CN104560771B - A kind of isolated culture method of anaerobic bacteria - Google Patents
A kind of isolated culture method of anaerobic bacteria Download PDFInfo
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- CN104560771B CN104560771B CN201310517811.4A CN201310517811A CN104560771B CN 104560771 B CN104560771 B CN 104560771B CN 201310517811 A CN201310517811 A CN 201310517811A CN 104560771 B CN104560771 B CN 104560771B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a kind of isolated culture method of anaerobic bacteria, including inflated with nitrogen before the preparation of culture medium, medium sterilization, the enrichment culture of strain and strain are separately cultured；Wherein strain is separately cultured by the way of fluid nutrient medium replaces inoculated and cultured with solid medium, the inoculation of Liquid Culture basal orientation solid medium uses glass capillary percutaneous puncture-inoculation, the smooth cylindric Anaerobic culturel chamber of an inoculating surfaces is formed in solid medium, the circular hole for being formed solid culture primary surface using not solidified culture medium after inoculation is sealed, so as to form sealed Anaerobic culturel chamber, the inwall of Anaerobic culturel chamber is the culture face of anaerobic bacteria.The inventive method forms the Anaerobic culturel chamber of inoculating surfaces flat smooth in solid medium, and anaerobic environment is provided for the culture of anaerobic bacteria, reduces the use of anaerobism equipment, simplifies separation process, saves separation process expense, shortens the acquisition single strain time.
Description
Technical field
The present invention relates to microorganism field, more particularly to detest in Petroleum Production recycle-water, oilfield sewage and oily sludge
The isolated culture method of oxygen bacterium.
Background technology
In petroleum chemical industry, corrosion caused by microorganism result in huge economic loss.There are document report, oil well
In more than 75% burn into buried pipeline and cable in 50% corrosion of the failure from microorganism（Mainly sulfate reduction mistake
Journey）；The another pertinent literature for having 2008 is reported, in China, is up to 200,000,000 yuan to the loss that oil field is caused due to corroding every year.
Exploitation with most onshore oil fields enters the intermediary and later stages, to improve recovery ratio water filling, steam injection and oil field reinjection water
Amount increasing year by year, extraction crude oil moisture content also increasing, in order to prevent microbiologic(al) corrosion be accomplished by oil field produce
With more chemical bactericides are added in water, because microbial evolution speed is fast, to the adaptable of environment, length is often led to
The bactericide effect that phase uses declines, while the difficulty for adding and causing disposing polluted water in oil of bulk sterilization agent strengthens.So,
The loss that microorganism is caused to oil field rises year by year.
The major class of anaerobic and aerobic two can be divided into by causing the microorganism of corrosion in process of oil production, in actual production environment
In, it is aerobic typically to occur simultaneously with Anaerobic Corrosion together with a variety of anaerobic bacterias coexist with aerobic bacteria in bacterial community, cause corrosion
Microorganism mainly have：Sulfate reducing bacteria（Generation sulfate-reducing process）, sulfur oxidizing bacterium, saprophytic bacteria, iron bacteria and fungi.
There is document report, sulfate reducing bacteria therein not only results in equipment corrosion, simultaneously because producing vulcanization in its metabolic process
Hydrogen, has potential personal safety to endanger, and because security incident caused by hydrogen sulfide is also appeared in the newspapers repeatly on oil field.
Corrosion caused by microorganism is prevented, while reducing chemical bactericide as far as possible to the pollution of environment it is necessary to from research
The physiological property of microorganism is set about, and finds the factor for suppressing its growth and breeding.So, first have to isolate from production environment and draw
The microorganism of harm is played, the separation of wherein aerobic microbiological is fairly simple.And traditional anaerobic bacteria isolated culture method, such as anaerobism
The method such as sandwich culfure, complex operation in tank, hungate rolling tube method and anaerobism work station, time-consuming for process, and costly.
