CN116103362B - Chromogenic medium for detecting vibrio parahaemolyticus - Google Patents
Chromogenic medium for detecting vibrio parahaemolyticus Download PDFInfo
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- 241000607272 Vibrio parahaemolyticus Species 0.000 title claims abstract description 47
- 239000003593 chromogenic compound Substances 0.000 claims abstract description 22
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 18
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 10
- 229930006000 Sucrose Natural products 0.000 claims abstract description 10
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 10
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 10
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 10
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 10
- 239000005720 sucrose Substances 0.000 claims abstract description 10
- 229920001817 Agar Polymers 0.000 claims abstract description 9
- 239000008272 agar Substances 0.000 claims abstract description 9
- 239000001103 potassium chloride Substances 0.000 claims abstract description 9
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 9
- 239000011780 sodium chloride Substances 0.000 claims abstract description 9
- 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 abstract description 8
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 7
- 239000008101 lactose Substances 0.000 claims abstract description 7
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 5
- BFPJYWDBBLZXOM-UHFFFAOYSA-L potassium tellurite Chemical compound [K+].[K+].[O-][Te]([O-])=O BFPJYWDBBLZXOM-UHFFFAOYSA-L 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 235000015278 beef Nutrition 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 229940054269 sodium pyruvate Drugs 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- CHRVKCMQIZYLNM-MBJXGIAVSA-N (2s,3r,4s,5r,6r)-2-[(5-bromo-6-chloro-1h-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol Chemical group O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC(Cl)=C(Br)C=C12 CHRVKCMQIZYLNM-MBJXGIAVSA-N 0.000 claims description 5
- CHRVKCMQIZYLNM-RKQHYHRCSA-N (2s,3r,4s,5s,6r)-2-[(5-bromo-6-chloro-1h-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC(Cl)=C(Br)C=C12 CHRVKCMQIZYLNM-RKQHYHRCSA-N 0.000 claims description 5
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 claims description 5
- OPIFSICVWOWJMJ-LNNRFACYSA-N 5-bromo-4-chloro-3-indolyl beta-D-glucoside Chemical group O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-LNNRFACYSA-N 0.000 claims description 5
- 239000002609 medium Substances 0.000 abstract description 28
- 239000001963 growth medium Substances 0.000 abstract description 23
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 150000008051 alkyl sulfates Chemical class 0.000 abstract description 6
- 239000003945 anionic surfactant Substances 0.000 abstract description 6
- 230000001580 bacterial effect Effects 0.000 abstract description 4
- SITVSCPRJNYAGV-UHFFFAOYSA-L tellurite Chemical compound [O-][Te]([O-])=O SITVSCPRJNYAGV-UHFFFAOYSA-L 0.000 abstract description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 19
- 241000607598 Vibrio Species 0.000 description 16
- 241000238557 Decapoda Species 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 238000012258 culturing Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 241000192125 Firmicutes Species 0.000 description 6
- -1 vibrio vulnificus Chemical compound 0.000 description 6
- 241000233866 Fungi Species 0.000 description 5
- 241000251468 Actinopterygii Species 0.000 description 4
- 235000019688 fish Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 239000007151 vibrio medium Substances 0.000 description 4
- 241000194032 Enterococcus faecalis Species 0.000 description 3
- 241000607265 Vibrio vulnificus Species 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 229940032049 enterococcus faecalis Drugs 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 208000019331 Foodborne disease Diseases 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000607291 Vibrio fluvialis Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 239000003833 bile salt Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229960002337 magnesium chloride Drugs 0.000 description 2
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 description 2
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 235000014102 seafood Nutrition 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 241000607528 Aeromonas hydrophila Species 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241000624562 Cololabis saira Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010017999 Gastrointestinal pain Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000588770 Proteus mirabilis Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000607594 Vibrio alginolyticus Species 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 208000012873 acute gastroenteritis Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 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
- 230000007423 decrease Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/63—Vibrio
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- 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
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Abstract
The invention discloses a chromogenic medium for detecting vibrio parahaemolyticus. The culture medium contains organic nutrients required by bacterial growth, sodium chloride, potassium chloride, magnesium chloride, sucrose, long-chain alkyl sulfate anionic surfactant, tellurite, beta-glucosidase chromogenic substrate, beta-galactosidase chromogenic substrate, isopropyl-beta-D-thiogalactoside, lactose and agar. The chromogenic medium has higher growth selectivity, chromogenic sensitivity to vibrio parahaemolyticus and higher specificity and sensitivity for detecting vibrio parahaemolyticus.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a chromogenic medium for detecting vibrio parahaemolyticus.
