CN102344901A - Screening method of bacillus subtilis with sodium chloride tolerance - Google Patents
Screening method of bacillus subtilis with sodium chloride tolerance Download PDFInfo
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- CN102344901A CN102344901A CN2011102220445A CN201110222044A CN102344901A CN 102344901 A CN102344901 A CN 102344901A CN 2011102220445 A CN2011102220445 A CN 2011102220445A CN 201110222044 A CN201110222044 A CN 201110222044A CN 102344901 A CN102344901 A CN 102344901A
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- subtilis
- sodium chloride
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Abstract
The invention belongs to the field of microbes, and relates to a screening method of bacillus subtilis with sodium chloride tolerance. The method comprises the following steps of: (1) screening, wherein bacillus subtilis with the CGMCC (China General Microbiological Culture Collection Center) preservation number of 1.0769 is subjected to test tube slant culture and a petri dish slant streak culture by using nutrient agar as a culture medium to screen a single colony of typical bacillus subtilis, and the typical bacillus subtilis is transformed to a slant of a nutrient agar test tube for culturing; and (2) acclimation, wherein sodium chloride is added into the nutrient agar culture medium to prepare petri dishes with different concentrations of sodium chloride. According to the method, the screening and acclimating cultivation are carried out by gradually increasing the concentration of sodium chloride, so that the bacillus subtilis capable of tolerating high-concentration sodium chloride can be screened. The bacillus subtilis capable of tolerating 12.5wt% sodium chloride can be prepared and can be used in a fertilizer.
Description
Technical field
The invention belongs to microorganism field, relate to screening acclimation method of a kind of subtilis (Bacillus subtilis) and the subtilis that obtains, is to be applied to improve in the fertilizer tolerance of subtilis to salt.
Background technology
Subtilis, Latin formal name used at school Bacillus subtilis is a kind of of bacillus.0.7～0.8 * 2～3 microns of individual cells, uniform coloring.No pod membrane, peritrichous can move.Gram-positive microorganism, 0.6～0.9 * 1.0～1.5 microns of gemma, oval to column, be positioned at thalline central authorities or inclined to one side slightly, gemma forms the back thalline and does not expand.The bacterium colony surface irregularity is opaque, dirty white or little yellow, and when growing in the liquid medium within, the normal wrinkle mould that forms.Aerophil.In genetics research, be widely used, clearer to route of synthesis and its regulation mechanism research of the purine nucleotides of this bacterium.Extensively be distributed in soil and the putrid organism, be prone to soak in the juice and breed, so name withered grass.
Subtilis has phosphate solubilization, can improve the plant utilization degree of phosphorus, therefore is applied in the fertilizer.CN101899405A disclosed " bacillus subtilis GU and in the application of preparation in the efficient biological compound phosphate fertilizer " for example; CN101643710A disclosed " bacillus subtilis and application thereof "; CN101200385A disclosed " preparing composite microbiological fertilizer with bacillus laterosporus, subtilis ", above-mentioned document has all related to subtilis in Application in Fertilizer.Yet because subtilis is relatively poor to the tolerance of salt, the salt of high density can suppress bacterial strain.For example Yang Qingxiang, Cao Weijun " Wuhan University's journal (natural science) " 02 phase in 1998 " height ooze environment to subtilis and serratia marcescens born of the same parents in the influence of free amino acid storehouse content and composition " in the literary composition, disclose subtilis (93151 and 93215) and can tolerate 8%NaCl.Therefore, subtilis all is to be used for biological organic fertilizer at present, or the composite fertilizer in the hyposaline.This has limited subtilis in Application in Fertilizer.The chemical fertilizer major ingredient is inorganic salt, makes that the major part subtilis can not survive when subtilis 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 subtilis to the sodium-chlor tolerance.
The present invention adopts following technical scheme: the screening method of a kind of subtilis to sodium-chlor tolerance comprises following process:
(1) screening: with subtilis CGMCC 1.0769, 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 subtilis after streak culture with the nutrient agar medium; Change typical subtilis 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 subtilis 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 subtilis 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.
The subtilis that method of the present invention obtains through screening, domestication, is a well-grown in 12.5% the sodium-chlor nutrient agar medium in mass concentration, shows that can tolerate mass concentration is 12.5% sodium-chlor.
Embodiment
Through specific embodiment the present invention is described in further detail below, to help understanding content of the present invention.
Subtilis subsp.subutilis 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 1.0769.
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 subtilis strain 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 subtilis to form.With single bacterium colony of typical subtilis, 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 2% 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 subtilis after the process screening domestication of surface can tolerate the sodium-chlor of 12.5% concentration.
(6) biological assay
The subtilis that can tolerate 20% concentration sodium-chlor that obtains after the domestication; Carry out biological assay; Qualification result: bacterial strain is the tubbiness bacillus of Gram-positive, the blunt circle in two ends; 2～8 * 0.7～1.0 μ m; Single or short chain, dynamic, no pod membrane; The oval gemma is bordering on thalline one end, greater than the thalline width.Very easily growth on the plain agar flat board, bacterium colony is irregular, grey, surface irregularity, drying have wrinkle, and the edge is not the curly hair shape.The liquid medium within surface forms thicker rugate mycoderm.Can decompose N.F,USP MANNITOL, pectinose and wood sugar, produce not aerogenesis of acid.Do not produce lecithinase.
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:
Subtilis survival before and after table 1 domestication
The survival rate of bacterial strain exists before the domestication: 41.1%, and the bacterial strain survival rate after the domestication can reach 72.8%, shows through the tolerance of domestication back subtilis to salt significantly to be strengthened.
Claims (4)
1. screening method to the subtilis (Bacillus subtilis) of sodium-chlor tolerance is characterized in that comprising following process:
(1) screening: with subtilis CGMCC 1.0769, 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 subtilis after streak culture with the nutrient agar medium; Change typical subtilis 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 subtilis 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 subtilis 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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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 |
| CN111647539A (en) * | 2020-07-06 | 2020-09-11 | 重庆工商大学 | Method for strengthening film forming capability of aerobic denitrifying bacteria |
| CN114990018A (en) * | 2022-06-09 | 2022-09-02 | 江苏农牧科技职业学院 | Domestication method and device of bacillus subtilis for changing bottom of aquatic products |
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| 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 |
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| 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 (3)
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| 《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 第17卷, 第1期 * |
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Cited By (4)
| 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 |
| CN111647539A (en) * | 2020-07-06 | 2020-09-11 | 重庆工商大学 | Method for strengthening film forming capability of aerobic denitrifying bacteria |
| CN111647539B (en) * | 2020-07-06 | 2022-07-08 | 重庆工商大学 | Method for strengthening film forming capability of aerobic denitrifying bacteria |
| CN114990018A (en) * | 2022-06-09 | 2022-09-02 | 江苏农牧科技职业学院 | Domestication method and device of bacillus subtilis for changing bottom of aquatic products |
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