CN104388522A - Selective chromogenic culture medium of listeria monocytogenes and preparation method of selective chromogenic culture medium - Google Patents
Selective chromogenic culture medium of listeria monocytogenes and preparation method of selective chromogenic culture medium Download PDFInfo
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- CN104388522A CN104388522A CN201410682559.7A CN201410682559A CN104388522A CN 104388522 A CN104388522 A CN 104388522A CN 201410682559 A CN201410682559 A CN 201410682559A CN 104388522 A CN104388522 A CN 104388522A
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Abstract
The invention provides a selective chromogenic culture medium of listeria monocytogenes. Every 1000ml of the culture medium comprises the following components by weight: 5-20g of heart-brain extract, 2-6g of agar, 2-5g of peptone, 0.1-1g of a specific enzyme chromogenic substrate, 0.05-20g of dimethylformamide, 0.2-0.5g of magnesium sulfate, 0.2-0.5g of lithium chloride, 0.3-0.6g of an oxygen scavenger, 0.2-1.0g of 3-morpholino-propane-1-sulfonic acid, 6-17g of a bacteria inhibitor and the balance being water; the specific enzyme chromogenic substrate is 5-bromo-4-chloro-3-indolyl-inositol-1-phosphate. A listeria monocytogene specific chromogenic substrate is made into the selective chromogenic culture medium, so that the specific selection and chromogenic functions of listeria monocytogenes are realized at low cost, and the problem that the listeria monocytogenes are difficult to isolate, culture and identify is solved.
Description
Technical field
The present invention relates to a kind of selective coloration culture medium being applicable to Listeria monocytogenes, and the preparation method of this selective coloration culture medium.
Background technology
Listeria bacteria is separated in rabbit and cavy body first in Britain nineteen twenty-six, and it is pathogenic to confirm people in nineteen twenty-nine.This bacterium has 6 kinds, is respectively listeria monocytogenes (being called for short Listeria monocytogenes, LM), Ying Nuoke listeria bacteria, Xi Er listeria bacteria, this listeria bacteria of Weir, sheep listeria bacteria and grignard Liszt mattress.Wherein Listeria monocytogenes is people and animals pathogenic bacterium, and sheep listeria bacteria is animal pathogen, all the other no pathogenicities.Listeria monocytogenes is extensively present in soil, animal and fishery products, wherein relates to the food that maximum food has soft cheese, milk-product, sausage, smoked fish, romaine lettuce food, cooked food shop, and freezing instant food.Because it has unique physiological property, can cause the illness such as mankind's meningitis, septicemia, especially with patient's more easy infection of pregnancy women, newborn infant and immunologic hypofunction, lethality rate reaches more than 30%.In recent years, the food poisoning caused because of food contamination Listeria monocytogenes day by day increased, and had caused the extensive concern of many countries, and had been listed in one of large food pathogenic nineties four in 20th century, become the essential items for inspection of many state food health.In China, this bacterium is also classified as must examining bacterium or monitoring pathogenic bacteria of import and export food by State General Administration for Quality Supervision at present.
Set up simple and efficient, that sensitivity good, specificity is high Listeria monocytogenes detection technique becomes numerous investigator common expectation.At present except traditional separation and Culture and identification method, also comprise color developing culture medium Rapid identification method, microbiological analysis, molecular biology method, Immunological Method.Desirable pathogen detection method at least should possess following characteristics: ease for operation, susceptibility, specificity, quick, cheap, automatization, and can detect the existence of microorganism that is alive, that have virulence.But, up to the present also do not have a kind of detection method can meet all standards listed above.Traditional method is time-consuming, and workload is large, be not suitable with present microorganism fast, the needs of Sensitive Detection.Molecular biology for detection is compared with traditional method, although greatly reduce workload, the positive detected will be reference in the conventional way, and false negative and false positive results easily appear in detection.
The separatory selective medium of current Listeria monocytogenes, comprise color developing culture medium optionally to cultivate Liszt's flora and selectivity can not cultivate Listeria monocytogenes, and listeria no pathogenicity beyond Listeria monocytogenes or only have pathogenic to animal, the microbiology of Listeria monocytogenes is differentiated to need complicated biochemical reaction test, discriminating required time is long, affects clinical diagnosis and treatment.
Summary of the invention
The invention provides a kind of Listeria monocytogenes selective coloration culture medium, and the preparation method of this selective coloration culture medium, to overcome the above-mentioned defect that prior art exists.
First the present invention relates to a kind of Listeria monocytogenes selective coloration culture medium, and wherein every 1000mL substratum is containing, for example the component of lower weight:
Described specific enzymes chromogenic substrate is the bromo-4-of 5-chloro-3-indoles-inositol-1-phosphoric acid salt, the bromo-4-of preferred 5-chloro-3-indoles-inositol-1-potassiumphosphate.
