CN104313122A - Toluidine blue-DNase agar culture medium and application thereof - Google Patents
Toluidine blue-DNase agar culture medium and application thereof Download PDFInfo
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- CN104313122A CN104313122A CN201410505132.XA CN201410505132A CN104313122A CN 104313122 A CN104313122 A CN 104313122A CN 201410505132 A CN201410505132 A CN 201410505132A CN 104313122 A CN104313122 A CN 104313122A
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- toluidine blue
- culture medium
- agar culture
- sodium chloride
- dnase
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Abstract
The invention relates to the field of microorganisms and in particular relates to a toluidine blue-DNase agar culture medium. The toluidine blue-DNase agar culture medium comprises the following components: 15.5g/L of 7.5% sodium chloride broth, 0.5g/L of DNA, 1.5g/L of calcium chloride, 6.3g/L of trihydroxymethyl aminomethane and 0.088g/L of toluidine blue, and pH value of the toluidine blue-DNase agar culture medium is 8.9-9.1. According to the toluidine blue-DNase agar culture medium, one litre of 7.5% sodium chloride broth contains the following components: 10.0g of tryptone, 5.0g of beef powder, 75.0g of sodium chloride and 2.0g of dipotassium phosphate. The toluidine blue-DNase agar culture medium is simple in composition, easy to prepare and operate, low in cost and beneficial to popularization and application; bacteria grow quickly, and mutation rate is low; and observation and analysis are facilitated.
Description
Technical field
The present invention is microorganism field, particularly a kind of toluidine blue-DNA enzymatic nutrient agar and application thereof.
Background technology
Streptococcus aureus, also claims " S. aureus L-forms ", and cell walls is containing the peptidoglycan of 90% and the teichoic acid of 10%.The reticulated structure of its peptidoglycan is finer and close than Gram-negative bacteria, do not decoloured by alcohol so present purple after Viola crystallina attachment during dyeing, on the contrary, the whole cell peptidoglycan layer of negative bacterium is thin, degree of crosslinking is poor, lipid content is high, so purple mixture is washed out by alcohol then attached to husky yellow redness.Streptococcus aureus and penicillin find that there is very large origin.Fleming is exactly in the culture dish of his streptococcus aureus, find that some coccus has been killed then, so found penicillin.And research also shows that penicillin is only obvious to the gram-positive microorganism effect taking streptococcus aureus as representative.This is also caused by the thickness of peptidoglycan layer and structure.Emerging methicillin-resistant staphylococcus aureus, is referred to as superbacteria, almost can resist all medicines of the mankind, but vancomycin can tackle it.Typical streptococcus aureus is ball-type, and diameter about 0.8 μm, is arranged in thyrsiform under microscope.
Streptococcus aureus is without brood cell, flagellum, most of without pod membrane, Gram-positive.Streptococcus aureus nutritional requirement is not high, well-grown on ordinary culture medium, aerobic or amphimicrobian, optimum growth temperature 37 DEG C, and the most suitable growth pH7.4 can viable for weeks under dry environment.Thick, glossy, the circular protrusions of bacterium colony on flat board, diameter 0.5 ~ 1.0mm.Blood agar periphery of bacterial colonies forms transparent zone of hemolysis.Streptococcus aureus has the salt tolerance of height, can grow in 10 ~ 15%NaCl meat soup.Decomposable asymmetric choice net glucose, maltose, lactose, sucrose, produce acid not aerogenesis.Clark and Lubsreaction is positive, and VP reacts the weak positive.Many bacterial strain decomposable asymmetric choice net arginine, hydrolyze urea, reduction nitrate, liquefy gelatin.Streptococcus aureus has stronger resistibility, to antibiotic sensitive such as sulfa drugs, penicillin, erythromycin, terramycin, Liu Suanyan NEOMYCIN SULPHATEs, but easily produces resistance, and reason is because these bacterial strains produce penicillinase etc.Responsive to basic dyestuff, 100,000/ Viola crystallina liquid get final product Developing restraint.
