CN112903406A - Method for making paraffin section of tree thin root - Google Patents
Method for making paraffin section of tree thin root Download PDFInfo
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- CN112903406A CN112903406A CN202110317793.XA CN202110317793A CN112903406A CN 112903406 A CN112903406 A CN 112903406A CN 202110317793 A CN202110317793 A CN 202110317793A CN 112903406 A CN112903406 A CN 112903406A
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Abstract
The invention discloses a method for making paraffin sections of tree thin roots, which relates to the technical field of plant dissection and comprises the following steps: step one, material taking and fixing: taking a tree thin root material, treating, fixing and exhausting; step two, storage and softening: storing the low-grade tender roots; softening the high-grade hard fine roots; step three, dehydration: dehydrating the low-grade tender roots in ethanol; dehydrating high-grade hard fine root in distilled water and ethanol; step four, transparent: the material is placed in mixed solution, dimethylbenzene and dimethylbenzene for transparency; step five, wax dipping: soaking the material in the mixed solution and soaking in wax; step six, embedding: embedding with liquid wax; step seven, slicing, sticking, spreading and baking: cutting wax strips out of the material, adhering the wax strips on a glass slide, stretching the wax strips, and baking; step eight, dewaxing and rehydration: putting slices into a mixed solution of xylene, xylene and dewaxing, and putting the slices into ethanol and distilled water for rehydration; step nine, dyeing and sealing: dyeing the slices, and sealing and drying.
Description
Technical Field
The invention relates to the technical field of plant anatomy, in particular to a method for manufacturing a paraffin section of a tree thin root.
Background
Paraffin sectioning is one of the most commonly used methods for observing plant organs, tissues and cells, and is one of the most classical experimental techniques in plant anatomy research.
The thin roots are roots with the diameter less than or equal to 2mm, are important organs for plants to obtain soil moisture and nutrients, have complex branch systems, and in recent years, researches on the thin roots are mainly based on root order classification, namely, the root with a root tip at the head end is defined as a grade 1 root, a mother root formed by the intersection of two grade 1 roots is a grade 2 root, a mother root of the grade 2 root is a grade 3 root, and the like, and the roots are generally classified into grade 5 roots. The fine roots play a key role in the functions of plants and the processes of an ecosystem, and the clarification of the relationship and the mechanism between the functional traits (morphology, anatomy, chemistry and physiological traits) has important significance for deeply understanding the structure and the functional relationship of the ecosystem under the functional traits of the plants and the climate change. The tree fine root paraffin slicing technology is an important means for researching tree fine root anatomical properties, but the wood part of a tree from a low-level fine root to a high-level fine root develops and perfects gradually, and the tissue structure of the tree has great difference, and the specific expression is that the thin-walled tissue of the low-level fine root (usually 1-3-level root) is developed and is extremely young and tender, the xylem and the phloem of the high-level fine root (usually 4-5-level root) are developed, the material is hard, and the hardness of the xylem, the phloem and the cambium is inconsistent, so that the conventional paraffin slicing technology easily causes the low-level young and tender fine root tissue to wrinkle and the high-level hard fine root tissue to wrinkle or break, the observation of the tree fine root anatomical properties is seriously influenced by the poor slicing effect, and the research on the tree fine.
Therefore, aiming at the extremely young and tender low-grade slender roots and the hard and high-grade slender roots with inconsistent hardness, if the paraffin section method can be improved and perfected according to the characteristics of the material, the method can provide technical support for the micro-anatomy research of the tree slender roots and has important significance for the research of revealing the relationship between the tree slender root functional properties and the mechanism thereof.
Disclosure of Invention
The invention solves the technical problem of providing the paraffin section manufacturing method of the tree thin root, which is simple and universal in operation, can ensure that the obtained section tissue structure is clear and complete and is convenient to observe.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for making paraffin sections of tree thin roots comprises the following steps:
step one, material taking and fixing: taking tree slender root materials outdoors by using a tool, placing the treated tree slender root materials in a fixing solution for fixing and exhausting air to obtain the fixed tree slender root materials, wherein the tree slender root materials comprise low-grade young slender roots and high-grade hard slender roots;
step two, storage and softening: storing the fixed low-grade tender fine root in 70% ethanol, and softening the high-grade hard fine root in 20% ethylenediamine to obtain softened high-grade hard fine root;
step three, dehydration: placing the fixed low-grade tender fine roots in 70% ethanol, 85% ethanol, 95% ethanol, absolute ethanol and absolute ethanol in sequence for dehydration to obtain dehydrated low-grade tender fine roots, and placing the softened high-grade hard fine roots in distilled water, 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 95% ethanol, absolute ethanol and absolute ethanol in sequence for dehydration to obtain dehydrated high-grade hard fine roots;
step four, transparent: placing the dehydrated material in a mixed solution of absolute ethyl alcohol and xylene with the volume ratio of 1:1, xylene and xylene in sequence for transparency to obtain a transparent material;
step five, wax dipping: soaking the transparent material in mixed solution of xylene and liquid wax in a volume ratio of 1:1, and then repeatedly soaking the transparent material in pure liquid wax to obtain a wax-soaked material;
step six, embedding: embedding the waxed material by using the same batch of liquid wax to obtain an embedded material;
step seven, slicing, sticking, spreading and baking: cutting the embedded material into wax strips by a slicer, adhering the wax strips on a glass slide uniformly coated with adhesive tablets, spreading the wax strips by distilled water, and then placing the wax strips on a baking sheet machine for baking to obtain dried slices;
step eight, dewaxing and rehydration: placing the dried slices in mixed solution of xylene, absolute ethyl alcohol and xylene with the volume ratio of 1:1 in sequence for dewaxing, and then placing the slices in the mixed solution of absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol, 70% ethyl alcohol, 50% ethyl alcohol, 30% ethyl alcohol and distilled water for rehydration;
step nine, dyeing and sealing: and dyeing the slices by adopting a safranin-fast green dyeing method, and then sealing the slices and naturally drying the slices.
