CN210001863U - device for controlling oxygen concentration in magnetotactic bacteria culture process - Google Patents
device for controlling oxygen concentration in magnetotactic bacteria culture process Download PDFInfo
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- CN210001863U CN210001863U CN201920779446.7U CN201920779446U CN210001863U CN 210001863 U CN210001863 U CN 210001863U CN 201920779446 U CN201920779446 U CN 201920779446U CN 210001863 U CN210001863 U CN 210001863U
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Abstract
The utility model relates to an device of control magnetotactic bacteria culture process oxygen concentration belongs to magnetotactic bacteria culture technical field, the utility model discloses a device of control magnetotactic bacteria culture process oxygen concentration includes gas source (1), xiLin bottle (2) and gas pipe (3), gas source (1) includes nitrogen gas source and oxygen source, the air inlet and the gas source (1) of gas pipe (3) are connected, the gas outlet of gas pipe (3) is connected with syringe (4) of getting rid of the piston, the syringe needle of syringe (4) of getting rid of the piston is used for linking to each other with xiLin bottle (2), pack cotton (5) in syringe (4) of getting rid of the piston.
Description
Technical Field
The utility model relates to a magnetic bacteria culture condition control technical field, concretely relates to kinds of devices of control magnetic bacteria culture process oxygen concentration, oxygen concentration control's device in microaerophilic magnetic bacteria growth environment promptly.
Background
Magnetotactic bacteria are group which can generate magnetosome nanoparticles in vivo by biomineralizationThe magnetosome research has become a research hotspot in a plurality of related fields due to the unique properties of the magnetosome, and the development and the application of the magnetosome have great scientific research value and commercial value, but the magnetotactic bacteria are types of microaerophilic bacteria with strict requirements on the growth environment, namely, different from the conventional aerobic bacteria (O)2About 20% concentration) and is different from anaerobic flora (no need of O)2) The oxygen concentration required for growth is only about 1%. If the oxygen concentration in its growth environment cannot be precisely controlled, the synthesis of magnetosomes is directly affected. Therefore, the control of the oxygen concentration in the growing environment is the first key problem to be solved for the cultivation of such bacteria.
SUMMERY OF THE UTILITY MODEL
An object of the utility model is to provide a device of kinds of control magnetism bacteria culture process oxygen concentration the device is simple easily operated, not only can satisfy the requirement of magnetic bacteria growth environment aerobe a little in the operation process, but also can guarantee that the bacterium is difficult for receiving the pollution in the operation process.
The utility model provides an device for controlling oxygen concentration in magnetotactic bacteria culture process, which comprises a gas source 1, a penicillin bottle 2 and a gas passing pipe 3, wherein the gas source 1 comprises a nitrogen source and an oxygen source, the gas inlet of the gas passing pipe 3 is connected with the gas source 1, the gas outlet of the gas passing pipe 3 is connected with a syringe 4 for removing a piston, the needle head of the syringe 4 for removing the piston is used for being connected with the penicillin bottle 2, and cotton 5 is filled in the syringe 4 for removing the piston.
Preferably, or more air outlets are formed in the air passing tube 3, and each air outlet is connected with penicillin bottles 2.
Preferably, the material of the air passing pipe 3 comprises rubber or silica gel.
Preferably, the air pipe 3 comprises an -shaped air pipe or an air pipe obtained by connecting a plurality of sections of air pipes through a tee pipe or a right-angle elbow.
Preferably, the penicillin bottle 2 comprises a bottle body and a bottle cap, wherein the bottle cap comprises a butyl rubber bottle plug and an aluminum plastic bottle cap.
Preferably, the device further comprises a bracket and a clamp for fixing the air duct 3.
Preferably, the device further comprises a needle extension hose.
The utility model provides a device of kinds of control magnetism bacteria culture process oxygen concentration that becomes magnetic, the device is simple easily to operate, not only can satisfy the requirement of the little aerobic in magnetism bacteria growth environment that becomes magnetic, but also can guarantee that the bacterium is difficult for receiving the pollution in the operation process, the device low cost, simple and easy, test result shows, the device can be used for magnetism bacteria growth environment's oxygen concentration in the accurate control blake bottle, can control more accurately to the special environment of the required 1% oxygen concentration of magnetism bacteria growth that becomes magnetic to the little aerobic environment of this kind of bacterium has been simulated well, consequently also can make the bacterium fully synthesize the required magnetism corpuscle of self, in addition, utilize the utility model the device, just can accomplish to stop magnetism bacteria culture process's pollution problem with clean workstation, consequently reduces greatly to the environmental requirement of experimental links such as inoculation.
