CN104388524A - Selective chromogenic medium for coliform and test paper with selective chromogenic medium - Google Patents
Selective chromogenic medium for coliform and test paper with selective chromogenic medium Download PDFInfo
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- CN104388524A CN104388524A CN201410682129.5A CN201410682129A CN104388524A CN 104388524 A CN104388524 A CN 104388524A CN 201410682129 A CN201410682129 A CN 201410682129A CN 104388524 A CN104388524 A CN 104388524A
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Abstract
The invention provides a selective chromogenic medium for coliform. Each 1,000mL of culture medium comprises the following components by weight: 15-25g of violet red bile agar, 5-25g of specific enzyme chromogenic substrates, 0.05-20g of dimethyl formamide, 1-5g of yeast powder, 1-8g of lactose, 5-10g of sodium chloride, 3-5g of dipotassium phosphate, 0.5-2g of monopotassium phosphate and 0.1-0.3g of sodium lauryl sulfate. The invention also provides a test paper containing the selective chromogenic medium for the coliform. Two specific enzyme chromogenic substrates are compounded to use; the specificity and sensitivity of the selective chromogenic medium on the coliform are significantly enhanced in comparison with those of any independently used chromogenic substrate; and the selective chromogenic medium is high in detection rate and short in detection time.
Description
Technical field
The present invention relates to a kind of selective coloration culture medium being applicable to coliform, and adopt the Test paper of this selective coloration culture medium.
Background technology
Coliform refers under specific culture condition can ferment lactose can produce the aerobic and amphimicrobian Gram-negative bactacin of a class of sour aerogenesis.The height of coliform group count, show the contaminated degree of food, also reflection takes in this food to the size of human health damage, so detection coliform or intestinal bacteria have broad sense in food hygiene, how easy, accurately, detect the coliform in food rapidly, also be the problem that domestic and international food sanitation person pays close attention to jointly, have many about the method detecting coliform at present, such as MTF method, membrane filtration technique, Enzyme assay method, paper disk method, immunological method (enzyme linked immunosorbent assay and immunofluorescence) and molecular biology method etc.These methods have certain relative merits, although such as Enzyme assay method is more time saving and energy saving, detection specificity is high, and cost is higher; Paper disk method compares and is applicable to field monitoring, save a large amount of material resources, manpower, but accuracy still has much room for improvement; PCR (Polymerase Chain React ion) detection method has the features such as quick, special, responsive, but has particular requirement to experimental situation, easily disturbs by some factors, occur false positive results.
The Kerma (unit of kinetic energy) company of praising of France have developed the color developing culture medium detected for coliform, apply comparatively extensive, this substratum mainly utilizes the feature of microorganism kind specific enzymes, add corresponding enzyme chromogenic substrate in the medium, dissociate chromogen substance after the enzymic hydrolysis that microorganism growth metabolism produces, and makes culture show particular color thus directly carry out target microorganism screening to differentiate.After this, occurred again some other coliform chromogenic medium product successively, wherein the color developing culture medium such as Coli ID, RapidE-coli, Chromocultcoliforms of Biomerieux SA also has larger market clout.A series of loaded down with trivial details confirmatory experiment is also needed after object bacteria being cultivated in traditional microbiological detection method, and color developing culture medium overcomes this shortcoming, realize Yi Walk and cultivate Direct Identification, substantially can meet the needs that coliform Chu Walk is identified, have great significance so coliform detects appearing in coliform laboratory qualification technology of color developing culture medium.Not only to coordinate mutually with the state of the art of the developmental level of country and the sector and active development as a new technique, also should have certain penetration and promotion simultaneously, a good Microbiological detection of foods technology must possess fast, accurately, specificity and highly sensitive, time saving and energy saving and low cost and other advantages, although color developing culture medium testing product conventional on the market at present may meet special, the testing requirements such as sensitivity, but most of color developing culture medium product all derives from foreign biomolecule Reagent Company, its expensive not convenient property causing testing cost to increase and to buy makes this new technology become the bottleneck of codex alimentarius detection method, also contain that some domestic food inspection industries are to the actual demand of this product.
Summary of the invention
The invention provides a kind of coliform selective coloration culture medium, and the Test paper adopting this selective coloration culture medium to make, to overcome the above-mentioned defect that prior art exists.
First the present invention relates to a kind of coliform 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 mixture of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside, and both weight proportions are 0.2-1:1, preferred 0.5-0.6:1.