Background
Vibrio parahaemolyticus is a gram-negative halophilic bacteria and widely exists in seafood such as fish, shrimp, shellfish and crabs. It is an important food-borne pathogenic microorganism for human beings, which can cause acute gastroenteritis with diarrhea, intestinal spasm and the like as main symptoms of patients, and can cause septicemia and even death in severe cases, especially high food poisoning rate of human beings in summer and autumn. The strain can also cause the diseases of marine cultured fishes, shrimps, shellfish and the like such as inflammation, congestion and the like to die in a large amount, is particularly easy to cause the death disease of the shrimps, and can cause huge economic loss for the marine culture industry. Therefore, detection of Vibrio parahaemolyticus is of great importance for preventing both food-borne diseases and aquaculture animal diseases.
Although TCBS (Thiosulfate citrate bile salt sucrose) medium is often used to detect Vibrio, it is poorly specific and cannot distinguish Vibrio parahaemolyticus from other Vibrio that is not fermented sucrose (e.g., vibrio vulnificus, etc.) and Vibrio-less bacteria (e.g., pseudomonas aeruginosa, etc.). At present, some vibrio chromogenic media capable of detecting vibrio parahaemolyticus more specifically have been developed, for example CHROMagar Vibrio medium and Bio-Chrome Vibrio medium. However, it has been reported in literature (Di Pinto et al. Food Control,2011,22,124-127;Su et al.Journal of Food Protection,2005,68,1454-145) that when they are used to detect Vibrio parahaemolyticus, false positive results of suspected Vibrio parahaemolyticus are given by a mixed bacterium such as Vibrio fluvialis (Vibrio fluvialis) which ferments sucrose on TCBS medium to present yellow colonies. Gram-positive bacteria such as enterococcus faecalis which are resistant to bile salts and potassium tellurite can also grow on the chromogenic medium and give false positive results of suspected vibrio parahaemolyticus. Furthermore, the above-mentioned chromogenic medium also has a certain proportion of false negative results, i.e., the colony of Vibrio parahaemolyticus should appear a specific color, but the result appears white, resulting in insufficient detection sensitivity. In addition, a development medium specially used for detection of Vibrio parahaemolyticus has been also reported in the literature (Lee et al food Control,2020,116,107308); it is relatively more specific and sensitive than CHROMagar Vibrio medium and TCBS culture medium in detecting vibrio parahaemolyticus. However, such specialized chromogenic media still have the problem that the specificity and sensitivity need to be further improved. Therefore, it is necessary to develop a chromogenic medium which can be used for detecting Vibrio parahaemolyticus more specifically and sensitively.
Disclosure of Invention
The present invention has been made in view of the above-mentioned drawbacks of the prior art, and an object of the present invention is to provide a chromogenic medium which is more excellent in selectivity and can detect Vibrio parahaemolyticus more specifically and sensitively.
The invention is realized by the following technical scheme:
A chromogenic medium for detecting vibrio parahaemolyticus contains 5-20 g/L of peptone, 3-15 g/L of beef extract powder, 0.05-2 g/L of sodium pyruvate, 10-30 g/L of sodium chloride, 0.5-6 g/L of potassium chloride, 0.1-10 g/L of magnesium chloride, 20-40 g/L of sucrose, 0.1-1 g/L of long-chain alkyl sulfate anionic surfactant, 0.1-3.5 mg/L of tellurite, 0.01-0.25 g/L of beta-glucosidase chromogenic substrate, 0.01-0.25 g/L of beta-galactosidase chromogenic substrate, 0.01-0.2 g/L of isopropyl-beta-D-thiogalactoside, 0.1-0.7 g/L of lactose, 10-20 g/L of agar and water as solvent.
Preferably, the chromogenic medium comprises 8-15 g/L of peptone, 3-10 g/L of beef extract powder, 0.1-0.5 g/L of sodium pyruvate, 10-20 g/L of sodium chloride, 1-5 g/L of potassium chloride, 0.5-6 g/L of magnesium chloride, 25-35 g/L of sucrose, 0.1-0.6 g/L of long-chain alkyl sulfate anionic surfactant, 0.5-3 mg/L of tellurite, 0.05-0.2 g/L of beta-glucosidase chromogenic substrate, 0.05-0.2 g/L of beta-galactosidase chromogenic substrate, 0.03-0.15 g/L of isopropyl-beta-D-thiogalactoside, 0.1-0.5 g/L of lactose, 10-15 g/L of agar and water as solvent.
