CN105950472A - High-throughput microorganism isolation culture method - Google Patents
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Abstract
The invention provides a high-throughput microorganism isolation culture method, which comprises the following steps: performing primary embedding on microorganisms to be isolated by taking agarose as an embedding material, performing secondary embedding to obtain microspheres by taking sodium alginate as an embedding material, and isolating the microspheres by virtue of a flow cytometer. According to the method, an in-situ sample can be directly added into a double-layer sphere culture system to implement co-culture of the microspheres and the in-situ sample. In such a co-culture manner, the difficult-to-culture microorganisms can be cultured more easily, and samples, particularly precious samples which are difficult to process such as nodules, can be fully utilized to increase the utilization rate of the precious samples.
Description
Technical field
The invention belongs to microorganism separation and Culture technical field, be specifically related to the high-throughput isolation of a kind of microorganism
Cultural method.
Background technology
Along with developing rapidly of Sampling techniques, scientists obtains increasing precious sample.How from
In these samples, efficient separation and Culture microorganism becomes the hot issue of world microbiologist research.1887
Year, since the invention of flat board cultural method, the method becomes the method separation and Culture for microorganism of classics.
Afterwards in 1979, scientist finds that obtaining bacterial strain by flat board cultural method is but portion few in sum
Point.People increasingly pay close attention to the weak point of flat board cultural method, first, the nutritional labeling in flat board since then
Ratio and quantity are typically all fixing, and for different sample of originating, plating method is not suitable for major part
The growth of bacterial strain；Secondly, microorganism growth in flat board is unbalanced, the growth of some non-severe bacteria
Rapidly, they can be bred rapidly within a short period of time, and some severe bacteria poor growths, in limited battalion
In the flat board supported, both unbalanced growths create again obvious competitive relation, quick such as non-severe bacteria
Growth consumes substantial amounts of nutrient substance so that the severe bacteria growth having nutritional requirement is more slow, simultaneously
The secondary metabolite that non-severe bacteria produces can change flat board microenvironment, limits severe bacteria to a certain extent
Growth；Finally, some kinds of microorganism itself will not form macroscopic bacterium colony thus can not get separating.
Therefore, scientists continuously attempts to carry out various method to improve microorganism separation and Culture efficiency, it is intended to obtain
Increasing microbe species.
Zengler etc. (2002) devise a kind of high-throughput isolation cultural method, and the method combines microsphere bag
Bury technology and low cytometric analysis, cultivate for selectivity and not cultivating in sorting manual simulation system
Antibacterial.Idiographic flow is, first passes through micro-embedded technology and is embedded in microsphere by single or several microbial cells,
Then about million microspheres are put into and chromatographic column is cultivated, chromatographic column is continually fed into filtration sterilization
Water body provides nutrient substance in situ, and the microbes that can grow forms monoclonal in microsphere, finally uses
Flow cytometer screening carries out separation and Culture containing monoclonal microsphere.This technology has 4 advantages, is first
A kind of state co-cultured is provided, from the microbial co culture of same environment in same system, they
Simply being physically isolated, signal conduction and material exchange between cell are not blocked；Secondly, whole
Culture environment is the culture medium of flowing continuously, is usually sea water etc. in situ, and lasting extra-nutrition is also simulated
In situ environment；Again, the flow cytometer in later stage screens the combination with microwell plate, it is easy to realize downstream
Amplification culture or selectivity cultivate.Compared with classical Marine microorganism isolated culture method, the method is first
Microorganism is separated from environment and cultivates again, and traditional method contrast, it is first by micro-life
Thing is cultivated together, waits and separates after growing single bacterium colony, and it is good that distinct thinking makes the method obtain
Good separation and Culture effect.
But, applicant repeatedly attempts mentioned microorganism high-throughput isolation culture technique, sends out in incubation
Existing above-mentioned method, in microsphere incubation, grows rapid microbes and grows microsphere, be diffused into cultivation
In environment, and the sea water circulated cannot fully and completely remove the microorganism that these are free.These broken balls and go out
Bacterial strain can stick on microsphere, the sorting of severe jamming flow cytometer.
