CN111808914B - Method for determining micro-plate detection inhibitor - Google Patents
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Abstract
The invention discloses a method for determining a micro-plate detection inhibitor, which adopts a self-made cutter to cut an agar culture medium micro-plate, a medicament sample and pathogenic bacteria spores are loaded on the micro-plate, and the inhibition effect of the medicament on the spore germination is detected, and the determination method comprises the following steps: 1) Preparing a qualified microplate; 2) Preparing a working-state micro-flat plate; 3) Loading a sample to be tested; 4) Loading pathogenic bacteria spores; 5) Culturing; 6) And (5) observing the result. The invention has the advantages that: 1) A plurality of samples can be tested on one glass slide, so that the test system is miniaturized; 2) One test unit only consumes 5 mul of sample liquid, and the micro-quantification of the detection sample is realized.
Description
Technical Field
The invention relates to a plant pathology technology, in particular to a method for measuring a micro-plate detection inhibitor.
Technical Field
Currently and for a considerable period of time in the future, pesticide control remains the main means for controlling plant diseases. Exploring and digging biogenic pesticides are important directions for pesticide control, and early exploration and digging of biogenic pesticides often collect or collect substances having inhibitory action on plant pathogenic bacteria from nature or separate active ingredients having inhibitory action from organisms or biological products. Obviously, the efficient and feasible inhibitor identification and detection means is beneficial to improving the exploration and excavation success and efficiency of the biological pesticide. The current common method for identifying the effect of the inhibitor on the pathogenic bacteria comprises a measuring method by utilizing an agar culture medium plate, and the main technical idea of the measuring method is to prepare a drug-containing culture plate by utilizing a common plate, then inoculate the pathogenic bacteria, and then culture and observe the inhibiting effect of the drug on the pathogenic bacteria. The technical scheme of the determination usually requires that the sample amount of a sample to be detected is large, the sample amount is too small, the detection cannot be performed, and the inhibitory active substances with small content are easy to miss or omit.
Disclosure of Invention
The invention aims to provide a determination method for detecting the inhibition effect of related substances on spore germination by utilizing a microplate technology.
The technical scheme for solving the technical problems is as follows:
a method for measuring the inhibitor of the microplate detection mainly comprises the following steps of cutting an agar culture medium microplate by a self-made cutter under the aseptic condition, loading a medicine sample and pathogenic bacteria spores on the microplate, and detecting the inhibition effect of the medicine on the spore germination:
1. cutting qualified micro-flat plate
1) Preparing a cutting tool: the sharp No. 23 surgical blades are arranged at intervals by using a material with the same thickness to form an equidistant blade group with the blade intervals of 4mm, and the equidistant blade group is used as a cutting tool for cutting the micro-flat plate.
2) Pouring the culture medium plate: heating and melting agar culture medium suitable for pathogenic bacteria spore germination, and pouring into a plate under aseptic condition to obtain culture medium plate.
3) Drawing of alignment drawing for cutting: two sets of mutually perpendicular and crossed line drawings are drawn on a piece of clean white paper and are prepared as aligning line drawings for cutting the micro-flat plate.
4) Cutting into a micro-flat plate: under aseptic conditions, padding the alignment chart paper drawn in the operation 3) below the flat plate dish inverted in the operation 2), opening a dish cover, and erecting a sterilized straight ruler at the edge of the dish, wherein the side line of the straight ruler is aligned with a group of straight lines of the alignment chart; sterilizing the blade of the cutting tool on alcohol lamp fire, cooling, gently inserting the blade close to the side line of the straight edge into a plate agar plate, pulling the cutting tool along the side line of the straight edge, and cutting the agar plate; then the alignment line drawing under the dish and the dish are rotated for 90 degrees, the movable ruler is aligned with the other group of straight lines vertical to the straight line direction just cut, the agar culture medium plate is cut in the same way, and the agar culture medium plate is cut into 4 multiplied by 4mm 2 The size of the square microplate is equal, the microplate is tidy and consistent, and the section is flat, so that the square microplate is qualified.