CN200710064715.3 discloses a kind of isolated culture method of anaerobic bacteria, and this method is during whole operation
Avoid contact of the purpose microorganism with oxygen, running cost relative moderate, particularly suitable for growth temperature higher than 50 DEG C and to detesting
Oxygen environment requirement strict difficult separating high-temp anaerobic bacteria, strict anaerobes are separately cultured.This method need to only be separated in anaerobic bacteria
Part operation when use anaerobism work station, standard incubator can be used during culture, without continue provide anaerobic environment, it is to avoid
An anaerobism work station is only used for a kind of situation of the bacterium of growth temperature when being separately cultured.But, the invention is not using
Solidify agar（Temperature is higher than 50 DEG C）Culture medium be inoculated with the mode that Enrichment of bacteria liquid is mixed, be less than for growing environment temperature
50 DEG C of anaerobic bacteria, the seeded process described in the invention can influence the activity of microorganism to be separated, even result in it dead.
The content of the invention
The present invention provides a kind of isolated culture method of anaerobic bacteria.The inventive method can form inoculation in solid medium
The Anaerobic culturel chamber of face flat smooth, anaerobic environment is provided for the culture of anaerobic bacteria, is reduced the use of anaerobism equipment, is simplified
Separation process, saves the expense of separation process, shortens the time for obtaining single strain.
Inflated with nitrogen before the isolated culture method of anaerobic bacteria of the present invention, including the preparation of culture medium, medium sterilization, strain
Enrichment culture and strain are separately cultured；Wherein being separately cultured for strain replaces inoculation with solid medium using fluid nutrient medium
The mode of culture, Liquid Culture basal orientation solid medium inoculation dips bacterium solution percutaneous puncture-inoculation to be separated using glass capillary,
The smooth cylindric Anaerobic culturel chamber of an inoculating surfaces is formed in solid medium, will be solid using not solidified culture medium after inoculation
The circular hole sealing of body media surface formation, so as to form sealed Anaerobic culturel chamber, the inwall of Anaerobic culturel chamber is anaerobic bacteria
Culture face；Solid culture basal orientation fluid nutrient medium inoculation be that transfer needle is extend into Anaerobic culturel intracavitary, from culture face on choose
The single bacterium colony to be formed is taken to access fluid nutrient medium.
In the present invention, the preparation of described culture medium, according to the original place environment of anaerobic bacteria to be separated, it is determined that corresponding culture
The composition and proportioning of base.
In the present invention, inflated with nitrogen before the medium sterilization, it is therefore an objective to remove the oxygen in culture medium is the life of anaerobic bacteria
It is long that anaerobic environment is provided.
In the present invention, the enrichment culture of the strain is first to be activated strain, then in corresponding fluid nutrient medium
Enrichment bacterium solution is obtained by 2 ~ 5 switchings.Inoculation is not used in carrying out in anaerobism work station, it is only necessary to be pierced into the needle tubing after sterilizing
The bacterium solution of respective amount is drawn in plug inoculation on blake bottle.To ensure success rate when primary activation is inoculated with, inoculum concentration accounts for culture medium
The 10% ~ 20% of volume.Obtain after bacterium solution, the inoculum concentration between fluid nutrient medium accounts for the 5% ~ 10% of culture volume.It is inoculated
Journey is rapid to reduce bacterium solution aerial open-assembly time, and inoculation is sealed with wax immediately after finishing.
In the present invention, being separately cultured for the strain is will to be enriched with the mistake being inoculated with after bacterium solution gradient dilution to solid medium
Journey in anaerobism work station, it is necessary to carry out, and inoculation adds plug after finishing, and is sealed with wax immediately after anaerobism work station is removed.Training
Supporting sealing with wax for bottle is used to be sealed between culture bottle plug and bottleneck after docking kind is finished, it is to avoid the entrance of air, maintains to grow
Anaerobic environment in time incubation in blake bottle.
