorganism identification methods cannot meet the requirements of rapidness and accuracy in sample detection. The specific (primer) molecular biology technology based on DNA sequence is one of the present methods for fast and accurate identification of bacterial species. However, when the number of the undesired bacteria in the environmental bacteria is excessive, the workload of identification is greatly increased, resulting in low work efficiency.
Bacillus circulans is a pathogenic bacterium of the Bacillus genus, and is often screened out from soil-contaminated foods and food packaging paper. In order to ensure the safety of paper for food packaging, and have strong application value and social significance for quickly and efficiently separating and identifying the bacillus circulans, no method for simply and quickly separating and identifying the bacillus circulans from environmental bacteria exists at present.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a simple and rapid bacillus circulans separation and identification method.
The specific technical scheme of the invention is as follows:
a bacillus circulans separation and identification method comprises the following steps:
(1) Selectively culturing environmental bacteria containing bacillus circulans by using a selective enrichment culture medium, reducing the proliferation of non-bacillus circulans and efficiently proliferating the bacillus circulans, wherein the selective enrichment culture medium is an improved tryptone soybean broth culture medium containing cephalosporin antibiotics and penicillin potassium;
(2) And (2) culturing the bacterial liquid obtained in the step (1) on a Columbia blood agar plate containing antibiotics, separating strains with hemolytic rings, and identifying according to colony morphology.
The present invention selects modified tryptic soy broth as the basal medium instead of the enrichment medium for bacillus in order to reduce the dominant proliferation of other high nutritional requirement bacilli.
Preferably, the selective enrichment culture medium in the step (1) can inhibit the growth of most of salt-tolerant and alkali-tolerant bacteria through high salt and pH adjustment, so that the proliferation of the bacillus circulans is enhanced efficiently. Preferably, 7% by mass of NaCl is used, and the selective enrichment medium pH =8 is adjusted.
The cephalosporin antibiotics are preferably ceftiofur or ceftiofur salt, for example, the ceftiofur sodium is preferably selected, the content of ceftiofur or ceftiofur salt in a selective enrichment medium is 50-500 mu g/mL, and the content of penicillin potassium is 10-30U/mL.
More preferably, the selective enrichment medium in step (1) can further contain polymyxin and/or nalidixic acid or a salt thereof, such as nalidixic acid sodium, so as to further inhibit the growth of infectious microbes.
The invention uses cephalosporin antibiotics, polymyxin and/or nalidixic acid or salts thereof to inhibit the growth of mixed bacteria, uses penicillin potassium to ensure that the bacillus circulans has advantages over other bacilli, and is beneficial to subsequent plate separation and identification.
According to the method for separating and identifying the bacillus circulans, the antibiotics in the step (2) are coumarin antibiotics and/or nalidixic acid or salts thereof, such as nalidixic acid sodium. Preferably, the coumarin antibiotic is novobiocin.
According to the method for separating and identifying the bacillus circulans, the culture temperature of the steps (1) and (2) is 36 ℃, and the bacillus circulans is cultured for 24 hours.
The bacillus circulans separation and identification method further comprises the step (3) of identifying the strain separated in the step (2) by using a molecular biology method.
The molecular biological method is a method conventionally used in the prior art, such as one or more of 16S rRNA/rDNA sequence analysis, random amplification polymorphic DNA, intergenic repetitive sequence analysis technology, denaturing gradient gel electrophoresis labeling technology, specific PCR, multiplex PCR and fluorescence quantitative PCR, and preferably 16S rRNA/rDNA sequence analysis.
The invention also aims to provide a bacillus circulans selective enrichment medium which comprises an improved tryptone soybean broth culture medium, cephalosporin antibiotics and penicillin potassium. The modified tryptone soy broth culture medium is a culture medium conventionally used in the prior art and can be purchased commercially or formulated according to a formula.
Preferably, the bacillus circulans selective enrichment culture medium comprises the following formula:
tryptone 17.0 g
Soy peptone 3.0 g
Potassium dihydrogen phosphate (anhydrous) 2.5 g
Glucose 2.5 g
Sodium chloride 70 g
Distilled water 1000.0 mL
Dissolving the above components in distilled water, heating to dissolve, sterilizing at 121 deg.C for 15 min, and adding antibiotics.
