CN108359707A - It is a kind of to be used to detect chromogenic culture medium of salmonella and preparation method thereof - Google Patents
It is a kind of to be used to detect chromogenic culture medium of salmonella and preparation method thereof Download PDFInfo
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- CN108359707A CN108359707A CN201810516950.8A CN201810516950A CN108359707A CN 108359707 A CN108359707 A CN 108359707A CN 201810516950 A CN201810516950 A CN 201810516950A CN 108359707 A CN108359707 A CN 108359707A
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- culture medium
- indoles
- chromogenic
- salmonella
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- 241000607142 Salmonella Species 0.000 title claims abstract description 77
- 239000001963 growth medium Substances 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000003593 chromogenic compound Substances 0.000 claims abstract description 26
- 108090000371 Esterases Proteins 0.000 claims abstract description 25
- 150000002148 esters Chemical class 0.000 claims abstract description 24
- 239000000654 additive Substances 0.000 claims abstract description 20
- 230000000996 additive effect Effects 0.000 claims abstract description 19
- 239000007640 basal medium Substances 0.000 claims abstract description 14
- -1 indoles nonyl ester Chemical class 0.000 claims abstract description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 58
- 241000894006 Bacteria Species 0.000 claims description 46
- 239000012452 mother liquor Substances 0.000 claims description 35
- 239000003112 inhibitor Substances 0.000 claims description 34
- 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 20
- 239000006184 cosolvent Substances 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 11
- 229930182470 glycoside Natural products 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 6
- 235000009508 confectionery Nutrition 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000000411 inducer Substances 0.000 claims description 4
- 102000002464 Galactosidases Human genes 0.000 claims description 3
- 108010093031 Galactosidases Proteins 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 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 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 abstract 6
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 abstract 6
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 abstract 6
- 229910052794 bromium Inorganic materials 0.000 abstract 6
- 229910052801 chlorine Inorganic materials 0.000 abstract 6
- 239000000460 chlorine Substances 0.000 abstract 6
- 150000002475 indoles Chemical class 0.000 abstract 3
- 239000000758 substrate Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 13
- 241000588769 Proteus <enterobacteria> Species 0.000 description 10
- 238000001914 filtration Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 238000004659 sterilization and disinfection Methods 0.000 description 7
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- 239000003153 chemical reaction reagent Substances 0.000 description 6
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- 238000002474 experimental method Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000006916 nutrient agar Substances 0.000 description 5
- 241000588767 Proteus vulgaris Species 0.000 description 4
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 229940007042 proteus vulgaris Drugs 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 241000943303 Enterococcus faecalis ATCC 29212 Species 0.000 description 3
- 241000588770 Proteus mirabilis Species 0.000 description 3
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000012046 mixed solvent Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 241000222122 Candida albicans Species 0.000 description 2
- 241000588919 Citrobacter freundii Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 235000004338 Syringa vulgaris Nutrition 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 239000003833 bile salt Substances 0.000 description 2
- 229940095731 candida albicans Drugs 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
- 239000000306 component Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- WMWTYOKRWGGJOA-CENSZEJFSA-N fluticasone propionate Chemical compound C1([C@@H](F)C2)=CC(=O)C=C[C@]1(C)[C@]1(F)[C@@H]2[C@@H]2C[C@@H](C)[C@@](C(=O)SCF)(OC(=O)CC)[C@@]2(C)C[C@@H]1O WMWTYOKRWGGJOA-CENSZEJFSA-N 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 239000008236 heating water Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 125000001400 nonyl 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])C([H])([H])[H] 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 229940127100 salmeterol-fluticasone Drugs 0.000 description 2
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].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)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
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- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193468 Clostridium perfringens Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 241001135265 Cronobacter sakazakii Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588697 Enterobacter cloacae Species 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 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
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102000002268 Hexosaminidases Human genes 0.000 description 1
- 108010000540 Hexosaminidases Proteins 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 241001104043 Syringa Species 0.000 description 1
- 244000297179 Syringa vulgaris Species 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 235000010633 broth Nutrition 0.000 description 1
- 229960004261 cefotaxime Drugs 0.000 description 1
- AZZMGZXNTDTSME-JUZDKLSSSA-M cefotaxime sodium Chemical compound [Na+].N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 AZZMGZXNTDTSME-JUZDKLSSSA-M 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- 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 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N ferric oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 150000008195 galaktosides Chemical class 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 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
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 239000006152 selective media Substances 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
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- NASFKTWZWDYFER-UHFFFAOYSA-N sodium;hydrate Chemical compound O.[Na] NASFKTWZWDYFER-UHFFFAOYSA-N 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000004094 surface-active agent Substances 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
- 201000008297 typhoid fever Diseases 0.000 description 1
Classifications
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- 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 kind of chromogenic culture mediums and preparation method thereof for detecting salmonella, it includes, basal medium, additive, the additive includes esterase chromogenic substrate, the esterase chromogenic substrate includes 3 indoles nonyl ester of 5 bromine, 6 chlorine, 3 indoles monooctyl ester and 5 bromine, 6 chlorine, and 5 bromine, 6 chlorine, 3 indoles monooctyl ester content is 0.1~0.4g/L, 5 bromine, 6 chlorine, 3 indoles nonyl ester content is 0.1~0.4g/L.Each additive component of chromogenic culture medium of the present invention mutually acts synergistically, and collectively as chromogenic substrate, collaboration promotes the generation of more Esterase reactions for 5 bromine, 6 chlorine, 3 indoles nonyl ester and 5 bromine, 6 chlorine, 3 indoles monooctyl ester, releases more colour developing groups.The chromogenic culture medium of the present invention has high sensitivity for detecting salmonella, and high specificity, identification higher, detection cycle are shorter, more adaptable, the advantages that being easy to industrialization production.
