CN102344895A - Method for screening sodium chloride-resistant bacillus licheniformis - Google Patents
Method for screening sodium chloride-resistant bacillus licheniformis Download PDFInfo
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- CN102344895A CN102344895A CN2011102220426A CN201110222042A CN102344895A CN 102344895 A CN102344895 A CN 102344895A CN 2011102220426 A CN2011102220426 A CN 2011102220426A CN 201110222042 A CN201110222042 A CN 201110222042A CN 102344895 A CN102344895 A CN 102344895A
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- bacillus licheniformis
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Abstract
The invention belongs to the field of microorganisms, and relates to a method for screening sodium chloride-resistant bacillus licheniformis, which comprises the following steps: (1) screening: through taking the nutrient agar as an agar medium, the slant culture of a test tube is firstly carried out on the bacillus licheniformis which has the number of 1.0518 and is preserved in the China general microbiological culture collection center (CGMCC), then after the slant streak cultivation of a culture vessel is carried out, the single bacterial colony of the typical bacillus licheniformis is screened; the typical bacillus licheniformis is transferred to the inclined plane of the nutrient agar test tube to culture; and (2) domestication: the sodium chloride is added in the nutrient agar culture medium to prepare the culture vessels with different sodium chloride concentrations, the sodium chloride concentration is gradually improved for screening and domestication culture, and the bacillus licheniformis which can resist the sodium chloride with higher concentration is screened. The bacillus licheniformis which can resist the sodium chloride with the mass concentration of 12.5% is obtained, and can be used for fertilizer.
Description
Technical field
The invention belongs to microorganism field, relate to screening acclimation method of a kind of Bacillus licheniformis (Bacillus licheniformis) and the Bacillus licheniformis that obtains, is to be applied to improve in the fertilizer tolerance of Bacillus licheniformis to salt.
Background technology
Bacillus licheniformis, Latin formal name used at school Bacillus licheniformis is a kind of of bacillus.Single Bacillus licheniformis cellular form and arrangement are shaft-like, Dan Sheng, can produce the activity resistent material, and have unique biological and take the oxygen mechanism of action by force, can suppress the growth and breeding of pathogenic bacterium.
Bacillus licheniformis can suppress the breeding of pathogenic bacteria in the soil and to the invasion and attack of plant root, reduce plant soil-borne diseases, and therefore the outburst of prevention various pests is applied in the fertilizer.CN101955903A disclosed " a kind of Bacillus licheniformis and application thereof " for example, CN101891548A disclosed " the multiple-effect microbial bacterial manure that a kind of three bacillus licheniformis are made ", above-mentioned document has all related to Bacillus licheniformis in Application in Fertilizer.Yet because Bacillus licheniformis is relatively poor to the tolerance of salt, the salt of high density can suppress bacterial strain.Therefore, Bacillus licheniformis all is to be used for biological organic fertilizer at present, or the composite fertilizer in the hyposaline.This has limited Bacillus licheniformis in Application in Fertilizer.And the chemical fertilizer main component is inorganic salt, makes that the major part Bacillus licheniformis can not survive when Bacillus licheniformis and chemical fertilizer used with.
Summary of the invention
The object of the invention just is to provide the screening method of a kind of Bacillus licheniformis to the sodium-chlor tolerance.
The present invention adopts following technical scheme: the screening method of a kind of Bacillus licheniformis to sodium-chlor tolerance comprises following process:
(1) screening: with Bacillus licheniformis CGMCC 1.0518, be substratum, carry out the test tube slant earlier and cultivate, and then carry out single bacterium colony that the culture dish inclined-plane filters out typical Bacillus licheniformis after streak culture with the nutrient agar medium; Change typical Bacillus licheniformis over to the nutrient agar medium test tube slant, cultivate;
(2) domestication: in nutrient agar, increase sodium-chlor, process sodium chloride concentration different culture ware, streak culture in the lower culture dish of phosphoric acid ammonia two ammonium concentrations the last Bacillus licheniformis elder generation that obtains of cultivating in the step (1); Single colony lift of cultivating is cultivated the back as bacterial classification to the test tube slant, streak culture in the higher culture dish of next sodium chloride concentration again, by that analogy, progressively improve sodium chloride concentration, filter out the Bacillus licheniformis that can tolerate higher concentration sodium-chlor.
The sodium-chlor mass concentration can be respectively 2%, 5%, 8%, 10%, 12%, 12.5%, 13% and 15% in the said step (2).
Said nutrient agar medium consists of: every 1000ml water is with peptone 10 grams, beef powder 3.0 grams and sodium-chlor 5.0 grams, and the pH value is 7.3 ± 0.1.
Culture condition described in each step be in the constant incubator 34 ℃ ± 0.5 ℃ cultivated 3-5 days.
