CN102660474B - The culture medium and preparation method of a kind of five kinds of foodborne bacterial pathogenses of Sync enrichment - Google Patents
The culture medium and preparation method of a kind of five kinds of foodborne bacterial pathogenses of Sync enrichment Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 25
- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 241000894006 Bacteria Species 0.000 claims abstract description 82
- 241000607142 Salmonella Species 0.000 claims abstract description 29
- 241000607768 Shigella Species 0.000 claims abstract description 28
- 241000588724 Escherichia coli Species 0.000 claims abstract description 26
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 26
- 241000186779 Listeria monocytogenes Species 0.000 claims abstract description 23
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims abstract description 22
- BFPJYWDBBLZXOM-UHFFFAOYSA-L potassium tellurite Chemical compound [K+].[K+].[O-][Te]([O-])=O BFPJYWDBBLZXOM-UHFFFAOYSA-L 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims abstract description 16
- 239000012153 distilled water Substances 0.000 claims abstract description 13
- 239000001888 Peptone Substances 0.000 claims abstract description 10
- 108010080698 Peptones Proteins 0.000 claims abstract description 10
- 229940099352 cholate Drugs 0.000 claims abstract description 10
- 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 claims abstract description 10
- 235000019319 peptone Nutrition 0.000 claims abstract description 10
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 9
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 8
- 229930195725 Mannitol Natural products 0.000 claims abstract description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 8
- 239000000594 mannitol Substances 0.000 claims abstract description 8
- 235000010355 mannitol Nutrition 0.000 claims abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 229940054269 sodium pyruvate Drugs 0.000 claims abstract description 8
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical group C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 claims description 6
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- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
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- 239000008187 granular material Substances 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
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- 101100098690 Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e) hly gene Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 239000006158 Baird–Parker agar Substances 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 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 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
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- 238000005660 chlorination reaction Methods 0.000 description 1
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- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound 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 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- OKBPCTLSPGDQBO-UHFFFAOYSA-L disodium;dichloride Chemical compound [Na+].[Na+].[Cl-].[Cl-] OKBPCTLSPGDQBO-UHFFFAOYSA-L 0.000 description 1
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- 239000008101 lactose Substances 0.000 description 1
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Classifications
-
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to for the simultaneously compound culture medium and preparation method for increasing bacterium of salmonella, Escherichia coli, staphylococcus aureus, five kinds of foodborne bacterial pathogenses of Listeria monocytogenes and Shigella.Foodborne bacterial pathogenses are the major reasons for causing food poisoning, rapidly and accurately detect food-borne pathogens to preventing and controlling food safety affair to have important practical significance.Basal culture medium feature is as follows：Peptone 10.0g, sodium chloride 10.0g, disodium hydrogen phosphate 9.0g, potassium dihydrogen phosphate 1.5g, cholate 0.1g, potassium tellurite 0.1mg, lithium chloride 1.0g, glucose 3.0g, mannitol 2.0g, Sodium Pyruvate 2.5g, aesculin 1.0g, distilled water 1000mL, pH7.1 7.5.Basal culture medium being capable of five kinds of target pathogenic bacterias of Sync enrichment, separation and identification available for object bacteria, it can also be used for Molecular Detection of multiple pathogenic bacteria in same detection platform, technical support is provided for five kinds of pathogen quick determination methods in food, and is met to five kinds of foodborne bacterial pathogenses while the need for detecting.
Description
Technical field
The invention belongs to the preceding enriched medium of foodborne bacterial pathogenses, and in particular to a kind of while to salmonella, large intestine
Compound culture medium for increasing bacterium of bacillus, staphylococcus aureus, Listeria monocytogenes and Shigella and preparation method thereof.
