A method for preparing a microplate of mixed medicine using a plastic culture dish

Abstract

The invention relates to microbiology and plant pathology technology, and specifically to a method of using a plastic culture dish to mix a small amount of drug sample liquid with water agar under the working condition of a micro-plate mixing station with a temperature system of 65°C to prepare an agar micro-plate containing drugs. The main components of the micro-plate mixing station include a temperature control body A, a plastic culture dish preheating component B for mixing samples, a drug sample preheating component C, a gun head preheating component D, and a glass slide preheating glass plate component E. The main steps of the preparation method of the drug micro-plate are as follows: 1) drug sample preparation, 2) sample mixing and water agar preparation, 3) gun head configuration, 4) starting the micro-plate mixing station to enter the working state, 5) sample preheating, 6) glass slide preheating, 7) absorbing sample liquid, 8) absorbing water agar, 9) mixing sample liquid with water agar, and 10) dripping to develop into a drug micro-plate. The advantages of the invention are as follows: 1) plastic culture dishes are conventional consumables in the field of biology and are easy to obtain; 2) the total amount of drug samples required is very small; 3) the prepared drug micro-plate is convenient for subsequent biological assays and microscope observations.

Description

Technical Field

[0001] The invention relates to microbiology and plant pathology technology, in particular to a method for preparing a drug microplate by mixing a plastic culture dish. Technical Background

[0002] Plant diseases caused by harmful microorganisms can cause huge economic losses and ecological damage. At present and for a long time to come, the main means of plant disease control is still pesticide control. Exploring and tapping biogenic drugs is an important direction for pesticide control. When searching for samples with inhibitory activity in a large number of microbial or plant samples, if the inhibitory activity can be detected and identified with very little metabolite (or tissue fluid) in the primary screening stage, the screening efficiency will be improved and the screening range will be expanded. In the research process of biogenic drugs, the inhibitory effect of metabolites produced by the cultivation process of beneficial microorganisms on harmful microorganisms is explored and analyzed. The current analysis and detection technology often requires the termination of the cultivation of some culture bottles so that the culture products can be filtered to prepare enough test samples. However, those skilled in the art most hope to extract trace samples directly from the culture bottles for bioassay without terminating the cultivation process. When it is found that microorganisms (or plants) can produce inhibitory metabolites (or plant tissue fluids), but the inhibitory active components are still unknown, it is generally necessary to separate and analyze the active substances. In the process of separating the active substances, it is often necessary to track the biological activity of a small amount of separated samples. Obviously, the research and development of biogenic pesticides requires technical support for the detection of trace or small amounts of drug samples.

[0003] The current conventional technical method for detecting the inhibitory activity of drugs against harmful microorganisms in this field is mainly the plate culture determination method. The technical key points of this method are that the drug is first mixed with water agar to make a drug-containing culture plate, and then harmful microorganisms are transplanted into the drug-containing plate and cultured under appropriate conditions. The growth of harmful microorganisms on the plate is observed and compared to achieve the measurement of the inhibitory activity of the drug. The basic technology of this method is the preparation of drug-containing culture plates, and the current conventional technology for preparing drug-containing culture plates is to mix the drug sample with water agar in a triangular flask and then pour it into a conventional culture dish to make a drug-containing culture plate. Although such a drug-containing culture plate preparation technology is simple and mature, it has an obvious feature, that is, the drug sample to be tested often needs to reach a sample volume of more than tens of milliliters, which is difficult to match with the aforementioned detection technology for trace or small amounts of samples desired in this field, and is therefore an important drawback of the current drug-containing culture plate preparation technology. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a drug-containing microplate by mixing a small amount of drug sample liquid with water agar using a plastic culture dish.

[0005] The present invention designs and adopts the technical conditions of the micro-plate sample mixing station to solve the above technical problems. The technical solutions are as follows:

[0006] 1. Design a microplate mixing table, the main components of which include a temperature control body A, a plastic culture dish preheating component B for mixing, a drug sample preheating component C, a gun tip preheating component D, and a slide preheating glass plate component E. The mixing table is suitable for use on a clean bench.