The invention still further relates to a kind of Test paper adopting this selective coloration culture medium to make, it is at least adsorbed with the absorbent filter of above-mentioned coliform selective coloration culture medium containing one.
The present invention adopts two species specificity enzyme chromogenic substrates to carry out composite use, and its specificity for coliform unexpected and susceptibility are comparatively used alone any one chromogenic substrate significantly to be strengthened, and recall rate is high, detection time is short.
Embodiment
Provide present pre-ferred embodiments below, to describe technical scheme of the present invention in detail.
Embodiment 1
A kind of coliform chromogenic medium, every 1000mL substratum contains: violet red bile agar 15g, specific enzymes chromogenic substrate 5g, dimethyl formamide 0.05g, yeast powder 1g, lactose 1g, sodium-chlor 5g, dipotassium hydrogen phosphate 5g, potassium primary phosphate 2g, Sodium Lauryl Sulphate BP/USP 0.3g.Described specific enzymes chromogenic substrate is the mixture of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside, and both weight proportions are 0.5:1.
By water-soluble for mentioned component to 1000mL, heated and boiled, 120 DEG C of sterilizing 15min, can use.
Embodiment 2
A kind of coliform chromogenic medium, every 1000mL substratum contains: violet red bile agar 25g, specific enzymes chromogenic substrate 25g, dimethyl formamide 20g, yeast powder 5g, lactose 8g, sodium-chlor 10g, dipotassium hydrogen phosphate 3g, potassium primary phosphate 0.5g, Sodium Lauryl Sulphate BP/USP 0.1g.Described specific enzymes chromogenic substrate is the mixture of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside, and both weight proportions are 0.6:1.
By water-soluble for mentioned component to 1000mL, heated and boiled, 120 DEG C of sterilizing 15min, can use.
Embodiment 3
A kind of coliform chromogenic medium, every 1000mL substratum contains: violet red bile agar 20g, specific enzymes chromogenic substrate 15g, dimethyl formamide 5g, yeast powder 3g, lactose 5g, sodium-chlor 8g, dipotassium hydrogen phosphate 4g, potassium primary phosphate 1g, Sodium Lauryl Sulphate BP/USP 0.2g.Described specific enzymes chromogenic substrate is the mixture of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside, and both weight proportions are 0.2:1.
By water-soluble for mentioned component to 1000mL, heated and boiled, 120 DEG C of sterilizing 15min, can use.
Comparative example 1
A kind of coliform chromogenic medium, every 1000mL substratum contains: violet red bile agar 15g, specific enzymes chromogenic substrate 5g, dimethyl formamide 0.05g, yeast powder 1g, lactose 1g, sodium-chlor 5g, dipotassium hydrogen phosphate 5g, potassium primary phosphate 2g, Sodium Lauryl Sulphate BP/USP 0.3g.Described specific enzymes chromogenic substrate is 4-methyl umbelliferone-β-D-galactoside.
By water-soluble for mentioned component to 1000mL, heated and boiled, 120 DEG C of sterilizing 15min, can use.
Comparative example 2
A kind of coliform chromogenic medium, every 1000mL substratum contains: violet red bile agar 15g, specific enzymes chromogenic substrate 5g, dimethyl formamide 0.05g, yeast powder 1g, lactose 1g, sodium-chlor 5g, dipotassium hydrogen phosphate 5g, potassium primary phosphate 2g, Sodium Lauryl Sulphate BP/USP 0.3g.Described specific enzymes chromogenic substrate is the mixture of the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside.
By water-soluble for mentioned component to 1000mL, heated and boiled, 120 DEG C of sterilizing 15min, can use.
Embodiment 4
The preparation of coliform selective coloration culture medium test paper: selected absorbent filter is immersed in coliform chromogenic medium to flood after 10-15 minute and take out oven dry, be cut into certain specification test paper after being cooled to room temperature for subsequent use.
Embodiment 5
The test paper upper and lower surface obtained in embodiment 4 covers respectively and covers a layer polypropylene protective membrane.
Embodiment 6
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.
Table 1 color developing culture medium specific assay result
As can be seen from the above table, observe when the Main Foods pathogenic bacterium such as the Shigella except coliform Pseudomonas, vibrios, salmonella cultivate 18h on substratum, bacterium colony does not occur.Visible substratum of the present invention detects coliform primary dcreening operation has good selectivity and specificity.
