CN110551620A - Special culture dish device of filamentous fungi separation and purification - Google Patents
Special culture dish device of filamentous fungi separation and purification Download PDFInfo
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- CN110551620A CN110551620A CN201910936687.2A CN201910936687A CN110551620A CN 110551620 A CN110551620 A CN 110551620A CN 201910936687 A CN201910936687 A CN 201910936687A CN 110551620 A CN110551620 A CN 110551620A
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- 238000000926 separation method Methods 0.000 title claims abstract description 87
- 241000233866 Fungi Species 0.000 title claims abstract description 33
- 238000000746 purification Methods 0.000 title claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 39
- 238000002955 isolation Methods 0.000 claims abstract description 8
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 239000003627 allelochemical Substances 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- IBVJWOMJGCHRRW-UHFFFAOYSA-N 3,7,7-Trimethylbicyclo[4.1.0]hept-2-ene Chemical compound C1CC(C)=CC2C(C)(C)C12 IBVJWOMJGCHRRW-UHFFFAOYSA-N 0.000 claims description 8
- CRPUJAZIXJMDBK-UHFFFAOYSA-N camphene Chemical compound C1CC2C(=C)C(C)(C)C1C2 CRPUJAZIXJMDBK-UHFFFAOYSA-N 0.000 claims description 8
- GRWFGVWFFZKLTI-UHFFFAOYSA-N α-pinene Chemical compound CC1=CCC2C(C)(C)C1C2 GRWFGVWFFZKLTI-UHFFFAOYSA-N 0.000 claims description 8
- UFLHIIWVXFIJGU-ONEGZZNKSA-N (E)-3-Hexenol Natural products CC\C=C\CCO UFLHIIWVXFIJGU-ONEGZZNKSA-N 0.000 claims description 4
- UFLHIIWVXFIJGU-ARJAWSKDSA-N (Z)-hex-3-en-1-ol Chemical compound CC\C=C/CCO UFLHIIWVXFIJGU-ARJAWSKDSA-N 0.000 claims description 4
- 239000000267 (Z)-hex-3-en-1-ol Substances 0.000 claims description 4
- 239000001169 1-methyl-4-propan-2-ylcyclohexa-1,4-diene Substances 0.000 claims description 4
- GRWFGVWFFZKLTI-IUCAKERBSA-N 1S,5S-(-)-alpha-Pinene Natural products CC1=CC[C@@H]2C(C)(C)[C@H]1C2 GRWFGVWFFZKLTI-IUCAKERBSA-N 0.000 claims description 4
- IBVJWOMJGCHRRW-DTWKUNHWSA-N 2-Carene Natural products C1CC(C)=C[C@H]2C(C)(C)[C@@H]12 IBVJWOMJGCHRRW-DTWKUNHWSA-N 0.000 claims description 4
- PXRCIOIWVGAZEP-UHFFFAOYSA-N Primaeres Camphenhydrat Natural products C1CC2C(O)(C)C(C)(C)C1C2 PXRCIOIWVGAZEP-UHFFFAOYSA-N 0.000 claims description 4
- XCPQUQHBVVXMRQ-UHFFFAOYSA-N alpha-Fenchene Natural products C1CC2C(=C)CC1C2(C)C XCPQUQHBVVXMRQ-UHFFFAOYSA-N 0.000 claims description 4
- MVNCAPSFBDBCGF-UHFFFAOYSA-N alpha-pinene Natural products CC1=CCC23C1CC2C3(C)C MVNCAPSFBDBCGF-UHFFFAOYSA-N 0.000 claims description 4
- 229930006739 camphene Natural products 0.000 claims description 4
- ZYPYEBYNXWUCEA-UHFFFAOYSA-N camphenilone Natural products C1CC2C(=O)C(C)(C)C1C2 ZYPYEBYNXWUCEA-UHFFFAOYSA-N 0.000 claims description 4
- UFLHIIWVXFIJGU-UHFFFAOYSA-N hex-3-en-1-ol Natural products CCC=CCCO UFLHIIWVXFIJGU-UHFFFAOYSA-N 0.000 claims description 4
- 150000007875 phellandrene derivatives Chemical class 0.000 claims description 4
- 239000000419 plant extract Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 13
- 239000002689 soil Substances 0.000 abstract description 12
- 241000186361 Actinobacteria <class> Species 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract description 6
- 230000001580 bacterial effect Effects 0.000 abstract description 2
- 239000008223 sterile water Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
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- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Clinical Laboratory Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A culture dish device special for separation and purification of filamentous fungi comprises a sample culture dish and a separation and purification cover, wherein the sample culture dish is provided with a bottom support, an isolation outer wall is arranged at the periphery of the bottom support, a central small ring is arranged in the center of the bottom support, a sample chamber is arranged in the central small ring, a culture medium is poured into the central small ring, a separation chamber is arranged on the lower surface of the separation and purification cover and is formed by separating a middle separating ring, the separation chamber is provided with a separation layer selective culture medium layer, and the middle separating ring is arranged between the central small ring and the isolation outer ring; the side part of the middle separating ring is provided with a separating hole. The high-pollution background soil sample can be directly planted in the sample culture ring, bacteria and fungi are separated by using a reverse spore ejection method, the fungi and actinomycetes can be separated by using a concentric ring device, and the fungi and the actinomycetes are separated in the separation ring by using aerial hypha creeping property and hypha penetrability. Can be used for fungus separation and purification analysis of high-pollution bacterial background.