[0007] The main components of the temperature control body A include a basin-shaped container A1, a water body A2, a water body electric heating temperature control component A3, and a water body surface cover A4. The water body electric heating temperature control component A3 can automatically heat the water body and stabilize the water body temperature to a working state of 65°C, and the water body surface cover A4 can prevent the water body from rapid evaporation and cooling.

[0008] The plastic culture dish preheating component B for mixing samples mainly includes a plastic culture dish B1 and a culture dish support B2. The bottom of the plastic culture dish B1 can be partially immersed in the water body A2. The drug sample preheating component C mainly includes a sample tube C1 and a sample tube support C2. The body of the sample tube C1 is directly immersed in the water body A2. The gun tip preheating component D mainly includes a gun tip box D1 and a gun tip box clamp D2. The body of the gun tip box (D1) is immersed in the water body A2. The slide preheating glass plate component E mainly includes a glass plate E1 and a glass plate support E2. Part of the glass plate E1 is immersed in the water body A2.

[0009] 2. A method for preparing a microplate containing a drug using the aforementioned microplate mixing table. The main technology is to place the mixing table in a clean bench, use a plastic culture dish as a mixing device for the drug and agar, use a glass plate as a preheating plate for the slide, and prepare the drug microplate in a technical system with a water body of 65°C. The preparation method steps are as follows:

[0010] 1) Drug sample preparation: Transfer a small amount of drug sample obtained during drug development work into a sterilized sample tube C1.

[0011] 2) Preparation of water agar for mixing: The ratio of water agar for mixing is: 40g agar and 1000ml water. After heating and melting, maintain it in a water bath above 90°C.

[0012] 3) Tip preparation: Use a conventional tip of 200 μl, first cut off a small section of the tip, then put the tip into a tip box, sterilize and dry it in a conventional manner, and then fix the tip box on the tip box holder D2.

[0013] 4) Start the sample mixing station and put it into working state: first start the clean bench normally, then place the sample mixing station on the working table inside the clean bench, then take the sterilized plastic culture dish, place it obliquely on the plastic culture dish pad rack B2, then turn on the power of the sample mixing station, electrically heat and adjust the temperature of the water body A2 to a working state of 65°C, and maintain operation until the plastic culture dish B1, the gun tip in the gun tip box D1, and the slide preheating glass plate E1 are in a working state balanced with the 65°C water temperature.

[0014] 5) Take the drug sample tube C1 of step 1) and insert it into the tube hole of the sample tube rack C2 floating on the water surface of the water body A2, and preheat it in the water body A2.

[0015] 6) Take a sterilized glass slide and place it flat on the surface of the glass slide preheating plate E1 for preheating.

[0016] 7) Put on the pipette tip in working state in step 4) and draw 100 μl of sample liquid from the preheated sample tube in step 5) and inject it into the lowest part of the plastic culture dish B1 in working state in step 4) to form a nearly spherical sample droplet.

[0017] 8) Put the pipette tip in the working state of step 4) on the pipette cover, aspirate 30 μl of the mixed water agar in step 2), inject it into the sample droplet formed in step 7), and keep the pipette tip in the droplet, repeatedly aspirate and discharge the liquid, wash the residual agar in the lower pipette tip, and obtain a mixed sample solution of the sample liquid and water agar.

[0018] 9) Use a pipette with a volume adjusted to 150 μl, put on the pipette tip in working state in step 4), insert the pipette tip into the mixed sample solution obtained in step 8), and perform repeated pipetting and discharging actions to ensure that the sample solution and water agar in the mixed sample solution are fully and evenly mixed.

[0019] 10) All the uniformly mixed sample solution in step 9) is sucked into the pipette tip, and then transferred to the preheated glass slide in step 6). The mixed sample solution of the pipette is discharged in the form of drops onto the glass slide surface at different positions on the glass slide, and the discharged liquid spots are moved and flattened with the pipette tip so that each liquid spot forms a thin layer of agar micro-block. The glass slide is then transferred to a sterile container at room temperature and kept moist. After the thin layer of agar micro-block is condensed, it becomes a drug micro-plate.

[0020] Advantages of the present invention

[0021] 1) Plastic culture dishes are common consumables in the field of biology and are easily available.

[0022] 2) The present invention can mix 0.1 ml of drug sample liquid into multiple drug microplates, and the total amount of drug sample required is very small.

