阅读说明：本技术 一种微平板检测抑制物的测定方法 (Method for determining micro-plate detection inhibitor ) 是由 张君成 王忠文 张正淳 于 2020-07-07 设计创作，主要内容包括：本发明公开了一种微平板检测抑制物的测定方法,采用自制刀具切制琼脂培养基微平板,在微平板上载入药物样品以及病原菌孢子,检测药物对孢子萌发的抑制作用,测定方法的步骤如下：1)制备合格的微平板；2)准备工作态微平板；3)载入待测样品；4)载入病原菌孢子；5)培养；6)结果观测。本发明的优点：1)可在一片载玻片上测试多个样品,实现测试体系微型化；2)一个测试单元只消耗样品液5μl,实现检测样本微量化。(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.)

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

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;

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;

drawing and cutting are performed by the drawing machine: drawing two groups of mutually perpendicular and crossed line drawings on a piece of clean white paper for preparing an alignment line drawing for cutting a micro-flat plate;

cutting into a micro-flat plate: under the aseptic condition, padding the alignment chart paper drawn by the operation III below the flat plate dish inverted by the operation III, opening a dish cover, putting a sterilized straight ruler on the edge of the dish, and aligning the side line of the straight ruler 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 4mm²The 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 working state micro-plate

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 microplate prepared in the step 3) under the condition that the head of the liquid transfer gun does not touch the surface of the microplate to naturally disperse the spore liquid;

5) culturing

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, and observing and recording the spore germination on each microplate. The inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.

Technical Field

The invention relates to a plant pathology technology, in particular to a method for measuring an inhibitor by using a micro-plate.

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 line drawing drawn in operation 3) below the flat plate dish inverted in 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 line drawing; 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 4mm²The 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 working state micro-plate

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 a 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 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 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 an 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 microplate prepared in the step (3) under the condition that the head of the liquid transfer gun does not touch the surface of the microplate to naturally disperse the spore liquid.

5. Culturing

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 spore germination 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 measurement technology usually needs to consume more than 10000 mul of sample liquid, while one test unit of the 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 4mm²The 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 original trial practice found that there are technical problems to be solved in order to prepare a qualified microplate, such as how to cut a microplate with a minute volume and an equivalent volume? 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 at intervals to expose the blade edge by 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 agar plates. 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 micro-plate, a printer (or a manual drawing) is used to draw 2 sets of mutually perpendicular crossed line drawings on a clean white paper, and an alignment line drawing for cutting the micro-plate is prepared. 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, the plate and the underlying alignment chart are not moved relatively, but only slightly rotatedMoving the alignment line drawing under the plate for 90 degrees, driving the plate and the ruler to rotate for 90 degrees, slightly rotating the ruler for 90 degrees, aligning with another group of straight lines vertical to the cutting direction, and cutting into multiple groups of 16 blocks (4 blocks multiplied by 4 blocks) with equal size and each block with size of 4 multiplied by 4mm²The 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 implement (such as a petri dish or a preservation box) in an aseptic state, in which a moist moisturizing material (such as 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 collecting prepared pathogenic bacteria spore liquid, suspending the oscillated spore completely and uniformly, and sucking with liquid-transfering gun on ultraclean bench1 mul of spore liquid, as with the spotted drug sample liquid, was spotted gently onto the middle of the drug-loaded microplate to allow the spore liquid to disperse naturally. Due to the small amount of spore liquid, the dispersion range does not usually exceed that of the drug sample. Practical work shows that each micro-plate surface is dispersed with about 100-200 spores to facilitate the observation of the later result, so that the concentration of the spores prepared in advance is adjusted to 1 x 10⁶～2×10⁶One 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.