CN109652316A - A kind of isolation medium and preparation method thereof of efficient peanut nodule azotobacter - Google Patents
A kind of isolation medium and preparation method thereof of efficient peanut nodule azotobacter Download PDFInfo
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- CN109652316A CN109652316A CN201910073254.9A CN201910073254A CN109652316A CN 109652316 A CN109652316 A CN 109652316A CN 201910073254 A CN201910073254 A CN 201910073254A CN 109652316 A CN109652316 A CN 109652316A
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- 235000017060 Arachis glabrata Nutrition 0.000 title claims abstract description 51
- 235000010777 Arachis hypogaea Nutrition 0.000 title claims abstract description 51
- 235000018262 Arachis monticola Nutrition 0.000 title claims abstract description 51
- 235000020232 peanut Nutrition 0.000 title claims abstract description 51
- 241000589151 Azotobacter Species 0.000 title claims abstract description 42
- 238000002955 isolation Methods 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 241001553178 Arachis glabrata Species 0.000 title abstract 4
- 239000002609 medium Substances 0.000 claims abstract description 34
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 230000001954 sterilising effect Effects 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims abstract description 8
- 239000010941 cobalt Substances 0.000 claims abstract description 6
- 229910017052 cobalt Inorganic materials 0.000 claims abstract description 6
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 244000105624 Arachis hypogaea Species 0.000 claims description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000008223 sterile water Substances 0.000 claims description 14
- 239000000725 suspension Substances 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 9
- 239000002689 soil Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 239000008156 Ringer's lactate solution Substances 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 238000003113 dilution method Methods 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 239000001569 carbon dioxide Substances 0.000 claims description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 5
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000012136 culture method Methods 0.000 claims description 4
- 239000001540 sodium lactate Substances 0.000 claims description 4
- 229940005581 sodium lactate Drugs 0.000 claims description 4
- 235000011088 sodium lactate Nutrition 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 239000000460 chlorine Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 238000004821 distillation Methods 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 241000589180 Rhizobium Species 0.000 abstract description 5
- 239000011573 trace mineral Substances 0.000 abstract description 3
- 235000013619 trace mineral Nutrition 0.000 abstract description 3
- 241000589157 Rhizobiales Species 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 2
- 239000012928 buffer substance Substances 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
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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
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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
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
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- Medicinal Chemistry (AREA)
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- Virology (AREA)
- Biochemistry (AREA)
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Abstract
The invention belongs to isolation medium technical fields, disclose a kind of isolation medium and preparation method thereof of efficient peanut nodule azotobacter, high-temperature sterilization is carried out to culture dish, each component is mixed according to a certain percentage, the solution of configuration is shaken up, buffer substance is added in mixed solution, solution ph is set to maintain 5.6, incubator temperature is adjusted simultaneously, culture medium is placed in 25 DEG C of incubator, the preparation for completing culture medium, bacterial strain is put into isolation medium, to be separately cultured to peanut nodule nitrogen-fixing bacteria.The isolation medium can carry out peanut nodule nitrogen-fixing bacteria efficiently, thoroughly to separate, by nodule azotobacter to the selectivity of culture medium, acidproof rhizobium are made to carry out quick separating, the trace element cobalt being added simultaneously can promote rhizobial growth and breeding after separation, accelerate the reproduction speed of rhizobium.
Description
Technical field
The invention belongs to being separately cultured for isolation medium technical field more particularly to a kind of efficient peanut nodule azotobacter
Base and preparation method thereof.
Background technique
Rhizobium are that one kind can form the nitrogen in root nodule homobium and fixed air for plant with specified plant symbiosis
A kind of bacillus of nutrition.When being separately cultured to nodule azotobacter, since artificial medium does not meet the life of bacterium colony
Elongate member causes bacterium colony to grow in the medium bad, therefore cannot be guaranteed that all peanut nodule nitrogen-fixing bacteria are totally separated
Out, follow-up work can not be carried out；And it is slower to nodule azotobacter progress separation process, separative efficiency is lower, to root nodule
When nitrogen-fixing bacteria are cultivated, since conventional medium ingredient is limited, the growth and breeding of nodule azotobacter is also influenced.