[0023] 3) Drug-containing microplates prepared on glass slides facilitate subsequent bioassays and microscopic observations. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the front view of the microplate mixing station. In the figure, A is the temperature control body component, B is the plastic culture dish preheating component for mixing, C is the drug sample preheating component, D is the gun tip preheating component, and E is the slide glass preheating component.

[0025] Figure 2 It is a schematic diagram of the side structure of the temperature control body A. In the figure, A1 is a basin-shaped container, A2 is a water body, A3 is a water body electric heating temperature control component, and A4 is a water body surface cover.

[0026] Figure 3 It is a schematic diagram of the side structure of the various components in the sample mixing table and its spatial relationship with the temperature-controlled water body. In the figure, A1 is the basin-shaped container of the temperature-controlled body, A5 is the bottom plane line of the basin-shaped container of the temperature-controlled body, and A6 is the horizontal line of the surface of the water body of the temperature-controlled body. B is a plastic culture dish preheating component for mixing samples, B1 is a plastic culture dish, and B2 is a culture dish pad. C is a drug sample preheating component, C1 is a sample tube, and C2 is a sample tube rack. D is a gun tip preheating component, D1 is a gun tip box, and D2 is a gun tip box clamp. E is a glass plate preheating component for a slide, E1 is a glass plate, and E2 is a glass plate bracket.

[0027] Figure 4 This is a schematic diagram of using a pipette to suck up the mixed sample liquid and drop it on the surface of a glass slide. The rectangular frame in the figure is the glass slide, and the gray-black circular spots are droplets of the mixed sample liquid.

[0028] Figure 5 The invention adopts a preparation method of mixing drug microplates with plastic culture dishes, wherein garlic tissue fluid is mixed with agar and then dropped on a common glass slide to form 6 drug microplates. DETAILED DESCRIPTION

[0029] When the amount of drug sample is small, the small amount of drug sample liquid is conventionally mixed with a small amount of water agar. The water agar often solidifies in the pipetting device before it can be mixed with the drug sample liquid, making it impossible to carry out the work. Obviously, under conventional technical conditions, it is difficult to prepare a drug-containing plate with a small amount of sample. The present invention cleverly designs a set of micro-plate mixing stations and establishes a proper mixing technical condition system, which can effectively and evenly mix a small amount of drug sample liquid with a small amount of high-concentration water agar, and form a thin layer on a glass slide to obtain a micro-plate containing the drug.

[0030] The main components of the microplate mixing station include a temperature control body A, a plastic culture dish preheating component B for mixing, a drug sample preheating component C, a gun tip preheating component D, and a slide preheating glass plate component E. Figure 1 The entire microplate sample mixing table can be placed on the inner desktop of the clean bench, and the relevant operations of the present invention can be carried out under the working state of the clean bench to ensure that the prepared drug-containing microplate is in a sterile state.

[0031] The cross-sectional structure of each main component is as follows: Figure 2 and Figure 3 shown.

[0032] The main components of the temperature control body A include a basin-shaped container A1, a water body A2 in the container, a water body electric heating temperature control component A3, and a water body surface cover A4. Figure 2 The water body electric heating temperature control component A3 can automatically heat the water body and stabilize the water body temperature to a working state of 65°C, and the water body surface cover A4 can prevent the water body from rapid evaporation and cooling.

[0033] The plastic culture dish preheating component B for mixing samples mainly includes a plastic culture dish B1 and a culture dish support B2. Figure 3 As shown in Figure B. Plastic culture dishes are commonly used in biological laboratories; the culture dish support B2 is buried in the water of the temperature-controlled water body A2, which can support the culture dish B1 in an inclined state and keep the bottom of the culture dish B1 partially immersed in the water body A2, so that the culture dish B1 can quickly balance to the temperature of the water body A2.

[0034] The drug sample preheating component C mainly includes a sample tube C1 and a sample tube rack C2. Figure 3 As shown in Figure C. The sample tube rack C2 has tube holes on it and floats on the water surface of the temperature-controlled water body A2. The sample tube C1 is preferably a 1.5ml small centrifuge tube; after the sample tube C1 is inserted into the tube hole of the tube rack C2, the sample tube body is directly immersed in the water of the water body A2, so that the sample tube and the sample liquid in the tube can quickly balance to the temperature of the water body A2.