Description
Technical Field
The invention relates to biotechnology detection, in particular to a culture dish device special for separation and purification of filamentous fungi.
Background
Fungi separation and purification research generally comes from samples with high environmental pollution background, wherein a large amount of bacillus is always accompanied with growth of filamentous fungi when the soil samples are separated by a conventional streaking and diluting method, so that strain separation and purification work are difficult.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a culture dish device special for separation and purification of filamentous fungi without repeated streaking separation or dilution separation.
The technical problem to be solved by the invention is realized by the following technical scheme, and the culture dish device special for separation and purification of the filamentous fungi is characterized in that: comprises a sample culture dish and a separation and purification cover,
The sample culture dish is provided with a bottom support, an isolation outer wall is arranged on the periphery of the bottom support, a central small ring is arranged in the center of the bottom support, a sample chamber is arranged in the central small ring, a culture medium layer is arranged in the central small ring, the height of the culture medium layer is 2/3 of the height of the central small ring,
The lower surface of the separation and purification cover is provided with a separation chamber which is formed by separating a middle separating ring,
The separation chamber is provided with a separation layer selective culture medium layer,
the middle separating ring is arranged between the central small ring and the isolating outer ring, the height of the middle separating ring is 1.5 ~ 2 times of the height of the central small ring, and the distance between the bottom edge of the middle separating ring and the upper edge of the central small ring is 1 ~ 1.5.5 times of the height of the central small ring;
The side part of the middle separating ring is provided with a separating hole.
The technical problem to be solved by the invention can be further realized by the following technical scheme, wherein the culture medium is water agar or semisolid agar. After the culture medium is solidified, directly pressing the soil to be detected into a sample chamber ring paved with the culture medium, and dripping a drop of sterile water or dilute water agar to prevent the soil from drifting;
the technical problem to be solved by the invention can be further realized by the following technical scheme, wherein the 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 solved by the following technical scheme that the middle separation ring is provided with an inner ring and an outer ring, and the distance between the inner ring and the outer ring is 10 ~ 20 mm.
The technical problem to be solved by the invention can be further realized by the following technical scheme that the separation holes on the inner ring are diagonally opened by 2, and the separation holes on the outer ring are diagonally opened by 4.
The technical problem to be solved by the invention can be further realized by the following technical scheme that the separation hole is opened from the bottom edge of the middle separation ring to the position 2/3 of the middle separation height, and the length of the separation hole is 8 ~ 12 mm.
compared with the prior art, the method can directly fix the high-pollution background soil sample in the sample culture ring, realize the separation of bacteria and fungi by using a reverse spore ejection method, realize the separation of the fungi and the actinomycetes by the isolation of a concentric ring device, and realize the separation of the fungi and the actinomycetes in the separation ring by using the creeping property and the penetrability of aerial hyphae. Can be used for fungus separation and purification analysis of high-pollution bacterial background.
Drawings
FIG. 1 is a schematic structural view of the present invention;
Fig. 2 is a view showing the structure of the spacer ring.