Drawings
Fig. 1 is a schematic structural diagram of a nitrogen gas workstation provided by the present invention;
FIG. 2 is a schematic structural view of a penicillin bottle and accessories for culturing magnetotactic bacteria according to the present invention;
FIG. 3 is an electron microscope image of the cultured magnetotactic bacterium AMB-1 provided by the present invention;
FIG. 4 is a partially enlarged magnetosome diagram of an electron microscope diagram of the cultured magnetotactic bacterium AMB-1 provided by the present invention;
fig. 5 is a statistical graph of the particle size of magnetosome provided by the present invention;
fig. 6 is a magnetosome film thickness statistical chart provided by the present invention.
Detailed Description
The utility model provides an device for controlling oxygen concentration in magnetotactic bacteria culture process, which comprises a gas source 1, a penicillin bottle 2 and a gas passing pipe 3, wherein the gas source 1 comprises a nitrogen source and an oxygen source, the gas inlet of the gas passing pipe 3 is connected with the gas source 1, the gas outlet of the gas passing pipe 3 is connected with a syringe 4 for removing a piston, the needle head of the syringe 4 for removing the piston is used for being connected with the penicillin bottle 2, and cotton 5 is filled in the syringe 4 for removing the piston.
The device for controlling the oxygen concentration in the magnetotactic bacteria culture process is shown in figure 1, wherein 1 is a gas source, 2 is a penicillin bottle, 3 is a gas passing pipe, 4 is an injector for removing a piston, and 5 is cotton.
The utility model has no special limit to the size and the character of the penicillin bottle, and the penicillin bottle which is well known by the technical personnel in the field can be adopted. In the specific embodiment of the utility model, for the convenience of calculation, the preferred xiLin bottle of choosing the total volume to be 100mL of xiLin bottle. The utility model discloses in, xiLin bottle 2 includes body and bottle lid, the bottle lid is preferred to include rubber bottle plug and plastic-aluminum bottle lid, more preferably includes butyl rubber bottle plug and plastic-aluminum bottle lid. The utility model discloses it is right butyl rubber bottle lid and plastic-aluminum bottle lid do not have special restriction, adopt xiLin bottle conventional bottle lid can. The utility model discloses prefer to use gland pincers, butyl rubber buffer and cavity aluminium lid to seal the xiLin bottle when appropriate. The penicillin bottle of the utility model is preferably a flat bottom penicillin bottle, and the specific structure is shown in figure 2.
In the present invention, or more air outlets of the air duct 3 are provided, each air outlet is connected to vials 2, in the present invention, the material of the air duct 3 comprises rubber or silica gel, in the present invention, the air duct 3 comprises body-formed air duct, or air duct obtained by connecting a plurality of sections of air ducts through a three-way pipe or a right angle bend, when the air duct is obtained by connecting a plurality of sections of air ducts through a three-way pipe or a right angle bend, the number of the three-way pipe is preferably plural, the number of the right angle bend is preferably 1, the total amount of the three-way pipe and the right angle bend is equal to of vials of the vials, in the present invention, the installation method of the air duct preferably comprises the steps of connecting air ducts, end of the air duct to an air tank, end to horizontal outlet end of the three-way pipe, horizontal outlet ends of another air ducts are connected to an air duct, an adhesive tape is connected to an air outlet of a nitrogen tank, another end of a third three-way pipe is connected to a needle holder, a needle holder is connected to a needle holder for keeping the needle head of a needle head, a needle head for keeping the needle head of a.