Further, the method comprises the following steps: in the first step, the low-grade tender and fine roots are placed in newly-prepared 50% FAA stationary liquid, air is pumped for 10-15 min, and then the fixed roots are fixed for 24 h; the volume ratio of 50% ethanol, glacial acetic acid and 37% formaldehyde in the 50% FAA fixing solution is 90:5: 5;
in the first step, the hard high-grade fine roots are placed in a newly-prepared 70% FAA fixing solution, air extraction is carried out for 20-25 min, then fixation is carried out for 24h, and the volume ratio of 70% ethanol, glacial acetic acid and 37% formaldehyde in the 70% FAA fixing solution is 90:5: 5.
Further, the method comprises the following steps: and in the second step, taking the high-grade hard fine roots out of the fixing solution, washing the high-grade hard fine roots with 70% ethanol for 3min, then soaking the high-grade hard fine roots in 70% ethanol for 12h, sequentially soaking the high-grade hard fine roots in 50% ethanol, 30% ethanol and distilled water for 2h respectively, then placing the high-grade hard fine roots in 20% ethylenediamine, exhausting air until no bubbles are generated, wherein the air is about 10min, and finally soaking the high-grade hard fine roots in the ethylenediamine for softening for 4-6 d.
Further, the method comprises the following steps: the dehydration time of the material in the third step is 1-2 h.
Further, the method comprises the following steps: the transparent time of the material in the fourth step is 30min-1.5 h.
Further, the method comprises the following steps: soaking the transparent material in mixed solution of xylene and liquid wax in a volume ratio of 1:1 for 12 hours at 40 ℃; and then, the material is placed in pure liquid wax for wax dipping, the liquid wax is replaced for 3 times, the wax dipping time is 4 hours each time, and the temperature is 65 ℃.
Further, the method comprises the following steps: and in the step eight, the dried slices are placed into dimethylbenzene to be soaked for 40min, then new dimethylbenzene is replaced, the soaking time is 20min, and then the slices are placed into a mixed solution of absolute ethyl alcohol and dimethylbenzene with the volume ratio of 1:1 to be soaked for 30min and are always controlled at the constant temperature of 30 ℃.
Further, the method comprises the following steps: and the time range of rehydration in the step eight is 10-30 min.
Further, the method comprises the following steps: in the ninth step, the slices are placed in 1% safranine solution for soaking for 12h, then the slices are washed by distilled water for 3 times to remove loose color, then the slices are sequentially placed in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol for soaking for 1min respectively, then 0.5% fast green is used for soaking and dyeing for 30s, then the slices are sequentially placed in 95% ethanol, absolute ethanol and absolute ethanol for soaking for 1min respectively to remove loose color, then the slices are sequentially placed in absolute ethanol and xylene mixed solution with the volume ratio of 1:1, xylene and xylene for soaking for 20s respectively, then neutral gum is immediately dropped, cover with a cover glass to seal the slices, and finally the slices are naturally dried.
The invention has the beneficial effects that: the invention can prevent the slice from folding or breaking by softening the high-grade hard fine root with 20 percent of ethylenediamine, and can ensure that the low-grade tender fine root can not shrink by adjusting the time of the steps of air extraction, dehydration, transparence, wax immersion, dyeing and the like and the temperature in partial steps, thereby ensuring the slicing effect in the whole process and cutting the slice with complete and clear tissue structure and clear dyeing. The technical method is suitable for slicing the rootlets of various trees at different levels, and can provide reliable technical support for anatomical research of the rootlets of the trees and disclosure of the relationship and mechanism between functional characters.
Drawings
In fig. 1, a, b, c, d, and e are schematic slices of fine roots of grade 1-5 of cinnamomum japonicum in example 1 of the present invention, respectively;
in FIG. 2, f, g, h, i, j are schematic slices of grade 1-5 fine roots of Zingiber zerumbet Hance in example 1 of the present invention;
in fig. 3, k, l, m, n, and o are schematic slices of thin roots of machilus 1-5 levels in embodiment 1 of the present invention;
in fig. 4, p, q, r, s, and t are schematic slices of 1-5-level slender roots of a phyllostachys pubescens in example 1 of the present invention.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
A method for making paraffin sections of tree thin roots comprises the following steps:
step one, material taking and fixing: taking tree slender root materials outdoors by using a tool, placing the treated tree slender root materials in a fixing solution for fixing and exhausting air to obtain the fixed tree slender root materials, wherein the tree slender root materials comprise low-grade young slender roots and high-grade hard slender roots;
step two, storage and softening: storing the fixed low-grade tender slender root in 70% ethanol, and softening the high-grade hard slender root in 20% ethylenediamine to obtain softened high-grade hard slender root;
step three, dehydration: placing the fixed low-grade tender fine roots in 70% ethanol, 85% ethanol, 95% ethanol, absolute ethanol and absolute ethanol in sequence for dehydration to obtain dehydrated low-grade tender fine roots, and placing the softened high-grade hard fine roots in distilled water, 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 95% ethanol, absolute ethanol and absolute ethanol in sequence for dehydration to obtain dehydrated high-grade hard fine roots;
step four, transparent: placing the dehydrated material in a mixed solution of absolute ethyl alcohol and xylene with the volume ratio of 1:1, xylene and xylene in sequence for transparency to obtain a transparent material;
step five, wax dipping: soaking the transparent material in mixed solution of xylene and liquid wax in a volume ratio of 1:1, and then repeatedly soaking the transparent material in pure liquid wax to obtain a wax-soaked material;
step six, embedding: embedding the waxed material by using the same batch of liquid wax to obtain an embedded material;
step seven, slicing, sticking, spreading and baking: cutting the embedded material into wax strips by a slicer, adhering the wax strips on a glass slide uniformly coated with adhesive tablets, spreading the wax strips by distilled water, and then placing the wax strips on a baking sheet machine for baking to obtain dried slices;
step eight, dewaxing and rehydration: placing the dried slices in mixed solution of xylene, absolute ethyl alcohol and xylene with the volume ratio of 1:1 in sequence for dewaxing, and then placing the slices in the mixed solution of absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol, 70% ethyl alcohol, 50% ethyl alcohol, 30% ethyl alcohol and distilled water for rehydration;
step nine, dyeing and sealing: and dyeing the slices by adopting a safranin-fast green dyeing method, and then sealing the slices and naturally drying the slices.