Background technology
Food origin disease is global most common public health problem.World Health Assembly has passed through on food for 2006
The resolution of safety, the resolution thinks that food origin disease has had a strong impact on the healthy and happiness of the people of the world, and to individual, family,
Community, industrial and commercial enterprises and country all cause serious economic loss [1].Wherein, the pathogenetic bacteria polluted in food is to cause
One of principal element of food origin disease, when food poisoning is broken out, identification foodborne bacterial pathogenses are most important [2].
Bacterium enteritidis, Escherichia coli O 157, staphylococcus aureus, monokaryon hyperplasia Listeria, Shigella
It is related to most common bacterial factor [3] in food origin disease Deng turning into.These pathogens always with welcome food such as
Poultry prod, instant meat, dairy products and fruits and vegetables are closely bound up, there is higher morbidity and mortality, several foods broken out in recent years
Thing poisoning is directed to their [4].It is pollution salmonella, monocyte increasing that the investigation carried out in Europe, which identifies meat,
Raw Listeria, the main media [5] of Escherichia coli O 157.It is also public attention that staphylococcus aureus, which produces enterotoxin,
One major health concern, staphylococcus aureus can be grown in hypersaline environment, and golden yellow Portugal is polluted in high protein food
The coccigenic food poisoning of grape happens occasionally [6].Shigella is the pathogen of human bacterial's property dysentery, is mainly passed through
Animal food propagates [7].Five kinds of pathogenic bacteria of the above are the essential items for inspection [8] that current China ensures multiclass food security.
The conventional method of detection foodborne bacterial pathogenses is cultivation, but cumbersome, time-consuming effort, it is difficult to meet current warp
The detection needs that Ji globalization food quickly circulates.In addition, the increasing with people's Consciousness of food security with the development of science and technology
By force, the foodborne bacterial pathogenses species of detection is needed also gradually to increase in food.While accordingly, with respect to a variety of pathogens, quickly
The research of detection technique increasingly gos deep into [9-12].Advanced Multiple detection technology reduces the sky handled needed for a large amount of samples
Between, time and labor, so as to save the cost of the single pathogen of detection；Moreover, Multiple detection technology is also rational, because
Numerous food product, such as milk and dairy products [13], meat and poultry [14] and fruits and vegetables [15], often a variety of pathogens take
Band person；In addition, Multiple detection technology can mitigate the detection specified risk material (SRM) that food industry and administrative department undertake（Easily by cause of disease
The food of bacterium pollution）Burden.
However, must account for two key factors of influence detection when with these technologies：First, food be by
What the processes such as processing, deep processing, freezing, packaging, storage were obtained, the pathogenic bacteria in food can be damaged with the change of environment
Wound, vigor declines.In this case, accuracy rate declines when being detected using PCR, genetic chip equimolecular biological method, in order to anti-
Stop-band has the missing inspection of pathogenic bacteria food, and food needs to carry out increasing bacterium before detection.Second, the test limit of many modern measure methods
All 102~104Cfu/ml scopes.Muting sensitivity is it is meant that when the contaminated degree of food is low, using immunology and molecule
Increasing bacterium [16] must be carried out before biology techniques detection object bacteria.
It can be seen that, Zengjing Granule processing altogether is essential for modern detecting, and the utilization of the step can not only
Increase the concentration of target pathogenic bacteria in sample, moreover it is possible to repair the cell oppressed or damaged by physiology.Generally, a kind of pathogen needs
It is enriched with one or more special culture mediums.If detecting that a variety of pathogens are accomplished by applying a variety of cultures simultaneously
Base is enriched with respectively, makes detection process complexity cumbersome.Sync enrichment then simplified operation is carried out using common enriched medium.
In domestic and international existing research, being related to the research of common enriched medium technology mainly includes：Salmonella and the common increasing of Shigella
Technology [17]；The common increasing technology of salmonella, Escherichia coli and staphylococcus aureus（BSB）[18]；Salmonella, golden yellow
Staphylococcus and single common increasing technology for increasing Listeria（SSL）[19]；Salmonella, Escherichia coli and single increasing Listeria
Common increasing technology（SEL）[20]；The common increasing technology of salmonella, vibrio parahemolyticus and comma bacillus（SVV）[21]；Can be simultaneously
Being enriched with the culture medium of a variety of pathogenetic bacterias includes general pre- Zengjing Granule meat soup（UPB）[22,23], albumen buffered water（BPW）、
Tryptic soy broth（TSB）And nutrient broth（NB）[7,23] etc..