2. Prepare the little flat plate of working state
Carrying a preparation working state micro-flat plate by using a common glass slide; a moist instrument capable of placing a slide glass in a sterile state is prepared in advance. Under the aseptic condition, opening the cover of the moisturizing appliance, flatly placing the sterilized glass slide on the surface of the moisturizing material, picking up the qualified microplate cut in the step (1) by using a blade, transferring the qualified microplate onto the sterilized glass slide, and separately placing the qualified microplate one by one to form a working-state microplate for testing; .
3. Loading a sample to be tested
Transferring the prepared drug sample solution to be detected and the test control treatment sample solution to an aseptic workbench, sucking 5 mu l of the sample solution by using a liquid transfer gun, placing the sample solution in the middle of the working micro-flat plate prepared in the step (2) under the condition that the gun head does not touch the micro-flat plate surface, automatically expanding and dispersing the sample solution on the micro-flat plate surface, and standing until the drug sample solution is not observed to obtain the drug-loaded micro-flat plate.
4. Loading of pathogenic spores
And (3) under the aseptic condition, fully and uniformly suspending the prepared pathogenic bacterium spore liquid, sucking 1 mu l of the spore liquid by using a liquid transfer gun, and placing the liquid in the middle of the drug-carrying micro-flat plate prepared in the step (3) under the condition that a gun head does not touch the surface of the micro-flat plate to naturally disperse the spore liquid.
5. Cultivation of
And 4, after the operation of the step 4 is finished, covering a cover of the moisturizing device, keeping the device in a stable state, and transferring to an incubator for constant-temperature culture until the spores treated by the blank control germinate sufficiently.
6. Observation of results
Taking out the culture material of the step 5 from the incubator, opening the cover of the moisturizing device, placing the glass slide carrying the microplate under a microscope, and observing and recording the germination of spores on each microplate. The inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.
The invention has the advantages that:
1) Test system miniaturization
The basic test unit of the conventional flat plate measurement technology is a set of plates, usually more than 3 sets of plates are arranged repeatedly, so that more than 3 sets of plates are needed for one sample, and more culture instruments, consumables and culture space are occupied; the basic test unit of the technology is a micro-flat plate, a plurality of samples can be tested on a glass slide, and the miniaturization of a test system can be realized.
2) Detection of sample miniaturisation
One test unit of the conventional plate measuring technology needs to consume more than 10000 mul of sample liquid, while one test unit of the invention technology only consumes 5 mul of sample liquid with little dosage.
Detailed description of the preferred embodiments
The present invention will be further described with reference to the following examples.
The invention cuts the agar culture medium plate by a self-made cutter to prepare the plate with the area of only 4 multiplied by 4mm 2 The microplate of (1), loading a drug sample and pathogenic spores on the microplate, and detecting the inhibitory action of the drug on spore germination.
The technical requirements of the micro-flat plate used in the invention are as follows: 1) The microplate size is the same and the smaller the volume the better. The reason is that the drug sample solution easily diffuses into the interior of the microplate to have a diluting effect of the sample solution. 2) The surface of the micro-flat plate is flat and has no deformation and damage. The reason is to ensure that the test results can be observed normally under a microscope. 3) The cut edge and the section of the cutting flat plate are flat and smooth. The reason is that the sample fluid is easily spread by seepage on rough and uneven cut edges and cut surfaces of the agar.
The inventor's initial trial practice found that there are technical problems to be solved in order to produce acceptable microplates, such as how to cut microplates with small volumes and equal volumes? How to ensure the trimming and the smooth cut surface of the microplate? The trial uses the blade to directly cut, and finds that the cut micro-flat plate can not meet the technical requirement of equal volume. The discovery of punching with ordinary hole puncher is tried on the go, and the hole puncher punches and belongs to extrusion and crushing formula cutting, and cutting edge and cutting plane are coarse and uneven, and the easy card of agar-agar piece that small-bore hole puncher was beaten moreover is in the hole puncher. How to solve the technical problems is not disclosed with a related technical scheme so far, so that how to prepare a qualified microplate becomes the technical key of the invention.
