A method for preparing a sapwood section of a spruce wood trunk
By softening the wood in ethylenediamine solution and then slicing it using a paraffin cryostat, the problem of poor softening effect in sapwood slices of spruce trunk xylem was solved, achieving rapid and efficient slice preparation and improving slice efficiency and quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG UNIVERSITY
- Filing Date
- 2026-03-05
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies suffer from poor softening effects and long preparation times when preparing sapwood sections of spruce trunk xylem, which affects the efficiency of rapidly preparing anatomical sections of spruce trunk xylem.
After fixation with a fixative, the sections were softened in ethylenediamine solution for 5-6 days, then sectioned using a paraffin cryostat. The sections were bleached with sodium hypochlorite solution, stained with safranin, and cleared with a mixture of ethanol and xylene. Finally, they were mounted with neutral resin and photographed under an optical microscope.
This technology enables rapid and efficient preparation of sapwood slices from the xylem of spruce, shortening the softening time from one month to 5-6 days, and improving the efficiency and quality of the slices.
Smart Images
Figure CN122108708A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of testing technology, specifically to a method for preparing sapwood slices of spruce wood. Background Technology
[0002] The xylem of a tree trunk is a major component of the transport system in plant tissues, serving as the primary pathway for water transport from the roots to the above-ground parts. The xylem consists of heartwood and sapwood. Heartwood, due to the deposition of large amounts of resin, gum, and pigments in the cavity walls, hinders water transport and primarily serves a mechanical support function; while sapwood is the main site of water transport. By studying the anatomical characteristics of xylem and sapwood and their role in water transport, the anatomical structure of xylem and sapwood can be linked to photosynthesis and transpiration, providing a basis for research on plant responses to global climate change. However, in preparing anatomical sections of xylem and sapwood, the high hardness of the xylem in the species *Picea spp.* results in poor softening effects and long preparation times in conventional procedures, thus affecting the rapid preparation of anatomical sections. Therefore, how to prepare xylem and sapwood sections more quickly and efficiently, and accurately measure their anatomical characteristics, is an urgent problem to be solved. Summary of the Invention
[0003] To address the shortcomings of the existing technology, this invention aims to provide a method for preparing sapwood slices of spruce wood, so as to prepare sapwood slices of spruce wood more quickly and efficiently.
[0004] To solve the above problems, the present invention adopts the following technical solution: A method for preparing sapwood slices of spruce wood includes: trimming a spruce wood sample and then fixing it in a fixative solution; The fixed sapwood samples of spruce xylem were softened in ethylenediamine solution for 5-6 days, and then sliced after softening. The sapwood samples of the xylem of the spruce were bleached, rinsed, stained, and eluted. Transparent sections of sapwood samples from the xylem of Picea spp. were prepared using a mixture of ethanol and xylene. Slices of the sapwood sample from the xylem of the spruce were sealed and photographed.
[0005] Furthermore, the fixative consists of 89 parts by volume of 70% ethanol, 6 parts by volume of glacial acetic acid, and 5 parts by volume of formalin; the fixation time is 24 hours.
[0006] Furthermore, the sapwood sample of the spruce wood is trimmed so that the thickness of the spruce wood sample is 0.5±0.2cm.
[0007] Furthermore, the concentration of the ethylenediamine solution is 10%.
[0008] Furthermore, the sections were prepared using a paraffin cryostat, and the thickness of the sections was 20~35 μm.
[0009] Furthermore, the bleaching process uses a 10% sodium hypochlorite solution for bleaching for 10 ± 2 minutes.
[0010] Furthermore, the staining was performed using a 1% safranin staining solution for 1 hour.
[0011] Furthermore, the elution is performed by gradient elution of safranin-stained sections with ethanol aqueous solutions of 30%, 45%, 60%, 70%, and 100% by mass percentage, with each gradient elution time being 3 ± 1 min.
[0012] Furthermore, the volume ratio of ethanol to xylene in the ethanol and xylene mixed solution is 1:1; and the time for achieving transparency is 5 ± 1 min.
[0013] Furthermore, the sealing agent is a neutral resin.
[0014] The beneficial effects of this invention are as follows: This invention provides a method for preparing sapwood slices of spruce wood, enabling rapid and efficient preparation of sapwood slices of spruce wood; it adds a sample trimming step, increasing the contact area between the sample and FAA solution and ethylenediamine solution, thus accelerating the sample fixation and softening speed; the softening time is shortened by using a 10% concentration ethylenediamine solution, from one month to 5-6 days. Attached Figure Description
[0015] Figure 1 An example image of a sapwood slice of spruce wood produced according to the method for producing sapwood slices of spruce wood according to the present invention. Detailed Implementation
[0016] The present invention will be further described in detail below with reference to specific embodiments.