In conclusion problem of the existing technology is: since artificial medium does not meet the growth conditions of bacterium colony, causing
Bacterium colony grow in the medium it is bad, therefore cannot be guaranteed all peanut nodule nitrogen-fixing bacteria be totally separated out, Wu Fakai
Open up follow-up work；And it is slower to nodule azotobacter progress separation process, separative efficiency is lower, trains to nodule azotobacter
When supporting, since conventional medium ingredient is limited, the growth and breeding of nodule azotobacter is also influenced；In culture solution pH value and when it is general
After benefit is neutralized with hydrochloric acid, but benefit is neutralized with hydrochloric acid, it need to sterilize again, complex procedures.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of isolation mediums of efficient peanut nodule azotobacter
And preparation method thereof.
The invention is realized in this way a kind of isolation medium of the efficient peanut nodule azotobacter of suitable acid soil,
The isolation medium of the efficient peanut nodule azotobacter is by 1.0g potassium dihydrogen phosphate, 1.54g dipotassium hydrogen phosphate, 0.05g chlorination
Sodium, 0.04g calcium chloride, 0.004g iron chloride, 0.1g magnesium sulfate, 0.8g yeast extract, 15.0g agar, 3.0g ironic citrate and
1000ml distilled water composition.
Another object of the present invention is to provide a kind of separation of the efficient peanut nodule azotobacter of suitable acid soil trainings
The preparation method of base is supported, the preparation method of the isolation medium of the efficient peanut nodule azotobacter of the suitable acid soil includes
Following steps:
The first step carries out high-temperature sterilization to culture dish,
Second step, by potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, yeast extract,
Agar, ironic citrate, distilled water are mixed in sterile test tube, and are shaken up, and mixed solution is poured into culture dish；
Culture medium is placed in 30 degrees Celsius of incubator by third step, and the carbonic acid generated using carbon dioxide high temperature is by pH
Value is adjusted to taking-up when 5.6；
4th step is put into 25 degrees Celsius of incubators, completes the configuration of isolation medium.
Further, the isolated culture method of the nodule azotobacter of the suitable acid soil includes:
(1) peanut nodule in acid soil, one section of root of clip are selected, and is cleaned with clear water；
(2) surface sterilizing is carried out to the peanut nodule after cleaning；
(3) peanut nodule is put into the test tube of high-temperature sterilization, and is crushed root nodule using aseptic nipper, be added 1ml without
Bacterium water is uniformly mixed, obtains suspension；
(4) nodule azotobacter bacterium colony is obtained using dilution method separation nodule azotobacter；
(5) bacterium colony is put into isolation medium, cobalt microelement and sodium lactate solution is added, to peanut nodule nitrogen-fixing bacteria
It is cultivated.
Further, the peanut nodule surface sterilizing, which is handled, includes:
Peanut nodule impregnates and 1 minute in 95% alcohol；
Peanut nodule is taken out with aseptic water washing 2-3 times；
The mercuric chloride solution that peanut nodule is immersed in 0.1% is impregnated 5 minutes；
Peanut nodule is taken out to be rinsed 3-4 times again with sterile water.
Further, the dilution method includes:
Prepare 4 sterile petri dish, take a ring suspension that 1ml sterile water is added to be put into first sterile petri dish, stirs
Uniformly；Take the suspension in 1 ring, first sterile petri dish that 1ml sterile water is added to be put into second sterile petri dish, stirring is equal
It is even；It takes the suspension in 1 ring, second sterile petri dish that 1ml sterile water is added to be put into third sterile petri dish, stirs evenly；
And so on, until occurring 3-4 independent bacterium colonies in culture dish.
Further, the sodium lactate solution preparation method includes: to weigh sodium lactate 10g, dissolves by heating and distills in 1000ml
In water, 121 DEG C of high pressure sterilization 15min obtain sodium lactate solution.
Advantages of the present invention and good effect are as follows: the isolation medium can carry out acidproof peanut nodule nitrogen-fixing bacteria high
Effect, thoroughly separation make rhizobium carry out quick separating by nodule azotobacter to the selectivity of culture medium, while being added
Trace element cobalt can promote rhizobial growth and breeding after separation, accelerate the reproduction speed of rhizobium；Utilize culture solution height
The carbonic acid that carbon dioxide generates when temperature processing reduces pH value, mildly and is not necessarily to degerming again；Nodule nitrogen fixation is separated using dilution method
Bacterium, good separating effect.
Detailed description of the invention
Fig. 1 is the preparation method process of the isolation medium of efficient peanut nodule azotobacter provided in an embodiment of the present invention
Figure；
Fig. 2 is the isolated culture method flow chart of efficient peanut nodule azotobacter provided in an embodiment of the present invention.