Summary of the invention
A kind of method that it is an object of the invention to provide high-throughput isolation cultivating microorganism, i.e. a kind of efficiently acquisition
The method of microbial resources, thus overcome the shortcoming in current method.
The method of the present invention effectively solves dominant bacteria present in single embedding method and " pollutes " culture systems
Problem, it is ensured that the microsphere surface for flow cytometer screening will not adhere to other microorganisms, adds separation
Efficiency.
The method of the present invention, is first to carry out wrapping for the first time with agarose for embedded material by microorganism to be separated
Bury, then carry out secondary embedding with sodium alginate for embedded material and obtain microsphere, then by thin for microsphere streaming
Born of the same parents' instrument separates.
Comprise the following steps that
1) prepared by bacteria suspension:
First it is enlarged cultivating by the flora separated from sample, it is thus achieved that bacteria suspension；
The method of one of which amplification culture, is to put in chromatographic column by sample, with sterilizing water body as cultivation
Liquid is cultivated, and after enrichment culture, filters cultivating bacterium solution, it is thus achieved that bacteria suspension；
2) Agarose embedding
Join agarose is mixed by the bacteria suspension prepared and make mixed liquor, then mixed liquor is added drop-wise to
In liquid paraffin, it is stirred；After stirring, immediately emulsion is poured in ice bath standing, treats micro-
After ball is formed, emulsion adds normal saline, then is centrifuged collecting microsphere；
Described normal saline is the NaCl aqueous solution of 0.85%；
3) sodium alginate micro ball is prepared
By step 2) microsphere prepared is added in sodium alginate soln, is added drop-wise to CaCl after being mixed2In solution,
After treating its whole droppings, standing processes, and collects microsphere, carries out chromatographic column cultivation after rinsing microsphere；Chromatography
The growth that the sample adding initial separation flora in post is microorganism provides growth desired substance in situ, and leads to
Enter sterilizing water body and nutrient substance is provided；
4) screening of microsphere after cultivating
By step 3) microsphere cultivate no less than after 3 weeks in chromatography strain, collect microsphere, microsphere be added to
50mM EDTA solution containing 0.85%NaCl make microsphere dissolve till being invisible to the naked eye；With filter
Film filters, and after filtration, microsphere can screen with flow cytometer；
Described filtration, is first to filter with the bolting silk of 20 μm, and the microsphere of collection is again by 70 μm
Silk cover filtering；
5) enrichment culture of microorganism and isolated and purified qualification
Microsphere after screening is cultivated by enriched medium, after making the internal bacterial multiplication of microsphere long broken microsphere and
Orifice plate is enriched with, then is purified.
Wherein enriched medium uses 2216E, R2A, SPGN, SPGS or K culture medium.
The method of the present invention can be directly added into original position sample in double-layer ball culture systems, it is achieved microsphere and former
Position sample co-cultures.This mode co-cultured not only is easier to make the microorganism of difficult cultivation obtain and cultivates, and
And sample can be made full use of, particular for as this amount of tuberculosis block sample little, the most tractable, improve
The utilization rate of precious sample.
Accompanying drawing explanation
Culture method schematic diagram is buried in Fig. 1: double-contracting,
(A, 4 times of object lens are observed in the microexamination of Fig. 2: double-layer ball；B, 10 times of object lens are observed),
The inside that Fig. 3: single investment microsphere microexamination (A) and double investment outer layer are collected after dissolving is micro-
Ball microexamination (B),
(A, normal light sem observation observes shape in internal microsphere in microexamination after the cultivation of Fig. 4: double investment microspheres
Become monoclonal；B, with fluorescence microscope after SYTO-9 fluorescent dyeing),
The broken internal microsphere microexamination (A and B) of Fig. 5: double investment microorganism length and outer layer microsphere are to environment
The barriering effect microexamination (C and D) of microorganism, (from A and B figure it can be seen that micro-life of being embedded
Spread after the broken internal microsphere of thing length；Scheme it can be seen that take from high-concentration bacterial pendular ring border from C and D
The double-layer ball gone out, microorganism is nearly all blocked on outside cladding material),
The bacterial strain cladogram that Fig. 6: Polymetallic Nodules high-throughput isolation is cultivated,
The detection of Fig. 7: Polymetallic Nodules high-throughput isolation part bacterial strain manganese microorganism manganese oxidability (A,
HK032；B, HK082；C, HK002-3；D, HK071；E, HK048；F, HK024-1；G,
Negative control；H, HRA130-1；I, HMA139-2).
