WO2017045274A1 - 一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法 - Google Patents

一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法 Download PDF

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WO2017045274A1
WO2017045274A1 PCT/CN2015/096050 CN2015096050W WO2017045274A1 WO 2017045274 A1 WO2017045274 A1 WO 2017045274A1 CN 2015096050 W CN2015096050 W CN 2015096050W WO 2017045274 A1 WO2017045274 A1 WO 2017045274A1
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nitrogen
free medium
bacteria
free
ammonia
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French (fr)
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杨国平
孙旭生
王亚君
杨盼盼
尹坤
张洪彬
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东莞市保得生物工程有限公司
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/045—Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)

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  • the invention relates to the field of medium and nitrogen fixation technology, in particular to a nitrogen-free medium and a method for identifying whether or not the autotrophic nitrogen-fixing bacteria secrete ammonia.
  • Biological nitrogen fixation is a biochemical property possessed by a very small number of prokaryotes. Microorganisms capable of nitrogen fixation can be divided into two major categories: symbiotic nitrogen-fixing bacteria and autotrophic nitrogen-fixing bacteria.
  • Symbiotic nitrogen-fixing bacteria refer to the ability to enter higher plants, live with host plants, and provide energy by plants. In return, bacteria convert nitrogen into ammonia for plant absorption and utilization. These nitrogen-fixing bacteria are usually not nitrogen-fixing when grown in vitro, and they usually exert their nitrogen-fixing function only on legumes.
  • Autotrophic nitrogen-fixing bacteria are bacteria that can fix nitrogen during the growth process. They do not need to enter the plant and can fix nitrogen in the soil, plant rhizosphere and plant roots. Therefore, autotrophic bacteria can fix nitrogen in the rhizosphere of any plant. In addition to its own nitrogen-fixing ability, autotrophic nitrogen-fixing bacteria can secrete fixed nitrogen nutrients to the cells for use by plants or other organisms, which is also an important factor for humans to consider.
  • the object of the present invention is to provide a nitrogen-free medium and a method for identifying whether or not the autotrophic nitrogen-fixing bacteria secrete ammonia.
  • the method is simple and easy, and can accurately identify whether the nitrogen-fixing bacteria are secreted. Nitrogen-containing compounds to the extracellular and secretion capacity.
  • a nitrogen-free medium each liter of nitrogen-free medium consisting of the following materials:
  • each liter of nitrogen-free medium consists of the following materials:
  • each liter of nitrogen-free medium consists of the following materials:
  • a method for identifying whether or not an autotrophic nitrogen-fixing bacteria secretes ammonia using a nitrogen-free medium comprising the steps of:
  • Step 1 Mix the formula amount of K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O, agar and distilled water, and mix well. Nitrogen-free medium;
  • Step 2 preparing a double-layer plate, sterilizing the nitrogen-free medium obtained in the first step at 118-124 ° C for 15-25 minutes, and adding the indicator bacteria when the temperature of the nitrogen-free medium after sterilization is lowered to 46-48 ° C.
  • Add 1 ml of indicator bacteria per 100 ml of nitrogen-free medium mix the nitrogen-free medium supplemented with indicator bacteria, and pour into a sterile culture dish. Control the thickness of the nitrogen-free medium to 2.2-2.8 mm. After solidification, pour a layer of water agar with a thickness of 0.5 to 1 mm.
  • the plate thus produced is a double-layer plate, the lower layer is a bacteria-free nitrogen-free solid medium, and the upper layer is a water-free agar containing no bacteria;
  • Step 3 The self-generated nitrogen-fixing bacteria to be detected are placed on the double-layer plate prepared in the second step, and cultured in a 30 ° C incubator for 2 to 5 days;
  • Step 4 Check the results and check whether the indicator bacteria in the lower layer of the double-layer plate grow. If the indicator bacteria grows, the self-generated nitrogen-fixing bacteria can be used to secrete ammonia.
  • the preparation method of the water agar in the second step is as follows: 1 liter of distilled water plus 10-15 g of agar, sterilized at 115-125 ° C for 15-25 minutes, and then placed in a 45-55 ° C incubator for heat preservation.
  • the indicator bacteria are Escherichia coli, and of course, microorganisms which cannot fix nitrogen can also be used.
