CN104099283B - 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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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 microbial culture medium and in particular to a kind of temperature-sensitive hydrogel microbiological culture media and its
Preparation method.
Background technology
Since the forties in 20th century, about the physicochemical property of hydrogel, synthesis, in biochemistry, field of medical applications
Research very active (referring to document: Zhai Maolin, goes away for some great undertakings in Kazakhstan；The synthesis of hydrogel, property and application, [j]. university chemistry,
2001；16 (5): 22-27).Hydrogel swelling can keep large quantity of moisture and undissolved cross-linked polymer, according to network in water
Close strong difference and be divided into physical gel and chemical gel, different according to material are divided into chemical synthesis gel and natural polymer again
Gel.Natural polymer gel has good biocompatibility, environmentally sensitive property, abundant raw material and cheap price,
Thus just causing the attention of more and more scholars.The aquagel of the developments such as Xu Bin, can be used for engineered cardiac muscle
Timbering material (referring to document: Xu Bin, Duan Cuimi, Hao Tong, etc.；The development of aquagel and its with c2c12 cell adherence,
The experimental study of supporting function, [j]. biomedical engineering research, 2008；27 (2): 122-124).Shitosan and phosphoglycerol
Trisodium mixes according to a certain percentage, makes temperature-sensitive hydrogel, for tissue engineering bracket material (referring to document: Yan Jihong, grandson
Hai Mei, still grand, etc.；The preparation of the chitosan-based thermo-responsive hydro gel of syringeability and its biocompatibility, [j]. Jilin agricultural is big
Journal, 2011；33 (5): 522-526).The main application of hydrogel includes: commodity, as a kind of high absorbency material,
It is widely used in sanltary towel, children's diaper, spacetabs type essence, paper handkerchief etc..Industrial goods is mainly used in solvent dehydration, metal
Ion concentration, air filtration, wastewater treatment etc..Water conservation, sludge curing, cement additire, greening are mainly used on agriculture civil engineering
Desert etc..Burn coating, drug delivery, dentures material, transplanting, contact lenses, cell curing are mainly used on biomedicine
Deng.
Traditional solid and semi-solid microbiological culture media are to add coagulator, such as agar, gelatin, silica gel in the medium
Deng its cost of material is more expensive.Due to bacteria resistance function known to shitosan, the application as microbiological culture media is there is not yet report.
Content of the invention
The researcher of the present invention it was unexpectedly observed that when shitosan is used as temperature-sensitive hydrogel microbiological culture media, through culture
Colony counts and growth speed basically identical with conventional method cultivation results, and then have developed the chitosan-based temperature sensitive water of one kind
Gel microbiological culture media and its preparation technology.
For achieving the above object, the present invention includes following technical scheme:
A kind of temperature-sensitive hydrogel microbiological culture media, this culture medium is made up of following compositions: shitosan 1-5 weight portion, hands over
Connection agent 0-2 weight portion, temperature sensitive dose of 20-50 weight portion, microorganism specific chromogenic agent 0.2-0.4 weight portion, microorganism differentiates training
Foster base 100 weight portion and ph conditioning agent regulation ph value are 7.0-7.2.
Temperature-sensitive hydrogel microbiological culture media as above it is preferable that described shitosan be n- deacetylation 70～
90% Chitosan-phospholipid complex.
Temperature-sensitive hydrogel microbiological culture media as above is it is preferable that described crosslinking agent is selected from polyvinyl alcohol, glutaraldehyde
With at least one in isobutanol.
Temperature-sensitive hydrogel microbiological culture media as above is it is preferable that described temperature sensitive dose sweet selected from α sodium glycero-phosphate, β
At least one in oleophosphoric acid sodium and anhydrous glycerol sodium phosphate.
Temperature-sensitive hydrogel microbiological culture media as above is it is preferable that described microorganism specific chromogenic agent refers to for ttc
Show agent, iodine, triple sugariron, phosphomolybdic acid, bismuth phosphate potassium, methyl red, Coomassie brilliant blue g-250, aesculin, the bromo- 4- of 5- chloro- 3- Yin
The bromo- 4- of diindyl-inositol monophosphate, 5- chloro- 3- indoles-glucopyranoside acid, ortho-nitrophenol β-d glucuronidase, propylene second
The bromo- 4- of glycol, 5- chloro- 3- indoles-β-d noside acid, toluidine blue, methyl green, acridine orange and the bromo- 4- of 5- are chloro-
At least one in 3- indoles-thymidine -3- phosphoric acid.