[0035] The gun tip preheating component D mainly includes a gun tip box D1 and a gun tip box clamp D2. Figure 3 As shown in Figure D. The gun tip box clamp D2 is also buried in the water body A2 of the temperature control body, which can clamp and fix the ordinary gun tip box and maintain the box body of the gun tip box D1 immersed in the water body A2, so that the gun tip box D1 and the gun tips placed inside can quickly balance to the temperature of the water body A2. The box cover of the gun tip box D1 is completely exposed outside the water body A2, and the gun tips can be opened at will.

[0036] The slide glass preheating glass plate component E mainly includes a slide glass preheating glass plate E1 and a glass plate bracket E2. Figure 3As shown in Figure E. The glass plate bracket E2 is immersed in the water body A2 of the temperature control body, which can stably support the glass plate E1; the glass plate E1 is composed of 3 ordinary glass plates with a thickness of 1 cm stacked together, and half of the thickness of the bottom glass plate is immersed in the water body A2. This glass plate can quickly balance to the temperature of the water body A2, but the heat conduction resistance of the two upper glass plates can cause the surface temperature of the top glass plate to drop to about 42°C.

[0037] The preparation method of the microplate containing the drug using the microplate sample mixing table designed above is as follows: the sample mixing table is placed in a clean bench, a plastic culture dish is used as a mixing device for the drug and agar, a glass plate is used as a preheating plate for the slide, and the drug microplate is prepared in a technical system with a water body of 65°C. The preparation method steps are as follows:

[0038] 1. Drug sample preparation: Transfer a small amount of drug sample obtained during drug development work into a sterilized sample tube C1.

[0039] 2. Preparation of water agar for mixing: The ratio of water agar for mixing is: 40g agar, 1000ml water. After conventional high-temperature sterilization, it is ready for use and heated to melt before use. The water agar is very viscous after melting. It is easy to produce a large number of bubbles when heated and boiled, and the bubbles are difficult to disappear. Since the current conventional microwave heating can quickly melt agar and cause a large number of bubbles, the water agar can be heated in boiling water when it is close to boiling in microwave heating to avoid the formation of a large number of bubbles in the water agar. When the water agar is completely melted, it is maintained in a water bath above 90°C and placed within the reach of the operator.

[0040] 3. Tip configuration: The preparation operation mainly uses 200μl tips. Since the tip of the 200μl tip has a small aperture, the highly viscous water agar cannot pass smoothly. Therefore, a cutting tool such as a blade is used to cut off about 5-8mm of the tip of the tip, thereby obtaining a larger aperture of the tip to ensure that the water agar can pass smoothly. The tip with the tip cut off is placed in a tip box for sterilization and drying in a conventional manner, and then fixed on the tip holder D2.

[0041] 4. Start the microplate mixing station and put it into working state: first start the clean bench normally, then place the microplate mixing station on the working table inside the clean bench, take a sterilized plastic culture dish, and place it obliquely on the culture dish pad rack B2 of the mixing station, then turn on the power of the microplate mixing station, electrically heat the water body A2 to a working state where the temperature reaches 65°C, and maintain this working state for more than 20 minutes, so that the plastic culture dish B1 and the gun tips in the gun tip box D1 reach equilibrium with the temperature of the water body A2 and are in a working state; also, the surface temperature of the slide preheating glass plate E1 is maintained at a working state of about 42°C.

[0042] 5. Take the drug sample tube C1 from step 1 and insert it into the tube hole of the sample tube rack C2 floating on the water surface of the water body A2. The tube body of the sample tube is immersed in the water body and preheated for more than 5 minutes.

[0043] 6. Place the sterilized slide flat on the slide preheating glass plate E1 and preheat for more than 5 minutes.

[0044] 7. Put on the pipette tip in the working state of step 4, draw 100 μl of sample liquid from the preheated sample tube in step 5, and inject it into the lowest part of the plastic culture dish B1 in the working state of step 4 to form a nearly spherical sample droplet.