[0017] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.
[0018] The process of preparing sapwood slices from the xylem of spruce mainly includes sapwood fixation, softening, slicing, bleaching, staining, clearing, sealing, and photography. The specific steps are as follows: (1) Wood fixation: Samples collected in the field should be sectioned as soon as possible to avoid moisture loss from the wood affecting the anatomical structure. Before sectioning, the sample should be trimmed with a scalpel to a thickness of about 0.5 cm to speed up subsequent softening steps. The sample should then be fixed in formalin-acetic acid-alcohol fixative (FAA) for 24 h. 100 ml of fixative can be prepared by the following ratio: 89 ml of 70% alcohol + 6 ml of glacial acetic acid + 5 ml of formalin (37%~40% formaldehyde).
[0019] Compared to traditional slide preparation methods, this step adds a sample trimming step: the purpose of which is to increase the contact area between the sample and the FAA solution and ethylenediamine solution, thereby accelerating the sample fixation and softening process.
[0020] (2) Softening: The wood was softened using the ethylenediamine softening method. The wood sapwood sample was placed in a 10% ethylenediamine solution for 5-6 days to soften. Pre-slicing began on the 5th day. When the sample was appropriately softened, softening was stopped and slicing was performed. If the sample was too hard, softening was continued, and slicing was performed again on the 6th day. It should be noted that the ethylenediamine solution contains ammonia gas, so it must be prepared and used immediately (operated in a fume hood). It is not advisable to prepare too much, otherwise the concentration of the ethylenediamine solution will decrease as the ammonia gas evaporates, resulting in a reduced softening effect on the wood sample.
[0021] Compared to traditional slide preparation methods, this step utilizes a freshly prepared 10% ethylenediamine solution. Compared to a 70% ethanol:propylene glycol (1:1) mixture, glycerol (glycerol) solution, or high-temperature softening, the softening time is significantly reduced, and the experimental procedure is safer. The time is shortened from one month to 5-6 days. The added pre-slicing step also avoids excessive softening of the samples.
[0022] When the concentration of ethylamine solution is greater than 10%, it will cause uneven softening of wood samples, surface segregation, and easy breakage of slices. When the concentration of ethylamine solution is less than 10%, the softening effect of wood samples is reduced, resulting in a relatively longer softening time.
[0023] (3) Sectioning: The wood samples were sectioned using a paraffin cryostat. After removing the samples from the softening solution, they were first washed with distilled water. Then, the sample blocks were trimmed again with a scalpel blade to make the cross-sections parallel, facilitating parallel fixation of the samples on the microtome. The paraffin cryostat was then adjusted to a thickness of 20-35 μm according to the hardness characteristics of the wood. After the samples were fixed, the cross-sections were trimmed again, making the cuts as perpendicular to the longitudinal axis as possible. The orientation could be judged by the wood grain on the core cross-section. If the orientation was inaccurate, the section could not be used. The correctly oriented and complete wood slices were placed in a petri dish containing a 50% ethanol aqueous solution, awaiting subsequent steps.
[0024] Compared to traditional slide preparation methods, this step utilizes a paraffin cryostat for slicing, which is relatively inexpensive and more suitable for laboratories with limited equipment. Cryo-embedding offers a short embedding time, and the sample blocks can be preserved after thawing for further processing such as preservation and softening. It also allows for initial selection of the slices, meeting subsequent slide preparation needs while reducing workload.
[0025] (4) Bleaching: Use 10% sodium hypochlorite solution for bleaching. The bleaching time should not be too long, about 10 minutes. When the slices turn white, take them out and rinse them with distilled water.
[0026] Compared to traditional slide preparation methods, this step uses a 10% sodium hypochlorite solution instead of 20% for bleaching, which better avoids over-bleaching of the slides and allows for more sufficient reaction time. Rinsing with distilled water afterwards prevents the sodium hypochlorite solution from damaging the safranin staining solution.
