CN110551642A - separation and purification inspection method for fungi with high pollution background - Google Patents
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- 241000233866 Fungi Species 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 title claims abstract description 18
- 238000007689 inspection Methods 0.000 title claims description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 59
- 238000002474 experimental method Methods 0.000 claims abstract description 16
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- 230000006698 induction Effects 0.000 claims abstract description 8
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- 238000005336 cracking Methods 0.000 claims abstract description 4
- 238000012136 culture method Methods 0.000 claims abstract description 4
- 208000031513 cyst Diseases 0.000 claims abstract description 4
- 241000223259 Trichoderma Species 0.000 claims description 15
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- 210000002993 trophoblast Anatomy 0.000 claims description 10
- 108010059892 Cellulase Proteins 0.000 claims description 8
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- 238000012258 culturing Methods 0.000 claims description 8
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- 238000012360 testing method Methods 0.000 claims description 7
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- CRPUJAZIXJMDBK-UHFFFAOYSA-N camphene Chemical compound C1CC2C(=C)C(C)(C)C1C2 CRPUJAZIXJMDBK-UHFFFAOYSA-N 0.000 claims description 6
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- 238000009395 breeding Methods 0.000 claims description 3
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- 229930006739 camphene Natural products 0.000 claims description 3
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- 239000011248 coating agent Substances 0.000 claims description 3
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- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 3
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- 239000000463 material Substances 0.000 abstract description 2
- 231100000614 poison Toxicity 0.000 abstract description 2
- 239000003440 toxic substance Substances 0.000 abstract description 2
- 241000223261 Trichoderma viride Species 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 3
- 230000035784 germination Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
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- 241000228143 Penicillium Species 0.000 description 1
- 241000378866 Trichoderma koningii Species 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
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- 230000004763 spore germination Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
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- 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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- 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
- C12N1/14—Fungi; Culture media therefor
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Abstract
A method for separating, purifying and inspecting fungi with high pollution background adopts a double-sided forward fungus purification and separation culture method, wherein the double-sided forward separation method is based on that two-sided culture media are arranged in a culture dish, air is used as a separation medium, the propagation of aerial hyphae to a target culture layer is promoted through induction of allelochemicals, or spores are produced through induction of a feeding layer, and the spores are ejected through cracking of fungal cysts, so that the separation and purification of the monospores are realized. The biological safety can be improved, the experiment time is reduced, the experiment efficiency is improved, and the consumption of consumed materials and the addition of toxic substances are reduced; the method has strong flexibility, can add one or more inhibitors or stimulators to any culture layer, and can intervene the growth of fungi by using allelochemicals as signals to achieve the purpose of separation and purification; can realize good separation of phage pollution of industrial fungi, and is beneficial to purifying bacterial strains polluted by the phage; the method is simple and easy to popularize, and has good fungus detection adaptability.
Description
Technical Field
The invention relates to a biological detection technology, in particular to a separation and purification inspection method for high-pollution background fungi.
background
It is known that trichoderma is difficult to purify and separate because of the growth of many symbiotic fungi and bacteria in the habitat, and the separation effect is poor and the biosafety risk is caused by the competitive growth among the fungi and the spore dispersion. And the traditional fungus culture experiment period is long, so that the experiment efficiency is greatly reduced. Trichoderma is one of the industrially used huge variety of microorganisms.
in the background of the separation of trichoderma, because various microorganisms are rich and the contents of spore bacteria, actinomycetes, penicillium, aspergillus, bacteriophage and the like are extremely high, the pollution of background interference bacteria cannot be reduced by adopting a liquid dilution method, and the separation purity requirement cannot be met by adopting a traditional streaking method, so that a better separation method and a better separation method are urgently needed in fungus separation and inspection.