The utility model discloses the gas outlet of gas passing pipe 3 is connected with syringe 4 of getting rid of the piston, gets rid of the syringe 4's of piston syringe 4 syringe needle and is used for linking to each other with xiLin bottle 2. the syringe remains the syringe needle, gets rid of the piston, preferably cuts off the overcoat turn-up with the scissors after, loosely fills into medical cotton, then will subtract the end of overcoat turn-up and connect the air duct, and end contains end of syringe needle and is used for being connected with xiLin bottle in addition, the utility model discloses it is right the syringe does not have special restriction, adopt the well-known conventional market aseptic syringe of technical personnel in the field can, when xiLin bottle's volume is 100mL, the syringe is the syringe of 1mL specification preferably, and is corresponding, the syringe of syringe needle preferred length of syringe is 2.5 cm. in the utility model discloses, syringe is preferred more than or equal to 23G (G: Gauge, international model, corresponds domestic 6 syringe needles), avoids leaving the not sealable hole of permanen.
In the present invention, the device further comprises a bracket and a clip for fixing the gas passing tube 3.
The utility model discloses in, based on above-mentioned technical scheme the method for culturing magnetotactic bacteria of device, including following step:
1) opening a bottle cap of a penicillin bottle, injecting a magnetotactic bacteria growth culture medium into the penicillin bottle, connecting an air inlet of the air passing pipe with a nitrogen source, introducing a needle head of an injector with a piston removed from an air outlet of the air passing pipe into the magnetotactic bacteria growth culture medium, introducing nitrogen for 20-40 min, covering the bottle cap, and sealing the penicillin bottle to obtain a th culture medium with dissolved oxygen removed;
2) removing the needle head of the injector of the piston from the air outlet of the air passing pipe, inserting the needle head into a butyl rubber plug of a penicillin bottle, immediately inserting another clean needle heads, introducing nitrogen into the Xilin bottle for 20-40 min, firstly pulling out the needle head of the injector of the air passing pipe air outlet for removing the piston, removing another needle heads after the pressure of the culture medium in the penicillin bottle is reduced to the atmospheric pressure, and performing high-pressure sterilization to obtain a sterilized second culture medium with oxygen removed;
3) connecting an oxygen source with another penicillin bottles, inserting the needle head of the injector with the piston removed from the air outlet of the air passing pipe into the penicillin bottle, immediately inserting another clean needle heads, introducing oxygen for 10-30 min, firstly pulling out the needle head of the injector with the piston removed from the air outlet of the air passing pipe, reducing the pressure of a culture medium in the penicillin bottles to atmospheric pressure, then removing another needle heads, and carrying out high-pressure sterilization to obtain the penicillin bottles containing pure oxygen;
4) using an aseptic syringe to suck oxygen with the volume of 1% of that of the penicillin bottle from the penicillin bottle containing pure oxygen in the step 3), injecting the oxygen into the second culture medium excluding oxygen in the step 2), using the aseptic syringe to inoculate magnetotactic bacteria, incubating for 2-4 h at 30-32 ℃, and culturing for 4-7 d at 28-30 ℃ to obtain a magnetotactic bacteria culture;
the step 3) and the step 1) are not limited in time sequence.
The utility model discloses open the bottle lid of xiLin bottle, inject magnetism convergence bacterial growth culture medium in xiLin bottle, will cross tracheal air inlet and nitrogen source connection, will the syringe needle that the piston was got rid of in the trachea gas outlet lets in let in nitrogen gas 20 ~ 40min in the magnetism convergence bacterial growth culture medium, the sealed xiLin bottle of bottle lid has been covered, obtains the culture medium of detaching dissolved oxygen preferred, the utility model discloses it is in to let in nitrogen gas 30min the utility model discloses in, sealed preferred is sealed with gland clamp and cavity aluminium lid with xiLin bottle bottleneck sealed for the preferred the utility model discloses in, magnetism convergence bacterial growth culture medium uses water as the solvent, and every 1L water adds 10.0mL vitamin supplementary solution, 5.0mL trace mineral substance supplementary solution, 0.68g KH's solution2PO40.848g of sodium succinate hexahydrate, 0.575g of disodium tartrate dihydrate, 0.083g of sodium acetate trihydrate, 0.45mL of 0.1% aqueous Resazurin