  • the viable concentration of the indicator bacteria is 10 million/ml to 30 million/ml.
  • the live bacteria concentration of the indicator bacteria in the second step is 20 million/ml, and the live bacteria concentration of the indicator bacteria refers to the concentration of the indicator bacteria contained in the aqueous solution.
  • K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O is analytically pure, and agar should be washed with distilled water before use.
  • the solid medium used in the present invention is a nitrogen-free medium containing no nitrogen nutrition, and bacteria which cannot be self-nurtating nitrogen cannot grow thereon.
  • the specific method is to use a bacteria that cannot fix nitrogen as an indicator bacteria, such as Escherichia coli, mixed in a nitrogen-free medium, and then point the autotrophic nitrogen-fixing bacteria to be tested onto the double-layer plate to observe whether the auto-a nitrogen-fixing bacteria can grow. If the autotrophic nitrogen-fixing bacteria can secrete the fixed ammonia to the extracellular, the E. coli around the colony can grow, and whether the auto-nitrogen-fixing bacteria is secreted and the strength can be judged according to whether the indicator grows or grows.
  • the beneficial effects of the present invention are as follows:
  • the present invention provides a nitrogen-free medium and a method for identifying whether or not the autotrophic nitrogen-fixing bacteria secrete ammonia.
  • the method is simple and easy, and can accurately identify whether the nitrogen-fixing bacteria secrete nitrogen-containing compounds to the extracellular, and the secretion capacity, which plays an important role in the development of the bio-fertilizer industry.
  • Figure 1 is a graph showing the growth of indicator bacteria around the colonies of autotrophic nitrogen-fixing bacteria capable of ammonia secretion.
  • Fig. 2 is a schematic diagram showing the growth of indicator bacteria around the colonies of autobiological nitrogen-fixing bacteria capable of secreting ammonia and capable of secreting ammonia.
  • a nitrogen-free medium consisting of the following raw materials per liter of nitrogen-free medium:
  • a method for identifying whether or not an autotrophic nitrogen-fixing bacteria secretes ammonia using a nitrogen-free medium comprising the steps of:
  • Step 1 Mix the formula amount of K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O, agar and distilled water, and mix well. Nitrogen-free medium;
  • Step 2 Prepare a double-layer plate, and sterilize the nitrogen-free medium obtained in the first step at 118 ° C for 15 minutes.
  • the indicator bacteria are added, and the nitrogen-free culture is performed per 100 ml. 1 ml of indicator bacteria in Kiga, immediately mix the nitrogen-free medium added to the indicator bacteria into a sterile culture dish, control the thickness of the nitrogen-free medium to be controlled at 2.2 mm, and then pour into the thickness after the nitrogen-free medium is solidified.
  • 0.5 mm layer of water agar the flat plate thus produced is a double-layer plate, the lower layer is a bacteria-free nitrogen-free solid medium, and the upper layer is a water-free agar containing no bacteria;
  • Step 3 The self-generated nitrogen-fixing bacteria to be detected are placed on the double-layer plate prepared in the second step, and cultured in a 30 ° C incubator for 2 to 5 days;
  • Step 4 Check the results and check whether the indicator bacteria in the lower layer of the double-layer plate grow. If the indicator bacteria grows, the self-generated nitrogen-fixing bacteria can be used to secrete ammonia.
  • the preparation method of the water agar in the second step is as follows: 1 liter of distilled water plus 10 grams of agar, sterilized at 115 ° C for 15 minutes, and then placed in a 45 ° C incubator for heat preservation.
  • the indicator bacteria is Escherichia coli.
  • the viable concentration of the indicator bacteria is 10 million/ml.
  • K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O is analytically pure, and agar should be washed with distilled water before use.
  • a nitrogen-free medium consisting of the following raw materials per liter of nitrogen-free medium:
  • a method for identifying whether or not an autotrophic nitrogen-fixing bacteria secretes ammonia using a nitrogen-free medium comprising the steps of:
  • Step 1 Mix the formula amount of K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O, agar and distilled water, and mix well. Nitrogen-free medium;
  • Step 2 Prepare a double-layer plate, and sterilize the nitrogen-free medium obtained in the first step at 121 ° C for 20 minutes.