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 culture dish device special for separating and purifying filamentous fungi, which comprises a sample culture dish 2 and a separating and purifying cover 1,
The sample culture dish is provided with a bottom support, an isolation outer wall 6 is arranged on the periphery of the bottom support, a central small ring 5 is arranged in the center of the bottom support, a sample chamber 3 is arranged in the central small ring, a culture medium layer 4 is arranged in the central small ring, the height of the culture medium layer is 2/3 of the height of the central small ring,
The lower surface of the separation and purification cover is provided with a separation chamber which is formed by separating a middle separating ring,
a separation layer selective culture medium layer 8 is arranged in the separation chamber,
the middle separating ring is arranged between the central small ring and the isolating outer ring, the height of the middle separating ring is 1.5 ~ 2 times of the height of the central small ring, and the distance between the bottom edge of the middle separating ring and the upper edge of the central small ring is 1 ~ 1.5.5 times of the height of the central small ring;
The side part of the middle separating ring is provided with a separating hole.
The culture medium is water agar or semisolid agar. After the culture medium is solidified, the soil to be detected is directly pressed into the sample chamber ring paved with the culture medium, and a drop of sterile water or dilute water agar can be dropped to prevent the soil from drifting.
The separating layer culture medium is a separating selective culture medium which takes a PDA culture medium as a substrate and is added with VB1, a target test substrate and allelochemicals.
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.
The middle separating ring is provided with an inner ring 7 and an outer ring 9, and the interval between the inner ring and the outer ring is 10 ~ 20 mm.
the separation holes 10 on the inner ring are diagonally opened by 2, and the separation holes on the outer ring are diagonally opened by 4.
the separation hole is formed from the bottom edge of the middle separation ring to 2/3 of the middle separation height, and the length of the separation hole is 8 ~ 12 mm.
the following specifically describes the embodiments of the present invention, and 1 example is provided, however, the practice of the present invention is not limited to the following example.
Specifically, the method comprises the following steps: the sample culture dish bottom support is formed by PE injection molding, the diameter of the bottom surface is 89mm, and the height of the isolation outer wall is 35 mm.
The diameter of the small central ring is 20mm, and the height of the small central ring is 8 mm.
the top separation chamber, outer circle diameter 92mm, the height is 35 mm. The spacing between the middle separating rings is 15mm, and the height of the separating rings is 11.2mm. A separation hole is arranged between the inner ring and the outer ring of the separation ring, the separation hole is 2/3 with the middle separation height, and the length of the hole is 10 mm. The inner ring is diagonally opened by 2, and the outer ring is diagonally opened by 4.
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.
(1) The device is simple in sample preparation and convenient to test, and is suitable for separating fungi from high-pollution samples;
(2) The device can separate fungi, actinomycetes and bacteria respectively, and is also suitable for separating bacteriophage or separating microorganisms which are not polluted by the bacteriophage;
(3) The manufacturing cost is low, and the use separation efficiency is high;
(4) The method is used for separation and purification analysis of fungi of more than thirty complex samples, and the coincidence rate is more than 95 percent;
(5) Purification and isolation of bacteria or phages in a sample of isolated contaminants can be used by inversion.
In order to illustrate the present invention more clearly, the following examples are given without any limitation to the scope of the present invention.
Example 1, soil separation of fungi as an example:
The culture dish device for separating and purifying the filamentous fungi is characterized in that a selective culture medium is flatly laid in a separating ring in a separating chamber, the height of the selective culture medium is not beyond a separating hole, water agar or semisolid agar is poured into a sample chamber, the height of the water agar or semisolid agar is 2/3 parts of the sample chamber ring, after the culture medium is solidified, soil to be detected is directly pressed into the sample chamber ring paved with the water agar, and one drop of sterile water or dilute water agar can be dropped to prevent soil from drifting. The separation chamber was partially covered on the sample cell, not to be turned upside down!
Placing the plate into an incubator to be cultured at a separation culture temperature of 22 ℃, observing through side view after the aerial hyphae of the thallus are formed, and immediately taking down the separation chamber to replace the sample chamber plate with a sterile plate to continue culturing and separating when the aerial hyphae contact the culture medium in the separation ring. Or when the filamentous fungi in the sample chamber produce spores, the spores are placed in the elastic spores, the sample chamber dish and the sample separation ring dish are separated in time, and the sterile plate bottom dish is used for replacing the sample chamber dish to continue culturing.