In the present invention, when enrichment bacterium solution is to solid medium percutaneous puncture-inoculation, inoculum concentration is to be diluted with glass capillary
Bacterium solution in dip once.Described glass capillary specification is 2.0 ~ 4.0mm of external diameter, 1.5 ~ 3.0mm of internal diameter, length 10
~ 20cm, can customize or buy standard specification to manufacturer.Because capillary glass tube wall is thin hollow, in percutaneous puncture-inoculation process
The middle culture medium that can be taken out of into glass capillary, without destroying the globality of solid medium, and can be trained in solid
Support the Anaerobic culturel chamber that inoculating surfaces flat smooth is formed in base, identification when so being conducive to choosing bacterium to single bacterium colony.Described hair
Bottom or the inwall of blake bottle are preferably touched during thin glass tube percutaneous puncture-inoculation, so do preferably to take out of by glass capillary
Culture medium therein, advantageously forms a complete, cylindric Anaerobic culturel chamber that inoculating surfaces are smooth.Glass capillary is punctured
Preferably tilted during inoculation with media surface, be so conducive to the picking of bacterium colony.
In the present invention, a circular hole, Circularhole diameter and capillary glass can be formed in solid culture primary surface after percutaneous puncture-inoculation
The internal diameter of pipe is relevant.The sealing of circular hole can be dripped few drops on media surface circular hole using dropper or liquid-transfering gun and not solidified culture
Base formation sealant, runs into after solid medium cooled and solidified quickly due to not solidifying culture medium, will not touch anaerobism cavity
Interior inoculating surfaces, so that Anaerobic culturel chamber will not be destroyed.For the relatively low anaerobic bacteria of growth temperature, seeded process of the invention is not
It can influence the activity of anaerobic bacteria to be separated because temperature is too high or cause its dead.The inventive method is applied to former growing environment and existed
10-60 DEG C of strain, separation process can be very good to keep former growth temperature with incubation.
In the present invention, during picking single bacterium colony, the sealant of the media surface circular hole can be first with the tweezers after sterilizing
Remove, then utilize transfer needle picking single bacterium colony.
In the present invention, cultivation temperature can be determined according to the former growing environment of strain to be separated, can cultivate 3 ~ 7 at 10 ~ 60 DEG C
My god.
In the present invention, blake bottle is using anaerobic bacteria culture bottle, preferably by medical discarded glass infusion bottle, to save training
50mL infusion bottle can be utilized by supporting the consumption enrichment culture process of base, and 100mL infusion bottle can be utilized by being separately cultured, used
Infusion bottle cleaning, sterilizing after can repeatedly use.
Compared with prior art, the present invention has following prominent effect：
1st, realized using glass capillary from Liquid Culture basal orientation solid medium percutaneous puncture-inoculation, the shape in solid medium
Not solidified culture medium is used into the cylindric Anaerobic culturel chamber that an inoculating surfaces are smooth, after inoculation by solid culture primary surface shape
Into circular hole sealing, so as to form sealed Anaerobic culturel chamber, present approach provides the anaerobic environment of anaerobism bacteria growing, no
The bioactivity of thalline can be influenceed, and is easy to identification and the picking of single bacterium colony, the time for obtaining single strain is shortened.
2nd, the present invention is only inoculated with from Liquid Culture basal orientation solid medium using glass capillary process and from solid
Cultivating the process of basal orientation fluid nutrient medium picking single bacterium colony needs to carry out in anaerobism work station, reduces to anaerobism work station
Use.The culture of strain need to only be cultivated in standard incubator after inoculation, it is not necessary to special Anaerobic culturel equipment, culture temperature
The regulation of degree is more convenient, it is to avoid an anaerobism equipment is only used for a kind of situation of the thalline of growth temperature when being separately cultured,
Save the expense of anaerobic bacteria separation process.
3rd, compared with plate sandwich culfure, present invention, avoiding activity of the high temperature of non-freezing solid culture medium to anaerobic bacteria
Influence, be conducive to keep strain separating, the uniformity of culture environment temperature.Compared with media surface seals with wax mode, save
Consumptive material, and the more convenient operation during strain transfer, accelerate the separation process of anaerobic bacteria.The present invention is manually being operated
Upper difficulty reduction, reduces the dependence to anaerobism work station in anaerobic bacteria culture separation process, so as to save consumptive material（Such as nitrogen
Gas）.
Embodiment
Below by embodiment, the invention will be further described.