  • the indicator bacteria are added, and the nitrogen-free culture is performed per 100 ml.
  • 1 ml of indicator bacteria in Kiga immediately mix the nitrogen-free medium added to the indicator bacteria into a sterile culture dish, control the thickness of the nitrogen-free medium to 2.5 mm, and then pour the thickness into the thickness after the nitrogen-free medium is solidified.
  • 0.8 mm layer of water agar the plate thus produced is a double-layer plate, the lower layer is a bacteria-free nitrogen-free solid medium, and the upper layer is a water-free agar containing no bacteria;
  • Step 3 The self-generated nitrogen-fixing bacteria to be detected are placed on the double-layer plate prepared in the second step, and cultured in a 30 ° C incubator for 2 to 5 days;
  • Step 4 Check the results and check whether the indicator bacteria in the lower layer of the double-layer plate grow. If the indicator bacteria grows, the self-generated nitrogen-fixing bacteria can be used to secrete ammonia.
  • the water agar is prepared by adding 1 liter of distilled water and 15 grams of agar, sterilizing at 121 ° C for 20 minutes, and then incubating at 50 ° C in an incubator for use.
  • the indicator bacteria is Escherichia coli.
  • the viable concentration of the indicator bacteria is 20 million/ml.
  • K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O is analytically pure, and agar should be washed with distilled water before use.
  • a nitrogen-free medium consisting of the following raw materials per liter of nitrogen-free medium:
  • a method for identifying whether or not an autotrophic nitrogen-fixing bacteria secretes ammonia using a nitrogen-free medium comprising the steps of:
  • Step 1 Mix the formula amount of K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O, agar and distilled water, and mix well. Nitrogen-free medium;
  • Step 2 Prepare a double-layer plate, and sterilize the nitrogen-free medium obtained in the first step at 118-124 ° C for 25 minutes.
  • the indicator bacteria are added, and each 100 ml is not added.
  • Add 1 ml of indicator bacteria to the nitrogen medium mix the nitrogen-free medium added with the indicator bacteria into the sterile culture dish, control the thickness of the nitrogen-free medium to 2.6 mm, and pour into the nitrogen-free medium.
  • the flat plate thus produced is a double-layer plate, the lower layer is a bacteria-free nitrogen-free solid medium, and the upper layer is a water-free agar containing no bacteria;
  • Step 3 The self-generated nitrogen-fixing bacteria to be detected are placed on the double-layer plate prepared in the second step, and cultured in a 30 ° C incubator for 2 to 5 days;
  • Step 4 Check the results and check whether the indicator bacteria in the lower layer of the double-layer plate grow. If the indicator bacteria grows, the self-generated nitrogen-fixing bacteria can be used to secrete ammonia.
  • the water agar is prepared by adding 1 liter of distilled water and 15 grams of agar, sterilizing at 125 ° C for 25 minutes, and then setting 55 °C incubator keep warm.
  • the indicator bacteria is Escherichia coli.
  • the viable concentration of the indicator bacteria is 20 million/ml.
  • said step in a K 2 HPO 4, MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O is analytically pure, and agar should be washed with distilled water before use.
  • a nitrogen-free medium consisting of the following raw materials per liter of nitrogen-free medium:
  • a method for identifying whether or not an autotrophic nitrogen-fixing bacteria secretes ammonia using a nitrogen-free medium comprising the steps of:
  • Step 1 Mix the formula amount of K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O, agar and distilled water, and mix well. Nitrogen-free medium;
  • Step 2 Prepare a double-layer plate, and sterilize the nitrogen-free medium obtained in the first step at 124 ° C for 25 minutes.
  • the indicator bacteria are added, and the nitrogen-free culture is performed per 100 ml.
  • 1 ml of indicator bacteria in Kiga immediately mix the nitrogen-free medium added to the indicator bacteria into a sterile culture dish, control the thickness of the nitrogen-free medium to 2.8 mm, and then pour into the thickness after the nitrogen-free medium is solidified.
