CN108359707B - Chromogenic medium for detecting salmonella and preparation method thereof - Google Patents
Chromogenic medium for detecting salmonella and preparation method thereof Download PDFInfo
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- CN108359707B CN108359707B CN201810516950.8A CN201810516950A CN108359707B CN 108359707 B CN108359707 B CN 108359707B CN 201810516950 A CN201810516950 A CN 201810516950A CN 108359707 B CN108359707 B CN 108359707B
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- indole
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- salmonella
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- 241000607142 Salmonella Species 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
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- 108090000371 Esterases Proteins 0.000 claims abstract description 31
- 239000003593 chromogenic compound Substances 0.000 claims abstract description 26
- 239000000654 additive Substances 0.000 claims abstract description 16
- 230000000996 additive effect Effects 0.000 claims abstract description 11
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 28
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- 229960003964 deoxycholic acid Drugs 0.000 claims description 20
- 238000011161 development Methods 0.000 claims description 20
- 239000010413 mother solution Substances 0.000 claims description 17
- XTRRFIYXAPRYES-NLFZDHTNSA-M sodium (6R,7R)-3-[(4-carbamoylpyridin-1-ium-1-yl)methyl]-8-oxo-7-[[(2R)-2-phenyl-2-sulfonatoacetyl]amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate hydrate Chemical compound O.[Na+].NC(=O)c1cc[n+](CC2=C(N3[C@H](SC2)[C@H](NC(=O)[C@@H](c2ccccc2)S([O-])(=O)=O)C3=O)C([O-])=O)cc1 XTRRFIYXAPRYES-NLFZDHTNSA-M 0.000 claims description 17
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
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- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 17
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- 241000588724 Escherichia coli Species 0.000 description 7
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- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- YJQPYGGHQPGBLI-UHFFFAOYSA-N Novobiocin Natural products O1C(C)(C)C(OC)C(OC(N)=O)C(O)C1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-UHFFFAOYSA-N 0.000 description 5
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 5
- 241000588770 Proteus mirabilis Species 0.000 description 4
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- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
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- YJQPYGGHQPGBLI-KGSXXDOSSA-M novobiocin(1-) Chemical compound O1C(C)(C)[C@H](OC)[C@@H](OC(N)=O)[C@@H](O)[C@@H]1OC1=CC=C(C([O-])=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-KGSXXDOSSA-M 0.000 description 4
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- REACMANCWHKJSM-DWBVFMGKSA-M Cefsulodin sodium Chemical compound [Na+].C1=CC(C(=O)N)=CC=[N+]1CC1=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)[C@@H](C=3C=CC=CC=3)S([O-])(=O)=O)[C@H]2SC1 REACMANCWHKJSM-DWBVFMGKSA-M 0.000 description 3
- 241000943303 Enterococcus faecalis ATCC 29212 Species 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
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- 239000004135 Bone phosphate Substances 0.000 description 2
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- 241000588919 Citrobacter freundii Species 0.000 description 2
- 241000588914 Enterobacter Species 0.000 description 2
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- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 2
- 206010016952 Food poisoning Diseases 0.000 description 2
- 208000019331 Foodborne disease Diseases 0.000 description 2
- 241000607715 Serratia marcescens Species 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
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- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 229940099352 cholate Drugs 0.000 description 2
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 229940032049 enterococcus faecalis Drugs 0.000 description 2
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- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical group [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 2
- 241000086254 Arnica montana Species 0.000 description 1
- 206010004022 Bacterial food poisoning Diseases 0.000 description 1
- 241001135265 Cronobacter sakazakii Species 0.000 description 1
- 241000588697 Enterobacter cloacae Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 102000002268 Hexosaminidases Human genes 0.000 description 1
- 108010000540 Hexosaminidases Proteins 0.000 description 1
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000240376 Proteus anguinus Species 0.000 description 1
- 241001138501 Salmonella enterica Species 0.000 description 1
- 241000531795 Salmonella enterica subsp. enterica serovar Paratyphi A Species 0.000 description 1
- 241000577483 Salmonella enterica subsp. enterica serovar Paratyphi B Species 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 241000607760 Shigella sonnei Species 0.000 description 1
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- 229960000484 ceftazidime Drugs 0.000 description 1
- NMVPEQXCMGEDNH-TZVUEUGBSA-N ceftazidime pentahydrate Chemical compound O.O.O.O.O.S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC(C)(C)C(O)=O)C=2N=C(N)SC=2)CC=1C[N+]1=CC=CC=C1 NMVPEQXCMGEDNH-TZVUEUGBSA-N 0.000 description 1