On the basis, in the step one, the low-grade tender and fine roots are placed into newly-prepared 50% FAA stationary liquid, air is pumped for 10-15 min, and then the fixation is carried out for 24 h; the volume ratio of 50% ethanol, glacial acetic acid and 37% formaldehyde in the 50% FAA fixing solution is 90:5: 5;
in the first step, the hard high-grade fine roots are placed in a newly-prepared 70% FAA fixing solution, air extraction is carried out for 20-25 min, then fixation is carried out for 24h, and the volume ratio of 70% ethanol, glacial acetic acid and 37% formaldehyde in the 70% FAA fixing solution is 90:5: 5.
On the basis, in the second step, the high-grade hard fine roots are taken out from the fixing solution, washed with 70% ethanol for 3min, then soaked in 70% ethanol for 12h, then sequentially soaked in 50% ethanol, 30% ethanol and distilled water for 2h respectively, then the high-grade hard fine roots are placed in 20% ethylenediamine, air is pumped until no bubbles are generated, the time is about 10min, and finally the high-grade hard fine roots are soaked in the ethylenediamine and softened for 4-6 d.
On the basis, the dehydration time of the material in the third step is 1-2 h.
On the basis, the material in the fourth step has the transparency time of 30min-1.5 h.
On the basis, the material which is transparent in the fifth step is placed in a mixed solution of xylene and liquid wax with the volume ratio of 1:1 for soaking for 12 hours at the temperature of 40 ℃; and then, the material is placed in pure liquid wax for wax dipping, the liquid wax is replaced for 3 times, the wax dipping time is 4 hours each time, and the temperature is 65 ℃.
On the basis, in the step eight, the dried slices are placed into dimethylbenzene to be soaked for 40min, then new dimethylbenzene is replaced, the slices are soaked for 20min, and then the slices are placed into a mixed solution of absolute ethyl alcohol and dimethylbenzene with the volume ratio of 1:1 to be soaked for 30min and are always controlled at the constant temperature of 30 ℃.
On the basis, the time range of the rehydration in the step eight is 10min-30 min.
On the basis, in the ninth step, the slices are placed in a 1% safranine solution to be soaked for 12h, then the slices are washed by distilled water for 3 times to remove floating color, then the slices are sequentially placed in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol to be respectively soaked for 1min, then 0.5% fast green is used for soaking and dyeing for 30s, then the slices are sequentially placed in 95% ethanol, absolute ethanol and absolute ethanol to be respectively soaked for 1min to remove floating color, then the slices are sequentially placed in an absolute ethanol and xylene mixed solution with a volume ratio of 1:1, xylene and xylene to be respectively soaked for 20s, then neutral gum is immediately dripped, a cover glass is covered to seal the slices, and finally the slices are naturally dried.
The first embodiment is as follows:
the illustration of the paraffin section of the fine roots of cinnamomum japonicum of this embodiment is shown in fig. 1,
a method for preparing paraffin sections of cinnamomum camphora cinnamomum japonicum 'Chenii' fine roots comprises the following steps:
step one, obtaining and fixing fine root materials of cinnamomum japonicum lour: the outdoor stainless steel fork is used for manually excavating at the soil depth of 0-20 cm within 2m from the trunk base of the cinnamomum japonicum, a complete root section containing at least 5-level root sequences is taken out, and the attached soil and impurities on the surface of the root are carefully cleaned. Carefully rinse the root with deionized water in the laboratory, take out complete 5 grades of root segments with sharp blade, then cut apart into different root prefaces with the root segment, every grade of root preface is cut into the short section of 3 ~ 8mm length. Placing low-grade tender and fine roots (grade 1-3) in newly-prepared 50% FAA stationary liquid (the volume ratio of 50% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), pumping the low-grade tender and fine roots for 10min by using an air pump until no bubbles are generated, and then placing the low-grade tender and fine roots in the stationary liquid for fixing for 24 h;
and (3) placing the high-grade hard fine roots (4-5 grade) into a newly-prepared 70% FAA fixing solution (the volume ratio of 70% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), extracting the high-grade hard fine roots for 25min by using an air extractor until no air bubbles are generated, and then placing the high-grade hard fine roots in the fixing solution for fixing for 24 h.