  • 1 mm of water agar the flat plate thus produced is a double-layer plate, the lower layer is a bacteria-free nitrogen-free solid medium, and the upper layer is a water-free agar containing no bacteria;
  • Step 3 The self-generated nitrogen-fixing bacteria to be detected are placed on the double-layer plate prepared in the second step, and cultured in a 30 ° C incubator for 2 to 5 days;
  • Step 4 Check the results and check whether the indicator bacteria in the lower layer of the double-layer plate grow. If the indicator bacteria grows, the self-generated nitrogen-fixing bacteria can be used to secrete ammonia.
  • the water agar is prepared by adding 1 liter of distilled water and 15 grams of agar, sterilizing at 121 ° C for 20 minutes, and then incubating at 50 ° C in an incubator for use.
  • the indicator bacteria is Escherichia coli.
  • the viable concentration of the indicator bacteria is 30 million / ml.
  • K 2 HPO 4 , MgSO 4 ⁇ 7H 2 O, CaCl 2 ⁇ 2H 2 O, glucose, FeSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O is analytically pure, and agar should be washed with distilled water before use.
  • FIG. 1 is a schematic view of the structure. It can be seen from the figure that the diameter of the indicator bacteria is gradually increased. The first one is the autotrophic nitrogen-fixing bacteria that cannot secrete ammonia, and the indicator bacteria around the colonies do not grow.
  • the second one is the ammonia secretion ability.
  • the weak autotrophic nitrogen-fixing bacteria have a slightly smaller indicator diameter around the colony, and the third is a self-generated nitrogen-fixing bacteria with a strong ammonia-carrying ability, and the diameter of the indicator bacteria around the colony is larger. Through the comparison of these three, the diameter of the indicator bacteria growing around the colonies of the autotrophic nitrogen-fixing bacteria with stronger ammonia-producing ability is also larger.
  • the nitrogenase activity of 72 strains of autotrophic nitrogen-fixing bacteria was tested. It was found that 68 strains had different levels of nitrogenase activity, and the nitrogen fixation activity was correlated with the diameter of indicator bacteria. The larger the indicator diameter, the more nitrogen-fixing enzyme activity. High, the stronger the ability to secrete ammonia to the outside of the cell, see Table 1.