The separation from bacteria is facilitated at 22 ℃, the obtained fungi can be further separated by turning over a separation ring at the speed of adherent growth of the hyphae, or the hyphae pass through the separation ring from a separation hole in a culture medium to realize the penetrating growth of a next-stage separation ring, and then single hyphae at different positions in the separation ring are picked in time for subculture to obtain the purified fungi hyphae.
Compared with the traditional method, the method has the advantages that:
The used time is less, the traditional method generally takes more than 20 days for purifying and separating the fungi, and the method can be completed in 5-7 days, so that the experimental efficiency is greatly improved.
the method can realize the separation of multiple strains, and is favorable for the separation of actinomycetes, bacteria and phage. This is difficult to achieve with conventional methods.
The method does not need to dilute the soil sample, can directly separate soil, has better detection effect compared with the traditional dilution method, and simultaneously avoids complicated experimental process and time.
The used reagent does not need to use antibiotics or heavy metal inhibitors, and is beneficial to the environmental control of a laboratory.
Claims (7)
1. the utility model provides a special culture dish device of filamentous fungi separation and purification which characterized in that: comprises a sample culture dish and a separation and purification cover,
the sample culture dish is provided with a bottom support, an isolation outer wall is arranged on the periphery of the bottom support, a central small ring is arranged in the center of the bottom support, a sample chamber is arranged in the central small ring, a culture medium layer is arranged in the central small ring, the height of the culture medium layer is 2/3 of the height of the central small ring,
The lower surface of the separation and purification cover is provided with a separation chamber which is formed by separating a middle separating ring,
The separation chamber is provided with a separation layer selective culture medium layer,
The middle separating ring is arranged between the central small ring and the isolating outer ring, the height of the middle separating ring is 1.5 ~ 2 times of the height of the central small ring, and the distance between the bottom edge of the middle separating ring and the upper edge of the central small ring is 1 ~ 1.5.5 times of the height of the central small ring;
The side part of the middle separating ring is provided with a separating hole.
2. the culture dish device special for filamentous fungi separation and purification according to claim 1, wherein: the culture medium is water agar or semisolid agar.
3. the culture dish device special for filamentous fungi separation and purification according to claim 1, wherein: the separating layer culture medium is a separating selective culture medium which takes a PDA culture medium as a substrate and is added with VB1, a target test substrate and allelochemicals.
4. The culture dish device special for filamentous fungi separation and purification according to claim 3, 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 allelochemicals added per 1000ml of the culture medium substrate.
5. The culture dish device special for filamentous fungi separation and purification according to claim 1, wherein the middle separating ring is provided with an inner ring and an outer ring, and the interval between the inner ring and the outer ring is 10 ~ 20 mm.
6. the culture dish device special for filamentous fungi separation and purification according to claim 5, wherein: the separation holes on the inner ring are diagonally opened by 2, and the separation holes on the outer ring are diagonally opened by 4.
7. The culture dish device special for filamentous fungi separation and purification as claimed in claim 1, 5 or 6, wherein the separation hole is opened from the bottom edge of the middle separation ring to 2/3 of the middle separation height, and the length of the separation hole is 8 ~ 12 mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910936687.2A CN110551620A (en) | 2019-09-29 | 2019-09-29 | Special culture dish device of filamentous fungi separation and purification |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910936687.2A CN110551620A (en) | 2019-09-29 | 2019-09-29 | Special culture dish device of filamentous fungi separation and purification |
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| Publication Number | Publication Date |
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| CN110551620A true CN110551620A (en) | 2019-12-10 |
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| CN201910936687.2A Pending CN110551620A (en) | 2019-09-29 | 2019-09-29 | Special culture dish device of filamentous fungi separation and purification |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116508646A (en) * | 2023-04-28 | 2023-08-01 | 十堰市农业科学院 | Lentinus edodes double-single hybridization device and method |
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2019
- 2019-09-29 CN CN201910936687.2A patent/CN110551620A/en active Pending
Patent Citations (4)
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| US20110210066A1 (en) * | 2009-02-27 | 2011-09-01 | Keisuke Takenouchi | Waste fluid treatment using filamentous fungus or actinomycete |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116508646A (en) * | 2023-04-28 | 2023-08-01 | 十堰市农业科学院 | Lentinus edodes double-single hybridization device and method |
| CN116508646B (en) * | 2023-04-28 | 2024-03-26 | 十堰市农业科学院 | Lentinus edodes double-single hybridization device and method |
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Application publication date: 20191210 |