Temperature-sensitive hydrogel microbiological culture media as above is it is preferable that described microorganism differential medium is nutrient meat
Soup nutrient solution, casein culture medium, gelatin culture medium, starch culture-medium, h2s test medium, sugared fermentation medium, remote Teng Shi training
Foster base or Yihong methylene blue culture medium.
Temperature-sensitive hydrogel microbiological culture media as above is it is preferable that described disodium hydrogen phosphate, potassium dihydrogen phosphate, chlorine
Change ammonium, sodium carbonate or sodium acid carbonate.
On the other hand, the present invention provides the preparation method of temperature-sensitive hydrogel microbiological culture media as above, the method
Comprise the steps:
A. weigh reagent according to composition described in claim 1 and proportioning, each reagent and container are carried out aseptic process；
B. shitosan is dissolved in microorganism differential medium；Add crosslinking agent toward in this culture medium at ambient temperature, continue
The lower cross-linking reaction 10-45 minute of stirring, forms clear liquid；
C. it is added dropwise over temperature sensitive dose toward in the liquid after cross-linking reaction under condition of ice bath, stirring 30-60min is extremely limpid molten
Liquid, adds microorganism specific chromogenic agent, stirring and dissolving, with ph conditioning agent regulation system ph value to 7.0-7.2.
Another further aspect, the present invention provides a kind of temperature-sensitive hydrogel microbiological culture media, and it is using method as defined above system
Standby.
The beneficial effects of the present invention is the following aspects: 1, temperature-sensitive hydrogel culture medium (0-30 under normal temperature condition
DEG C) it is liquid, under physiological temp or microorganism cultivation temperature (33-38 DEG C), 5-10min interior energy is frozen into solid, this characteristic
Culture bacterium in terms of be mainly used for isolating and purifying bacterium, bacterium solidification culture medium on grow bacterium colony, isolate and purify
When choose single bacterium colony.2nd, all through aseptic process before each component packing, can directly use for microorganism culture, need not
To sterilize again, easy to use.3rd, added with microorganism specific chromogenic agent, microorganism specific chromogenic in microbial cultivation process
Agent can make bacterium colony colour developing it is easy to distinguish bacterium colony and sample solid residue, convenient counting.4th, shitosan abundant raw material is easy to get, preparation
Hydrogel culture medium is nontoxic, good biocompatibility, can degrade.And the coagulator raw material price in short supply of conventional medium is high.
Specific embodiment
With reference to embodiment, the invention will be further described, and following illustrated embodiment is for ease of more fully understanding this
Invention, but be not used to limit the present invention.Experimental technique in following embodiments, if no special instructions, is conventional method.Under
State experiment material used in embodiment, if no special instructions, be and be commercially available from routine biochemistry reagent shop or manufacturer.
Reagent source used in following examples, experimental example is:
Shitosan, Jinan Haidebei Marine Organism Engineering Co., Ltd. produces, 60 mesh；
Sodium glycero-phosphate, AlfaAesar Tianjin Chemical Co., Ltd.；
Nutrient broth nutrient solution, Beijing overpass Technical responsibilities Co., Ltd；
Nutrient agar, Beijing overpass Technical responsibilities Co., Ltd, lot number 20121123.
Embodiment 1
2g water soluble chitosan, 45g β sodium glycero-phosphate, through co-60 radiation sterilizing；0.3g ttc is dissolved in 30ml distilled water
In, degerming through 0.45 μm of degerming membrane filtration, standby.