Substratum (Medium) is the nutriment of the artificial preparation growing for microorganism, plant tissue and animal tissues and maintain, and generally all contains carbohydrate, nitrogenous substances, inorganic salt (comprising trace element) and VITAMIN and water etc.Some substratum also contain antibiotic and pigment, for single microorganism culturing and qualification.
Substratum (Medium) is for microorganism, plant and animal tissue growth and the nutriment of artificial preparation that maintains, generally all contains carbohydrate, nitrogenous substances, inorganic salt (comprising trace element) and growth hormone and water etc.Some substratum are also containing antibiotic, pigment, hormone and serum.
Substratum is due to the raw material difference of preparation, and service requirements is different, and storage and custody aspect is also slightly different.General substratum, being heated, after the moisture absorption, being easily contaminated by bacterial or decomposing rotten, therefore general substratum must protection against the tide, lucifuge, the preservation of shady and cool place.Some are needed to the substratum (as tissue culture medium (TCM)) of stringent sterilization, the storage of long period, must be placed in the refrigerator of 3 ~ 6 DEG C.Because liquid nutrient medium is not easily taken care of for a long time, all change system into powder.
Can't infer from the ultimate principle of biochemical reaction completely and calculate the culture medium prescription of applicable a certain bacterial classification at present, the basic theories of biological chemistry, cytobiology, microbiology etc. can only be used, with reference to the empirical formula being comparatively applicable to a certain class bacterial classification that forefathers use, again in conjunction with the characteristic of bacterial classification used and product, adopt the small-sized fermentation equipment such as shaking flask, glass pot, select the substratum be comparatively applicable to according to certain experimental design and experimental technique.
The basic step of substratum design is:
Some problems that must consider when determining according to the experience of forefathers and medium component, tentatively determine by single factor experiment, possible medium component finally determines that medium component the most suitable is after medium component is determined, remaining issues is exactly the suitableeest concentration of each composition, because medium component is a lot, often adopt some rational experimental design methods for reducing experiment number.These experiments, often based on factorial experiment, comprise homogeneous design, orthogonal, response surface analysis etc.
Summary of the invention
The present invention overcomes deficiency of the prior art, provides a kind of toluidine blue-DNA enzymatic nutrient agar and application thereof.
The present invention uses biological chemistry, the principle of microbiology and inorganic, organic, analytical chemistry and fluorescence technique, row filter is combined into all class nutrition compositions such as different carbon sources, nitrogenous source, inorganic salts, VITAMIN, growth stimulants, the materialization factor such as pH value, ionic strength, oxidation-reduction potential, surface tension, atmosphere surrounding of substratum is compared, investigated substratum of the present invention.
The technical solution adopted in the present invention is:
A kind of toluidine blue-DNA enzymatic nutrient agar, the component of described substratum comprises 15.5g/L7.5% sodium chloride broth, 0.5g/LDNA, 1.5g/L calcium chloride, 6.3g/L Tutofusin tris, 0.088g/L toluidine blue, and pH value is 8.9-9.1.
On the basis of above scheme, described toluidine blue-DNA enzymatic nutrient agar, described 7.5% sodium chloride broth is containing Tryptones 10.0g, beef powder 5.0g, sodium-chlor 75.0g, dipotassium hydrogen phosphate 2.0g in often liter of 7.5% sodium chloride broth.
The invention also discloses the application of described toluidine blue-DNA enzymatic nutrient agar, detect for rnase.
There is the thymus nucleic acid (DNA) in the bacterial degradation substratum of deoxyribonuclease, make DNA long-chain be hydrolyzed into the oligonucleotide short chain of several mononucleotide, make indicator toluidine blue effect pulverize red; Calcium chloride provides positively charged ion as activator; Sodium-chlor maintains balanced osmotic pressure; Tutofusin tris is buffer reagent; Agar is the peptizer of substratum.
Take this product 27.58g, heating for dissolving in 1000ml distilled water, packing triangular flask.
Sterilizing can not be needed as used immediately.If do not used immediately, 121 DEG C of autoclavings 15 minutes.
