Separation method based on single spore localization

Abstract

The invention relates to the technical field of microorganisms, in particular to a separation method based on single spore localization. The method comprises the following steps: S1, disinfecting and sterilizing used instruments; s2, single spore selection is carried out on the spores; and S3, guiding the agar blocks to be accurately cut and transferred. According to the separation method based on single spore localization, a sterile water dilution method is adopted, a low-power monospore localization technology is combined, agar block cutting is carried out through light-through hole light source observation, and the single spore separation process is remarkably simplified.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a separation method based on single spore positioning. Background Art

[0002] Pure cultures obtained from single spore culture are the basis for fungal isolation and identification as well as basic physiological and biochemical research. Therefore, the isolation of single fungal spores is particularly important. Currently, there are many methods for single spore isolation, such as fixed insect pins, eyebrow pins, glass capillaries, and handmade glass pins.

[0003] To date, although many methods have been developed for the isolation of single spores, they all have certain limitations in operation, application, and preservation. In particular, traditional methods for the isolation of single fungal spores are complicated (especially for small spores such as anthrax and stem fungi) and have low separation efficiency. Traditional methods (such as dilution purification, spore picking on agar plates, glass capillary separation, dried spore separation, and micromanipulator separation) require expensive equipment or multiple purification steps.

[0004] To this end, a separation method based on single spore positioning was designed. By optimizing the spore suspension preparation process and simplifying the spore separation procedure on the plate, rapid and efficient single spore separation was achieved, providing a technical solution to the above technical problems. Summary of the Invention

[0005] Based on this, it is necessary to provide a separation method based on single spore positioning to solve the technical problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A separation method based on single spore localization, the steps are as follows:

[0008] S1: Disinfect and sterilize the instruments used;

[0009] S2: single spore picking;

[0010] S3: Guide the agar block for precise cutting and transfer.

[0011] As a preferred embodiment of the single spore localization isolation method provided by the present invention, in step S1, the environmental disinfection and sterilization steps are as follows:

[0012] When entering the laboratory, turn on the ultraviolet sterilization half an hour in advance;

[0013] Clean and disinfect the workbench surface with alcohol and alcohol cotton balls;

[0014] Use alcohol cotton balls to wipe the surface of the microscope to disinfect and sterilize;

[0015] Use an alcohol lamp to heat the needle repeatedly until it turns red, and then use it after cooling.

[0016] As a preferred embodiment of the isolation method based on single spore localization provided by the present invention, in step S2, the steps are as follows:

[0017] 1) Take a sterile bottle filled with 50 ml of sterile water, use a cooled needle to pick up a small amount of spores and place them into the sterile water bottle, gently shake to mix thoroughly with the water to prepare a spore suspension;

[0018] 2) Dilute the spore suspension with sterile water and observe under a 400x microscope to ensure that the number of spores per field of view is less than 50;

[0019] 3) Use an inoculating loop to smear the spores onto the surface of a water agar plate, being careful not to damage the agar surface. Place the plate near an alcohol lamp and wait for the water to evaporate, but be careful not to place it too close to the alcohol lamp to avoid inactivation of spores due to high temperatures.

[0020] 4) Place the dried culture medium under a microscope to look for spores;

[0021] 5) Find the spores under a microscope and determine the number (1).

[0022] As a preferred embodiment of the single spore localization separation method provided by the present invention, in step 3), after fusion, the number of spores is checked on a glass slide to see if the number of spores per field of view is less than 50 under a 400x microscope. If the number of spores is large, sterile water is added; if the number of spores is small, spore suspension is added.

[0023] As a preferred embodiment of the single spore localization-based separation method provided by the present invention, in step S3, the steps are as follows:

[0024] A. Place a glass slide on the microscope stage. Place the cut agar block on the slide. Observe the slide and reconfirm the spore count.

