CN104099283A - Temperature-sensitive hydrogel microorganism culture medium and preparation method thereof - Google Patents
Temperature-sensitive hydrogel microorganism culture medium and preparation method thereof Download PDFInfo
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- CN104099283A CN104099283A CN201410350404.3A CN201410350404A CN104099283A CN 104099283 A CN104099283 A CN 104099283A CN 201410350404 A CN201410350404 A CN 201410350404A CN 104099283 A CN104099283 A CN 104099283A
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 42
- 239000001963 growth medium Substances 0.000 title claims abstract description 33
- 244000005700 microbiome Species 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 229920001661 Chitosan Polymers 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 239000003002 pH adjusting agent Substances 0.000 claims abstract description 6
- 238000009629 microbiological culture Methods 0.000 claims description 24
- 235000015097 nutrients Nutrition 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 15
- 229960002901 sodium glycerophosphate Drugs 0.000 claims description 11
- REULQIKBNNDNDX-UHFFFAOYSA-M sodium;2,3-dihydroxypropyl hydrogen phosphate Chemical compound [Na+].OCC(O)COP(O)([O-])=O REULQIKBNNDNDX-UHFFFAOYSA-M 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
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- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical group [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 5
- 235000019800 disodium phosphate Nutrition 0.000 claims description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
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- 239000002253 acid Substances 0.000 claims description 4
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- 235000019322 gelatine Nutrition 0.000 claims description 3
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 claims description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 2
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- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 claims description 2
- QMIDDQFZXOSYIL-UHFFFAOYSA-K [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O.OCC(O)CO Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O.OCC(O)CO QMIDDQFZXOSYIL-UHFFFAOYSA-K 0.000 claims description 2
- VSTHNGLPHBTRMB-UHFFFAOYSA-N acridine orange Chemical compound [H+].[Cl-].C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 VSTHNGLPHBTRMB-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 239000005018 casein Substances 0.000 claims description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 238000003381 deacetylation reaction Methods 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 2
- DWCZIOOZPIDHAB-UHFFFAOYSA-L methyl green Chemical compound [Cl-].[Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)[N+](C)(C)C)=C1C=CC(=[N+](C)C)C=C1 DWCZIOOZPIDHAB-UHFFFAOYSA-L 0.000 claims description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 2
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 235000021317 phosphate Nutrition 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 2
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 235000013772 propylene glycol Nutrition 0.000 claims description 2
- 150000003214 pyranose derivatives Chemical class 0.000 claims description 2
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- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 claims description 2
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- 241000193830 Bacillus <bacterium> Species 0.000 description 2
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- MWPPHUFLMXDSDV-UHFFFAOYSA-N [Na].[Na].[Na].P(=O)(O)(O)OCC(O)CO Chemical compound [Na].[Na].[Na].P(=O)(O)(O)OCC(O)CO MWPPHUFLMXDSDV-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a temperature-sensitive hydrogel microorganism culture medium and a preparation method thereof. The culture medium is prepared from the components as follows: 1-5 parts of chitosan by weight, 0-2 parts of a cross-linking agent by weight, 20-50 parts of a temperature-sensitive agent by weight, 0.2-0.4 part of a microorganism specificity color developing agent by weight, 100 parts of a microorganism differential culture medium by weight and a pH adjusting agent for adjusting the pH value to be in a range of 7.0-7.2. The temperature-sensitive hydrogel culture medium is a liquid under the normal temperature condition (0-30 DEG C) and can be solidified into a solid within 5-10 min at the physiological temperature or the microorganism culture temperature (33-38 DEG C), and the microorganism specificity color developing agent is added in the culture medium and can enable a bacterial colony to develop the color during microorganism culture, so that the bacterial colony and sample solid residues are easy to differentiate, and counting is facilitated. The raw material, namely, the chitosan, is rich, easy to obtain and low in price, and the prepared hydrogel culture medium is nontoxic, good in biocompatibility and degradable.
