CN114149900B - Cutting head and mycelium cutting method - Google Patents
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- CN114149900B CN114149900B CN202111442956.3A CN202111442956A CN114149900B CN 114149900 B CN114149900 B CN 114149900B CN 202111442956 A CN202111442956 A CN 202111442956A CN 114149900 B CN114149900 B CN 114149900B
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- 238000005520 cutting process Methods 0.000 title claims abstract description 182
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000002028 Biomass Substances 0.000 claims abstract description 16
- 244000005700 microbiome Species 0.000 claims abstract description 11
- 238000009826 distribution Methods 0.000 claims abstract description 9
- 230000009471 action Effects 0.000 claims abstract description 3
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 11
- 230000004151 fermentation Effects 0.000 abstract description 11
- 239000011148 porous material Substances 0.000 abstract description 9
- 238000013537 high throughput screening Methods 0.000 abstract description 8
- 241000233866 Fungi Species 0.000 description 9
- 238000012360 testing method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 3
- 241000186361 Actinobacteria <class> Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000012431 wafers Nutrition 0.000 description 2
- 238000004804 winding Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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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
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
- C12M37/02—Filters
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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
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Sustainable Development (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
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- Immunology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a cutting head and a mycelium cutting method, wherein the cutting head comprises a cutting head body, the cutting head body is a hollow cylinder body, the cutting head body comprises a cutting part and a connecting part, the cutting part is connected to the bottom end of the connecting part and is used for executing cutting action, and the connecting part is used for being connected with a mechanical arm. The cutting head and the method for cutting the hyphae can realize uniform cutting and distribution of the hyphae, so that biomass is uniformly distributed in the pore plates, the consistency of biomass in each pore is ensured, the comparability of fermentation results is improved, and the optimal strain is found out by a high-throughput screening method of microorganisms.
Description
Technical Field
The invention relates to the field of experimental equipment, in particular to a cutting head and a mycelium cutting method.
Background
The microorganism can be used for producing various products such as enzyme preparations, organic acids, amino acids, antibiotics, vitamins, hormone, ethanol, biological materials and the like, and the excellent strain is the root for realizing the high yield of microorganism fermentation. Mutagenesis and high-throughput editing both produce a large number of candidate strains, and high-throughput screening is an important means for efficiently and rapidly screening more excellent strains from among a plurality of candidate strains.
The high throughput screening (High throughput screening, HTS) technology of microorganisms is to use an orifice plate or an agar plate as a culture carrier of strains, perform an experimental process by using an advanced modern automatic operation system, collect experimental data by using a sensitive and rapid detection instrument, culture and test a large number of microbial strains in a short time, and find more excellent strains.
Biomass is an important influencing factor of fermentation, fermentation performance of the well plate method for testing microorganisms is closely related to biomass in the wells, and if biomass cannot be uniformly distributed to the same batch of well plates, comparability of fermentation results is poor, so that consistency of biomass in each well should be ensured.
However, uniform mycelium/mycelium selection and distribution is a bottleneck for high throughput screening of actinomycetes, filamentous fungi, and large fungi (actinomycetes, filamentous fungi, and large fungi are collectively referred to herein as "filamentous fungi"). Due to morphological characteristics of the filamentous fungi, the existing pipetting workstations, flow cytometry, microfluidic technologies and other instruments and methods suitable for single cells cannot achieve uniform picking and distribution of hyphae. Some take the hypha of the filiform fungus by taking the fungus thread winding method, then distribute the hypha into the deep hole plate, the winding method can not realize the even taking and distributing of the hypha.
Disclosure of Invention
In view of the above problems, a first object of the present invention is to provide a cutting head capable of uniformly cutting mycelium or mycelia. Another object of the present invention is to provide a mycelium cutting method, which can uniformly cut mycelium, and put the mycelium into a well plate for comparison experiments, so as to screen out an optimal strain.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a cutting head, a cutting head body, the cutting head body being a hollow cylinder, the cutting head body including a cutting portion and a connecting portion, the cutting portion being connected to a bottom end of the connecting portion, a cutting edge being formed at the bottom end of the cutting portion for performing a cutting operation, the connecting portion being for connection with a robot arm.
