CN104560771A - Separation culture method of anaerobic bacteria - Google Patents
Separation culture method of anaerobic bacteria Download PDFInfo
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- CN104560771A CN104560771A CN201310517811.4A CN201310517811A CN104560771A CN 104560771 A CN104560771 A CN 104560771A CN 201310517811 A CN201310517811 A CN 201310517811A CN 104560771 A CN104560771 A CN 104560771A
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Abstract
The invention discloses a separation culture method of anaerobic bacteria. The method comprises the following steps: preparing a culture medium; filling nitrogen before sterilizing the culture medium; carrying out enrichment culture of a strain; and carrying out separation culture on the strain, wherein the separation culture on the strain adopts a way of alternate inoculated culture by a liquid culture medium and a solid culture medium, and the liquid culture medium inoculates the solid culture medium by capillary glass tube puncture, a cylindrical anaerobic culture cavity with a smooth inoculating face is formed in the solid culture medium, and round holes formed on the surface of the solid culture medium are sealed after inoculation by adopting an unset culture medium so as to form a sealed anaerobic culture cavity, and the inner wall of the anaerobic culture cavity is a culture face of anaerobic bacteria. According to the method disclosed by the invention, the anaerobic culture cavity with the smooth inoculating face is formed in the solid culture medium to provide an anaerobic environment for culturing anaerobic bacteria, so that the use of anaerobic equipment is reduced, the separating process is simplified, the cost in the separating process is lowered, and the time of acquiring single strain is shortened.
Description
Technical field
The present invention relates to microorganism field, particularly relate to the isolation cultivation method of anerobe in oil production reuse water, oilfield sewage and oily sludge.
Background technology
In petroleum chemical industry, the corrosion that microorganism causes result in huge financial loss.Have bibliographical information, in oil well more than 75% burn into buried pipeline and cable in 50% fault from the corrosion (mainly sulfate-reducing process) of microorganism; Separately have the pertinent literature of 2008 to report, in China, the loss caused oil field due to corrosion is every year up to 200,000,000 yuan.
Along with the exploitation of most onshore oil field enters the intermediary and later stages, for improving recovery ratio water filling, the amount of steam treatment and oil field reinjection water all increasing year by year, the water ratio of extraction crude oil is also increasing, just need to prevent microbiological corrosion to add more chemical bactericide in field produces water, because microbial evolution speed is fast, adaptable to environment, often causes the sterilant effect of life-time service to decline, the adding and cause the difficulty of disposing polluted water in oil to strengthen of bulk sterilization agent simultaneously.So microorganism rises year by year to the loss that oil field is caused.
Cause the microorganism of corrosion can be divided into the large class of anaerobic and aerobic two in process of oil production, in actual production environment, together with in bacterial population, multiple anerobe coexists with aerobic bacteria, aerobicly generally to occur with Anaerobic Corrosion simultaneously, to cause the microorganism of corrosion to mainly contain: sulphate reducing bacteria (generation sulfate-reducing process), sulfur oxidizing bacterium, saprophytic microorganism, iron bacteria and fungi.Have bibliographical information, sulphate reducing bacteria wherein not only can cause equipment corrosion, simultaneously owing to producing hydrogen sulfide in its metabolic process, have potential personnel safety harm, and the security incident that oil field causes due to hydrogen sulfide is also appeared in the newspapers repeatly.
Prevent the corrosion that microorganism causes, reduce chemical bactericide to the pollution of environment simultaneously as far as possible, will set about from the physiological property of microorganisms, and find the factor suppressing its growth and breeding.So first will isolate the microorganism causing harm from production environment, wherein the separation of aerobic microbiological is fairly simple.And traditional anerobe isolation cultivation method, as anaerobic jar, hungate roll the method such as sandwich culfure in tube method and anaerobism workstation, complicated operation, process length consuming time, and costly.