[0027] (5) Staining: a) Stain wood slices with 1% safranin staining solution (1 g safranin mixed with 100 mL 50% alcohol solution filtrate) for 1 hour to obtain safranin-stained slices; b) The safranin-stained sections were sequentially eluted with ethanol aqueous solutions of 30%, 45%, 60%, 70%, and 100% by mass percentage, with each step lasting about 3 minutes (too long a time will destroy the staining effect), to obtain destained sections. Compared to traditional slide preparation methods, this step involves gradient elution of safranin-stained sections using five gradient ethanol-water solutions, with each step requiring only about 3 minutes. Firstly, sufficient gradient elution cleanses the red dye outside the tracheids without damaging the dye inside. Secondly, the 3-minute elution time for each gradient avoids over-elution of the sections, which could compromise the staining effect. Thirdly, the 100% ethanol-water solution not only performs elution but also provides preliminary dehydration to the sections.
[0028] (6) Clearing: Xylene is used as a clearing agent to make the sections transparent. First, soak the sections in a 1:1 ethanol-xylene solution for 5 minutes to dehydrate them and avoid making them cloudy. Second, clear the sections in pure xylene solution for about 5 minutes. This step requires that the petri dish containing the xylene is dry and free of moisture, otherwise the sections may become blurry. Because xylene solution is volatile, the entire clearing process needs to be carried out in a fume hood.
[0029] Compared to traditional slide preparation methods, this step first involves soaking the slides in a 1:1 ethanol-xylene solution for 5 minutes to dehydrate them. The 5-minute time is controlled to achieve dehydration while minimizing damage to the slide's color, thus striking a balance. Excessive clearing time can lead to further decolorization of the destained slides, completely destroying the staining.
[0030] At the same time, ensure that the petri dish containing xylene is dry and free of moisture. It is advisable to cover the petri dish with a transparent lid to reduce the evaporation of xylene and to prevent water from accidentally falling into the xylene solution.
[0031] (7) Sealing: Place the slide on a glass slide with tweezers, drop a drop of neutral resin (Canada resin), and first observe the integrity of the slide with an optical microscope (subsequent processes will also damage the slide) and whether the staining is uniform. After meeting the requirements, cover with a coverslip and press to seal, allowing the remaining resin to overflow, and ensure that the slide is on the same horizontal plane. Label it and air dry or dry at low temperature for observation.
[0032] Compared to traditional slide preparation methods, this step involves preliminary screening using an optical microscope before covering with a coverslip, ensuring the quality of slide mounting.
[0033] Compared to traditional slide preparation methods, this step involves preliminary screening using an optical microscope before covering with a coverslip, ensuring the quality of the slide mounting. This preliminary screening serves as a prerequisite for subsequent microscopic imaging, reducing the workload.
[0034] (8) Photography: Use an optical microscope to observe the slides at an appropriate magnification and field of view, and take photos. The photos should be saved in TIF format for subsequent analysis; the sample images processed by this method are as follows. Figure 1 As shown.
[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims
1. A method for preparing sapwood slices from the xylem of spruce, characterized in that, include: After trimming, the sapwood samples of the xylem of the spruce were fixed in a fixative solution; The fixed sapwood samples of spruce xylem were softened in ethylenediamine solution for 5-6 days, and then sliced after softening. The sapwood samples of the xylem of the spruce were bleached, rinsed, stained, and eluted. Transparent sections of sapwood samples from the xylem of Picea spp. were prepared using a mixture of ethanol and xylene. Slices of the sapwood sample from the xylem of the spruce were sealed and photographed.
2. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The fixative consists of 89 parts by volume of 70% ethanol, 6 parts by volume of glacial acetic acid, and 5 parts by volume of formalin; the fixation time is 24 hours.
3. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The sapwood sample of the spruce wood was trimmed so that the thickness of the spruce wood sample was 0.5±0.2cm.
4. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The concentration of the ethylenediamine solution is 10%.
5. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The sections were prepared using a paraffin cryostat, and the thickness of the sections was 20-35 μm.
6. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The bleaching process was performed using a 10% sodium hypochlorite solution for 10 ± 2 minutes.
7. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The staining was performed using a 1% safranin staining solution for 1 hour.
8. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The elution was performed by gradient elution of safranin-stained sections with ethanol aqueous solutions of 30%, 45%, 60%, 70%, and 100% by mass percentage, with each gradient elution time being 3 ± 1 min.
9. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The volume ratio of ethanol to xylene in the ethanol and xylene mixed solution is 1:1; the time for achieving transparency is 5 ± 1 min.
10. The method for preparing sapwood slices of spruce wood according to claim 1, characterized in that, The sealing agent is a neutral resin.