In-situ sediment microcell fidelity collection method and application thereof in uncultured microorganism research

Abstract

The invention discloses an in-situ sediment microcell fidelity collection method and application thereof in uncultured microorganism research. The handheld rod, the sampling thickness control accessory, the cutting blade and the fixing component are assembled into the portable collecting device, so that accurate sampling of in-situ sediment micro-area samples is realized; the problems that in-situ sediment collecting, transferring and flaking processes in the prior art are prone to physical disturbance, in-situ sediment electron transfer conditions cannot be kept, and the original state of microorganisms is difficult to maintain are solved. By utilizing the device disclosed by the invention, a required amount of in-situ sediment sample can be accurately collected, and the obtained sediment micro-area sample can be transported and flaked without disturbance, so that the morphological behavior research and physiological status observation of uncultured microorganisms, particularly long-line microorganisms, are facilitated. The method provides key technical support for researching in-situ physiological functions of the microorganisms, and plays an important role in scientific research work for researching physiological ecology of the uncultured microorganisms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of environmental microbiology and microbial ecology, and particularly relates to a method for in-situ sediment micro-area faithful collection and application thereof in uncultured microbial research. BACKGROUND

[0002] In the field of microbiology, traditional culture techniques face major limitations, and can only touch 0.1%-1.0% of the total microbial population in the environment, meaning that the vast majority of microbial resources are still unknown and have not been fully explored. Although advanced technologies such as high-throughput sequencing have greatly advanced our understanding of microbial diversity and genomic information, how to visually and dynamically observe the behavior and activity patterns of these microorganisms in natural environments remains a major challenge in current research. Currently, a small amount of sediment sample is inoculated onto a glass slide with a specific groove (such as a groove with a length of 20 mm, a width of 10 mm, and a thickness of 1 mm), and microscopic observation of uncultured microorganisms that can be transferred to the gap between the cover glass and the glass slide is achieved. However, in the implementation process, a small amount of sediment needs to be laid flat in the groove, and this laying process can cause physical disturbance to the in-situ sediment, especially for long linear microorganisms that are structurally fragile and easily broken by external interference. This disturbance seriously affects the accurate observation of the natural state and physiological behavior of microorganisms. The current sampling method is mainly grab sampling and column sampling, and these sampling devices are large in size and not easy to carry. In multi-point sampling, the sampling device needs to be reused, and in the process of sediment transfer and transportation, disturbance is likely to occur, and the collected sediment cannot maintain the in-situ electron transfer conditions. Therefore, a method is needed that is low in cost, easy to operate, easy to carry, and can achieve non-disturbance collection of in-situ sediment. SUMMARY

[0003] The present application provides a method for in-situ sediment micro-area faithful collection and application thereof in uncultured microbial research. By assembling a handheld rod, a sampling thickness control accessory, a cutting piece, and a fixing component into a portable collection device, precise sampling, non-disturbance fixation, and transportation and preservation of in-situ sediment micro-area samples are achieved, solving the problems of physical disturbance, difficulty in maintaining in-situ electron transfer conditions, and easy contamination in the process of in-situ sediment collection, transfer, and slide preparation in the prior art.

[0004] The first object of the present application is to provide an in-situ sediment micro-area fidelity acquisition device, comprising a hand-held rod, a sampling thickness control accessory, cutting pieces and a fixing component; the sampling thickness control accessory comprises a baffle and a positioning rod passing through the baffle, one end of the positioning rod is a thin piece, the thin piece is clamped between two cutting pieces; the fixing component is arranged outside the two cutting pieces and tightly fixes the two cutting pieces and the thin piece to form a micro-area sample fidelity bin; the micro-area sample fidelity bin is detachably connected with the hand-held rod through the positioning rod.

[0005] The sampling thickness control accessory is used to limit the sampling thickness of the sediment micro-area sample, and the micro-area sample fidelity bin is used to clamp and maintain the in-situ structural characteristics of the sediment micro-area sample.

[0006] Preferably, the fixing component comprises but is not limited to a magnetic fixing component, and can also be other structures capable of realizing detachable fixation, which is used to detachably fix the cutting pieces and the sampling thickness control accessory. Preferably, one end of the hand-held rod has a threaded hole, the other end of the positioning rod is provided with an external thread capable of being screwed into the threaded hole, and the positioning rod and the hand-held rod are detachably connected by screwing the external thread into the threaded hole.

[0007] Preferably, the size of the cutting piece is greater than the corresponding size of the sampling thickness control accessory, so that a limited space is formed inside the micro-area sample fidelity bin, thereby regulating the amount of oxygen entering during storage or transportation.

