CN115232713A - Portable laboratory simple and easy device of oxygen in getting rid of culture medium - Google Patents
Portable laboratory simple and easy device of oxygen in getting rid of culture medium Download PDFInfo
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- CN115232713A CN115232713A CN202211069882.8A CN202211069882A CN115232713A CN 115232713 A CN115232713 A CN 115232713A CN 202211069882 A CN202211069882 A CN 202211069882A CN 115232713 A CN115232713 A CN 115232713A
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- 239000001963 growth medium Substances 0.000 title claims abstract description 128
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 239000001301 oxygen Substances 0.000 title claims abstract description 39
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 39
- 238000009630 liquid culture Methods 0.000 claims abstract description 81
- 238000003860 storage Methods 0.000 claims abstract description 69
- 239000007789 gas Substances 0.000 claims abstract description 58
- 229910000831 Steel Inorganic materials 0.000 claims abstract description 13
- 239000010959 steel Substances 0.000 claims abstract description 13
- 238000007789 sealing Methods 0.000 claims description 79
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 3
- 239000001569 carbon dioxide Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 description 23
- 238000000034 method Methods 0.000 description 17
- 239000002609 medium Substances 0.000 description 14
- 244000005700 microbiome Species 0.000 description 12
- 238000005273 aeration Methods 0.000 description 6
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 5
- 229960002433 cysteine Drugs 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 4
- 238000005086 pumping Methods 0.000 description 3
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 229940123973 Oxygen scavenger Drugs 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 238000011177 media preparation Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004173 biogeochemical cycle Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 229940099259 vaseline Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
- C12M29/22—Oxygen discharge
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a portable laboratory simple device for removing oxygen in a culture medium, which comprises a gas conveying pipe with a partial pressure valve, wherein the gas conveying pipe is connected to the outlet at the top of an oxygen-free gas steel cylinder; the top of the first liquid culture medium storage bottle is provided with a first bottle opening and a second bottle opening, the first conveying pipe, the first exhaust pipe and the second conveying pipe penetrate through the first bottle opening and are in sealed connection with the first bottle opening, and the third conveying pipe penetrates through the second bottle opening and is in sealed connection with the second bottle opening; the top of the second liquid culture medium storage bottle is provided with a third bottle opening and a fourth bottle opening, a fourth conveying pipe, a second exhaust pipe and a fifth conveying pipe penetrate through the third bottle opening and are in sealed connection with the third bottle opening, the fourth conveying pipe is connected with the second conveying pipe, the fifth conveying pipe is communicated with the third conveying pipe, and a sixth conveying pipe is in sealed connection with the fourth bottle opening; the first conveying pipe is communicated with the first exhaust pipe through a first branch pipe. The device has small volume, simple operation and low cost.
Description
Technical Field
The invention relates to the technical field of microorganism experimental equipment, in particular to a portable simple laboratory device for removing oxygen in a culture medium.
Background
The anaerobic microorganisms are widely distributed in natural environment, including water, soil, sediments and the like. The anaerobic microorganisms are various in types, and commonly comprise denitrifying bacteria, zymophyte, methanogen and the like, which not only drive the biogeochemical cycle of carbon and nitrogen elements in the environment, but also are closely related to the generation of greenhouse gases in the atmosphere, so that the preparation of an anaerobic microorganism liquid culture medium is the key for culturing the anaerobic microorganisms.
In the past, a liquid culture medium for anaerobic microorganisms has been generally prepared by a meat-in-place culture method, pyrogallic acid method, vacuum-pumping method, anaerobic jar culture method, and anaerobic glove box method.
The principle of the medium method of meat preparation: unsaturated fatty acid, sulfydryl and other reducing substances contained in meat residues in the culture medium can absorb oxygen in the culture medium to reduce the redox potential, and a layer of sterile vaseline or liquid paraffin is covered on the liquid surface to isolate air, so that good anaerobic conditions are formed to meet the requirement of anaerobic bacteria growth. However, this method is applicable only to the culture of specific anaerobic microorganisms.