Detailed description of the invention
Below in conjunction with example, the method for the present invention is described in detail.
Embodiment 1
1) prepared by bacteria suspension
Polymetallic Nodules gathers from East Pacific nodule province PRZ1302-MC12 in 2013
(154 ° of 49.8218 ' W, 10 ° of 29.2573 ' N) stands, and the depth of water has 5098m.After sampler obtains sample, vertical
I.e. put in sterile sealing bag, 4 DEG C of dark preservations.At laboratory, take one piece of tuberculosis sample (diameter 1～2cm),
Put in 15ml chromatographic column, add aseptic filtration sea water and (supplement 1mM NH4Cl), 10 DEG C of dark enrichments 6
Individual month, monthly (supplement 1mM NH with the sea water of 7ml fresh sterile4Cl) replacing of culture fluid is carried out.
After enrichment culture, take culture fluid 1ml and be filled on 0.22 μm microporous filter membrane and carry out microbial cell meter
Number.According to count results, obtain concentration by sample concentration and be about 107The bacteria suspension of cells/ml.Carry out
Double embedding cultures (flow process such as Fig. 1).
2) Agarose embedding
20ml liquid paraffin (Span 80 containing 1%) is added, afterwards in advance in the big centrifuge tube of 50ml
Prepare and after the thawing of degerming agarose (3.5%, w/v), hang with big centrifuge tube and the bacterium equipped with liquid paraffin
Liquid is unified is placed on room temperature (28 DEG C) or 28 DEG C of water-baths carry out being incubated that (Sigma IX-A type agarose belongs to super
Low temperature agarose, solidification temperature less than 28 DEG C), stirring and evenly mixing.Rotating speed is 1000rpm, first prerotation 2
Min, 200 μ l bacteria suspensions add in the agarose of 1ml liquid and mix, and avoid the occurrence of bubble by period.
Adjustment rotating speed is to 1800rpm afterwards, and is added dropwise over agarose-bacterium mixing in liquid paraffin with 1ml rifle head
Liquid 1ml, keeps the constant stirring 10min that continues of rotating speed, the 50ml small beaker of period preparation ice bath.Stirring
After, immediately emulsion is poured in the small beaker of ice bath, stands 20min, after microsphere is formed,
Adding the most aseptic normal saline (0.85%NaCl aqueous solution) in emulsion, 2000rpm, 6min enter
Row is centrifugal collects microsphere, removes supernatant oil phase, and lower sediment cleans microsphere, transfer with a small amount of physiological saline solution
In new centrifuge tube, so it is repeated several times, it is thus achieved that remove the microsphere of oil phase.
3) sodium alginate micro ball is prepared
Prepare 0.5% sodium alginate aqueous solution 10ml, the microsphere obtained be added in sodium alginate soln,
Fully after mixing, it is added drop-wise to 1%CaCl with 5ml syringe (band syringe needle)2In, shake the most gently during dropping
Rolling CaCl2Aqueous solution, prevents the microsphere front and back formed to be sticked together, and treats that it all drips complete, stands
10min.Remove remaining CaCl2Aqueous solution, the most remaining double-layer ball, carry out after rinsing microsphere with antiseptic sea water
Chromatographic column is cultivated.Adding one piece of former tuberculosis sample in chromatographic column, the growth for microorganism provides growth in situ
Desired substance, and sterilising filtration sea water offer nutrient substance is provided.