[0025] B. Observe the spores under a microscope to determine their location. Use light to guide the precise cutting and transfer of the agar block. Align the illuminated area of ​​the light hole and use a sterilized scalpel to cut the corresponding agar block.

[0026] C. Place the agar block obtained in step B into the PDA medium (spore side up).

[0027] As a preferred embodiment of the single spore localization-based separation method provided by the present invention, the PDA culture medium obtained in step C is labeled, sealed, bagged, and placed in an incubator.

[0028] It can be seen without a doubt that the above-mentioned technical solution of this application can definitely solve the technical problem to be solved by this application.

[0029] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0030] 1. The present invention provides a separation method based on single spore localization, which adopts the sterile water dilution method, combined with the single spore localization technology under a high-power microscope, and agar block cutting by observation through a light source of a light hole. This significantly simplifies the single spore separation process, thereby shortening the operation time by 90% and reducing the requirements for hand stability. It is particularly suitable for the efficient separation of microspore fungi such as Anthrax and Phoma.

[0031] 2. The present invention achieves rapid and efficient single spore separation by optimizing the spore suspension preparation process and simplifying the spore separation procedure on the plate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a schematic diagram of the mycelium of the present invention entering a sterile water bottle;

[0034] Figure 2 A schematic diagram of searching for spores under a microscope according to the present invention;

[0035] Figure 3 A schematic diagram of finding the number of spores under a microscope of the present invention;

[0036] Figure 4 Schematic diagram of the agar block cut out of the present invention on a glass slide;

[0037] Figure 5 A schematic diagram of a scalpel of the present invention cutting an agar block;

[0038] Figure 6 This is a schematic diagram of spores after cutting in an agar block on a microscope of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0041] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions therein may be combined with each other.

[0042] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0043] Reference Figures 1-6 , a separation method based on single spore localization, the steps are as follows:

[0044] Step 1: Disinfection and sterilization of personnel and experimental equipment, the steps are as follows:

[0045] When entering the laboratory, the ultraviolet sterilization should be turned on half an hour in advance. Personnel entering the laboratory should clean and disinfect themselves and wear laboratory clothes;

[0046] Clean and disinfect the workbench surface with alcohol and alcohol cotton balls;

[0047] Use an alcohol cotton ball to wipe the surface of the microscope for disinfection and sterilization, but do not wipe the mirror surface and light hole;

[0048] Use an alcohol lamp to heat the needle until it turns red (3 times) and use it after it cools down.

[0049] At the same time, after the experiment, the table surface needs to be wiped with 75% ethanol, and antibiotics (such as chloramphenicol 50 μg / mL) should be added to the culture medium to reduce the risk of contamination.

[0050] Step 2: Single spore picking of spores, the steps are as follows:

[0051] 1) Take a spore water bottle (pre-filled with 50ml of sterile water), use a cooled needle to pick the mycelium from the spore-forming area and place it in the sterile water bottle;

[0052] In this example, if the spores do not germinate, the culture medium composition is optimized or a germination inducer (such as 0.1% glucose) is added.

[0053] 2) Gently shake the sterile water bottle to fully integrate the mycelium, spores, and water. Use sterile water to gradually dilute the spore suspension. Observe under a 400x microscope to ensure that the number of spores per field of view is within 50. If the number of spores is high, add more sterile water; if the number of spores is low, add more spores.

[0054] 3) Use an inoculating loop to smear the solution onto the surface of the water agar plate, taking care not to damage the agar surface;

[0055] 4) Place the culture medium near an alcohol lamp to use the high temperature of the alcohol to quickly evaporate the water. At the same time, pay attention to the distance between the water culture medium and the alcohol lamp to prevent the spores from being burned to death. Therefore, confirm the distance specifically during implementation.

[0056] 5) Place the clean water culture medium under a microscope to look for spores. If there are too many spores, reduce the amount of spore water; if there are too few, add one or two drops of spore water.

[0057] 6) Find the spores under a microscope and determine their number. Ideally, one or two spores should be found, with some distance between them to facilitate cutting.

