CN102286380B - Method for enriching microbes by using solid culture media - Google Patents
Method for enriching microbes by using solid culture media Download PDFInfo
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- CN102286380B CN102286380B CN201110190931.9A CN201110190931A CN102286380B CN 102286380 B CN102286380 B CN 102286380B CN 201110190931 A CN201110190931 A CN 201110190931A CN 102286380 B CN102286380 B CN 102286380B
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- bacterium
- enrichment
- enriching
- culture media
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- 239000007787 solid Substances 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 title abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 229920002148 Gellan gum Polymers 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 41
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 19
- 235000010413 sodium alginate Nutrition 0.000 claims description 19
- 239000000661 sodium alginate Substances 0.000 claims description 19
- 229940005550 sodium alginate Drugs 0.000 claims description 19
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 230000003068 static effect Effects 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract 2
- 238000000746 purification Methods 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000002609 medium Substances 0.000 description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 238000009533 lab test Methods 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 241000282461 Canis lupus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000626621 Geobacillus Species 0.000 description 1
- 241001576832 Geobacillus sp. Y4.1MC1 Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 1
- 241000822976 bacterium M-1 Species 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Abstract
The invention discloses a method for enriching microbes by using solid culture media. The solid culture media with different substrates are prepared, solidifying agents are added and adopt 1.5 to 2 percent phytagel or solidifying agents incapable of being utilized by any substrates, and target strains are obtained through further separation and purification. Different substrates are used for enriching different microbes in the same liquid environment on different culture media, and a fast and efficient enriching method is provided for the microbe screening.
Description
Technical field:
The invention belongs to biological technical field, particularly, relate to the method for microorganism that utilizes the different substrate utilizations of solid medium enrichment, and the sodium alginate decomposer that utilizes aforesaid method, the application of cellulose-decomposing bacterium and saccharomycetic concentration and separation.
Background technology:
The screening of microorganism refers to carry out selected operating process by intended target with regard to the bacterial classification that certain has special properties in multiple-microorganism.Its operation steps generally is divided into, primary dcreening operation, and multiple sieve separates several steps of purifying.The purpose of primary dcreening operation is to leave out clear and definite undesirable most of bacterial strain, and the similar bacterial strain of production shape is remained as far as possible, strain excellent is unlikely slips through the net.But, may be because the quantity of purpose bacterial strain be few and failed in the primary dcreening operation process, thus first carry out enrichment at primary dcreening operation as last, seek a kind of fast, enriching method will be conducive to next step screening efficiently.Adopt the solid medium enrichment, not only can make microorganism fast at the solid medium surface enrichment, but also can be in same enrichment environment the dissimilar microorganism of enrichment.In prior art there are no the report of this method.
Summary of the invention:
The purpose of this invention is to provide a kind of method with solid medium enrichment different microorganisms, relate to the making of solid culture matrix, enriching method and the application that utilizes the concentration and separation of aforesaid method.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Utilize the method for solid medium enriched microorganism, comprise the steps:
(1) the different substrate cultivation bases of preparation, add peptizer 1.5-2%phytagel or anyly can not be the peptizer of substrate utilization, and sterilization after being down flat plate, is got difform solid piece with punch tool;
(2) solid piece is joined in aqua sterilisa the bacterium liquid of access 10%;
(3) 42 ℃, 150rpm, shaking table is cultivated; Or 55 ℃, static cultivation;
(4) cultivate 1-2d, solid piece is taken out, cultivate 1-2d on the corresponding flat board of putting into different substrates successively, or put on liquid nutrient medium and cultivate.
Description of drawings:
Fig. 1 and Fig. 2 are one group of parallel laboratory test of Xylo-Mucine decomposer, can find out around the enrichment piece from two picture groups and surface growth goes out bacterium colony (bacterium I Xylo-Mucine decomposer) and without other varied bacteria growing;
Fig. 3 and Fig. 4 are one group of parallel laboratory test, only have Yeast Growth (bacterium II yeast) on the YPD substratum, and the growth fraction comparatively dense, almost do not see single bacterium colony.
Fig. 5 is the experiment of sodium alginate decomposer, only have sodium alginate to utilize the growth (bacterium III sodium alginate decomposer) of bacterium on flat board, as seen from Figure 5, the sodium alginate decomposer growth is not only arranged on enrichment piece surface and around the enrichment piece growth also relatively good.
