CN105907839A - Efficient separation and identification method of ammonia oxidizing bacteria of mulberry field soil - Google Patents
Efficient separation and identification method of ammonia oxidizing bacteria of mulberry field soil Download PDFInfo
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- CN105907839A CN105907839A CN201610366826.9A CN201610366826A CN105907839A CN 105907839 A CN105907839 A CN 105907839A CN 201610366826 A CN201610366826 A CN 201610366826A CN 105907839 A CN105907839 A CN 105907839A
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- oxidizing bacteria
- ammonia oxidizing
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- soil
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Abstract
The invention discloses an efficient separation and identification method of the ammonia oxidizing bacteria of mulberry field soil. The method comprises the following steps: enrichment of the ammonia oxidizing bacteria; separation and purification of the ammonia oxidizing bacteria; and identification of the separated microorganisms. According to the invention, based on the characteristic of the ammonia oxidizing bacteria preferring a slightly alkaline growth environment, the pH value of the enrichment culture solution is regulated to 8.5 while the pH value of the separation culture solution is regulated to 8.0; by adopting the method, 10 kinds of ammonia oxidizing bacteria can be enriched and cultured from the mulberry field soil, and the enrichment efficiency is remarkably improved; and a Biolog microorganism identification system is adopted for rapid identification, so that a foundation is laid for utilizing the ammonia oxidizing bacteria of the mulberry field soil.
Description
Technical field
The invention belongs to microbial ecology technical field, specifically, relate to efficiently dividing of a kind of Soil of Mulberry Garden ammonia oxidizing bacteria
From with authentication method.
Background technology
Anaerobic ammonium oxidation antibacterial (AOB) is can be by NH3It is converted into NO2-, and only with in this reaction produce energy be only one
Energy source carries out the microorganism of ammoxidation reaction, and in total number of bacteria, proportion is minimum, but is widely present in soil, sea
In the environment such as ocean and lake.Anaerobic ammonium oxidation antibacterial is gram negative bacteria, and the energy only generated with chemical reaction is carried out certainly
Health preserving is long, and the growth conditions of preference slightly alkalescence, using NH3 as sole energy source, with CO2As sole carbon source, therefore its
Capacity usage ratio is on the low side, grows the slowest, it is difficult to separation and Culture.Research shows that at pH be 7.0-8.5, temperature 24 DEG C-28
Most beneficial for the growth of this bacterium under conditions of DEG C, when pH is less than 6.5, typically it is unfavorable for that the growth of this bacterium, experiment prove
Increasing the situation of ammonia oxidizing bacteria, this ammonia oxidizing bacteria can promote the ammoxidation process in soil environment, adds ammonia in soil
Oxidizing bacteria, the ammoxidation activity of soil is significantly improved, and soil nitrification strengthens.
Forefathers indicate multiformity and the importance of function of ammonia oxidizing bacteria group based on the research separating ammonia oxidation microbiological,
But ammonia oxidizing bacteria difficulty is cultivated and the speed of growth is very slow, the concentration and separation culture medium that forefathers use pH value to be 7.8-8.0 is respectively
It is separated to a strain and two strain ammonia oxidation microbiologicals from mud with aquiculture waste water, is checked order by 16S rDNA, at GenBank
Upper comparison reaches 99% with the similarity of Nitrosomonas (Nitrosomonas) and pseudomonas (Pseudomonas), this
The ammonia oxidizing bacteria that method is separated to is less, and 16S rDNA order-checking can accurately not identify kind, has some limitations,
And the coverage of the ammonia oxidizing bacteria being separated to is the highest, hinder its multiformity and the analysis of function and utilization, the most urgently
The high efficiency separation of a kind of soil ammonia oxidizing bacteria and authentication method.