So far, have no on Sync enrichment salmonella, staphylococcus aureus, single increasing Listeria, large intestine
The report of the common Zengjing Granule technology of bacillus and Shigella.In order to meet during background microorganism complex situations to specific objective disease
Opportunistic pathogen progressive increases the requirement of bacterium, the common enriched medium of research and development energy five kinds of pathogens of Sync enrichment, to realizing simultaneously altogether
Detect that a variety of pathogens have great importance, be the important prior art platform of Fast Detection Technique.
The content of the invention
The present invention is intended to provide salmonella, Escherichia coli, staphylococcus aureus, single increasing Liszt can be supported simultaneously
The culture medium that five kinds of foodborne bacterial pathogenses of bacterium and Shigella grow simultaneously, by the preceding increasing bacterium step in common detection methods and choosing
Selecting property increases bacterium step and united two into one, and multiple pathogens are detected for identical platform, can improve detection efficiency, and saving is detected into
This.
Buffer system containing disodium hydrogen phosphate and potassium dihydrogen phosphate in basal culture medium is complicated for food samples
Acid-base environment, can prevent increase bacterium during pH value acute variation, be conducive to fast breeding and the growth of target pathogenic bacteria.This
Culture medium inhibitor includes cholate, potassium tellurite and lithium chloride.The cholate of low concentration does not influence salmonella and Shigella
Other three kinds of bacterium are grown the inhibitory action existed slightly by growth.Growth of the potassium tellurite to five kinds of object bacterias is all served
Inhibitory action, inhibition and concentration close association.Lithium chloride is better than to the inhibitory action of Gram-negative bacteria to gram sun
Property bacterium, the appropriate additions of three kinds of inhibitor can effective coordination positive bacteria and negative bacterium syntrophism state, while also functioning to one
Surely the effect of the growth of background microorganism is suppressed.Five kinds of bacterium add the chlorination of higher concentration to the tolerance better performances of sodium chloride
Sodium, can also suppress the growth of other parts non-targeted microorganism.Accelerator glucose, mannitol and pyruvic acid in basal culture medium
Growth of the sodium to five kinds of bacterium all serves preferable facilitation.Growth of the aesculin to Listeria monocytogenes is shown significantly
Facilitation, and it is little to the growth effect of other four kinds of bacterium.Because Listeria monocytogenes are in five kinds of bacterium syntrophisms
Weak tendency is competed, therefore, appropriate aesculin can ensure that Listeria monocytogenes are in harmonious growth with other four kinds of bacterium
State.
The preparation method is that：
（1）By peptone, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, cholate, lithium chloride, glucose, sweet
Dew alcohol, Sodium Pyruvate and aesculin are added in 990mL distilled water, are heated to dissolving, are cooled to after room temperature, regulation pH value to
7.1-7.3；
（2）Mix, 121 DEG C of sterilizing 20min；
（3）Weigh potassium tellurite 0.1mg to be added in the sterilized distilled water of 10mL, obtain potassium tellurite solution, dispense
It is stored in standby at 4-6 DEG C;
（4）Aseptically, add（3）The mL of potassium tellurite solution 10 of preparation.
The present invention has the advantage that：
（1）SSSLE can reach the enriching effect of the respective selective enrichment medium of five kinds of target pathogenic bacterias, Ke Yiti
High detection efficiency, it is cost-effective, simplify procedures.
（2）Compared with other collective medias, SSSLE can finally be obtained to five kinds of object bacteria Sync enrichments and harmonic growth
Relatively uniform cellular biomass is obtained, with optimal enriching effect, the demand of modern Fast Detection Technique can be preferably met.