The microsphere obtained by above-mentioned steps, is detected it:
1, the double-layer ball cultivating acquisition is carried out microexamination:
Be can be observed by ordinary optical microscope, the diameter of double-layer ball about 2～4mm, wherein contain
Hundreds and thousands of microspheres, microspherulite diameter is about 20～50 μm, and microsphere is more evenly distributed in double-layer ball
Face (such as Fig. 2).Internal the agarose microbeads such as Fig. 3 of the form before embedding, A, microsphere form is rounded,
Edge divider, dispersibility is preferable.Bury and the dissolving of calcium alginate outer layer through double-contracting, the agarose collected
Substantially it is not changed in before the relatively second time embedding of microsphere (Fig. 3, B) form size.In preliminary experiment, bacterium is buried in double-contracting
Strain sed016 (Arthrobacter agilis), is added to double-layer ball in chromatographic column cultivate, and uses 2216E
After 2d cultivated by fluid medium (table 1), obtain internal layer microsphere by dissolving outer layer calcium alginate, show
Microcosmic is examined, and finds to obtain the microsphere that form is complete, and has part microsphere to contain monoclonal (Fig. 4).
2, the restriction to internal spreading
With double investments, one strain pure bacterium sed016 (Arthrobacter agilis) is embedded, use 2216E
Culture medium is cultivated, after 10 DEG C are cultivated 6d, with form and the position of antibacterial of fluorescence microscope microsphere.
Finding that bacterial strain length still rests in microsphere after breaking microsphere, be not the most moved (Fig. 5, A and B).
3, the double-layer embedment isolation to inoculating microbe
Preparing double-layer ball by the double-contracting method of burying, inside does not embed antibacterial, during cultivation in peripheral 2216E culture medium
Add bacterial strain sed016 (Arthrobacter agilis), after 10 DEG C are cultivated 6d, find culture medium the most more
Muddiness, is diluted coating counting to peripheral bacterium solution, double-layer ball is carried out microexamination.Find when microsphere is trained
Support and be with about 10 outside7Time in the collarium border of cell/ml concentration, double-layer ball plays partition effect well, fluorescence
Basis of microscopic observation finds almost without bacterial invasion to double-layer ball internal (Fig. 5, C and D).
4) tuberculosis sample double-layer ball is cultivated and screening
Double-contracting buried the sample of culture method incubation step 3 after 6 weeks, collected double-layer ball, prepared 50mM EDTA
(NaCl containing 0.85%) is used for dissolving outer layer microsphere.Double-layer ball is added to 1L 50mM EDTA (contain
The NaCl of 0.85%) in, magnetic agitation 30min, double-layer ball is slowly dissolved, till being invisible to the naked eye.
Carrying out sucking filtration with manual pump and filter, filter membrane uses the bolting silk of 20 μm, and pressure is less than 15kPa.Filter
After bolting silk put in the big centrifuge tube of 50ml, the filtering sea aseptic with 2ml is carried out, it is thus achieved that micro-
Ball is again by i.e. can use flow cytometer to screen after the silk cover filtering of 70 μm.
5) enrichment culture of microorganism and isolated and purified qualification
Microsphere after screening is cultivated by enriched medium, after making the internal bacterial multiplication of microsphere long broken microsphere and
Orifice plate is enriched with, determines whether bacterial growth by absorbance in detection orifice plate afterwards, it is simple to later separation is pure
Change.Enriched medium uses 2216E, R2A, SPGN, SPGS and K culture medium (such as table 1).