Step two, storing the low-grade tender and fine roots of the cinnamomum japonicum, and softening the high-grade hard and fine roots: taking the low-grade tender roots out of the fixing solution, washing with 50% ethanol for 3min, soaking in 50% ethanol for 2h, and storing in 70% ethanol. Taking out the high-grade hard fine root from the fixed solution, washing with 70% ethanol for 3min, soaking in 70% ethanol for 12h, sequentially soaking in 50% ethanol, 30% ethanol and distilled water for 2h, placing the material in 20% ethylenediamine, exhausting for 10min, and soaking in 20% ethylenediamine for 5d to obtain good softening effect.
Step three, dehydrating the fine root material of the cinnamomum japonicum thunb: taking out the softened high-grade hard fine root from the softening agent, washing with distilled water for 3min, and sequentially soaking in distilled water, 30% ethanol, 50% ethanol, and 70% ethanol for 2 hr respectively. Then, the low-grade tender and fine roots and the high-grade hard and fine roots which are preserved in advance are synchronously subjected to experimental operation, the materials are sequentially placed in 85 percent, 95 percent and absolute ethyl alcohol to be respectively soaked for 2 hours, 2 hours and 1.5 hours, and finally, new absolute ethyl alcohol is replaced to be soaked for 1 hour.
Step four, carrying out transparency on the fine root material of the cinnamomum japonicum lour: after dehydration is finished, adding equal volume of xylene into the original absolute ethyl alcohol to form a mixed solution of the absolute ethyl alcohol and the xylene with the volume ratio of 1:1, soaking the materials for 2 hours, then placing the materials into the xylene to soak for 2 hours, and then replacing new xylene to soak for 2 hours.
Step five, waxing the fine root material of the cinnamomum japonicum thunb: after the transparency is finished, adding liquid wax with the same volume into the original xylene to form a mixed solution of the xylene and the liquid wax with the volume ratio of 1:1, placing the mixed solution in a thermostat with the temperature of 40 ℃ for soaking for 12 hours, then placing the material in pure liquid wax in a thermostat with the temperature of 65 ℃ for heat preservation, replacing 1 time of pure liquid wax every 4 hours of soaking, and soaking 3 times of pure liquid wax in total.
Step six, embedding the fine root material of the cinnamomum japonicum: opening an embedding machine in advance, setting the temperature of an instrument to be 70 ℃, adding liquid wax for preheating, preparing an embedding box, filling the embedding box with the liquid wax, moving the embedding box to an ice platform, standing the cross section of the material upwards in the middle of the embedding box when the liquid wax at the bottom layer is slightly solidified, and finally standing the material until the liquid wax is solidified.
Step seven, slicing, sticking, spreading and baking the fine root material of the cinnamomum japonicum lour: after the wax dipping is finished, the wax block is trimmed into a regular trapezoid shape, the cross section of the material is parallel to the bottom surface of the wax block, the lower bottom surface of the wax block is pasted on a wooden candle stick by liquid wax, and finally the wooden candle stick is installed on a rotary manual slicing machine for slicing, wherein the thickness of the low-grade tender fine root slice is 10um, and the thickness of the high-grade hard fine root slice is 14 um. Uniformly coating a sticky tablet (the volume ratio of egg white to glycerin is 1:1) on the smooth surface of a glass slide, then dropwise adding a proper amount of boiled distilled water by using a rubber head dropper to cover the smooth surface on the glass slide, then placing a wax tape cut in advance on the distilled water on the surface of the glass slide by using flat tweezers, absorbing the distilled water by using absorbent paper after the wax tape is fully stretched on the distilled water, finally placing the glass slide on a 40 ℃ baking machine for baking for 30min, and then naturally drying.
Step eight, dewaxing and rehydrating the fine root slices of the cinnamomum japonicum lour: soaking the slices in xylene at 30 deg.C for 40min, then replacing with new xylene, soaking at 30 deg.C for 20min, and soaking in mixed solution of anhydrous ethanol and xylene at a volume ratio of 1:1 at 30 deg.C for 30 min. Dewaxing, and sequentially soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol and distilled water for 30min, 15min, 10min and 10 min.
Step nine, the fine root sections of the cinnamomum japonicum are dyed and sliced: soaking the slices in 1% safranine solution for 12h, washing the slices with distilled water for 3 times to remove floating color, sequentially soaking the slices in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol for 1min, soaking and dyeing the slices in 0.5% fast green for 30s, sequentially soaking the slices in 95% ethanol, absolute ethanol and absolute ethanol for 1min to remove floating color, sequentially soaking the slices in a mixed solution of absolute ethanol and xylene in a volume ratio of 1:1, xylene and xylene for 20s, immediately dripping neutral gum, covering with a cover glass to seal the slices, and naturally drying.
Example two:
the paraffin section of the cinnamomum japonicum root of this example is shown in figure 2,
a method for making paraffin sections of cinnamomum japonicum fine roots comprises the following steps:
step one, obtaining and fixing fine root materials of cinnamomum japonicum: the method comprises the following steps of manually excavating at a soil depth of 0-20 cm within a range of 2m from the root of a trunk of the cinnamomum japonicum by using an outdoor stainless steel fork, taking out a complete root section containing at least 5-level root sequences, and carefully cleaning out attached soil and impurities on the surface of the root. Carefully rinse the root with deionized water in the laboratory, take out complete 5 grades of root segments with sharp blade, then cut apart into different root prefaces with the root segment, every grade of root preface is cut into the short section of 3 ~ 8mm length. Placing low-grade tender and fine roots (grade 1-2) in newly-prepared 50% FAA fixing solution (the volume ratio of 50% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), pumping the low-grade tender and fine roots for 10min by using an air pump until no bubbles are generated, and then placing the low-grade tender and fine roots in the fixing solution for fixing for 24 h;
and (3) placing the high-grade hard fine roots (3-5 grade) into a newly-prepared 70% FAA fixing solution (the volume ratio of 70% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), extracting the high-grade hard fine roots for 25min by using an air extractor until no air bubbles are generated, and then placing the high-grade hard fine roots in the fixing solution for fixing for 24 h.