The culture of the present invention is used directly for being separately cultured and molecular Biological Detection experiment for object bacteria.
Brief description of the drawings
Fig. 1：The sample of five kinds of object bacterias of artificial contamination is schemed through being combined the PCR after enriched medium increases bacterium and detecting, single to increase Lee
Si Te（hlyA）, Shigella（ipaH）, salmonella（invA）, staphylococcus aureus（16S）And Escherichia coli（uidA）.
Fig. 2：After pork sample increases bacterium through compound enriched medium, salmonella is detected（invA）And Escherichia coli
（uidA）.
Fig. 3：After beef sample increases bacterium through compound enriched medium, Listeria monocytogenes are detected（hlyA）, Shigella
（ipaH）, staphylococcus aureus（16S）, Escherichia coli（uidA）.
Fig. 4：After chicken meat sample increases bacterium through compound enriched medium, staphylococcus aureus is detected（16S）With will Hayes
Bacterium（ipaH）.
Embodiment
To be best understood from the present invention, the present invention is elaborated below with reference to embodiment, but application claims are protected
The scope of shield is not limited to the scope that embodiment is represented.
Embodiment 1:
By peptone 10.0g, disodium hydrogen phosphate 9.0g, potassium dihydrogen phosphate 1.5g, sodium chloride 10.0g, glucose
3.0g, aesculin 1.0g, cholate 0.1g, lithium chloride 1.0g, mannitol 2.0g and Sodium Pyruvate 2.5g, distilled water add to 990mL,
Dissolving is heated to, is cooled to after room temperature, correction pH value to 7.1-7.3；Mix, 121 DEG C of sterilizing 20min；Weigh potassium tellurite
0.10mg is added in the sterilized distilled water of 10mL, obtains potassium tellurite solution；Aseptically, the tellurious prepared is added
Sour potassium solution；Packing is stored in standby at 4-6 DEG C.
The compound culture medium for increasing bacterium that is used for of preparation is respectively connected to be diluted to 10-4Salmonella, Escherichia coli, golden yellow
Color staphylococcus, the bacterium solution 0.5mL, 37 DEG C of culture 24h of five kinds of object bacterias of Listeria monocytogenes and Shigella.Use UV, visible light
OD values under spectrophotometric determination 540nm.Bacterium solution is accessed into the respective selective enrichment culture of every kind of target pathogenic bacteria simultaneously
Compareed in base.
The growing state of the object bacteria of table 1 individually in the medium
In table, salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes and Shigella do three
Repeat.As shown in Table 1, five kinds of object bacterias are linked into enriched medium SSSLE, after increasing bacterium through 24h, with respective selectivity
Enriched medium has equal enriching effect.
Embodiment 2:
By peptone 10.0g, disodium hydrogen phosphate 9.0g, potassium dihydrogen phosphate 1.5g, sodium chloride 15.0g, glucose
3.0g, aesculin 1.0g, cholate 0.1g, lithium chloride 1.5g, mannitol 6.0g and Sodium Pyruvate 1.0g, distilled water add to 990mL,
Dissolving is heated to, is cooled to after room temperature, correction pH value to 7.1-7.3；Mix, 121 DEG C of sterilizing 20min；Weigh potassium tellurite
0.20mg is added in the sterilized distilled water of 10mL, obtains potassium tellurite solution；Aseptically, the tellurious prepared is added
Sour potassium solution；Packing is stored in standby at 4-6 DEG C.
The compound culture medium for increasing bacterium that is used for of preparation is respectively connected to be diluted to 10-4Salmonella, Escherichia coli, golden yellow
Color staphylococcus, the bacterium solution 0.5mL, 37 DEG C of culture 24h of five kinds of object bacterias of Listeria monocytogenes and Shigella.Use UV, visible light
OD values under spectrophotometric determination 540nm.Bacterium solution is accessed into the respective selective enrichment culture of every kind of target pathogenic bacteria simultaneously
Compareed in base.
