CN110747113A - Culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and use method thereof - Google Patents
Culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and use method thereof Download PDFInfo
- Publication number
- CN110747113A CN110747113A CN201911223565.5A CN201911223565A CN110747113A CN 110747113 A CN110747113 A CN 110747113A CN 201911223565 A CN201911223565 A CN 201911223565A CN 110747113 A CN110747113 A CN 110747113A
- Authority
- CN
- China
- Prior art keywords
- culture dish
- pathogenic bacteria
- screening
- culture
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
-
- 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
- C12M23/34—Internal compartments or partitions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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
- C12Q1/18—Testing for antimicrobial activity of a material
- C12Q1/20—Testing for antimicrobial activity of a material using multifield media
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Sustainable Development (AREA)
- Clinical Laboratory Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a culture dish and a method for efficiently screening antagonistic bacteria of pathogenic bacteria, wherein the culture dish comprises a dish cover l, a dish cover 2 and a dish body, the culture dish body is made of glass or polycarbonate, and the middle of the culture dish body contains a partition layer with a microporous structure. The invention also provides a method for efficiently screening pathogenic bacteria antagonistic bacteria, which comprises the following steps: pouring solid culture mediums required for separating strains and culturing pathogenic bacteria into the surface A and the surface B of the culture dish in parts, then coating diluent with appropriate dilution gradient to the surface A culture medium, and culturing for a certain number of days under appropriate conditions; and coating the pathogenic bacteria on a B surface culture medium, continuously culturing under a proper condition, observing the position of a B surface inhibition zone, and then selecting strains at the corresponding position of the inhibition zone on the A surface, so that the strains can be used for a re-screening experiment. Therefore, the culture dish for efficiently screening the antagonistic bacteria of the pathogenic bacteria has a simple structure, and the provided method can simultaneously carry out strain separation and preliminary screening experiments, thereby greatly improving the screening efficiency and reducing the screening cost.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture dish for efficiently screening antagonistic bacteria of pathogenic bacteria and a using method thereof.
Background
There are a variety of pathogenic bacteria in the natural environment that can cause plant, animal and human diseases. Although scientists have invented a plurality of medicaments which can effectively control pathogenic bacteria, long-term use of a single medicament can easily cause drug resistance of the pathogenic bacteria, thereby reducing the control effect. Screening for pathogenic bacteria antagonistic bacteria and discovery of new agents is therefore a long and important task.
The conventional screening process of the antagonistic bacteria mainly comprises the following steps: strain separation, primary screening and secondary screening. Firstly, separating strains by adopting a dilution coating plate method, and inoculating all obtained single colonies to a slant for preservation; secondly, inoculating the obtained single colony to a corresponding culture medium plate, and performing primary screening by using an agar block method; and finally, re-screening the preliminarily screened antagonistic bacteria by using an oxford cup method to finally obtain the optimal antagonistic bacteria. The existing bacteria separation and primary screening methods are time-consuming and labor-consuming and have low efficiency.
Disclosure of Invention
In order to overcome the defects, the invention aims to provide a culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and a using method thereof, so that the screening work efficiency is improved, and the experiment cost is reduced.
In order to achieve the above object, the present invention provides a culture dish for efficiently screening antagonistic bacteria against pathogenic bacteria, which is characterized in that: the culture dish comprises a dish cover l, a dish cover 2 and a dish body, wherein the middle of the culture dish body comprises a partition layer with a microporous structure; the front and back sides of the culture dish can be used, wherein one side is used for strain separation, and the other side is used for culturing corresponding pathogenic bacteria.
The invention also provides a use method of the culture dish for efficiently screening the pathogenic bacteria antagonistic bacteria, which comprises the following steps: pouring a solid culture medium required by separating strains into the surface A of the culture dish, and pouring a solid culture medium required by culturing pathogenic bacteria into the surface B of the culture dish; diluting the collected sample by using a gradient dilution method, then coating the diluent with a proper dilution gradient on a culture medium on the surface A of a culture dish, and culturing under proper conditions; taking out the culture dish for culturing for a certain number of days, coating the pathogenic bacteria on the surface B of the culture dish, and continuously culturing under proper conditions; observing the position of the inhibition zone on the surface B, selecting the strain at the corresponding position on the surface A, storing the strain in a slant culture medium, and performing subsequent re-screening.