Embodiment 6
The detection sensitivity of testing example 1-3 and employing National Standard Method respectively, wherein National Standard Method is the method adopting GB47893-2010 " national food safety standard food microbiological analysis enumeration of coliforms ".
Table 2 color developing culture medium detection sensitivity
| Sample | 18h susceptibility (%) | 24h susceptibility (%) | 48h susceptibility (%) |
| Embodiment 1 | 98 | 98 | 98 |
| Embodiment 2 | 97 | 97 | 97 |
| Embodiment 3 | 94 | 96 | 98 |
| Comparative example 1 | 75 | 90 | 98 |
| Comparative example 2 | 85 | 95 | 98 |
| National Standard Method | 10 | 65 | 98 |
As can be seen from Table 2, from 18h to 48h, color developing culture medium detection sensitivity of the present invention does not have too large change, and when 18h, the susceptibility detecting pure fungi pollution sample just can reach 94-98%, and National Standard Method is at 18h, and detection sensitivity is very low, along with incubation time is increased to 48h, susceptibility just can reach 98%.And than being used alone the susceptibility of any one chromogenic substrate, especially 18 hours susceptibilitys all will significantly improve.Color developing culture medium of the present invention eliminates the biochemical identification experiment in National Standard Method after VRBA separation and Culture, greatly shortens detection time, reduces testing cost.
Claims (5)
1. a coliform selective coloration culture medium, is characterized in that, wherein every 1000mL substratum is containing, for example the component of lower weight:
2. coliform selective coloration culture medium as claimed in claim 1, it is characterized in that, described specific enzymes chromogenic substrate is the mixture of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside.
3. coliform selective coloration culture medium as claimed in claim 2, it is characterized in that, in described specific enzymes chromogenic substrate, the weight proportion of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside is 0.2-1:1.
4. coliform selective coloration culture medium as claimed in claim 2, it is characterized in that, in described specific enzymes chromogenic substrate, the weight proportion of 4-methyl umbelliferone-β-D-galactoside and the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside is 0.5-0.6:1.
5. a coliform selective coloration culture medium Test paper, is characterized in that, is wherein at least adsorbed with the absorbent filter of coliform selective coloration culture medium described in any one of claim 1-4 containing one.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811404A (en) * | 2017-01-22 | 2017-06-09 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of quick detection coliform and preparation method thereof, detection method |
| CN111304280A (en) * | 2020-02-26 | 2020-06-19 | 成都海关技术中心 | Culture medium for color development detection of coliform group in food and seasoning containing pepper |
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| CN1796568A (en) * | 2004-12-28 | 2006-07-05 | 重庆食品工业研究所 | Quick detecting coliform group and medium for culturing coliforms and preparation method |
| CN101294190A (en) * | 2007-12-05 | 2008-10-29 | 姚毓才 | Composite quick colour-developing examination and check agent for coliform group bacteria, researching and developing flow scheme thereof |
| CN101580870A (en) * | 2009-06-26 | 2009-11-18 | 青岛佳明测控仪器有限公司 | Method for fast and continuously detecting faecal coliform and colon bacillus group |
| CN102424832A (en) * | 2011-11-23 | 2012-04-25 | 广东环凯微生物科技有限公司 | Chromogenic medium used for detecting esherichia coli O157:H7 |
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Patent Citations (4)
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| CN1796568A (en) * | 2004-12-28 | 2006-07-05 | 重庆食品工业研究所 | Quick detecting coliform group and medium for culturing coliforms and preparation method |
| CN101294190A (en) * | 2007-12-05 | 2008-10-29 | 姚毓才 | Composite quick colour-developing examination and check agent for coliform group bacteria, researching and developing flow scheme thereof |
| CN101580870A (en) * | 2009-06-26 | 2009-11-18 | 青岛佳明测控仪器有限公司 | Method for fast and continuously detecting faecal coliform and colon bacillus group |
| CN102424832A (en) * | 2011-11-23 | 2012-04-25 | 广东环凯微生物科技有限公司 | Chromogenic medium used for detecting esherichia coli O157:H7 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811404A (en) * | 2017-01-22 | 2017-06-09 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of quick detection coliform and preparation method thereof, detection method |
| CN111304280A (en) * | 2020-02-26 | 2020-06-19 | 成都海关技术中心 | Culture medium for color development detection of coliform group in food and seasoning containing pepper |
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Application publication date: 20150304 |