CN200710064715.3 discloses the isolation cultivation method of a kind of anerobe, the method avoids the contact of object microorganism and oxygen in whole operating process, running cost relative moderate, is particularly suited for the separation and Culture of growth temperature higher than 50 DEG C and to the strict difficult separating high-temp anerobe of anaerobic environment requirement, strictly anaerobic bacterium.Use anaerobism workstation during the part operation that the method only need be separated anerobe, can using standard incubator, providing anaerobic environment without the need to continuing during cultivation, when avoiding separation and Culture, anaerobism workstation can only be used for the situation of a kind of bacterium of growth temperature.But, the mode that this invention utilizes the substratum not solidifying agar (temperature is higher than 50 DEG C) to mix with Enrichment of bacteria liquid is inoculated, for the anerobe of growing environment temperature lower than 50 DEG C, the seeded process described in this invention can affect microbic activity to be separated, even causes it dead.
Summary of the invention
The invention provides the isolation cultivation method of a kind of anerobe.The inventive method can form the Anaerobic culturel chamber of inoculating surfaces flat smooth in solid medium, for the cultivation of anerobe provides anaerobic environment, decrease the use of anaerobism equipment, simplify sepn process, save the expense of sepn process, shorten the time obtaining single strain.
The isolation cultivation method of anerobe of the present invention, comprises inflated with nitrogen, the enrichment culture of bacterial classification and the separation and Culture of bacterial classification before the preparation of substratum, medium sterilization; Wherein the separation and Culture of bacterial classification adopts liquid nutrient medium and solid medium to replace the mode of inoculation culture, the inoculation of liquid culture basal orientation solid medium adopts glass capillary to dip bacterium liquid percutaneous puncture-inoculation to be separated, the smooth cylindric Anaerobic culturel chamber of an inoculating surfaces is formed in solid medium, the circular hole sealing that solid culture primary surface is formed by not solidified substratum is adopted after inoculation, thus forming the Anaerobic culturel chamber of sealing, the inwall in Anaerobic culturel chamber is the cultivation face of anerobe; The inoculation of solid culture basal orientation liquid nutrient medium is extend into by inoculating needle in Anaerobic culturel chamber, single bacterium colony access liquid nutrient medium that picking is formed from cultivation face.
In the present invention, the preparation of described substratum, according to the original place environment of anerobe to be separated, determines composition and the proportioning of corresponding substratum.
In the present invention, inflated with nitrogen before described medium sterilization, object is the oxygen in removing substratum, for the growth of anerobe provides anaerobic environment.
In the present invention, the enrichment culture of described bacterial classification is first activated by bacterial classification, then obtains enrichment bacterium liquid at corresponding liquid nutrient medium through 2 ~ 5 switchings.Inoculation not be used in anaerobism workstation to be carried out, and only needs the plug inoculation of thrusting on culturing bottle of the needle tubing after with sterilizing to draw the bacterium liquid of respective amount.For ensureing success ratio during primary activation inoculation, inoculum size accounts for 10% ~ 20% of culture volume.After obtaining bacterium liquid, the inoculum size between liquid nutrient medium accounts for 5% ~ 10% of culture volume.Seeded process is wanted, rapidly to reduce bacterium liquid aerial open-assembly time, to seal with wax immediately after inoculation.
In the present invention, the separation and Culture of described bacterial classification be by after enrichment bacterium liquid gradient dilution to the process of solid medium inoculation, need to carry out in anaerobism workstation, add plug after inoculation, and seal with wax immediately after shifting out anaerobism workstation.Sealing with wax of culturing bottle seals between culturing bottle plug and bottleneck after docking kind, avoids entering of air, maintains the anaerobic environment in culturing bottle in long-time culturing process.
