CN111808912B - Determination method of microcarrier detection inhibitor - Google Patents
Determination method of microcarrier detection inhibitor Download PDFInfo
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Abstract
The invention relates to a plant pathology technology, and particularly discloses a method for preparing 25 mul agar culture medium microcarriers by adopting a concave glass slide, loading a medicine sample and pathogenic bacteria spores on the microcarriers, and detecting the inhibition effect of the medicines on spore germination, wherein the measuring method comprises the following steps: 1) Preparing qualified microcarrier; 2) Loading a sample to be detected into a microcarrier; 3) Pathogenic bacteria spores are dripped; 4) Spore germination and culture; 5) And (5) observing the result. The invention has the advantages that: 1) A plurality of samples can be tested on one plate, so that the miniaturization of a test system is realized; 2) One test unit only consumes 10 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 microcarrier 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 microcarrier technology.
The technical scheme for solving the technical problems is as follows:
a method for measuring the inhibitor of microcarrier detection mainly includes preparing 25 mul agar culture microcarrier by using a concave slide, loading a drug sample and pathogenic spore on the microcarrier, and measuring the inhibition effect of the drug on spore germination, wherein the method includes the following steps:
1. preparation of qualified microcarriers
1) And (3) sterilizing the concave glass slide: and (4) sterilizing the clean concave glass at high temperature for later use.
2) Placing the concave glass sheet: preparing a moisturizing device capable of containing a moisturizing material and a concave glass sheet in advance, opening a cover of the moisturizing device on an aseptic bench, and smoothly placing the sterilized concave glass sheet of operation 1) on the surface of the moisturizing material.
3) Melting of agar medium: heating and melting agar culture medium in a triangular flask suitable for pathogenic bacteria spore germination.
4) 80 ℃ treatment of the medium: heating a common water bath kettle in advance to be constant at 80 ℃, putting the culture medium melted in the operation 3) into the water bath kettle, and balancing to 80 ℃.
5) And (3) treatment of bubble-free pipetting by a pipetting gun: and (3) transferring the culture medium balanced to 80 ℃ in the operation 4) to a sterile workbench, adjusting the liquid taking amount of the liquid-transferring gun to 25 mu l, and extending the gun head into the culture medium at 80 ℃ to repeatedly suck and discharge the culture medium until no bubble phenomenon occurs in the liquid discharging operation.
6) Instillation of microcarriers: operation 5) after no bubble phenomenon, excessively pressing a liquid-transferring gun to suck the culture medium, moving the gun head and attaching the gun head to the wall of the bottle mouth of the triangular flask to remove the culture medium adhered to the outside of the gun head, transferring and dripping 25 mul to the bottom of the recess of the concave glass sheet, and after the culture medium liquid drops are expanded and balanced in the recess, forming a 25 mul tiny block which has no bubbles and is flat in surface and becomes a qualified microcarrier used for testing; and (5) continuing the operation until all the concave glass slides are instilled.
2. Loading the sample to be tested into the microcarrier
Transferring the prepared drug sample solution to be detected and the test control treatment sample solution to an aseptic workbench, sucking 10 mu l of the sample solution by using a pipette, placing the pipette in the middle of the surface of the qualified microcarrier prepared in the step (1) under the condition that the pipette tip does not touch the microcarrier surface, automatically expanding and dispersing the pipette on the microcarrier surface, and standing until no drug sample solution is observed to obtain the drug-loaded microcarrier.
3. Pathogenic bacteria spore is dripped
And (3) 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 spore liquid in the middle of the drug-carrying microcarrier prepared in the step (2) under the condition that a gun head does not touch the surface of the microcarrier to naturally disperse the spore liquid.
4. Spore germination culture
And (3) after the operation of the step 3 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 subjected to blank control treatment fully germinate.
5. Observation of results
Taking out the culture material obtained in the step 4 from the incubator, opening the cover of the moisturizing device, placing the concave glass carrying the microcarriers under a microscope, and observing and recording the germination condition of the spores on each microcarrier; the inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.
The invention has the advantages that:
1) The test system is miniaturized: the basic test unit of the conventional flat plate measurement technology is a set of plates, more than 3 repeats are usually arranged, and more than 3 sets of plates are needed for one sample, so that more culture instruments, more consumables and more culture space are occupied; the basic test unit of the technology of the invention is a microcarrier, a plurality of microcarriers can be loaded on a plurality of concave slides, and a set of dish can contain a plurality of concave slides, so that a plurality of samples can be tested in a set of dish, and the miniaturization of a test system can be realized.
2) Detection sample micro-quantification: 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 10 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.