- JEEWDSDYUSEQML-ROMZVAKDSA-M ceftazidime sodium Chemical class [Na+].S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC(C)(C)C([O-])=O)C=2N=C(N)SC=2)CC=1C[N+]1=CC=CC=C1 JEEWDSDYUSEQML-ROMZVAKDSA-M 0.000 description 1
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- 230000000813 microbial effect Effects 0.000 description 1
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- PAVKBQLPQCDVNI-UHFFFAOYSA-N n',n'-diethyl-n-(9-methoxy-5,11-dimethyl-6h-pyrido[4,3-b]carbazol-1-yl)propane-1,3-diamine Chemical class N1C2=CC=C(OC)C=C2C2=C1C(C)=C1C=CN=C(NCCCN(CC)CC)C1=C2C PAVKBQLPQCDVNI-UHFFFAOYSA-N 0.000 description 1
- YJQPYGGHQPGBLI-KGSXXDOSSA-N novobiocin Chemical compound O1C(C)(C)[C@H](OC)[C@@H](OC(N)=O)[C@@H](O)[C@@H]1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-KGSXXDOSSA-N 0.000 description 1
- 229960004781 novobiocin sodium Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229960005264 piperacillin sodium Drugs 0.000 description 1
- WCMIIGXFCMNQDS-IDYPWDAWSA-M piperacillin sodium Chemical compound [Na+].O=C1C(=O)N(CC)CCN1C(=O)N[C@H](C=1C=CC=CC=1)C(=O)N[C@@H]1C(=O)N2[C@@H](C([O-])=O)C(C)(C)S[C@@H]21 WCMIIGXFCMNQDS-IDYPWDAWSA-M 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 229940115939 shigella sonnei Drugs 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- PODWXQQNRWNDGD-UHFFFAOYSA-L sodium thiosulfate pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[O-]S([S-])(=O)=O PODWXQQNRWNDGD-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000012137 tryptone Substances 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
- C12Q1/10—Enterobacteria
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a chromogenic culture medium for detecting salmonella and a preparation method thereof, wherein the chromogenic culture medium comprises a basic culture medium and an additive, the additive comprises an esterase chromogenic substrate, the esterase chromogenic substrate comprises 5-bromo-6-chloro-3-indole-octyl ester and 5-bromo-6-chloro-3-indole-nonyl ester, the content of the 5-bromo-6-chloro-3-indole-octyl ester is 0.1-0.4 g/L, and the content of the 5-bromo-6-chloro-3-indole-nonyl ester is 0.1-0.4 g/L. The additive components of the chromogenic medium of the invention act synergistically, and 5-bromo-6-chloro-3-indole-nonyl ester and 5-bromo-6-chloro-3-indole-octyl ester are jointly used as chromogenic substrates to synergistically promote the generation of more esterase reactions and release more chromogenic groups. The chromogenic medium disclosed by the invention has the advantages of high sensitivity, strong specificity, higher identification degree, shorter detection period, stronger adaptability, easiness in industrial production and the like when used for detecting salmonella.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a chromogenic medium for detecting salmonella and a preparation method thereof.
Background
Salmonella (Salmonella) is a common food-borne pathogenic bacterium, enterobacteriaceae, a gram-negative bacterium. Feces from people infected with salmonella or carriers contaminate food and can cause food poisoning. According to statistics, the food poisoning caused by salmonella is often listed as the top in various countries in the world of bacterial food poisoning. Salmonella is also the first place in inland areas of China.
At present, the most common method for detecting salmonella in China is still the traditional national standard method. The identification medium commonly used in the national standard method has the advantages of good selectivity of the chromogenic medium and the like, and has the best use effect. The salmonella chromogenic medium is a novel medium which utilizes the reaction and color development of enzyme generated by the self metabolism of salmonella and a corresponding chromogenic substrate. The corresponding chromogenic substrates are composed of chromogenic genes and microorganism part metabolisable substances, the chromogenic genes are dissociated to display certain color under the action of specific enzyme, and the strains can be identified by directly observing the color of a bacterial colony.
At present, commonly used substrates of a salmonella chromogenic medium of a certain manufacturer in China are 5-bromo-4-chloro-3-indole-beta-galactoside, 5-bromo-4-chloro-3-indole-N-acetyl-beta-D-glucosaminide and 5-bromo-6-chloro-3-indole-octyl ester, wherein the beta-galactoside aims at distinguishing enterobacter (such as Citrobacter freundii) containing beta-galactosidase, and the hexosaminidase chromogenic substrate aims at distinguishing candida; the octanase chromogenic substrate is intended to distinguish salmonella. However, some salmonella (such as salmonella typhi and salmonella typhi) have weak esterase activity, so that the color of the salmonella is light after the culture time is up, and the salmonella is not easy to observe.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Accordingly, in one aspect, the present invention provides a chromogenic medium for detecting Salmonella.
In order to solve the technical problems, the invention provides the following technical scheme: a chromogenic culture medium for detecting salmonella comprises a basic culture medium and an additive, wherein the additive comprises an esterase chromogenic substrate, the esterase chromogenic substrate comprises 5-bromo-6-chloro-3-indole-octyl ester and 5-bromo-6-chloro-3-indole-nonyl ester, the content of the 5-bromo-6-chloro-3-indole-octyl ester is 0.1-0.4 g/L, and the content of the 5-bromo-6-chloro-3-indole-nonyl ester is 0.1-0.4 g/L.