Step two, storing the low-grade tender and fine roots of the cinnamomum japonicum, and softening the high-grade hard and fine roots: taking the low-grade tender roots out of the fixing solution, washing with 50% ethanol for 3min, soaking in 50% ethanol for 2h, and storing in 70% ethanol. Taking out the high-grade hard fine root from the fixed solution, washing with 70% ethanol for 3min, soaking in 70% ethanol for 12h, sequentially soaking in 50% ethanol, 30% ethanol and distilled water for 2h, placing the material in 20% ethylenediamine, exhausting for 10min, and soaking in 20% ethylenediamine for 6d to obtain good softening effect.
Step three, dehydrating the fine root material of cinnamomum japonicum: taking out the softened high-grade hard fine root from the softening agent, washing with distilled water for 3min, and sequentially soaking in distilled water, 30% ethanol, 50% ethanol, and 70% ethanol for 2 hr respectively. Then, the low-grade tender and fine roots and the high-grade hard and fine roots which are preserved in advance are synchronously subjected to experimental operation, the materials are sequentially placed in 85 percent, 95 percent and absolute ethyl alcohol to be respectively soaked for 2 hours, 2 hours and 1.5 hours, and finally, new absolute ethyl alcohol is replaced to be soaked for 1 hour.
Step four, carrying out transparency on the fine root material of cinnamomum japonicum: after dehydration is finished, adding equal volume of xylene into the original absolute ethyl alcohol to form a mixed solution of the absolute ethyl alcohol and the xylene with the volume ratio of 1:1, soaking the materials for 2 hours, then placing the materials into the xylene to soak for 2 hours, and then replacing new xylene to soak for 2 hours.
Step five, waxing the fine root material of the cinnamomum japonicum: after the transparency is finished, adding liquid wax with the same volume into the original xylene to form a mixed solution of the xylene and the liquid wax with the volume ratio of 1:1, placing the mixed solution in a thermostat with the temperature of 40 ℃ for soaking for 12 hours, then placing the material in pure liquid wax in a thermostat with the temperature of 65 ℃ for heat preservation, replacing 1 time of pure liquid wax every 4 hours of soaking, and soaking 3 times of pure liquid wax in total.
Step six, embedding the fine root material of cinnamomum japonicum: opening an embedding machine in advance, setting the temperature of an instrument to be 70 ℃, adding liquid wax for preheating, preparing an embedding box, filling the embedding box with the liquid wax, moving the embedding box to an ice platform, standing the cross section of the material upwards in the middle of the embedding box when the liquid wax at the bottom layer is slightly solidified, and finally standing the material until the liquid wax is solidified.
Step seven, slicing, sticking, spreading and baking the fine root material of the cinnamomum japonicum: after the wax dipping is finished, the wax block is trimmed into a regular trapezoid shape, the cross section of the material is parallel to the bottom surface of the wax block, the lower bottom surface of the wax block is pasted on a wooden candle stick by liquid wax, and finally the wooden candle stick is installed on a rotary manual slicing machine for slicing, wherein the thickness of the low-grade tender fine root slice is 10um, and the thickness of the high-grade hard fine root slice is 14 um. Uniformly coating a sticky tablet (the volume ratio of egg white to glycerin is 1:1) on the smooth surface of a glass slide, then dropwise adding a proper amount of boiled distilled water by using a rubber head dropper to cover the smooth surface on the glass slide, then placing a wax tape cut in advance on the distilled water on the surface of the glass slide by using flat tweezers, absorbing the distilled water by using absorbent paper after the wax tape is fully stretched on the distilled water, finally placing the glass slide on a 40 ℃ baking machine for baking for 30min, and then naturally drying.
Step eight, dewaxing and rehydrating the fine root slices of cinnamomum japonicum: soaking the slices in xylene at 30 deg.C for 40min, then replacing with new xylene, soaking at 30 deg.C for 20min, and soaking in mixed solution of anhydrous ethanol and xylene at a volume ratio of 1:1 at 30 deg.C for 30 min. Dewaxing, and sequentially soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol and distilled water for 30min, 15min, 10min and 10 min.
Step nine, the thin root section of the cinnamomum japonicum is dyed and sealed: soaking the slices in 1% safranine solution for 12h, washing the slices with distilled water for 3 times to remove floating color, sequentially soaking the slices in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol for 1min, soaking and dyeing the slices in 0.5% fast green for 30s, sequentially soaking the slices in 95% ethanol, absolute ethanol and absolute ethanol for 1min to remove floating color, sequentially soaking the slices in a mixed solution of absolute ethanol and xylene in a volume ratio of 1:1, xylene and xylene for 20s, immediately dripping neutral gum, covering with a cover glass to seal the slices, and naturally drying.