The theory of pyrogallic acid method: then pyrogallic acid is added into alkaline solution to rapidly absorb a large amount of oxygen to generate dark brown pyrogallic orange, thereby effectively absorbing oxygen in any closed container to form conditions favorable for anaerobic bacteria growth. But it will produce a small amount of carbon monoxide during the oxidation process and will poison the microorganisms to some extent, so that the growth of some anaerobic bacteria sensitive to CO will be inhibited.
The principle of the vacuum pumping method is as follows: the preparation of the anaerobic culture medium is completed by discharging the culture medium and the gas in the headspace of the container through a vacuum pumping system to realize the replacement of oxygen and other anaerobic bacteria. However, this method is time-consuming and it is difficult to remove oxygen from the liquid medium to the maximum extent.
Principle of anaerobic jar method: the oxygen in the closed container is removed by the oxygen-scavenging particles to provide an anaerobic environment for the growth of anaerobic microorganisms. But the anaerobic environment of culture medium preparation and microorganism inoculation links can not be realized, and only the anaerobic environment in the culture process can be ensured.
Principle of anaerobic glove box method: the anaerobic glove box with good sealing performance is made of transparent hard plastics, and is externally connected with an anaerobic gas cylinder, and the inside of the box is kept in an anaerobic state by a gas extraction and exchange method. In the whole process, the culture is not contacted with the air through the operation and treatment of the rubber gloves on the box body in the box, so that the anaerobic environment is always kept. However, the apparatus is expensive, large in size and not suitable for culturing extreme anaerobic microorganisms (such as seabed strains and deep soil strains).
Disclosure of Invention
The invention aims to overcome the problems in the prior art, and provides a portable laboratory simple device for removing oxygen in a culture medium, which does not depend on an anaerobic glove box and a vacuum pump with high price, has small volume, simple operation and low price, and can be freely operated, and has very important significance.
The invention provides a portable laboratory simple device for removing oxygen in a culture medium, which comprises: the top outlet of the oxygen-free gas steel cylinder is connected with a gas pipe with a partial pressure valve; the top of the first liquid culture medium storage bottle is provided with a first bottle opening and a second bottle opening, a first conveying pipe with a first valve, a first exhaust pipe with a second valve and a third valve and a second conveying pipe with a fourth valve all penetrate through the first bottle opening and are in sealed connection with the first bottle opening, the top end of the first conveying pipe is communicated with the gas conveying pipe, the bottom ends of the first conveying pipe and the second conveying pipe are immersed in the liquid culture medium, a third conveying pipe with a fifth valve penetrates through the second bottle opening and is in sealed connection with the second bottle opening, the top end of the third conveying pipe is located above the second bottle opening, and the bottom end of the third conveying pipe is immersed in the liquid culture medium; a third bottle opening and a fourth bottle opening are formed in the top of the second liquid culture medium storage bottle, a fourth conveying pipe with a sixth valve, a second exhaust pipe with a seventh valve and a fifth conveying pipe with an eighth valve penetrate through the third bottle opening and are in sealed connection with the third bottle opening, the top end of the fourth conveying pipe above the third bottle opening is connected with the top end of the second conveying pipe, the top end of the fifth conveying pipe is communicated with the top end of the third conveying pipe, the bottom ends of the fourth conveying pipe and the fifth conveying pipe are immersed in the liquid culture medium, and the sixth conveying pipe with a ninth valve is in sealed connection with the fourth bottle opening; the first conveying pipe is communicated with a first exhaust pipe between the second valve and the third valve through a first branch pipe with a tenth valve.
Preferably, the second delivery pipe is communicated with the first delivery pipe through a second branch pipe with an eleventh valve, and the fourth delivery pipe is communicated with a second exhaust pipe positioned below the seventh valve through a third branch pipe with a twelfth valve.