In conclusion, the culture dish for efficiently screening the pathogenic bacteria antagonistic bacteria has a simple structure, and the provided method can simultaneously carry out strain separation and preliminary screening experiments, so that the screening efficiency is greatly improved, and the screening cost is reduced.
Drawings
FIG. 1 is a culture dish for efficiently screening antagonistic bacteria of pathogenic bacteria.
FIG. 2 plastic petri dish modified double-sided petri dish.
FIG. 3B shows the inhibition zone of bacterial blight of rice.
FIG. 4 shows the antibacterial activity of antagonistic bacteria obtained by preliminary screening.
Detailed Description
Example 1A culture dish for efficiently screening antagonistic bacteria of pathogenic bacteria
As shown in figure 1, the biological culture dish comprises a dish cover l, a dish cover 2 and a dish body, wherein the dish body is made of glass or polycarbonate, and a partition layer with a microporous structure is arranged in the middle of the dish body. The significant difference from the traditional culture dish is that: solid culture mediums can be added into both sides of the culture dish, and the middle partition layer can isolate the culture mediums on both sides and does not influence the passing of active substances secreted by the strains. The partition layer of the microporous structure in the middle of the dish body can be a glass sand core or other materials with the same function.
With reference to the above method, it is also possible to use existing plastic culture dishes for the transformation, as shown in FIG. 2. The two plastic petri dishes were first glued together in a reversed manner, and then the bottom of the glued dishes were perforated. When the culture dish is used, sterilized filter paper is added to the bottom of the culture dish, and then a solid culture medium is poured. The filter paper may function as a glass sand core.
Example 2 separation and screening of antagonistic bacteria against bacterial blight of rice from silkworm excrement Using a culture dish for efficient screening of antagonistic bacteria against pathogenic bacteria
The culture medium:
LB agar medium (1L): 5 g of yeast extract, 10g of peptone, 10g of NaCl, 20 g of agar and pH 7.0-7.2.
Modified potato dextrose agar medium (1L): 300 g of potato, 15 g of cane sugar and Ca (NO)3)2·4H2O0.5g，Na2HPO4·12H2O2.0 g, Tryptone 5.0g, agar 20 g, pH natural.
Silkworm excrement sample sampling
In a silkworm breeding farm, sterile kraft paper is laid on the vacant space beside the silkworm breeding farm, then fresh mulberry leaves are laid, a batch of five-instar silkworms are collected on the mulberry leaves from a breeding bed, and silkworm excrement is generated while silkworm babies eat the mulberry leaves. Folium Mori is added continuously during the process. After one and a half hours, wearing sterile gloves, lightly moving the five-instar silkworms back to a culture bed, removing the residual mulberry leaves and residual branches, collecting fresh silkworm excrement, subpackaging the fresh silkworm excrement in a 50ml sterile centrifugal tube, putting the sterile centrifugal tube into a sterile plastic bag, taking the sterile plastic bag back to a laboratory, and storing the sterile plastic bag in a refrigerator at the temperature of minus 80 ℃ for later use.
(III) preparing silkworm excrement diluent
Weighing 10g faeces Bombycis sample on super clean bench, placing into a triangular flask containing 90ml sterile normal saline and glass beads, shaking for 20min to mix the sample with water, and dispersing cells to obtain 10-1Diluting the solution; aspirate 1ml 10 with 1ml pipette-1Adding the diluted solution into a test tube containing 9ml of sterile water, and fully and uniformly mixing to obtain 10-2Diluting the solution; by analogy, preparing 10 in sequence-3、10-4、10-5、10-6、10-7And diluting the solution.
(IV) inverted plate
Heating and melting an LB culture medium, slowly pouring the culture medium into the surface A of the culture dish in an aseptic operation mode on a super clean bench when the culture medium is cooled to about 50 ℃, and standing; heating and melting the improved potato glucose agar culture medium, after the surface A is solidified, slowly pouring the culture medium cooled to about 50 ℃ into the surface B of a culture dish on a super clean bench in an aseptic operation mode, and standing and solidifying.
(V) coating Diluent
The name of the medium and the dilution used are indicated on the A-side lid of the dish and 200. mu.L pipetting is usedThe guns sucking 10 respectively-5、10-6、10-7And (3) diluting the solution by 0.1mL to the surface A of the culture dish with the corresponding dilution, uniformly coating the solution by using an aseptic coating rod, standing the solution at room temperature for 5-10 min, and adsorbing the bacterial solution into a culture medium. The plate was placed upside down in a 37 ℃ incubator for culture.