by mass, 0.17g of NaNO30.04g ascorbic acid and 3.0mL quinolinate iron stock solution with a concentration of 10 mM. In the present invention, the water is preferably ultrapure water. The vitamin supplement solution preferably contains biotin 2.0mg, folic acid 2.0mg, pyridoxine hydrochloride 10mg, thiamine hydrochloride 5.0mg, riboflavin 5.0mg, nicotinic acid 5.0mg, calcium D-pantothenate 5.0mg, p-aminobenzoic acid 5.0mg, lipoic acid 5.0mg, vitamin B per 1L of water120.1 mg; the trace mineral supplement solution is prepared by the following preparation method: 1.5g of nitrilotriacetic acid was dissolved in 1L of water and adjusted to pH 6.5 with potassium hydroxide solution, followed by addition of MgSO4·7H2O 3g，MnSO4·H2O 0.5g，NaCl 1.0g，FeSO4·7H2O 0.1g，CoSO4·7H2O 0.18g，CaCl2·2H2O 0.1g，ZnSO4·7H2O 0.18g，CuSO4·5H2O 0.01g，Kal(SO4)2·12H2O0.02g，H3BO30.01g，Na2MoO4·2H2O 0.01g，NiCl2·6H2O 0.03g，Na2SeO3·5H2O0.3 g and Na2WO4·2H20.4g of O; finally, regulating the pH value to 7.0 by using a potassium hydroxide solution; the preparation method of the quinoline iron stock solution comprises the following steps: 1.9g of quinic acid was dissolved in 1L of water, followed by the addition of 4.5g of FeCl3·6H2O, dissolved by stirring, and sterilized by filtration through a 0.02 μm syringe filter. In the present invention, the water in the vitamin supplement solution, the trace mineral supplement solution, and the quinoline iron stock solution is preferably ultrapure water. The utility model discloses preferably store quinoline iron stock solution at room temperature in the dark place, as aseptic stock solution, abandon the solution when the precipitate becomes obvious. In the utility model, the magnetotactic bacteria culture medium is preferably stirred during the preparation process. In the present invention, the trace mineral supplement solution and the quinolinate iron(III) stock solution need to be kept sterile, to avoid contamination, uses standard aseptic technique (open the bottle top using bunsen burner) and uses sterile pipettor tip to dispense when in use the utility model discloses preferably store trace mineral make-up solution and vitamin make-up solution in 4 ℃ refrigerator the utility model discloses in, the pH value of magnetotactic bacteria medium growth medium is preferably 6.75, the pH value condition preferably adopts 1M naoh when the volume of xiLin bottle is 100mL, preferably pour into 50 ~ 80mL magnetotactic bacteria growth medium into xiLin bottle, more preferably 60mL the utility model discloses when letting in the syringe needle of trachea gas outlet to let in nitrogen gas in the magnetotactic bacteria growth medium, preferably deepening the syringe needle under the magnetotactic bacteria growth medium liquid level, when the utility model discloses the syringe needle is not long enough, preferably use sterile syringe needle extension hose to operate, in the utility model embodiment, the length of syringe needle extension hose is preferably 10 ~ 15cm, more preferably 10cm when the utility model discloses when letting in the bacterium gas outlet of trachea is not enough, preferably the bubble is left in the bubble in the nitrogen gas flow adjustment bottle to avoid the bubble to produce the corresponding small bubble through the utility model discloses a nitrogen gas flow adjustment bubble is preferably used to produce the utility model discloses a bubble.
After obtaining the th culture medium of getting rid of oxygen, will the syringe needle of the syringe of piston is got rid of to the trachea gas outlet, insert on the butyl rubber stopper of xiLin bottle, and insert another clean syringe needles immediately, let in nitrogen gas 20 ~ 40min in the xiLin bottle, extract earlier the syringe needle of the syringe of piston is got rid of to the trachea gas outlet, culture medium pressure in the xiLin bottle removes another syringe needles after dropping to atmospheric pressure again, autoclaving, obtains the sterilized second culture medium of getting rid of oxygen, the utility model discloses melt nitrogen gas in to xiLin bottle and can expel remaining oxygen in the xiLin bottle, in the utility model discloses, the speed of letting in of nitrogen gas is preferably 50mL/min, the utility model discloses when letting in the xiLin bottle, preferably insert in the butyl rubber stopper of the same bottle with clean syringe needles in addition promptly to make manyThe remaining gas was vented and the air in the bottle was eventually replaced with nitrogen. After removing the nitrogen spike, a few seconds were allowed until the pressure in the media bottle was reduced to atmospheric pressure and the second spike was removed. To prevent O2After oxygen removal, the vessel is returned to the penicillin bottle culture medium, preferably in a rapid continuous operation.
