CN102660459A - Anaerobic culture box and using method thereof - Google Patents
Anaerobic culture box and using method thereof Download PDFInfo
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- CN102660459A CN102660459A CN2012101464413A CN201210146441A CN102660459A CN 102660459 A CN102660459 A CN 102660459A CN 2012101464413 A CN2012101464413 A CN 2012101464413A CN 201210146441 A CN201210146441 A CN 201210146441A CN 102660459 A CN102660459 A CN 102660459A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/04—Bioreactors or fermenters specially adapted for specific uses for producing gas, e.g. biogas
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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Abstract
The invention discloses an anaerobic culture box and a using method thereof and belongs to a medical experimental device used for culturing animal cells in an anaerobic environment; and according to the anaerobic culture box, the oxygen in the cultivation environment is rapidly removed, and the pressures inside and outside the box can be maintained to be balanced. The anaerobic culture box is composed of three parts, namely a sealed box cover (1), a box body (2) and an alkaline pyrogallic acid solution bag (3), wherein a pressure buffer air bag (12) is arranged at the inner side of the sealed box cover; and the wall of the box body (2) is provided with a liquid adding valve (23), a valve and channel (24) for adding and taking a culture medium, and a liquid adding coil pipe (25). The method comprises the following steps of: preparing the alkaline pyrogallic acid solution in a sealed plastic bag; removing oxygen, and then adding a citric acid solution, so as to form a buffer solution; and adding carbon dioxide gas, and maintaining cell or bacterial metabolism demand and the pH value in the cultivation environment. The anaerobic culture box disclosed by the invention has the advantages of realizing rapid anaerobic speed, counting the anaerobic time, being simple in structure and operation, affordable and small in volume, and realizing mass production.
Description
Technical field
The invention belongs to medicine technology field, relate to a kind of medical experiment equipment and method of use thereof, be specifically related to a kind of anoxic and cultivate box and method of use thereof.
Background technology
Histanoxia is clinical common pathological phenomenon, is the common factor that causes cell injury in the multiple disease.For the cell of research vitro culture to the reaction of oxygen free condition; The various countries scientist takes multiple mode to set up cell anaerobic culture condition; Typical several kinds of anoxia model are following: have the investigator in conventional incubator, to charge into nitrogen replacement oxygen, and detect oxygen concentration in the incubator; Have the investigator in encloses container, to put into cell, carry out vacuum take-off after, charge into 95%N
2+ 5%CO
2
Some shortcoming below above design of the prior art exists: 1, nitrogen replacement oxygen is insufficient; Need the long period just can reach the anaerobic requirement; The switch cell culture incubator needs the long period to reach oxygen free condition again in addition, and required time can not be held the difficult accurately calculating anoxic time; 2, vacuumize at encloses container, but the negative pressure pair cell causes damage in the container, influences experimental result; 3, required equipment is expensive, and complex design is not easy to operate.
The oxygen free condition of setting up culturing cell quickly and easily helps the laboratory accurately to study pathologic-physiological reaction and the molecular mechanism thereof of cell to anaerobic, and existing condition can't satisfy this requirement.
Summary of the invention
In order to solve the problems of the technologies described above, the present invention provides a kind of anoxic to cultivate box and method of use thereof, and anoxic of the present invention is cultivated box can reach anoxia condition fast; And can satisfy oxygen free condition for a long time, and the simple easy handling of the present invention, cost is low; But mass production is reusable, can sterilize.
Concrete technical scheme is:
A kind of anoxic is cultivated box; Comprise sealing lid 1, box body 2 and alkaline pyrogallol solution bag 3; It is characterized in that: sealing lid 1 inboard is provided with pressure buffer air bag 12, and air bag communicates with the external world through gas valve 11, and box body 2 bottoms are provided with cultivates vessel support 25; Box body 2 walls are provided with liquid feeding valve 23, add and get substratum valve and passage 24, liquid feeding dish intestines 25, and solution join anoxic through transfusion device defeated 32 and liquid feeding valve 23 and cultivate in the box in the alkaline pyrogallol solution bag 3.
Be provided with pressure buffer air bag 12 in sealing lid 1 inboard and communicate with the external world, come the balance anoxic to cultivate the pressure in box and the external world, and said pressure buffer capsule 12 is detachable through air bag gas valve 11.
Said liquid feeding dish intestines 25 have the flushing liquor aperture.
Said box body 2 walls are provided with to add gets nutrient solution valve and passage 24.