Example three:
a schematic representation of a paraffin section of fine roots of photinia fraseri of this example is shown in figure 3,
a paraffin section making method of fine roots of photinia fraseri comprises the following steps:
step one, obtaining and fixing a fine root material of the photinia fraseri: the method comprises the steps of manually excavating at a soil depth of 0-20 cm within a range of 2m from the base of a Photinia fraseri trunk by using an outdoor stainless steel fork, taking out a complete root section containing at least 5-level root sequences, and carefully cleaning out the attached soil and impurities on the surface of the root. Carefully rinse the root with deionized water in the laboratory, take out complete 5 grades of root segments with sharp blade, then cut apart into different root prefaces with the root segment, every grade of root preface is cut into the short section of 3 ~ 8mm length. Placing low-grade tender and fine roots (grade 1-3) in newly-prepared 50% FAA stationary liquid (the volume ratio of 50% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), pumping the low-grade tender and fine roots for 10min by using an air pump until no bubbles are generated, and then placing the low-grade tender and fine roots in the stationary liquid for fixing for 24 h;
and (3) placing the high-grade hard fine roots (4-5 grade) into a newly-prepared 70% FAA fixing solution (the volume ratio of 70% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), extracting the high-grade hard fine roots for 25min by using an air extractor until no air bubbles are generated, and then placing the high-grade hard fine roots in the fixing solution for fixing for 24 h.
Step two, storing the low-grade tender and fine roots of the photinia fraseri, and softening the high-grade hard and fine roots: taking the low-grade tender roots out of the fixing solution, washing with 50% ethanol for 3min, soaking in 50% ethanol for 2h, and storing in 70% ethanol. Taking out the high-grade hard fine root from the fixed solution, washing with 70% ethanol for 3min, soaking in 70% ethanol for 12h, sequentially soaking in 50% ethanol, 30% ethanol and distilled water for 2h, placing the material in 20% ethylenediamine, exhausting for 10min, and soaking in 20% ethylenediamine for 5d to obtain good softening effect.
Step three, dehydrating the fine root material of the photinia fraseri: taking out the softened high-grade hard fine root from the softening agent, washing with distilled water for 3min, and sequentially soaking in distilled water, 30% ethanol, 50% ethanol, and 70% ethanol for 2 hr respectively. Then, the low-grade tender and fine roots and the high-grade hard and fine roots which are preserved in advance are synchronously subjected to experimental operation, the materials are sequentially placed in 85 percent, 95 percent and absolute ethyl alcohol to be respectively soaked for 2 hours, 2 hours and 1.5 hours, and finally, new absolute ethyl alcohol is replaced to be soaked for 1 hour.
Step four, carrying out transparency on the fine root material of the photinia fraseri: after dehydration is finished, adding equal volume of xylene into the original absolute ethyl alcohol to form a mixed solution of the absolute ethyl alcohol and the xylene with the volume ratio of 1:1, soaking the materials for 2 hours, then placing the materials into the xylene to soak for 2 hours, and then replacing new xylene to soak for 2 hours.
Step five, carrying out wax dipping on the fine root material of the photinia fraseri: after the transparency is finished, adding liquid wax with the same volume into the original xylene to form a mixed solution of the xylene and the liquid wax with the volume ratio of 1:1, placing the mixed solution in a thermostat with the temperature of 40 ℃ for soaking for 12 hours, then placing the material in pure liquid wax in a thermostat with the temperature of 65 ℃ for heat preservation, replacing 1 time of pure liquid wax every 4 hours of soaking, and soaking 3 times of pure liquid wax in total.
Step six, embedding the fine root material of the photinia fraseri: opening an embedding machine in advance, setting the temperature of an instrument to be 70 ℃, adding liquid wax for preheating, preparing an embedding box, filling the embedding box with the liquid wax, moving the embedding box to an ice platform, standing the cross section of the material upwards in the middle of the embedding box when the liquid wax at the bottom layer is slightly solidified, and finally standing the material until the liquid wax is solidified.
Step seven, slicing, sticking, spreading and baking the fine root material of the photinia fraseri: after the wax dipping is finished, the wax block is trimmed into a regular trapezoid shape, the cross section of the material is parallel to the bottom surface of the wax block, the lower bottom surface of the wax block is pasted on a wooden candle stick by liquid wax, and finally the wooden candle stick is installed on a rotary manual slicing machine for slicing, wherein the thickness of the low-grade tender fine root slice is 10um, and the thickness of the high-grade hard fine root slice is 14 um. Uniformly coating a sticky tablet (the volume ratio of egg white to glycerin is 1:1) on the smooth surface of a glass slide, then dropwise adding a proper amount of boiled distilled water by using a rubber head dropper to cover the smooth surface on the glass slide, then placing a wax tape cut in advance on the distilled water on the surface of the glass slide by using flat tweezers, absorbing the distilled water by using absorbent paper after the wax tape is fully stretched on the distilled water, finally placing the glass slide on a 40 ℃ baking machine for baking for 30min, and then naturally drying.
Eighthly, slicing the fine roots of the photinia fraseri, dewaxing and rehydrating: soaking the slices in xylene at 30 deg.C for 40min, then replacing with new xylene, soaking at 30 deg.C for 20min, and soaking in mixed solution of anhydrous ethanol and xylene at a volume ratio of 1:1 at 30 deg.C for 30 min. Dewaxing, and sequentially soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol and distilled water for 30min, 15min, 10min and 10 min.
Step nine, slicing and dyeing the fine roots of the photinia fraseri, and sealing: soaking the slices in 1% safranine solution for 12h, washing the slices with distilled water for 3 times to remove floating color, sequentially soaking the slices in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol for 1min, soaking and dyeing the slices in 0.5% fast green for 30s, sequentially soaking the slices in 95% ethanol, absolute ethanol and absolute ethanol for 1min to remove floating color, sequentially soaking the slices in a mixed solution of absolute ethanol and xylene in a volume ratio of 1:1, xylene and xylene for 20s, immediately dripping neutral gum, covering with a cover glass to seal the slices, and naturally drying.