Summary of the invention
It is an object of the invention to the defect overcoming above-mentioned technology to exist, it is provided that the high efficiency separation of a kind of Soil of Mulberry Garden ammonia oxidizing bacteria
With authentication method, the method utilizes ammonia oxidizing bacteria to prefer the characteristic of meta-alkalescence growing environment, by the pH value of enrichment culture liquid
Be transferred to 8.5, the pH value of separation and Culture liquid be transferred to 8.0 simultaneously, the method can from Soil of Mulberry Garden enrichment culture to 10 class ammonia
Oxidizing bacteria, greatly improves bioaccumulation efficiency, and uses Biolog microbial identification system that it is carried out Rapid identification, for
The utilization of Soil of Mulberry Garden ammonia oxidizing bacteria is laid a good foundation.
Its concrete technical scheme is:
The high efficiency separation of a kind of Soil of Mulberry Garden ammonia oxidizing bacteria and authentication method, comprise the following steps:
Step 1, the enrichment of ammonia oxidizing bacteria: take 1.0g fresh Soil of Mulberry Garden sample, be equipped with in the test tube of enrichment culture liquid,
In 28 DEG C of constant incubators cultivate 14 days, draw culture fluid 5 in white plaque cave, be sequentially added into Griess reagent A liquid,
B liquid 1 or 2, if any nitrous acid: NO2 -Exist, then take on a red color, indicate that nitrite bacteria exists；If be detected that it is red
Color, adds the nutritional supplementation liquid of 1mL in this pipe, continues to cultivate.Detected once with Griess reagent every 3～4 days, when
When the culture fluid of detection is peony, the culture fluid drawing 1mL is further cultured for fresh enrichment culture liquid, is deep to detection liquid
Redness, this is highly enriched ammonia oxidizing bacteria culture fluid；
Step 2, isolated and purified ammonia oxygen antibacterial: take secondary described in 100mL step 1 and be enriched with to being detected as wine-colored culture fluid,
It is applied on isolation medium flat board, is cultivated 1-2 week more than for 28 DEG C, the most constantly observe colony growth situation, then choose
Taking circular water white transparency, the single bacterium colony line showing slightly yellow identifies cultivation in culture medium to BUG；
Step 3, the qualification of separate microorganism: identifying, with inoculating loop picking, the single bacterium colony separated of ruling in culture medium, to identifying
On culture fluid IF-A tube wall, with inoculating loop, bacterium is broken up, then it is mixed, to nephelometer with qualification liquid so that it is mixed degree
Between 90～95%, then the inoculation liquid volley of rifle fire is received on qualification culture plate, according to the speed of growth of each bacterium, at 28 DEG C
Incubator is cultivated 1～7 day, then use Biology Microststion (MicroStationTM, USA) and microbial identification system
Carry out Rapid identification, identify that the ammonia oxidizing bacteria being separated to includes Pseudomonas, Arthrobacter, Ochrobactrum,
Roseomonas, Enterobacter, Gordonia, Delftia, Pseudoxanthomonas, Mycobacterium,
Stenotrophomonas。
Further, described Pseudomonas is the main groups cultivating ammonia oxidizing bacteria.
Further, the compound method of enrichment culture liquid described in step 1 is particularly as follows: weigh (NH4)2SO42.50g, K2HPO4
0.35g, MgSO4·7H2O 0.25g, CaCl20.18g, with the Na of 5%2CO3Adjust pH to 8.5, be settled to 500mL, point
Dress 10mL/ test tube, 121 DEG C of sterilizing 30min.
Further, the compound method of nutritional supplementation liquid described in step 1 is particularly as follows: weigh (NH4)2SO45.50g, it is settled to
100mL, 121 DEG C of sterilizing 30min.
Further, the compound method of Griess reagent described in step 1 is particularly as follows: A liquid, and 0.5g p-aminobenzene sulfonic acid is added to
In the acetic acid solution of 150mL 200g/L；B liquid: 1g alpha-naphthylamine is added to 20mL distilled water and the acetic acid of 150mL 200g/L
In solution.
Further, the compound method of isolation medium described in step 2 is particularly as follows: weigh (NH4)2SO42.00g, KH2PO4
0.75g, NaH2PO40.25g, MgSO4·7H2O 0.03g, MnSO4·4H2O, CaCO35.00g, with the Na of 5%2CO3
Adjusting pH to 8.0, be settled to 1000mL, then add 16g agar strip, described agar strip to shift to an earlier date three days and soak with pure water, every
Within one day, change a water, until agar strip is dipped to transparent, be down flat plate after 121 DEG C of sterilizing 30min standby.