Alkalescence pyrogallol solution bag 3 is vacuum seal bag or medical 250ml infusion bag of abandoning, and behind distilled water flushing, is evacuated with syringe.Its method of use characteristic is: earlier with the 18.87g pyrogallol 100ml of being made into solution soluble in water; Join in the vacuum seal bag with syringe, again that 17.96g sodium hydroxide is soluble in water, be made into 50ml solution; Join in the vacuum seal bag with syringe; Mixing places 25 ℃ of cleansing bath tubs, uses after the preheating.
A kind of anoxic is cultivated the method for use of box; May further comprise the steps: in adding alkaline pyrogallol solution removal box behind the oxygen; And in the anoxic box, add citric acid solution through liquid feeding valve 23 with syringe, make Hydrocerol A, sodium hydroxide and pyrogallol constitute damping fluid.Its proportioning characteristic is: if experimental design need not add carbonic acid gas, not adding citric acid; If experiment needs to remove too much carbon dioxide accumulation, but adding citric acid 24g is dissolved in the solution that is made in the 50ml water; Need not remove the carbonic acid gas of accumulating like experiment, but the solution that is made in the adding citric acid 28g solution 50ml water.
Behind adding citric acid solution, if the invention designing institute uses sodium hydroxide to be analytical pure, contain yellow soda ash 1.5%, then need not add carbonic acid gas, it is required that the carbonic acid gas that is generated can satisfy cell cultures; If used sodium hydroxide purity is higher, then need further to add dioxide gas 60ml.
Anoxic is cultivated the box principle of work: alkaline pyrogallol can react with oxygen fast, absorbs airborne oxygen, reaches and removes the effect of cultivating the interior oxygen of box, and do not contact with substratum.
3NaOH+C
6H
3(OH)
3→C
6H
3(ONa)
3+H
2O
4C
6H
3(ONa)
3+O
2→2(KO)
3C
6H
2-H
2C
6(OK)
3+2H
2O
Anoxic is cultivated the box working process: prepare oxygen uptake liquid in advance, sodium hydroxide 17.96g is dissolved in the 40ml water, and pyrogallol 18.87g is dissolved in the 80ml water, successively is injected in the vacuum-packed plastics bag, and mixing places 25 ℃ of water-bath preheatings.Culturing bottle or culture plate are placed on the anoxic box inner support, cover lid, open anoxic box breather valve; In the pressure buffer bag, inject the 100ml air with the 50ml syringe, close breather valve, the oxygen uptake liquid bag for preparing is connected in the liquid feeding valve with transfusion device; Open the liquid feeding valve; Open pressure buffer bag gas valve, use transfusion device that the oxygen uptake liquid in the oxygen uptake liquid bag is joined anoxic through the liquid feeding valve and cultivate in the box, close the liquid feeding valve.Anoxic is cultivated box place on the shaking table, shook 5 minutes, oxygen concn can be lower than 0.6% in the culture environment at this moment.Hypoxia culture box is placed 37 ℃ of environment, after half a hour.Add citric acid solution (preheating temperature is 20 ℃) 50ml with the 50ml syringe through liquid filling hole and be dissolved with the 24g Hydrocerol A.Make it to constitute damping fluid, pH value is between 7-8, in order in the buffer pocket and the carbonic acid gas of too much accumulating.Can not consider without adding citric acid if need not add carbonic acid gas in the test,, can add Hydrocerol A 28g if need not remove the carbonic acid gas of accumulating in the experiment.The shaking table concussion, 1 minute.Add dioxide gas 60ml (need not add the experiment of carbonic acid gas, this step can save) through gas valve.Place 37 ℃ to cultivate hypoxia culture box.
Beneficial effect of the present invention: anoxic of the present invention is cultivated box can reach anoxia condition fast, and can satisfy oxygen free condition for a long time, and the simple easy handling of the present invention, and cost is low, but mass production is reusable, can sterilize.
Description of drawings
Fig. 1 cultivates the structural diagrams intention of box for anoxic of the present invention.
Embodiment
Below in conjunction with the accompanying drawing embodiment method of the present invention is done explanation in further detail.