Description
Technical field
The present invention relates to a kind of novel microorganism substratum, be specifically related to a kind of temperature-sensitive hydrogel microbiological culture media and preparation method thereof.
Background technology
Since the forties in 20th century, about the physico-chemical property of hydrogel, synthetic, in the research of biological chemistry, field of medical applications, very active (referring to document: Zhai Maolin, go away for some great undertakings in Kazakhstan; Synthetic, the character of hydrogel and application, [J]. university chemistry, 2001; 16 (5): 22-27).Hydrogel is the swelling maintenance large quantity of moisture of energy and undissolved cross-linked polymer in water, is divided into physical gel and chemical gel according to the strong difference of complexing, is divided into again chemosynthesis gel and natural polymer gel according to the difference of material.Natural polymer gel has good biocompatibility, to environmental sensitivity, abundant raw material and cheap price, thereby just causing more and more scholars' attention.The aquagel of Xu Bin etc. development, can be used for engineered cardiac muscle timbering material (referring to document: Xu Bin, Duan Cuimi, Hao Tong, etc.; The development of aquagel and with the experimental study of C2C12 cell adhesion, supporting function, [J]. biomedical engineering research, 2008; 27 (2): 122-124).Chitosan mixes according to a certain percentage with phospho-glycerol trisodium, makes temperature-sensitive hydrogel, for tissue engineering bracket material (referring to document: Yan Jihong, Sun Haimei, still grand, etc.; The preparation of the chitosan-based thermo-responsive hydro gel of syringeability and biocompatibility thereof, [J]. Jilin Agriculture University's journal, 2011; 33 (5): 522-526).The main application of hydrogel comprises: daily necessities, as a kind of high absorbency material, are widely used in sanitary napkin, children's diaper, slow release type essence, paper handkerchief etc.Industrial goods is mainly used in that solvent dehydration, metal ion concentrate, air filtration, wastewater treatment etc.In agricultural civil engineering, be mainly used in water conservation, sludge curing, cement additire, afforestation desert etc.In biomedicine, be mainly used in burn and be coated with application, drug delivery, dentures material, transplanting, contact lenses, cell curing etc.
Traditional solid and semi-solid microbiological culture media are in substratum, to add peptizer, and as agar, gelatin, silica gel etc., its cost of material is more expensive.Due to the known bacteria resistance function of chitosan, there is not yet report as the application of microbiological culture media.
Summary of the invention
Investigator of the present invention is surprised to find that, in the time that chitosan is used as temperature-sensitive hydrogel microbiological culture media, colony counts through cultivating and speed and the ordinary method cultivation results of growth are basically identical, and then have researched and developed a kind of chitosan-based temperature-sensitive hydrogel microbiological culture media and preparation technology thereof.
For achieving the above object, the present invention includes following technical scheme:
A kind of temperature-sensitive hydrogel microbiological culture media, this substratum is made up of following compositions: chitosan 1-5 weight part, linking agent 0-2 weight part, temperature sensitive dose of 20-50 weight part, microorganism specificity developer 0.2-0.4 weight part, it is 7.0-7.2 that microorganism differential medium 100 weight parts and pH adjusting agent regulate pH value.
Temperature-sensitive hydrogel microbiological culture media as above, preferably, described chitosan is N-deacetylation at 70～90% chitosan and derivative thereof.
Temperature-sensitive hydrogel microbiological culture media as above, preferably, described linking agent is selected from least one in polyvinyl alcohol, glutaraldehyde and isopropylcarbinol.
Temperature-sensitive hydrogel microbiological culture media as above, preferably, described temperature sensitive dose of at least one being selected from α Sodium Glycerophosphate, β Sodium Glycerophosphate and anhydrous glycerol sodium phosphate.