Repeated research shows that the technical problems can be solved by adopting the following technical method: a set of sharp blades arranged at equal intervals is self-made as a cutter, pulling type cutting and vertical cutting are carried out on the agar plate, and the qualified microplate can be cut out, and the specific operation method comprises the following steps:
preparing 5 pieces of common surgical blades with the specification of No. 23; the method comprises the steps of cutting 4 small films with the size close to that of a blade from a uniform-thickness rubber plate with the thickness of 3.6mm, arranging the 4 small films and the 5 surgical blades alternately to expose the blade edges for about 1cm, tightly clamping the small films and the 5 surgical blades by using a long tail clamp, and keeping 5 blade edge tips arranged on a straight line during clamping operation to form a group of equidistant blade groups with the blade edge spacing of 4mm as a cutter for cutting the agar plate. After the surface of the cutter is disinfected by 75% alcohol, the cutter is stored in a clean small container for later use.
The plates of agar medium for cutting were prepared by the usual procedure. The agar medium, which is melted by heating, is poured into a large flat-bottom plate (preferably over 12cm in size) under aseptic conditions on a clean bench to form a medium plate with a thickness of about 1 mm.
Before cutting the microplate, drawing 2 sets of mutually perpendicular cross straight line drawings on clean white paper by a printer (or manual drawing) to prepare an alignment straight line drawing for cutting the microplate. The drawing is placed under an inverted plate through which the alignment line can be seen. Taking a small sterilized steel ruler to be erected on the edge of a plate which is opened, aligning the side line of the ruler with a group of straight lines on an alignment line drawing, taking out a prepared cutting tool, sterilizing 5 blades on an alcohol burner, slightly cooling, slightly inserting the blades close to the side line of the ruler into the plate agar plate, pulling and cutting the agar plate along the side line of the ruler, cutting out a group of 4 agar strips with equal width, and repeating the same operation to cut multiple groups of agar strips. Because the blade is sharp, the flat agar cutting surfaces cutting two sides are neat and smooth and can be overlapped naturally, so that the cutting trace formed after the blade passes through is not easy to see, namely the cutting line trace and the direction of the cutting operation on the flat plate are not easy to identify. At this time, without relatively moving the plate and the alignment line drawing under the plate, the alignment line drawing under the plate is slightly rotated by 90 degrees, the plate and the ruler are driven to rotate by 90 degrees, the ruler is slightly rotated by 90 degrees to align with another group of straight lines vertical to the cutting direction just before, and the cutter is used for the same cutting operation, so that the vertical cutting operation can be accurately performed, and the plate and the ruler are cutMake multiple groups of 16 blocks (4 blocks × 4 blocks), equal in size, each block being 4 × 4mm in size 2 The square microplate becomes a qualified microplate.
The cut micro-plate needs to be transferred to a glass slide to be placed as a working micro-plate for testing. A general moisturizing device (e.g., a petri dish or a preservation box) in an aseptic state, in which a moist moisturizing material (e.g., absorbent paper or gauze) is placed, is prepared in advance. Opening a cover of a moisturizing device on an ultraclean workbench, flatly placing sterilized glass slides on a moisturizing material surface, then opening a dish cover cut into micro-plates, lightly picking out agar sheets outside the micro-plates by a blade to expose 4 orderly-multiplied by 4 micro-plates, lightly picking up the micro-plates by the blade one by one, transferring the micro-plates onto the sterilized glass slides to be orderly placed, separating the micro-plates from each other, arranging the micro-plates in a row of 3 blocks, arranging more than 7 rows of glass slides, and forming the working-state micro-plates which can be tested and used after being arranged.