Preferably, the beta-glucosidase chromogenic substrate is 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside or 5-bromo-6-chloro-3-indolyl-beta-D-glucopyranoside; the beta-galactosidase chromogenic substrate is 5-bromo-6-chloro-3-indolyl-beta-D-galactopyranoside or 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.
Preferably, when the beta-glucosidase chromogenic substrate is 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, the beta-galactosidase chromogenic substrate is 5-bromo-6-chloro-3-indolyl-beta-D-galactopyranoside.
Preferably, when the beta-glucosidase chromogenic substrate is 5-bromo-6-chloro-3-indolyl-beta-D-glucopyranoside, the beta-galactosidase chromogenic substrate is 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.
Preferably, the long-chain alkyl sulfate anionic surfactant is a ten to eighteen carbon long-chain alkyl sulfate anionic surfactant.
More preferably, the long-chain alkyl sulfate anionic surfactant is sodium dodecyl sulfate.
Preferably, the tellurite is potassium tellurite.
Compared with the prior art, the invention has the advantages that:
The chromogenic medium has high growth selectivity, has higher growth selectivity inhibition effect on non-target bacteria and fungi, and has more excellent chromogenic effect on vibrio parahaemolyticus, thereby displaying higher detection specificity and sensitivity of vibrio parahaemolyticus.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
The culture medium is based on the following formula: 10g/L peptone, 5g/L beef extract powder, 20g/L sodium chloride, 0.5g/L potassium chloride, 0.1g/L magnesium chloride hexahydrate, 2g/L sodium pyruvate, 0.1g/L Sodium Dodecyl Sulfate (SDS) and 10g/L agar, wherein the solvent is water to examine the influence of potassium tellurite with different addition concentrations on the detection sensitivity of vibrio parahaemolyticus (the preparation method of the culture medium comprises the steps of adding other components except the potassium tellurite into quantitative water, heating, boiling and completely dissolving, and then adding sterile potassium tellurite), and the result is shown in Table 1.
TABLE 1 influence of Potassium tellurite with different addition concentrations on Vibrio parahaemolyticus detection sensitivity
Note that: culturing Vibrio parahaemolyticus in alkaline peptone water at 37deg.C under oxygen for 15-18 hr, diluting with sterile physiological saline to desired concentration, and coating 0.25mL on the culture medium; "+" indicates detected and "-" indicates undetected.
As seen from the results of Table 1, when the concentration of potassium tellurite added is too high, the limit of detection of Vibrio parahaemolyticus increases, that is, the detection sensitivity thereof decreases. In addition, the team of the invention also finds that when the added potassium tellurite is increased from low concentration to 5mg/L, the vibrio colonies of parahaemolyticus vibrio, vibrio vulnificus, vibrio cholerae, vibrio alginolyticus and the like are changed from white or yellow-white to more obvious grey brown or brownish black, which is consistent with the phenomenon reported in some documents; however, this phenomenon may seriously interfere with the detection effect of the added chromogenic substrate. Therefore, in order to achieve sensitive detection of low-concentration Vibrio parahaemolyticus and to avoid as much as possible the occurrence of color interference to the added chromogenic substrate, the potassium tellurite is suitably added at a concentration of 0.1 to 3.5mg/L.
Example 2
The culture medium is based on the following formula: 10g/L peptone, 5g/L beef extract powder, 10g/L sodium chloride, 2g/L potassium chloride, 6g/L magnesium chloride hexahydrate, 0.05g/L sodium pyruvate, 0.1mg/L potassium tellurite, 15g/L agar, water as solvent, and examined the sensitivity of Sodium Dodecyl Sulfate (SDS) of different concentrations to detection of Vibrio parahaemolyticus and the effect on inhibiting gram-positive bacteria (the preparation method of the culture medium is that other components except potassium tellurite are added into quantitative water, and after the components are heated and boiled to be completely dissolved, sterile potassium tellurite is added), and the results are shown in Table 2.
TABLE 2 Effect of SDS at various additive concentrations on Vibrio parahaemolyticus detection sensitivity and on inhibition of gram-positive bacteria
Note that: culturing vibrio parahaemolyticus and non-vibrio bacteria in alkaline peptone water and TSB broth at 37deg.C under oxygen condition for 15-18 hr, diluting with sterile physiological saline to desired concentration, respectively inoculating 0.25mL of the culture medium, and culturing at 37deg.C under oxygen condition for 24 hr; "+" indicates detected or grown, "-" indicates undetected or non-grown.
As seen from the results of Table 2, when the concentration of SDS added is too low, the inhibitory effect on gram-positive bacteria is poor, and therefore, the concentration of SDS added may be 0.1 to 1g/L while suppressing the growth of gram-positive bacteria as much as possible and without affecting the sensitivity of detecting the target pathogenic vibrio as much as possible.