Preferably, the first bottle opening is detachably connected with a first sealing cover, the first sealing cover is respectively connected with the first conveying pipe, the first exhaust pipe and the second conveying pipe in a sealing manner, the second bottle opening is detachably connected with a second sealing cover, the second sealing cover is connected with the third conveying pipe in a sealing manner, the third bottle opening is detachably connected with a third sealing cover, the third sealing cover is respectively connected with the fourth conveying pipe, the second exhaust pipe and the fifth conveying pipe in a sealing manner, the fourth bottle opening is detachably connected with a fourth sealing cover, and the fourth sealing cover is connected with the sixth conveying pipe in a sealing manner.
Preferably, the first sealing cover and the third sealing cover both include a first cover body and three pagoda-shaped sealing pipes arranged at the bottom of the first cover body and connected with the first cover body in a sealing manner, the first conveying pipe, the first exhaust pipe and the second conveying pipe or the fourth conveying pipe, the second exhaust pipe and the fifth conveying pipe respectively penetrate through the three pagoda-shaped sealing pipes, the second sealing cover and the fourth sealing cover both include a second cover body and a pagoda-shaped connecting pipe arranged at the bottom of the second cover body and connected with the second cover body in a sealing manner, and the third conveying pipe or the sixth conveying pipe respectively penetrates through the connecting pipe.
Preferably, the bottoms of the first sealing cover, the second sealing cover, the third sealing cover and the fourth sealing cover are all provided with O-shaped sealing rings.
Preferably, the volume fraction ratio of nitrogen to carbon dioxide in the oxygen-free gas cylinder is 80.
Preferably, the pressure of the pressure division valve is 0.1-0.2MPa.
Preferably, first liquid culture medium storage bottle and second liquid culture medium storage bottle are made by the glass material, the gas-supply pipe is stereoplasm PU pipe, first conveyer pipe, first blast pipe, second conveyer pipe, third conveyer pipe, second blast pipe, fourth conveyer pipe, first minute pipe, second minute pipe, third minute pipe are the silica gel hose, pagoda shaped sealing tube and pagoda shaped connecting pipe are made by stainless steel material.
Preferably, the first conveying pipe and the gas conveying pipe, the fourth conveying pipe and the second conveying pipe, and the fifth conveying pipe and the third conveying pipe are detachably connected.
Preferably, the first liquid culture medium storage bottle has a volume of 1L-5L, and the second liquid culture medium storage bottle has a volume of 50mL-200mL.
Compared with the prior art, the invention has the beneficial effects that:
1. the aeration oxygen removal device can realize aeration oxygen removal of the liquid culture medium in the first liquid culture medium storage bottle. The method comprises the following specific operations: open first valve, second valve and third valve for anaerobic gas passes through during gas-supply pipe and first conveyer pipe get into the liquid culture medium of first liquid culture medium storage bottle, thereby will exist originally in the anaerobic gas that oxygen in liquid culture medium and headspace was input and pass through first exhaust pipe release, finally reach the purpose of culture medium aeration deoxidization.
2. The invention can realize aeration, oxygen removal and anaerobic split charging of the liquid culture medium in the second liquid culture medium storage bottle. The method comprises the following specific operations: opening a tenth valve, a third valve, a fourth valve, a sixth valve and a seventh valve; the anaerobic gas enters the headspace of the liquid in the first liquid culture medium storage bottle through the gas transmission pipe, the first conveying pipe, the first branch pipe and the first exhaust pipe; allowing the culture medium in the first liquid culture medium storage bottle to flow into the second liquid culture medium storage bottle through the second delivery pipe and the fourth delivery pipe under the pressure of the gas; along with the volume of the culture medium in the second liquid culture medium storage bottle is gradually increased, the original oxygen in the second liquid culture medium storage bottle is discharged through the second exhaust pipe. When the volume of the culture medium in the second liquid culture medium storage bottle reaches the maximum, opening an eleventh valve, a twelfth valve, an eighth valve, a fifth valve, a second valve and a third valve on a fifth conveying pipe, and allowing the oxygen-free gas to enter the headspace of the second liquid culture medium storage bottle through a gas conveying pipe, a first conveying pipe, a second branch pipe, a second conveying pipe, a fourth conveying pipe, a third branch pipe and a second exhaust pipe; under the pressure of gas, the liquid culture medium in the first liquid culture medium storage bottle flows back into the first liquid culture medium storage bottle through the fifth conveying pipe and the third conveying pipe, and the gas in the headspace of the first liquid culture medium storage bottle is exhausted through the first exhaust pipe along with the pressure of the backflow liquid; and when the volume of the culture medium in the second liquid culture medium storage bottle reaches the target scale, completing the anaerobic subpackaging of the culture medium.