The method comprises the following steps of placing a prepared drug sample to be detected and a test contrast treatment sample into a workbench, sucking 5 mu l of sample liquid by using a liquid transfer gun, slightly placing the sample liquid in the middle of the surface of the microplate, taking care to avoid the gun head from touching the surface of the microplate, and adopting the practical operation technique that when the gun head is close to the surface of the microplate, the liquid transfer gun is slightly pressed, the sample liquid is discharged into small liquid drops which are hung on the gun head, the small liquid drops are slightly close to the surface of the microplate, when the small liquid drops touch the surface of the microplate, the small liquid drops of the sample automatically fall down and expand on the surface of the microplate, and then the microplate is allowed to stand on the workbench for a period of time (about 20 minutes) until the liquid sample cannot be observed. The microplate after sample application becomes a drug-loaded microplate.
The drug-loaded microplates can then be loaded with spores of the pathogenic bacteria. The specific method comprises the steps of taking the prepared pathogenic bacteria spore liquid, oscillating the spores to be fully and uniformly suspended, sucking 1 mu l of the spore liquid on a superclean bench by using a liquid transfer gun, slightly putting the spore liquid in the middle of a drug-carrying microplate just like putting a drug sample liquid in a spot manner, and naturally dispersing the spore liquid. Due to the small amount of spore liquid, the dispersion range does not usually exceed that of the drug sample. The practical work finds that each micro-flat plate surface is dispersedThe later observation is facilitated by about 100-200 spores, so that the concentration of the spores prepared in advance is adjusted to 1 × 10 6 ～2×10 6 One per ml.
After the above steps are finished, the cover of the moisturizing device is covered, the device is kept in a stable state, and the device is transferred to an incubator for constant-temperature culture. Because of different germination characteristics of the spores of different pathogenic bacteria, the culture time of different pathogenic bacteria is different, and the spores are cultured until the blank control treated spores are fully germinated in principle. And then, observing results, placing the glass slide carrying the micro-plate under a microscope, and observing and recording the spore germination condition on each micro-plate. The inhibitory effect of the tested drug on spore germination was calculated from the results of the control treatment.
Example 1
The method for detecting the inhibitor by using the micro-plate is used for determining the inhibition effect of 25 percent of benzaprochloraz on the spore germination of the fusarium oxysporum f-4 of banana, and is implemented and operated according to the following steps:
1. cutting qualified micro-flat plate
1) Preparing a cutting tool: the sharp No. 23 surgical blades are arranged at intervals by using a material with the same thickness to form an equidistant blade group with blade edges at intervals of 4mm, and the equidistant blade group is used as a cutting tool for cutting a micro-flat plate.
2) Pouring a culture medium plate: heating and melting potato agar culture medium suitable for spore germination of banana vascular wilt pathogen, and pouring into a 12cm dish under aseptic condition to obtain culture medium plate.
3) Drawing of alignment drawing for cutting: two sets of mutually perpendicular and crossed line drawings are drawn on a piece of clean white paper and are prepared as aligning line drawings for cutting the micro-flat plate.
4) Cutting into a micro-flat plate: under the aseptic condition, padding the alignment chart paper drawn in the operation 3) below the plate inverted in the operation 2), opening a dish cover, and erecting a sterilized steel ruler at the edge of the plate, wherein the side line of the ruler is aligned with a group of straight lines of the alignment chart; sterilizing the blade of the cutting tool on alcohol burner, cooling, gently inserting the blade close to the edge line of the ruler into the plate agar plate, and pulling the cutting tool along the edge line of the rulerCutting the agar plate by a cutter; a12 cm plate can be cut 3 times in parallel, then the alignment drawing under the plate is rotated 90 degrees, the straight edge is moved to align with another set of lines perpendicular to the direction of the straight line just cut, and the same cutting operation is performed to cut groups of micro-plates 9, each group having 4X 4 blocks =16 blocks each having a size of 4X 4mm 2 The square micro-flat plate has the same size, is tidy and consistent, and has a flat section.