Description
Technical field
The invention belongs to technical field of microbial detection, and in particular to a kind of chromogenic culture medium for detecting salmonella
And preparation method thereof.
Background technology
Salmonella (Salmonella) is a kind of common food-borne pathogens, enterobacteriaceae, Gram-negative bacteria.Sense
The people of salmonella or the fecal pollution food of carrier are contaminated, can make one to poison by food.According to statistics in the kind of countries in the world
In class food posioning, the salmonellal normal row umber one of food poisoning.China hinterland is also with salmonella
It is the first.
It is most common in country's Salmeterol fluticasone propionate at present to be still traditional national standard method.National standard method commonly identifies training
It supports best with chromogenic culture medium good selective using effect in base.Salmonella color culture medium is to utilize salmonella
The novel culture medium for the enzyme and corresponding chromogenic substrate reaction solution that own metabolism generates.These corresponding chromogenic substrates are by chromogenic
Gene and the metabolizable material composition of microbial portion, under enzyme-specific effect, free color base of producing is because showing certain color, directly
Strain can be made identification by connecing observation colony colour.
At present the country's certain producer sramana's chromogenic culture medium common substrate be the chloro- 3- indoles-beta galactose glycosides of the bromo- 4- of 5-,
The chloro- 3- indoles-N- acetyl-β-D- glucosaminides of the bromo- 4- of 5- and the chloro- 3- indoles of the bromo- 6- of 5--monooctyl ester, wherein beta galactose
Glycosides purpose is to discriminate between the Enterobacter (such as C. freundii bacillus) containing beta galactosidase, hexosaminidase chromogenic substrate
Purpose is to discriminate between candida albicans；Monooctyl ester enzyme chromogenic substrate purpose is to discriminate between salmonella.But due to part salmonella (such as typhoid fever
Salmonella and first salmonella typhi etc.) esterase activity it is weaker, incubation time reach after, colour generation is thin, is not easy to observe.
Invention content
The purpose of this part is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferably to implement
Example.It may do a little simplified or be omitted to avoid our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, and this simplification or omit and cannot be used for limiting the scope of the invention.
In view of above-mentioned technological deficiency, it is proposed that the present invention.
Therefore, as in terms of one of present invention, the present invention overcomes the deficiencies in the prior art, provides one kind
Chromogenic culture medium for detecting salmonella.
In order to solve the above technical problems, the present invention provides following technical solutions：It is a kind of to be used to detect the aobvious of salmonella
Color culture medium comprising, basal medium, additive, the additive include esterase chromogenic substrate, the esterase chromogenic substrate
Including the chloro- 3- indoles-monooctyl esters of the bromo- 6- of 5- and the chloro- 3- indoles-nonyl esters of the bromo- 6- of 5-, the chloro- 3- indoles-monooctyl esters of the bromo- 6- of 5- contain
Amount is that the chloro- 3- indoles-nonyl ester content of 0.1~0.4g/L, the bromo- 6- of the 5- is 0.1~0.4g/L.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The basis culture
Base includes peptone, beef extract, sodium chloride, bacterial agar, wherein the peptone content is 8~15g/L, the beef
Cream content is 1~5g/L, the sodium chloride content is 5~6g/L, the bacteria Agr powder content is 15~18g/L.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The additive is also
Including inhibitor, galactosidase chromogenic substrate, enzyme inducer, cosolvent, wherein the inhibitor include NaTDC,
Ovobiocin, Cefsulodin sodium-salt hydrate, the deoxycholic acid sodium content are 0.4~0.6g/L, the ovobiocin content
It is 5~10mg/L for 10~15mg/L, the Cefsulodin sodium-salt hydrate content.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The galactoside
Enzyme chromogenic substrate includes the chloro- 3- indoles-beta galactose glycosides of the bromo- 4- of 5-, the chloro- 3- indoles of the bromo- 4- of the 5--beta galactose glycosides content
For 0.04~0.1g/L.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The enzyme inducer
Including isopropyl-β-D-thiogalactoside, the content of the isopropyl-β-D-thiogalactoside is 0.03~0.06g/L.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The cosolvent packet
Dimethyl sulfoxide (DMSO), polysorbas20 are included, the dimethyl sulfoxide (DMSO) content is 1~3ml/L, the polysorbas20 content is 2~4ml/L.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The cosolvent
In, dimethyl sulfoxide (DMSO) volume content is 40%, polysorbas20 volume content is 60%.
As a kind of preferred embodiment of the present invention for detecting the chromogenic culture medium of salmonella：The culture medium packet
It includes peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, bacterial agar 15g/L, NaTDC 0.55g/L, newly mildew
The chloro- chloro- 3- Yin of 3- indoles-bromo- 6- of monooctyl ester 0.2g/L, 5- of plain 15mg/L, the bromo- 6- of Cefsulodin sodium-salt hydrate 6mg/L, 5-
The chloro- 3- indoles of diindyl-bromo- 4- of nonyl ester 0.3g/L, 5--beta galactose glycosides 0.08g/L, isopropyl-β-D-thiogalactoside
0.05g/L, dimethyl sulfoxide (DMSO) 2ml/L, polysorbas20 3ml/L.