Take the nutrient broth nutrient solution through 121 DEG C of autoclaving 20min for the 100ml, first dissolve in 2g under room temperature condition water-soluble
Shitosan, then adds crosslinking agent polyvinyl alcohol 0.8g under the conditions of 25 DEG C toward in shitosan nutrient broth nutrient solution, continuously stirred
Become clear liquid, be added dropwise over glutaraldehyde 0.2ml, cross-linking reaction 10min, form clear liquid.Toward crosslinking under condition of ice bath
It is gradually added 45g β sodium glycero-phosphate in reacted liquid, 45min stirring while adding to clear solution, then plus 20ml ttc
Solution, stirring, with disodium hydrogen phosphate saturated solution regulation system ph value to 7.1 about, mix packing to sterile centrifugation tube, often
Pipe 20ml, temperature-sensitive hydrogel culture medium ().
Add 1ml during use according to a conventional method and treat culture sample in culture dish, take a pipe temperature-sensitive hydrogel culture medium, pour into
In culture dish, shake up, put in 37 DEG C of incubators culture 48h, have red colonies to manifest, you can count.
Embodiment 2
1.5g water soluble chitosan, 30g β sodium glycero-phosphate, through co-60 radiation sterilizing；0.3gttc is dissolved in 30ml distilled water
In, degerming through 0.45 μm of degerming membrane filtration, standby.
Take the nutrient broth nutrient solution through 121 DEG C of autoclaving 20min for the 100ml, first dissolve in 1.5g under room temperature condition water-soluble
Property shitosan, then under the conditions of 25 DEG C toward in shitosan nutrient broth nutrient solution plus crosslinking agent polyvinyl alcohol 0.4g, persistently stir
Mix clear liquid, cross-linking reaction 40min, form clear liquid.Under condition of ice bath toward in the liquid after cross-linking reaction progressively
Then plus 20ml bacterium indicator ttc solution add 30g β sodium glycero-phosphate, 45min stirring while adding to clear solution, stir
Mix, with disodium hydrogen phosphate saturated solution regulation system ph value to 7.1 about, mix packing to sterile centrifugation tube, often pipe 20ml,
Temperature-sensitive hydrogel culture medium (two).
Embodiment 3
4g water soluble chitosan, 50g β sodium glycero-phosphate, through co-60 radiation sterilizing；0.3gttc is dissolved in 30ml distilled water,
Degerming through 0.45 μm of degerming membrane filtration, standby.
Take the nutrient broth nutrient solution through 121 DEG C of autoclaving 20min for the 100ml, first dissolve in 4g under room temperature condition water-soluble
Shitosan, then adds crosslinking agent polyvinyl alcohol 1.4g under the conditions of 25 DEG C toward in shitosan nutrient broth nutrient solution, continuously stirred
Become clear liquid, be added dropwise over glutaraldehyde 0.6ml, cross-linking reaction 10min, form clear liquid.Toward crosslinking under condition of ice bath
It is gradually added 50g β sodium glycero-phosphate in reacted liquid, 45min stirring while adding to clear solution, then plus 20ml bacterium
Indicator ttc solution, stirring, with disodium hydrogen phosphate saturated solution regulation system ph value to 7.1 about, mix packing to aseptic from
In heart pipe, often pipe 20ml, temperature-sensitive hydrogel culture medium (three).
Experimental example 1
1. experimental technique
By salmonella atcc13311, streptococcus fecalis cmcc1.1030, pseudomonas aeruginosa atcc25619 and large intestine angstrom
The fresh broth culture of uncommon Salmonella atcc29522 carries out 10 times of gradient dilutions to 10-8, draw 1ml dilution respectively, add no
Bacterium plate, pours into the temperature-sensitive hydrogel culture medium of 20ml embodiment 1-3 preparation, mixes, and is just putting culture 24h～48h in 37 DEG C, is seeing
Examine result.Comparison culture is carried out with conventional nutrient agar medium simultaneously.
2. result of the test
Table 1
Table 2
Table 3
Table 4
3. experiment conclusion
The explanation of above experimental result, the temperature-sensitive hydrogel culture medium of embodiment 1-3 preparation to salmonella atcc13311,
The culture of streptococcus fecalis cmcc1.1030, pseudomonas aeruginosa atcc25619 and ETEC atcc29522, in bacterium colony
The speed of quantity and growth is basically identical with conventional medium cultivation results.