Further, the cutter head body is made of resin or plastic.
Further, the cutting head body is a hollow cylinder.
Further, a filter layer is further arranged in the hollow cylinder body, and can only pass through the air, so that microorganisms in the air can be filtered, and the sterile state is realized.
Further, the diameter of the cutting portion is different from the diameter of the connecting portion.
Further, the cutting head further comprises an outer layer cutting sleeve sleeved outside the cutting head body, the inner wall of the outer layer cutting sleeve is attached to the outer wall of the cutting head body, the outer layer cutting sleeve and the outer wall of the cutting head body can rotate mutually, and a cutting edge is formed at the bottom of the outer layer cutting head body.
The invention also provides a mycelium cutting method based on the cutting head, which is characterized by comprising the following steps:
mounting the cutting head on an automatic cutting machine, and aligning the cutting portion of the cutting head to an area of the colony edge;
cutting a circular sheet from the bacterial colony together with the culture medium to form bacterial colony, and simultaneously extracting gas in the cutting head body to form negative pressure in the cutting head body so as to prevent the bacterial colony from falling off;
the colony plates which are cut by the cutting head are moved to a culture hole or an agar plate to be inoculated, so that a small amount of air is introduced into the cutting head pipe, and the colony plates are blown into the culture hole or blown onto the agar plate;
and repeating the operation, and finishing the cutting and distribution of the mycelium.
Further, the cutting head further comprises an outer layer cutting sleeve sleeved outside the cutting head body, the inner wall of the outer layer cutting sleeve is attached to the outer wall of the cutting head body, the outer layer cutting sleeve and the outer wall of the cutting head body can rotate mutually, and a cutting edge is formed at the bottom of the outer layer cutting head body;
the step of cutting a circular sheet from the colony together with the culture medium to form a colony sheet is specifically as follows:
the cutting head is depressed toward the colony by the robotic arm and rotated by the cutting head body or outer cutting sleeve to ensure severing of the mycelium.
Due to the adoption of the technical scheme, the invention has the following advantages:
the cutting head provided by the invention has the advantages that the inner diameters of the cutting parts are the same, the diameters of the cut wafers are the same, and the even cutting and distribution of hyphae are realized;
the cutting head provided by the invention comprises a cutting head body and an outer layer cutting sleeve sleeved on the outer side of the cutting head body, and relative rotation is formed between the cutting head body and the outer layer cutting sleeve, so that hypha cutting is ensured by rotation of the cutting head body or the outer layer cutting head body in the cutting process.
The invention can be used for transferring the filamentous fungi, cutting hyphae and inoculating the hyphae into agar plates/pore plates/deep pore plates/test tubes/triangular flasks or other suitable culture containers. The cut hyphae can also be transferred into a test tube/a freezing tube/an ampoule tube to implement automatic operation of strain preservation.
Drawings
FIG. 1 is a schematic cross-sectional view of a first embodiment of a cutting head according to the present invention;
FIG. 2 is a schematic cross-sectional view of a cutting head according to the present invention;
FIG. 3 is a schematic cross-sectional view of a second embodiment of a cutting head according to the present invention;
reference numerals illustrate:
1-cutting head body, 2-filter layer, 11-cutting portion, 12-connecting portion, 3-outer cutting sleeve.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the description of the present invention, it should be noted that the azimuth or positional relationship indicated by the terms "upper", "lower", "inner", "outer", etc. are based on the azimuth or positional relationship shown in the drawings, and are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the system or element referred to must have a specific azimuth, be configured and operated in a specific azimuth, and thus should not be construed as limiting the present invention. Moreover, the use of the terms first, second, etc. to define elements is merely for convenience in distinguishing the elements from each other, and the terms are not specifically meant to indicate or imply relative importance unless otherwise indicated.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
The embodiment of the invention provides a cutting head and a mycelium cutting method, which can realize uniform cutting and distribution of mycelium, ensure that biomass is uniformly distributed in a pore plate, ensure the consistency of biomass in each pore, improve the comparability of fermentation results and find out an optimal strain by a high-throughput screening method of microorganisms.