As a preferred embodiment of the chromogenic medium for detecting Salmonella of the present invention: the basic culture medium comprises peptone, beef extract, sodium chloride and bacterial agar powder, wherein the content of the peptone is 8-15 g/L, the content of the beef extract is 1-5 g/L, the content of the sodium chloride is 5-6 g/L, and the content of the bacterial agar powder is 15-18 g/L.
As a preferred embodiment of the chromogenic medium for detecting Salmonella of the present invention: the additive also comprises an inhibitor, a galactosidase chromogenic substrate, an enzyme inducer and a cosolvent, wherein the inhibitor comprises sodium deoxycholate, neomycin and cefsulodin sodium salt hydrate, the content of the sodium deoxycholate is 0.4-0.6 g/L, the content of the neomycin is 10-15 mg/L, and the content of the cefsulodin sodium salt hydrate is 5-10 mg/L.
As a preferred embodiment of the chromogenic medium for detecting Salmonella of the present invention: the galactosidase chromogenic substrate comprises 5-bromo-4-chloro-3-indole-beta-galactoside, and the content of the 5-bromo-4-chloro-3-indole-beta-galactoside is 0.04-0.1 g/L.
As a preferred embodiment of the chromogenic medium for detecting Salmonella of the present invention: the enzyme inducer comprises isopropyl-beta-D-thiogalactoside, and the content of the isopropyl-beta-D-thiogalactoside is 0.03-0.06 g/L.
As a preferred embodiment of the chromogenic medium for detecting Salmonella of the present invention: the cosolvent comprises dimethyl sulfoxide and tween 20, wherein the content of the dimethyl sulfoxide is 1-3 ml/L, and the content of the tween 20 is 2-4 ml/L.
As a preferable scheme of the chromogenic medium for detecting salmonella in the invention: in the cosolvent, the volume content of dimethyl sulfoxide is 40%, and the volume content of tween 20 is 60%.
As a preferable scheme of the chromogenic medium for detecting salmonella in the invention: the culture medium comprises 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 15g/L of bacterial agar powder, 0.55g/L of sodium deoxycholate, 15mg/L of neomycin, 6mg/L of cefsulodin sodium salt hydrate, 0.2g/L of 5-bromo-6-chloro-3-indole-octyl ester, 0.3g/L of 5-bromo-6-chloro-3-indole-nonyl ester, 0.08g/L of 5-bromo-4-chloro-3-indole-beta-galactoside, 0.05g/L of isopropyl-beta-D-thiogalactoside, 2ml/L of dimethyl sulfoxide and 3ml/L of Tween 20.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art and provides a method for preparing the chromogenic medium for detecting Salmonella according to any one of claims 3 to 8.
In order to solve the technical problems, the invention provides the following technical scheme: the method for preparing the chromogenic medium for Salmonella according to any one of claims 3 to 8, which comprises preparing a mother liquor 1: weighing a basic culture medium and uniformly mixing the basic culture medium with sodium deoxycholate to obtain mother solution 1; preparing a mother solution 2: dissolving an esterase chromogenic substrate in a cosolvent, adding the esterase chromogenic substrate into the mother liquor 1, and boiling to obtain a mother liquor 2; preparing a chromogenic culture medium: dissolving the neomycin and the cefsulodin sodium salt hydrate in water, adding the solution into the mother liquor 2, and adding the galactose glycolase substrate and the isopropyl-beta-D-thiogalactoside into the mother liquor 2.
As a preferable scheme of the preparation method of the chromogenic medium for detecting salmonella provided by the invention: and uniformly mixing the basic culture medium with sodium deoxycholate at the speed of 2800rpm for 1min, and preparing a mother solution 2, wherein after boiling, the mother solution 2 is cooled to 45 +/-1 ℃, and the inhibitor is added into the mother solution 2.
The invention has the beneficial effects that: the additive components of the chromogenic medium of the invention act synergistically, 5-bromo-6-chloro-3-indole-nonyl ester and 5-bromo-6-chloro-3-indole-octyl ester are jointly used as chromogenic substrates to synergistically promote the generation of more esterase reactions and release more chromogenic groups, and meanwhile, the selection and the proportion of the cosolvent ensure that the chromogenic medium of the invention does not generate the phenomenon of oily appearance on the surface of a flat plate due to excessive emulsifying agents, can completely dissolve the substrates, and the synergistic effect of the cosolvent ensures that the chromogenic effect of the enzyme chromogenic substrate of the chromogenic medium of the invention reaches the best.