In the present invention, a drug sample and pathogenic spores are loaded on a micro-block of an agar medium to perform an inhibition test, and thus the micro-block is referred to as a microcarrier.
Since the drug sample is directly loaded onto the surface of the microcarrier, the surface is technically required to be relatively flat. And because the drug is easy to diffuse into the interior of the microcarrier through surface penetration, the diluting effect of the sample liquid exists, the smaller the prepared microcarrier is, the smaller the diluting effect is, and the size of the prepared microcarrier is also required to be equal in technology. The observation of the test results on the microcarriers requires the observation by means of a microscope, which cannot normally observe when bubbles exist on the microcarriers, so that the microcarriers prepared by the technical requirements cannot have bubbles. Microcarriers meeting these specifications are acceptable microcarriers according to the invention.
The liquid-transfering gun is adopted to quantitatively suck the culture medium for trial production practice, and some technical problems and technical obstacles are found: (1) the agar medium in a hot-melt state is transferred to a planar device (such as a plate or a glass slide) and is either flattened into a liquid state or condensed into a spherical shape, so that microcarriers with relatively flat and uniform shapes cannot be obtained. (2) When agar medium in a hot-melt state is transferred by a conventional pipetting operation, air bubbles are easily formed on the microcarriers. (3) When the temperature of the culture medium is lower, the culture medium sucked in the gun head is easy to solidify and cannot be discharged, or the culture medium is solidified quickly and cannot be flattened after being discharged. (4) On the other hand, when the temperature of the culture medium is too high, bubbles are more likely to form in the discharged agar medium. (5) More culture medium liquid is adhered to the outer wall of the pipette head in the liquid suction operation, and the pipette head is transferred and released along with the pipette head, so that the accuracy of the liquid transfer amount is influenced. How to solve the technical problems is not disclosed with relevant technical schemes so far, so that the preparation of qualified microcarriers becomes the technical key of the invention.
Through repeated groping, a method for solving the technical problems is found, namely, a pipette gun is used for adopting proper operation, 25 mu l of agar culture medium with the temperature of 80 ℃ is sucked and injected into a concave cavity of a concave glass slide, and the micro-carrier is prepared by condensation.
Preparing a matched moisturizing device (such as a culture dish or a plastic box) in advance, and placing a moisturizing material (such as moist absorbent paper or gauze) in a sterile state in the device. The moisturizing appliance is placed on an aseptic workbench, the cover of the appliance is opened, and the prepared sterilized concave glass is stably placed on the surface of the moisturizing material. Heating and melting agar culture medium suitable for pathogenic bacteria spore germination in a triangular flask, and placing in a 80 deg.C water bath to balance to 80 deg.C; then transferring the culture medium with the temperature of 80 ℃ to a sterile workbench; adjusting the liquid transfer amount of a liquid transfer gun to 25 mu l, extending a gun head into a culture medium at 80 ℃, repeatedly carrying out liquid suction/liquid discharge operation, observing the bubble emergence condition of the liquid level of the culture medium, repeatedly carrying out the liquid suction/liquid discharge operation until no bubble phenomenon appears, generally carrying out liquid suction/liquid discharge for about 20 times to reach balance, at the moment, pressing down the liquid suction amount excessively, slightly moving the gun head, slightly sticking the gun head on the wall of the mouth of a triangular flask and slightly staying for about 1 second, removing the culture medium adhered to the outside of the gun head, then transferring to a concave glass slide concave cavity, slightly pressing the gun to discharge 25 mu l of culture medium to the bottom of the concave cavity, and naturally expanding the culture medium in the cavity and quickly cooling and solidifying the culture medium into a tiny block with a flat surface; keeping the pipette gun pressed down, returning to the culture medium in a triangular flask at 80 ℃, sucking and moving out a next culture medium tiny block by spot injection, repeating the operation until all recesses of the concave glass slide are pipetted to form the culture medium tiny block, paying attention to the fact that the operation cannot be stopped for a long time in the process, otherwise, solidifying the culture medium at the gun head; once solidification occurs, the tip needs to be replaced, the bubble removal operation described above is repeated, and then the next tiny piece of the recessed medium is pipetted. And checking after injection, and obtaining the qualified microcarrier by flattening the tiny blocks without bubbles.