As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provide claim 3~
The preparation method of 8 any chromogenic culture mediums for detecting salmonella.
In order to solve the above technical problems, the present invention provides following technical solutions：Any use of claim 3~8
In the preparation method of the chromogenic culture medium of detection salmonella comprising, prepare mother liquor 1：Weigh basal medium and and deoxidation
Sodium taurocholate mixing, as mother liquor 1；Prepare mother liquor 2：Esterase chromogenic substrate is dissolved in cosolvent, and is added in mother liquor 1, is boiled
Boiling, as mother liquor 2；Prepare chromogenic culture medium：Ovobiocin, Cefsulodin sodium-salt hydrate is soluble in water, mother liquor 2 is added
In, and the sweet zymolyte of galactolipin and isopropyl-β-D-thiogalactoside are added in mother liquor 2.
As a kind of preferred embodiment of the present invention for detecting the preparation method of the chromogenic culture medium of salmonella：Institute
State basal medium and with NaTDC mixing, speed 2800rpm, time 1min, the preparation mother liquor 2, wherein institute
It states after boiling, the mother liquor 2 is cooled to 45 ± 1 DEG C, the inhibitor is added in mother liquor 2.
Beneficial effects of the present invention：Each additive component of chromogenic culture medium of the present invention mutually acts synergistically, the bromo- 6- of 5-
Collectively as chromogenic substrate, collaboration promotes more Esterase reactions for chloro- 3- indoles-nonyl ester and the chloro- 3- indoles-monooctyl esters of the bromo- 6- of 5-
It generates, releases more colour developing groups, meanwhile, selection and the proportioning of cosolvent were so that the present invention had both been not in due to emulsifier
Excessively planar surface is made oily phenomenon occur, and substrate can be made to be completely dissolved, and the synergistic effect of cosolvent makes of the present invention group
The color developing effect of synthase chromogenic substrate reaches best.
The chromogenic culture medium of the present invention has high sensitivity, high specificity, identification higher, inspection for detecting salmonella
It is shorter, more adaptable to survey the period, the advantages that being easy to industrialization production.
Specific implementation mode
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment pair
The specific implementation mode of the present invention is described in detail.
Many details are elaborated in the following description to facilitate a thorough understanding of the present invention, still the present invention can be with
Implemented different from other manner described here using other, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to that may be included at least one realization side of the present invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiment.
The selection of G+ bacteria inhibitors：
The chromogenic culture medium basis of the present invention is based on ordinary nutrient agar, by adding inhibitor, to exclude completely
The interference of the gram-positive bacterias such as enterococcus faecalis corresponds to growth of the strain in culture medium in order to facilitate statistical analysis using score
Situation and other influences situation：
The selection of the most suitable additive amount of G+ bacteria inhibitors：
《4789.28-2013 the quality requirement of culture medium and reagent》6.1.2.2 it is the selection of qualitative assessment culture medium in
Property, should the Working Culture of appropriate test strain be seeded to by selective medium with proper method according to the rules and reference is cultivated
In base.This patent is based on ordinary nutrient agar, by adding the additive of various dose, after preparing tablet, according to
《4789.28-2013 the quality requirement of culture medium and reagent》6.1.2.2 middle sxemiquantitative scribble method measures inhibitor to gram
The selectivity of positive bacteria.Testing bacterium is：Staphylococcus aureus ATCC6538 and enterococcus faecalis ATCC29212, it is suppressed to observe its
Situation processed.
The selection of the most suitable inhibitor of proteus：
Proteus also has interference since with flagellum, easily sprawling is grown on tablet, and to the detection of sramana, therefore finds
Go out to can inhibit the additive of growth.Different inhibitor is added in basal medium, according to《4789.28-2013 culture medium
With the quality requirement of reagent》6.1.2.2 middle sxemiquantitative scribble method measures selectivity of the inhibitor to proteus.It is pressed after culture
Following methods calculate growth index G to culture medium.Every has denser bacterium colony to grow, G=1；The line of only half has dense bacterium
It is born length, G=0.5；No bacterium colony growth, increment grow faint, G=0 less than the half or bacterium colony of scribing line in scribing line；Record
The score summation of each tablet just obtains G.
The selection of proteus inhibitor optium concentration：
Based on ordinary nutrient agar, by adding the additive of various dose, after preparing tablet, according to
《4789.28-2013 the quality requirement of culture medium and reagent》6.1.2.2 middle sxemiquantitative scribble method measures inhibitor to gram
The selectivity of positive bacteria.Testing bacterium is：Proteus vulgaris CMCC49027 and proteus mirabilis CMCC49005, observe its by
Inhibit situation.
The selection of the most suitable inhibitor of pseudomonas aeruginosa：
Since pseudomonas aeruginosa also has esterase, there is interference to the selection of salmonella, therefore Salmonella develops the color
It needs that the inhibitor that can inhibit such bacterium is added in culture medium.Different inhibitor is added in basal medium, according to
《4789.28-2013 the quality requirement of culture medium and reagent》6.1.2.2 middle sxemiquantitative scribble method measures inhibitor to deformed rod
The selectivity of bacterium.