3. The anaerobic microorganism culture medium preparation device provided by the invention does not depend on an anaerobic glove box and a vacuum pump which are high in price, and the whole device is small in size, simple to operate, low in price and free to operate; in addition, the device can be combined with a cysteine deoxidant to prepare a strict anaerobic culture medium.
Drawings
FIG. 1 is a schematic view of the overall structure of the present invention;
FIG. 2 is a schematic view of the structure of a first liquid medium storage bottle according to the present invention;
FIG. 3 is a schematic view of a second liquid medium storage bottle according to the present invention;
FIG. 4 is a schematic view of the structure of a first and third seal cap of the present invention (wherein a is a bottom view; b is a longitudinal sectional view);
fig. 5 is a schematic structural view (wherein a is a bottom view and b is a longitudinal sectional view) of a second sealing cap and a fourth sealing cap according to the present invention.
Description of reference numerals:
1. the oxygen-free gas storage device comprises an oxygen-free gas steel cylinder, 2. A gas conveying pipe, 3. A first liquid culture medium storage bottle, 4. A first conveying pipe, 5. A first exhaust pipe, 6. A second conveying pipe, 7. A third conveying pipe, 8. A second liquid culture medium storage bottle, 9. A fourth conveying pipe, 10. A second exhaust pipe, 11. A fifth conveying pipe, 12. A sixth conveying pipe, 13. A first branch pipe, 14. A second branch pipe, 15. A third branch pipe, 16. A first cover body, 17. A sealing pipe, 18. A second cover body, 19. A connecting pipe and 20. An O-shaped sealing ring.
Detailed Description
Detailed description of the preferred embodimentsthe following detailed description of the present invention will be made with reference to the accompanying drawings 1-5, although it should be understood that the scope of the present invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Examples
This embodiment provides a portable laboratory-friendly device for removing oxygen from a culture medium, which achieves the objects of the present invention through essential technical features.
Specifically, as shown in fig. 1 to 5, the present invention provides a portable laboratory simple device for removing oxygen from a culture medium, comprising: the anaerobic culture medium system comprises an anaerobic gas steel cylinder 1, a first liquid culture medium storage bottle 3 and a second liquid culture medium storage bottle 8, wherein a gas conveying pipe 2 with a partial pressure valve is connected to an outlet at the top of the anaerobic gas steel cylinder 1; the top of the first liquid culture medium storage bottle 3 is provided with a first bottle mouth and a second bottle mouth, a first conveying pipe 4 with a first valve, a first exhaust pipe 5 with a second valve and a third valve and a second conveying pipe 6 with a fourth valve all penetrate through the first bottle mouth and are in sealing connection with the first bottle mouth, wherein the top end of the first conveying pipe 4 is communicated with the gas conveying pipe 2, the bottom ends of the first conveying pipe 4 and the second conveying pipe 6 are immersed in the liquid culture medium, a third conveying pipe 7 with a fifth valve penetrates through the second bottle mouth and is in sealing connection with the second bottle mouth, the top end of the third conveying pipe 7 is positioned above the second bottle mouth, and the bottom end of the third conveying pipe 7 is immersed in the liquid culture medium; a third bottle opening and a fourth bottle opening are formed in the top of the second liquid culture medium storage bottle 8, a fourth conveying pipe 9 with a sixth valve, a second exhaust pipe 10 with a seventh valve and a fifth conveying pipe 11 with an eighth valve penetrate through the third bottle opening and are in sealed connection with the third bottle opening, the top end of the fourth conveying pipe 9 located above the third bottle opening is connected with the top end of the second conveying pipe 6, the top end of the fifth conveying pipe 11 is communicated with the top end of the third conveying pipe 7, the bottom ends of the fourth conveying pipe 9 and the fifth conveying pipe 11 are immersed in a liquid culture medium, and a sixth conveying pipe 12 with a ninth valve is in sealed connection with the fourth bottle opening; the first transfer pipe 4 communicates with the first exhaust pipe 5 between the second valve and the third valve through a first branch pipe 13 with a tenth valve.