The chromogenic medium for detecting salmonella has the advantages of high sensitivity, strong specificity, higher identification degree, shorter detection period, stronger adaptability, easy industrial production and the like.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying specific embodiments of the present invention are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Selection of G + bacteria inhibitors:
the chromogenic medium of the invention is based on common nutrient agar, and by adding an inhibitor, the interference of gram-positive bacteria such as enterococcus faecalis and the like is completely eliminated, and for the convenience of statistical analysis, the growth condition and other influence conditions of strains in the medium corresponding to the scores are adopted:
selection of optimal addition amount of the G + bacteria inhibitor:
for quantitative assessment of the selectivity of the medium in "quality requirements for medium 4789.28-2013 and reagents" 6.1.2.2, an appropriate amount of working culture of the test strain should be inoculated in an appropriate manner into the selective medium and the reference medium as specified. According to the method, after a flat plate is prepared by adding additives with different dosages on the basis of common nutrient agar, the selectivity of an inhibitor on gram-positive bacteria is determined according to a semi-quantitative streaking method in ' 4789.28-2013 quality requirements of culture media and reagents ' 6.1.2.2 '. The test bacteria are: staphylococcus aureus ATCC6538 and enterococcus faecalis ATCC29212, which were observed to be inhibited.
Selection of the most suitable inhibitor of proteus:
since proteus bacteria have flagella, are easy to spread and grow on a plate, and interfere with the detection of salmonella, an additive capable of inhibiting the growth of proteus bacteria is found. Adding different inhibitors into a basic culture medium, and determining the selectivity of the inhibitors to proteus according to a semi-quantitative streaking method in 4789.28-2013 quality requirements of culture medium and reagents 6.1.2.2. After the cultivation, the growth index G was calculated for the medium as follows. Each had a relatively dense colony growth, G =1; only half of the lines had dense colony growth, G =0.5; no colony growth, less than half of the streaked growth or weak colony growth on the streaks, G =0; record the sum of the scores for each plate to get G.
Selection of optimal concentration of proteus inhibitor:
after preparing a flat plate by adding additives with different dosages based on common nutrient agar, and determining the selectivity of the inhibitor on gram-positive bacteria according to a semi-quantitative streaking method in ' quality requirements of 4789.28-2013 culture medium and reagent ' 6.1.2.2 '. The test bacteria are: proteus vulgaris CMCC49027 and Proteus mirabilis CMCC49005, and the inhibition thereof was observed.
Selection of the most suitable inhibitor of pseudomonas aeruginosa:
since Pseudomonas aeruginosa also has esterase, which interferes with the selection of Salmonella, an inhibitor for inhibiting this type of bacteria needs to be added to the chromogenic Salmonella medium. Adding different inhibitors into a basic culture medium, and determining the selectivity of the inhibitors on proteus according to a semi-quantitative streaking method in '4789.28-2013 quality requirements of culture medium and reagents' 6.1.2.2.
Selection of optimal concentration of pseudomonas aeruginosa inhibitor:
based on common nutrient agar, adding additives with different dosages, preparing a flat plate, and determining the selectivity of the inhibitor on gram-positive bacteria according to a semi-quantitative streaking method in quality requirements of 4789.28-2013 culture medium and reagent 6.1.2.2. The test bacteria are: pseudomonas aeruginosa ATCC9027, which was observed to be inhibited.
And (4) analyzing results:
selection of G + bacteria inhibitors
The inhibitory capacity of the inhibitor against different indicator bacteria and other performance results are given in the following table:
* Note: the addition amount of each inhibitor is shown in a reference part commercial culture medium composition table
Sodium dodecyl sulfate, tribasic cholate and sodium deoxycholate can inhibit gram-positive bacteria, because sodium dodecyl sulfate is a surfactant and a substrate added in a culture medium is fat-soluble, oil drops are easily formed in the culture medium to influence the dissolution of an esterase substrate, and the sodium deoxycholate is considered to be an important one of the cholates, a large number of researches show that the sodium deoxycholate can reduce the stability of cell membranes, enhance the fluidity, reduce the orderliness of the membrane structure, inhibit the growth of mycelia, and select the sodium deoxycholate desalted as a gram-positive bacteria inhibitor.
Results of the inhibitory potency of different inhibitors on proteus:
* Note: the addition amount of each inhibitor is shown in a reference part commercial culture medium composition table
According to the above results, ammonium citrate, sodium thiosulfate and crystal violet have no inhibitory effect on the spread of proteus vulgaris, and novobiocin has an inhibitory effect on the spread of proteus vulgaris CMCC49027 and proteus mirabilis CMCC 49005.
Effect of different concentrations of novobiocin on growth of various target bacteria:
as can be seen from the above table, with the increase of the content of the neomycin, when the content exceeds 20mg/L, the four target bacteria are inhibited more and more, and the growth capacity is weakened, but the sensitivity of the pseudomonas aeruginosa to the neomycin is smaller. Only when the concentration of the neomycin is 15mg/L, the growth of the salmonella typhimurium and the escherichia coli can not be influenced, and the spread of the interfering strain proteus mirabilis can be inhibited.
Inhibitory capacity of different inhibitors on the growth of pseudomonas aeruginosa:
* Note: the addition amount of each inhibitor is shown in a reference part commercial culture medium composition table
The results in the table show that the cefsulodin sodium salt hydrate has no inhibition ability on pseudomonas aeruginosa, and has partial inhibition ability. Further studies on the concentration of cefsulodin sodium salt hydrate were carried out.