Sterilized substratum room temperature can deposit 4 months unchanged, and still can to use after for several times fusing.
Beneficial effect of the present invention:
1, substratum prescription is simple, and preparation manipulation is convenient, and cost is low, is beneficial to and applies;
2, bacteria growing is rapid, and aberration rate is low;
3, be convenient to observe, analyze.
Embodiment
Embodiment 1:
A kind of toluidine blue-DNA enzymatic nutrient agar, the component of described substratum comprises 15.5g/L7.5% sodium chloride broth, 0.5g/LDNA, 1.5g/L calcium chloride, 6.3g/L Tutofusin tris, 0.088g/L toluidine blue, and pH value is 8.9-9.1.
Described toluidine blue-DNA enzymatic nutrient agar, described 7.5% sodium chloride broth is containing Tryptones 10.0g, beef powder 5.0g, sodium-chlor 75.0g, dipotassium hydrogen phosphate 2.0g in often liter of 7.5% sodium chloride broth.
There is the thymus nucleic acid (DNA) in the bacterial degradation substratum of deoxyribonuclease, make DNA long-chain be hydrolyzed into the oligonucleotide short chain of several mononucleotide, make indicator toluidine blue effect pulverize red; Calcium chloride provides positively charged ion as activator; Sodium-chlor maintains balanced osmotic pressure; Tutofusin tris is buffer reagent; Agar is the peptizer of substratum.
Take this product 27.58g, heating for dissolving in 1000ml distilled water, packing triangular flask.
Sterilizing can not be needed as used immediately.If do not used immediately, 121 DEG C of autoclavings 15 minutes.
Sterilized substratum room temperature can deposit 4 months unchanged, and still can to use after for several times fusing.
35 DEG C of cultivation 4h results are as follows:
| Bacterium name | Cultural characteristic |
| Streptococcus aureus | Pink chromosphere |
| Staphylococcus epidermidis | Without pink chromosphere |
Claims (3)
1. toluidine blue-DNA enzymatic nutrient agar, it is characterized in that the component of described substratum comprises 15.5g/L7.5% sodium chloride broth, 0.5g/LDNA, 1.5g/L calcium chloride, 6.3g/L Tutofusin tris, 0.088g/L toluidine blue, pH value is 8.9-9.1.
2. toluidine blue according to claim 1-DNA enzymatic nutrient agar, is characterized in that described 7.5% sodium chloride broth is for containing Tryptones 10.0g, beef powder 5.0g, sodium-chlor 75.0g, dipotassium hydrogen phosphate 2.0g in often liter of 7.5% sodium chloride broth.
3. an application for toluidine blue according to claim 1-DNA enzymatic nutrient agar, is characterized in that detecting for rnase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410505132.XA CN104313122A (en) | 2014-09-28 | 2014-09-28 | Toluidine blue-DNase agar culture medium and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410505132.XA CN104313122A (en) | 2014-09-28 | 2014-09-28 | Toluidine blue-DNase agar culture medium and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497915A (en) * | 2008-12-30 | 2009-08-05 | 广东省微生物研究所 | Reagent and method for fast detecting Staphylococcus aureus |
| CN104099283A (en) * | 2014-07-22 | 2014-10-15 | 张维 | Temperature-sensitive hydrogel microorganism culture medium and preparation method thereof |
| CN104651477A (en) * | 2013-11-25 | 2015-05-27 | 刘汉斌 | Toluidine blue-DNA enzyme agar medium and use thereof |
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2014
- 2014-09-28 CN CN201410505132.XA patent/CN104313122A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497915A (en) * | 2008-12-30 | 2009-08-05 | 广东省微生物研究所 | Reagent and method for fast detecting Staphylococcus aureus |
| CN104651477A (en) * | 2013-11-25 | 2015-05-27 | 刘汉斌 | Toluidine blue-DNA enzyme agar medium and use thereof |
| CN104099283A (en) * | 2014-07-22 | 2014-10-15 | 张维 | Temperature-sensitive hydrogel microorganism culture medium and preparation method thereof |
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Application publication date: 20150128 |