Example 3
A chromogenic medium for detecting vibrio parahaemolyticus comprises 10g/L of peptone, 5g/L of beef extract powder, 15g/L, KCl 2.0.0 g/L, mgCl 2·6H2 O2.0 g/L of sodium chloride, 0.2g/L of sodium pyruvate, 25g/L, SDS 0.4.4 g/L of sucrose, 1.0mg/L of potassium tellurite, 0.1g/L of 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, 0.1g/L of 5-bromo-6-chloro-3-indolyl-beta-D-galactopyranoside, 0.05g/L of isopropyl-beta-D-thiogalactoside (IPTG), 0.2g/L of lactose and 15g/L of agar, and the solvent is water. The preparation method of the culture medium comprises the following steps: adding other components except potassium tellurite into quantitative water, heating, boiling to dissolve completely, and adding sterile potassium tellurite.
Example 4
A chromogenic medium for detecting vibrio parahaemolyticus comprises 10g/L of peptone, 5g/L of beef extract powder, 15g/L, KCl 2.0.0 g/L, mgCl 2·6H2 O2.0 g/L of sodium chloride, 0.2g/L of sodium pyruvate, 25g/L, SDS 0.4.4 g/L of sucrose, 1.0mg/L of potassium tellurite, 0.1g/L of 5-bromo-6-chloro-3-indolyl-beta-D-glucopyranoside, 0.1g/L, IPTG 0.05.05 g/L of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, 0.2g/L of lactose, 15g/L of agar and water as a solvent. The preparation method of the culture medium comprises the following steps: adding other components except potassium tellurite into quantitative water, heating, boiling to dissolve completely, and adding sterile potassium tellurite.
Example 5
The detection effects of different culture mediums on bacteria and fungi are compared (see tables 3 and 4).
TABLE 3 comparison of the detection effects of different media on bacteria
Note that: culturing vibrio and non-vibrio bacteria in alkaline peptone water and TSB broth at 37 deg.C under oxygen condition for 15-18 hr, inoculating to the culture medium, and culturing at 37 deg.C under oxygen condition for 24 hr; the TCBS, CHROMagar vibrio culture medium is commercially available in China and is prepared according to the specification; "+++++++" means the growth is good, the quality of the product is good, 1-6 "+" indicates that growth is inhibited to different degrees, "+/-" indicates sparse or little growth, and "-" indicates no growth.
TABLE 4 comparison of the detection effects of different media on fungi
Note that: streaking each fungus lawn grown on MEA culture medium, inoculating to each culture medium, and culturing at 28deg.C under aerobic condition for 5d; the TCBS, CHROMagar vibrio culture medium is commercially available in China and is prepared according to the specification; "+" indicates significant growth or growth in flakes, "+/-" indicates sparse growth or little growth, "-" indicates no growth.
From the results shown in Table 3, compared with the conventional TCBS culture medium and the conventional CHROMagar vibrio culture medium for detecting vibrio parahaemolyticus, the chromogenic culture medium provided by the invention has a better selective inhibition effect on common non-vibrio bacteria, can better inhibit the growth of bacteria such as bacillus proteus, aeromonas hydrophila which are common in aquatic products, and can also better inhibit the growth of gram-positive bacteria such as cholate (juice), potassium tellurite-resistant enterococcus faecalis and staphylococcus aureus. In the aspect of detecting the specificity of vibrio parahaemolyticus, the TCBS culture medium has the chromogenic false positive interference of vibrio vulnificus, pseudomonas aeruginosa and proteus mirabilis, the CHROMagar vibrio culture medium has the chromogenic false positive interference of enterococcus faecalis, and the chromogenic culture medium does not have the chromogenic false positive interference, so that the invention has higher specificity. In addition, the chromogenic medium of the present invention also shows a more sensitive chromogenic effect, i.e., a higher sensitivity, than the chromogenic medium of Vibrio CHROMagar.
As shown in Table 4, the chromogenic medium of the present invention can inhibit the growth of fungi such as yeasts and molds better than the TCBS medium and the CHROMagar vibrio medium.
Example 6
The sensitivity of the different media to detection of Vibrio parahaemolyticus was compared (see Table 5).