Determination of the optimal bacteriostatic content of the pseudomonas aeruginosa inhibitor:
influence of different contents of cefsulodin sodium salt hydrate on the indicator:
according to the experimental results in the table above, when the cefsulodin sodium salt hydrate is more than 10mg/L, pseudomonas aeruginosa is inhibited, and the optimum concentration range of the cefsulodin sodium salt hydrate can be preliminarily determined to be 5-10 mg/L
Influence of 5-10 mg/L cefsulodin sodium salt hydrate on the indicator:
from the above table, the concentration of cefsulodin sodium salt hydrate was determined to be in the range of 6mg/L.
Example 1:
a chromogenic medium for detecting Salmonella, comprising the following medium components per 1000ml of the medium:
33g of basic culture medium and 0.55g of sodium deoxycholate are weighed and added into 1000ml of water to be mixed uniformly to obtain mother liquor 1. The octaesterase substrate is completely dissolved in the cosolvent according to the proportion, added into the mother liquor 1, shaken up and boiled to be dissolved to be used as a mother liquor 2, and cooled to 45 +/-1 ℃. Fully dissolving the neomycin and the cefsulodin sodium hydrate in sterile water, filtering and sterilizing, and adding the filtrate into the mother liquor 2 which is boiled and cooled to 45 +/-1 ℃; dissolving galactose glycolase substrate and isopropyl-beta-D-thiogalactoside in solvent respectively, filtering to remove bacteria, adding the above solution, boiling, and cooling to 45 + -1 deg.C mother liquor 2. Shaking up, pouring into a sterile plate, and storing the prepared plate at 2-8 ℃ in the dark.
In the preparation process, 2ml of dimethyl sulfoxide and 3ml of Tween 20 are uniformly mixed by shaking at 2800rpm for 1min, then octyl esterase substrate is added into the mixed solvent and uniformly mixed by shaking at 2800rpm for 1min, after complete dissolution, mother liquor 1 is added, and after shaking uniformly, boiling is carried out. The octyl esterase substrate has thermal stability, and needs to be heated and boiled after the mother liquor 1 is added, and if the substrate is directly added into a basic culture medium cooled to 45 +/-1 ℃, the dissolved substrate is easy to precipitate again. According to example 1, to maintain the effectiveness of the inhibitor, the novobiocin and cefsulodin sodium hydrate are dissolved in sterile water in a proper proportion to prepare a solution, and the solution is filtered and sterilized and added into mother liquor 2 which is boiled and cooled to 45 +/-1 ℃.
The method is characterized in that salmonella and gram-positive bacteria growth are screened as targets, and bacteriostatic experiments are further performed on proteus, serratia and pseudomonas aeruginosa, and in inhibitors such as sodium dodecyl sulfate, sodium deoxycholate, tribasic cholate, sodium citrate, sodium thiosulfate, crystal violet, novobiocin, piperacillin sodium, ceftazidime, gentamicin, ciprofloxacin, ceftazidime sodium salt hydrate and the like, 3 selective additives are finally determined, wherein the selective additives are respectively: sodium deoxycholate, novobiocin and cefsulodin sodium salt hydrate. In the invention, sodium deoxycholate can inhibit the growth of gram-positive bacteria and mould hyphae and can increase the membrane permeability of microorganisms; the neomycin can inhibit the spread growth of proteus while not influencing the growth of salmonella and escherichia coli; the cefsulodin sodium salt hydrate can inhibit the growth of the pseudomonas aeruginosa which is a false positive bacterium.
Experiments prove that after 40% of dimethyl sulfoxide and 60% of Tween 20 are mixed and dissolved, the caprylase substrate is dissolved, the phenomenon that the surface of a flat plate is oily due to excessive emulsifying agents is avoided, the substrate can be completely dissolved, and the chromogenic effect of the combined enzyme chromogenic substrate is optimal. Experiments prove that when 0.55g of sodium deoxycholate is contained in each 1000ml of culture medium, the sodium deoxycholate can inhibit the growth of gram-positive bacteria and mould hyphae, can increase the membrane permeability of microorganisms, and is favorable for transporting metabolites out of the membrane. Experiments prove that when each 1000ml of culture medium contains 15mg of neomycin, the growth of salmonella and escherichia coli is not influenced, and the spread growth of proteus can be inhibited. Experiments prove that when each 1000ml of culture medium contains 6mg of cefsulodin sodium salt hydrate, the growth of salmonella and escherichia coli is not influenced, and the growth of pseudomonas aeruginosa serving as a false positive bacterium can be inhibited.
Since 5-br-6-cl-3-indole-nonyl ester + 5-br-6-cl-3-indole-octyl ester is fat-soluble and difficult to dissolve, and further influences the color development effect, it is very important to find a cosolvent and a dissolution mode which are suitable for dissolving the ester and can better develop color. For convenience of statistical analysis, score is adopted to score the color development condition of the corresponding strain in the culture medium.