The utility model discloses get xiLin bottles in addition, be connected oxygen source and mistake trachea, will cross the syringe needle that the syringe of trachea gas outlet got rid of the piston and insert this xiLin bottle to insert clean syringe needles in addition immediately, let in oxygen 10 ~ 30min, extract earlier cross the syringe needle that the syringe of piston was got rid of to the trachea gas outlet, culture medium pressure in xiLin bottle drops to and removes another syringe needles after the atmospheric pressure again, and autoclaving obtains the xiLin bottle that contains pure oxygen.
The utility model discloses use sterile injector, follow draw xiLin bottle volume 1% oxygen in containing pure oxygen, inject into in getting rid of the second culture medium of oxygen, use sterile injector inoculation magnetotactic bacterium, 30 ~ 32 ℃ incubate 2 ~ 4h, 28 ~ 30 ℃ culture 4 ~ 7d, obtain magnetotactic bacterium culture the utility model discloses preferably incubate 3h at 30 ℃, 29 ℃ culture 5d the utility model discloses when injecting oxygen, preferably insert needles in the butyl rubber stopper and do benefit to unnecessary gas and discharge, after the operation is accomplished immediately extract the syringe needle and prevent gas leakage the utility model discloses in, if inoculate from the magnetotactic bacterium that grows in xiLin bottle, need to drip several drops of 70% ethanol solution on the stopper of xiLin bottle that contains fresh magnetotactic bacterium growth culture medium and old culture bottle and make them sterilize through the flame of this bunsen lamp, and inoculate fresh bacterium magnetotactic bacterium culture in fresh bacteria growth culture medium if inoculate, deposit its magnetotactic bacterium in the tube, only need to deposit in this kind of sterile bacteria in the sterile syringe in the cryotactic tube, only the cryotactic bacterium inoculation room temperature is sealed in the DMSO of this kind of syringe (1 mL-100 mL does not have the bacterium to freeze the bacterium inoculation, and use the DMSO
700264TM))。
In the process of magnetotactic bacteria culture, the utility model discloses the preferred growth condition of observing the magnetotactic bacterium, the utility model discloses the method of observing preferably includes ① under the flame of bunsen burner, uses aseptic syringe needle and aseptic syringe to extract the magnetotactic bacterium, ② uses the hanging drop method (guaranteeing that bacterium AMB-1 has both magnetism and mobility) to drip the magnetotactic bacterium under the microscope and observe, the utility model discloses the microscope is preferred to include that phase contrast microscope magnification factor 10X is suitable to 60X, ③ the utility model discloses preferably use Transmission Electron Microscope (TEM) to observe cell structure and magnetosome in detail.
In the utility model, the operation of each step is aseptic operation; the aseptic operation preferably comprises sterilization using a bunsen burner.
The following will describe in detail the steps of the apparatus for controlling oxygen concentration in magnetotactic bacteria culture process according to the present invention with reference to specific embodiments, and the technical solution of the present invention includes but is not limited to the following embodiments.
Example 1
The method for controlling the oxygen concentration in the magnetotactic bacteria growth environment comprises the following steps:
the method comprises the following steps of A, designing and installing the device, wherein the device comprises a nitrogen tank, a workbench, a laboratory square seat support (with parallel clamps), a rubber air passing pipe (long), a 5cm air passing pipe (multiple), a 20cm air passing pipe (multiple), a support, a three-way pipe (multiple), a right-angle bent pipe, a -time syringe (with the capacity of 1 mL), a needle head retaining tube, a piston removing tube, cotton filling, a plurality of needle head extending tubes (multiple), a penicillin bottle (with the diameter of 52mm, the height of 95mm, the total volume of 100mL, the diameter of an opening part of 20mm, a flat bottom), a 20mm butyl rubber bottle plug, an aluminum plastic bottle cap with the same specification and a gland clamp.
The installation method comprises the following steps:
① rubber air passing pipes are connected, wherein end of the rubber air passing pipe (long) is connected with an air outlet of a nitrogen tank, the other end is connected with the horizontal outlet end of the th three-way pipe, the other horizontal outlet ends of the th three-way pipe are connected with the th 5cm air passing pipe, and so on, until the rubber air passing pipes are connected to the last right-angle bent pipes, the 20cm air passing pipes are respectively connected with the vertical outlet ends of the connecting three-way pipes.