The selection of P. aeruginosa bacteria inhibitor optium concentration：
Based on ordinary nutrient agar, by adding the additive of various dose, after preparing tablet, according to
《4789.28-2013 the quality requirement of culture medium and reagent》6.1.2.2 middle sxemiquantitative scribble method measures inhibitor to gram
The selectivity of positive bacteria.Testing bacterium is：Pseudomonas aeruginosa ATCC9027 observes its suppressed situation.
Interpretation of result：
The selection of G+ bacteria inhibitors
Rejection ability and other results of property such as following table of the inhibitor to different indicator bacterias：
* it notes：The additive amount reference section commercial medium component list of each inhibitor
Dodecyl sodium sulfate, no. 3 bile salt and NaTDC can inhibit gram-positive bacteria, due to dodecyl
Sodium sulfonate is surfactant, and the substrate added in culture medium is fat-soluble, therefore oil droplet is easily formed in culture medium, influences esterase
The dissolving of substrate, and consider that NaTDC is very important one kind in cholate, numerous studies show that NaTDC can
Cell membrane stability is set to decline, the order of mobility enhancing, membrane structure reduces, and can inhibit mycelial growth, selects desalination
Sodium taurocholate is as Gram-positive bacteria inhibitor.
Different inhibitor are to proteus rejection ability result：
* it notes：The additive amount reference section commercial medium component list of each inhibitor
According to the above results, ammonium citrate, sodium thiosulfate and crystal violet to the no inhibiting effect of the sprawling of proteus,
Ovobiocin has inhibiting effect to the sprawling of proteus vulgaris CMCC49027 and proteus mirabilis CMCC49005.
The influence that various concentration ovobiocin grows various object bacterias：
As seen from the above table, with the increase of ovobiocin content, when more than 20mg/L, four kinds of object bacterias are suppressed intensity
Increasing, growth ability weakens, but pseudomonas aeruginosa is smaller to ovobiocin sensitivity.Only when ovobiocin is dense
When degree is 15mg/L, the growth of salmonella typhimurium and escherichia coli is neither influenced, and can inhibit to interfere bacterial strain unusual
The sprawling of proteus.
Rejection ability of the different inhibitor to P. aeruginosa growth：
* it notes：The additive amount reference section commercial medium component list of each inhibitor
There is result in table to can be seen that pseudomonas aeruginosa unrestraint ability, Cefsulodin sodium-salt hydrate has part
Rejection ability.It needs further to study the concentration of Cefsulodin sodium-salt hydrate.
The determination of the most preferably antibacterial content of P. aeruginosa bacteria inhibitor：
Influence of the different content Cefsulodin sodium-salt hydrate to indicator：
According to upper table experimental result, when Cefsulodin sodium-salt hydrate is more than 10mg/L, pseudomonas aeruginosa is pressed down
System, can primarily determine most suitable Cefsulodin sodium-salt hydrate concentration range in 5~10mg/L
Influence of 5~10mg/L Cefsulodins sodium-salt hydrate to indicator：
As can be known from the above table, determine Cefsulodin sodium-salt hydrate concentration range in 6mg/L.
Embodiment 1：
It is as follows to contain medium component table per 1000ml culture mediums for a kind of chromogenic culture medium for detecting salmonella：
It weighs basal medium 33g and mixing in 1000ml water is added in NaTDC 0.55g, as mother liquor 1.Monooctyl ester enzyme
Substrate is proportionally completely dissolved in cosolvent, is added in mother liquor 1, dissolving is boiled after shaking up, as mother liquor 2, is cooled to 45
±1℃.Ovobiocin and Cefsulodin sodium-salt hydrate salt hydrate fully dissolve in sterile water, filtration sterilization, in addition
It states to boil and be cooled in 45 ± 1 DEG C of mother liquor 2；The sweet zymolyte of galactolipin and isopropyl-β-D-thiogalactoside are respectively at molten
It is dissolved in agent, after filtration sterilization, above-mentioned boil is added and is cooled to 45 ± 1 DEG C of mother liquors 2.It is poured into sterilized petri dishes after shaking up, prepares
Tablet be kept in dark place in 2-8 DEG C.
In above-mentioned preparation process, 2ml dimethyl sulfoxide (DMSO)s and 3ml polysorbas20s, after 2800rpm shakes 1min mixings, by monooctyl ester
Mixed solvent is added in zymolyte, and 2800rpm shakes 1min mixings, and mother liquor 1 is added after being completely dissolved, is boiled after shaking up.This monooctyl ester
Zymolyte has thermal stability, needs heating to boil after mother liquor 1 is added, 45 ± 1 DEG C are cooled to if being directly added to this substrate
In basal medium, the substrate dissolved is easily precipitated again.According to embodiment 1, to keep inhibitor effectiveness, ovobiocin and head
Spore sulphur pyridine sodium-salt hydrate salt hydrate is dissolved in wiring solution-forming in sterile water, filtration sterilization according to proper ratio, and addition is boiled cold
But in 45 ± 1 DEG C of mother liquors 2.
