CN102286605A - General culture medium for detecting Gram negative bacteria - Google Patents
General culture medium for detecting Gram negative bacteria Download PDFInfo
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- CN102286605A CN102286605A CN2011102229793A CN201110222979A CN102286605A CN 102286605 A CN102286605 A CN 102286605A CN 2011102229793 A CN2011102229793 A CN 2011102229793A CN 201110222979 A CN201110222979 A CN 201110222979A CN 102286605 A CN102286605 A CN 102286605A
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Abstract
The invention discloses a general culture medium for detecting Gram negative bacteria. The general culture medium comprises 5.0 to 7.0g of peptone, 2.5 to 3.0g of yeast extract, 1.0g of glucose, 0 to 5.0g of sodium chloride, 15.0g of agar, 1,000mL of distilled water and 0.5 to 1.0mg/L vancomycin hydrochloride. The culture medium has high selectivity, can specifically selectively culture the Gram negative bacteria, and is easy and convenient to prepare; vancomycin is singly used as a selective agent; the simplest formula can be formed by adding 0.5 to 1.0mg/L vancomycin hydrochloride into a plate count agar; the general culture medium is friendly to human bodies and environment, and the use concentration of the vancomycin is not more than 1.0mg/L; and the working efficiency is improved, and the Gram negative bacteria can be directly counted.
Description
Technical field
The invention belongs to the general detection substratum of Gram-negative bacteria field.
Background technology
Exist the microorganism of many monoids in the culture environment of aquatic products, these microorganisms interact in water body, and growth and decline each other affect the variation of water quality, are determining the success or failure of culturing; Therefore, the dynamic change of microbe groups, kind and quantity in the research aquaculture water has crucial meaning to instructing aquifer cultivation, protection environmental ecology.
Microorganism in the culture environment of aquatic products mainly is a bacterium, bacterium is divided into Gram-negative and Gram-positive two big classes again, this two big bacterioid is because the difference of cell walls structure, disinfecting drug there is different susceptibility, therefore, understand the variation of bacteria flora in the water body fast, can instruct water body correctly to sterilize.On the other hand, because rapid detection is passed through in the popularization day by day of bioremediation technology; may find that negative bacterium and positive bacteria in the water body are in a benign equilibrium state; thereby not needing blindly to go to implement water body disinfection, this realizes that to the protection environmental ecology healthy aquaculture is equally very important.
Differentiate the flora kind, need suitable medium, but the collective media that does not also have Gram-negative bacteria to detect at present, it is the substratum of fungistat that the substratum that Gram-negative bacteria that can be for reference detects has with the cholate, as violet red bile agar (VRBA) (seeing GB 4789.3-2010 food safety national standard food microbiological analysis coliform counting), Viola crystallina neutrality is through cholate agar glucose (VRBGA) (seeing Enterobacter sakazakii method of inspection part 1 in the SN 1632.1-2005 milk powder: separate and method of counting); With the vancomycin is the substratum of fungistat, as improve lauryl sulfate tryptose broth-vancomycin (Modified lauryl sulfate tryptose broth-vancomycine medium, mLST-Vm) (see the check of GB 4789.40-2010 food safety national standard food microbiological analysis Enterobacter sakazakii), the disclosed selective isolation medium for enterobacter sakazakii of Chinese patent " selective isolation medium for enterobacter sakazakii " (patent No. 200710075355.7).
Cholate (Bile salt) is to be combined with glycine or taurine and the sodium salt or the sylvite that form by liver cell excretory bile acide, can suppress gram-positive microorganism, and the consumption of cholate is 1.5g/L in this bile salt culture-medium; Test finds that the cholate under this concentration has certain restraining effect (biocidal property 20%) to Gram-negative bacteria, and concentration is high more, and restraining effect is strong more; Cholate under this concentration has stronger restraining effect to gram-positive microorganism; But studies show that, some Bacterium lacticum (is seen " the acidproof and cholate capability study of lactobacillus rhamnosus " Food science, 2008,29 (12), 449-451) and faecalis (see " 2 strain pig sources benefits natural disposition faecalis is to acid and cholate and hot tolerance studies " Beijing Agricultural College's journal, 2008,23 (4), 29-32) can tolerate the cholate of higher concentration, tolerance level reaches 0.5% (being 5g/L) and 0.4% (being 4g/L) respectively.Therefore, improper with cholate as the selectivity of the general detection substratum of Gram-negative bacteria.