Described oxygen scavenger is selected from pyruvate salt, hydrogen peroxide, thioglycolate salt, halfcystine, sodium sulphite or Iron sulfuret.
Described pyruvate salt, thioglycolate salt can be its sodium salt or sylvite.
Described miscellaneous bacteria inhibitor is selected from Nalidixic Acid, cycloheximide or flavicid, the mixture of preferred three, and the weight proportion of three is as follows: Nalidixic Acid 0.01-0.05 part, cycloheximide 0.015-0.05 part, flavicid 6-15 part.
The preparation method of substratum of the present invention is as follows:
1) specific enzymes chromogenic substrate is dissolved in dimethyl formamide is made into mixed solution;
2) heart-brain extract, agar, peptone, magnesium sulfate, lithium chloride, oxygen scavenger, 3-morpholino propane-1-sulfonic acid, miscellaneous bacteria inhibitor are mixed in proportion;
3) by step 1) mixed solution add step 2) in mixed solution, add water and be made into certain concentration solution;
4) cool after 120 DEG C of sterilizing 15min for subsequent use.
The present invention adopts Listeria monocytogenes specific chromogenic substrate to make Listeria monocytogenes selective coloration culture medium, achieves the single Li Site of increasing bacterium specificity and selects and coloring function, solve the problem that list increases the equal separation and Culture of Li Site and differentiates difficulty with low cost.
Embodiment
Provide present pre-ferred embodiments below, to describe technical scheme of the present invention in detail.
Embodiment 1
Listeria monocytogenes selective coloration culture medium, wherein every 1000mL substratum is containing, for example the component of lower weight: the chloro-3-indoles of the heart-brain extract 5g, agar 2g, the bromo-4-of peptone 2g, 5--inositol-1-potassiumphosphate 0.1g, dimethyl formamide 0.05g, magnesium sulfate 0.2g, lithium chloride 0.2g, Potassium pyruvate 0.3g, 3-morpholino propane-1-sulfonic acid 0.2g, cycloheximide 0.015g, surplus are water.
Concrete preparation method is as follows:
1) specific enzymes chromogenic substrate is dissolved in dimethyl formamide is made into mixed solution;
2) heart-brain extract, agar, peptone, magnesium sulfate, lithium chloride, oxygen scavenger, 3-morpholino propane-1-sulfonic acid, miscellaneous bacteria inhibitor are mixed in proportion;
3) by step 1) mixed solution add step 2) in mixed solution, add water and be made into certain concentration solution;
4) cool after 120 DEG C of sterilizing 15min for subsequent use.
Embodiment 2
Listeria monocytogenes selective coloration culture medium, wherein every 1000mL substratum is containing, for example the component of lower weight: the chloro-3-indoles of the heart-brain extract 20g, agar 6g, the bromo-4-of peptone 5g, 5--inositol-1-potassiumphosphate 1g, dimethyl formamide 20g, magnesium sulfate 0.5g, lithium chloride 0.5g, halfcystine 0.6g, 3-morpholino propane-1-sulfonic acid 1.0g, flavicid 15g, surplus are water.
Concrete preparation method is as follows:
1) specific enzymes chromogenic substrate is dissolved in dimethyl formamide is made into mixed solution;
2) heart-brain extract, agar, peptone, magnesium sulfate, lithium chloride, oxygen scavenger, 3-morpholino propane-1-sulfonic acid, miscellaneous bacteria inhibitor are mixed in proportion;
3) by step 1) mixed solution add step 2) in mixed solution, add water and be made into certain concentration solution;
4) cool after 120 DEG C of sterilizing 15min for subsequent use.
Embodiment 3
Listeria monocytogenes selective coloration culture medium, wherein every 1000mL substratum is containing, for example the component of lower weight: the chloro-3-indoles of the heart-brain extract 12g, agar 5g, the bromo-4-of peptone 3g, 5--inositol-1-potassiumphosphate 0.6g, dimethyl formamide 0.2g, magnesium sulfate 0.3g, lithium chloride 0.4g, Thioglycolic acid sodium salt 0.5g, 3-morpholino propane-1-sulfonic acid 0.8g, Nalidixic Acid 0.02 part, cycloheximide 0.05 part, flavicid 7 parts, surplus are water.
Concrete preparation method is as follows:
1) specific enzymes chromogenic substrate is dissolved in dimethyl formamide is made into mixed solution;
2) heart-brain extract, agar, peptone, magnesium sulfate, lithium chloride, oxygen scavenger, 3-morpholino propane-1-sulfonic acid, miscellaneous bacteria inhibitor are mixed in proportion;
3) by step 1) mixed solution add step 2) in mixed solution, add water and be made into certain concentration solution;
4) cool after 120 DEG C of sterilizing 15min for subsequent use.