Further, BUG described in step 2 identify culture medium compound method particularly as follows: with Biolog identification systems outfit
Biolog BUGTM Agar prepares qualification culture medium according to the ratio of every 100mL distilled water 6g, after 121 DEG C of sterilizing 30min
It is down flat plate standby.
Compared with prior art, the invention have the benefit that
The present invention is directed to Soil of Mulberry Garden and the characteristic of ammonia oxidizing bacteria self, by the culture fluid of enrichment Soil of Mulberry Garden ammonia oxidizing bacteria
PH value is transferred to 8.5, the pH value of separation and Culture liquid is transferred to 8.0 simultaneously, the method from Soil of Mulberry Garden enrichment culture to 10 classes
Ammonia oxidizing bacteria, including Pseudomonas, Arthrobacter, Ochrobactrum, Roseomonas, Enterobacter,
Gordonia, Delftia, Pseudoxanthomonas, Mycobacterium, Stenotrophomonas, greatly improve
Bioaccumulation efficiency.And the enrichment that different tests material is used pH value to be 7.8-8.0 by forefathers is only separated to 2 genus with separation and Culture liquid
Ammonia oxidizing bacteria, and through 16S rDNA order-checking with GenBank comparison identify kind the most accurately, in the present invention separate
Ammonia oxidizing bacteria uses Biolog microbial identification system that it is carried out Rapid identification, simple and easy to do, and result is accurately and reliably,
Can be the separation offer reference of ammonia oxidizing bacteria in other plant soil, and base has been established in the utilization for Soil of Mulberry Garden ammonia oxidizing bacteria
Plinth.
Detailed description of the invention
For the technological means making the present invention realize, creation characteristic, reach purpose and be easy to understand with effect, below in conjunction with tool
Body example is expanded on further the present invention.
The high efficiency separation of a kind of Soil of Mulberry Garden ammonia oxidizing bacteria and authentication method, comprise the following steps:
Step 1, the enrichment of ammonia oxidizing bacteria: take 1.0g fresh Soil of Mulberry Garden sample, be equipped with in the test tube of enrichment culture liquid,
In 28 DEG C of constant incubators cultivate 14 days, draw culture fluid 5 in white plaque cave, be sequentially added into Griess reagent A liquid,
B liquid 1 or 2, if any nitrous acid: NO2 -Exist, then take on a red color, indicate that nitrite bacteria exists；If be detected that it is red
Color, adds the nutritional supplementation liquid of 1mL in this pipe, continues to cultivate.Detected once with Griess reagent every 3～4 days, when
When the culture fluid of detection is peony, the culture fluid drawing 1mL is further cultured for fresh enrichment culture liquid, is deep to detection liquid
Redness, this is highly enriched ammonia oxidizing bacteria culture fluid；
Step 2, isolated and purified ammonia oxygen antibacterial: take secondary described in 100mL step 1 and be enriched with to being detected as wine-colored culture fluid,
It is applied on isolation medium flat board, is cultivated 1-2 week more than for 28 DEG C, the most constantly observe colony growth situation, then choose
Taking circular water white transparency, the single bacterium colony line showing slightly yellow identifies cultivation in culture medium to BUG；
Step 3, the qualification of separate microorganism: identifying, with inoculating loop picking, the single bacterium colony separated of ruling in culture medium, to identifying
On culture fluid IF-A tube wall, with inoculating loop, bacterium is broken up, then it is mixed, to nephelometer with qualification liquid so that it is mixed degree
Between 90～95%, then the inoculation liquid volley of rifle fire is received on qualification culture plate, according to the speed of growth of each bacterium, at 28 DEG C
Incubator is cultivated 1～7 day, then use Biology Microststion (MicroStationTM, USA) and microbial identification system
Carry out Rapid identification, identify that the ammonia oxidizing bacteria being separated to includes Pseudomonas, Arthrobacter, Ochrobactrum,
Roseomonas, Enterobacter, Gordonia, Delftia, Pseudoxanthomonas, Mycobacterium,
Stenotrophomonas。
Described Pseudomonas is the main groups cultivating ammonia oxidizing bacteria.