In the present invention, when enrichment bacterium liquid is to solid medium percutaneous puncture-inoculation, inoculum size is dip once in the bacterium liquid diluted with glass capillary.Described glass capillary specification is external diameter 2.0 ~ 4.0mm, internal diameter 1.5 ~ 3.0mm, length 10 ~ 20cm, to manufacturer's customization or can buy standard specifications.Due to the thin hollow of capillary glass tube wall, the substratum entered in glass capillary can be taken out of in percutaneous puncture-inoculation process, and the globality of solid medium can not be destroyed, and the Anaerobic culturel chamber of inoculating surfaces flat smooth can be formed in solid medium, the identification to single bacterium colony when being conducive to like this choosing bacterium.Preferably touch bottom or the inwall of culturing bottle during described glass capillary percutaneous puncture-inoculation, do like this and can take substratum wherein out of by glass capillary better, be conducive to formation one complete, cylindric Anaerobic culturel chamber that inoculating surfaces is smooth.During glass capillary percutaneous puncture-inoculation preferably and media surface tilt, be conducive to the picking of bacterium colony like this.
In the present invention, can form a circular hole at solid culture primary surface after percutaneous puncture-inoculation, Circularhole diameter is relevant with the internal diameter of glass capillary.The sealing of circular hole can adopt dropper or liquid-transfering gun on media surface circular hole, drip several not solidify substratum and form sealing ply, due to do not solidify substratum run into solid medium after very fast cooled and solidified, the inoculating surfaces in anaerobism cavity can not be touched, thus Anaerobic culturel chamber can not be destroyed.For the anerobe that growth temperature is lower, seeded process of the present invention or will cause it dead because of the activity of the too high impact of temperature anerobe to be separated.The inventive method is applicable to former growing environment at the bacterial classification of 10-60 DEG C, and sepn process and culturing process all can well keep former growth temperature.
In the present invention, during picking list bacterium colony, the sealing ply of described media surface circular hole, first can remove with the tweezers after sterilizing, then utilize inoculating needle picking list bacterium colony.
In the present invention, culture temperature can be determined according to the former growing environment of bacterial classification to be separated, can cultivate 3 ~ 7 days at 10 ~ 60 DEG C.
In the present invention, culturing bottle adopts anaerobic bacteria culture bottle, preferably utilize medical discarded glass infusion bottle, for the consumption enrichment culture process saving substratum can utilize the infusion bottle of 50mL, separation and Culture can utilize the infusion bottle of 100mL, can repeatedly use with after the infusion bottle cleaning of crossing, sterilizing.
Compared with prior art, the present invention has following outstanding effect:
1, glass capillary is utilized to realize from liquid culture basal orientation solid medium percutaneous puncture-inoculation, the smooth cylindric Anaerobic culturel chamber of an inoculating surfaces is formed in solid medium, the circular hole sealing that solid culture primary surface is formed by not solidified substratum is adopted after inoculation, thus form the Anaerobic culturel chamber of sealing, present approach provides the anaerobic environment of anerobe growth, the biological activity of thalline can not be affected, and be easy to identification and the picking of single bacterium colony, shorten the time obtaining single strain.
2, the present invention's process of only utilizing glass capillary to inoculate from liquid culture basal orientation solid medium and carrying out anaerobism workstation from the process need of solid culture basal orientation liquid nutrient medium picking list bacterium colony, decreases the use to anaerobism workstation.After inoculation, the cultivation of bacterial classification only need be cultivated at standard incubator, do not need special Anaerobic culturel equipment, the adjustment of culture temperature is more convenient, and when avoiding separation and Culture, anaerobism equipment can only be used for a kind of situation of thalline of growth temperature, saves the expense of anerobe sepn process.
3, compared with plate sandwich culfure, the high temperature that present invention, avoiding non-freezing solid substratum, on the impact of the activity of anerobe, is conducive to the consistence keeping strain separating, culture environment temperature.Seal with wax compared with mode with media surface, save consumptive material, and in strain transfer process more convenient operation, accelerate the sepn process of anerobe.The present invention's difficulty in manual operation reduces, and decreases the dependence to anaerobism workstation in anaerobic bacteria culture sepn process, thus has saved consumptive material (as nitrogen).
Embodiment
Below by embodiment, the invention will be further described.
First the water sample water quality at anerobe source to be separated place is analyzed, obtain the information of corresponding original place environment, and then adjust existing culture medium prescription, to keep separating obtained bacterial classification residing environment in physiological property research process consistent as far as possible with original place environment.Do the time that can reduce on the one hand and obtain single strain like this, on the other hand acquisition data are more conformed to reality.