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Abstract

提供了一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法。每升无氮培养基由以下原料组成：K2HPO40.5-2g、MgSO4·7H2O 0.1-0.5g、CaCl2·2H2O 0.05-0.15g、葡萄糖7-13g、FeSO4·7H2O 0.005-0.02g、NaMoO4·2H2O、0.005-0.01g、琼脂10-20g、蒸馏水余量。

Description

一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法 技术领域：
本发明涉及培养基及固氮技术领域，具体涉及一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法。
背景技术：
生物固氮是极少数原核生物才具备的生物化学特性，能够固氮的微生物可分为2大类：共生固氮菌和自生固氮菌。
共生固氮菌是指能够进入高等植物体内，与寄主植物共同生活，由植物提供能量物质，作为回报，细菌则把氮气转化为氨供植物吸收利用。这类固氮菌在寄主植物体外生长时通常是不固氮的，它们通常只在豆科作物上才能发挥其固氮功能。
自生固氮菌是指在生长过程中就能够固氮的菌，它无需进入植物体内，在土壤、植物根际和植物根表等生态环境下均能固氮。因此自生固氮菌可以在任何植物根际固氮。自生固氮菌除本身固氮能力强弱之外，能否把固定的氮营养分泌到细胞外供植物或其它生物利用，也是人类需要考虑的重要因素。
在将自生固氮菌用于微生物肥料生产时，如果所用的菌株不能将固定的氮分泌出来，那它促进植物生长的效果就很弱。如何鉴定自生固氮菌是否把固定的氮分泌到胞外，对筛选真正有效的固氮菌用于微生物肥料生产具有重要意义。
发明内容：
为了克服现有技术中存在的缺点和不足，本发明的目的在于提供一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法，该方法简单易行，可准确鉴定固氮菌是否分泌含氮化合物到胞外以及分泌能力大小。
本发明的目的通过下述技术方案实现：一种无氮培养基，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000001
Figure PCTCN2015096050-appb-000002
优选的，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000003
更为优选的，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000004
Figure PCTCN2015096050-appb-000005
一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，它包括以下步骤：
步骤一：将配方量的K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O、琼脂和蒸馏水混合后搅拌均匀得到无氮培养基；
步骤二：制备双层平板，将步骤一得到的无氮培养基在118-124℃灭菌15-25分钟，待灭菌后的无氮培养基温度降至46-48℃时加入指示菌，每100毫升无氮培养基加1毫升指示菌，将加入指示菌的无氮培养基立即混匀倒入无菌培养皿，控制无氮培养基的厚度控制在2.2-2.8毫米，待无氮培养基凝固后再倒入厚度为0.5至1毫米的一层水琼脂，这样制作的平板即为双层平板，下层为含菌的无氮固体培养基，上层为不含菌的水琼脂；
步骤三：将待检测的自生固氮菌点到步骤二制备的双层平板上，置30℃恒温箱培养2至5天；
步骤四：结果检查，检查双层平板下层的指示菌是否有生长，如果发现指示菌出现生长，则待检测自生固氮菌能分泌氨。
所述步骤二中水琼脂的制备方法为：1升蒸馏水加10-15克琼脂，115-125℃灭菌15-25分钟，然后置45-55℃恒温箱保温备用。
所述步骤二中指示菌为大肠杆菌，当然也可以用不能固氮的微生物。
所述步骤二中指示菌的活菌浓度为1千万/毫升-3千万/毫升。
优选的，所述步骤二中指示菌的活菌浓度为2千万/毫升，指示菌的活菌浓度指的是水溶液中含有指示菌的浓度。
为了避免残留的微量氮源对试验的影响，所述步骤一中K2HPO4、MgSO4·7H2O、CaCl2·2H2O、 葡萄糖、FeSO4·7H2O、NaMoO4·2H2O均为分析纯，琼脂使用前需用蒸馏水洗。
本发明所用的固体培养基是不含氮素营养的无氮培养基，不能自生固氮的细菌不能在上面生长。具体方法是把不能固氮的细菌作为指示菌，如大肠杆菌，混在无氮培养基中，然后将待测的自生固氮菌点到双层平板上，观察自生固氮菌是否能够生长。如果自生固氮菌能够把固定的氨分泌到胞外，那么该菌落周围的大肠杆菌就能够生长，根据指示菌是否生长以及生长的多少可以判断自生固氮菌是否泌氨以及强度。
本发明的有益效果在于：本发明提供了一种无氮培养基及利用其鉴定自生固氮菌是否分泌氨的方法。该方法简单易行，可以准确鉴定固氮菌是否分泌含氮化合物到胞外，以及分泌能力大小，为生物肥料行业的发展起到重要作用。
附图说明：
图1能够泌氨的自生固氮菌其菌落周围的指示菌生长图。
图2不能泌氨的和能够泌氨的自生固氮菌其菌落周围的指示菌生长比较示意图。
具体实施方式：
