CN104388525A - Chromogenic medium used for detecting escherichia coli O157:H7 - Google Patents
Chromogenic medium used for detecting escherichia coli O157:H7 Download PDFInfo
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- CN104388525A CN104388525A CN201410682521.XA CN201410682521A CN104388525A CN 104388525 A CN104388525 A CN 104388525A CN 201410682521 A CN201410682521 A CN 201410682521A CN 104388525 A CN104388525 A CN 104388525A
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Abstract
The invention discloses a chromogenic medium used for detecting escherichia coli O157:H7 and other intestinal floras. The medium contains agar, peptone, a yeast extract, sodium chloride, sorbitol, inositol, neutral red, a beta-galactosidase chromogenic substrate, 4-methyl sector ketone-beta-D-glucuronide, isopropyl-beta-D-thiopyran galactoside, bile salt, potassium tellurite and cefixime. The chromogenic medium disclosed by the invention can be used for identifying escherichia coli O157:H7 in coliforms rapidly, simply and conveniently.
Description
Technical field
The present invention relates to a kind of substratum of microorganism detection, particularly relating to a kind of for detecting Escherichia coli O 157: the color developing culture medium of H7.
Background technology
Enterohemorrhagic E.coli (enterohemorrhage E.Coli, EHEC) be a colibacillary hypotype, EHEC is divided into 157,26,111 serotypes, and principal causative bacterial strain is O157:H7, can infectious diarrhea be caused, gain the name because the hemorrhagic enteritis of the mankind can be caused.
EHEC O157:H7 is except nonfermented or delayed reaction sorbyl alcohol, and other common biochemical characters and colon bacillus basic simlarity, but also have some biochemical reaction not quite identical, have discriminating meaning.Although EHECO157:H7 has uidA gene, the GRD beta-glucuronidase non-activity of its coding, can not decompose 4-methyl umbelliferone-β-D-Glucose aldehydic acid glycosides (MUG) and produce fluorescence, and namely MUG is negative.
The color developing culture medium of EHEC O157:H7 utilizes principle for Escherichia coli O 157: H7 nonfermented or nonfermented sorbyl alcohol and expression beta-galactosidase enzymes in 2 days, adds the chromogenic substrate of sorbyl alcohol and beta-galactosidase enzymes in basic medium; In this color developing culture medium, the color and luster of E. coli clones display fermentation sorbyl alcohol, and EHEC O157:H7 presents in beta-galactosidase enzymes chromogenic substrate the color of the group that develops the color.
Research finds, there is nonfermented sorbyl alcohol in intestinal bacteria, and GRD beta-glucuronidase is positive, but EHECO157:H7 serum also inagglutinable bacterial strain, cultivate to the colour developing of EHEC O157:H7 and cause false positive.At present, also will not be used for detecting Escherichia coli O 157: the color developing culture medium of H7.
Summary of the invention
The object of the present invention is to provide a kind of for detecting Escherichia coli O 157: the color developing culture medium of H7.
Of the present invention adopted technical scheme is:
A kind of for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 15-20g, peptone 5-15g, yeast extract 4-6g, sodium-chlor 5-10g, also containing sorbyl alcohol 10-20g, inositol 4-7g, the red 0.005-0.03g of property, beta-galactosidase enzymes chromogenic substrate 0.05-3g, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1-0.2g, isopropyl-beta D-thio galactopyranoside 0.05-0.2g, bovine bile 1-5g, potassium tellurite 1-5mg, Cefixime Micronized 0.01-0.06mg.
Preferably, beta-galactosidase enzymes chromogenic substrate is the chloro-3-indoles-beta galactose of the bromo-4-of 5-.
Preferably, the content of the chloro-3-indoles-beta galactose of the bromo-4-of beta-galactosidase enzymes chromogenic substrate 5-in 1000mL substratum is 0.1g.
Preferably, beta-galactosidase enzymes chromogenic substrate is O-nitrophenyl-β-D-galactoside.
Preferably, the content of beta-galactosidase enzymes chromogenic substrate O-nitrophenyl-β-D-galactoside in 1000mL substratum is 2g.