Substratum prepare complete point install after, first inflated with nitrogen, then carries out high-temperature sterilization to substratum and experiment appliance.Plug on culturing bottle will be built before sterilization, clamp rope between plug and bottleneck, to prevent plug in autoclaving process to be ejected because of gas expansion, after sterilizing, first will take out cotton rope and build plug.
To the first enrichment of anerobe in oil field reinjection water, inoculum size wants large, accounts for 10% ~ 20% of liquid nutrient medium volume.The enrichment bacterium liquid of anerobe is obtained for 2 ~ 5 times by switching between liquid medium within, enrichment switching process utilizes common syringe that enrichment bacterium liquid is transferred to fresh culture, whole process is carried out on normal operations platform, carry out process of sealing with wax after inoculation, then go to incubator and cultivate at a set temperature.
In strain separating process, the bacterium liquid aqua sterilisa gradient dilution first enrichment obtained in anaerobism workstation is to 10 of original content
-2, 10
-3, 10
-4, 10
-5, the bacterium liquid then utilizing glass capillary to dip to have diluted, to solid medium percutaneous puncture-inoculation.Adopt not solidified substratum to seal after inoculation, then go to incubator and cultivate at a set temperature.By solid culture basal orientation liquid nutrient medium seeded process, with the single bacterium colony in inoculating needle picking solid medium Anaerobic culturel chamber, be seeded to liquid nutrient medium, cultivated through 3 ~ 5 days and obtain enrichment bacterium liquid.Repeat above-mentioned steps 2 ~ 5 times, namely can obtain single strain.
The method for culturing and separating of anerobe of the present invention, separates the inoculation in anerobe sepn process and culture environment, and the inoculation between liquid bacteria liquid does not need to carry out in anaerobism workstation yet, makes the cultivation of anerobe, separation method more flexible.Anerobe isolation cultivation method provided by the invention is equally applicable to the related experiment in anerobe physiological property research process, and culturing bottle can be replaced by plug test tube to save substratum consumption, can save the expense in anerobe physiological property research process and time equally.
Embodiment 1
The inventive method is adopted to carry out separation and Culture to the anerobe sulphate reducing bacteria in certain oil field reinjection water.
(1), analyze the water quality of certain oil field reinjection water water sample, in conjunction with water quality information, existing sulfate reduction bacteria culture medium is adjusted accordingly.Substratum consists of: K
2hPO
40.5g, (NH
4)
2sO
42.5g, Na
2sO
41.0g, CaCl
20.1g, MgSO
41.0g, Vc 0.1g, (NH
4)
2fe (SO
4)
26H
2o(indicator, substratum blackening when having Growth of Sulfate Reducing Bacteria to breed) 0.5g, Sodium.alpha.-hydroxypropionate 2.0mL, L-cysteine hydrochloride 0.5g, yeast extract paste 1.5g, pure water 1.0L; PH6.0 ~ 6.5; Solid medium adds agar by 1.5% ~ 2%.
(2), in the substratum that preparing and packaging is complete inflated with nitrogen, carry out high-temperature sterilization.
(3), utilize needle tubing to inoculation of medium oil field reinjection water water sample, primary vaccination amount accounts for 20% of culture volume, inoculates complete substratum and carries out sealing with wax and turn incubator 30 DEG C of cultivations.Inoculate to fresh culture after obtaining bacterium liquid, inoculum size accounts for the volume 10% of substratum.The operation of enrichment culture process is all carried out on normal operations platform, needs bacterium liquid switch over operation speed fast, after 3 switchings, obtains enrichment bacterium liquid.
(4), in anaerobism workstation by enrichment bacterium liquid gradient dilution, then utilize external diameter 3.5 ± 0.1mm, the glass capillary of internal diameter 2.5 ± 0.1mm dip 10
-2, 10
-3, 10
-4the bacterium liquid of concentration carries out percutaneous puncture-inoculation respectively to solid medium, inoculates complete solid medium and produces anaerobism workstation and seal with wax, and then goes to standard incubator and cultivates.
