A method for preparing a microplate for mixing medicine using a centrifuge tube

Abstract

The present invention relates to microbiology and plant pathology technology, and specifically to a method of using a small centrifuge tube to mix a small amount of drug sample liquid with water agar under the working condition of a microplate mixing station with a temperature system of 65°C to prepare an agar microplate containing drugs. The main components of the microplate mixing station include a temperature control body A, a centrifuge tube preheating component B for mixing samples, a drug sample preheating component C, a gun head preheating component D, and a glass slide preheating iron box component E. The main steps of the preparation method of the drug microplate are as follows: 1) preparing drug samples, 2) preparing water agar for mixing samples, 3) preparing gun heads, 4) starting the microplate mixing station to enter the working state, 5) preheating samples, 6) preheating glass slides, 7) transferring sample liquid, 8) transferring water agar, 9) mixing sample liquid and water agar, and 10) dripping and developing into a drug microplate. The advantages of the present invention are as follows: 1) small centrifuge tubes are commonly used consumables in biological laboratories and are convenient for use. 2) the total amount of drug samples required is very small; 3) the obtained drug microplate is convenient for subsequent biological assays and microscopic observations.

Description

Technical Field

[0001] The invention relates to microbiology and plant pathology technology, in particular to a preparation method of mixing a drug microplate using a centrifuge tube. 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 invention aims to provide a method for preparing a drug-containing microplate by mixing a small amount of drug sample liquid with water agar using a small centrifuge tube.

[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 centrifuge tube preheating component B for mixing, a drug sample preheating component C, a gun tip preheating component D, and a slide preheating iron box component E. The mixing table is suitable for use on an ultra-clean workbench.

[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 centrifuge tube preheating component B for mixing samples mainly includes a centrifuge tube B1 and a centrifuge tube fixing rack B2, and the body of the centrifuge tube B1 is directly immersed in the water body A2. The drug sample preheating component C mainly includes a sample tube C1 and a sample tube rack C2, and 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, and the body of the gun tip box D1 is immersed in the water body A2. The slide preheating iron box component E is mainly composed of an ordinary iron box, the upper cover plate E1 of the iron box cover has a flat upper surface, the bottom plate E2 of the iron box body has perforations, and part of the iron box body 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 centrifuge tube as a mixing device for the drug and agar, use an iron box as a preheating plate for the slide, and prepare the drug-containing microplate in a technical system with a water body of 65°C. The preparation method steps are as follows:

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

[0011] 2) Prepare 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) Prepare the gun tip: Use a conventional gun tip with a volume of 200 μl, first cut off a small section of the tip, then put the gun tip with the tip cut off into a gun tip box in a conventional manner, sterilize and dry it, and then fix the gun tip box on the gun tip box clamp D2.

[0013] 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 small centrifuge tube, insert it into the centrifuge tube fixing rack B2, and then turn on the power of the sample mixing table, electrically heat and adjust the temperature of the water body A2 to a working state of 65°C, and maintain operation until the centrifuge tube B1, the gun tip in the gun tip box D1, and the upper cover E1 of the slide preheating iron box 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 upper cover plate E1 of the glass slide preheating iron box 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 centrifuge tube B1 in working state in step 4) to form a 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 segmented dots on 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) Microcentrifuge tubes are commonly used consumables in biological laboratories and are easy to use.

[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 centrifuge tube preheating component for mixing, C is the drug sample preheating component, D is the gun tip preheating component, and E is the slide preheating iron box 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 their 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 water surface of the temperature-controlled body. B is a centrifuge tube preheating component for sample mixing, B1 is a centrifuge tube, and B2 is a centrifuge tube fixing rack. 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 slide preheating iron box component, E1 is the upper cover of the iron box lid, and E2 is the bottom plate of the iron box body.