Disclosure of Invention
the invention aims to solve the technical problem of the prior art and provides a separation and purification inspection method for high-pollution background fungi, which improves the efficiency, reduces the biological safety risk and protects the safety of testers.
the technical problem to be solved by the invention is realized by the following technical scheme, and the separation and purification inspection method of the fungi with high pollution background is characterized in that:
adopts a double-sided forward fungus purification and separation culture method,
The double-sided forward separation method is based on that two-sided culture media are arranged in a culture dish, air is used as a separation medium, the growth of aerial hyphae towards a target culture layer is promoted through allelopathy substance induction, or spores are produced through induction of a breeding layer, and spores are ejected through cracking of fungal cysts, so that the separation and purification of the monospores are realized.
The technical problem to be solved by the invention can be further realized by the following technical scheme, and the specific operation method comprises the following steps:
Sterilizing water agar culture medium at 115 ~ 125 deg.C for 10 ~ 20min, spreading to a thickness of 3 + -1 mm, and solidifying to form trophoblast culture medium;
paving a target culture layer to a thickness of 3 +/-1 mm, and cooling to form a separation layer culture medium for later use;
Weighing 2g of soil, and paving the soil on a trophoblast culture medium into a round shape with the diameter of 20mm by using an aseptic coating rod; removing the peripheral water agar to prepare a sample separation dish;
sterilizing the glass ring, then pasting the sterilized glass ring on a separation layer culture medium to prepare a separation dish, oppositely buckling the separation dish with a sample separation dish, and placing the separation dish into an incubator for culture;
When observing that the filamentous fungus aerial hyphae generated by the soil on the trophoblast culture medium in the sample separation dish just contact the separation layer culture medium at the top through the culture dish, moving the separation layer culture medium separation dish with the upside down, covering the separation layer culture medium separation dish with a sterile flat dish, and performing inverted culture,
the sample separation vessel is buckled on the separation vessel again according to the requirement to continue the separation;
Separation and identification:
the fungal plaque grown on the medium in the separation dish, cultured under the above conditions, has a transparent ring, and the fungus having this ring demonstrates the ability to break down cellulose and produce cellulase, to isolate a functional fungus;
Picking hypha with transparent rings, inoculating the hypha into a new cellulose culture medium again, culturing, making an insert, observing the hypha and spores grown by the insert, and proving that the hypha and the spores belong to trichoderma and produce cellulase to hydrolyze cellulose;
the purified trichoderma is further used for fungus biochemical experiments or molecular identification experiments.
the technical problem to be solved by the invention can be further realized by the following technical scheme that the nutrient layer culture medium is water agar with the mass ratio of 1 ~ 2.5.5%.
the technical problem to be solved by the invention can be further solved by the following technical scheme that an inhibiting substance with the mass ratio of 0.5 ~ 1.5.5% is added into the nutrient layer culture medium.
the technical problem to be solved by the invention can be further realized by the following technical scheme, wherein the target separation layer culture medium is a separation selective culture medium which takes a PDA culture medium as a substrate and is added with VB1, a target test substrate and allelochemicals.
The technical problem to be solved by the invention can be further realized by adopting the following technical scheme that the allelochemicals are one or more of plant extract, 3-hexen-1-ol, phellandrene, 2-carene, alpha-pinene or camphene, and the adding amount of the allelochemicals is 1 mu g of the culture medium substrate added per 1000 ml.
the technical problem to be solved by the invention can be further realized by the following technical scheme, and the culture temperature is 2-25 ℃.
The technical problem to be solved by the invention can be further realized by the following technical scheme, wherein the culture temperature is 22 ℃ as the general temperature.
Compared with the prior art, the invention has the beneficial effects that:
(1) The biological safety can be improved, the experiment time is reduced, the experiment efficiency is improved, and the consumption of consumed materials and the addition of toxic substances are reduced;
(2) the method has the advantages that the method is established, the reliability of the separation experiment result is high, and the separation of multiple filamentous fungus mixed samples can be realized;
(3) The method has strong flexibility, can add one or more inhibitors or stimulators to any culture layer, and can intervene the growth of fungi by using allelochemicals as signals to achieve the purpose of separation and purification;
(4) Can realize good separation of phage pollution of industrial fungi, and is beneficial to purifying bacterial strains polluted by the phage;
(5) the method is simple and easy to popularize, and has good fungus detection adaptability.