Vancomycin is the glycopeptide antibiotics that is produced by a kind of streptomycete, and vancomycin hydrochloride is its hydrochloride, and gram-positive microorganism is had stronger germicidal action, and is invalid to Gram-negative bacteria, is a kind of ideal Gram-negative bacteria selective agent therefore.Detect the Gram-negative bacteria of new research and development such as Enterobacter sakazakii and also to use the vancomycin agent that elects in the substratum, but the working concentration of vancomycin is higher, concentration as vancomycin among the mLST is 10mg/L, the concentration of vancomycin also has 3.0-5.0mg/L in the selective isolation medium for enterobacter sakazakii, and will be used in combination with cefoxitin (Cephalothin) simultaneously.Test finds that when vancomycin concentration is 2.0mg/L, Gram-negative bacteria is also had certain restraining effect (biocidal property 10%), concentration is high more, and restraining effect is big more.In addition, because vancomycin has ototoxicity and certain renal toxicity, and drug toxicity is bigger, therefore, should control on working concentration, makes and can be effective, again to the human and environment close friend.
Summary of the invention
The technical problem to be solved in the present invention be seek a kind of selectivity height, preparation easy, to the general detection substratum of human and environment close friend's Gram-negative bacteria, be used for studying and monitoring the dynamic change of aquaculture system Gram-negative bacteria.
The present invention solves the problems of the technologies described above with following technical scheme: a kind of Gram-negative bacteria detects collective media, is made by the following weight proportion raw material: peptone 5.0g～7.0g, yeast extract 2.5g～3.0g, glucose 1.0g, sodium-chlor 0g～5.0g, vancomycin hydrochloride 0.5mg～1.0mg, agar 15.0g, distilled water 1000mL, pH 7.0 ± 0.2.
Outstanding advantage of the present invention is: 1, selectivity height: can single-mindedly optionally cultivate Gram-negative bacteria.2, preparation is easy: use vancomycin as selective agent separately, do not need to be used in combination with other any microbiotic; Simple formulation even as long as in plate count agar, add the vancomycin hydrochloride of 0.5mg/L～1.0mg/L.3, to the human and environment close friend: the working concentration of vancomycin is no more than 1.0mg/L, is significantly less than the used 10.0mg/L of reference standard, and is very friendly to human and environment.4, increase work efficiency: need not choosing colony and carry out dyeing microscopic examination, but direct census Gram-negative bacteria bacterium colony.
Embodiment
Following examples are to further describe of the present invention, but the present invention is not limited to following examples.
Embodiment 1
The prescription of embodiment 1 substratum is as shown in table 1.
Peptone 5.0g, yeast extract 2.5g, glucose 1.0g, agar 15.0g are added in the 1000mL distilled water, boil dissolving, regulate pH to 7.0 ± 0.2, divide to be filled to Erlenmeyer flask, every bottle of 200mL, 121 ℃ of autoclaving 15min get basic medium.Accurately take by weighing the 10.0mg vancomycin hydrochloride, be dissolved in (amounting to concentration is 100mg/L) in the 100mL distilled water, the vancomycin hydrochloride storage liquid, the packing test tube, it is standby to put-20 ℃ of preservations.