The second delivery pipe 6 is communicated with the first delivery pipe 4 through a second branch pipe 14 with an eleventh valve, and the fourth delivery pipe 9 is communicated with a second exhaust pipe 10 positioned below a seventh valve through a third branch pipe 15 with a twelfth valve.
Specifically, can dismantle on the first bottleneck and be connected with first sealed lid, first sealed lid respectively with first conveyer pipe 4, first blast pipe 5 and second conveyer pipe 6 sealing connection, can dismantle on the second bottleneck and be connected with the sealed lid of second, the sealed lid of second and third conveyer pipe 7 sealing connection, the sealed lid of third can be dismantled and be connected with the sealed lid of third, the sealed lid of third respectively with fourth conveyer pipe 9, second blast pipe 10 and fifth conveyer pipe 11 sealing connection, can dismantle on the fourth bottleneck and be connected with the sealed lid of fourth, the sealed lid of fourth and sixth conveyer pipe 12 sealing connection.
Specifically, the first sealing cover and the third sealing cover each include a first cover 16 and three pagoda-shaped sealing pipes 17 arranged at the bottom of the first cover 16 and connected with the first cover 16 in a sealing manner, the first delivery pipe 4, the first exhaust pipe 5, the second delivery pipe 6 or the fourth delivery pipe 9, the second exhaust pipe 10 and the fifth delivery pipe 11 respectively penetrate through the three pagoda-shaped sealing pipes 17, the second sealing cover and the fourth sealing cover each include a second cover 18 and one pagoda-shaped connecting pipe 19 arranged at the bottom of the second cover 18 and connected with the second cover 18 in a sealing manner, and the third delivery pipe 7 or the sixth delivery pipe 12 respectively penetrate through the connecting pipe 19.
Specifically, the bottoms of the first sealing cover, the second sealing cover, the third sealing cover and the fourth sealing cover are all provided with O-shaped sealing rings 20.
Specifically, the volume fraction ratio of nitrogen to carbon dioxide in the oxygen-free gas cylinder 1 is 80.
Specifically, the pressure of the pressure dividing valve is 0.1MPa.
Specifically, first liquid culture medium storage bottle 3 and second liquid culture medium storage bottle 8 are made by glass material, gas-supply pipe 2 is stereoplasm PU pipe, first conveyer pipe 4, first blast pipe 5, second conveyer pipe 6, third conveyer pipe 9, second blast pipe 10, fourth conveyer pipe 12, first minute pipe 13, second minute pipe 14, third minute pipe 15 are the silica gel hose, pagoda shaped sealing tube and pagoda shaped connecting pipe are made by stainless steel material.
Specifically, the first conveying pipe 4 and the gas conveying pipe 2, and the fourth conveying pipe 9 and the second conveying pipe 6 can be detachably connected.
Specifically, the volume of the first liquid culture medium storage bottle 3 is 1L-5L, and the volume of the second liquid culture medium storage bottle 8 is 50mL-200mL.
The use method of the portable laboratory simple device for removing oxygen in the culture medium comprises the following steps:
aeration and oxygen removal of liquid culture medium in the first liquid culture medium storage bottle 3:
1. liquid culture medium is filled into a first liquid culture medium storage bottle 3, and then a simple laboratory device for removing oxygen in the culture medium is set up as shown in figure 1.