Detailed Description
The following further describes particular embodiments of the present invention to facilitate further understanding of the present invention by those skilled in the art, and does not constitute a limitation to the right thereof.
A separation and purification inspection method of fungi with high pollution background,
adopts a double-sided forward fungus purification and separation culture method,
The double-sided forward separation method is based on that two-sided culture media are arranged in a culture dish, air is used as a separation medium, the growth of aerial hyphae towards a target culture layer is promoted through allelopathy substance induction, or spores are produced through induction of a breeding layer, and spores are ejected through cracking of fungal cysts, so that the separation and purification of the monospores are realized.
The specific operation method comprises the following steps:
Sterilizing water agar culture medium at 115 ~ 125 deg.C for 10 ~ 20min, spreading to a thickness of 3 + -1 mm, and solidifying to form trophoblast culture medium;
Paving a target culture layer to a thickness of 3 +/-1 mm, and cooling to form a separation layer culture medium for later use;
weighing 2g of soil, and paving the soil on a trophoblast culture medium into a round shape with the diameter of 20mm by using an aseptic coating rod; removing the peripheral water agar to prepare a sample separation dish;
sterilizing the glass ring, then pasting the sterilized glass ring on a separation layer culture medium to prepare a separation dish, oppositely buckling the separation dish with a sample separation dish, and placing the separation dish into an incubator for culture;
When observing that the filamentous fungus aerial hyphae generated by the soil on the trophoblast culture medium in the sample separation dish just contact the separation layer culture medium at the top through the culture dish, moving the separation layer culture medium separation dish with the upside down, covering the separation layer culture medium separation dish with a sterile flat dish, and performing inverted culture,
The sample separation vessel is buckled on the separation vessel again according to the requirement to continue the separation;
Separation and identification:
The fungal plaque grown on the medium in the separation dish, cultured under the above conditions, has a transparent ring, and the fungus having this ring demonstrates the ability to break down cellulose and produce cellulase, to isolate a functional fungus;
Picking hypha with transparent rings, inoculating the hypha into a new cellulose culture medium again, culturing, making an insert, observing the hypha and spores grown by the insert, and proving that the hypha and the spores belong to trichoderma and produce cellulase to hydrolyze cellulose;
the purified trichoderma is further used for fungus biochemical experiments or molecular identification experiments.
The nutrient layer culture medium is water agar with the mass ratio of 1 ~ 2.5.5%.
the nutrient layer culture medium is added with 0.5 ~ 1.5.5 wt% of inhibitor such as glucose.
The technical problem to be solved by the invention can be further realized by the following technical scheme, wherein the target separation layer culture medium is a separation selective culture medium which takes a PDA culture medium as a substrate and is added with VB1, a target test substrate and allelochemicals.
VB1, adding 6 ~ 10mg/L, adding 4 ~ 6% of cellulose for a target test substrate by mass percent, wherein the allelochemicals are one or more of plant extract, 3-hexen-1-ol, phellandrene, 2-carene, alpha-pinene or camphene, and the adding amount of the allelochemicals is 1 mu g of the culture medium substrate added per 1000 ml.
The culture temperature is 2-25 ℃.
The incubation temperature was 22 ℃ as the general temperature.
wherein 22 ℃ can be used as the general temperature, and 2-8 ℃ can also be used as the separation culture temperature under special conditions. The temperature of 2-8 ℃ is realized by adopting a refrigeration incubator or a safe refrigerator.
Taking trichoderma viride as an example to illustrate:
The trichoderma viride hyphae are white and fine, and the width of the trichoderma viride hyphae is 1.5-2.4 micrometers. Conidia are produced. Conidiophores are vertically and symmetrically diverged, conidiophores grow singly or in clusters, and are round and green. The appearance of the trichoderma viride colony is dark green or blue-green; trichoderma koningii colonies were pale green, yellowish green or green in appearance.