Adding antibiotics:
polymyxin solution: weighing 10mg polymyxin B in 10mL sterilized distilled water, shaking and mixing uniformly, fully dissolving, and filtering for sterilization. 10mL polymyxin solution was added per 1000mL selective enrichment medium. Ceftiofur sodium solution: 50mg of ceftiofur sodium is weighed into 10mL of sterilized distilled water, uniformly mixed by shaking, fully dissolved and filtered for sterilization. 10mL of ceftiofur sodium solution was added per 1000mL of the medium. Nalidixic acid sodium solution: weighing 10mg nalidixic acid in 10mL0.05mol/L sodium hydroxide solution, shaking, mixing uniformly, fully dissolving, and filtering for sterilization. 10mL of Nalidinone acid sodium solution was added per 1000mL of medium.
Penicillin potassium solution: weighing 10mg of penicillin potassium 40 million U in 10mL of sterilized distilled water, shaking and mixing uniformly, fully dissolving, and filtering for sterilization. 5mL of penicillin potassium solution was added per 1000mL of the medium.
The invention also aims to provide a plate culture medium for separating and identifying the bacillus circulans, which comprises Columbia blood agar, coumarin antibiotics and/or nalidixic acid or salts thereof.
Columbia blood agar is a medium conventionally used in the art, and may be purchased commercially or formulated.
Preferably, the formula of the plate culture medium is as follows:
tryptone 12.0 g
Animal tissue protein digestive juice 5.0 g
Yeast extract 3.0 g
Beef extract 3.0 g
Corn starch 1.0 g
Sodium chloride 5.0 g
Agar 13.5 g
Distilled water 1000.0 mL
Dissolving the above components in distilled water, heating to dissolve, sterilizing at 121 deg.C for 15 min, adding 5mL/100mL of aseptic defibrinated sheep blood and antibiotic, shaking, and packaging.
Adding antibiotics:
nongthromycin 0.01 g
Naphthyridinone sodium salt 0.01 g.
The invention has the advantages that:
the method can effectively and quickly screen the bacillus circulans from the environmental bacteria, and has higher screening precision.
Drawings
FIG. 1 shows the result of PCR amplification electrophoresis in example 4.
FIG. 2 is a photograph showing the state of culture after 24 hours of culture in example 4.
FIG. 3 shows the sequencing results of the strain of example 4.
FIG. 4 shows strain X constructed according to software MEGA 1 The evolutionary tree of (1).
FIG. 5 is a strain X constructed according to software MEGA 2 The evolutionary tree of (1).
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1: establishment of optimal culture conditions
Generally, the selective enrichment medium is selected by adding nutrient components or antibiotic components which are sensitive to other bacteria and insensitive to target bacteria on the basis of a basic medium. The basic culture medium of the embodiment is modified trypticase soy broth, and a enrichment culture medium of bacillus is not selected, so that the advantage proliferation of other bacillus with high nutritional requirement is reduced.
Preparation of samples: the screening subject of the experiment is food contact paper purchased in the market, used for daily supervision and spot check and submitted for inspection by enterprises.
Comparison products: taking 25g of a sample which is verified to have no growth of any mixed bacteria by experiments, and respectively adding the positive strains: bacillus circulans, lysine bacillus, bacillus subtilis and hemolytic streptococcus to obtain 1~9 × 10 4 The bacterial solution (bacterial strain inoculated into nutrient broth, cultured at 36 ℃ for 24 hours, and the concentration range is 1~9X 10) 8 Dilution 10 4 Multiple) 100 µ L was added. A single positive sample was prepared.
Basal positive samples: 25g of the sample with the bacteria is taken as a basic positive sample.
Preparation of a culture medium:
tryptone 17.0 g
Soy peptone 3.0 g
Potassium dihydrogen phosphate (anhydrous) 2.5 g
Glucose 2.5 g
Additive/conditions:
NaCl%（W/V）：3%，5%，7%
pH：6，7，8，9
distilled water 1000.0 mL
Dissolving the above components in distilled water, heating to dissolve, and sterilizing at 121 deg.C for 15 min.
And (3) experimental operation:
respectively adding 25g of single positive sample and basic positive sample added with different positive strains into 225mL of enrichment medium added with 3%,5%,7% NaCl and the pH 6,7,8,9 for 18-hour enrichment culture, observing the concentration of the enrichment medium by naked eyes, and respectively inspecting the growth conditions of the mixed bacteria, the bacillus circulans, the bacillus lysinate, the bacillus subtilis and the hemolytic streptococcus.