Comparative example:
the paraffin section of the leaflet frame thin root of this example is shown in figure 4,
step one, obtaining and fixing a fine root material of a small leaf-frame Machilus: manually excavating the outdoor stainless steel fork at the soil depth of 0-20 cm within 2m from the base of the root of the phyllanthus niveus tree, taking out a complete root segment containing at least 5-level root sequences, and carefully cleaning the attached soil and impurities on the surface of the root. Carefully rinse the root with deionized water in the laboratory, take out complete 5 grades of root segments with sharp blade, then cut apart into different root prefaces with the root segment, every grade of root preface is cut into the short section of 3 ~ 8mm length. Placing the low-grade tender and fine roots (grade 1-3) in newly-prepared 50% FAA stationary liquid (the volume ratio of 50% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), exhausting the low-grade tender and fine roots for 25min by using an air extractor, and then placing the low-grade tender and fine roots in the stationary liquid for fixing for 24 h;
and (3) placing the high-grade hard fine roots (4-5 grade) into a newly-prepared 70% FAA fixing solution (the volume ratio of 70% ethanol to glacial acetic acid to 37% formaldehyde is 90:5:5), extracting the high-grade hard fine roots for 25min by using an air extractor until no air bubbles are generated, and then placing the high-grade hard fine roots in the fixing solution for fixing for 24 h.
Step two, storing the low-grade tender and fine roots of the small leaf phoenix: taking the low-grade tender roots out of the fixing solution, washing with 50% ethanol for 3min, soaking in 50% ethanol for 2h, and storing in 70% ethanol. Taking out high-grade hard fine root from the fixed solution, washing with 70% ethanol for 3min, and storing in 70% ethanol for 5 days to obtain good softening effect.
Step three, dehydrating the fine root material of the phyllostachys pubescens: taking out the softened high-grade hard fine root from the softening agent, washing with distilled water for 3min, and sequentially soaking in distilled water, 30% ethanol, 50% ethanol, and 70% ethanol for 2 hr respectively. Then, the low-grade tender and fine roots and the high-grade hard and fine roots which are preserved in advance are synchronously subjected to experimental operation, the materials are sequentially placed in 85 percent, 95 percent and absolute ethyl alcohol to be respectively soaked for 2 hours, 2 hours and 1.5 hours, and finally, new absolute ethyl alcohol is replaced to be soaked for 1 hour.
Step four, carrying out transparency on the fine root material of the phyllostachys pubescens: after dehydration is finished, adding equal volume of xylene into the original absolute ethyl alcohol to form a mixed solution of the absolute ethyl alcohol and the xylene with the volume ratio of 1:1, soaking the materials for 2 hours, then placing the materials into the xylene to soak for 2 hours, and then replacing new xylene to soak for 2 hours.
Step five, waxing the fine root material of the phyllostachys pubescens: after the transparency is finished, adding liquid wax with the same volume into the original xylene to form a mixed solution of the xylene and the liquid wax with the volume ratio of 1:1, placing the mixed solution in a thermostat with the temperature of 40 ℃ for soaking for 12 hours, then placing the material in pure liquid wax in a thermostat with the temperature of 65 ℃ for heat preservation, replacing 1 time of pure liquid wax every 4 hours of soaking, and soaking 3 times of pure liquid wax in total.
Step six, embedding the fine root material of the phyllostachys pubescens: opening an embedding machine in advance, setting the temperature of an instrument to be 70 ℃, adding liquid wax for preheating, preparing an embedding box, filling the embedding box with the liquid wax, moving the embedding box to an ice platform, standing the cross section of the material upwards in the middle of the embedding box when the liquid wax at the bottom layer is slightly solidified, and finally standing the material until the liquid wax is solidified.
Seventhly, slicing, sticking, spreading and baking the thin root material of the phyllostachys pubescens: after the wax dipping is finished, the wax block is trimmed into a regular trapezoid shape, the cross section of the material is parallel to the bottom surface of the wax block, the lower bottom surface of the wax block is pasted on a wooden candle stick by liquid wax, and finally the wooden candle stick is installed on a rotary manual slicing machine for slicing, wherein the thickness of the low-grade tender fine root slice is 10um, and the thickness of the high-grade hard fine root slice is 14 um. Uniformly coating a sticky tablet (the volume ratio of egg white to glycerin is 1:1) on the smooth surface of a glass slide, then dropwise adding a proper amount of boiled distilled water by using a rubber head dropper to cover the smooth surface on the glass slide, then placing a wax tape cut in advance on the distilled water on the surface of the glass slide by using flat tweezers, absorbing the distilled water by using absorbent paper after the wax tape is fully stretched on the distilled water, finally placing the glass slide on a 40 ℃ baking machine for baking for 30min, and then naturally drying.
And step eight, dewaxing and rehydrating the fine root slices of the Machilus kusnezoffii: soaking the slices in xylene at 30 deg.C for 40min, then replacing with new xylene, soaking at 30 deg.C for 20min, and soaking in mixed solution of anhydrous ethanol and xylene at a volume ratio of 1:1 at 30 deg.C for 30 min. Dewaxing, and sequentially soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol and distilled water for 30min, 15min, 10min and 10 min.
And step nine, dyeing and mounting the thin root section of the Machilus kusnezoffii: soaking the slices in 1% safranine solution for 12h, washing the slices with distilled water for 3 times to remove floating color, sequentially soaking the slices in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol for 1min, soaking and dyeing the slices in 0.5% fast green for 30s, sequentially soaking the slices in 95% ethanol, absolute ethanol and absolute ethanol for 1min to remove floating color, sequentially soaking the slices in a mixed solution of absolute ethanol and xylene in a volume ratio of 1:1, xylene and xylene for 20s, immediately dripping neutral gum, covering with a cover glass to seal the slices, and naturally drying.