The water sample water quality at anaerobic bacteria source to be separated is analyzed first, the information of corresponding original place environment is obtained,
And then existing culture medium prescription is adjusted, to keep separating obtained strain local environment and original place in physiological property research process
Environment is as consistent as possible.On the one hand so do can reduce the time for obtaining single strain, on the other hand make acquisition data and reality
More it is consistent.
Culture medium, which is prepared, to be finished after packing well, first inflated with nitrogen, then carries out high-temperature sterilization to culture medium and experiment appliance.
Plug on blake bottle will be covered before sterilization, clamp rope between plug and bottleneck, with prevent in autoclaving process plug because
Gas expansion is ejected, and after sterilizing is finished, is first had to take out cotton rope and is covered plug.
First enrichment to anaerobic bacteria in oil field reinjection water, inoculum concentration is big, accounts for the 10% ~ 20% of fluid nutrient medium volume.
The enrichment bacterium solution of anaerobic bacteria utilizes common syringe by richness by 2 ~ 5 acquisitions of being transferred between liquid medium within, enrichment switching process
Collection bacterium solution is transferred to fresh culture, and whole process is carried out on normal operations platform, and inoculation carries out processing of sealing with wax after finishing, then
Incubator is gone to cultivate at a set temperature.
During strain separating, obtained bacterium solution aqua sterilisa gradient dilution to original will be enriched with first in anaerobism work station
The 10 of concentration-2、10-3、10-4、10-5, the bacterium solution diluted is then dipped using glass capillary, punctures and connects to solid medium
Kind.It is inoculated with after finishing using the sealing of not solidified culture medium, then goes to incubator and cultivates at a set temperature.By solid medium
To fluid nutrient medium seeded process, with the single bacterium colony in transfer needle picking solid medium Anaerobic culturel chamber, liquid training is seeded to
Base is supported, enrichment bacterium solution is obtained by culture in 3 ~ 5 days.Repeat the above steps 2 ~ 5 times, you can to obtain single strain.
The method for culturing and separating of anaerobic bacteria of the present invention, by the inoculation in anaerobic bacteria separation process and culture environment point
Leave and, the inoculation between liquid bacterium solution is carried out also without in anaerobism work station, makes the culture of anaerobic bacteria, separation method cleverer
It is living.The correlation that the anaerobic bacteria isolated culture method that the present invention is provided is equally applicable in anaerobic bacteria physiological property research process is real
Test, and blake bottle can be replaced by plug test tube to save culture medium consumption, can equally save anaerobic bacteria physiological property research
During expense and the time.
Embodiment 1
The anaerobic bacteria sulfate reducing bacteria in certain oil field reinjection water is separately cultured using the inventive method.
（1）, the water quality of certain oil field reinjection water water sample is analyzed, with reference to water quality information to existing sulfate reduction bacteria culture medium
Adjust accordingly.Culture medium is constituted：K2HPO40.5g, (NH4)2SO42.5g, Na2SO41.0g, CaCl20.1g, MgSO4
1.0g, Vc 0.1g, (NH4)2Fe(SO4)2·6H2O（Indicator, culture medium blackening when having Growth of Sulfate Reducing Bacteria breeding）
0.5g, sodium lactate 2.0mL, L-cysteine hydrochloride 0.5g, yeast extract 1.5g, pure water 1.0L；PH6.0~6.5；Solid culture
Base adds agar by 1.5% ~ 2%.
（2）, in the culture medium that preparing and packaging is finished inflated with nitrogen, carry out high-temperature sterilization.
（3）, using needle tubing to inoculation of medium oil field reinjection water water sample, primary vaccination amount accounts for the 20% of culture volume,
The culture medium finished is inoculated with to be sealed with wax and turn incubator in 30 DEG C of cultures.Inoculate to fresh culture, connect after obtaining bacterium solution
The amount of kind accounts for the volume 10% of culture medium.The operation of enrichment culture process is carried out, it is necessary to bacterium solution switch over operation on normal operations platform
Speed is fast, after 3 times are transferred, and obtains being enriched with bacterium solution.