Example 1
The cutting head provided in one embodiment 1 of the present invention includes a cutting head body 1, the cutting head body 1 is a hollow cylinder, the cutting head body 1 includes a cutting portion 11 and a connecting portion 12, the cutting portion 11 is connected to a bottom end of the connecting portion, for performing a cutting action, and the connecting portion 12 is used for connecting with a mechanical arm (not shown in the figure).
The cutting head body 1 is preferably made of resin or plastic, and the cutting head body 1 may be made of stainless steel or the like.
The cutter head body 1 is preferably a hollow cylinder. It will be appreciated that the cutter head body 1 is not limited to a hollow cylinder, but may be a square or polygonal cylinder.
The bottom of the cutting head body 1 is a cutting part 11, and the cutting part 11 is a sharp point, and can press in a culture medium and a colony under a certain pressure condition and cut the colony together with the culture medium.
In order to avoid being polluted by microorganisms and the like in the process of cutting and transferring hyphae, a filter layer 2 is fixedly arranged inside the hollow cylinder, and the filter layer 2 can pass through gas and is used for filtering microorganisms in the gas.
The top of the connection part 12 is connected to a robot arm (not shown) which performs cutting and transferring operations on the cutting head. The top of the connection portion 12 may be connected to the mechanical arm by a bolt, a plug-in connection, or other detachable connection, preferably a plug-in connection.
As one embodiment, the diameter of the cutting portion 11 is different from the diameter of the connecting portion 12, and as shown in fig. 2, the outer diameter and the inner diameter of the cutting portion 11 are larger than those of the connecting portion 12.
Based on the cutting head, another embodiment of the present invention further provides a method for cutting mycelium, which is characterized by comprising the steps of:
(1) The spores or hyphae of the candidate strains are inoculated on agar plates;
(2) The candidate strain grows into colonies on an agar plate;
(3) The cutting head is arranged on a mechanical arm on the automatic cutting machine;
(4) Cutting a wafer (called a 'colony piece') from a colony together with a culture medium by using a cutting head aiming at a region of the edge of the colony, and simultaneously extracting gas in the cutting head body 1 to form negative pressure in the cutting head body 1 so as to prevent the colony piece from falling off;
(5) The cutting head moves to a culture hole or an agar plate to be inoculated with the cut colony, a small amount of air is introduced into the barrel body of the cutting head, and the colony is blown into the culture hole or blown onto the agar plate;
(6) And repeating the operation, so as to complete the cutting and distribution of hyphae, replacing the sterile cutting head each time, and selecting mycelium areas with the same or similar density on the edge of the colony each time through digital photographing and machine vision so as to ensure that the biomass on the cut colony pieces is the same or similar.
When the mycelium density at the edge of the colony is different, the percentage of mycelium on the colony to the area on the colony is controlled to realize the same or similar biomass on each cut colony. If the mycelium density at the edge of the colony is too low, two or more pieces may be cut so that the biomass obtained from the strain is the same or similar to that of other bacteria.
The automatic cutting machine mainly comprises a mechanical arm and the like, and can automatically change a cutting head according to a program to cut colony pieces at the colony edge selected by digital photographing and machine vision. The mechanical arm of the automatic cutting machine controls the falling force (the pressure contacted with the flat plate) of the cutting head through a spring and/or a lever, so that the cutting head cuts to the surface of a vessel under the agar flat plate to finish cutting of colony pieces, and how to control the movement, transfer and force of the cutting head specifically belongs to the prior art in the field, so that the details are not repeated.
The excised colony pieces are used for inoculation into agar plates/well plates/deep well plates/tubes/flasks or other suitable culture vessels. The excised colony can also be transferred to a test tube/a freezing tube/an ampoule tube to implement automatic operation of strain preservation.
The cutting head and the method for cutting the hyphae can realize uniform cutting and distribution of the hyphae, so that biomass is uniformly distributed in the pore plates, the consistency of biomass in each pore is ensured, the comparability of fermentation results is improved, and the optimal strain is found out by a high-throughput screening method of microorganisms.