Isolated and purified bacterial strain 382 strain altogether, carries out 16S rRNA gene by picking 75 strain bacterium after HaeIII enzyme action
Order-checking, it is thus achieved that sequence compare in Korea S's database website (http://eztaxon-e.ezbiocloud.net/)
Right, 3 classes of final acquisition, are Bacteroidetes respectively, and γ-deformation Gammaproteobacteria and α-deformation Gammaproteobacteria belong to altogether
There are 11 genus, 15 kinds.(such as table 2, Fig. 6), what wherein quantity was most is γ-deformation Gammaproteobacteria, has 283
Strain, accounts for 74%；Next to that α-deformation Gammaproteobacteria, having 93 strains, Bacteroidetes has 6 strains.Dominant genera is γ-deformation
The sulphite Bacillus of Ke Beite Bordetella Cobetia (22.5%) of Gammaproteobacteria and α-deformation Gammaproteobacteria
Sulfitobacter (22.5%).Bacterial strain HRA130-1 and HRA191 wherein obtained and similar strain
Polaribacter huanghezhanensis SM1202T similarity is 96.22%, belongs to potential novel species.With knot
The flat board coating cultural method at core district PRZ1302-MC12 station is compared, in 11 genus that high-throughput isolation is cultivated,
8 genus (72%) are had not obtain (such as table 2) from flat board cultural method
Table 1 enriched medium
The identification of strains result that table 2 Polymetallic Nodules high-throughput isolation is cultivated
6) strain characteristics detection
(1) microorganism manganese toleration detection
Carry out manganese toleration detection to separating the bacterial strain obtained, prepare manganese Concentraton gradient culture medium, 2.0mg/ml
Peptone, 0.5mg/ml yeast extract, 75% antiseptic sea water, after sterilizing add 20mM HEPES (pH 7.6) and
1～100mM MnCl2(1,5,10,25,50 and 100mM).190 μ l are added respectively in 96 orifice plates,
Take a certain amount of well-grown thalline to add in 200 μ l normal saline, make bacteria suspension, by 10 μ l bacterium
Suspension adds 10 μ l in corresponding aperture to, every strain bacterium do three parallel, in blank add dissolve bacterium solution physiology salt
Water.After interpolation, 96 orifice plates are used to detect under 590nm wavelength, as initial value immediately.28℃
Cultivating and detect absorbance under 3d, 590nm wavelength, OD value increases by 0.1 as positive findings.
Analyzed by absorbance it can be seen that control strain LZB001 Yu LZB002 tolerance manganese concentration model
Enclose is 0～25mM.And bacterial strain HK082, HRA130-1 and HRA126 of obtaining from this separation and Culture,
Low compared with control strain toleration, it is 0～10mM, 0～1mM and 0～5mM respectively.But also there are some bacterial strains
The higher manganese ion toleration, HK048, HK071 and HMA139-2 manganese ion toleration is had to be respectively
0～50mM, 0～more than 100mM and 0～more than 100mM.Other bacterial strains refer to table 3.
Table 3 Polymetallic Nodules high-throughput isolation part bacterial strain manganese tolerability results
(2) microorganism manganese oxidability detection
Choose bacterial strain HK032, HK082, HK002-3, HK071, HK048, HK024-1, HRA130-1
With the detection that HMA139-2 carries out manganese oxidability, by bacterial strain dibbling in K culture medium.After cultivating 6 weeks,
LBB blobs of dye is added on bacterium colony, bacterium colony be blueness then for the positive, color is not changed in as feminine gender.
Use the manganese oxidability (such as Fig. 7) of LBB development process detection different strains, find carrying out testing
In 9 strain bacterium, there are 5 strain bacterium to have manganese oxidation activity, are HK032, HK082, HK071, HK048 respectively
And HMA139-2.
The method of the present invention, on the basis of making full use of tuberculosis sample, separates and obtains flat board coating process not
Some the Deep-Sea Microorganisms kinds obtained, part has manganese toleration and manganese oxidability, and two strains are doubtful
The bacterial strain of the new bacterium in ocean.Cultivation results reflects feasibility and the advantage of high-throughput isolation cultural method, greatly
Compensate for flat board coating cultural method deficiency.
Claims (6)
1. the method for a high-throughput isolation cultivating microorganism, it is characterised in that described method is to treat
The microorganism separated first carries out embedding for the first time, then with sodium alginate for embedding with agarose for embedded material
Material carries out secondary embedding and obtains microsphere, is then separated by microsphere flow cytometer.