The results of the experiment are shown in table 1:
the medium was divided into +++, ++, + and-No growth observed-depending on the concentration of the broth.
The results show that the bacillus circulans can achieve high-efficiency enrichment at NaCl content of 7%, pH value of 8 and culture temperature of 36 ℃, and the growth of most of non-salt-tolerant bacteria is inhibited.
Example 2: antibiotic and bacteriostatic compound selection
Because the composition of environmental microorganisms is complex, and the excessive mixed bacteria in the bacteria enrichment process can interfere more in the later identification process, the aim of the embodiment is to realize the screening of the bacillus circulans in a plurality of microorganisms by adding antibiotics in a previous bacteria enrichment culture medium, and inhibit the growth of the mixed bacteria according to the sensitivity of different microorganisms to different antibiotics, thereby ensuring the advantage growth of the bacillus circulans. The following antibiotics and bacteriostatic compounds were selected for the experiments:
(1) The cephalosporin antibiotics have wide antibacterial spectrum and are effective on gram-positive bacteria and gram-negative bacteria. The sensitive bacteria are pasteurella multocida, pasteurella hemolyticus, salmonella, escherichia coli, streptococcus, staphylococcus and the like, and some pseudomonas aeruginosa and enterococcus are resistant to drugs.
(2) Lincomycin hydrochloride belongs to bacteriostatic agents, and sensitive bacteria comprise staphylococcus aureus, streptococcus, pneumococcus, clostridium and most actinomycetes and the like.
(3) The antibiotic effect of neomycin on gram-positive cocci and bacilli (including anaerobes) is basically the same as that of penicillin, and the antibiotic effect on enterococcus faecalis is stronger than that of penicillin. Gram-negative bacteria are sensitive to neisseria meningitidis, neisseria gonorrhoeae, haemophilus influenzae, bordetella pertussis, brucella, proteus mirabilis, salmonella and the like. Some Escherichia coli and some Shigella species are also sensitive, but most Shigella species are resistant to drugs, and others are resistant to them, such as Enterobacteriaceae, pseudomonas aeruginosa, bacteroides fragilis, and the like.
(4) Polymyxin has a strong effect on gram-negative bacteria. Has antibacterial effect on gram-negative bacteria such as Pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, haemophilus, salmonella, shigella, bordetella pertussis, pasteurella, and Vibrio.
(5) Sodium naphthyridinone: one of the antibacterial synthetic compounds has a broad antibacterial spectrum and is particularly effective against gram-negative bacteria.
Control and basic positive samples were prepared according to the method of example 1 by adding the corresponding antibiotics/bacteriostats to the medium containing 7% NaCl, pH8 of example 1, respectively, according to Table 2:
comparison products: taking 25g of a sample which is verified to have no growth of any mixed bacteria by experiments, and respectively adding the positive strains: pseudomonas aeruginosa (gram negative bacteria), staphylococcus aureus (gram positive bacteria), bacillus circulans, bacillus lysin and bacillus subtilis to obtain 1~9X 10 4 The bacterial solution (bacterial strain inoculated into nutrient broth, cultured at 36 ℃ for 24 hours, and the concentration range is 1~9X 10) 8 Dilution 10 4 Multiple) was added 100 μ L. A single positive sample was prepared.
Basal positive samples: 25g of the sample with the bacteria is taken as a basic positive sample.
The growth of various bacteria, as well as Pseudomonas aeruginosa (gram negative bacteria), staphylococcus aureus (gram positive bacteria), bacillus circulans, bacillus lysinate, and Bacillus subtilis under different antibiotic environments was examined, and the experimental procedures were as in example 1. The results of the experiment are shown in table 2.
TABLE 2
The concentration of the bacterial liquid in the culture medium is divided into +++, ++, + and the value in which no growth is observed.
The results show that ceftiofur sodium can obviously inhibit the growth of most of mixed bacteria, and the effect is better when the ceftiofur sodium is combined with neomycin, sodium naphthyridone and polymyxin, but the antibiotics have no inhibition effect on bacillus circulans, bacillus lysinate and bacillus subtilis. The penicillin potassium 10-30U/mL can obviously inhibit the growth of the lysine bacillus and the bacillus subtilis, but has no inhibition effect on the bacillus circulans.