[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 medicine microplates in a centrifuge tube, wherein garlic tissue fluid is mixed with agar, and the mixture is drop-spread on a common glass slide to form 6 medicine 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 small centrifuge tube preheating component B for mixing, a drug sample preheating component C, a gun tip preheating component D, and a slide preheating iron box component E. Figure 1The entire 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 centrifuge tube preheating component B for mixing samples mainly includes a centrifuge tube B1 and a centrifuge tube fixing frame B2. Figure 3 As shown in Figure B. The centrifuge tube is preferably a 1.5ml small centrifuge tube; the centrifuge tube holder B2 floats on the surface of the temperature-controlled water body A2, and the tube body of the centrifuge tube B1 is buried in the water body A2, which 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 a tube hole 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 preheating iron box component E is mainly composed of an ordinary iron box and a water body A2 entering the iron box. The water body A2 enters the iron box through the perforation of the bottom plate E2 of the iron box body. The upper surface of the upper cover plate E1 of the iron box lid is a flat surface. The upper cover plate E1 is 3-4 cm away from the horizontal plane of the water body A2. Figure 3 As shown in Figure E. The 60°C water temperature is conducted through the cavity between the upper surface of the water body A2 and the upper cover E1, and the temperature reaching the surface of the upper cover can be reduced 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 small centrifuge tube is used as a mixing device for the drug and agar, an iron box is used as a preheating plate for the slide, and the drug-containing microplate is prepared in a technical system with a water body of 65°C. The preparation method steps are as follows:

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

[0039] 2. Prepare 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, it can be heated in boiling water when the microwave heating water agar is close to boiling 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. Prepare the gun tip: The preparation operation mainly uses a 200μ.l gun tip. Since the tip of the 200μl gun tip has a small aperture, the highly viscous water agar cannot pass smoothly. Therefore, use a cutting tool such as a blade to cut off about 5-8mm of the tip of the gun tip, thereby obtaining a larger aperture of the gun tip opening to ensure that the water agar can pass smoothly. The gun tip with the tip cut off is placed in a gun tip box for sterilization and drying in the conventional way, and then fixed on the gun 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 the sterilized centrifuge tube, insert it into the centrifuge tube fixing rack B2 of the mixing station, then turn on the power of the mixing station, electrically heat the water body A2 to a working state with a temperature of 65°C, and maintain this working state for more than 20 minutes, so that the centrifuge tube B1 and the gun tips in the gun tip box D1 are in equilibrium with the temperature of the water body A2 and are in working state; also keep the plate surface temperature of the slide preheating iron box E1 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 upper cover plate E1 of the slide preheating iron box 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 centrifuge tube B1 in the working state of step 4. The sample liquid is concentrated into a sample droplet at the bottom of the centrifuge tube.

[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. Aspirate all the mixed sample solution evenly mixed in step 9 into the pipette tip, transfer to the preheated slide in step 6, and discharge the mixed sample solution from the pipette gun onto the slide surface in segmented dots 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 small centrifuge tube as a sample mixing device, an ordinary iron box as a preheating plate for a glass slide, and the water body of the microplate sample mixing table is designed to be 65°C; the preheating of the sample mixing device, the preheating of the sample, the preheating of the gun tip, and the preheating of the glass slide are all organically integrated into a sample mixing table system. Such a thermal balance technology system can not only prevent the mixed sample liquid in the centrifuge tube 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 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.

[0051] Example 1

[0052] 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 centrifuge tube, 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 centrifuge tube preheating component for mixing (B), a drug sample preheating component (C), a gun tip preheating component (D), and a slide preheating iron box component (E). The 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 centrifuge tube preheating component (B) for mixing samples mainly comprises a centrifuge tube (B1) and a centrifuge tube fixing frame (B2), and the tube body of the centrifuge tube (B1) is directly immersed in the water body (A2); the drug sample preheating component (C) mainly comprises a sample tube (C1) and a sample tube frame (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 frame (D2), and the box body of the gun tip box (D1) is immersed in the water body (A2); the slide glass preheating iron box component (E) is mainly composed of an ordinary iron box, the upper cover plate (E1) of the iron box cover has a flat upper surface, the bottom plate (E2) of the iron box body has perforations, and part of the iron box body 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 centrifuge tube as a mixing device for drugs and agar, use an iron box 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) Prepare drug samples: transfer a small amount of drug samples obtained during drug development work into a sterilized sample tube (C1); 2) Prepare water agar for mixing: the water agar ratio for mixing is: 40g agar and 1000ml water, heat and melt, and maintain in a water bath above 90°C; 3) Prepare the gun tip: Use a conventional gun tip with a volume of 200 μl, first cut off a small section of the tip tip, then put the gun tip with the tip cut off into a gun tip box in a conventional manner, sterilize and dry it, and then fix the gun tip box on the gun tip box clamp (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 centrifuge tube, insert it into the centrifuge tube fixing 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 centrifuge tube (B1), the gun tip in the gun tip box (D1), and the upper cover plate (E1) of the slide preheating iron box are in a working state balanced with the 65°C water body 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 upper cover plate (E1) of the glass slide preheating iron box 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 centrifuge tube (B1) in working state in step 4) to form a 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 segmented dots on 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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