2. Opening a main valve of the oxygen-free gas steel cylinder 1, and adjusting the gas pressure on the valve to 0.1-0.2MPa; simultaneously open first valve on the first conveyer pipe 4, second valve and third valve on the first blast pipe 5 respectively to keep other valves to close, the gas in the anaerobic gas steel bottle 1 passes through gas-supply pipe 2 and first conveyer pipe 4 and gets into the bottom of liquid medium in the first liquid medium storage bottle 3 this moment, and the oxygen that originally exists in liquid medium and headspace is inputed and anaerobic gas passes through first blast pipe 5 and releases.
3. For example, after 2 operation aeration is carried out for 1 hour, all valves are closed, after the pressure of a partial pressure valve is adjusted to 0, a main valve of the anaerobic gas steel cylinder 1 is closed, and therefore efficient oxygen removal of the culture medium in the first liquid culture medium storage bottle 3 can be achieved.
And (II) subpackaging a culture medium:
1. after the operation of the first part is completed, the main valve of the oxygen-free gas steel cylinder 1 is kept open, and other valves are closed; the tenth valve in the first branch pipe 13, the third valve in the first exhaust pipe 5, the fourth valve in the second delivery pipe 6, the sixth valve in the fourth delivery pipe 9, and the seventh valve in the second exhaust pipe 10 are opened, respectively.
2. Gas output from the oxygen-free gas steel cylinder 1 sequentially enters the headspace of the first liquid culture medium storage bottle 3 through the gas conveying pipe 2, the first conveying pipe 4, the first branch pipe 13 and the first exhaust pipe 5; the liquid culture medium of the first liquid culture medium storage bottle 3 flows into the second liquid culture medium storage bottle 8 through the second delivery pipe 6 and the fourth delivery pipe 9 due to the increasing pressure of the headspace gas, and at the same time, the gas originally present in the second liquid culture medium storage bottle 8 is discharged by the inflowing liquid through the second exhaust pipe 10.
3. When the medium in the second liquid medium storage bottle 8 reaches its maximum capacity, this means that all the gas originally present in the headspace of the second liquid medium storage bottle 8 is vented, so that all the headspace oxygen in the second liquid medium storage bottle 8 can be removed.
4. The main valve of the oxygen-free gas steel cylinder 1 is still kept open, and other valves are closed at the same time; and opening an eleventh valve on the second branch pipe 14, a twelfth valve on the third branch pipe 15, an eighth valve on the fifth conveying pipe 11, a fifth valve on the third conveying pipe 7, a second valve (upper) on the first exhaust pipe 5 and a third valve (lower) in sequence.
5. The gas in the oxygen-free gas steel cylinder 1 passes through the gas conveying pipe 2 and enters the headspace of the second liquid culture medium storage bottle 8 through the gas conveying pipe 2, the first conveying pipe 4, the second branched pipe 14, the second conveying pipe 6, the fourth conveying pipe 9, the third branched pipe 15 and the second exhaust pipe 10; thereafter, the liquid culture medium in the second liquid culture medium storage bottle 8 flows back into the first liquid culture medium storage bottle 3 through the fifth transport pipe 11 and the third transport pipe 7 due to the pressure of the headspace gas therein, while the headspace gas is released through the first exhaust pipe 5 by the first liquid culture medium storage bottle 3 due to the increase in liquid volume. When the remaining volume of the second liquid culture medium storage bottle 8 is reduced to the target volume (for example, 20-30 mL of culture medium should be contained in 50mL of the second liquid culture medium storage bottle 8; 40-60 mL of culture medium should be contained in 100mL of the second liquid culture medium storage bottle 8; 100-140 mL of culture medium should be contained in 200mL of the second liquid culture medium storage bottle 8), all valves are closed; thereby realizing the anaerobic split charging of the liquid culture medium.
6. The connection between fourth delivery pipe 9 and second delivery pipe 6, and between fifth delivery pipe 11 and third delivery pipe 7 is then broken.