Not influence salmonella and gram-positive bacteria is inhibited to be grown to object filtering, further to proteus, sand
Thunder Salmonella and pseudomonas aeruginosa carry out bacteriostatic experiment, the present invention dodecyl sodium sulfate, NaTDC, no. 3 bile salt,
Sodium citrate, sodium thiosulfate, crystal violet, ovobiocin, avocin, cefotaxime, gentamicin, Ciprofloxacin, head
It is final to determine 3 kinds of selective additives of the invention in the inhibitor such as spore sulphur pyridine sodium-salt hydrate, be respectively：It is NaTDC, new
Mildew element and Cefsulodin sodium-salt hydrate.In the present invention, NaTDC can inhibit gram-positive bacteria and fungal hyphae
Growth, and the membrane permeability of microorganism can be increased；Ovobiocin is not influencing the growth of salmonella and Escherichia coli while can
Inhibit the sprawling growth of proteus；Cefsulodin sodium-salt hydrate salt hydrate can inhibit false positive bacterium pseudomonas aeruginosa
Growth.
Tests prove that dissolving monooctyl ester zymolyte after 40% dimethyl sulfoxide (DMSO) and 60% polysorbas20 are miscible, be both not in
Since emulsifier excessively makes planar surface oily phenomenon occur, and substrate can be made to be completely dissolved, and such group of synthase chromogenic substrate
Color developing effect it is best.It is demonstrated experimentally that when containing NaTDC 0.55g per 1000ml culture mediums, NaTDC can inhibit
The growth of gram-positive bacteria and fungal hyphae, and the membrane permeability of microorganism can be increased, be conducive to metabolite and transport film
Outside.It is demonstrated experimentally that when containing ovobiocin 15mg per 1000ml culture mediums, the growth of salmonella and Escherichia coli is neither influenced
And and it can inhibit the sprawling growth of proteus.It is demonstrated experimentally that containing Cefsulodin sodium-salt hydrate 6mg per 1000ml culture mediums
When, it neither influences the growth of salmonella and Escherichia coli and can inhibit the growth of false positive bacterium pseudomonas aeruginosa.
Since 5-br-6-cl-3- indoles-nonyl ester+5-br-6-cl-3- indoles-monooctyl ester is fat-soluble, it is difficult to dissolve, in turn
Color developing effect is influenced, therefore seeks to be suitble to the cosolvent of its dissolving and the more preferable colour generation of energy and dissolution mechanism extremely important.For convenience
Statistical analysis, using score score corresponding strain culture medium colour developing situation.
The selection of cosolvent and dissolution mechanism see the table below：
By many experiments, heating water bath solute effect is poor, can be soluble after tween heating water bath, but prepares flat
Plate surface has grease, wherein being better than Tween 80 using the dissolved color developing effect of polysorbas20.Dimethyl sulfoxide (DMSO) (DMSO) is
Polar solvent easily dissolves esterase substrate, but color developing effect is poor, after the two is combined, first dissolve esterase substrate with DSMO
Then it is that polysorbas20 is miscible, colour developing and solute effect are preferable.It is not up to best due to developing the color, therefore carry out the two proportions most
Good experiment, it is as a result as follows：
Wherein there is damage to cell due to DMSO, and Twin-20 contents keep tablet appearance grease more more, when with 40%
When DMSO and 60%Twin-20 mixed dissolution substrates, not only dissolving is convenient, and appearance meets, and color developing effect is best, and color is purple
It is red.
Embodiment 2：
It is as follows to contain medium component table per 1000ml culture mediums for a kind of chromogenic culture medium for detecting salmonella：
Basal medium 42g is weighed, NaTDC 0.6g is added mixing in 1000ml water, dissolving is boiled, as mother liquor
1.Monooctyl ester zymolyte is proportionally completely dissolved with cosolvent, is added in mother liquor 1, dissolving is boiled after shaking up, as mother liquor 2.
Ovobiocin and Cefsulodin sodium-salt hydrate salt hydrate fully dissolve in sterile water, filtration sterilization, and above-mentioned boil is added
It is cooled in 45 ± 1 DEG C of mother liquors 2；The sweet zymolyte of galactolipin and isopropyl-β-D-thiogalactoside respectively at being dissolved in solvent,
After filtration sterilization, above-mentioned boil is added and is cooled to 45 ± 1 DEG C of mother liquors 2.Sterilized petri dishes are poured into after shaking up, the tablet prepared is in 2-
8 DEG C are kept in dark place.
Monooctyl ester zymolyte is added but after 20% dimethyl sulfoxide (DMSO) and 80% polysorbas20 in the mixed solvent are completely dissolved to 45
± 1 DEG C of mother liquor 1.
Embodiment 3：
A kind of chromogenic culture medium for detecting salmonella contains medium component table per 1000ml culture mediums：
Basal medium 41g is weighed, mixing in 1000ml water is added in NaTDC 0.4g, as mother liquor 1.Monooctyl ester enzyme bottom
Object is proportionally completely dissolved with cosolvent, is added in mother liquor 1, dissolving is boiled after shaking up, as mother liquor 2.Ovobiocin and
Cefsulodin sodium-salt hydrate salt hydrate fully dissolves in sterile water, filtration sterilization, and above-mentioned boil is added and is cooled to 45 ± 1
In DEG C mother liquor 2；The sweet zymolyte of galactolipin and isopropyl-β-D-thiogalactoside in solvent respectively at dissolving, after filtration sterilization,
Above-mentioned boil is added and is cooled to 45 ± 1 DEG C of mother liquors 2.Sterilized petri dishes are poured into after shaking up, the tablet prepared is protected from light guarantor in 2-8 DEG C
It deposits.