Embodiment 4
Reference culture is made respectively the standard bacteria suspension of 100cfu/mL, draw 1mL bacterium liquid and detect, cultivate 18 hours for 37 DEG C.Result is as table 1, and "+" represents positive findings, and " one " represents negative findings.Pathogenic Listeria monocytogenes presents green bacterium colony on color developing culture medium of the present invention, and no pathogenicity Listeria is white colony.
Table 1 color developing culture medium specific assay result
As can be seen from the above table, observe when the pathogenic bacterium except Listeria monocytogenes cultivate 18h on substratum, do not occur specific chromogenic, and can be separated from listera innocua and identify Listeria monocytogenes.Visible substratum of the present invention detects Listeria monocytogenes primary dcreening operation has excellent selectivity and specificity.
Claims (9)
1. a Listeria monocytogenes selective coloration culture medium, is characterized in that, wherein every 1000mL substratum is containing, for example the component of lower weight:
Described specific enzymes chromogenic substrate is the bromo-4-of 5-chloro-3-indoles-inositol-1-phosphoric acid salt.
2. Listeria monocytogenes selective coloration culture medium as claimed in claim 1, it is characterized in that, described specific enzymes chromogenic substrate is the bromo-4-of 5-chloro-3-indoles-inositol-1-potassiumphosphate.
3. Listeria monocytogenes selective coloration culture medium as claimed in claim 1, it is characterized in that, described oxygen scavenger is selected from pyruvate salt, hydrogen peroxide, thioglycolate salt, halfcystine, sodium sulphite or Iron sulfuret.
4. Listeria monocytogenes selective coloration culture medium as claimed in claim 3, it is characterized in that, described pyruvate salt is its sodium salt or sylvite.
5. Listeria monocytogenes selective coloration culture medium as claimed in claim 3, it is characterized in that, described, thioglycolate salt is its sodium salt or sylvite.
6. Listeria monocytogenes selective coloration culture medium as claimed in claim 1, it is characterized in that, described miscellaneous bacteria inhibitor is selected from Nalidixic Acid, cycloheximide or flavicid.
7. Listeria monocytogenes selective coloration culture medium as claimed in claim 6, it is characterized in that, described miscellaneous bacteria inhibitor is the mixture of Nalidixic Acid, cycloheximide and flavicid.
8. Listeria monocytogenes selective coloration culture medium as claimed in claim 7, it is characterized in that, in described miscellaneous bacteria inhibitor, the weight proportion of each component is as follows: Nalidixic Acid 0.01-0.05 part, cycloheximide 0.015-0.05 part, flavicid 6-15 part.
9. prepare a method for Listeria monocytogenes selective coloration culture medium as described in any one of claim 1-8, it is characterized in that, comprise the steps:
1) specific enzymes chromogenic substrate is dissolved in dimethyl formamide is made into mixed solution;
2) heart-brain extract, agar, peptone, magnesium sulfate, lithium chloride, oxygen scavenger, 3-morpholino propane-1-sulfonic acid, miscellaneous bacteria inhibitor are mixed in proportion;
3) by step 1) mixed solution add step 2) in mixed solution, add water and be made into certain concentration solution;
4) cool after 120 DEG C of sterilizing 15min, namely obtain described Listeria monocytogenes selective coloration culture medium.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506054A (en) * | 2016-02-05 | 2016-04-20 | 夏淑平 | Listeria monocytogenes selective medium |
| CN106337027A (en) * | 2015-07-13 | 2017-01-18 | 深圳市检验检疫科学研究院 | Medium used for separating and detecting listeria monocytogens |
| CN110869490A (en) * | 2017-06-09 | 2020-03-06 | 日水制药株式会社 | Culture medium for detecting listeria |
-
2014
- 2014-11-24 CN CN201410682559.7A patent/CN104388522A/en active Pending
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| 刘雅莉等: "九种单核细胞增生性李斯特菌检测技术效果比较及评价", 《中国生物工程杂志》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106337027A (en) * | 2015-07-13 | 2017-01-18 | 深圳市检验检疫科学研究院 | Medium used for separating and detecting listeria monocytogens |
| CN106337027B (en) * | 2015-07-13 | 2020-04-14 | 深圳市检验检疫科学研究院 | Culture medium for isolating and detecting listeria monocytogenes |
| CN105506054A (en) * | 2016-02-05 | 2016-04-20 | 夏淑平 | Listeria monocytogenes selective medium |
| CN110869490A (en) * | 2017-06-09 | 2020-03-06 | 日水制药株式会社 | Culture medium for detecting listeria |
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