(VI) coating with pathogenic bacteria
Taking out the culture dish after 3 days of culture, coating the bacterial leaf blight original bacteria on the surface B of the culture dish, and then placing the culture dish in a constant temperature incubator at 28 ℃ for culture.
(VII) observing the bacteriostatic results
After further 2 days, the dish was removed. Observing the inhibition zone condition of the surface B (figure 3), and selecting the strain at the corresponding position of the surface A to the inclined surface for storage.
(VIII) verification of antibacterial Activity of antagonistic bacteria obtained by Primary Screen
Inoculating the primary screened antagonistic strain preserved on the inclined plane to an LB solid medium flat plate, culturing at 37 ℃ for 2 days, then picking out thalli by using an inoculating loop to a 250 ml triangular flask filled with 30 ml of LB liquid medium, placing the triangular flask in a shaking table with the rotating speed of 200 r/min and the temperature of 37 ℃ for culturing, centrifuging after 2 days to obtain a strain fermentation supernatant, and detecting the bacteriostatic activity of the strain on the rice bacterial blight by an Oxford cup method, wherein the result is shown in figure 4.
The above description is only a preferred embodiment of the present invention, and any other improvements and modifications without departing from the principle of the present invention should be considered as the protection scope of the present invention.
Claims (6)
1. The utility model provides a culture dish of high-efficient screening pathogenic bacteria antagonistic bacteria which characterized in that: the culture dish includes ware lid l, ware lid 2 and the ware body, wherein contains a microporous structure's partition layer in the middle of the culture dish body, and the culture dish tow sides all can use.
2. The culture dish for efficiently screening antagonistic bacteria against pathogenic bacteria according to claim 1, wherein: the culture dish is made of glass or polycarbonate.
3. The culture dish for efficiently screening antagonistic bacteria against pathogenic bacteria according to claim 1, wherein: the culture dish is circular.
4. The culture dish for efficiently screening antagonistic bacteria against pathogenic bacteria according to claim 1, wherein: the partition layer of the microporous structure in the middle of the dish body is a glass sand core or other materials with the same function.
5. The culture dish for efficiently screening antagonistic bacteria against pathogenic bacteria according to claim 1, wherein: the culture dish can be modified by using the existing plastic culture dish.
6. The use method of the culture dish for efficiently screening pathogenic bacteria antagonistic bacteria according to claim 1, comprising the steps of: pouring a solid culture medium required by separating strains into the surface A of the culture dish, and pouring a solid culture medium required by culturing pathogenic bacteria into the surface B of the culture dish; diluting the collected sample by using a gradient dilution method, then coating the diluent with a proper dilution gradient on a culture medium on the surface A of a culture dish, and culturing under proper conditions; taking out the culture dish for culturing for a certain number of days, coating the pathogenic bacteria on the surface B of the culture dish, and continuously culturing under proper conditions; observing the bacteriostasis condition of the surface B, selecting the strain at the corresponding position of the surface A, storing the strain in a slant culture medium, and using the strain for subsequent re-screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911223565.5A CN110747113A (en) | 2019-12-06 | 2019-12-06 | Culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911223565.5A CN110747113A (en) | 2019-12-06 | 2019-12-06 | Culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and use method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110747113A true CN110747113A (en) | 2020-02-04 |
Family
ID=69285508
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911223565.5A Pending CN110747113A (en) | 2019-12-06 | 2019-12-06 | Culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110747113A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553064A (en) * | 2020-12-24 | 2021-03-26 | 广东省微生物研究所（广东省微生物分析检测中心） | Device and method for high-throughput screening of active strains with potential biological corrosion prevention function |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1026253A (en) * | 1964-11-27 | 1966-04-14 | St Luke S Hospital Res Foundat | Anaerobic culturing device |