Temperature-sensitive hydrogel microbiological culture media as above, preferably, described microorganism specificity developer is at least one in TTC indicator, iodine, triple sugariron, phospho-molybdic acid, bismuth phosphate potassium, methyl red, Xylene Brilliant Cyanine G G-250, polychrom, the chloro-3-indoles-inositol monophosphate of the bromo-4-of 5-, the chloro-3-indoles-glucose of the bromo-4-of 5-pyranose thuja acid, o-nitrophenol β-D glucuronidase, propyleneglycoles, the chloro-3-indoles-β-D of the bromo-4-of 5-semi-lactosi pyranose thuja acid, toluidine blue, methyl green, Sumitomo Acridine Orange RK conc and the chloro-3-indoles-thymidine-3-of the bromo-4-of 5-phosphoric acid.
Temperature-sensitive hydrogel microbiological culture media as above, preferably, described microorganism differential medium is nutrient broth nutrient solution, casein substratum, gelatine culture, starch culture-medium, H2S test medium, sugar-fermenting substratum, Endo's medium or Yihong methylene blue substratum.
Temperature-sensitive hydrogel microbiological culture media as above, preferably, described Sodium phosphate dibasic, potassium primary phosphate, ammonium chloride, sodium carbonate or sodium bicarbonate.
On the other hand, the invention provides the preparation method of temperature-sensitive hydrogel microbiological culture media as above, the method comprises the steps:
A. take reagent according to composition described in claim 1 and proportioning, each reagent and container are carried out to aseptically process;
B. chitosan is dissolved in microorganism differential medium; In this substratum, add linking agent at ambient temperature, continue to stir lower crosslinking reaction 10-45 minute, form limpid liquid;
C. under condition of ice bath, in the liquid after crosslinking reaction, dropwise add temperature sensitive dose, stir 30-60min to clear solution, add microorganism specificity developer, stirring and dissolving, by pH adjusting agent regulation system pH value to 7.0-7.2.
On the one hand, the invention provides a kind of temperature-sensitive hydrogel microbiological culture media again, it adopts method described above to prepare.
Beneficial effect of the present invention is the following aspects: 1, temperature-sensitive hydrogel substratum (0-30 DEG C) under normal temperature condition is liquid, at physiological temp or microorganism culturing temperature (33-38 DEG C), in 5-10min, can be frozen into solid, the main application of this characteristic aspect culturing bacterium is separation and purification bacterium, bacterium grows bacterium colony on curing substratum, chooses single bacterium colony when separation and purification.2, before each component packing all through aseptically process, can directly use for microorganism culturing, sterilizing again, easy to use.3, be added with microorganism specificity developer, in microbial cultivation process, microorganism specificity developer can make bacterium colony colour developing, is easy to distinguish bacterium colony and sample solid residue, convenient counting.4, chitosan abundant raw material is easy to get, and the hydrogel substratum of preparation is nontoxic, good biocompatibility, can degrade.And the peptizer raw material price in short supply of traditional substratum is high.
Embodiment
Below in conjunction with embodiment, the invention will be further described, below illustrated embodiment be for ease of understanding better the present invention, but be not used for limiting the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Experiment material used in following embodiment, if no special instructions, is from routine biochemistry reagent shop or manufacturer buys and obtains.
The reagent source using in following examples, experimental example is:
Chitosan, Jinan Haidebei Marine Organism Engineering Co., Ltd. produces, 60 orders;
Sodium Glycerophosphate, A Faaisha Tianjin Chemical Co., Ltd.;
Nutrient broth nutrient solution, Beijing overpass Technical responsibilities company limited;
Nutrient agar, Beijing overpass Technical responsibilities company limited, lot number 20121123.
Embodiment 1
2g water-soluble chitosan, 45g β Sodium Glycerophosphate, through co-60 radiation sterilizing; 0.3g TTC is dissolved in 30ml distilled water, through 0.45 μ m degerming membrane filtration degerming, for subsequent use.