② the airway device after connection is mounted on the laboratory square base bracket and fixed by parallel clamps.
③ A plurality of -time disposable syringes (needle retained, piston removed) with a 1mL capacity were cut with scissors, crimped, loosely packed into medical cotton, and connected one by one to the outlet of the 20cm air duct.
④ the respective ports were checked for leaks with soapy water and if necessary, sealed with Teflon tape to ensure no nitrogen leaks.
And B: preparing a magnetotactic bacteria growth culture medium;
① stock solutions of 10mM quinolinate iron were prepared by dissolving 1.9g of quinic acid in 1L of ultrapure water, adding 4.5g of FeCl 3.6H2O, dissolving with stirring, filter sterilizing through a 0.02 μm syringe filter, and using the standard cycle (i.e., autoclaving at 121 ℃ for 15 min).
Note that: the quinoline iron stock solution was stored at room temperature protected from light as a sterile stock solution and discarded when the precipitate became apparent.
② in a beaker containing 1L of ultrapure water, 10.0mL of a vitamin supplement solution, 5.0mL of a trace mineral supplement solution, and 0.68g of KH were added in this order with stirring2PO40.848g of sodium succinate hexahydrate, 0.575g of disodium tartrate dihydrate, 0.083g of sodium acetate trihydrate, 0.45mL of 0.1% aqueous Resazurin, 0.17g of NaNO30.04g ascorbic acid and 3.0mL of a 10mM stock solution of iron (III) quinolinate.
Note that: the trace mineral supplement solution and the quinoline iron (III) stock solution need to be kept sterile. To avoid contamination, use is made of standard aseptic techniques (using bunsen burner to open the top of the bottle) and dispensing is done using a sterile pipette tip. The mineral and vitamin solutions were stored in a refrigerator at 4 ℃.
③ after all chemicals were added, the pH was adjusted to 6.75 with 1M NaOH solution and the freshly prepared medium was distributed into 100mL vials into each of which 60mL of medium was poured.
④ Nitrogen gas was blown into the medium for 30min using a 15cm needle extension hose connected to the needle of the device described in step A to remove dissolved O2Place a butyl rubber stopper on top of each bottle, leaving small openings to allow excess gas to exit the bottles.
Note that: bubbles may form when bubbling with nitrogen and the gas flow is adjusted accordingly to avoid bubbles.
⑤ the prepared butyl rubber stopper and aluminum plastic bottle cap are used for sealing and the same specification of gland clamp is used for each bottle.
⑥ the needle and the narrow tube were disconnected from the nitrogen station and a clean needle (2.5 cm long,. gtoreq.23G) was inserted into the rubber stopper of the closed vial, the valve of the nitrogen tank was adjusted to allow gentle and continuous nitrogen gas to expel the residual oxygen (about 50mL/min) from the vial, another clean needles were immediately inserted into the same vials, and the procedure was repeated for the other vials to allow nitrogen gas to flow for about 25min to replace the air in the vial with nitrogen gas.
Note that: needles smaller than 23 gauge may leave a permanent, non-sealable hole in the stopper.
⑦ disconnect the vial from the nitrogen station by removing the corresponding needle, wait for a few seconds until the pressure in the vial drops to atmospheric pressure and remove the second needle.
Note that: to prevent O2After step B ④, re-enter vial medium, steps B ④ - ⑦ should be performed in rapid succession if all vials cannot be connected to the apparatus of the present invention at the same time, please operate on group vials according to steps B ④ - ⑦, and then repeat these steps for the remaining vials.
⑧ the bottles in step B ⑦ were autoclaved, allowed to cool to room temperature, and then stored at room temperature.
And C: inoculating magnetotactic bacteria AMB-1.
Note that under the flame of a bunsen burner, sterile conditions perform all of the following steps except ①.
① A empty 100mL vial was sealed using a butyl rubber stopper and gland clamp.A device of the present invention was connected to an oxygen cylinder, the needle of the device was inserted into the cylinder, and another needles were inserted over the butyl rubber stopper for venting.
② let O2Flow through the bottle for about 30min to ensure all air in the bottle is O2Instead, the syringe needle and another needle were removed, slightly over-pressurized in the bottle, and then autoclaved.