2. Preparing a working-state microplate:
carrying a preparation working state micro-flat plate by using a common glass slide; a moist plastic box in a sterile state in which slides can be placed is prepared in advance. Under the aseptic condition, opening the cover of the moisture-preserving plastic box, flatly placing the sterilized glass slide on the surface of a moisture-preserving material, picking up the qualified micro-flat plates cut in the step 1 by using a blade, transferring the qualified micro-flat plates onto the sterilized glass slide, and separately placing the micro-flat plates one by one, wherein 2 rows of micro-flat plates are arranged in each 3 micro-flat plates to form a working-state micro-flat plate for testing; .
3. Loading a sample to be tested
The 25% benzalkonium chloride is diluted 1000 times by sterile water to be used as a medicine sample to be detected, and the sterile water is used as a control to treat the sample. And (3) under an aseptic condition, sucking 5 mu l of sample liquid by using a liquid transfer gun, dispensing the sample liquid in the middle of the working-state micro-flat plate prepared in the step (2) under the condition that the gun head does not touch the micro-flat plate surface, automatically expanding and dispersing the sample liquid on the micro-flat plate surface, repeatedly dispensing 3 micro-flat plates (one row) on the sample to be detected and the control sample, and standing until no drug sample liquid is observed, thus obtaining the drug-loaded micro-flat plate.
4. Loading of pathogenic spores
Under aseptic condition, the prepared solution with the concentration of 10 6 Fully and uniformly suspending the spore liquid of the banana fusarium wilt bacterium strain Fo-4 with each spore/ml, sucking 1 mu l of the spore liquid by using a liquid transfer gun, and placing the liquid transfer gun in the middle of the drug-carrying micro-plate prepared in the step (3) under the condition that the gun head does not touch the surface of the micro-plate to naturally disperse the spore liquid.
5. Cultivation of
And 4, after the operation of the step 4 is finished, covering a cover of the moisturizing device, keeping the device in a stable state, transferring the device into an incubator, and culturing for 12 hours at the constant temperature of 28 ℃.
6. Observation of results
Taking out the culture material of the step 5 from the incubator, opening the cover of the moisturizing device, and placing the glass slide carrying the microplate under a microscope to observe the spore germination condition on each microplate.
As a result, the spore germination rate of the sterile water blank control treatment is 97.37%; the germination rate of the 25% benzalkonium chloride treatment is 3.33%; the tested benzyl prochloraz sample has 96.58 percent of inhibition effect on spore germination of banana fusarium wilt.
Example 2
The method for determining the inhibitory substances detected by the microplate is used for determining the inhibitory effect of 25% propiconazole on the germination of Uv-111 spores of ustilaginoidea virens strain and is implemented according to the operations from step 1 to step 5 of the embodiment 1, except that the drug sample loaded in the step 2 is 500 times of the dilution of 25% propiconazole, and the spores loaded in the step 3 are 10-fold in concentration 6 The culture time of step 4 was 48 hours per ml of the spore liquid of Uv-111, a strain of Ustilaginoidea virens.
Results the spore germination rate of the sterile water blank control treatment was 95.09%; while the germination rate of the 25% propiconazole treatment is 0; the propiconazole sample tested had an inhibitory effect on ustilaginoidea virens spore germination of 100%.