(5), cultivate after about 3 ~ 5 days, in solid medium, grow bacterium colony.In anaerobism workstation, utilize inoculating needle picking list bacterium colony afterwards and be forwarded to liquid nutrient medium, inoculating complete liquid nutrient medium and produce anaerobism workstation and seal with wax, then going to standard incubator and cultivate, cultivating and obtain isolate liquid in 3 ~ 5 days.
(6) after, repeating 4,5 steps 3 time, DNA extraction is carried out to being separated the anerobe obtained, PCR instrument is utilized to increase to its 16S rDNA, by amplified production sequencing analysis, sequencing result is contrasted in ncbi database, find that itself and a fusobacterium anaerobic sulfate reducer homology are the highest, reach 98%.
Embodiment 2
Analyze longitudinal microbiology distribution character of the oily sludge of (16 months) after certain oil field microbial method process, in order to can with the separation and Culture of aerobic microbiological simultaneously, carry out with under condition, have employed the separation and Culture that the inventive method carries out anerobe.
(1), in anaerobism workstation, prepare soil supension (90% sterilized water, 10% soil) 100mL, then suspension liquid is diluted to 10 of original content
-1, 10
-2, 10
-3, as inoculation liquid.
(2) substratum, used consists of: K
2hPO
40.5g, (NH
4)
2sO
41.5g, (NH
4)
2cl 1.5g, Na
2sO
40.5g, NaCl 0.5g, CaCl
20.1g, MgSO
41.0g, Vc 0.1g, (NH
4)
2fe (SO
4) 26H
2o 0.5g, Sodium.alpha.-hydroxypropionate 2.0mL, L-cysteine hydrochloride 0.5g, yeast extract paste 1.5g, resazurin (0.1%) 10, pure water 1.0L; PH value 6.0 ~ 6.5; Solid medium adds agar by 1.5% ~ 2%.
(3), in the substratum that preparing and packaging is complete inflated with nitrogen, carry out high-temperature sterilization.
(4), the inoculation liquid that utilizes needle tubing to prepare in inoculation of medium step 1, primary vaccination amount accounts for 18% of culture volume, inoculates complete substratum and carries out sealing with wax and turn incubator 30 DEG C of cultivations.Inoculate to fresh culture after obtaining bacterium liquid, inoculum size accounts for the volume 8% of substratum.Inoculate complete substratum to carry out sealing with wax and turn in incubator and cultivate under similarity condition with aerobic bacteria.The operation of enrichment process is all carried out on normal operations platform, needs bacterium liquid switch over operation speed fast, after 3 switchings, obtains enrichment bacterium liquid.
(5), in anaerobism workstation by enrichment bacterium liquid gradient dilution, then utilize external diameter 2.8 ± 0.1mm, internal diameter 1.8 ± 0.1mm glass capillary dips the bacterium liquid of respective concentration and carry out percutaneous puncture-inoculation to solid medium, inoculate complete solid medium produce anaerobism workstation and seal with wax, then go to incubator and aerobic bacteria is cultivated under the same conditions.
(6), cultivate 3 ~ 5 days time after, grow bacterium colony in solid medium.In anaerobism workstation, utilize inoculating needle picking list bacterium colony afterwards and be forwarded to liquid nutrient medium, inoculating complete liquid nutrient medium and produce anaerobism workstation and seal with wax, then going to standard incubator and cultivate, cultivating and obtain isolate liquid in 3 ~ 5 days.
(7), DNA extraction is carried out to being separated the anerobe obtained, utilize PCR instrument to increase to its 16S rDNA, by amplified production sequencing analysis, sequencing result is contrasted in ncbi database, find that itself and a strain anaerobic methanogens homology are the highest, reach 99%.