Mother liquor 1 is added in monooctyl ester zymolyte after 60% dimethyl sulfoxide (DMSO) and 40% polysorbas20 in the mixed solvent are completely dissolved,
It is boiled after shaking up.
Comparative example 1：
Specificity experiments：
According to it is demonstrated experimentally that embodiment 1 is most preferred embodiment, the tablet of preparation is faint yellow translucent appearance, is easy to see
It examines.By salmonella typhimurium ATCC14028, Salmonella choleraesuls ATCC 13312, Bacterium enteritidis ATCC13076,
Moscow' paratyphi B CM50094, salmonella typhi CM50071, A type pair salmonella CM50093, duck Salmonella
Bacterium ATCC9150, escherichia coli ATCC25922, Enterobacter sakazakii ATCC25944, citrobacter freundii ATCC43864,
Pseudomonas aeruginosa ATCC9027, shigella flexneri CMCC51572, Song Shi Shigella, clostridium perfringen ATCC13048,
Enterobacter cloacae CMCC45301, cement Sha Leibai bacterium CMCC41002, proteus vulgaris CMCC49027, proteus mirabilis
20 kinds of reference cultures such as CMCC49005, staphylococcus aureus ATCC25923 and enterococcus faecalis ATCC29212 and 5 kinds are collected into
Montevideo salmonella, Argonne receive salmonella, Newport salmonella, salmonella 1# and salmonella 2# sramana
Salmonella positive strain, after nutrient agar activation, respectively on sramana's chromogenic culture medium made of streak inoculation to embodiment 1 (referred to as
S2) and in certain domestic producer, for 24 hours, testing result is shown in Table 1 for 37 DEG C of cultures.
The comparison of 1 S2 chromogenic culture medium specific detection results of table and developing time
1 is the results are shown in Table, specificity aspect, seven plants of salmonella reference cultures and five plants of positive strains are in S2 and certain domestic factory
Equal well-grown on family's colour developing tablet, positive bacterium colony is purple, and purple is vivid compared with domestic manufacturer's colour developing color on S2, easily sees
It examines；Many of salmonella shows lilac on the good colour developing tablet of Kerma (unit of kinetic energy) and domestic manufacturer's colour developing tablet, and purple is shown on S2
Color；Purple is shown on other two kinds of tablets can show darkviolet on S2 tablets, and can be in more short time view result.For
Darkviolet can be presented on S2 tablets, cultivate on certain tablet just present for 24 hours at home by the strong salmonella of enzyme activity, culture 18h
Purple；After the slightly weak salmonella of enzyme activity equally culture for 24 hours, purple can be presented on S2 tablets, but at home certain
Lilac can only be presented on tablet.Incubation time and colour generation experiment more can prove that S2 tablets more and can promote the hydrolysis of esterase substrate.
Blue-green, staphylococcus aureus and excrement intestines are presented in Escherichia coli and other strains for containing the sweet enzyme of galactolipin on two kinds of tablets
The bacterium colonies such as coccus are suppressed on both brand culture mediums, and other bacterial strains are colourless.It is noisy to Salmeterol fluticasone propionate
The main pseudomonas aeruginosa of bacterial strain and serratia marcescens etc. are suppressed through specific inhibitor.
By representative and strong interference salmonella typhi CM50071, proteus vulgaris CMCC49027, large intestine
Plastc ring is made in Escherichia ATCC25922, pseudomonas aeruginosa ATCC9027, enterococcus faecalis ATCC29212, takes a ring
Enrichment liquid streak inoculation is mixed on S2 and certain domestic producer's chromogenic culture medium tablet, the results are shown in Table 2.
Separating resulting of 2 plastc ring of table in various brands
As shown in Table 2, S2 and certain domestic producer are all easily separated for other interference miscellaneous bacterias, three kinds of colors of appearance on tablet
Bacterium colony, colourless, darkviolet and blue-green；Object bacteria colour developing is more easy to recognize wherein on S2 culture mediums, and especially develop the color slow wound
Cold salmonella.
Comparative example 2：
Growth rate：
Mouse salmonella ATCC14028 and Bacterium enteritidis ATCC13076 are made to the bacteria suspension (concentration of suitable concentration
It is 108-109CFU/ml), it is 10 to take dilution-5With 10-6Bacteria suspension 0.1ml be spread evenly across S21, certain domestic producer and
On tryptose soya agar (TSA), 36 DEG C of cultures are for 24 hours.It is that the tablet of 20CFU~200CFU is counted to take inoculation level,
Calculate respective growth rate.
Comparison of 3 chromogenic culture medium of table to salmonella sensitivity and colonial morphology
As shown in Table 3, S2 and certain domestic producer's sensitivity are relatively high, and salmonella typhimurium and Bacterium enteritidis exist
The growth rate of S2 is higher than certain domestic producer, and far above the regulation of PR >=0.5 in GB4789.28-2013.In terms of bacterium colony size,
The colony diameter of salmonella typhimurium and Bacterium enteritidis on S2 is more than the colony diameter of certain producer at home.S2 tablets
The growth of salmonella preferably.
Comparative example 3：
The detection of salmonella in artificial contamination's sample：
1. strain：Standard bacteria suspension will be made in mouse salmonella ATCC14028 inoculation nutrient broths, preparation contains target
The bacteria suspension of bacterium 10CFU-100CFU and 1-10CFU, it is spare.It is counted and is used with TSA simultaneously.