Fig. 6 is not for accessing the 0.5%AVicel substratum before the enrichment culture piece;
Fig. 7 and Fig. 8 are one group of parallel test, after access crystalline cellulose AVicel enrichment piece substratum 24h, as can be seen from the figure after 24h, flavescence of AVicel in liquid, and the enrichment culture matrix is flavescence also, illustrates that AVicel is utilized, AVicel enrichment piece substratum enrichment success;
Fig. 9 is the cellulose-decomposing bacterium M116srDNA sequence that obtains after the enrichment screening.
Embodiment:
Below in conjunction with accompanying drawing, further explain content of the present invention and effect thereof with following illustrated embodiment, but should not be considered as limitation of the scope of the invention.
Embodiment 1:
Different culture media, the experiment of three kinds of different microorganisms bacterial classifications of different substrate solid piece enrichment:
Bacterial classification used is described below:
Bacterium I: the sodium alginate decomposer, can grow on sodium alginate is the substratum of sole carbon source, and not utilize CMC or AVicel and sucrose, this bacterial strain to belong to Cellvibio to belong to;
Bacterium II: cellulose-decomposing bacterium, can grow on CMC or AVicel, can not utilize sodium alginate and sucrose; This bacterial strain belongs to Geobacillus and belongs to;
Bacterium III: yeast can utilize the various saccharides such as glucose, sucrose, but because bacterium I and bacterium II also can utilize glucose, selecting sucrose when therefore testing is substrate.The similarity of this bacterial strain and Oqataea polymorpha strainATCC66057 is 99%.
Culture medium prescription is as follows:
Sodium alginate substratum: K
2HPO
4: 1.0g; NaH
2PO
4: 4.0g; Mg SO
46H
2O； NH
4Cl:1.0g, 1% sodium alginate; PH 7.0
Mierocrystalline cellulose substratum: KH
2PO
4: 0.75g; K
2HPO
4.3H
2O:1.50g; CaCl
2: 0.15g; MgCl
2.6H
2O:0.4g; NH
4Cl:0.9g; NaCl:0.9g; FeSO
47H
2O:3mg; 0.5%CMC or 0.5%AVicel; 0.10% resazurin; Wolf ' s solution:1mL; Be settled to 1L; PH 7.0
Yeast YPD substratum: yeast extract:1%; Peptone:2%; Sucrose: 2%.
Prepare respectively sodium alginate decomposer substratum, cellulose decomposition bacterium culture medium and yeast YPD substratum add 2%phytagel, and sterilization after being down flat plate, is got difform solid piece with punch tool; Solid piece is joined in aqua sterilisa, respectively bacterium liquid (the bacterium I: sodium alginate decomposer of corresponding access 10%; Bacterium II: cellulose-decomposing bacterium; Bacterium III: yeast), 42 ℃, 150rpm, shaking table is cultivated, or 55 ℃, static cultivation; Cultivate 1-2d, solid piece is taken out, cultivate 1-2d on the corresponding flat board of putting into above-mentioned different culture media substrate successively, observe the growth situation.
The growing state of bacterium as shown in the figure, figure (1) (2) be one group of parallel laboratory test of Xylo-Mucine decomposer, can find out from two picture groups at the enrichment piece on every side and surface growth goes out bacterium colony (bacterium I Xylo-Mucine decomposer) and without other varied bacteria growing; Figure (3) (4) are one group of parallel laboratory test, only have Yeast Growth (bacterium III yeast) on the YPD substratum, and the growth fraction comparatively dense, almost do not see single bacterium colony.Figure (5) is the experiment of sodium alginate decomposer, only have sodium alginate to utilize the growth (bacterium II sodium alginate decomposer) of bacterium on flat board, as seen from Figure 5, the sodium alginate decomposer growth is not only arranged on enrichment piece surface and around the enrichment piece growth also relatively good.
Result shows: (Fig. 1 and Fig. 2) only has cellulose decomposition bacteria growing (bacterium I) on the substratum of cellulose-decomposing bacterium; Only have growth (bacterium II) that sodium alginate utilizes bacterium (Fig. 5) on the sodium alginate flat board; Only has saccharomycetic growth (bacterium III) on the YPD substratum, (Fig. 3 and Fig. 4).
Experiment can be found out thus, utilizes different substratum, can enrichment according to the specificity of different substrate utilizations, and separate to a certain extent different strain.