Preferably, the 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.05-0.2g.
Preferably, the 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.15g.
Preferably, the 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.1g.
Preferably, for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 15g, peptone 10g, yeast extract 5g, sodium-chlor 5g, sorbyl alcohol 15g, inositol 5g, the red 0.01g of property, O-nitrophenyl-β-D-galactoside 2g, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1g, isopropyl-beta D-thio galactopyranoside 0.1g, bovine bile 1.5g, potassium tellurite 2mg, Cefixime Micronized 0.04mg.
Preferably, for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 15g, peptone 10g, yeast extract 5g, sodium-chlor 5g, the chloro-3-indoles-beta galactose 0.1g of sorbyl alcohol 15g, inositol 5g, the bromo-4-of property red 0.01g, 5-, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1g, isopropyl-beta D-thio galactopyranoside 0.1g, bovine bile 1.5g, potassium tellurite 2mg, Cefixime Micronized 0.04mg.
The present invention with the addition of sorbyl alcohol and toluylene red in the medium, except Escherichia coli O 157: the coliform fermentation sorbyl alcohol except H7 produces acid, red shade is demonstrated under the effect of pH indicator toluylene red, and Escherichia coli O 157: H7 does not utilize sorbyl alcohol and presents colourless bacterium colony.
The present invention with the addition of beta-galactosidase enzymes chromogenic substrate I and beta-galactosidase enzymes chromogenic substrate II in the medium, and coliform decomposes substrate under beta-galactosidase enzymes effect, and dissociate developer, makes bacterium colony present the color and luster of developer.
Cholate can suppress most gram-positive cocci in sample, comprises bovine bile, pig cholate, no. 3 bile salt, sodium deoxycholate and mixing cholate.The present invention with the addition of cholate in the medium.
The present invention with the addition of in the medium, and the gram negative bacillus of part in sample can be suppressed as coliform and the microbiotic potassium tellurite and the Cefixime Micronized that easily spread the modification bacillus grown.
Color developing culture medium of the present invention can identify Escherichia coli O 157 quickly and easily from coliform: H7.
Embodiment
A kind of for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 15-20g, peptone 5-15g, yeast extract 4-6g, sodium-chlor 5-10g, also containing sorbyl alcohol 10-20g, inositol 4-7g, the red 0.005-0.03g of property, beta-galactosidase enzymes chromogenic substrate 0.05-3g, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1-0.2g, isopropyl-beta D-thio galactopyranoside 0.05-0.2g, bovine bile 1-5g, potassium tellurite 1-5mg, Cefixime Micronized 0.01-0.06mg.
Preferably, beta-galactosidase enzymes chromogenic substrate is the chloro-3-indoles-beta galactose of the bromo-4-of 5-, and the content in 1000mL substratum is 0.1g.
Preferably, beta-galactosidase enzymes chromogenic substrate is O-nitrophenyl-β-D-galactoside, and the content in 1000mL substratum is 2g.
Preferably, the 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.05-0.2g.
Preferably, the 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.15g.
Preferably, the 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.1g.
Color developing culture medium of the present invention is for detecting Escherichia coli O 157: the method for H7, comprises the steps:
(1) preparation colour developing is dull and stereotyped: add the raw material of above-mentioned color developing culture medium except potassium tellurite, Cefixime Micronized in every 1000mL deionized water, stir, heated and boiled is to dissolving completely, wait to be chilled to about 50 DEG C, add the potassium tellurite of filtration sterilization, Cefixime Micronized, mixing, be down flat plate, for subsequent use;
(2) inoculation culture: by sample or be inoculated on colour developing flat board containing the enrichment liquid of sample, cultivates 22 ~ 26h for 37 DEG C;
(3) interpretation of result: if colour developing is dull and stereotyped upper occur smooth, slightly projection, diameter 1 ~ 3mm, glaucous bacterium colony, and bacterium colony under 366nm without pearl opal fluorescence, illustrate that this sample exists Escherichia coli O 157: H7, other entero-bacte or suppressed or for display is red or colour developing blue-greenish colour but present pearl opal fluorescence under 366nm.