| US4326028A (en) * | 1980-09-10 | 1982-04-20 | Brown Lewis R | Antibiotic testing vessel |
| JPS59106289A (en) * | 1982-12-11 | 1984-06-19 | Terumo Corp | Multi-layered tool and device for inspecting culture of microorganism |
| JPS6359878A (en) * | 1986-08-28 | 1988-03-15 | Gunze Sangyo Kk | Petri dish for search of antibiotic substance |
| FR2605330A1 (en) * | 1986-10-20 | 1988-04-22 | Millipore Sa | CONTAINER FOR RECEIVING ONE OR MORE CULTURE MEDIA FOR MICROORGANISMS. |
| CN1446903A (en) * | 2002-03-26 | 2003-10-08 | 贝克顿迪肯森公司 | Pitot culture dish with dual-side use |
| DE10329996A1 (en) * | 2003-07-02 | 2005-01-27 | Universität Tübingen | Composite culture vessel |
| CN105670914A (en) * | 2016-04-26 | 2016-06-15 | 湖南生物机电职业技术学院 | Petri dish capable of continuously supplementing impact factors |
-
2019
- 2019-12-06 CN CN201911223565.5A patent/CN110747113A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1026253A (en) * | 1964-11-27 | 1966-04-14 | St Luke S Hospital Res Foundat | Anaerobic culturing device |
| US4326028A (en) * | 1980-09-10 | 1982-04-20 | Brown Lewis R | Antibiotic testing vessel |
| JPS59106289A (en) * | 1982-12-11 | 1984-06-19 | Terumo Corp | Multi-layered tool and device for inspecting culture of microorganism |
| JPS6359878A (en) * | 1986-08-28 | 1988-03-15 | Gunze Sangyo Kk | Petri dish for search of antibiotic substance |
| FR2605330A1 (en) * | 1986-10-20 | 1988-04-22 | Millipore Sa | CONTAINER FOR RECEIVING ONE OR MORE CULTURE MEDIA FOR MICROORGANISMS. |
| CN1446903A (en) * | 2002-03-26 | 2003-10-08 | 贝克顿迪肯森公司 | Pitot culture dish with dual-side use |
| DE10329996A1 (en) * | 2003-07-02 | 2005-01-27 | Universität Tübingen | Composite culture vessel |
| CN105670914A (en) * | 2016-04-26 | 2016-06-15 | 湖南生物机电职业技术学院 | Petri dish capable of continuously supplementing impact factors |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553064A (en) * | 2020-12-24 | 2021-03-26 | 广东省微生物研究所（广东省微生物分析检测中心） | Device and method for high-throughput screening of active strains with potential biological corrosion prevention function |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103667187B (en) | A kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank | |
| Hajek et al. | Methods for study of the Entomophthorales | |
| CN104004708B (en) | The separation and purification of a kind of sheep stem spermatogonium, the long-term cultivation that goes down to posterity, frozen and recovery method | |
| CN101591638B (en) | Method for constructing kidney cell line of scophthalmus maximus | |
| CN105713871A (en) | Human chorion mesenchymal stem cell isolated culture method | |
| CN107372361A (en) | A kind of honeybee ovum superfreeze method and its kit | |
| CN110747113A (en) | Culture dish for efficiently screening pathogenic bacteria antagonistic bacteria and use method thereof | |
| CN107937444A (en) | The method of somatic cell clone dog | |
| CN104178451B (en) | From milk, separate and cultivate method and the special culture media of cow mammary gland epithelial cells | |
| WO2009078637A2 (en) | Cell culture container and cell culture system | |
| CN106754364A (en) | A kind of cell culture container for improving lung cancer stem cell enrichment efficiency | |
| CN116218762A (en) | Tubular organ culture medium, application thereof and tubular organ culture method | |
| CN203613177U (en) | Culture device applicable to microorganism separation and purification | |
| CN105532583A (en) | Living body breeding method for entomopathogenic nematodes | |
| CN113481108B (en) | Nutritional matrix for stimulating growth of nematode-trapping fungi on trunk, and preparation method and application method thereof | |
| CN109402041A (en) | A kind of people's renal cells isolation and culture method | |
| CN106754672A (en) | A kind of cultural method of attached cell | |
| CN112280733A (en) | Extraction method of amniotic stem cells | |
| JPH0551276B2 (en) | ||
| CN104845931A (en) | Acquisition method for human milk stem cells and construction method for human milk stem cell bank | |
| CN206219580U (en) | The raising ware of bacterium feeding habits mite | |
| CN104894145A (en) | Ukraine carp PEPCK gene and method for calibrating blood glucose of cyprinid fishes | |
| CN110592005A (en) | Method for separating mesenchymal stem cells | |
| CN105670991A (en) | Human bone marrow cell processing kit and cell processing method | |
| CN117143799B (en) | Culture method of deep sea pen body cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200204 |
|
| WD01 | Invention patent application deemed withdrawn after publication |