Experimental example 2
1. experimental technique
Draw 1ml Escherichia coli 8099 respectively, the fresh broth culture of staphylococcus aureus adds sterilized petri dishes, incline
The temperature-sensitive hydrogel culture medium of note 20ml embodiment 1-3 preparation, mixes, and is just putting culture 48h in 37 DEG C, is observing result.Simultaneously with
Conventional nutrient agar medium carries out comparison culture.
2. result of the test
Table 5
Table 6
Table 7
3. experiment conclusion
Above experimental result explanation, the temperature-sensitive hydrogel culture medium of embodiment 1-3 preparation is to Escherichia coli 8099, golden yellow
Staphylococcic culture, substantially suitable with conventional medium cultivation results in the speed of colony counts and growth.
Claims (8)
1. a kind of temperature-sensitive hydrogel microbiological culture media is it is characterised in that this culture medium is made up of following compositions: shitosan 1-5
Weight portion, crosslinking agent 0.4-2 weight portion, temperature sensitive dose of 20-50 weight portion, microorganism specific chromogenic agent 0.2-0.4 weight portion, micro-
Bioassay culture medium 100 weight portion and ph conditioning agent regulation ph value are 7.0-7.2；Wherein, temperature sensitive dose is selected from α phosphoglycerol
At least one in sodium, β sodium glycero-phosphate and anhydrous glycerol sodium phosphate.
2. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1 is it is characterised in that described shitosan is that n- is deacetylated
Spend the Chitosan-phospholipid complex 70～90%.
3. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1 is it is characterised in that described crosslinking agent is selected from polyethylene
At least one in alcohol, glutaraldehyde and isobutanol.
4. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1 is it is characterised in that described microorganism specific chromogenic
Agent is ttc indicator, iodine, triple sugariron, phosphomolybdic acid, bismuth phosphate potassium, methyl red, Coomassie brilliant blue g-250, aesculin, the bromo- 4- of 5-
Chloro- 3- indoles-inositol monophosphate, the bromo- 4- of 5- chloro- 3- indoles-glucopyranoside acid, ortho-nitrophenol β-d glucuronidase,
The bromo- 4- of propyleneglycoles, 5- chloro- 3- indoles-β-d noside acid, toluidine blue, methyl green, acridine orange and 5- are bromo-
At least one in 4- chloro- 3- indoles-thymidine -3- phosphoric acid.
5. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1 is it is characterised in that described microorganism differential medium
For nutrient broth nutrient solution, casein culture medium, gelatin culture medium, starch culture-medium, h2s test medium, sugared fermentation medium,
Endo's medium or Yihong methylene blue culture medium.
6. temperature-sensitive hydrogel microbiological culture media as claimed in claim 1 is it is characterised in that described ph conditioning agent is phosphoric acid hydrogen
Disodium, potassium dihydrogen phosphate, ammonium chloride, sodium carbonate or sodium acid carbonate.
7. the temperature-sensitive hydrogel microbiological culture media as any one of claim 1-6 preparation method it is characterised in that
The method comprises the steps:
A. weigh reagent according to composition described in claim 1 and proportioning, each reagent and container are carried out aseptic process；
B. shitosan is dissolved in microorganism differential medium；Crosslinking agent is added at ambient temperature toward in this culture medium, continuously stirred
Lower cross-linking reaction 10-45 minute, forms clear liquid；
C. it is added dropwise over temperature sensitive dose toward in the liquid after cross-linking reaction under condition of ice bath, 30-60min is to clear solution for stirring,
Add microorganism specific chromogenic agent, stirring and dissolving, with ph conditioning agent regulation system ph value to 7.0-7.2.
8. a kind of temperature-sensitive hydrogel microbiological culture media is it is characterised in that it is using the preparation of claim 7 methods described.
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| TWI673056B (en) * | 2016-07-22 | 2019-10-01 | 大江生醫股份有限公司 | Bacterium-containing hydrogel and method of making the same |
| CN107460230A (en) * | 2017-10-20 | 2017-12-12 | 河南科谱特医药科技研究院有限公司 | A kind of quick determination method of staphylococcus aureus |
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