Claims (1)
1. A method for measuring the inhibitor of the microplate detection is characterized in that under the aseptic condition, a self-made cutter is used for cutting an agar culture medium microplate, a medicine sample and pathogenic bacteria spores are loaded on the microplate, and the inhibition effect of the medicine on the spore germination is detected, wherein the measuring method comprises the following steps:
1) Preparing qualified microplate
(1) Preparing a cutting tool: arranging sharp No. 23 surgical blades at intervals by using a material with equal thickness to form an equidistant blade group with blade intervals of 4mm, and using the equidistant blade group as a cutting tool for cutting a micro-flat plate;
(2) pouring a culture medium plate: heating and melting agar culture medium suitable for pathogenic bacteria spore germination, and pouring into a plate under aseptic condition to obtain culture medium plate;
(3) drawing of alignment drawing for cutting: drawing two groups of straight line drawings which are mutually perpendicular and crossed on a piece of clean white paper to prepare an alignment drawing for cutting the micro-flat plate;
(4) cutting a microplate: under the aseptic condition, padding the alignment chart paper drawn in the operation (3) below the flat plate dish inverted in the operation (2), opening a dish cover, and taking a sterilized straight ruler to be erected on the edge of the dish, wherein the side line of the straight ruler is aligned with a group of straight lines of the alignment chart; sterilizing the blade of the cutting tool on alcohol lamp fire, cooling, gently inserting the blade close to the side line of the straight edge into a plate agar plate, pulling the cutting tool along the side line of the straight edge, and cutting the agar plate; then the alignment line drawing under the dish and the dish are rotated for 90 degrees, the movable ruler is aligned with the other group of straight lines vertical to the straight line direction just cut, the agar culture medium plate is cut in the same way, and the agar culture medium plate is cut into 4 multiplied by 4mm 2 The size of the square micro-flat plate is equal, the micro-flat plate is tidy and consistent, and the section is flat, so that the square micro-flat plate is qualified;
2) Prepare the little flat plate of working state
Carrying a preparation working state micro-flat plate by using a common glass slide; preparing a moisturizing device which is in a sterile state and can be used for placing a glass slide in advance; under the aseptic condition, opening a cover of a moisturizing appliance, flatly placing the sterilized glass slide on the surface of a moisturizing material, picking up the qualified microplate cut in the step 1) by using a blade, transferring the qualified microplate onto the sterilized glass slide, and placing the qualified microplate and the sterilized glass slide block by block to form a working-state microplate for testing;
3) Loading a sample to be tested
Transferring the prepared drug sample solution to be detected and the test control treatment sample solution to an aseptic workbench, sucking 5 mu l of the sample solution by using a liquid transfer gun, placing the sample solution in the middle of the upper surface of the working-state micro-flat plate prepared in the step 2) under the condition that the gun head does not touch the surface of the micro-flat plate, automatically expanding and dispersing the sample solution on the surface of the micro-flat plate, and standing until no drug sample solution is observed to form a drug-loaded micro-flat plate;
4) Loading of pathogenic spores
Under the aseptic condition, fully and uniformly suspending the prepared pathogenic bacteria spore liquid, sucking 1 mu l of the spore liquid by using a liquid transfer gun, and placing the liquid in the middle of the drug-carrying micro-plate prepared in the step 3) under the condition that a gun head does not touch the surface of the micro-plate to naturally disperse the spore liquid;
5) Cultivation of
Step 4), after the operation is finished, covering a cover of the moisturizing device, keeping the device in a stable state, and transferring the device to an incubator for constant-temperature culture until the spores subjected to blank control treatment fully germinate;
6) Observation of results
Taking out the culture material of the step 5) from the incubator, opening the cover of the moisturizing device, placing the glass slide carrying the microplate under a microscope, observing and recording the spore germination condition on each microplate, and calculating the inhibitory effect of the measured drug on the spore germination according to the data of the control treatment.
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| CN2104101U (en) * | 1991-08-11 | 1992-05-13 | 丁晓东 | Kitchen knife |
| CN208215410U (en) * | 2018-05-17 | 2018-12-11 | 福建农林大学 | A kind of edible mushroom plate bacterial standard block cutter |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2104101U (en) * | 1991-08-11 | 1992-05-13 | 丁晓东 | Kitchen knife |
| CN208215410U (en) * | 2018-05-17 | 2018-12-11 | 福建农林大学 | A kind of edible mushroom plate bacterial standard block cutter |
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