The conidiophores of the trichoderma viride have septa and are vertically branched oppositely, the end of a conidiophores bottle body is tapered and slightly bent, conidiophores mass is generated at the tip, the conidiophores mass contains 4 ~ 12 spores, and the conidiophores are colorless, spherical to oval and have the diameter of 2.5 ~ 4.5.5 multiplied by 2 ~ 4 mu m.
the green trichoderma has strong adaptability, spores germinate on a PDA culture medium plate at 24 ℃, and colonies rapidly expand. Culturing for 2 days, wherein the diameter of a bacterial colony is 3.5-5.0 cm; culturing for 3 days, wherein the diameter of a bacterial colony is 7.3-8.0 cm; culturing for 4 days, wherein the diameter of a bacterial colony is 8.1-9.0 cm.
Observation by a microscope oil microscope shows that: the spore 1-2mm under the mirror is in a scattered sac static state, dark green or blue-green color, metallic luster, more spores, less white or dark green hyphae, and sectioned hyphae.
Usually the colonies spread rapidly, and especially in high temperature and humidity conditions within a few days the trichoderma colonies can spread over the entire surface. The growth temperature of hyphae is 4-42 ℃, the growth is fastest at 25-30 ℃, the germination temperature of spores is 10-35 ℃, the germination rate is highest at 15-30 ℃, the growth of bacterial colonies at 25-27 ℃ is changed from white to green only by 4-5 days, and the high temperature is favorable for the growth and germination of the hyphae. Spore germination requires more than 95% of relative humidity, but spore can also grow in a dry environment, the pH value of hypha growth is 3.5-5.8, and the growth is fastest under the condition of pH value 4-5.
The following description specifically describes a specific embodiment of the present invention suitable for experiments of isolating and purifying trichoderma, and provides 1 example, but the practice of the present invention is not limited to the following examples.
example 1 isolation, screening and purification of Trichoderma species was used. It was used as follows:
sterilizing the water agar with the agar content of 1.5% at 121 ℃ for 15min, paving a flat plate with the thickness of about 3mm, and solidifying for later use.
the separation layer medium was based on PDA and then VB 1: 8mg/L, 5ml of corydalis edulis extract and 5 percent of cellulose. The thickness of the paving plate is 3mm, and the paving plate is cooled for standby.
2g of soil was weighed and spread with a sterile spreading rod into a circle having a diameter of 20 mm. The outer water agar was removed and a sample separation dish was prepared.
Sterilizing the glass ring, attaching the sterilized glass ring to a separation layer culture medium to prepare a separation dish, oppositely buckling the separation dish with a sample separation dish, and culturing in a 22 ℃ incubator.
When the culture dish is observed that the aerial hyphae of the filamentous fungi generated by the soil on the trophoblast culture medium in the separation dish just contact with the separation identification culture medium at the top, the separation culture medium dish with the upside-down top is moved away and is covered by a sterile plate to cause culture, and the sample culture dish can be buckled on the separation plate again as required to be continuously separated.
example of isolation and identification:
the mold plaque growing on the medium in the separation plate cultured at 22 ℃ has a transparent ring, and the fungus having this ring demonstrates the ability to decompose cellulose and produce cellulase, and a functional fungus is isolated. And (3) selecting hypha with a transparent ring, inoculating the hypha into a new cellulose culture medium again, culturing, making an insert, and observing the hypha and spores grown on the insert to prove that the hypha and the spores belong to trichoderma and have the function of producing cellulase to hydrolyze cellulose. The purified trichoderma can be further used for fungus biochemical experiments or molecular identification experiments.
Claims (8)
1. A separation and purification inspection method for high-pollution background fungi is characterized by comprising the following steps:
Adopts a double-sided forward fungus purification and separation culture method,
the double-sided forward separation method is based on that two-sided culture media are arranged in a culture dish, air is used as a separation medium, the growth of aerial hyphae towards a target culture layer is promoted through allelopathy substance induction, or spores are produced through induction of a breeding layer, and spores are ejected through cracking of fungal cysts, so that the separation and purification of the monospores are realized.