Getting the 1mL bacteria concentration is 10
3The intestinal bacteria of CFU/mL, salmonella, shigella, proteus vulgaris, Proteus mirabilis, citrobacter freundii, Bacterium lacticum, faecium, bacillus licheniformis, streptococcus aureus add dull and stereotyped each 4 ware (totally 40 wares) of 9cm, pour into the basic medium that is chilled to 45 ℃ after the heating to 2 wares wherein, every ware 15mL, cultivate 24h for 36 ℃, counting, in contrast; To be chilled to 50 ℃ after the basic medium 200mL heating for dissolving in addition, add 1mL vancomycin hydrochloride storage liquid, shake up, making its final concentration is 0.5mg/L, the every ware of other 2 wares pours into 15mL, cultivates 24h for 36 ℃, counting, compared with the control, calculate growth rate, being growth more than 95%, be expressed as "+" with growth rate, with growth rate is 0% for not growing, and is expressed as "-".The gained result is as implementing shown in 1 data in the table 2.
As can be seen from the table, intestinal bacteria, salmonella, shigella, proteus vulgaris, Proteus mirabilis, these Gram-negative bacterias of citrobacter freundii are all grown, and Bacterium lacticum, faecium, bacillus licheniformis, these gram-positive microorganisms of streptococcus aureus are not all grown.
Embodiment 2
Repeat the method for embodiment 1, just the basic medium composition is different, shown in embodiment in the table 12.Basic medium is made up of peptone 7.0g, yeast extract 3.0g, glucose 1.0g, sodium-chlor 5.0g, agar 15.0g.The final concentration of vancomycin hydrochloride still is 0.5mg/L.Cultivation results is shown in 2 data of embodiment in the table 2.The result is identical with embodiment 1, and Gram-negative bacteria is all grown, and gram-positive microorganism is not all grown.
Embodiment 3
Repeat the method for embodiment 1, just the concentration of basic medium composition and vancomycin hydrochloride is all different, shown in embodiment in the table 13.Basic medium is made up of peptone 5.0g, yeast extract 3.0g, glucose 1.0g, sodium-chlor 5.0g, agar 15.0g.The addition of vancomycin hydrochloride storage liquid is 2mL, and its final concentration is 1.0mg/L.Cultivation results is shown in 3 data of embodiment in the table 2.The result is identical with embodiment 1, and Gram-negative bacteria is all grown, and gram-positive microorganism is not all grown.
Table 1 Gram-negative bacteria detects the collective media prescription
The general detection substratum of table 2 Gram-negative bacteria detected result
The a:1-intestinal bacteria; The 2-salmonella; The 3-shigella; The 4-proteus vulgaris; The 5-Proteus mirabilis; The 6-citrobacter freundii; The 7-Bacterium lacticum; The 8-faecium; The 9-bacillus licheniformis; The 10-streptococcus aureus
B:+ represents that bacterial growth is arranged, and growth rate is more than 95%.
C:-represents not have bacterial growth, and growth rate is 0%.
Claims (2)
1. general detection substratum of Gram-negative bacteria is characterized in that described substratum is made up of basic medium and vancomycin hydrochloride, and concrete component is:
2. the general detection substratum of Gram-negative bacteria according to claim 1, the pH value that it is characterized in that the general detection substratum of described Gram-negative bacteria is 7.0 ± 0.2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| US6037140A (en) * | 1995-10-13 | 2000-03-14 | Pasteur Sanofi Diagnostics | Selective media for the culture and isolation of gram bacteria, antibiotic composition |
| CN101481663A (en) * | 2008-01-09 | 2009-07-15 | 天津市金章科技发展有限公司 | One-time selective XV agar medium and preparation thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6037140A (en) * | 1995-10-13 | 2000-03-14 | Pasteur Sanofi Diagnostics | Selective media for the culture and isolation of gram bacteria, antibiotic composition |
| CN101481663A (en) * | 2008-01-09 | 2009-07-15 | 天津市金章科技发展有限公司 | One-time selective XV agar medium and preparation thereof |
Non-Patent Citations (3)
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| 杨万颖等: "新型阪崎肠杆菌选择性分离培养基的研制", 《食品工业科技》 * |
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Cited By (1)
| 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 |
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