The compound method of enrichment culture liquid described in step 1 is particularly as follows: weigh (NH4)2SO42.50g, K2HPO40.35g,
MgSO4·7H2O 0.25g, CaCl20.18g, with the Na of 5%2CO3Adjust pH to 8.5, be settled to 500mL, subpackage 10mL/
Test tube, 121 DEG C of sterilizing 30min.
The compound method of nutritional supplementation liquid described in step 1 is particularly as follows: weigh (NH4)2SO45.50g, is settled to 100mL, and 121
DEG C sterilizing 30min.
The compound method of Griess reagent described in step 1 is particularly as follows: A liquid, and 0.5g p-aminobenzene sulfonic acid is added to 150mL 200g/L
Acetic acid solution in；B liquid: 1g alpha-naphthylamine is added in the acetic acid solution of 20mL distilled water and 150mL 200g/L.Ge Lisi
Reagent can generate a kind of mauve compound (C with nitrite reaction10H6NH2-N:N-C6H4-SO3H), whether culture fluid becomes
Red and shade may indicate that NO2-generation and concentration.
The compound method of isolation medium described in step 2 is particularly as follows: weigh (NH4)2SO42.00g, KH2PO40.75g,
NaH2PO40.25g, MgSO4·7H2O 0.03g, MnSO4·4H2O, CaCO35.00g, with the Na of 5%2CO3PH is adjusted to arrive
8.0, it is settled to 1000mL, then adds 16g agar strip, described agar strip to shift to an earlier date three days and soak with pure water, every other day changes one
Secondary water, until agar strip is dipped to transparent, is down flat plate after 121 DEG C of sterilizing 30min standby.
BUG described in step 2 identify culture medium compound method particularly as follows: with Biolog identification systems outfit Biolog
BUGTM Agar prepares qualification culture medium according to the ratio of every 100mL distilled water 6g, is down flat plate after 121 DEG C of sterilizing 30min
Standby.
The above, only best mode for carrying out the invention, any those familiar with the art is at present disclosure
In technical scope, the simple change of the technical scheme that can become apparent to or equivalence are replaced and are each fallen within protection scope of the present invention
In.
Claims (7)
1. the high efficiency separation of a Soil of Mulberry Garden ammonia oxidizing bacteria and authentication method, it is characterised in that comprise the following steps:
Step 1, the enrichment of ammonia oxidizing bacteria: take 1.0g fresh Soil of Mulberry Garden sample, be equipped with in the test tube of enrichment culture liquid,
In 28 DEG C of constant incubators cultivate 14 days, draw culture fluid 5 in white plaque cave, be sequentially added into Griess reagent A liquid,
B liquid 1 or 2, if any nitrous acid: NO2 -Exist, then take on a red color, indicate that nitrite bacteria exists；If be detected that it is red
Color, adds the nutritional supplementation liquid of 1mL in this pipe, continues to cultivate, detected once with Griess reagent every 3～4 days, when
When the culture fluid of detection is peony, the culture fluid drawing 1mL is further cultured for fresh enrichment culture liquid, is deep to detection liquid
Redness, this is highly enriched ammonia oxidizing bacteria culture fluid；
Step 2, isolated and purified ammonia oxygen antibacterial: take secondary described in 100mL step 1 and be enriched with to being detected as wine-colored culture fluid,
It is applied on isolation medium flat board, is cultivated 1-2 week more than for 28 DEG C, the most constantly observe colony growth situation, then choose
Taking circular water white transparency, the single bacterium colony line showing slightly yellow identifies cultivation in culture medium to BUG；
Step 3, the qualification of separate microorganism: identifying, with inoculating loop picking, the single bacterium colony separated of ruling in culture medium, to identifying
On culture fluid IF-A tube wall, with inoculating loop, bacterium is broken up, then it is mixed, to nephelometer with qualification liquid so that it is mixed degree
Between 90～95%, then the inoculation liquid volley of rifle fire is received on qualification culture plate, according to the speed of growth of each bacterium, at 28 DEG C
Incubator is cultivated 1～7 day, then use Biology Microststion microbial identification system to carry out Rapid identification, identify and divide
From to ammonia oxidizing bacteria include Pseudomonas, Arthrobacter, Ochrobactrum, Roseomonas, Enterobacter,
Gordonia, Delftia, Pseudoxanthomonas, Mycobacterium, Stenotrophomonas.