[0008] Preferably, the material of the cutting piece is transparent glass or transparent plastic.

[0009] The second object of the present application is to provide an in-situ sediment micro-area fidelity acquisition method, which is used for microscopic observation of uncultured microorganisms in sediments, and adopts the above-mentioned in-situ sediment micro-area fidelity acquisition device, and specifically comprises the following steps:

[0010] 1) In-situ sampling: inserting the micro-area sample fidelity bin into the sediment and lifting it to obtain a sediment micro-area sample;

[0011] 2) Sample processing: sealing and storing the micro-area sample fidelity bin containing the sediment micro-area sample or directly performing subsequent slicing; the sealing and storing is: sealing the side and bottom of the micro-area sample fidelity bin with in-situ sediment, and covering the collected in-situ water on the upper part of the micro-area sample fidelity bin to maintain the in-situ electron transfer condition of the sample;

[0012] 3) Slicing observation: transferring the sediment micro-area sample from the micro-area sample fidelity bin to a glass slide, performing cover slipping and microscopic observation.

[0013] During in-situ sampling, the sediment micro-area sample is held in the micro-area sample preservation chamber by the cutting blade. After sampling, the micro-area sample preservation chamber is removed from the handheld rod and sealed as needed to maintain the in-situ characteristics of the sediment micro-area sample during preservation and transportation. If transportation is not required, slide preparation can be carried out directly. When preparing the slide for observation, first open the micro-area sample preservation chamber, cut and transfer the sediment micro-area sample into the groove of the glass slide, then seal the glass slide, and observe the microorganisms in the sediment micro-area sample under a microscope.

[0014] Preferably, in step 1), when sampling does not require a handheld rod, an iron sheet or a magnetic sheet with the same thickness as the groove of the glass slide is used instead of the thickness control accessory.

[0015] Preferably, in step 1), the sampling thickness matches the depth of the groove in the glass slide.

[0016] Preferably, in step 1), the micro-area sample preservation chamber is quickly inserted into the sediment. When the sediment has a high sand content, the micro-area sample preservation chamber can be tilted appropriately and kept approximately horizontal to reduce disturbance to the in-situ sediment structure.

[0017] Preferably, in step 2), the micro-area sample preservation chamber is selectively sealed according to the redox state of the sediment micro-area sample to maintain the in-situ environmental conditions of the sample: when the sediment is in an anaerobic state, the micro-area sample preservation chamber is completely sealed; when the sediment surface is exposed to air, the sides and bottom can be sealed while the top remains open.

[0018] Preferably, in step 2), the chemical conditions of the collected in-situ water are maintained by connecting external aeration or circulation equipment. The chemical conditions include dissolved oxygen, nitrate concentration, sulfate concentration, etc.

[0019] Preferably, in step 3), petrolatum or polydimethylsiloxane is used as a sealing material for sealing the slide. Petrolatum sealing prevents oxygen from penetrating (between the slide and coverslip). Polydimethylsiloxane is a breathable but waterproof adhesive that allows oxygen to enter, creating an oxygen gradient that attracts aerobic microorganisms.

[0020] Preferably, in step 3), if it is necessary to maintain anaerobic conditions, the slide preparation and sealing process can be completed in an anaerobic chamber.

[0021] Preferably, in step 3), the microscopic observation refers to observing the microorganisms that have moved to the gap between the slide and the coverslip outside the groove.

[0022] The present invention has the following advantages:

[0023] 1. This invention provides an in-situ sediment micro-area authentic sampling device. The device includes a handheld rod, a sampling thickness control accessory, a cutting blade, and a fixing component. By assembling the sampling thickness control accessory, the cutting blade, and the fixing component to form a micro-area sample authentication chamber, precise acquisition and fixation of sediment micro-area samples are achieved, significantly reducing disturbance to the in-situ structure during sampling and transfer. The micro-area sample authentication chamber combines collection and preservation functions, reducing the number of sample transfers, lowering the risk of contamination, and facilitating the maintenance of in-situ electron transfer conditions. The in-situ sediment micro-area authentic sampling device is simple in structure, low in cost, easy to operate, and portable. It is suitable for multi-point sampling in the field and laboratory microscopic observation, and has broad application prospects in the fields of environmental microbiology and uncultured microorganism research.

[0024] 2. The device of this invention enables precise collection of the required amount of in-situ sediment samples. The obtained sediment micro-region samples can be transported and prepared without disturbance, preserving their original structural characteristics. Direct transfer of the sediment micro-region samples to the grooves of a glass slide for microscopic observation is beneficial for the study of the morphology and behavior of uncultured microorganisms, especially filamentous microorganisms, and for observing their physiological state. Furthermore, the oxygen conditions of the collected sediment micro-region samples can be adjusted as needed.