Get the nutrient broth nutrient solution of 100ml through 121 DEG C of autoclaving 20min, under room temperature condition, first dissolve in 2g water-soluble chitosan, then under 25 DEG C of conditions, in chitosan nutrient broth nutrient solution, add linking agent polyvinyl alcohol 0.8g, continue to stir into limpid liquid, dropwise add glutaraldehyde 0.2ml, crosslinking reaction 10min, forms limpid liquid.Under condition of ice bath, in the liquid after crosslinking reaction, progressively add 45g β Sodium Glycerophosphate, limit edged stirs 45min to clear solution, then add 20ml TTC solution, stir, by Sodium phosphate dibasic saturated solution regulation system pH value to 7.1 left and right, mix and point be filled in aseptic centrifuge tube, every pipe 20ml, temperature-sensitive hydrogel substratum ().
When use, in culture dish, add according to a conventional method 1ml and treat culture sample, get a pipe temperature-sensitive hydrogel substratum, pour in culture dish, shake up, put in 37 DEG C of incubators and cultivate 48h left and right, have red bacterium colony to manifest, i.e. count enable.
Embodiment 2
1.5g water-soluble chitosan, 30g β Sodium Glycerophosphate, through co-60 radiation sterilizing; 0.3gTTC is dissolved in 30ml distilled water, through 0.45 μ m degerming membrane filtration degerming, for subsequent use.
Get the nutrient broth nutrient solution of 100ml through 121 DEG C of autoclaving 20min, under room temperature condition, first dissolve in 1.5g water-soluble chitosan, then under 25 DEG C of conditions, in chitosan nutrient broth nutrient solution, add linking agent polyvinyl alcohol 0.4g, continue to stir into limpid liquid, crosslinking reaction 40min, forms limpid liquid.Under condition of ice bath, in the liquid after crosslinking reaction, progressively add 30g β Sodium Glycerophosphate, limit edged stirs 45min to clear solution, then add 20ml bacterium indicator TTC solution, stir, by Sodium phosphate dibasic saturated solution regulation system pH value to 7.1 left and right, mix and point be filled in aseptic centrifuge tube, every pipe 20ml, temperature-sensitive hydrogel substratum (two).
Embodiment 3
4g water-soluble chitosan, 50g β Sodium Glycerophosphate, through co-60 radiation sterilizing; 0.3gTTC is dissolved in 30ml distilled water, through 0.45 μ m degerming membrane filtration degerming, for subsequent use.
Get the nutrient broth nutrient solution of 100ml through 121 DEG C of autoclaving 20min, under room temperature condition, first dissolve in 4g water-soluble chitosan, then under 25 DEG C of conditions, in chitosan nutrient broth nutrient solution, add linking agent polyvinyl alcohol 1.4g, continue to stir into limpid liquid, dropwise add glutaraldehyde 0.6ml, crosslinking reaction 10min, forms limpid liquid.Under condition of ice bath, in the liquid after crosslinking reaction, progressively add 50g β Sodium Glycerophosphate, limit edged stirs 45min to clear solution, then add 20ml bacterium indicator TTC solution, stir, by Sodium phosphate dibasic saturated solution regulation system pH value to 7.1 left and right, mix and point be filled in aseptic centrifuge tube, every pipe 20ml, temperature-sensitive hydrogel substratum (three).
Experimental example 1
1. experimental technique
The fresh broth culture of Salmonellas ATCC13311, streptococcus faecium CMCC1.1030, Pseudomonas aeruginosa ATCC25619 and colon bacillus ATCC29522 is carried out to 10 times of gradient dilutions to 10
-8, draw respectively 1mL diluent, add aseptic plate, pour into temperature-sensitive hydrogel substratum prepared by 20mL embodiment 1-3, mix, just putting and cultivating 24h～48h, observations in 37 DEG C.Carry out contrast culture with conventional nutrient agar simultaneously.