[0045] 8. Put the pipette tip in the working state of step 4 on the pipette cover, aspirate 30 μl of the mixed water agar in step 2, inject it into the sample droplet formed in step 7, and keep the pipette tip in the droplet. Repeat the aspiration and discharge action about 5 times to wash the residual agar in the lower pipette tip to obtain a mixed sample solution of sample liquid and water agar.

[0046] 9. Use a pipette with the liquid aspiration volume adjusted to 150 μl, put on the pipette tip in the working state of step 4, insert the pipette tip into the mixed sample obtained in step 8, and repeat the liquid aspiration and discharge action about 5 times to ensure that the sample liquid and water agar in the mixed sample are fully and evenly mixed.

[0047] 10. Pipette all the mixed solution evenly mixed in step 9 into the pipette tip, transfer to the preheated slide in step 6, and discharge the mixed solution from the pipette onto the slide surface in the form of drops at different locations on the slide. Figure 4 As shown, use the gun tip to spread the discharged liquid spots flat so that each liquid spot forms a thin layer of agar micro-block, and then transfer the glass slide to a sterile container that is kept moist at room temperature. After the thin layer of agar micro-block condenses, it becomes a drug micro-plate. Usually, 5-10 drug micro-plates can be formed on the glass slide at one time.

[0048] After the preparation of the drug microplate is completed, turn off the power and stop the operation of the microplate mixing station, clean and dry the relevant components of the mixing station, and store them properly for future use.

[0049] The present invention is designed to be implemented in a laboratory at a room temperature of 25°C. The main technology is to use a plastic culture dish as a sample mixing device, and an ordinary glass plate as a preheating plate for a glass slide. The water body of the microplate sample mixing table is designed to be 65°C, and the preheating of the sample mixing equipment, sample preheating, gun tip preheating, and glass slide preheating technology are all organically integrated into a microplate sample mixing table system. Such a thermal balance technology system can not only prevent the mixed sample liquid in the plastic culture dish from being in an excessively high temperature state and causing rapid evaporation of water, but also prevent the rapid evaporation of water in the microplate unfolded on the glass slide, and at the same time prevent agar condensation during the operation. Since the total amount of the mixed sample liquid (130μl) is relatively small, the aspiration and drainage mixing operations of step 9 of the above-mentioned preparation method steps are prone to generate bubbles. It is necessary to pay attention to observe and control the speed of the aspiration and drainage actions to reduce the formation of bubbles, which is conducive to obtaining a bubble-free agar microplate.

[0050] The purpose of designing the plastic culture dish for mixing to be in an inclined state is to drip the sample liquid and water agar at the lowest position in the culture dish, avoid the flow and expansion of the mixed liquid, make it easier to maintain the mixed liquid as relatively concentrated droplets, and reduce the bubbles generated by the suction and discharge mixing operation in step 9.

[0051] Since one portion of the mixed sample liquid occupies a smaller space in the culture dish, the remaining blank positions on the inner surface of the culture dish that have not come into contact with the mixed sample liquid can be used for mixing other samples. This can be done by simply turning the blank positions of the plastic culture dish on the culture dish stand B2 to the lowest position.

[0052] The operations of steps 7 to 10 of the method steps are for a small amount of sample liquid and water agar, and are performed under high temperature conditions, so the operation needs to be continuous and long pauses should be avoided during the operation, which can reduce the condensation probability of water agar during the operation and reduce the evaporation and loss of water in the mixed sample liquid. In actual operation, three pipettes with fixed liquid aspiration can be used to adjust and fix 100 μl of drug sample liquid, 30 μl of water agar, and 150 μl for mixing the mixed sample liquid, respectively, so as to avoid the need to adjust the liquid aspiration volume when using a single pipette.

[0053] Example 1

[0054] Take 1 ml of garlic tissue grinding extract (extraction ratio: 1 g garlic, 5 ml water) as the drug sample solution. Using the method for preparing a drug microplate using a plastic culture dish of the present invention, the drug sample solution is mixed with water agar to prepare a microplate containing garlic tissue fluid. As a result, a microplate containing garlic tissue fluid without bubbles can be obtained. Figure 5 shown.