The growing state of the object bacteria of table 2 individually in the medium
In table, salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes and Shigella do three
Repeat.As shown in Table 2, five kinds of object bacterias are linked into enriched medium SSSLE, after increasing bacterium through 24h, with respective selectivity
Enriched medium has equal enriching effect.
Embodiment 3:
By peptone 10.0g, disodium hydrogen phosphate 9.0g, potassium dihydrogen phosphate 1.5g, sodium chloride 15.0g, glucose
3.0g, aesculin 1.0g, cholate 0.1g, lithium chloride 1.5g, mannitol 6.0g and Sodium Pyruvate 5.0g, distilled water add to 990mL,
Dissolving is heated to, is cooled to after room temperature, correction pH value to 7.1-7.3；Mix, 121 DEG C of sterilizing 20min；Weigh potassium tellurite
0.05mg is added in the sterilized distilled water of 10mL, obtains potassium tellurite solution；Aseptically, the tellurious prepared is added
Sour potassium solution；Packing is stored in standby at 4-6 DEG C.
The compound culture medium for increasing bacterium that is used for of preparation is respectively connected to be diluted to 10-4Salmonella, Escherichia coli, golden yellow
Color staphylococcus, the bacterium solution 0.5mL, 37 DEG C of culture 24h of five kinds of object bacterias of Listeria monocytogenes and Shigella.Use UV, visible light
OD values under spectrophotometric determination 540nm.Bacterium solution is accessed into the respective selective enrichment culture of every kind of target pathogenic bacteria simultaneously
Compareed in base.
The growing state of the object bacteria of table 3 individually in the medium
In table, salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes and Shigella do three
Repeat.As shown in Table 3, five kinds of object bacterias are linked into enriched medium SSSLE, after increasing bacterium through 24h, with respective selectivity
Enriched medium has equal enriching effect.
Embodiment 4：
LB, nutrient broth NB, pancreas peptone soybean broth TSB, buffered peptone water BPW and SSSLE are prepared respectively（According to
It is formulated in embodiment 1）Each 1000mL of culture medium.
The culture medium of preparation is respectively connected to be diluted to 10-4Salmonella, Escherichia coli, staphylococcus aureus, list
Increase the bacterium solution 0.5mL, 37 DEG C of culture 24h of five kinds of object bacterias of Listeria and Shigella.Surveyed with ultraviolet-uisible spectrophotometer
Determine the OD values under 540nm.
The independent enriching effect of the culture medium of table 4
In table, salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes and Shigella do two
Repeat.As shown in Table 4, five kinds of culture mediums are imitated to the increasing bacterium of salmonella, Escherichia coli, staphylococcus aureus and Shigella
Fruit is all relatively good, after 37 DEG C of culture 24h, OD540It has been above 1.2.And for Listeria monocytogenes, five kinds of culture mediums are to it
Enriching effect is widely different.Listeria monocytogenes are not grown almost in BPW and NB, and nutrient solution is clarified very much；Grown in LB
It is faint；Grown in TSB and SSSLE it is preferable, to be optimal in TSB.Therefore, TSB and SSSLE are suitable for being total to for five kinds of object bacterias
Zengjing Granule.Further, since not only containing abundant nutritional ingredient in SSSLE, also containing cholate, potassium tellurite and lithium chloride etc.
Inhibitor, can play a part of necessarily suppressing non-targeted varied bacteria growing.So, when the feelings of background microorganism numerous and complicated in sample
, can preferably five kinds of object bacterias of selective enrichment using SSSLE under condition.
Embodiment 5：
Culture medium is prepared according to embodiment 1, by salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes
Ratio with Shigella is 1:1:1:1:In 1 access culture medium, after 37 DEG C of culture 24h, bacterium solution is suitably diluted, is respectively coated
To deoxycholate hydrogen sulfide lactose agar（DHL）, eosin methylene blue agar（EMB）, Baird-Parker agar, PALCAM agar and SS agar plates
On, it is separately cultured.