Color developing culture medium of the present invention is for detecting Escherichia coli O 157: the method for H7, comprises the steps:
(1) preparation colour developing is dull and stereotyped: add the raw material of above-mentioned color developing culture medium except beta-galactosidase enzymes chromogenic substrate O-nitrophenyl-β-D-galactoside, potassium tellurite, Cefixime Micronized in every 1000mL deionized water, stir, heated and boiled is to dissolving completely, wait to be chilled to about 50 DEG C, add the O-nitrophenyl-β-D-galactoside of filtration sterilization, potassium tellurite, Cefixime Micronized, mixing, is down flat plate, for subsequent use;
(2) inoculation culture: by sample or be inoculated on colour developing flat board containing the enrichment liquid of sample, cultivates 22 ~ 26h for 37 DEG C;
(3) interpretation of result: if smooth, slightly projection, diameter 1 ~ 3mm, yellow the bacterium colony of the dull and stereotyped upper appearance that develops the color, and bacterium colony under 366nm without pearl opal fluorescence, illustrate that this sample exists Escherichia coli O 157: H7, other entero-bacte or suppressed or for display is red or colour developing is yellow but present pearl opal fluorescence under 366nm.
Below in conjunction with specific embodiment, the present invention is further illustrated, but do not limit to so.
Embodiment 1
A kind of for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 10g, peptone 10g, yeast extract 5g, sodium-chlor 5g, also containing sorbyl alcohol 15g, inositol 5g, the chloro-3-indoles-beta galactose 0.1g of the bromo-4-of property red 0.1g, 5-, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1g, isopropyl-beta D-thio galactopyranoside 0.1g, bovine bile 1.5g, potassium tellurite 2mg, Cefixime Micronized 0.04mg.
Embodiment 2
A kind of for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 10g, peptone 10g, yeast extract 5g, sodium-chlor 5g, also containing sorbyl alcohol 15g, inositol 5g, the red 0.1g of property, O-nitrophenyl-β-D-galactoside 2g, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1g, isopropyl-beta D-thio galactopyranoside 0.1g, bovine bile 1.5g, potassium tellurite 2mg, Cefixime Micronized 0.04mg.
Embodiment 3 specificity experiments
8 kinds of reference cultures such as E.coli O157:H7NCTC12900, E.coli O157:H7882364, E.coli O157:H7ATCC43895, E.coli O157:H7ATCC43888, E.coli ATCC8739, E.coli CMCC (B) 44102, E.coli CMCC (B) 44103, P.vulgaris CMCC (B) 49027 are made respectively the standard bacteria suspension (concentration is 108 ~ 109cfu/mL) of proper concn, streak inoculation is on the flat board made with embodiment 1 color developing culture medium respectively, cultivates 18 ~ 24h for 37 DEG C.Specificity experiments the results are shown in Table 1.
The specific chromogenic test-results of table 1 E. coli O157:H7 color developing culture medium
Embodiment 4 specificity experiments
8 kinds of reference cultures such as E.coli O157:H7NCTC12900, E.coli O157:H7882364, E.coli O157:H7ATCC43895, E.coli O157:H7ATCC43888, E.coli ATCC8739, E.coli CMCC (B) 44102, E.coli CMCC (B) 44103, P.vulgaris CMCC (B) 49027 are made respectively the standard bacteria suspension (concentration is 108 ~ 109cfu/mL) of proper concn, streak inoculation is on the flat board made with embodiment 2 color developing culture medium respectively, cultivates 18 ~ 24h for 37 DEG C.Specificity experiments the results are shown in Table 2.
The specific chromogenic test-results of table 2 E. coli O157:H7 color developing culture medium
From embodiment 3, embodiment 4 experimental result, 4 strain E.coil 0157:H7 all show beta-galactosidase enzymes chromogenic substrate colour developing group color, without pearl opal fluorescence under 366mm ultraviolet.For non-targeted bacterium, colony colour can be distinguished with object bacteria E.coil 0157:H7 phase, and aobvious pearl opal fluorescence under 366mm ultraviolet; Miscellaneous bacteria is suppressed.What above experimental result showed that the present invention announces has higher specificity for the color developing culture medium detecting intestinal bacteria E.coil 0157:H7.