Note that to save time, steps C ① - ② were performed during media preparation and the vials were autoclaved with the media or stock solution .
③ bottle for fresh culture medium and O contained in step C ②2The stopper of the vial was topped with a few drops of 70% ethanol solution and then sterilized by the flame of a bunsen burner.
④ containing O from step C ② using a sterile syringe and needle2Extracting 1mL of O from penicillin bottle2And transferred to the vial containing fresh medium obtained in step B ⑧, the needle is ensured to be tightly attached to the syringe during this step to avoid any air in the syringe.
⑤ if inoculating AMB-1 from growth in penicillin bottles, several drops of 70% ethanol solution are dropped onto the stoppers of fresh medium penicillin bottles and old bottles and they are sterilized by passing them through the flame of a bunsen burner, and 1mL of older AMB-1 culture is inoculated into fresh medium, if inoculating AMB-1 of a frozen tube, it is only necessary to warm its frozen tube to room temperature while sealing it in the flame of a bunsen burner, and only 0.1mL is inoculated to dilute glycerol or dimethyl sulfoxide (DMSO) used in the freezing process.
⑥ AMB-1 cultures were incubated at 30 ℃ for 4h, incubated at 30 ℃ for 5d and inoculated into fresh medium.
Step D: the growth of AMB-1 was observed.
① bacteria AMB-1 was extracted under the flame of a bunsen burner using a sterile needle and a sterile syringe.
② the use of hanging drops ensures that the bacteria AMB-1 is both magnetic and motile.
Using a phase contrast microscope, magnification of 10X to 60X is suitable.
③ the cell structure and magnetosome were observed in detail using a Transmission Electron Microscope (TEM), and the results of the observation are shown in FIGS. 3 and 4.
FIG. 5 is a statistical plot of the particle size of magnetosomes, which shows a particle size of 46.72. + -. 5.87 nm; FIG. 6 is a statistical plot of magnetosome membrane thickness, which statistical analysis shows a thickness of 5.02. + -. 1.15 nm. As can be seen from FIGS. 3 to 6, the magnetosomes are arranged in a chain shape, the size of the magnetosomes is about 46.72 + -5.87 nm, the outside of the magnetosomes is visible as a film structure, and the thickness of the film is about 5.02 + -1.15 nm.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
- The device for controlling the oxygen concentration in the magnetotactic bacteria culture process is characterized by comprising a gas source (1), a penicillin bottle (2) and a gas passing pipe (3), wherein the gas source (1) comprises a nitrogen source and an oxygen source, a gas inlet of the gas passing pipe (3) is connected with the gas source (1), a gas outlet of the gas passing pipe (3) is connected with an injector (4) for removing a piston, a needle head of the injector (4) for removing the piston is used for being connected with the penicillin bottle (2), and cotton (5) is filled in the injector (4) for removing the piston.
- 2. The device according to claim 1, characterized in that the air passing pipe (3) has or more air outlets, and each air outlet is connected with penicillin bottles (2).
- 3. The device according to claim 1, characterized in that the material of the air duct (3) comprises rubber or silica gel.
- 4. The device according to claim 1, characterized in that the air duct (3) comprises an -shaped air duct or a plurality of air ducts connected by a tee or elbow.
- 5. The device according to claim 1, characterized in that the vial (2) comprises a body and a cap, the cap comprising a butyl rubber stopper and an aluminum plastic cap.
- 6. Device according to claim 1, characterized in that it further comprises brackets and clamps for fixing the air duct (3).
- 7. The device of claim 1, further comprising a needle extension hose.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201920779446.7U CN210001863U (en) | 2019-05-28 | 2019-05-28 | device for controlling oxygen concentration in magnetotactic bacteria culture process |
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| CN201920779446.7U CN210001863U (en) | 2019-05-28 | 2019-05-28 | device for controlling oxygen concentration in magnetotactic bacteria culture process |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029050A (en) * | 2019-05-28 | 2019-07-19 | 甘肃中医药大学 | A kind of device and cultural method controlling magnetotactic bacteria incubation oxygen concentration |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110029050A (en) * | 2019-05-28 | 2019-07-19 | 甘肃中医药大学 | A kind of device and cultural method controlling magnetotactic bacteria incubation oxygen concentration |
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