[0025] 3. The device of the present invention uses low-cost materials and can be used as a disposable sampling tool. Compared with the sampling tools in the prior art that need to be reused and are cumbersome to maintain, it ensures the independence and purity of each sample collected, eliminates cross-contamination between samples, and significantly improves the efficiency of field sampling.

[0026] 4. This invention simulates and maintains the in-situ biochemical environment of sediments in a laboratory setting. Specifically, by sealing the sides and bottom of the in-situ sediment micro-sample fidelity chamber and sealing the top with in-situ water, the original structural characteristics of the sediments are maintained to the greatest extent. This high fidelity is beneficial for maintaining the electron transport activity of uncultured microorganisms, providing key technical support for studying their in-situ physiological functions, and playing an important role in scientific research on the physiological ecology of uncultured microorganisms. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the in-situ sediment micro-area fidelity acquisition device of the present invention, wherein, Figure 1 A represents the various components of the data acquisition device; Figure 1 B is a top view of the micro-area sample fidelity chamber used in this invention, which is assembled from a sampling thickness control accessory, a cutting blade, and a magnetic fixing component. From bottom to top, the top view shows one magnetic fixing component, one cutting blade, one sampling thickness control accessory, one cutting blade, and one magnetic fixing component.

[0028] Figure 2This is a schematic diagram of a glass slide with grooves.

[0029] Figure 3 This is a schematic diagram of uncultured microorganisms observed under a dark-field microscope in Example 2.

[0030] Explanation of reference numerals in the attached drawings: 1. Handheld rod; 2. Sampling thickness control accessory; 3. Cutting disc; 4. Magnetic fixing component; 2.1. Baffle; 2.2. Positioning rod; 2.2.1. Thin sheet. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0032] Example 1: In-situ Sediment Micro-area Fidelity Acquisition Device

[0033] This invention discloses an in-situ sediment micro-area fidelity acquisition device, such as... Figure 1 As shown in Figure A, the sampling device includes a handheld rod 1, a sampling thickness control accessory 2, a cutting disc 3 (two glass plates), and a magnetic fixing component 4 (two magnet plates). One end of the handheld rod 1 has a screw hole with a diameter of 2.5 mm, and the handheld rod 1 is 1 m long. The sampling thickness control accessory 2 includes a baffle 2.1 and a positioning rod 2.2 passing through the baffle. One end of the positioning rod 2.2 has an external thread for connecting to the handheld rod 1, with a thread diameter of 2.5 mm. The other end of the positioning rod 2.2 is fixed with a rectangular thin plate 2.2.1, which is 1 mm thick, 7.5 mm long, and 4.5 mm wide. The cutting disc 3 is a glass plate with a length of 60 mm, a width of 24 mm, and a thickness of 0.15 mm. The magnetic fixing component 4 is a magnet plate with a length of 24 mm, a width of 7.5 mm, and a thickness of 1 mm.

[0034] Assembly and fixation of the in-situ sediment micro-area fidelity acquisition device:

[0035] 1. Assembly of the micro-area sample fidelity chamber: such as Figure 1 As shown in B (top view of the micro-area sample fidelity chamber), the five components are stacked in the following order: magnet sheet, cutting disc, thin sheet, cutting disc, and magnet sheet. Specifically, a magnet sheet is placed horizontally, a cutting disc is aligned and placed flat on the surface of the magnet sheet, the thin sheet of the sampling thickness control accessory is placed in the center of the cutting disc, another cutting disc is aligned and placed over the thin sheet, and finally, another magnet sheet is placed on top of the cutting disc. The two magnet sheets generate magnetic force to press the "cutting disc-thin sheet-cutting disc" in the middle together, assembling the micro-area sample fidelity chamber.

[0036] 2. Assembling the sampling device: Tighten the screw hole of the handheld rod to the external thread of the positioning rod in the sampling thickness control accessory to construct an in-situ sediment micro-area fidelity sampling device. The thickness control accessory and the handheld rod are detachably connected.

[0037] This device is simple to install, easy to carry, suitable for in-situ micro-area sampling of sediments, and facilitates subsequent observation of uncultured microorganisms on grooved glass slides.