为了便于本领域技术人员的理解，下面结合实施例和附图对本发明作进一步的说明，实施方式提及的内容并非对本发明的限定。
实施例1。
一种无氮培养基，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000006
Figure PCTCN2015096050-appb-000007
一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，它包括以下步骤：
步骤一：将配方量的K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O、琼脂和蒸馏水混合后搅拌均匀得到无氮培养基；
步骤二：制备双层平板，将步骤一得到的无氮培养基在118℃灭菌15分钟，待灭菌后的无氮培养基温度降至46℃时加入指示菌，每100毫升无氮培养基加1毫升指示菌，将加入指示菌的无氮培养基立即混匀倒入无菌培养皿，控制无氮培养基的厚度控制在2.2毫米，待无氮培养基凝固后再倒入厚度为0.5毫米的一层水琼脂，这样制作的平板即为双层平板，下层为含菌的无氮固体培养基，上层为不含菌的水琼脂；
步骤三：将待检测的自生固氮菌点到步骤二制备的双层平板上，置30℃恒温箱培养2至5天；
步骤四：结果检查，检查双层平板下层的指示菌是否有生长，如果发现指示菌出现生长，则待检测自生固氮菌能分泌氨。
所述步骤二中水琼脂的制备方法为：1升蒸馏水加10克琼脂，115℃灭菌15分钟，然后置45℃恒温箱保温备用。
所述步骤二中指示菌为大肠杆菌。
所述步骤二中指示菌的活菌浓度为1千万/毫升。
为了避免残留的微量氮源对试验的影响，所述步骤一中K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O均为分析纯，琼脂使用前需用蒸馏水洗。
实施例2。
一种无氮培养基，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000008
Figure PCTCN2015096050-appb-000009
一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，它包括以下步骤：
步骤一：将配方量的K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O、琼脂和蒸馏水混合后搅拌均匀得到无氮培养基；
步骤二：制备双层平板，将步骤一得到的无氮培养基在121℃灭菌20分钟，待灭菌后的无氮培养基温度降至47℃时加入指示菌，每100毫升无氮培养基加1毫升指示菌，将加入指示菌的无氮培养基立即混匀倒入无菌培养皿，控制无氮培养基的厚度控制在2.5毫米，待无氮培养基凝固后再倒入厚度为0.8毫米的一层水琼脂，这样制作的平板即为双层平板，下层为含菌的无氮固体培养基，上层为不含菌的水琼脂；
步骤三：将待检测的自生固氮菌点到步骤二制备的双层平板上，置30℃恒温箱培养2至5天；
步骤四：结果检查，检查双层平板下层的指示菌是否有生长，如果发现指示菌出现生长，则待检测自生固氮菌能分泌氨。
所述步骤二中水琼脂的制备方法为：1升蒸馏水加15克琼脂，121℃灭菌20分钟，然后置50℃恒温箱保温备用。
所述步骤二中指示菌为大肠杆菌。
所述步骤二中指示菌的活菌浓度为2千万/毫升。
为了避免残留的微量氮源对试验的影响，所述步骤一中K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O均为分析纯，琼脂使用前需用蒸馏水洗。
实施例3。
一种无氮培养基，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000010
一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，它包括以下步骤：
步骤一：将配方量的K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O、琼脂和蒸馏水混合后搅拌均匀得到无氮培养基；
步骤二：制备双层平板，将步骤一得到的无氮培养基在118-124℃灭菌25分钟，待灭菌后的无氮培养基温度降至47℃时加入指示菌，每100毫升无氮培养基加1毫升指示菌，将加入指示菌的无氮培养基立即混匀倒入无菌培养皿，控制无氮培养基的厚度控制在2.6毫米，待无氮培养基凝固后再倒入厚度为0.6毫米的一层水琼脂，这样制作的平板即为双层平板，下层为含菌的无氮固体培养基，上层为不含菌的水琼脂；
步骤三：将待检测的自生固氮菌点到步骤二制备的双层平板上，置30℃恒温箱培养2至5天；
步骤四：结果检查，检查双层平板下层的指示菌是否有生长，如果发现指示菌出现生长，则待检测自生固氮菌能分泌氨。
所述步骤二中水琼脂的制备方法为：1升蒸馏水加15克琼脂，125℃灭菌25分钟，然后置55 ℃恒温箱保温备用。
所述步骤二中指示菌为大肠杆菌。
所述步骤二中指示菌的活菌浓度为2千万/毫升。
为了避免残留的微量氮源对试验的影响，所述步骤一中K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O均为分析纯，琼脂使用前需用蒸馏水洗。
实施例4。
一种无氮培养基，每升无氮培养基由以下原料组成：
Figure PCTCN2015096050-appb-000011
一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，它包括以下步骤：