The selection of co-solvents and dissolution modes are given in the following table:
through a plurality of experiments, the dissolving effect of heating in a water bath is poor, and the Tween can be easily dissolved after heating in the water bath, but the surface of the prepared flat plate has oily substances, wherein the color development effect after dissolving the Tween 20 is better than that of the Tween 80. Dimethyl sulfoxide (DMSO) is a polar solvent, an esterase substrate is very easy to dissolve, but the color development effect is poor, then the two are combined, the esterase substrate is dissolved by DSMO firstly, and then the mixture is dissolved in Tween 20, so that the color development and dissolution effects are good. Because the color development is not optimal, the experiment of optimal proportion of the color development and the color development is carried out, and the result is as follows:
wherein DMSO damages cells, and the amount of Twin-20 is so large that the apparent oil content of the plate is large, and when the substrate is dissolved by mixing 40% DMSO and 60% Twin-20, the dissolution is convenient, the appearance is consistent, and the color development effect is best, and the color is purple red.
Example 2:
a chromogenic medium for detecting Salmonella comprising, per 1000ml of medium, the following composition table:
42g of basal culture medium and 0.6g of sodium deoxycholate are weighed and added into 1000ml of water to be mixed evenly, and the mixture is boiled and dissolved to be used as mother liquor 1. The octaesterase substrate is completely dissolved in the cosolvent according to the proportion, added into the mother solution 1, shaken up and boiled to be dissolved to be used as a mother solution 2. Fully dissolving the neomycin and the cefsulodin sodium salt hydrate in sterile water, filtering and sterilizing, and adding the mixture into the mother liquor 2 which is boiled and cooled to 45 +/-1 ℃; dissolving galactose substrate and isopropyl-beta-D-thiogalactoside in solvent respectively, filtering, sterilizing, adding above solution, boiling, and cooling to 45 + -1 deg.C mother liquor 2. Shaking up and pouring into a sterile plate, and storing the prepared plate at the temperature of 2-8 ℃ in the dark.
And (3) completely dissolving the octyl esterase substrate in a mixed solvent of 20% of dimethyl sulfoxide and 80% of Tween 20, and adding the mother liquor 1 to 45 +/-1 ℃.
Example 3:
a chromogenic medium for detecting Salmonella comprising, per 1000ml of medium, the following table of media components:
41g of basal culture medium and 0.4g of sodium deoxycholate are weighed and added into 1000ml of water to be mixed uniformly to obtain mother liquor 1. And (3) completely dissolving the octanase substrate and the cosolvent according to the proportion, adding the dissolved octaesterase substrate into the mother solution 1, shaking uniformly, and boiling to dissolve the dissolved octaesterase substrate to obtain the mother solution 2. Fully dissolving the hydrate of the neomycin and the cefsulodin sodium hydrate in sterile water, filtering and sterilizing, and adding the filtrate into the mother liquor 2 which is boiled and cooled to 45 +/-1 ℃; dissolving galactose glycolase substrate and isopropyl-beta-D-thiogalactoside in solvent respectively, filtering to remove bacteria, adding the above solution, boiling, and cooling to 45 + -1 deg.C mother liquor 2. Shaking up, pouring into a sterile plate, and storing the prepared plate at 2-8 ℃ in the dark.
And (3) completely dissolving the octyl esterase substrate in a mixed solvent of 60% of dimethyl sulfoxide and 40% of tween-20, adding the mother solution 1, shaking uniformly and boiling.
Comparative example 1:
specific experiments:
according to experiments, example 1 is the best example, and the prepared flat plate has a light yellow semitransparent appearance and is easy to observe. Salmonella typhimurium ATCC14028, salmonella choleraesuis ATCC 13312, salmonella enteritidis ATCC13076, salmonella paratyphi B CM50094, salmonella typhi CM50071, salmonella paratyphi A CM50093, salmonella anatipestifer ATCC9150, escherichia coli ATCC25922, enterobacter sakazakii ATCC25944, citrobacter freundii ATCC43864, pseudomonas aeruginosa ATCC9027, shigella flexneri CMCC51572, shigella sonnei, enterobacter aerogenes ATCC13048, enterobacter cloacae CMCC45301, serratia marcescens CMCC41002, proteus vulgaris CMCC49027, proteus mirabilis CMCC49005, staphylococcus aureus ATCC25923, enterococcus faecalis ATCC29212 and other 20 standard strains and 5 Salmonella collected by Montmorida, salmonella arnica, salmonella newans, salmonella typhimurium 1# and Salmonella salmonella 2# are inoculated into a Botrychizogenes and cultured at a certain temperature of a culture medium S1 and 24 ℃ respectively (see Table 1, S37 ℃ for short).