Claims (10)
1. an isolation cultivation method for anerobe, comprises inflated with nitrogen, the enrichment culture of bacterial classification and the separation and Culture of bacterial classification before the preparation of substratum, medium sterilization; Wherein the separation and Culture of bacterial classification adopts liquid nutrient medium and solid medium to replace the mode of inoculation culture, the inoculation of liquid culture basal orientation solid medium adopts glass capillary to draw bacterium liquid percutaneous puncture-inoculation to be separated, the smooth cylindric Anaerobic culturel chamber of an inoculating surfaces is formed in solid medium, the circular hole sealing that solid culture primary surface is formed by not solidified substratum is adopted after inoculation, thus forming the Anaerobic culturel chamber of sealing, the inwall in Anaerobic culturel chamber is the cultivation face of anerobe; The inoculation of solid culture basal orientation liquid nutrient medium is extend into by inoculating needle in Anaerobic culturel chamber, single bacterium colony access liquid nutrient medium that picking is formed from cultivation face.
2. in accordance with the method for claim 1, it is characterized in that: the preparation of described substratum, according to the original place environment of anerobe to be separated, determine composition and the proportioning of corresponding substratum.
3. in accordance with the method for claim 1, it is characterized in that: the enrichment culture of described bacterial classification is first activated by bacterial classification, then between corresponding liquid nutrient medium, obtain enrichment bacterium liquid through 2 ~ 5 switchings.
4. in accordance with the method for claim 3, it is characterized in that: during primary activation inoculation, inoculum size accounts for 10% ~ 20% of culture volume; After obtaining the bacterium liquid cultivated, inoculum size accounts for 5% ~ 10% of culture volume.
5. in accordance with the method for claim 1, it is characterized in that: the separation and Culture of described bacterial classification be by after enrichment bacterium liquid gradient dilution to the process of solid medium inoculation, need to carry out in anaerobism workstation, add plug after inoculation, and seal with wax immediately after shifting out anaerobism workstation.
6. in accordance with the method for claim 1, it is characterized in that: described glass capillary specification is external diameter 2.0 ~ 4.0mm, internal diameter 1.5 ~ 3.0mm, length 10 ~ 20cm.
7. according to the method described in claim 1 or 6, it is characterized in that: during percutaneous puncture-inoculation, inoculum size is dip once in the bacterium liquid diluted with glass capillary.
8. according to the method described in claim 1 or 6, it is characterized in that: the bottom or the inwall that touch culturing bottle during glass capillary percutaneous puncture-inoculation.
9., according to the method described in claim 1 or 6, it is characterized in that: during glass capillary percutaneous puncture-inoculation and media surface tilt.
10. in accordance with the method for claim 1, it is characterized in that: culture temperature can be determined according to bacterial classification original place to be separated growing environment, cultivate 3 ~ 7 days at 10 ~ 60 DEG C.
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| CN105274002A (en) * | 2015-11-23 | 2016-01-27 | 桂林理工大学 | Enrichment culturing method for anaerobic microorganisms |
| CN106635863A (en) * | 2016-07-19 | 2017-05-10 | 桂林理工大学 | Culturing method of clostridium strain YB-7 for anaerobically degrading oil-field wastewater |
| CN107699519A (en) * | 2017-10-18 | 2018-02-16 | 三峡大学 | One strain of sulfate reduction bacteria, isolation and identification method and its application |
| CN115824932A (en) * | 2022-12-07 | 2023-03-21 | 瑞芯智造(深圳)科技有限公司 | Method and kit for detecting concentration of sulfate reducing bacteria |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105274002A (en) * | 2015-11-23 | 2016-01-27 | 桂林理工大学 | Enrichment culturing method for anaerobic microorganisms |
| CN106635863A (en) * | 2016-07-19 | 2017-05-10 | 桂林理工大学 | Culturing method of clostridium strain YB-7 for anaerobically degrading oil-field wastewater |
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| CN115824932A (en) * | 2022-12-07 | 2023-03-21 | 瑞芯智造(深圳)科技有限公司 | Method and kit for detecting concentration of sulfate reducing bacteria |
| CN115824932B (en) * | 2022-12-07 | 2024-06-04 | 瑞芯智造(深圳)科技有限公司 | Method and kit for detecting concentration of sulfate reducing bacteria |
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|---|---|
| CN104560771B (en) | 2017-08-22 |
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