Embodiment 2:
Enrichment cellulosic decomposer in compost sample:
Embodiment is similar to example 1, and difference is that the substrate of used medium solid piece is 0.5%Avicel (figure (6)), accesses the solid culture matrix in liquid nutrient medium after enrichment.Cultivate after one day, Avicel flavescence gradually in liquid illustrates that Avicel is utilized, cultivates two days later, and Avicel enrichment piece also begins flavescence, and illustrating enrichment piece surfaces A vicel decomposer also has growth (Fig. 7 and Fig. 8).Separate the bacterial strain process blast comparison of screening after order-checking (sequencing result is seen Fig. 9) and find that the similarity of this bacterial strain and Geobacillus sp.Y4.1MC1 reaches 99%, M1 is a strain cellulose-decomposing bacterium that filters out under 55 ℃, can utilize glucose, the multiple substrate such as CMC.The feasibility of solid culture matrix enrichment purpose bacterial classification has further been verified in this experiment.Growing state such as Fig. 6,7,8 of bacterium after access enrichment piece.
Before and after access enrichment piece, substratum contrast: figure (6) is not for accessing the 0.5%AVicel substratum before the enrichment culture piece, figure (7) (8) are one group of parallel test, after access crystalline cellulose AVicel enrichment piece substratum 24h, as can be seen from the figure after 24h, flavescence of AVicel in liquid, and the enrichment culture matrix is flavescence also, illustrates that AVicel is utilized, AVicel enrichment piece substratum enrichment success.
The cellulose-decomposing bacterium M1 16srDNA sequence that obtains after the enrichment screening:
GGTTACCTTGTTACGACTTCACCCCAATCACTTGCCCCACCTTCGGCGGCTGGCTCCCTTGCGGGTTACCTCACCGACTTCGGGTGTTGCAAGCTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAGCGGCTTTTTGGGATTCGCTCCCCCTCGCGGGTTCGCAGCCCTTTGTACCGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGACTTTTAGCCGGCAGTCCCCCTAGAGTGCCCAACTGAATGCTGGCAACTAGTGGCGAGGGTTGCGCTCGTTGCGGGACTTAA?CCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCCTGTCCTCCCGCAAGGGAGGAACGCCCTGTCTCCAGGGTTGTCAGGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAACGCGTTAGCTACAGCACTAAAGGGAATAACCCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCGCTCTCCTCTTCTGCACTCAAGTCCCCCAGTTTCCAATGACCCTCCACGGTTAAGCCGTGGGCTTTCACATCAGACTTAAGGGACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTCGCCCCCTATGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTTCTCGTTAGGTACCGTCACCGCGCCGCCCTGTTCGAACGGCACTTCTTCTTCCCTAACAACAGAGCTTTACGATCCGAAGACCTTCTTCGCTCACGCGGCGTCGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGCCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGCCCATCCGTAAGTGGCAGCCGGAGCCGCCTTTCAACCGAAGACCATGCGGTCTTCGGTGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCGGTCTTACGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACCAAGCGGAAGCAAGCTCCCGCCCGGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCT?。
Claims (1)
1. utilize the method for solid medium enriched microorganism, it is characterized in that comprising the steps:
(1) the different substrate cultivation bases of preparation, add peptizer 1.5-2% phytagel or anyly can not be the peptizer of substrate utilization, and sterilization after being down flat plate, is got difform solid piece with punch tool;
(2) solid piece is joined in aqua sterilisa the bacterium liquid of access 10%;
(3) 42 ℃, 150rpm, shaking table is cultivated; Or 55 ℃, static cultivation;
(4) cultivate 1-2d, solid piece taken out, cultivate 1-2d on the corresponding flat board of putting into different substrates successively, or put on liquid nutrient medium and cultivate,
Described bacterium liquid is sodium alginate decomposer, cellulose-decomposing bacterium or yeast.
?
?
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| CN103525717B (en) * | 2013-06-04 | 2014-12-31 | 昆明理工大学 | Anaerobic alginate decomposing bacterium and application thereof |
| CN114104482A (en) * | 2021-11-05 | 2022-03-01 | 西南大学 | Kit for separating and decomposing cellulose microorganisms for teaching and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1167148A (en) * | 1997-05-19 | 1997-12-10 | 刘志强 | Technology for preparing polymerized gel biological compatibility carrier products |
| US6365407B1 (en) * | 2001-03-05 | 2002-04-02 | Council Of Scientific & Industrial Research | Culture medium composition useful for induction and proliferation of Taxus calli |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1167148A (en) * | 1997-05-19 | 1997-12-10 | 刘志强 | Technology for preparing polymerized gel biological compatibility carrier products |
| US6365407B1 (en) * | 2001-03-05 | 2002-04-02 | Council Of Scientific & Industrial Research | Culture medium composition useful for induction and proliferation of Taxus calli |
Non-Patent Citations (1)
| Title |
|---|
| 吴清平，等.微生物固体培养基凝固剂研究进展.《微生物学通报》.2006,第33卷(第5期),145－149. * |
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