TABLE 1 S2 detection results of the specificity of the chromogenic Medium and comparison of the chromogenic time
The results are shown in table 1, in the aspect of specificity, seven salmonella standard strains and five positive strains grow well on the S2 and a color development plate of a certain domestic manufacturer, the positive bacterial colonies are purple, and the purple color is brighter than the color development color of the domestic manufacturer on the S2 and is easy to observe; wherein, a plurality of salmonella are light purple on a Colaga color development plate and a color development plate of a domestic manufacturer, and are purple on S2; purple on the other two plates a dark purple on the S2 plate was observed and the results were viewed in a shorter time. For salmonella with strong enzyme activity, the salmonella can be dark purple on an S2 plate after being cultured for 18h, and the salmonella can be purple after being cultured on a certain plate in China for 24h; after the salmonella with slightly weak enzyme activity is cultured for 24 hours, the salmonella can be purple on an S2 plate, but only light purple can be presented on a certain plate in China. The culture time and the color development experiment can prove that the S2 plate can promote the hydrolysis of esterase substrates. Coli and other galactoglycase-containing strains show blue-green on two plates, and colonies of staphylococcus aureus, enterococcus faecalis and the like are inhibited on the two brand culture media, and other strains are colorless. The strains which interfere with the detection of salmonella, such as pseudomonas aeruginosa, serratia marcescens and the like, are inhibited by specific inhibitors.
Representative Salmonella typhi CM50071 with strong interference, common proteus CMCC49027, escherichia coli ATCC25922, pseudomonas aeruginosa ATCC9027 and enterococcus faecalis ATCC29212 are prepared into mixed bacterial suspension, a ring of mixed enrichment liquid is taken and inoculated on S2 and a color development medium plate of a certain domestic manufacturer in a streak manner, and the result is shown in Table 2.
TABLE 2 isolation of mixed bacterial suspensions on various brands
As can be seen from Table 2, S2 and a certain domestic manufacturer are easy to separate from other interfering mixed bacteria, and colonies with three colors, namely colorless, deep purple and blue-green, appear on the flat plate; wherein the color development of target bacteria on the S2 culture medium is easier to identify, especially the slow-developing salmonella typhi.
Comparative example 2:
growth rate:
salmonella murine ATCC14028 and Salmonella enteritidis ATCC13076 were prepared as bacterial suspensions (concentration 10) 8 -10 9 CFU/ml), take dilution 10 -5 And 10 -6 0.1ml of the bacterial suspension was uniformly applied to S21, a certain domestic manufacturer and Tryptone Soy Agar (TSA), and cultured at 36 ℃ for 24 hours. Counting the number of plates with inoculation level of 20-200 CFU, and calculating the growth rate of each plate.
TABLE 3 comparison of sensitivity of chromogenic Medium to Salmonella and colony morphology
As can be seen from Table 3, the sensitivity of S2 and a certain domestic manufacturer is higher, the growth rate of the salmonella typhimurium and the salmonella enteritidis in S2 is higher than that of the certain domestic manufacturer, and is far higher than the specification that PR is more than or equal to 0.5 in GB 4789.28-2013. In the aspect of colony size, the colony diameter of the salmonella typhimurium and the salmonella enteritidis on S2 is larger than that of a certain domestic manufacturer. The S2 plate is more suitable for the growth of salmonella.
Comparative example 3:
detection of salmonella in artificially contaminated samples:
1. strain: salmonella typhimurium ATCC14028 is inoculated into nutrient broth to prepare a standard bacterial suspension, and bacterial suspensions containing target bacteria 10CFU-100CFU and 1-10CFU are prepared for standby. And the TSA count is used.
2. Pre-enrichment and post-enrichment: weighing 25g of milk powder sample, adding into 225ml of BPW enrichment liquid, and uniformly mixing. Inoculating the prepared bacterial suspension into the sample suspension, and culturing at 36 ℃ for 8-18 h. The incubated sample mixture was gently shaken, 1mL was removed,
3. inoculating and culturing: one ring of the enrichment medium after culture is taken, four zones are streaked and inoculated on a chromogenic medium plate of example 1 (S2 for short), XLD, HE and a chromogenic medium of a certain domestic manufacturer, the mixture is cultured for 18 to 24 hours at 36 ℃, and the color and the morphology of a colony are recorded according to the growth condition in a streaking area, which is shown in a table 4.
TABLE 4 detection of artificially contaminated samples by S2
As can be seen from Table 4, the larger the amount of Salmonella inoculated, the larger the streaked growth area, and when the amount of Salmonella inoculated is 1-100 CFU, all the target bacteria grow in the 4 zones, but due to the competition relationship of the bacteria for the nutrient components, distinguishable typical colonies can only appear in the third and fourth zones on the XLD medium. On the S2 flat plate, the target bacteria are the bacterial colonies as long as the bacterial colonies are purple, the identification degree is high, and the identification and the detection are easy; when the inoculation amount is 1-10CFU, XLD and S2 can grow in 3 areas, typical colonies appear in the third area in XLD, and purple-red colonies can appear in all the areas in S2. Therefore, S2 can detect salmonella in a low-pollution area, and can reach the detection limit of 1-10CFU, which is equivalent to the detection limit of a certain domestic manufacturer.
Comparative example 4:
detection of salmonella in actual samples:
1. collecting samples
139 portions of each food product were collected from the supermarket and the raw material supplier.
2. Enrichment culture
Weighing 25g of sample, adding the sample into 225ml of BPW enrichment solution, mixing uniformly, and culturing for 18h at 36 ℃.