（4）, in anaerobism work station will be enriched with bacterium solution gradient dilution, then using 3.5 ± 0.1mm of external diameter, internal diameter 2.5 ±
0.1mm glass capillary dips 10-2、10-3、10-4The bacterium solution of concentration carries out percutaneous puncture-inoculation, inoculation to solid medium respectively
The solid medium finished produces anaerobism work station and sealed with wax, and then goes to standard incubator culture.
（5）, culture about 3 ~ 5 days after, bacterium colony is grown in solid medium.Chosen afterwards in anaerobism work station using transfer needle
Take single bacterium colony and be forwarded to fluid nutrient medium, be inoculated with the fluid nutrient medium finished and produce anaerobism work station and seal with wax, then go to
Standard incubator culture, cultivates and obtains within 3 ~ 5 days separation bacterium solution.
（6）, repeat after 4,5 steps 3 time, DNA extractions are carried out to isolated anaerobic bacteria, using PCR instrument to its 16S
RDNA is expanded, and sequencing result is contrasted by amplified production sequencing analysis in ncbi database, itself and a shuttle is found
Pseudomonas anaerobic sulfate reducer homology highest, reaches 98%.
Embodiment 2
Analyze after certain oil field handles with microbial method（16 months）Oily sludge longitudinal microbiology distribution character, be
It can be separately cultured simultaneously, carry out with the conditions of with aerobic microbiological, employ point that the inventive method carries out anaerobic bacteria
From culture.
（1）, in anaerobism work station prepare soil supension（90% sterilized water, 10% soil）100mL is then dilute by suspension
Release the 10 of original content-1、10-2、10-3, it is used as inoculation liquid.
（2）, use culture medium composition be：K2HPO4 0.5g, (NH4)2SO4 1.5g, (NH4)2Cl 1.5g, Na2SO4
0.5g, NaCl 0.5g, CaCl2 0.1g, MgSO4 1.0g, Vc 0.1g, (NH4)2Fe(SO4)2·6H2O 0.5g, sodium lactate
2.0mL, L-cysteine hydrochloride 0.5g, yeast extract 1.5g, resazurin（0.1%）10 drops, pure water 1.0L；PH value 6.0~6.5；
Solid medium adds agar by 1.5% ~ 2%.
（3）, in the culture medium that preparing and packaging is finished inflated with nitrogen, carry out high-temperature sterilization.
（4）, the inoculation liquid that is prepared into inoculation of medium step 1 using needle tubing, primary vaccination amount accounts for medium body
Long-pending 18%, is inoculated with the culture medium finished and is sealed with wax and turn incubator in 30 DEG C of cultures.Inoculated after obtaining bacterium solution to fresh training
Base is supported, inoculum concentration accounts for the volume 8% of culture medium.The culture medium finished is inoculated with to be sealed with wax and turned in incubator with aerobic bacteria same
Cultivated under the conditions of sample.The operation of enrichment process is carried out on normal operations platform, it is necessary to which bacterium solution switch over operation speed is fast, by 3
After secondary switching, obtain being enriched with bacterium solution.
（5）, in anaerobism work station will be enriched with bacterium solution gradient dilution, then using 2.8 ± 0.1mm of external diameter, internal diameter 1.8 ±
0.1mm glass capillaries dip the bacterium solution of respective concentration and carry out percutaneous puncture-inoculation to solid medium, are inoculated with the solid training finished
Foster base produces anaerobism work station and sealed with wax, and then goes to incubator and is cultivated under the same conditions with aerobic bacteria.
（6）, culture 3 ~ 5 days when after, bacterium colony is grown in solid medium.Chosen afterwards in anaerobism work station using transfer needle
Take single bacterium colony and be forwarded to fluid nutrient medium, be inoculated with the fluid nutrient medium finished and produce anaerobism work station and seal with wax, then go to
Standard incubator culture, cultivates and obtains within 3 ~ 5 days separation bacterium solution.
（7）, DNA extractions are carried out to isolated anaerobic bacteria, its 16S rDNA is expanded using PCR instrument, will be expanded
Increase production thing sequencing analysis, sequencing result is contrasted in ncbi database, it is found that it is homologous with one plant of anaerobic methanogens
Property highest, reaches 99%.