7. The culture medium in the second liquid culture medium storage bottle 8 can be combined with a cysteine oxygen scavenger (0.5 g/L cysteine hydrochloride combined with 0.002g/L resazurin) to realize the preparation of a strict anaerobic culture medium. Cysteine is a reducing agent, so that trace oxygen in the liquid can be removed; the resazurin is an oxygen concentration indicator, and when the oxygen indicator resazurin is colorless in the culture medium, the culture medium can be considered to be in a strict anaerobic state; when the resazurin is pink in the culture medium, the culture medium is in a micro-aerobic state; when resazurin is blue in the culture medium, the culture medium is in an aerobic state. For the operation of adding a cysteine oxygen scavenger to the medium in the second liquid medium storage bottle 8, the step of inoculating the cells was referred to (iii).
(III) inoculating thalli:
1. sucking the bacteria liquid to be inoculated by using a sterile oxygen-free injector, and removing gas in the liquid as much as possible by moving a piston in the injector;
2. removing a needle head of the injector, communicating the injector with the sixth conveying pipe 12, opening a ninth valve on the sixth conveying pipe 12, and transferring the bacterial liquid in the injector to the second liquid culture medium storage bottle 8;
3. the ninth valve on the sixth delivery tube 12 is closed and the syringe is pulled off.
(IV) Collection of liquid or gas sample in culture flask
1. Placing the second liquid culture medium storage bottle 8 upright (for collecting gas samples) or upside down (for collecting liquid samples);
2. the syringe is communicated with the sixth conveying pipe 12 by using a syringe without a needle head and sterile oxygen, a ninth valve on the sixth conveying pipe 12 is opened, and the syringe is used for sucking the gas in the headspace of the second liquid culture medium storage bottle 8 or the liquid in the bottle;
3. after completion, the second liquid medium storage bottle 8 is placed upright, the ninth valve of the sixth transport tube 12 is closed, and the syringe is pulled out.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A portable laboratory simplified apparatus for removing oxygen from a culture medium, comprising:
the oxygen-free gas steel cylinder (1) is connected with a gas pipe (2) with a partial pressure valve at the top outlet;
the liquid culture medium storage bottle comprises a first liquid culture medium storage bottle (3), wherein a first bottle opening and a second bottle opening are formed in the top of the first liquid culture medium storage bottle, a first conveying pipe (4) with a first valve, a first exhaust pipe (5) with a second valve, a first exhaust pipe (5) with a third valve and a second conveying pipe (6) with a fourth valve penetrate through the first bottle opening and are in sealing connection with the first bottle opening, the top end of the first conveying pipe (4) is communicated with an air conveying pipe (2), the bottom ends of the first conveying pipe (4) and the second conveying pipe (6) are immersed in a liquid culture medium, a third conveying pipe (7) with a fifth valve penetrates through the second bottle opening and is in sealing connection with the second bottle opening, the top end of the third conveying pipe (7) is located above the second bottle opening, and the bottom end of the third conveying pipe (7) is immersed in the liquid culture medium;
a second liquid culture medium storage bottle (8), wherein a third bottle opening and a fourth bottle opening are arranged at the top of the second liquid culture medium storage bottle, a fourth conveying pipe (9) with a sixth valve, a second exhaust pipe (10) with a seventh valve and a fifth conveying pipe (11) with an eighth valve penetrate through the third bottle opening and are in sealing connection with the third bottle opening, the top end of the fourth conveying pipe (9) above the third bottle opening is connected with the top end of the second conveying pipe (6), the top end of the fifth conveying pipe (11) is communicated with the top end of the third conveying pipe (7), the bottom ends of the fourth conveying pipe (9) and the fifth conveying pipe (11) are immersed in a liquid culture medium, and a sixth conveying pipe (12) with a ninth valve is in sealing connection with the fourth bottle opening;
the first conveying pipe (4) is communicated with a first exhaust pipe (5) positioned between the second valve and the third valve through a first branch pipe (13) with a tenth valve.