Specific embodiment
In order to further understand the content, features and effects of the present invention, the following examples are hereby given, and cooperate attached drawing
Detailed description are as follows.
Structure of the invention is explained in detail with reference to the accompanying drawing.
The isolation medium of efficient peanut nodule azotobacter provided in an embodiment of the present invention by 1.0g potassium dihydrogen phosphate,
1.54g dipotassium hydrogen phosphate, 0.05g sodium chloride, 0.04g calcium chloride, 0.004g iron chloride, 0.1g magnesium sulfate, 0.8g yeast extract,
15.0g agar, 3.0g ironic citrate and 1000ml distilled water composition.
As shown in Figure 1, the preparation method of the isolation medium of efficient peanut nodule azotobacter provided in an embodiment of the present invention
The following steps are included:
S101: carrying out high-temperature sterilization to culture dish,
S102: by potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, yeast extract, fine jade
Rouge, ironic citrate, distilled water are mixed in sterile test tube, and are shaken up, and mixed solution is poured into culture dish；
S103: culture medium being placed in 30 DEG C of incubator, is adjusted pH value using the carbonic acid that carbon dioxide high temperature generates
It is taken out when being 5.6；
S104: 25 DEG C of incubators are put into, the configuration of isolation medium is completed.
As shown in Fig. 2, the isolated culture method of nodule azotobacter provided in an embodiment of the present invention includes:
S201: the peanut nodule in selection acid soil, one section of root of clip, and cleaned with clear water；
S202: surface sterilizing is carried out to the peanut nodule after cleaning；
S203: peanut nodule is put into the test tube of high-temperature sterilization, and is crushed root nodule using aseptic nipper, and 1ml is added
Sterile water is uniformly mixed, obtains suspension；
S204: nodule azotobacter bacterium colony is obtained using dilution method separation nodule azotobacter；
S205: bacterium colony is put into isolation medium, the cobalt microelement and sodium lactate solution of appropriate concentration is added, to flower
Raw nodule azotobacter is cultivated.
In step S202, peanut nodule surface sterilizing processing provided in an embodiment of the present invention includes:
Peanut nodule impregnates and 1 minute in 95% alcohol；
Peanut nodule is taken out with aseptic water washing 2-3 times；
The mercuric chloride solution that peanut nodule is immersed in 0.1% is impregnated 5 minutes；
Peanut nodule is taken out to be rinsed 3-4 times again with sterile water.
In step S204, dilution method provided in an embodiment of the present invention includes:
Prepare 4 sterile petri dish, take a ring suspension that 1ml sterile water is added to be put into first sterile petri dish, stirs
Uniformly；Take the suspension in 1 ring, first sterile petri dish that 1ml sterile water is added to be put into second sterile petri dish, stirring is equal
It is even；It takes the suspension in 1 ring, second sterile petri dish that 1ml sterile water is added to be put into third sterile petri dish, stirs evenly；
And so on, until occurring 3-4 independent bacterium colonies in culture dish.
In step S205, sodium lactate solution provided in an embodiment of the present invention includes:
Weigh sodium lactate 10g, dissolve by heating in 1000ml distilled water, 121 DEG C high pressure sterilization 15 minutes, it is molten to obtain sodium lactate
Liquid.
The working principle of the invention: by 1.0g potassium dihydrogen phosphate, 1.54g dipotassium hydrogen phosphate, 0.05g sodium chloride, 0.04g chlorine
Change calcium, 0.004g iron chloride, 0.1g magnesium sulfate, 0.8g yeast extract, 15.0g agar, 3.0g ironic citrate, 1000ml distilled water
It is mixed, provides nitrogen source by yeast extract for bacterium colony, sodium chloride maintains balanced osmotic pressure, and agar block is the solidifying of culture medium
Gu agent adjusts incubator temperature, weighs a certain amount of culture medium, load weighted culture medium is placed in 30 DEG C of incubator, benefit
PH value is adjusted with the carbon source that carbon dioxide generates, then culture medium is placed in 25 DEG C of incubator, makes media environment temperature
Degree meets the optimum temperature of bacterium colony separation, breeding, separates nodule azotobacter using dilution method, bacterial strain is put into isolation medium
In, the growth of nodule azotobacter is maintained by the nutriment and trace element cobalt of culture medium, promotes the breeding of nodule azotobacter.
The above is only the preferred embodiments of the present invention, and is not intended to limit the present invention in any form,
Any simple modification made to the above embodiment according to the technical essence of the invention, equivalent variations and modification, belong to
In the range of technical solution of the present invention.