2. test-results
Table 1
Table 2
Table 3
Table 4
3. experiment conclusion
Above experimental result explanation, temperature-sensitive hydrogel substratum prepared by the embodiment 1-3 cultivation to Salmonellas ATCC13311, streptococcus faecium CMCC1.1030, Pseudomonas aeruginosa ATCC25619 and colon bacillus ATCC29522, basically identical in speed and the conventional medium cultivation results of colony counts and growth.
Experimental example 2
1. experimental technique
The fresh broth culture of drawing respectively 1mL intestinal bacteria 8099, streptococcus aureus adds aseptic plate, pours into temperature-sensitive hydrogel substratum prepared by 20mL embodiment 1-3, mixes, and is just putting and is cultivating 48h, observations in 37 DEG C.Carry out contrast culture with conventional nutrient agar simultaneously.
2. test-results
Table 5
Table 6
Table 7
3. experiment conclusion
Above experimental result explanation, temperature-sensitive hydrogel substratum prepared by the embodiment 1-3 cultivation to intestinal bacteria 8099, streptococcus aureus, substantially suitable in speed and the conventional medium cultivation results of colony counts and growth.
Claims (9)
1. a temperature-sensitive hydrogel microbiological culture media, it is characterized in that, this substratum is made up of following compositions: chitosan 1-5 weight part, linking agent 0-2 weight part, temperature sensitive dose of 20-50 weight part, microorganism specificity developer 0.2-0.4 weight part, it is 7.0-7.2 that microorganism differential medium 100 weight parts and pH adjusting agent regulate pH value.
2. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1, is characterized in that, described chitosan is N-deacetylation at 70～90% chitosan and derivative thereof.
3. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1, is characterized in that, described linking agent is selected from least one in polyvinyl alcohol, glutaraldehyde and isopropylcarbinol.
4. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1, is characterized in that, described temperature sensitive dose of at least one being selected from α Sodium Glycerophosphate, β Sodium Glycerophosphate and anhydrous glycerol sodium phosphate.
5. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1, it is characterized in that, described microorganism specificity developer is TTC indicator, iodine, triple sugariron, phospho-molybdic acid, bismuth phosphate potassium, methyl red, Xylene Brilliant Cyanine G G-250, polychrom, the chloro-3-indoles-inositol monophosphate of the bromo-4-of 5-, the chloro-3-indoles-glucose of the bromo-4-of 5-pyranose thuja acid, o-nitrophenol β-D glucuronidase, propyleneglycoles, the chloro-3-indoles-β-D of the bromo-4-of 5-semi-lactosi pyranose thuja acid, toluidine blue, methyl green, at least one in the chloro-3-indoles-thymidine-3-of the bromo-4-of Sumitomo Acridine Orange RK conc and 5-phosphoric acid.
6. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1, it is characterized in that, described microorganism differential medium is nutrient broth nutrient solution, casein substratum, gelatine culture, starch culture-medium, H2S test medium, sugar-fermenting substratum, Endo's medium or Yihong methylene blue substratum.
7. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1, is characterized in that, described pH adjusting agent is Sodium phosphate dibasic, potassium primary phosphate, ammonium chloride, sodium carbonate or sodium bicarbonate.
8. the preparation method of the temperature-sensitive hydrogel microbiological culture media as described in any one in claim 1-7, is characterized in that, the method comprises the steps:
A. take reagent according to composition described in claim 1 and proportioning, each reagent and container are carried out to aseptically process;
B. chitosan is dissolved in microorganism differential medium; In this substratum, add linking agent at ambient temperature, continue to stir lower crosslinking reaction 10-45 minute, form limpid liquid;
C. under condition of ice bath, in the liquid after crosslinking reaction, dropwise add temperature sensitive dose, stir 30-60min to clear solution, add microorganism specificity developer, stirring and dissolving, by pH adjusting agent regulation system pH value to 7.0-7.2.
9. a temperature-sensitive hydrogel microbiological culture media, is characterized in that, it adopts method described in claim 8 to prepare.
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