A kind of Bacillus licheniformis of the salt tolerant that obtains with aforesaid method screening domestication is gram positive bacterium, the long 2.5-3.5 μ of bar m, the wide 0.8-1.5 μ of bar m, produce near in living ellipticity gemma, sporangiocyst expands slightly; Cellular form and arrangement are shaft-like, Dan Sheng; No pod membrane, atrichia can move; Nos Poly-salt is very easily grown on the plain agar flat board in the cell, colonial morphology be flat, the edge is irregular, white, surface irregularity gauffer; Bacillus licheniformis can produce acetyl methyl carbinol, the property run concurrently anoxybiosis; But decomposition glucose, N.F,USP MANNITOL and wood sugar produce acid, produce acid but can not decompose pectinose; Litmus milk is reduction and peptonizes reaction, and ability starch-splitting and casein can utilize Citrate trianion; The tolerance mass concentration is 12.5% sodium-chlor.
The Bacillus licheniformis that method of the present invention obtains through screening, domestication, is a well-grown in 20% the sodium-chlor nutrient agar medium in mass concentration, shows that can tolerate mass concentration is 20% sodium-chlor.
Embodiment
Through specific embodiment the present invention is described in further detail below, to help understanding content of the present invention.
Bacillus licheniformis Bacillus licheniformis source, the Chinese common micro-organisms culture presevation administrative center (CGMCC) of Institute of Micro-biology of the Chinese Academy of Sciences buys, and is numbered 1.0518.
Used separation basic medium: nutrient agar
Peptone: 10 grams
Beef powder: 3.0 grams
Sodium-chlor: 5.0 grams
Water: 1000ml
PH value: 7.3 ± 0.1
The screening acclimation method:
(1) prepares: prepare substratum according to the nutrient agar standard, together with auxiliary implements such as the culture dish of wrapping, test tube substratum, sterilized water, glass slicker, transfer pipets, through for use behind the high-temperature sterilization.The nutrient agar of bacterium of having gone out falls dull and stereotyped, test tube substratum pendulum inclined-plane, and cooling, subsequent use.
(2) rejuvenation: Bechtop is opened ultraviolet lamp sterilization 30 minutes; The laboratory formaldehyde fumigation; After preparation work is carried out; The sterilized water of inhaling 0.2 milliliter adds in the freeze-drying pipe; Bacillus licheniformis bacterial classification dry powder is made bacteria suspension,, bacteria suspension is tiled on the slant medium pouring under the state of flame sealing on the test tube slant for preparing; Put into 34 ℃ ± 0.5 ℃ of constant incubator, cultivated 3-5 days.Microscopy is observed form, gemma rate.
(3) screening: at the bacterial classification operation room; Bechtop is opened ultraviolet lamp sterilization 30 minutes; Carry out aseptic technique; With inoculating needle in cultured test tube slant bacterial classification provoke bacterium colony; In ready culture dish, rule; Whether the culture dish of drawing good line is put into 34 ℃ ± 0.5 ℃ of constant incubator, cultivated 3-5 days, observing has single bacterium colony of typical Bacillus licheniformis to form.With single bacterium colony of typical Bacillus licheniformis, change on the ready nutrient agar medium test tube slant, put into 34 ℃ ± 0.5 ℃ of constant incubator, cultivated 3-5 days, for use.
(4) domestication: the concentration of corresponding increase sodium-chlor in nutrient agar according to massfraction 2%, 5%, 8%, 10%, 12%, 12.5%, 13%, 15% each concentration, is prepared into culture dish; On cultured nutrient agar medium test tube slant; It is earlier streak culture in 10% sodium-chlor nutrient agar medium culture dish to choose bacterial classification, and single bacterium colony of one batch is deposited the test tube slant in the past then, after cultivation finishes; Streak culture in the culture dish of next concentration as bacterial classification, and the like.The concentration (massfraction) that shows sodium-chlor according to experimental result is at 12.5% o'clock, and this bacterial strain can well-grown, as the bacterial classification after the domestication.
(5), detect: choosing nutrient agar adding massfraction is the sodium-chlor of 12.5% concentration, behind the autoclaving, falls dull and stereotyped with the culture dish after the sterilization.Line or dilution plate coating, 34 ℃ ± 0.5 ℃ cultivation grew single bacterium colony in 3-5 days, and the Bacillus licheniformis after the process screening domestication of surface can tolerate the sodium-chlor of 12.5% concentration.
(6) biological assay
The Bacillus licheniformis that can tolerate 12.5% concentration sodium-chlor that obtains after the domestication, carry out biological assay, qualification result: be gram positive bacterium, the long 2.5-3.5 μ of bar m, the wide 0.8-1.5 μ of bar m produces nearly middle ellipticity gemma of giving birth to, and sporangiocyst expands slightly.Cellular form and arrangement are shaft-like, Dan Sheng.No pod membrane, atrichia can move.Nos Poly-salt is very easily grown on the plain agar flat board in the cell, colonial morphology be flat, the edge is irregular, white, surface irregularity gauffer.Bacillus licheniformis can produce acetyl methyl carbinol, the property run concurrently anoxybiosis; But decomposition glucose, N.F,USP MANNITOL and wood sugar produce acid, produce acid but can not decompose pectinose; Litmus milk is reduction and peptonizes reaction, and ability starch-splitting and casein can utilize Citrate trianion.