Claims (10)
1. one kind for detecting Escherichia coli O 157: the color developing culture medium of H7, every 1000mL substratum contains agar 15-20g, peptone 5-15g, yeast extract 4-6g, sodium-chlor 5-10g, it is characterized in that, also containing sorbyl alcohol 10-20g, inositol 4-7g, the red 0.005-0.03g of property, beta-galactosidase enzymes chromogenic substrate 0.05-3g, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1-0.2g, isopropyl-beta D-thio galactopyranoside 0.05-0.2g, bovine bile 1-5g, potassium tellurite 1-5mg, Cefixime Micronized 0.01-0.06mg.
2. according to claim 1 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, beta-galactosidase enzymes chromogenic substrate is the chloro-3-indoles-beta galactose of the bromo-4-of 5-.
3. according to claim 2 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, the content of the chloro-3-indoles-beta galactose of the bromo-4-of 5-in 1000mL substratum is 0.1g.
4. according to claim 1 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, beta-galactosidase enzymes chromogenic substrate is O-nitrophenyl-β-D-galactoside.
5. according to claim 4 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, the content of O-nitrophenyl-β-D-galactoside in 1000mL substratum is 2g.
6. according to any one of claim 1-5 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, the described 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.05-0.2g.
7. according to claim 6 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, the described 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.15g.
8. according to claim 6 for detecting Escherichia coli O 157: the color developing culture medium of H7, is characterized in that, the described 4-methyl fan ketone-β-content of D-Glucose thuja acid in 1000mL substratum is 0.10g.
9. according to claim 8 for detecting Escherichia coli O 157: the color developing culture medium of H7, it is characterized in that, every 1000mL substratum contains agar 15g, peptone 10g, yeast extract 5g, sodium-chlor 5g, sorbyl alcohol 15g, inositol 5g, the red 0.01g of property, O-nitrophenyl-β-D-galactoside 2g, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1g, isopropyl-beta D-thio galactopyranoside 0.1g, bovine bile 1.5g, potassium tellurite 2mg, Cefixime Micronized 0.04mg.
10. according to claim 8 for detecting Escherichia coli O 157: the color developing culture medium of H7, it is characterized in that, every 1000mL substratum contains agar 15g, peptone 10g, yeast extract 5g, sodium-chlor 5g, the chloro-3-indoles-beta galactose 0.1g of sorbyl alcohol 15g, inositol 5g, the bromo-4-of property red 0.01g, 5-, 4-methyl fan ketone-β-D-Glucose thuja acid 0.1g, isopropyl-beta D-thio galactopyranoside 0.1g, bovine bile 1.5g, potassium tellurite 2mg, Cefixime Micronized 0.04mg.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763709A (en) * | 2020-06-28 | 2020-10-13 | 浙江泰林生命科学有限公司 | Preparation method of coliform group detection reagent by enzyme substrate method |
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| CN1141344A (en) * | 1995-07-27 | 1997-01-29 | 南京铁道医学院 | Coliform bacteria membrane fluorescence method on-site rapid quantitative detection system |
| US20040166557A1 (en) * | 2001-06-20 | 2004-08-26 | Lawrence Restaino | Plasting media for the identification of Salmonella |
| 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 |
| CN103725746A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Coli group and colibacillus testing medium |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141344A (en) * | 1995-07-27 | 1997-01-29 | 南京铁道医学院 | Coliform bacteria membrane fluorescence method on-site rapid quantitative detection system |
| US20040166557A1 (en) * | 2001-06-20 | 2004-08-26 | Lawrence Restaino | Plasting media for the identification of Salmonella |
| 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 |
| CN103725746A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Coli group and colibacillus testing medium |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763709A (en) * | 2020-06-28 | 2020-10-13 | 浙江泰林生命科学有限公司 | Preparation method of coliform group detection reagent by enzyme substrate method |
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Application publication date: 20150304 |