[0058] 7) The microscope aperture is cut into squares; at the same time, if the objective lens is too close, the stage can be lowered. This operation should be done with caution and without excessive shaking.

[0059] Step 3: Guide the agar block to accurately cut and transfer. The steps are as follows:

[0060] A. Place a glass slide on the microscope stage. Place the cut agar block on the slide. Observe the slide and recheck the spore count. Find a field of view with only one spore at 400x magnification.

[0061] B Observe the spores under a microscope to determine their location, and use light to guide the precise cutting and transfer of the agar block. That is, align the light-irradiated area of ​​the light hole and use a sterilized scalpel to cut the corresponding agar block.

[0062] If there is only one spore in the agar block under a microscope, simply trim the edge (the spore is usually the center when cutting a single spore). If there are two spores in the agar block, observe it more carefully to determine the center line, and then use a spore cutter to divide it into two parts.

[0063] C. Place the square agar block obtained in step B in the PDA culture medium (spore side facing up), label it, seal it with film, bag it, and place it in an incubator.

[0064] In this embodiment, the clear water culture medium and the PDA culture medium are both existing materials prepared in advance by those skilled in the art, and their preparation is a common technique used by those skilled in the art.

[0065] In this embodiment, during the operation, ensure that the entire process is completed in a clean bench to avoid contamination by miscellaneous bacteria. At the same time, attention should be paid to the spore concentration. If it is too high, it will lead to overlapping colonies, and if it is too low, the efficiency will be low. Before the operation, practice micromanipulation in advance to avoid damaging the spores or inhaling multiple spores.

[0066] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A separation method based on single spore localization, characterized in that, Here are the steps: S1: Disinfect and sterilize the instruments used; S2: single spore picking; S3: Guide the agar block for precise cutting and transfer.

2. A separation method based on single spore positioning according to claim 1, characterized in that, In step S1, the environmental disinfection and sterilization steps are as follows: When entering the laboratory, turn on the ultraviolet sterilization half an hour in advance; Clean and disinfect the workbench surface with alcohol and alcohol cotton balls; Use alcohol cotton balls to wipe the surface of the microscope to disinfect and sterilize; Use an alcohol lamp to heat the needle repeatedly until it turns red, and then use it after cooling.

3. A separation method based on single spore positioning according to claim 1, characterized in that, In step S2, the steps are as follows: 1) Take a sterile bottle filled with 50 ml of sterile water, use a cooled needle to pick up a small amount of spores and place them into the sterile water bottle, gently shake and mix thoroughly with the water to prepare a spore suspension; 2) Dilute the spore suspension with sterile water and observe under a 400x microscope to ensure that the number of spores per field of view is less than 50; 3) Use an inoculating loop to smear the solution onto the surface of a water agar plate and place it near an alcohol lamp to wait for the water to evaporate. 4) Place the dried culture medium under a microscope to look for spores; 5) Locate and count the spores under a microscope.

4. A separation method based on single spore positioning according to claim 3, characterized in that, In step 3), after fusion, check the number of spores on the slide to see if the number of spores per field of view is less than 50 under a 400x microscope. If the number of spores is large, add sterile water; if the number of spores is small, add spore suspension.

5. A separation method based on single spore positioning according to claim 1, characterized in that, In step S3, the steps are as follows: A. Place a glass slide on the microscope stage. Place the cut agar block on the slide. Observe the slide and reconfirm the spore count. B. Observe the spores under a microscope to determine their location. Use light to guide the precise cutting and transfer of the agar block. Align the illuminated area of ​​the light hole and use a sterilized scalpel to cut the corresponding agar block. C. Place the agar block obtained in step B into the PDA medium with the spore side facing up.

6. A separation method based on single spore positioning according to claim 5, characterized in that, The PDA culture medium obtained in step C is labeled, sealed, bagged, and placed in an incubator.

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