2. the method for claim 1, it is characterised in that described method comprises the steps:
1) prepared by bacteria suspension:
First it is enlarged cultivating by the flora separated from sample, it is thus achieved that bacteria suspension；
2) Agarose embedding:
Join agarose is mixed by the bacteria suspension prepared and make mixed liquor, then mixed liquor is added drop-wise to
In liquid paraffin, it is stirred；After stirring, immediately emulsion is poured in ice bath standing, treats micro-
After ball is formed, emulsion adds normal saline, then is centrifuged collecting microsphere；
3) sodium alginate micro ball is prepared:
By step 2) microsphere prepared is added in sodium alginate soln, is added drop-wise to CaCl after being mixed2In solution,
After treating its whole droppings, standing processes, and collects microsphere, carries out chromatographic column cultivation after rinsing microsphere；Chromatography
The growth that the sample adding initial separation flora in post is microorganism provides growth desired substance in situ, and leads to
Enter sterilizing water body and nutrient substance is provided；
4) screening of microsphere after cultivating:
By step 3) microsphere cultivate no less than after 3 weeks in chromatography strain, collect microsphere, microsphere be added to
50mM EDTA solution containing 0.85%NaCl make microsphere dissolve till being invisible to the naked eye；With filter
Film filters, and after filtration, microsphere can screen with flow cytometer；
5) enrichment culture of microorganism and isolated and purified qualification:
Microsphere after screening is cultivated by enriched medium, after making the internal bacterial multiplication of microsphere long broken microsphere and
Orifice plate is enriched with, then is purified.
3. method as claimed in claim 2, it is characterised in that described step 1) in amplification culture,
It is that sample is put in chromatographic column, cultivates as culture fluid with sterilizing water body, after enrichment culture, to training
Bacteria liquid filters, it is thus achieved that bacteria suspension.
4. method as claimed in claim 2, it is characterised in that described step 2) in normal saline
It it is the NaCl aqueous solution of 0.85%.
5. method as claimed in claim 2, it is characterised in that described step 3) in filtration, be
First filtering with the bolting silk of 20 μm, the microsphere of collection is again by the silk cover filtering of 70 μm.
6. method as claimed in claim 2, it is characterised in that described step 5) in enrichment incubation
Base uses 2216E, R2A, SPGN, SPGS or K culture medium.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107008516A (en) * | 2017-05-08 | 2017-08-04 | 北京旌准医疗科技有限公司 | The sorting enriching apparatus and its sorting enrichment method of a kind of intestinal wall cast-off cells |
| CN109097415A (en) * | 2018-08-08 | 2018-12-28 | 浙江海洋大学 | A method of saxitoxin is prepared using malicious ocean sulfurous acid bacillus fermentation is produced |
| CN114958817A (en) * | 2022-06-24 | 2022-08-30 | 江苏大学 | Binuclear shell loaded microbial material and preparation method and application thereof |
| CN116286366A (en) * | 2023-03-15 | 2023-06-23 | 齐齐哈尔大学 | Microbial separation method for alkaline microbial detection |
| CN117106678A (en) * | 2023-10-24 | 2023-11-24 | 中国海洋大学 | Enrichment culture of marine organism film bacteria and method for obtaining genome thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629136A (en) * | 2009-08-06 | 2010-01-20 | 中国海洋大学 | High-efficient culture and sorting method and culture device of microorganisms |
| CN104673777A (en) * | 2014-12-31 | 2015-06-03 | 浙江至美环境科技有限公司 | Microbial microspheres and preparation method and application thereof |
-
2016
- 2016-07-12 CN CN201610547354.7A patent/CN105950472B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629136A (en) * | 2009-08-06 | 2010-01-20 | 中国海洋大学 | High-efficient culture and sorting method and culture device of microorganisms |
| CN104673777A (en) * | 2014-12-31 | 2015-06-03 | 浙江至美环境科技有限公司 | Microbial microspheres and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| EITAN BEN-DOV等: "An in situ method for cultivating microorganisms using a double encapsulation technique", 《FEMS MICROBIOL ECOL》 * |
| 袁东芳等: "海洋微生物高通量培养方法和分选技术的研究进展", 《微生物学通报》 * |
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| CN109097415A (en) * | 2018-08-08 | 2018-12-28 | 浙江海洋大学 | A method of saxitoxin is prepared using malicious ocean sulfurous acid bacillus fermentation is produced |
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| CN117106678A (en) * | 2023-10-24 | 2023-11-24 | 中国海洋大学 | Enrichment culture of marine organism film bacteria and method for obtaining genome thereof |
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