Claims

1. A drug microplate mixing station, characterized in that: The main components include a temperature control body (A), a plastic culture dish preheating component for mixing samples (B), a drug sample preheating component (C), a gun tip preheating component (D), and a slide preheating glass plate component (E). The sample mixing table is suitable for use on a clean bench. The temperature control body (A) mainly comprises a basin-shaped container (A1), a water body (A2), a water body electric heating temperature control component (A3), and a water body surface cover (A4). The water body electric heating temperature control component (A3) can automatically heat the water body and stabilize the water body temperature to a working state of 65°C, and the water body surface cover (A4) can prevent the water body from rapid evaporation and cooling. The sample mixing plastic culture dish preheating component (B) mainly comprises a plastic culture dish (B1) and a culture dish support (B2), and the bottom of the plastic culture dish (B1) can be partially immersed in the water body (A2); The drug sample preheating component (C) mainly comprises a sample tube (C1) and a sample tube rack (C2), and the tube body of the sample tube (C1) is directly immersed in the water body (A2); the gun tip preheating component (D) mainly comprises a gun tip box (D1) and a gun tip box clamping rack (D2), and the box body of the gun tip box (D1) is immersed in the water body (A2); the slide glass preheating glass plate component (E) mainly comprises a glass plate (E1) and a glass plate bracket (E2), and part of the glass plate (E1) is immersed in the water body (A2).

2. A method for preparing a microplate containing a drug using the drug microplate mixing station of claim 1, characterized in that: Place the mixing table in the clean bench, use a plastic culture dish as a mixing device for the drug and agar, use a glass plate as a preheating plate for the slide, and prepare the drug-containing microplate in a technical system with a water temperature of 65°C. The preparation method steps are as follows: 1) Drug sample preparation: transfer a small amount of drug sample obtained during drug development work into a sterilized sample tube (C1); 2) Preparation of water agar for mixing: The ratio of water agar for mixing is: 40g agar and 1000ml water. After heating and melting, maintain it in a water bath above 90℃; 3) Tip preparation: Use a conventional tip of 200 μl, first cut off a small section of the tip, then put the tip-cut tip into a tip box in a conventional manner, sterilize and dry it, and then fix the tip box on the tip box holder (D2); 4) Start the sample mixing table and put it into working state: first start the clean bench normally, then place the sample mixing table on the working table inside the clean bench, then take the sterilized plastic culture dish, place it obliquely on the plastic culture dish pad rack (B2), then turn on the power of the sample mixing table, electrically heat and adjust the water body (A2) temperature to a working state of 65°C, and maintain the operation until the plastic culture dish (B1), the gun tip in the gun tip box (D1), and the slide preheating glass plate (E1) are in a working state balanced with the 65°C water temperature; 5) inserting the drug sample tube (C1) obtained in step 1) into a tube hole of a sample tube rack (C2) floating on the surface of the water body (A2), and preheating the sample tube in the water body (A2); 6) Place the sterilized glass slide flat on the glass slide preheating plate (E1) for preheating; 7) Put the pipette tip in working state in step 4) on the pipette cover, draw 100 μl of sample liquid from the preheated sample tube in step 5), and inject it into the lowest part of the plastic culture dish (B1) in working state in step 4) to form a nearly spherical sample droplet; 8) Put the pipette tip in the working state of step 4) on the pipette, suck up 30 μl of the mixed water agar in step 2), inject it into the sample drop formed in step 7), and keep the pipette tip in the drop, repeatedly suck and discharge the liquid, wash the residual agar in the lower pipette tip, and obtain a mixed sample solution of the sample liquid and water agar; 9) Use a pipette with a liquid aspiration volume adjusted to 150 μl, put on the pipette tip in the working state of step 4), insert the pipette tip into the mixed sample solution obtained in step 8), and perform repeated liquid aspiration and liquid discharge actions to ensure that the sample solution and water agar in the mixed sample solution are fully and evenly mixed; 10) All the uniformly mixed sample solution in step 9) is sucked into the pipette tip, and then transferred to the preheated glass slide in step 6). The mixed sample solution of the pipette is discharged in the form of drops onto the glass slide surface at different positions on the glass slide, and the discharged liquid spots are moved and flattened with the pipette tip so that each liquid spot forms a thin layer of agar micro-block. The glass slide is then transferred to a sterile container at room temperature and kept moist. After the thin layer of agar micro-block is condensed, it becomes a drug micro-plate.

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