This embodiment is compared with the first embodiment: it can be seen that the long air-pumping time causes the low-grade tender and thin root to shrink, and the high-grade hard and thin root is not softened to cause the tissue to break.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for making paraffin sections of tree thin roots is characterized by comprising the following steps: the method comprises the following steps:
step one, material taking and fixing: using a tool to obtain a tree thin root material outdoors, placing the treated tree thin root material in a fixing solution for fixing and exhausting air to obtain the fixed tree thin root material;
step two, storage and softening: storing the fixed low-grade tender roots in 70% ethanol; softening the high-grade hard fine root in 20% ethylene diamine to obtain a softened high-grade hard fine root;
step three, dehydration: placing the fixed low-grade tender fine roots in 70% ethanol, 85% ethanol, 95% ethanol, absolute ethanol and absolute ethanol in sequence for dehydration to obtain dehydrated low-grade tender fine roots, and placing the softened high-grade hard fine roots in distilled water, 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol, 95% ethanol, absolute ethanol and absolute ethanol in sequence for dehydration to obtain dehydrated high-grade hard fine roots;
step four, transparent: placing the dehydrated material in a mixed solution of absolute ethyl alcohol and xylene with the volume ratio of 1:1, xylene and xylene in sequence for transparency to obtain a transparent material;
step five, wax dipping: soaking the transparent material in mixed solution of xylene and liquid wax in a volume ratio of 1:1, and then repeatedly soaking the transparent material in pure liquid wax to obtain a wax-soaked material;
step six, embedding: embedding the waxed material by using the same batch of liquid wax to obtain an embedded material;
step seven, slicing, sticking, spreading and baking: cutting the embedded material into wax strips by a slicer, adhering the wax strips on a glass slide uniformly coated with adhesive tablets, spreading the wax strips by distilled water, and then placing the wax strips on a baking sheet machine for baking to obtain dried slices;
step eight, dewaxing and rehydration: placing the dried slices in mixed solution of xylene, absolute ethyl alcohol and xylene with the volume ratio of 1:1 in sequence for dewaxing, and then placing the slices in the mixed solution of absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol, 70% ethyl alcohol, 50% ethyl alcohol, 30% ethyl alcohol and distilled water for rehydration;
step nine, dyeing and sealing: and dyeing the slices by adopting a safranin-fast green dyeing method, and then sealing the slices and naturally drying the slices.
2. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: in the first step, the low-grade tender and fine roots are placed in newly-prepared 50% FAA stationary liquid, air is pumped for 10-15 min, and then the fixed roots are fixed for 24 h; the volume ratio of 50% ethanol, glacial acetic acid and 37% formaldehyde in the 50% FAA fixing solution is 90:5: 5;
in the first step, the hard high-grade fine roots are placed in a newly-prepared 70% FAA fixing solution, air extraction is carried out for 20-25 min, then fixation is carried out for 24h, and the volume ratio of 70% ethanol, glacial acetic acid and 37% formaldehyde in the 70% FAA fixing solution is 90:5: 5.
3. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: and in the second step, taking the high-grade hard fine roots out of the fixing solution, washing the high-grade hard fine roots with 70% ethanol for 3min, then soaking the high-grade hard fine roots in 70% ethanol for 12h, sequentially soaking the high-grade hard fine roots in 50% ethanol, 30% ethanol and distilled water for 2h respectively, then placing the high-grade hard fine roots in 20% ethylenediamine, exhausting air until no bubbles are generated, wherein the air is about 10min, and finally soaking the high-grade hard fine roots in the ethylenediamine for softening for 4-6 d.
4. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: the dehydration time of the material in the third step is 1-2 h.
5. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: the transparent time of the material in the fourth step is 30min-1.5 h.
6. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: soaking the transparent material in mixed solution of xylene and liquid wax in a volume ratio of 1:1 for 12 hours at 40 ℃; and then, the material is placed in pure liquid wax for wax dipping, the liquid wax is replaced for 3 times, the wax dipping time is 4 hours each time, and the temperature is 65 ℃.
7. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: and in the step eight, the dried slices are placed into dimethylbenzene to be soaked for 40min, then new dimethylbenzene is replaced, the soaking time is 20min, and then the slices are placed into a mixed solution of absolute ethyl alcohol and dimethylbenzene with the volume ratio of 1:1 to be soaked for 30min and are always controlled at the constant temperature of 30 ℃.
8. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: and the time range of rehydration in the step eight is 10-30 min.
9. The method for making paraffin wax slices of tree rootlets as claimed in claim 1, wherein: in the ninth step, the slices are placed in 1% safranine solution for soaking for 12h, then the slices are washed by distilled water for 3 times to remove loose color, then the slices are sequentially placed in 30% ethanol, 50% ethanol, 70% ethanol, 85% ethanol and 95% ethanol for soaking for 1min respectively, then 0.5% fast green is used for soaking and dyeing for 30s, then the slices are sequentially placed in 95% ethanol, absolute ethanol and absolute ethanol for soaking for 1min respectively to remove loose color, then the slices are sequentially placed in absolute ethanol and xylene mixed solution with the volume ratio of 1:1, xylene and xylene for soaking for 20s respectively, then neutral gum is immediately dropped, cover with a cover glass to seal the slices, and finally the slices are naturally dried.
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