Claims (6)
1. a kind of isolation medium of the efficient peanut nodule azotobacter of suitable acid soil, which is characterized in that the efficient flower
The isolation medium of raw nodule azotobacter is by 1.0g potassium dihydrogen phosphate, 1.54g dipotassium hydrogen phosphate, 0.05g sodium chloride, 0.04g chlorine
Change calcium, 0.004g iron chloride, 0.1g magnesium sulfate, 0.8g yeast extract, 15.0g agar, 3.0g ironic citrate and 1000ml distillation
Water composition.
2. a kind of preparation method of the isolation medium of efficient peanut nodule azotobacter as described in claim 1, which is characterized in that
The preparation method of the isolation medium of the efficient peanut nodule azotobacter the following steps are included:
The first step carries out high-temperature sterilization to culture dish；
Second step, by potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, yeast extract, agar,
Ironic citrate, distilled water are mixed in sterile test tube, and are shaken up, and mixed solution is poured into culture dish；
Culture medium is placed in 30 DEG C of incubator by third step, is adjusted to pH value using the carbonic acid that carbon dioxide high temperature generates
It is taken out when 5.6；
4th step is put into 25 DEG C of incubators, completes the configuration of isolation medium.
3. the preparation method of the isolation medium of the efficient peanut nodule azotobacter of suitable acid soil as claimed in claim 2,
It is characterized in that, the isolated culture method of the nodule azotobacter includes:
(1) peanut nodule in acid soil, one section of root of clip are selected, and is cleaned with clear water；
(2) surface sterilizing is carried out to the peanut nodule after cleaning；
(3) peanut nodule is put into the test tube of high-temperature sterilization, and is crushed root nodule using aseptic nipper, 1ml sterile water is added,
It is uniformly mixed, obtains suspension；
(4) nodule azotobacter bacterium colony is obtained using dilution method separation nodule azotobacter；
(5) bacterium colony is put into isolation medium, cobalt microelement and sodium lactate solution is added, peanut nodule nitrogen-fixing bacteria are carried out
Culture.
4. the preparation method of the isolation medium of efficient peanut nodule azotobacter as claimed in claim 2, which is characterized in that institute
Stating the processing of peanut nodule surface sterilizing includes:
(1) peanut nodule impregnates and 1 minute in 95% alcohol；
(2) peanut nodule is taken out with aseptic water washing 2-3 times；
(3) mercuric chloride solution that peanut nodule is immersed in 0.1% is impregnated 5 minutes；
(4) peanut nodule is taken out to be rinsed 3-4 times again with sterile water.
5. the preparation method of the isolation medium of efficient peanut nodule azotobacter as claimed in claim 2, which is characterized in that institute
Stating dilution method includes:
Prepare 4 sterile petri dish, takes a ring suspension that 1ml sterile water is added to be put into first sterile petri dish, stir evenly；
It takes the suspension in 1 ring, first sterile petri dish that 1ml sterile water is added to be put into second sterile petri dish, stirs evenly；Take 1
Suspension in second sterile petri dish of ring adds 1ml sterile water to be put into third sterile petri dish, stirs evenly；With such
It pushes away, until occurring 3-4 independent bacterium colonies in culture dish.
6. the preparation method of the isolation medium of efficient peanut nodule azotobacter as claimed in claim 2, which is characterized in that institute
Stating sodium lactate solution preparation method includes: to weigh sodium lactate 10g, dissolves by heating 121 DEG C of high pressure sterilizations in 1000ml distilled water
15 minutes, obtain sodium lactate solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113174346A (en) * | 2021-05-21 | 2021-07-27 | 中国农业科学院农业资源与农业区划研究所 | Method for efficiently separating corn endophytic azotobacter |
| CN113265367A (en) * | 2021-05-17 | 2021-08-17 | 中国科学院昆明植物研究所 | Culture for rapidly obtaining morchella conidia and preparation method thereof |
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| CN106754481A (en) * | 2016-11-26 | 2017-05-31 | 安徽省农业科学院园艺研究所 | The method and related culture medium of the azotobacter strain of resistance to high salt are screened from ice dish |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113174346A (en) * | 2021-05-21 | 2021-07-27 | 中国农业科学院农业资源与农业区划研究所 | Method for efficiently separating corn endophytic azotobacter |
| CN113174346B (en) * | 2021-05-21 | 2022-04-15 | 中国农业科学院农业资源与农业区划研究所 | Method for efficiently separating corn endophytic azotobacter |
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