Increase bacterium and rear increasing bacterium before 2.：Milk powder sample 25g is weighed, is added in 225mlBPW enrichment liquids, is uniformly mixed.It will prepare
Good bacterial suspension inoculation is in this sample suspension, 36 DEG C of culture 8h-18h.The sample mixture cultivated is gently shaken, is pipetted
1mL,
3. inoculated and cultured：The enrichment liquid after a ring culture, four rides is respectively taken to be inoculated in embodiment 1 (abbreviation S2) colour developing training
On foster base tablet, XLD, HE and certain domestic producer's chromogenic culture medium, 36 DEG C of cultures 18~for 24 hours, grow feelings according in scribe area
Condition records colony colour and form, is shown in Table 4.
Detections of 4 S2 of table to artificial contamination's sample
As shown in Table 4, salmonella inoculum concentration is more, and scribing line growth district is bigger, and inoculum concentration is in 1~100CFU, 4th area
All there is an object bacteria growth, but since bacterium is to the competitive relation of nutritional ingredient, can just go out in third and fourth area on XLD culture mediums
Existing differentiable colonies typical.On S2 tablets, as long as it is object bacteria that bacterium colony, which is purple, identification is big, easy to identify and detection；
When inoculum concentration is 1-10CFU, XLD and S2 can the growth of 3rd area, there is colonies typical in third area in XLD, and S2 may be used in whole sections
There is aubergine bacterium colony.It can be seen that S2 can also detect salmonella in low stain area, the detection limit of 1-10CFU is can reach, with
Certain domestic producer's detection limit is suitable.
Comparative example 4：
The detection of salmonella in actual sample：
1. acquiring sample
Various totally 139 parts of food are acquired from supermarket and material supplier.
2. Zengjing Granule
Sample 25g is weighed, is added in 225mlBPW enrichment liquids, is uniformly mixed, 36 DEG C of culture 18h.
3. inoculated and cultured
The enrichment liquid after a ring culture is taken, streak inoculation is on sramana's chromogenic culture medium tablet made of embodiment 1,37 DEG C
18-24h is cultivated, colony colour and form are observed.The sample mixture cultivated is gently shaken, pipettes 1mL, transferred species is in 10mL
In TTB, in 42 DEG C of ± 1 DEG C of culture 18h~for 24 hours；Meanwhile 1mL is separately taken, transferred species cultivates 18h in 10mL SC in 36 DEG C ± 1 DEG C
~for 24 hours.
4. result is observed and analysis
There are 23 parts of sample detection salmonellas in 139 parts of samples, while being reflected using GB4789.4-2010 and API20E biochemistry
Determine system detectio and compare sample, consistent with chromogenic culture medium result, two methods testing result is consistent with expected results.
To sum up, the present invention is added to various inhibitors first in sramana's chromogenic culture medium, and there is no the senses of candida albicans to disturb；
Spore sulphur pyridine sodium-salt hydrate is added, the interference for easily causing the pseudomonas aeruginosa of false positive containing esterase activity is can avoid, but
The growth of salmonella and Escherichia coli is not interfered with；The chloro- 3- indoles-beta galactose glycosides of the bromo- 4- of 5- is added, enterobacteria can be distinguished
Belong to such as Escherichia coli and the coliform containing beta galactosidase.
The present invention with the chloro- 3- indoles-nonyl esters of the bromo- 6- of 5- and the chloro- 3- indoles-monooctyl esters of the bromo- 6- of 5- collectively as chromogenic substrate,
Increase zymolyte to promote the production of more Esterase reactions, makes up the disadvantage that part salmonella color is slow, colour developing is weak, the present invention
In, salmonella hydrolyzes chromogenic substrate under the action of esterase, releases more colour developing groups, bacterium colony is made to show more obviously
Feature.
It can make salmonella that deeper color-darkviolet be presented, and colour generation becomes apparent from, faster.In addition, the bromo- 6- of 5- are chloro-
3- indoles-nonyl ester is that one kind containing novel substrate, has many advantages, such as that purity is high, is easily-synthesized, at low cost and thermal stability compensates for
The most of limitation by import of esterase substrate.The present invention is the study found that salmonella has height except the esterase in C8 levels
Also contain C9 esterases (nonyl esterase), hydrolyzable nonyl ester chromogenic substrate outside consistency.The present invention be added in the medium nonyl esterase and
Monooctyl ester enzyme chromogenic substrate, salmonella all hydrolyzable substrates generate colour developing alcohols.
Each additive component of chromogenic culture medium of the present invention mutually acts synergistically, the chloro- 3- indoles-nonyl esters of the bromo- 6- of 5- and 5-
The bromo- chloro- 3- indoles-monooctyl esters of 6- promote the generation of more Esterase reactions, release more colour developings collectively as chromogenic substrate, collaboration
Group, meanwhile, selection and the proportioning of cosolvent were so that the present invention had both been not in since emulsifier excessively makes planar surface occur
Oily phenomenon, and substrate can be made to be completely dissolved, and the synergistic effect of cosolvent is so that the present invention organizes the colour developing of synthase chromogenic substrate
Effect reaches best.
The chromogenic culture medium of the present invention has high sensitivity, high specificity, identification higher, inspection for detecting salmonella
It is shorter, more adaptable to survey the period, the advantages that being easy to industrialization production.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to preferable
Embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the technology of the present invention
Scheme is modified or replaced equivalently, and without departing from the spirit of the technical scheme of the invention and range, should all be covered in this hair
In bright right.