Contrast before and after the domestication:
Bacterial strain after bacterial strain before the domestication and the domestication is inserted liquid nutrient medium respectively cultivate, treat that 95% above gemma forms down shaking table and prepares plate count.Result such as table 1:
Bacillus licheniformis survival before and after table 1 domestication
The survival rate of bacterial strain exists before the domestication: 45.7%, and the bacterial strain survival rate after the domestication can reach 68.8%, shows through the tolerance of domestication back Bacillus licheniformis to sodium-chlor significantly to be strengthened.
Claims (4)
1. screening method to the Bacillus licheniformis (Bacillus licheniformis) of sodium-chlor tolerance is characterized in that comprising following process:
(1) screening: with Bacillus licheniformis CGMCC 1.0518, be substratum, carry out the test tube slant earlier and cultivate, and then carry out single bacterium colony that the culture dish inclined-plane filters out typical Bacillus licheniformis after streak culture with the nutrient agar medium; Change typical Bacillus licheniformis over to the nutrient agar medium test tube slant, cultivate;
(2) domestication: in nutrient agar, increase sodium-chlor, process sodium chloride concentration different culture ware, streak culture in the lower culture dish of phosphoric acid ammonia two ammonium concentrations the last Bacillus licheniformis elder generation that obtains of cultivating in the step (1); Single colony lift of cultivating is cultivated the back as bacterial classification to the test tube slant, streak culture in the higher culture dish of next sodium chloride concentration again, by that analogy, progressively improve sodium chloride concentration, filter out the Bacillus licheniformis that can tolerate higher concentration sodium-chlor.
2. the method for claim 1 is characterized in that: the sodium-chlor mass concentration is respectively 2%, 5%, 8%, 10%, 12%, 12.5%, 13% and 15% in the said step (2).
3. method as claimed in claim 1 or 2 is characterized in that: said nutrient agar medium consists of: every 1000ml water is with peptone 10 grams, beef powder 3.0 grams and sodium-chlor 5.0 grams, and the pH value is 7.3 ± 0.1.
4. method as claimed in claim 1 or 2 is characterized in that: culture condition described in each step in constant incubator 34 ℃ ± 0.5 ℃ cultivated 3-5 days.
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| CN201110222042.6A CN102344895B (en) | 2011-08-04 | 2011-08-04 | Method for screening sodium chloride-resistant bacillus licheniformis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305451A (en) * | 2013-06-20 | 2013-09-18 | 双赢集团有限公司 | Screening method for ammonium dihydrogen phosphate-tolerant bacillus laterosporus |
| CN103320375A (en) * | 2013-06-20 | 2013-09-25 | 双赢集团有限公司 | Method for screening sodium chloride tolerant bacillus laterosporus |
| CN112961817A (en) * | 2021-04-06 | 2021-06-15 | 广西中医药大学 | Method for screening high-yield Macrolactins marine bacillus by using osmotic pressure stress of sea salt |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563354A (en) * | 2004-03-24 | 2005-01-12 | 河北科技大学 | Strain in use sensor of measuring oxygen quantity needed by industrial wastewater or seawater biochemistry-method for cultivating bacillus licheniformis |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563354A (en) * | 2004-03-24 | 2005-01-12 | 河北科技大学 | Strain in use sensor of measuring oxygen quantity needed by industrial wastewater or seawater biochemistry-method for cultivating bacillus licheniformis |
Non-Patent Citations (4)
| Title |
|---|
| 《Malaysian Journal of Biochemistry and Molecular Biology》 20091231 Vasanthy Arasaratnam等 Substitution of GPR Grade Salts in the Production of alpha-Amylase by Bacillus lichenif ormis 6346 in Solid State Fermentation 1-4 1-4 第17卷, 第1期 * |
| VASANTHY ARASARATNAM等: "Substitution of GPR Grade Salts in the Production of α-Amylase by Bacillus lichenif ormis 6346 in Solid State Fermentation", 《MALAYSIAN JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY》 * |
| 李俊叶等: "耐盐菌的筛选及初步鉴定", 《安徽农业科学》 * |
| 黄红英等: "一株地衣芽孢杆菌碱性蛋白酶的研究Ⅰ", 《微生物学通报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305451A (en) * | 2013-06-20 | 2013-09-18 | 双赢集团有限公司 | Screening method for ammonium dihydrogen phosphate-tolerant bacillus laterosporus |
| CN103320375A (en) * | 2013-06-20 | 2013-09-25 | 双赢集团有限公司 | Method for screening sodium chloride tolerant bacillus laterosporus |
| CN112961817A (en) * | 2021-04-06 | 2021-06-15 | 广西中医药大学 | Method for screening high-yield Macrolactins marine bacillus by using osmotic pressure stress of sea salt |
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Granted publication date: 20140507 Termination date: 20200804 |