The reasonable enrichment medium is determined according to the experimental result
Selective enrichment culture medium:
tryptone 17.0 g
Soy peptone 3.0 g
Potassium dihydrogen phosphate (anhydrous) 2.5 g
Glucose 2.5 g
Sodium chloride 70 g
Distilled water 1000.0 mL
The pH of the culture medium is adjusted to 8,
dissolving the above components in distilled water, heating to dissolve, sterilizing at 121 deg.C for 15 min, and adding antibiotics.
And (3) adding antibiotics:
polymyxin solution: weighing 10mg polymyxin B in 10mL sterilized distilled water, shaking, mixing, dissolving completely, and filtering for sterilization. 10mL of polymyxin solution was added per 1000mL of medium. Ceftiofur sodium solution: weighing 50mg ceftiofur sodium in 10mL sterilized distilled water, shaking and mixing uniformly, fully dissolving, and filtering and sterilizing. 10mL of ceftiofur sodium solution was added per 1000mL of the medium. Nalidixic acid sodium solution: weighing 10mg nalidixic acid in 10mL0.05mol/L sodium hydroxide solution, shaking, mixing well, dissolving well, filtering and sterilizing. 10mL of Nalidinone acid sodium solution was added per 1000mL of medium.
Penicillin potassium solution: weighing 40 million U of 10mg penicillin potassium in 10mL sterilized distilled water, shaking and mixing uniformly, fully dissolving, and filtering and sterilizing. 5mL of penicillin potassium solution was added per 1000mL of the medium.
Example 3: columbia blood agar plate separation and identification
According to the experimental results of examples 1 and 2, the growth status of different positive strains in antibiotics is determined, and a columbia blood agar plate added with antibiotics of neomycin and nalidixic acid sodium is determined as a separation and identification medium.
Columbia blood agar
Tryptone 12.0 g
Animal tissue protein digestive juice 5.0 g
Yeast extract 3.0 g
Beef extract 3.0 g
Corn starch 1.0 g
Sodium chloride 5.0 g
Agar 13.5 g
Distilled water 1000.0 mL
Dissolving the above components in distilled water, heating to dissolve, sterilizing at 121 deg.C for 15 min, adding 5mL/100mL of aseptic defibrinated sheep blood and antibiotic, shaking, and packaging.
Adding antibiotics:
nongthromycin 0.01 g
Naphthyridinone sodium salt 0.01 g
Sample preparation: and taking 25g of the sample with the detected mixed bacteria as a basic positive sample, and adding pseudomonas aeruginosa, staphylococcus aureus, hemolytic streptococcus, lysine bacillus and bacillus subtilis positive strains into the basic positive sample to serve as experimental samples for verification for later use.
Inoculating enrichment medium, culturing at 36 deg.C for 24 hr, growing on improved culture medium, selecting single colony with 1-3 mm hemolytic ring, beige, opaque, slight bulge in the middle, and irregular edge, and determining by 16S rRNA method.
Since bacillus subtilis also develops a hemolytic ring after 48 hours of culture, observation must be performed after 24 hours of culture.
After the enrichment culture medium and the separation and identification culture medium are determined, the verification sample is used, the experimental steps are repeated, finally, the colony X1 which can not grow dominantly and can generate the hemolytic cycle and the colony X2 which grows dominantly are obtained on the blood agar plate, and the bacillus circulans is suspected to be X2 according to the colony morphology. Further characterization was performed using 16S rRNA.
Example 4: amplification and sequencing of 16S rRNA
X1 and X2 are respectively purified and cultured by nutrient broth and streaked on nutrient agar plates to form single colonies, as shown in figure 2. Selecting unknown bacteria X1 (beige colonies protruding at noon with smooth edges) and X2 (colonies meeting the characteristics of bacillus circulans) of single colonies from a plate, transferring the unknown bacteria X1 and the unknown bacteria X2 into an NB liquid culture medium for culture, taking fresh bacteria liquid 1mL at logarithmic growth phase, centrifuging 12000g for 3min, collecting bacteria, washing the bacteria once by double distilled water, centrifuging the suspended cells by 60 mu L of double distilled water, using water bath 2min at 100 ℃ and 13000g for 1min at high speed, using supernatant as a DNA template at-20 ℃ for later use, referring to a universal primer sequence of bacteria, using an upstream primer F1:5'-CAGAGTTTGATCCTGGCT-3' (SEQ ID No: 3) and a downstream primer R1:5'-AGGAGGTGATCCAGCCGCA-3' (SEQ ID No: 4) and synthesizing by the company Limited in the biological engineering (Shanghai).