With reference to Fig. 1, a kind of anoxic is cultivated box and is processed by the transparent box plastics, and the box internal volume is 1.4 liters; Comprise sealing lid 1, box body 2 and alkaline pyrogallol solution bag 3, sealing lid 1 inboard is provided with pressure buffer air bag 12, and the sealing lid is fixed on the box body through the snap close 13 that is connected with box body; The sealing lid is connected the side that places seal box lid 1 in snap close 13 designs with box body; Being provided with draw-in groove can interlock with the draw-in groove on box body 2 sidewalls, and sealing lid 1 periphery has and box body connecting groove 14, and groove is equipped with rubber weather strip 15; Pressure buffer air bag 12 has sealed plastic bag to process, and has to connect to emit with pressure buffer air bag gas valve 11 to link to each other; Box body 2 walls are provided with gas valve 21, liquid taking valve door and passage 22, liquid feeding valve 23, add and get substratum valve and passage 24, liquid feeding dish enteron aisle 26; The liquid 27 that joins in the box is to join the interior alkaline pyrogallol solution of box and the mixing liquid of citric acid solution through liquid feeding valve 23 and liquid feeding dish intestines 26; Add and get the substratum passage, get the liquid passage and liquid feeding dish enteron aisle has plastic hose to process; Evenly have the about 1mm aperture of diameter on the liquid feeding dish enteron aisle 26, Open Side Down for aperture, sticks at box body 2 inner side-walls under the dish enteron aisle screw; The box body bottom is provided with cultivates vessel support 25, and the petridish support has plastics to process and welding and box body 2 bottoms; The sealing bag that alkalescence pyrogallol solution bag 3 has plastics to process, volume 250ml, an end is provided with soft rubber ball; Add and get reagent and use injection to realize; Or use medical depleted infusion bag to replace plastic sealing bag, add zero(ppm) water with syringe before using and wash, and extract the inside air out.
During use, prepare alkaline pyrogallol solution 31 in advance, sodium hydroxide 17.96g is dissolved in the 40ml water, and pyrogallol 18.87g is dissolved in the 80ml water, successively is injected in the alkaline pyrogallol solution bag 3, and mixing places 25 ℃ of water-bath preheatings.Culturing bottle, culture plate or petridish are placed on the anoxic box inner support 25; Cover sealing lid 1, open anoxic box breather valve 21, in pressure buffer capsule 12, inject the 100ml air through pressure buffer air bag gas valve 11 with the 50ml syringe; Close breather valve 11; The alkaline pyrogallol solution bag 3 that will prepare with transfusion device is connected in liquid feeding valve 23, opens liquid feeding valve 23, opens pressure buffer capsule gas valve 11; Use transfusion device that alkaline pyrogallol solution 31 is joined anoxic through the liquid feeding valve and cultivate in the box, close liquid feeding valve 23.Anoxic is cultivated box place on the shaking table, shook 5 minutes.Hypoxia culture box is placed 37 ℃ of environment, after half a hour.Add citric acid solution 50ml (20 ℃ of preheating temperatures) with the 50ml syringe through liquid filling hole and be dissolved with the 24g Hydrocerol A.Can not consider without adding citric acid if need not add carbonic acid gas in the test,, can add Hydrocerol A 28g if need not remove the carbonic acid gas of accumulating in the experiment.The shaking table concussion, 1 minute.Add dioxide gas 60ml (if need not add the experiment of carbonic acid gas, this step can save) through gas valve.Place 37 ℃ to cultivate hypoxia culture box.In culturing process, can get substratum valve and passage 24 and under the condition that guarantees anaerobic, add and get substratum, the line correlation of going forward side by side experiment through adding.
The above; Be merely the preferable embodiment of the present invention; Protection scope of the present invention is not limited thereto; Any technician who is familiar with the present technique field is in the technical scope that the present invention discloses, and the simple change of the technical scheme that obtains or equivalence replacement all fall in protection scope of the present invention with may be obvious that.
Claims (7)
1. an anoxic is cultivated box; It is characterized in that; Comprise sealing lid (1), box body (2) and alkaline pyrogallol solution bag (3); It is characterized in that: sealing lid (1) inboard is provided with pressure buffer air bag (12), and air bag communicates with the external world through gas valve (11), and box body (2) bottom is provided with cultivates vessel supports (25); Box body (2) wall is provided with liquid feeding valve (23), add and get substratum valve and passage (24), liquid feeding dish intestines (25), and the interior solution of alkaline pyrogallol solution bag (3) joins anoxic through transfusion device defeated (32) and liquid feeding valve (23) and cultivates in the box.
2. anoxic according to claim 1 is cultivated box, it is characterized in that, is provided with pressure buffer air bag (12) in sealing lid (1) inboard and communicates with the external world through air bag gas valve (11), and said pressure buffer capsule (12) is detachable.