Authentication method：Salmonella is water white transparency bacterium colony on DHL agar, has black center, Escherichia coli are on DHL
For pink bacterium colony, Shigella is water white transparency bacterium colony on DHL, and staphylococcus aureus and Listeria monocytogenes are not given birth to
It is long；Escherichia coli have the bacterium colony of metallic luster on EMB flat boards for atropurpureus, and salmonella and Shigella are ash on EMB
White translucent bacterium colony；Staphylococcus aureus is to have precipitation ring, Dan Zeng around black colonies on Baird-Parker flat boards
Listeria is for black colonies but without precipitation ring, and salmonella, Escherichia coli and Shigella do not grow on B-P；It is single to increase Lee
This special bacterium is celadon bacterium colony on PALCAM agar, around has dark brown colour circle, staphylococcus aureus is yellow color colonies, sramana
Salmonella, Escherichia coli and Shigella do not grow；Shigella is water white transparency bacterium colony on SS agar, and salmonella is colourless
Bacterium colony, there is black center, and Escherichia coli are red colonies, and staphylococcus aureus and Listeria monocytogenes do not grow.Same luck
With five kinds of pathogens, each specific gene enters performing PCR detection and identified, occur purpose band for positive findings, be otherwise the moon
Property result.
The compound enriching effect of the culture medium of table 5
As shown in Table 4, bacterium is increased by 24h, thalline, which can breed, reaches 106-108, the bacterial concentration of PCR detections can be met
It is required that.
Embodiment 6：
Culture medium is prepared according to embodiment 1.Aseptically, by 10g porks（Be free of through GB/T 4789-2008 identifications
Five kinds of object bacterias）Comminution is prepared into, respectively by 101Cfu/g and 103Cfu/g fresh cultured thing is inoculated in sample, room temperature
Lower processing 15min, is absorbed bacterium liquid, is then put it into sterile triangular flask, adds 90mL self-control enrichment liquids, mixing
2min.Uniform good sample is placed at 37 DEG C with 150 rpm shaken cultivations 24h.Bacterium is extracted with phenol/chloroform/isoamyl alcohol method
DNA, the PCR for salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes and Shigella is detected, is detected
As a result Fig. 1 is seen.It can be seen that obvious DNA cloning band occur in five kinds of object bacterias, illustrate that compound enriched medium causes in sample
The five kinds of object bacterias manually accessed increase simultaneously, and reach the bacteria concentration requirement of PCR detections.Prove to increase through compound enriched medium
After bacterium culture, available for PCR detections, the requirement of its test limit is met.
Embodiment 7：
Culture medium is prepared according to embodiment 1.Aseptically, 25g porks, beef and chicken meat sample are prepared into respectively
Comminution, then puts it into sterile triangular flask, adds 225mL self-control enrichment liquids, mixes 2min.Uniform good sample is placed in
With 150 rpm shaken cultivations 24h at 37 DEG C.The DNA of bacterium is extracted with phenol/chloroform/isoamyl alcohol method, for salmonella, large intestine
Bacillus, staphylococcus aureus, the PCR detections of Listeria monocytogenes and Shigella, testing result are shown in Fig. 2, Fig. 3 and figure respectively
4。
Claims (3)
1. one kind is used for Sync enrichment salmonella（Salmonella）, Escherichia coli（Escherichia coli）, it is golden yellow
Staphylococcus（Staphylococcus aureus）, Listeria monocytogenes（Listeria monocytogenes）With will Hayes
Bacterium（Shigella）The culture medium of five kinds of foodborne bacterial pathogenses, its formula is：The g of peptone 5.0 ~ 20.0, sodium chloride 5.0 ~ 15.0
G, the g of disodium hydrogen phosphate 5.0 ~ 15.0, the g of potassium dihydrogen phosphate 0.5 ~ 3.0, the g of cholate 0.05 ~ 0.2, potassium tellurite 0.05 ~
0.15 mg, the g of lithium chloride 0.5 ~ 1.5, the g of glucose 1.0 ~ 5.0, the g of mannitol 1.0 ~ 6.0, Sodium Pyruvate 1.0 ~ 5.0 g, seven
The g of leaf glycosides 0.5 ~ 1.5, distilled water 1000 mL, pH7.1-7.5.