The invention greatly simplifies the operation, realizes the uniform sampling and uniform distribution of hyphae, and has several advantages: (1) As no bacterial strain is required to grow spores, the method is suitable for bacterial strains which do not produce spores; (2) The strain is not required to grow spores, so that the culture time of the strain on an agar plate is short; (3) Cutting mycelia at the edge of a colony, wherein the mycelia are new mycelia, so that the mycelia fermentation of different strains is easy to realize synchronization; (4) The biomass on each cut colony is the same or similar with the assistance of digital photographing and machine vision, so that the consistency of the biomass in each fermentation pore plate can be realized, and parallel experiments or comparison of different experiments can be implemented; (5) cross-contamination is not favored; (6) In the subsequent fermentation stage, the spore germination stage is not needed, so that the experimental period of fermentation is shortened.
Example 2
The embodiment 2 of the present invention is different from the embodiment 1 in that the cutting head in the present embodiment is a double layer, the outer layer of the cutting head body in the embodiment 1 is further sleeved with an outer layer cutting sleeve 3, the inner wall of the outer layer cutting sleeve 3 is attached to the outer wall of the cutting head body 1, the inner wall and the outer wall can rotate with each other, and the bottom of the outer layer cutting sleeve 3 is sharp.
Thus, in the mycelium cutting process, the step of cutting a circular plate from the colony together with the culture medium to form a colony plate is specifically as follows:
the cutter head is pressed down toward the colony by the robot arm, and the cutter head body 1 or the outer layer cutting sleeve 3 is rotated to ensure cutting of hyphae.
Therefore, in the present invention, by providing the cutter head in a double-layered structure with the inner and outer sleeves, the cutting of hyphae can be ensured by rotating the cutter head body 1 of the inner layer or the outer cutting sleeve 3 of the outer layer.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (3)
1. The cutting head is characterized by comprising a cutting head body, wherein the cutting head body is a hollow cylinder, the cutting head body comprises a cutting part and a connecting part, the cutting part is connected to the bottom end of the connecting part, a cutting edge is formed at the bottom end of the cutting part and is used for executing cutting action, the connecting part is used for being connected with a mechanical arm, and the mechanical arm controls the falling force of the cutting head through a spring and/or a lever so that the cutting head is cut to the surface of a vessel under an agar plate;
the cutting head also comprises an outer layer cutting sleeve sleeved outside the cutting head body, the inner wall of the outer layer cutting sleeve is attached to the outer wall of the cutting head body, the outer layer cutting sleeve and the outer wall of the cutting head body can rotate mutually, and a cutting edge is formed at the bottom of the outer layer cutting sleeve;
the mycelium cutting method based on the cutting head comprises the following steps:
mounting the cutting head on an automatic cutting machine, and aligning the cutting portion of the cutting head to an area of the colony edge;
cutting a circular sheet from the bacterial colony together with the culture medium to form bacterial colony, and simultaneously extracting gas in the cutting head body to form negative pressure in the cutting head body so as to prevent the bacterial colony from falling off;
the colony plates which are cut by the cutting head are moved to a culture hole or an agar plate to be inoculated, so that a small amount of air is introduced into the cutting head pipe, and the colony plates are blown into the culture hole or blown onto the agar plate;
repeating the operation to finish the distribution of hyphae;
the step of cutting a circular sheet from the colony together with the culture medium to form a colony sheet is specifically as follows:
selecting mycelium areas with the same or similar density from the edges of the colonies through digital photographing and machine vision so as to ensure that the biomass on the cut colony pieces is the same or similar;
pressing down the cutting head toward the colony by the mechanical arm and rotating by the cutting head body or an outer cutting sleeve to ensure cutting of hyphae;
the cutting head body is a hollow cylinder;
the inside of hollow barrel still is equipped with the filter layer, the filter layer can only be through by the gas for filter the microorganism in the gas realizes aseptic condition.
2. The cutting head of claim 1, wherein the cutting head body is made of resin or plastic.
3. The cutting head of claim 1, wherein the diameter of the cutting portion is different from the diameter of the connecting portion.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111442956.3A CN114149900B (en) | 2021-11-30 | 2021-11-30 | Cutting head and mycelium cutting method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111442956.3A CN114149900B (en) | 2021-11-30 | 2021-11-30 | Cutting head and mycelium cutting method |
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| CN114149900A CN114149900A (en) | 2022-03-08 |
| CN114149900B true CN114149900B (en) | 2024-03-29 |
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