The reaction system is 25 μ l: PCR master 12.5. Mu.L, upstream and downstream primers 0.5. Mu.L, DNA template (100 ng/mL) 0.5. Mu.L, and double distilled water to 25. Mu.L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min; 1min at 95 ℃, 1min at 59.4 ℃, 1min at 72 ℃ and 30 cycles; extension at 70 ℃ for 10min.
mu.L of the PCR product was detected by electrophoresis on a 1% agarose gel using a DNA Marker as a reference, and the results are shown in Table 1. The PCR product was subjected to sequence sequencing. And (3) carrying out homology analysis on the similar sequences in GeneBank software according to the sequencing result, and constructing a phylogenetic tree.
As can be seen from FIG. 1, a specific band of about 1500bp was amplified.
The sequencing results are shown in FIG. 3. The sequencing results were aligned by Blast analysis in the NCBI GeneBank database, and the size of the 16S rRNA gene fragment of unknown strain X1 was found to be 1494 bp (the sequence of 16SrDNA is shown in SEQ ID No: 1), and the size of the 16S rRNA gene fragment of unknown strain X2 was found to be 1491 bp (the sequence of 16SrDNA is shown in SEQ ID No: 2).
Sequencing results Blast comparison analysis was performed in NCBI database, as shown in tables 3 and 4, and found to be related to unknown bacteria X 1 The concentrated complete sequence with the highest matching degree is the 16s rRNA sequence of each strain of lysine bacillus, and unknown bacterium X 2 The highest matching degree is bacillus circulans, and the matching degree is as high as 99%.
TABLE 3
TABLE 4
Phylogenetic Tree the phylogenetic tree was constructed according to software MEGA as shown in FIGS. 4 and 5, and Strain X was analyzed 1 Has a close homology with the bacteria of the genus Lysinibacillus. X 2 Has a close homology with Bacillus circulans. By combining the morphological and physiological and biochemical test identification results, the unknown bacteria X1 obtained by test separation can be judged to be the lysine bacillus, and the unknown bacteria X2 can be judged to be the bacillus circulans.
And (5) successfully screening the bacillus circulans through verification. Although both bacillus lysinate and bacillus circulans formed hemolytic rings on blood plates, there was a clear difference between the colony morphology of bacillus lysinate and bacillus circulans, and the difference could also be finally confirmed in 16S rRNA.
Sequence listing
<110> Nanjing products quality supervision and inspection institute
<120> method for separating and identifying bacillus circulans
<160> 4
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<211> 1494
<212> DNA
<213> lysine bacillus (Lysinibacillus)
<400> 1
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gctggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt 1380
acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtggg gtaacctttt 1440
ggagccagcc gcctaaggtg ggatagatga ttgggggtga agtcgtaaca a 1491
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cagagtttga tcctggct 18
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aggaggtgat ccagccgca 19
Claims (4)
1. The bacillus circulans separation and identification method is characterized by comprising the following steps:
(1) Selectively culturing environmental bacteria containing bacillus circulans by using a selective enrichment medium, reducing the proliferation of non-bacillus circulans and efficiently proliferating the bacillus circulans, wherein the selective enrichment medium is a modified tryptone soybean broth culture medium which has pH =8 and contains 7% of NaCl, 50-500 mu g/mL of ceftiofur or a salt thereof and 10-30U/mL of penicillin potassium by mass percentage;
(2) Culturing the bacterial liquid obtained in the step (1) on a Columbia blood agar plate containing coumarin antibiotics and/or nalidixic acid or salts thereof, separating strains with hemolytic rings, and identifying according to colony morphology.
2. The method for separating and identifying Bacillus circulans as claimed in claim 1, wherein the selective enrichment medium of step (1) further contains polymyxin and/or nalidixic acid or a salt thereof.
3. The method for separating and identifying Bacillus circulans according to claim 1, wherein the culturing temperature in steps (1) and (2) is 36 ℃ and the culturing time is 24 hours.
4. The method for separating and identifying Bacillus circulans according to claim 1, further comprising the step (3) of identifying the strain separated in step (2) by using a molecular biological method selected from one or more of 16S rRNA/rDNA sequence analysis, random amplified polymorphic DNA, intergenic repeat sequence analysis technique, denaturing gradient gel electrophoresis labeling technique, specific PCR, multiplex PCR and fluorescent quantitative PCR.
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