After a qualified microcarrier is prepared, a drug can be loaded into the microcarrier, and the specific method is that a prepared drug sample to be detected and a test control treatment sample are placed in a workbench, a liquid transfer gun is used for sucking 10 mu l of sample liquid, the sample liquid is lightly spotted in the middle of the surface of the microcarrier, the gun head is prevented from touching the surface of the microcarrier, the actual operation skill is that when the gun head is close to the surface of the microcarrier, the liquid transfer gun is lightly pressed down, the sample liquid is discharged into small liquid drops and hung on the gun head, the small liquid drops are slightly close to the surface of the microcarrier, when the small liquid drops touch the surface of the microcarrier, the small liquid drops of the sample automatically fall and expand on the surface of the microcarrier, and then the microcarrier is allowed to stand on the workbench for a period of time (about 20 minutes) until no liquid sample can be observed. The micro carrier after sample adding becomes a drug-carrying micro carrier.
The loaded microcarriers can then be loaded with spores of pathogenic bacteria. The specific method comprises collecting prepared pathogenic bacteria spore solution, shaking to suspend spore completely and uniformly, sucking 1 μ l spore solution on aseptic work bench with pipette, just like spotting medicine sample solution,gently put in the middle of the drug-loaded microcarrier to naturally disperse 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 shows that the spore concentration of the prepared spore liquid is adjusted to 1 x 10 because each microcarrier surface is dispersed by about 100-200 spores to be beneficial to the subsequent result observation 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, namely placing the concave slide carrying the microcarrier under a microscope, and observing and recording the germination condition of the spores on the surface of the microcarrier. The inhibitory effect of the tested drug on spore germination was calculated from the results of the control treatment.
Example 1
The determination method for detecting the inhibitor by using the microcarrier 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. preparation of qualified microcarrier
1) And (3) sterilizing the concave glass slide: and (4) sterilizing the clean concave glass at high temperature for later use.
2) Placing the concave glass sheet: preparing a moisturizing device capable of containing a moisturizing material and a concave glass sheet in advance, opening a cover of the moisturizing device on an aseptic work bench, and stably placing the sterilized concave glass sheet of operation 1) on the surface of the moisturizing material.
3) Melting of agar medium: heating and melting agar culture medium in a triangular flask suitable for pathogenic bacteria spore germination.
4) Treatment of the medium at 80 ℃: heating a common water bath kettle in advance to be constant at 80 ℃, putting the culture medium melted in the operation 3) into the water bath kettle, and balancing to 80 ℃.
5) And (3) treatment of bubble-free pipetting by a pipetting gun: and (3) transferring the culture medium balanced to 80 ℃ in the operation 4) to a sterile workbench, adjusting the liquid taking amount of the liquid-transferring gun to 25 mu l, and extending the gun head into the culture medium at 80 ℃ to repeatedly suck and discharge the culture medium until no bubble phenomenon occurs in the liquid discharging operation.
6) Instillation of microcarriers: operation 5) after no bubble phenomenon, excessively pressing a liquid-transferring gun to suck the culture medium, moving the gun head and attaching the gun head to the wall of the bottle mouth of the triangular flask to remove the culture medium adhered to the outside of the gun head, transferring and dripping 25 mul to the bottom of the recess of the concave glass sheet, and after the culture medium liquid drops are expanded and balanced in the recess, forming a 25 mul tiny block which has no bubbles and is flat in surface and becomes a qualified microcarrier used for testing; and (5) continuing the operation until all the concave glass slides are instilled.
2. Loading the sample to be tested into the microcarrier
The drug sample to be detected is prepared by diluting 25% of benzalkonium chloride 1000 times with sterile water, and the sample is treated with sterile water as a control. And (2) sucking 10 mu l of sample liquid by using a pipette under the aseptic condition, placing the sample liquid in the middle of the surface of the qualified microcarrier prepared in the step (1) under the condition that the pipette head does not touch the surface of the microcarrier, automatically expanding and dispersing the sample liquid on the surface of the microcarrier, repeatedly spotting 2 microcarriers on the sample to be detected and the control sample, and standing until no drug sample liquid is observed, thus obtaining the drug-carrying microcarrier.
3. Pathogenic bacteria spore is dripped
Under aseptic condition, the prepared solution with the concentration of 10 6 And (3) fully and uniformly suspending the spore liquid of the banana fusarium oxysporum strain Fo-4 in each ml of spore, sucking 1 mu l of the spore liquid by using a liquid transfer gun, and placing the point in the middle of the drug-loaded microcarrier prepared in the step (2) to naturally disperse the spore liquid.
4. Spore germination culture
And (3) after the operation of the step 3 is finished, covering a cover of the moisturizing device, keeping the device in a stable state, transferring the device to an incubator, and culturing for 12 hours at the constant temperature of 28 ℃.
5. Observation of results
Taking out the culture material of the step 4 from the incubator, opening the cover of the moisturizing device, and placing the microcarrier-carrying concave glass sheet under a microscope to observe the spore germination condition on each microcarrier.