2. the method for separating, purifying and inspecting the high-pollution background fungi according to claim 1, which is characterized by comprising the following specific operation methods:
Sterilizing water agar culture medium at 115 ~ 125 deg.C for 10 ~ 20min, spreading to a thickness of 3 + -1 mm, and solidifying to form trophoblast culture medium;
Paving a target culture layer to a thickness of 3 +/-1 mm, and cooling to form a separation layer culture medium for later use;
weighing 2g of soil, and paving the soil on a trophoblast culture medium into a round shape with the diameter of 20mm by using an aseptic coating rod; removing the peripheral water agar to prepare a sample separation dish;
sterilizing the glass ring, then pasting the sterilized glass ring on a separation layer culture medium to prepare a separation dish, oppositely buckling the separation dish with a sample separation dish, and placing the separation dish into an incubator for culture;
When observing that the filamentous fungus aerial hyphae generated by the soil on the trophoblast culture medium in the sample separation dish just contact the separation layer culture medium at the top through the culture dish, moving the separation layer culture medium separation dish with the upside down, covering the separation layer culture medium separation dish with a sterile flat dish, and performing inverted culture,
the sample separation vessel is buckled on the separation vessel again according to the requirement to continue the separation;
Separation and identification:
the fungal plaque grown on the medium in the separation dish, cultured under the above conditions, has a transparent ring, and the fungus having this ring demonstrates the ability to break down cellulose and produce cellulase, to isolate a functional fungus;
Picking hypha with transparent rings, inoculating the hypha into a new cellulose culture medium again, culturing, making an insert, observing the hypha and spores grown by the insert, and proving that the hypha and the spores belong to trichoderma and produce cellulase to hydrolyze cellulose;
The purified trichoderma is further used for fungus biochemical experiments or molecular identification experiments.
3. the method for separating, purifying and testing high-pollution background fungi as claimed in claim 2, wherein the culture medium of the nutrient layer culture medium is water agar with a mass ratio of 1 ~ 2.5.5%.
4. the method for separating, purifying and inspecting high-pollution background fungi according to claim 2, wherein 0.5 ~ 1.5.5% by mass of inhibiting substances are added into the nutrient layer culture medium.
5. the method for separating, purifying and inspecting high-pollution background fungi according to claim 2, wherein the method comprises the following steps: the target separation layer culture medium is a separation selective culture medium which takes a PDA culture medium as a substrate and is added with VB1, a target test substrate and allelochemicals.
6. The method for separating, purifying and testing high-pollution background fungi according to claim 5, wherein the method comprises the following steps: the allelochemicals are one or more of plant extract, 3-hexen-1-ol, phellandrene, 2-carene, alpha-pinene or camphene, and the adding amount of the allelochemicals is 1 mu g of the allelochemicals added per 1000ml of the culture medium substrate.
7. The method for separating, purifying and inspecting high-pollution background fungi according to claim 2, wherein the method comprises the following steps: the culture temperature is 2-25 ℃.
8. The method for separating, purifying and testing high-pollution background fungi according to claim 7, wherein the method comprises the following steps: the incubation temperature was 22 ℃ as the general temperature.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN204224588U (en) * | 2014-11-12 | 2015-03-25 | 西北农林科技大学 | A kind of filamentous fungus purifying vessel and filamentous fungus culture dish |
| US20160369226A1 (en) * | 2013-11-07 | 2016-12-22 | Tianjin Institute of Industrial Biotechnology, Chinese Acadamy of Sciences | Solid-state biological reaction device and method for preparing filamentous organism spores by using the same |
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Patent Citations (2)
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| US20160369226A1 (en) * | 2013-11-07 | 2016-12-22 | Tianjin Institute of Industrial Biotechnology, Chinese Acadamy of Sciences | Solid-state biological reaction device and method for preparing filamentous organism spores by using the same |
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Application publication date: 20191210 |