2. A portable laboratory facility for the removal of oxygen from culture media according to claim 1, wherein said second delivery pipe (6) is connected to said first delivery pipe (4) through a second branch pipe (14) with an eleventh valve, and said fourth delivery pipe (9) is connected to a second exhaust pipe (10) located below said seventh valve through a third branch pipe (15) with a twelfth valve.
3. The portable simple laboratory device for removing oxygen from a culture medium according to claim 2, wherein a first sealing cover is detachably connected to the first bottle opening, the first sealing cover is respectively connected with the first delivery pipe (4), the first exhaust pipe (5) and the second delivery pipe (6) in a sealing manner, a second sealing cover is detachably connected to the second bottle opening, the second sealing cover is connected with the third delivery pipe (7) in a sealing manner, a third sealing cover is detachably connected to the third bottle opening, the third sealing cover is respectively connected with the fourth delivery pipe (9), the second exhaust pipe (10) and the fifth delivery pipe (11) in a sealing manner, a fourth sealing cover is detachably connected to the fourth bottle opening, and the fourth sealing cover is connected with the sixth delivery pipe (12) in a sealing manner.
4. The laboratory simple apparatus for portable oxygen removal from a culture medium according to claim 3, wherein the first sealing cover and the third sealing cover each comprise a first cover (16) and three pagoda-shaped sealing tubes (17) disposed at the bottom of the first cover (16) and hermetically connected to the first cover (16), the first delivery tube (4), the first exhaust tube (5), the second delivery tube (6) or the fourth delivery tube (9), the second exhaust tube (10), and the fifth delivery tube (11) respectively extend through the three pagoda-shaped sealing tubes (17), the second sealing cover and the fourth sealing cover each comprise a second cover (18) and a pagoda-shaped connecting tube (19) disposed at the bottom of the second cover (18) and hermetically connected to the second cover (18), and the third delivery tube (7) or the sixth delivery tube (12) respectively extend through the connecting tubes (19).
5. The laboratory simple device for removing oxygen from culture medium according to claim 3, wherein the first sealing cover, the second sealing cover, the third sealing cover and the fourth sealing cover are provided with O-shaped sealing rings (20) at the bottom.
6. The laboratory mounted device for portable removal of oxygen from culture medium according to claim 1, wherein the volume fraction ratio of nitrogen to carbon dioxide in said oxygen-free gas cylinder (1) is 80.
7. The laboratory simplified apparatus for removing oxygen from a culture medium according to claim 1, wherein the pressure of the partial pressure valve is 0.1-0.2MPa.
8. The simple laboratory device for portable removal of oxygen from a culture medium according to claim 4, wherein the first liquid culture medium storage bottle (3) and the second liquid culture medium storage bottle (8) are made of glass material, the gas pipe (2) is a hard PU pipe, the first conveying pipe (4), the first exhaust pipe (5), the second conveying pipe (6), the third conveying pipe (9), the second exhaust pipe (10), the fourth conveying pipe (12), the first branch pipe (13), the second branch pipe (14) and the third branch pipe (15) are silica gel hoses, and the pagoda-shaped sealing pipe and the pagoda-shaped connecting pipe are made of stainless steel material.
9. The laboratory simple device for portable removal of oxygen from culture medium according to claim 3, wherein the first delivery pipe (4) and the gas pipe (2), the fourth delivery pipe (9) and the second delivery pipe (6), and the fifth delivery pipe (11) and the third delivery pipe (7) are detachably connected.
10. The laboratory simplified apparatus for portable removal of oxygen from a culture medium as claimed in claim 1, wherein the first liquid culture medium storage bottle (3) has a volume of 1L to 5L, and the second liquid culture medium storage bottle (8) has a volume of 50mL to 200mL.
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| CN202211069882.8A CN115232713A (en) | 2022-09-02 | 2022-09-02 | Portable laboratory simple and easy device of oxygen in getting rid of culture medium |
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| CN202211069882.8A CN115232713A (en) | 2022-09-02 | 2022-09-02 | Portable laboratory simple and easy device of oxygen in getting rid of culture medium |
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Application publication date: 20221025 |