As a result, the spore germination rate of the sterile water blank control treatment is 96.61%; the germination rate of the 25% benzalkonium chloride treatment is 3.70%; the tested benzamidine prochloraz sample has 96.17 percent of inhibition effect on banana fusarium oxysporum spores.
Example 2
The determination method for detecting the inhibitor by using the microcarrier of the invention is used for determining the inhibition effect of 25 percent 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 the 25 percent 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.
As a result, the germination rate of the spores treated by the sterile water blank control is 94.00%; while the germination rate of the 25% propiconazole treatment is 0; the propiconazole sample tested had an inhibitory effect on ustilaginoidea virens spores of 100%.
Claims (1)
1. A method for measuring a microcarrier detection inhibitor is characterized in that a 25 microliter agar culture medium microcarrier is prepared by using a concave slide, a drug sample and pathogenic bacteria spores are loaded on the microcarrier, and the inhibition effect of the drug on the spore germination is detected, wherein the measuring method comprises the following steps:
1) Preparation of qualified microcarriers
(1) And (3) sterilizing the concave glass sheet: sterilizing the clean concave glass sheet at high temperature for later use;
(2) placing the concave glass sheet: preparing a moisturizing device capable of containing a moisturizing material and the concave glass sheet in advance, opening a cover of the moisturizing device on an aseptic workbench, and stably placing the sterilized concave glass sheet in the operation (1) on the surface of the moisturizing material;
(3) melting of agar medium: heating and melting agar culture medium contained in a triangular flask suitable for pathogenic spore germination;
(4) treatment of the medium at 80 ℃: heating a common water bath kettle to a constant temperature of 80 ℃ in advance, putting the culture medium melted in the operation (3) into the water bath kettle, and balancing to 80 ℃;
(5) and (3) treatment of bubble-free pipetting by a pipetting gun: transferring the culture medium which is balanced to 80 ℃ in the operation (4) to a sterile workbench, adjusting the liquid taking amount of a liquid-transferring gun to 25 mu l, and extending a gun head into the culture medium at 80 ℃ to repeatedly suck and discharge the culture medium until no bubble phenomenon occurs in liquid discharging operation;
(6) instillation of microcarriers: after no bubble phenomenon exists in the operation (5), the pipette tip is pressed down excessively to suck the culture medium, the pipette tip is moved up and is attached to the wall of the bottle mouth of the triangular flask to remove the culture medium adhered to the outside of the pipette tip, then 25 mu l of the culture medium is transferred and dripped to the bottom of the recess of the concave glass, after the culture medium drops are expanded and balanced in the recess, a tiny block of 25 mu l is formed, the tiny block has no bubbles, the surface is flat, and the tiny block becomes a qualified microcarrier used for testing; continuously operating until all the concave slides are instilled;
2) Loading the sample to be tested into the microcarrier
Transferring the prepared drug sample liquid to be detected and the test control treatment sample liquid to an aseptic workbench, sucking 10 mu l of the sample liquid by using a liquid transfer gun, placing the sample liquid in the middle of the surface of the qualified microcarrier prepared in the step 1) under the condition that the gun head does not touch the microcarrier surface, automatically expanding and dispersing the sample liquid on the microcarrier surface, and standing until the drug sample liquid cannot be observed to obtain the drug-loaded microcarrier;
3) Pathogenic bacteria spore is dripped
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 microcarrier prepared in the step 2) under the condition that a gun head does not touch the surface of the microcarrier to naturally disperse the spore liquid;
4) Spore germination culture
Step 3), 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;
5) Observation of results
Taking out the culture material obtained in the step 4) from the incubator, opening a cover of a moisturizing appliance, placing the concave glass carrying the microcarriers under a microscope, and observing and recording the germination condition of spores on each microcarrier; the inhibitory effect of the tested drugs on spore germination was calculated from the control-treated data.
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| US3941658A (en) * | 1973-05-31 | 1976-03-02 | Gist-Brocades N.V. | Method for determination of the presence of antibiotics |
| CN102492616A (en) * | 2011-11-14 | 2012-06-13 | 福建农林大学 | Fungus spore germination carrying membrane and production method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3941658A (en) * | 1973-05-31 | 1976-03-02 | Gist-Brocades N.V. | Method for determination of the presence of antibiotics |
| CN102492616A (en) * | 2011-11-14 | 2012-06-13 | 福建农林大学 | Fungus spore germination carrying membrane and production method thereof |
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| 8种杀菌剂对苦瓜枯萎病菌的室内毒力测定;何美仙等;《金华职业技术学院学报》;20130628(第03期);第36-37页 * |
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