2. culture medium according to claim 1, it is characterised in that：The g of peptone 10.0, the g of sodium chloride 10.0,12 water phosphorus
The sour g of disodium hydrogen 9.0, the g of potassium dihydrogen phosphate 1.5, the g of cholate 0.1, the mg of potassium tellurite 0.1, the g of lithium chloride 1.0, glucose 3.0
G, the g of mannitol 2.0, the g of Sodium Pyruvate 2.5, the g of aesculin 1.0, distilled water 1000 mL, pH7.1-7.3.
3. it is used for salmonella, Escherichia coli, staphylococcus aureus, Listeria monocytogenes and will Hayes described in claim 1
The preparation method of the culture medium of bacterium, it is characterised in that in accordance with the following steps：
（1）According to the formula described in claim 1, by peptone, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, courage
Salt, lithium chloride, glucose, mannitol, Sodium Pyruvate and aesculin are added in 990 mL distilled water, are heated to dissolving, cooling
To room temperature, regulation pH value to 7.1-7.3；
（2）Mix, 121 DEG C of 20 min of sterilizing；
（3）Potassium tellurite is weighed by claim 1 to be added in the sterilized distilled water of 10 mL, obtains potassium tellurite solution, point
Dress is stored in standby at 4-6 DEG C；
（4）Aseptically, step is added（3）The potassium tellurite solution 10mL of middle preparation.
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| CN104694434A (en) * | 2015-03-13 | 2015-06-10 | 黑龙江出入境检验检疫局检验检疫技术中心 | Culture medium capable of simultaneously proliferating multiple pathogenic bacteria in meat products and preparation method of culture medium |
| CN105039202B (en) * | 2015-06-10 | 2018-01-12 | 舟山出入境检验检疫局综合技术服务中心 | A kind of salmonella, Listeria monocytogenes and the compound selective medium for increasing bacterium of vibrio parahaemolytious and preparation method thereof |
| CN104894040B (en) * | 2015-06-30 | 2019-07-12 | 刘少伟 | Salmonella in Food one-step method selective enrichment medium and preparation method thereof |
| CN105506054A (en) * | 2016-02-05 | 2016-04-20 | 夏淑平 | Listeria monocytogenes selective medium |
| CN106011297A (en) * | 2016-07-30 | 2016-10-12 | 天津市农业质量标准与检测技术研究所 | Real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber |
| CN106047781B (en) * | 2016-08-22 | 2018-05-18 | 四川华汉三创生物科技有限公司 | A kind of enriched medium and preparation method thereof, Zengjing Granule method |
| CN106434842B (en) * | 2016-11-22 | 2019-09-27 | 上海市农业科学院 | A kind of enriched medium SES altogether |
| CN106591421A (en) * | 2017-02-27 | 2017-04-26 | 新疆农业大学 | Escherichia coli and/or salmonella and/or listeria monocytogenes enzyme production co-culture medium |
| CN107385010B (en) * | 2017-08-28 | 2021-09-03 | 河北科技师范学院 | Novel selective separation culture medium for listeria, preparation method and application |
| CN109055514A (en) * | 2018-04-27 | 2018-12-21 | 曾小敏 | Single rapid detection method for increasing listeria spp viable bacteria |
| CN110066752A (en) * | 2019-05-07 | 2019-07-30 | 天津商业大学 | It is used for the compounded culture medium and preparation method of four kinds of food-borne pathogens simultaneously |
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