The high efficiency separation of Soil of Mulberry Garden ammonia oxidizing bacteria the most according to claim 1 and authentication method, it is characterised in that
Described Pseudomonas is the main groups cultivating ammonia oxidizing bacteria.
The high efficiency separation of Soil of Mulberry Garden ammonia oxidizing bacteria the most according to claim 1 and authentication method, it is characterised in that
The compound method of enrichment culture liquid described in step 1 is particularly as follows: weigh (NH4)2SO42.50g, K2HPO40.35g,
MgSO4·7H2O 0.25g, CaCl20.18g, with the Na of 5%2CO3Adjust pH to 8.5, be settled to 500mL, subpackage 10mL/
Test tube, 121 DEG C of sterilizing 30min.
The high efficiency separation of Soil of Mulberry Garden ammonia oxidizing bacteria the most according to claim 1 and authentication method, it is characterised in that
The compound method of nutritional supplementation liquid described in step 1 is particularly as follows: weigh (NH4)2SO45.50g, is settled to 100mL, 121 DEG C
Sterilizing 30min.
The high efficiency separation of Soil of Mulberry Garden ammonia oxidizing bacteria the most according to claim 1 and authentication method, it is characterised in that
The compound method of Griess reagent described in step 1 is particularly as follows: A liquid, and 0.5g p-aminobenzene sulfonic acid is added to 150mL 200g/L
Acetic acid solution in；B liquid: 1g alpha-naphthylamine is added in the acetic acid solution of 20mL distilled water and 150mL 200g/L.
The high efficiency separation of Soil of Mulberry Garden ammonia oxidizing bacteria the most according to claim 1 and authentication method, it is characterised in that
The compound method of isolation medium described in step 2 is particularly as follows: weigh (NH4)2SO42.00g, KH2PO40.75g, NaH2PO4
0.25g, MgSO4·7H2O 0.03g, MnSO4·4H2O, CaCO35.00g, with the Na of 5%2CO3Adjust pH to 8.0, fixed
Holding to 1000mL, then add 16g agar strip, described agar strip to shift to an earlier date three days and soak with pure water, every other day changes a water,
Until agar strip is dipped to transparent, it is down flat plate after 121 DEG C of sterilizing 30min standby.
The high efficiency separation of Soil of Mulberry Garden ammonia oxidizing bacteria the most according to claim 1 and authentication method, it is characterised in that
BUG described in step 2 identify culture medium compound method particularly as follows: with Biolog identification systems outfit Biolog BUGTM
Agar prepares qualification culture medium according to the ratio of every 100mL distilled water 6g, is down flat plate standby after 121 DEG C of sterilizing 30min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570427A (en) * | 2018-03-22 | 2018-09-25 | 鄂尔多斯市亿鼎生态农业开发有限公司 | A kind of preparation method of microbial bacterial agent and the fertilizer prepared using microbial bacterial agent |
| NL2031454B1 (en) * | 2021-12-30 | 2023-07-05 | Inst Of Economic Crops Hubei Academy Of Agricultural Sciences | Method for efficient separation and identification of ammonia-oxidizing bacteria in mulberry field soil |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570427A (en) * | 2018-03-22 | 2018-09-25 | 鄂尔多斯市亿鼎生态农业开发有限公司 | A kind of preparation method of microbial bacterial agent and the fertilizer prepared using microbial bacterial agent |
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