[0038] Example 2: Observation of Uncultured Microorganisms in Sediments

[0039] A method for in-situ sediment micro-area fidelity acquisition, using the in-situ sediment micro-area fidelity acquisition device of Example 1, includes the following steps:

[0040] Step 1 Sampling: Insert the micro-area sample preservation chamber into the sediment, lift it to obtain an in-situ micro-area sample of the sediment. The sediment micro-area sample is retained in the micro-area sample preservation chamber by the clamping action of the cutting disc. Note that when the sediment collection does not require a handheld rod, an iron sheet or magnetic sheet of the same thickness as the groove can be used instead of a thickness control device.

[0041] Step 2: Sealing and preservation: Seal the sides and bottom of the micro-area sample preservation chamber with in-situ sediment, and cover the top of the micro-area sample preservation chamber with collected in-situ water to maintain the in-situ electron transfer conditions of the sample.

[0042] Step 3: Slide Preparation: Place the micro-area sample fidelity chamber horizontally, remove the magnetic retainer and upper cutting blade, and cut the deposit to the size of the groove on the slide to be observed. Transfer the deposit area into the groove on the slide. Note that the micro-area sample fidelity chamber should be placed in a non-ferrous tray to prevent magnet and iron attraction, which would make the transfer operation difficult and cause in-situ sample disturbance.

[0043] Step 4: Cover with a coverslip and seal with polydimethylsiloxane. Note that this example uses a breathable adhesive for sealing.

[0044] Step 5: Observe the environmental microorganisms in the sediment using a dark-field microscope.

[0045] Experimental results: Figure 3 This is a diagram showing the effect of observing uncultured microorganisms using the above method.

Claims

1. A device for in-situ sediment micro-area high-fidelity data acquisition, characterized in that, The device includes a handheld lever, a sampling thickness control accessory, cutting blades, and a fixing component. The sampling thickness control accessory includes a baffle plate and a positioning rod passing through the baffle plate. One end of the positioning rod is a thin sheet, which is sandwiched between two cutting blades. The fixing component is placed outside the two cutting blades and presses the two cutting blades and the thin sheet together to form a micro-area sample fidelity chamber. The micro-area sample fidelity chamber is detachably connected to the handheld lever via the positioning rod.

2. The data acquisition device according to claim 1, characterized in that, The fixing component is a magnetic fixing component, preferably a magnet sheet.

3. The data acquisition device according to claim 1, characterized in that, The size of the cutting disc is larger than the corresponding size of the sampling thickness control accessory; preferably, the material of the cutting disc is transparent glass or transparent plastic; preferably, one end of the handheld rod has a screw hole, and the other end of the positioning rod has an external thread that can be screwed into the screw hole, so that the positioning rod and the handheld rod can be detachably connected by screwing the external thread into the screw hole.

4. A method for in-situ micro-area high-fidelity collection of sediments, characterized in that, The in-situ sediment micro-area fidelity acquisition device according to any one of claims 1-3 specifically includes the following steps: 1) In-situ sampling: Insert the micro-area sample preservation chamber into the sediment and lift it up to obtain micro-area samples of the sediment; 2) Sample processing: The micro-area sample fidelity chamber containing sediment micro-area samples is sealed and preserved or directly used for subsequent slide preparation; the sealing and preservation is: the sides and bottom of the micro-area sample fidelity chamber are sealed with in-situ sediment, and in-situ water is collected and covered on the top of the micro-area sample fidelity chamber to maintain the in-situ electron transfer conditions of the sample. 3) Slide preparation and observation: Transfer the sediment micro-area sample from the micro-area sample preservation chamber to a glass slide, seal it, and observe it under a microscope.

5. The method according to claim 4, characterized in that, In step 1), when sampling does not require a handheld rod, an iron sheet or a magnetic sheet with the same thickness as the groove of the glass slide is used instead of the thickness control accessory.

6. The method according to claim 4, characterized in that, In step 1), the sampling thickness is matched with the depth of the groove in the glass slide.

7. The method according to claim 4, characterized in that, In step 1), when the sediment has a high sand content, the micro-area sample preservation chamber is tilted and kept approximately horizontal overall when extracting the micro-area sample.

8. The method according to claim 4, characterized in that, In step 2), the micro-area sample preservation chamber is selectively sealed according to the redox state of the sediment micro-area sample.

9. The method according to claim 4, characterized in that, In step 2), the chemical conditions of the collected in-situ water are maintained by connecting external aeration or circulation equipment. Preferably, the chemical conditions include dissolved oxygen, nitrate concentration, and sulfate concentration.

10. The method according to claim 4, characterized in that, In step 3), petrolatum or polydimethylsiloxane is used as a sealing material for sealing; anaerobic conditions are maintained by preparing and sealing the sheets inside the anaerobic chamber.

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