3. anoxic according to claim 1 is cultivated box, it is characterized in that said liquid feeding dish intestines (25) have the flushing liquor aperture.
4. anoxic according to claim 1 is cultivated box, it is characterized in that said box body (2) wall is provided with to add gets nutrient solution valve and passage (24).
5. anoxic according to claim 1 is cultivated box, it is characterized in that alkaline pyrogallol solution bag (3) is vacuum seal bag or medical 250ml infusion bag of abandoning, and behind distilled water flushing, is evacuated with syringe.
6. anoxic according to claim 1 is cultivated the method for use of box; It is characterized in that; May further comprise the steps: in adding alkaline pyrogallol solution removal box behind the oxygen; And in the anoxic box, add citric acid solution through liquid feeding valve (23) with syringe, make Hydrocerol A, sodium hydroxide and pyrogallol constitute damping fluid.
7. anoxic according to claim 6 is cultivated the method for use of box, it is characterized in that, behind adding citric acid solution, adds dioxide gas 60ml through liquid feeding valve (23).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101464413A CN102660459A (en) | 2012-05-14 | 2012-05-14 | Anaerobic culture box and using method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101464413A CN102660459A (en) | 2012-05-14 | 2012-05-14 | Anaerobic culture box and using method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102660459A true CN102660459A (en) | 2012-09-12 |
Family
ID=46770041
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|---|---|---|---|
| CN2012101464413A Pending CN102660459A (en) | 2012-05-14 | 2012-05-14 | Anaerobic culture box and using method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694389A (en) * | 2013-12-04 | 2015-06-10 | 中国科学院大连化学物理研究所 | Cell oxygen-deficient environment system constructed based on micro-numerical control living cell culture chamber |
| CN107523496A (en) * | 2017-10-18 | 2017-12-29 | 安徽理工大学 | A kind of installation for fermenting of comprehensive environmental experiment |
| CN110106082A (en) * | 2019-05-28 | 2019-08-09 | 中南大学湘雅医院 | Embedded cell hypoxia culture box |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1278531A (en) * | 1969-06-03 | 1972-06-21 | Pfizer | Process and culturing device with means for controlling atmosphere |
| CN2441809Y (en) * | 2000-09-05 | 2001-08-08 | 阎侗有 | Micro anaerobic culture device |
| CN101260366A (en) * | 2008-04-15 | 2008-09-10 | 湖南师范大学 | Gas concentration and gas pressure adjustable cell culture case |
| CN101397546A (en) * | 2008-10-17 | 2009-04-01 | 杭州百思生物技术有限公司 | Quick-speed generation method of anaerobic microorganism culture environment |
| CN201797949U (en) * | 2010-05-26 | 2011-04-20 | 刘明璇 | Air pressure balanced type pickle jar |
| CN102061258A (en) * | 2010-11-11 | 2011-05-18 | 天津开发区合普工贸有限公司 | Cell exposed anaerobic environment experiment device |
-
2012
- 2012-05-14 CN CN2012101464413A patent/CN102660459A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1278531A (en) * | 1969-06-03 | 1972-06-21 | Pfizer | Process and culturing device with means for controlling atmosphere |
| CN2441809Y (en) * | 2000-09-05 | 2001-08-08 | 阎侗有 | Micro anaerobic culture device |
| CN101260366A (en) * | 2008-04-15 | 2008-09-10 | 湖南师范大学 | Gas concentration and gas pressure adjustable cell culture case |
| CN101397546A (en) * | 2008-10-17 | 2009-04-01 | 杭州百思生物技术有限公司 | Quick-speed generation method of anaerobic microorganism culture environment |
| CN201797949U (en) * | 2010-05-26 | 2011-04-20 | 刘明璇 | Air pressure balanced type pickle jar |
| CN102061258A (en) * | 2010-11-11 | 2011-05-18 | 天津开发区合普工贸有限公司 | Cell exposed anaerobic environment experiment device |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694389A (en) * | 2013-12-04 | 2015-06-10 | 中国科学院大连化学物理研究所 | Cell oxygen-deficient environment system constructed based on micro-numerical control living cell culture chamber |
| CN107523496A (en) * | 2017-10-18 | 2017-12-29 | 安徽理工大学 | A kind of installation for fermenting of comprehensive environmental experiment |
| CN110106082A (en) * | 2019-05-28 | 2019-08-09 | 中南大学湘雅医院 | Embedded cell hypoxia culture box |
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Application publication date: 20120912 |