Claims (7)
1. a kind of isolated culture method of anaerobic bacteria, including inflated with nitrogen, the enrichment of strain before the preparation of culture medium, medium sterilization
Culture is separately cultured with strain；The enrichment culture of the strain is first to be activated strain, is then trained in corresponding liquid
Enrichment bacterium solutions are obtained by 2 ~ 5 switchings between foster base, is inoculated with and is not used in the progress of anaerobism work station；Wherein strain is separately cultured
By the way of fluid nutrient medium replaces inoculated and cultured with solid medium, the inoculation of Liquid Culture basal orientation solid medium uses hair
Thin glass tube draws bacterium solution percutaneous puncture-inoculation to be separated, and the smooth cylindric anaerobism training of an inoculating surfaces is formed in solid medium
Chamber is supported, and the circular hole for solid culture primary surface being formed using not solidified culture medium after inoculation is sealed, and is sealedly detested so as to be formed
Oxygen culture chamber, the inwall of Anaerobic culturel chamber is the culture face of anaerobic bacteria；It will be enriched with after bacterium solution gradient dilution to solid culture first
Base is inoculated with, it is necessary to be carried out in anaerobism work station, and inoculation adds plug, and the wax immediately after anaerobism work station is removed after finishing
Envelope, goes to standard incubator culture, and after cultivating 3-7 days, bacterium colony is grown in solid medium；Solid culture basal orientation fluid nutrient medium
Inoculation is that transfer needle is extend into Anaerobic culturel intracavitary in anaerobism work station, from culture face on picking formation single bacterium colony access
Fluid nutrient medium, inoculation, which is finished, to be produced anaerobism work station and seals with wax, and then goes to standard incubator culture, is cultivated 3-7 days and is obtained
Separate bacterium solution；For former growing environment being separately cultured in 10 ~ 60 DEG C of strain.
2. in accordance with the method for claim 1, it is characterised in that：The preparation of the culture medium, according to anaerobic bacteria to be separated
Original place environment, it is determined that the composition and proportioning of corresponding culture medium.
3. in accordance with the method for claim 1, it is characterised in that：When strain enrichment culture primary activation is inoculated with, inoculum concentration is accounted for
The 10% ~ 20% of culture volume；After the bacterium solution for obtaining culture, inoculum concentration accounts for the 5% ~ 10% of culture volume.
4. in accordance with the method for claim 1, it is characterised in that：Described glass capillary specification is 2.0 ~ 4.0mm of external diameter,
1.5 ~ 3.0mm of internal diameter, 10 ~ 20cm of length.
5. according to the method described in claim 1 or 4, it is characterised in that：During percutaneous puncture-inoculation, inoculum concentration is to be existed with glass capillary
Dipped once in the bacterium solution diluted.
6. according to the method described in claim 1 or 4, it is characterised in that：Blake bottle is touched during glass capillary percutaneous puncture-inoculation
Bottom or inwall.
7. according to the method described in claim 1 or 4, it is characterised in that：During glass capillary percutaneous puncture-inoculation and media surface
Tilt.
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| CN105274002A (en) * | 2015-11-23 | 2016-01-27 | 桂林理工大学 | Enrichment culturing method for anaerobic microorganisms |
| CN106635863B (en) * | 2016-07-19 | 2018-09-11 | 桂林理工大学 | Anaerobic degradation handles the cultural method of the Clostridium strain YB-7 of oil extraction waste water |
| CN107699519B (en) * | 2017-10-18 | 2020-10-02 | 三峡大学 | Sulfate reducing bacteria, separation and identification method and application thereof |
| 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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| CN101050438B (en) * | 2007-03-09 | 2010-11-10 | 吉林大学 | New method for separating and purifying strain of bacteria of sulfate reducing bacteria |
| CN101270334B (en) * | 2007-03-23 | 2011-04-20 | 中国科学院过程工程研究所 | Isolated culture method for anaerobic microorganism |
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