Claims (10)
1. a kind of chromogenic culture medium for detecting salmonella, it is characterised in that：Including basal medium, additive are described
Additive includes esterase chromogenic substrate, and the esterase chromogenic substrate includes the chloro- 3- indoles-monooctyl esters of the bromo- 6- of 5- and the bromo- 6- of 5- chloro-
3- indoles-nonyl ester, the chloro- 3- indoles-monooctyl ester contents of the bromo- 6- of 5- are the chloro- 3- indoles-of 0.1~0.4g/L, the bromo- 6- of the 5-
Nonyl ester content is 0.1~0.4g/L.
2. the chromogenic culture medium as described in claim 1 for detecting salmonella, it is characterised in that：The basal medium
Including peptone, beef extract, sodium chloride, bacterial agar, wherein the peptone content is 8~15g/L, the beef extract
Content is 1~5g/L, the sodium chloride content is 5~6g/L, the bacteria Agr powder content is 15~18g/L.
3. the chromogenic culture medium as claimed in claim 1 or 2 for detecting salmonella, it is characterised in that：The additive
Further include inhibitor, galactosidase chromogenic substrate, enzyme inducer, cosolvent, wherein the inhibitor includes deoxycholic acid
Sodium, ovobiocin, Cefsulodin sodium-salt hydrate, the deoxycholic acid sodium content is 0.4~0.6g/L, the ovobiocin contains
Amount is 10~15mg/L, the Cefsulodin sodium-salt hydrate content is 5~10mg/L.
4. the chromogenic culture medium as claimed in claim 3 for detecting salmonella, it is characterised in that：The galactosidase
Chromogenic substrate includes the chloro- 3- indoles-beta galactose glycosides of the bromo- 4- of 5-, and the chloro- 3- indoles of the bromo- 4- of the 5--beta galactose glycosides content is
0.04~0.1g/L.
5. the chromogenic culture medium as claimed in claim 4 for detecting salmonella, it is characterised in that：The enzyme inducer packet
Isopropyl-β-D-thiogalactoside is included, the content of the isopropyl-β-D-thiogalactoside is 0.03~0.06g/L.
6. the chromogenic culture medium for detecting salmonella as described in claim 4 or 5, it is characterised in that：The cosolvent
Including dimethyl sulfoxide (DMSO), polysorbas20, the dimethyl sulfoxide (DMSO) content is 1~3ml/L, the polysorbas20 content is 2~4ml/L.
7. the chromogenic culture medium as claimed in claim 6 for detecting salmonella, it is characterised in that：In the cosolvent,
Dimethyl sulfoxide (DMSO) volume content is 40%, polysorbas20 volume content is 60%.
8. the chromogenic culture medium for detecting salmonella as described in claim 1,2,4,5 or 7 are any, it is characterised in that：
The culture medium includes peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, bacterial agar 15g/L, NaTDC
The chloro- 3- indoles of 0.55g/L, ovobiocin 15mg/L, the bromo- 6- of Cefsulodin sodium-salt hydrate 6mg/L, 5--monooctyl ester 0.2g/L, 5-
The chloro- 3- indoles of the bromo- chloro- 3- indoles-bromo- 4- of nonyl ester 0.3g/L, 5- of 6--beta galactose glycosides 0.08g/L, isopropyl-beta D-thio
Galactoside 0.05g/L, dimethyl sulfoxide (DMSO) 2ml/L, polysorbas20 3ml/L.
9. the preparation method of any chromogenic culture medium for detecting salmonella of claim 3~8, feature exist
In：Including,
Prepare mother liquor 1：Weigh basal medium and with NaTDC mixing, as mother liquor 1；
Prepare mother liquor 2：Esterase chromogenic substrate is dissolved in cosolvent, and is added in mother liquor 1, is boiled, as mother liquor 2；
Prepare chromogenic culture medium：Ovobiocin, Cefsulodin sodium-salt hydrate is soluble in water, it is added in mother liquor 2, and by gala
The sweet zymolyte of sugar and isopropyl-β-D-thiogalactoside are added in mother liquor 2.
10. the preparation method as claimed in claim 9 for detecting the chromogenic culture medium of salmonella, it is characterised in that：Institute
State basal medium and with NaTDC mixing, speed 2800rpm, time 1min, the preparation mother liquor 2, wherein institute
It states after boiling, the mother liquor 2 is cooled to 45 ± 1 DEG C, the inhibitor is added in mother liquor 2.
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| CN109355352A (en) * | 2018-12-10 | 2019-02-19 | 湖南长沙天地人生物科技有限公司 | A kind of fluid nutrient medium detecting beta galactosidase and its application in Bacteria Identification |
| CN109706214A (en) * | 2019-03-04 | 2019-05-03 | 中国检验检疫科学研究院 | A kind of identification of resistance to quinolone antibiotics Salmonella strains and separation method |
| CN112980919A (en) * | 2019-12-17 | 2021-06-18 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
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Cited By (4)
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| CN112980919A (en) * | 2019-12-17 | 2021-06-18 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
| CN112980919B (en) * | 2019-12-17 | 2023-09-12 | 杭州远大生物制药有限公司 | Culture medium for detecting mixed bacteria in live bacteria preparation |
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