步骤一：将配方量的K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O、琼脂和蒸馏水混合后搅拌均匀得到无氮培养基；
步骤二：制备双层平板，将步骤一得到的无氮培养基在124℃灭菌25分钟，待灭菌后的无氮培养基温度降至48℃时加入指示菌，每100毫升无氮培养基加1毫升指示菌，将加入指示菌的无氮培养基立即混匀倒入无菌培养皿，控制无氮培养基的厚度控制在2.8毫米，待无氮培养基凝固后再倒入厚度为1毫米的一层水琼脂，这样制作的平板即为双层平板，下层为含菌的无氮固体培养基，上层为不含菌的水琼脂；
步骤三：将待检测的自生固氮菌点到步骤二制备的双层平板上，置30℃恒温箱培养2至5天；
步骤四：结果检查，检查双层平板下层的指示菌是否有生长，如果发现指示菌出现生长，则待检测自生固氮菌能分泌氨。
所述步骤二中水琼脂的制备方法为：1升蒸馏水加15克琼脂，121℃灭菌20分钟，然后置50℃恒温箱保温备用。
所述步骤二中指示菌为大肠杆菌。
所述步骤二中指示菌的活菌浓度为3千万/毫升。
为了避免残留的微量氮源对试验的影响，所述步骤一中K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O均为分析纯，琼脂使用前需用蒸馏水洗。
实验数据
本发明按照实施例1-4的方法，对200株不同的自生固氮菌进行测试，发现72株自生固氮菌不同程度的能使指示菌生长，指示菌生长可参考图1，实验还发现这72株自生固氮菌均能不同程度的促进植物生长，这说明这72株自生固氮菌均有不同程度分泌氨到细胞外的能力。图2是结构示意图，从图中可以看出指示菌形成的直径逐渐增大，第一个是不能泌氨的自生固氮菌，其菌落周围的指示菌没有生长，第二个是泌氨能力稍弱的自生固氮菌，其菌落周围的指示菌直径稍小，第三个是泌氨能力较强的自生固氮菌，其菌落周围的指示菌直径较大。通过这三者的比较，说明泌氨能力越强的自生固氮菌其菌落周围的指示菌生长形成的直径也越大。
接下来又对这72株自生固氮菌进行固氮酶活性测试，结果发现68株均有不同程度的固氮酶活性，而且固氮活性与指示菌直径具有相关性，指示菌直径越大，固氮酶活性越高，分泌氨到细胞外的能力越强，详见表1。
表1
Figure PCTCN2015096050-appb-000012
Figure PCTCN2015096050-appb-000013
上述实施例为本发明较佳的实现方案，除此之外，本发明还可以其它方式实现，在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。

Claims (8)

  1. 一种无氮培养基，其特征在于：每升无氮培养基由以下原料组成：
    Figure PCTCN2015096050-appb-100001
  2. 根据权利要求1所述的一种无氮培养基，其特征在于：每升无氮培养基由以下原料组成：
    Figure PCTCN2015096050-appb-100002
    Figure PCTCN2015096050-appb-100003
  3. 根据权利要求1所述的一种无氮培养基，其特征在于：每升无氮培养基由以下原料组成：
    Figure PCTCN2015096050-appb-100004
  4. 一种利用权利要求1-3任一项所述的无氮培养基鉴定自生固氮菌是否分泌氨的方法，其特征在于：它包括以下步骤：
    步骤一：将配方量的K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O、琼脂和蒸馏水混合后搅拌均匀得到无氮培养基；
    步骤二：制备双层平板，将步骤一得到的无氮培养基在118-124℃灭菌15-25分钟，待灭菌后的无氮培养基温度降至46-48℃时加入指示菌，每100毫升无氮培养基加1毫升指示菌，将加入指示菌的无氮培养基立即混匀倒入无菌培养皿，控制无氮培养基的厚度控制在2.2-2.8毫米，待无氮培养基凝固后再倒入厚度为0.5至1毫米的一层水琼脂，这样制作的平板即为双层平板，下层为含菌的无氮固体培养基，上层为不含菌的水琼脂；
    步骤三：将待检测的自生固氮菌点到步骤二制备的双层平板上，置30℃恒温箱培养2至5天；
    步骤四：结果检查，检查双层平板下层的指示菌是否有生长，如果发现指示菌出现生长，则待检测自生固氮菌能分泌氨。
  5. 根据权利要求4所述的一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，其特征在于：所述步骤二中水琼脂的制备方法为：1升蒸馏水加10-15克琼脂，115-125℃灭菌15-25分钟，然后置45-55℃恒温箱保温备用。
  6. 根据权利要求4所述的一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，其特征在于：所述步骤二中指示菌为大肠杆菌。
  7. 根据权利要求4所述的一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，其特征在于：所述步骤一中K2HPO4、MgSO4·7H2O、CaCl2·2H2O、葡萄糖、FeSO4·7H2O、NaMoO4·2H2O均为分析纯，琼脂使用前需用蒸馏水洗。
  8. 根据权利要求4所述的一种利用无氮培养基鉴定自生固氮菌是否分泌氨的方法，其特征在于：所述步骤二中指示菌的活菌浓度为1千万/毫升-3千万/毫升。
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