TABLE 5 comparison of the detection sensitivity of different media to Vibrio parahaemolyticus
Note that: culturing vibrio parahaemolyticus in alkaline peptone water at 37 ℃ under oxygen for 15-18h, diluting the vibrio parahaemolyticus to a required concentration by using sterile physiological saline, then coating and inoculating 0.25mL of the vibrio parahaemolyticus to the culture medium, and culturing the vibrio parahaemolyticus at 37 ℃ under oxygen for 24h; the TCBS, CHROMagar vibrio culture medium is commercially available in China and is prepared according to the specification; "+" indicates that there is target bacterial growth, and "-" indicates that there is no target bacterial growth.
As shown in Table 5, the chromogenic medium of the present invention was found to be visible from the viewpoint of the presence or absence of bacterial growth in 10 1～102 CFU/mL of Vibrio parahaemolyticus ATCC 33847, and the chromogenic medium of the present invention was found to be clear (green or purplish red) in the chromogenic medium of the present invention when cultured for 24 hours, whereas the chromogenic of Vibrio parahaemolyticus was not clear but white in the chromogenic medium of the present invention.
Example 7
The chromogenic medium and national standard method are used for detecting and controlling the vibrio parahaemolyticus in a natural sample (see table 6).
TABLE 6 detection control of Vibrio parahaemolyticus in Natural sample by using the chromogenic medium of the invention and national standard method
Note that: sea water fish (pacific saury, salmon, large yellow croaker, garter, multi-treasure fish), sea water shrimp (basic shrimp, sea shrimp, bamboo shrimp, lute shrimp, prawn) and seafood water samples are purchased from the market and 5 parts each, vibrio parahaemolyticus is detected by applying the chromogenic medium of the invention, vibrio parahaemolyticus is detected according to the national standard method GB 4789.7-2013 for comparison, and the identification method adopts MALDI-TOF-MS.
From the results of Table 6, the color development medium of the present invention was used to detect Vibrio parahaemolyticus with the national standard method for the natural actual samples taken, and the detection results were consistent.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (2)
1. A chromogenic medium for detecting vibrio parahaemolyticus is characterized by comprising 5-20 g/L of peptone, 3-15 g/L of beef extract powder, 0.05-2 g/L of sodium pyruvate, 10-30 g/L of sodium chloride, 0.5-6 g/L of potassium chloride, 0.1-10 g/L of magnesium chloride, 20-40 g/L of sucrose, 0.1-1 g/L of sodium dodecyl sulfate, 0.1-3.5 mg/L of potassium tellurite, 0.01-0.25 g/L of beta-glucosidase chromogenic substrate, 0.01-0.25 g/L of beta-galactosidase chromogenic substrate, 0.01-0.2 g/L of isopropyl-beta-D-thiogalactoside, 0.1-0.7 g/L of lactose and 10-20 g/L of agar, wherein a solvent is water; the beta-glucosidase chromogenic substrate is 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside or 5-bromo-6-chloro-3-indolyl-beta-D-glucopyranoside; the beta-galactosidase chromogenic substrate is 5-bromo-6-chloro-3-indolyl-beta-D-galactopyranoside or 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.
2. The chromogenic medium according to claim 1, wherein the chromogenic medium comprises 8-15 g/L peptone, 3-10 g/L beef extract powder, 0.1-0.5 g/L sodium pyruvate, 10-20 g/L sodium chloride, 1-5 g/L potassium chloride, 0.5-6 g/L magnesium chloride, 25-35 g/L sucrose, 0.1-0.6 g/L sodium dodecyl sulfate, 0.5-3 mg/L potassium tellurite, 0.05-0.2 g/L beta-glucosidase chromogenic substrate, 0.05-0.2 g/L beta-galactosidase chromogenic substrate, 0.03-0.15 g/L isopropyl-beta-D-thiogalactoside, 0.1-0.5 g/L lactose, 10-15 g/L agar, and the solvent is water; the beta-glucosidase chromogenic substrate is 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside or 5-bromo-6-chloro-3-indolyl-beta-D-glucopyranoside; the beta-galactosidase chromogenic substrate is 5-bromo-6-chloro-3-indolyl-beta-D-galactopyranoside or 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside.
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| RU2711914C1 (en) * | 2019-02-14 | 2020-01-23 | Федеральное казенное учреждение здравоохранения "Ростовский-на-Дону ордена Трудового Красного Знамени научно-исследовательский противочумный институт" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека | Nutrient medium for separation and identification of para-hemolytic vibrios (embodiments) and method for production thereof (embodiments) |
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| RU2711914C1 (en) * | 2019-02-14 | 2020-01-23 | Федеральное казенное учреждение здравоохранения "Ростовский-на-Дону ордена Трудового Красного Знамени научно-исследовательский противочумный институт" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека | Nutrient medium for separation and identification of para-hemolytic vibrios (embodiments) and method for production thereof (embodiments) |
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