3. Inoculating and culturing
The enrichment broth after one-cycle culture was streaked on the Salmonella chromogenic medium plate prepared in example 1, cultured at 37 ℃ for 18-24 hours, and the color and morphology of the colonies were observed. Gently shaking the cultured sample mixture, transferring 1mL of the sample mixture, transferring the sample mixture into 10mL of TTB, and culturing the sample mixture for 18-24h at 42 +/-1 ℃; meanwhile, another 1mL of the suspension is transferred into 10mL of SC, and cultured for 18-24h at 36 +/-1 ℃.
4. Results Observation and analysis
23 samples in 139 samples are used for detecting salmonella, and the samples are detected and compared by using GB4789.4-2010 and API20E biochemical identification systems, and the results are consistent with those of a chromogenic medium, and the detection results of the two methods are consistent with expected results.
In conclusion, various inhibitors are added into the salmonella chromogenic medium firstly, so that the infection of candida does not exist; the sodium sulfadiazine salt hydrate is added, so that the interference of pseudomonas aeruginosa containing esterase activity which is easy to cause false positive can be avoided, and the growth of salmonella and escherichia coli can not be influenced; by adding 5-bromo-4-chloro-3-indole-beta-galactoside, enterobacter such as Escherichia coli and Escherichia coli containing beta-galactosidase can be distinguished.
The invention takes 5-bromo-6-chloro-3-indole-nonyl ester and 5-bromo-6-chloro-3-indole-octyl ester as chromogenic substrates, increases enzyme substrates to promote more esterase reaction production, and makes up for the defects of slow color development and weak color development of part of salmonella.
The salmonella can be in a darker color, namely dark purple, and the color is more obvious and faster. In addition, the 5-bromo-6-chloro-3-indole-nonyl ester contains a novel substrate, has the advantages of high purity, easiness in synthesis, low cost, thermal stability and the like, and makes up for the limitation that most of esterase substrates pass through import. The research of the invention finds that the salmonella also contains C9 esterase (nonanesterase) besides esterase at the C8 level, and the esterase can hydrolyze nonane ester chromogenic substrates. According to the invention, the culture medium is added with the nonane esterase and the octyl esterase chromogenic substrates, and the salmonella can hydrolyze the substrates to generate chromogenic alcohols.
The additive components of the chromogenic medium of the invention act synergistically, 5-bromo-6-chloro-3-indolyl-nonyl ester and 5-bromo-6-chloro-3-indolyl-octyl ester are jointly used as chromogenic substrates, the generation of more esterase reactions is synergistically promoted, more chromogenic groups are released, and meanwhile, the selection and the proportion of the cosolvent enable the chromogenic medium of the invention to not cause the oily phenomenon on the surface of a flat plate due to excessive emulsifying agent, but also enable the substrates to be completely dissolved, and the synergistic effect of the cosolvent enables the chromogenic effect of the enzyme chromogenic substrate of the chromogenic medium of the invention to achieve the best.
The chromogenic medium disclosed by the invention has the advantages of high sensitivity, strong specificity, higher identification degree, shorter detection period, stronger adaptability, easiness in industrial production and the like when used for detecting salmonella.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (1)
1. A preparation method of a chromogenic medium for detecting salmonella is characterized by comprising the following steps: comprises a basic culture medium and an additive, wherein the additive comprises an esterase chromogenic substrate, and the esterase chromogenic substrate comprises 5-bromo-6-chloro-3-indole-octyl ester and 5-bromo-6-chloro-3-indole-nonyl ester;
the method comprises the following steps:
preparing a mother solution 1: weighing a basic culture medium and uniformly mixing the basic culture medium with sodium deoxycholate to obtain mother solution 1;
preparing a mother solution 2: dissolving an esterase chromogenic substrate in a cosolvent, adding the esterase chromogenic substrate into the mother liquor 1, and boiling to obtain a mother liquor 2;
preparing a color development culture medium: dissolving neomycin and cefsulodin sodium salt hydrate in water, adding the solution into the mother solution 2, and adding a galactose glycolase substrate and isopropyl-beta-D-thiogalactoside into the mother solution 2;
the galactosidase chromogenic substrate comprises 5-bromo-4-chloro-3-indole-beta-galactoside;
the culture medium comprises 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride, 15g/L of bacterial agar powder, 0.55g/L of sodium deoxycholate, 15mg/L of neomycin, 6mg/L of cefsulodin sodium salt hydrate, 0.2g/L of 5-bromo-6-chloro-3-indole-octyl ester, 0.3g/L of 5-bromo-6-chloro-3-indole-nonyl ester, 0.08g/L of 5-bromo-4-chloro-3-indole-beta-galactoside, 0.05g/L of isopropyl-beta-D-thiogalactoside, 2ml/L of dimethyl sulfoxide and 3ml/L of Tween 20;
in the cosolvent, the volume content of dimethyl sulfoxide is 40%, and the volume content of tween 20 is 60%;
and uniformly mixing the basic culture medium and sodium deoxycholate at the speed of 2800rpm for 1min to prepare a mother solution 2, wherein after boiling, the mother solution 2 is cooled to 45 +/-1 ℃.
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