CN116754336B - Dyeing reagent and method for obtaining microscopic structure of large seaweed with section fiber small filaments - Google Patents
Dyeing reagent and method for obtaining microscopic structure of large seaweed with section fiber small filaments Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 241001474374 Blennius Species 0.000 title claims abstract description 24
- 238000004043 dyeing Methods 0.000 title claims description 36
- 239000000835 fiber Substances 0.000 title claims description 29
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000012128 staining reagent Substances 0.000 claims abstract description 21
- 239000012445 acidic reagent Substances 0.000 claims abstract description 20
- 239000002904 solvent Substances 0.000 claims abstract description 16
- 229960000583 acetic acid Drugs 0.000 claims abstract description 10
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 9
- 241000512259 Ascophyllum nodosum Species 0.000 claims description 45
- OSDLLIBGSJNGJE-UHFFFAOYSA-N 4-chloro-3,5-dimethylphenol Chemical compound CC1=CC(O)=CC(C)=C1Cl OSDLLIBGSJNGJE-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- 238000009835 boiling Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000001467 acupuncture Methods 0.000 claims description 5
- 239000004310 lactic acid Substances 0.000 claims description 5
- 235000014655 lactic acid Nutrition 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 206010008583 Chloroma Diseases 0.000 claims description 2
- 239000000975 dye Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 230000001054 cortical effect Effects 0.000 abstract description 26
- 238000011161 development Methods 0.000 abstract description 15
- LYOFHYLVYHTGBK-UHFFFAOYSA-N 4-chloro-1,5-dimethylcyclohexa-2,4-dien-1-ol Chemical compound CC1=C(Cl)C=CC(C)(O)C1 LYOFHYLVYHTGBK-UHFFFAOYSA-N 0.000 abstract description 6
- 239000012472 biological sample Substances 0.000 abstract description 2
- 238000005464 sample preparation method Methods 0.000 abstract description 2
- 210000003449 plasmodesmata Anatomy 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 64
- 210000004027 cell Anatomy 0.000 description 47
- 210000003668 pericyte Anatomy 0.000 description 15
- 241000195493 Cryptophyta Species 0.000 description 14
- 206010011732 Cyst Diseases 0.000 description 9
- 208000031513 cyst Diseases 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 244000205754 Colocasia esculenta Species 0.000 description 6
- 235000006481 Colocasia esculenta Nutrition 0.000 description 6
- 241001646834 Mesona Species 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000012188 paraffin wax Substances 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241001344092 Tetraspora <Myxozoa> Species 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
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- 238000005260 corrosion Methods 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 235000012405 Artemisia integrifolia Nutrition 0.000 description 2
- 241000584312 Artemisia integrifolia Species 0.000 description 2
- 241000080997 Ceramium gardneri Species 0.000 description 2
- 241000192584 Synechocystis Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000005257 cortical tissue Anatomy 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 208000001076 sarcopenia Diseases 0.000 description 2
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 description 2
- 235000008658 Artemisia capillaris Nutrition 0.000 description 1
- 241000092668 Artemisia capillaris Species 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- 241000057282 Centroceras Species 0.000 description 1
- 241000206641 Ceramiales Species 0.000 description 1
- 241000195634 Dunaliella Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000151096 Herposiphonia Species 0.000 description 1
- 241001519524 Kappaphycus alvarezii Species 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 241000145125 Neosiphonia Species 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明公开了一种染色试剂以及获得具节纤小丝状大型海藻显微结构的方法。涉及生物体样品制片方法领域。上述染色试剂，包括以下组分：对氯间二甲苯酚；酸性试剂；洋红试剂；溶剂；其中，对氯间二甲苯酚、酸性试剂和溶剂的体积比为5：(45‑80)：(55‑80)；所述酸性试剂包括冰醋酸。本发明的染色试剂，可染色出凸显结构中某些重要特征，如纹孔连接的节点、胞间连丝等，这些特征有利于更清晰的展示皮层发育和四分孢子囊的发育。
The present invention discloses a staining reagent and a method for obtaining a microscopic structure of a large seaweed with noded filamentous filaments. The invention relates to the field of biological sample preparation methods. The staining reagent comprises the following components: p-chloro-meta-xylenol; an acidic reagent; a magenta reagent; a solvent; wherein the volume ratio of p-chloro-meta-xylenol, the acidic reagent and the solvent is 5:(45-80):(55-80); the acidic reagent comprises glacial acetic acid. The staining reagent of the present invention can stain out certain important features in the structure, such as nodes connecting pits, plasmodesmata, etc. These features are conducive to a clearer display of cortical development and tetrasporangium development.
Description
Technical Field
The invention relates to the field of biological sample preparation methods, in particular to a staining reagent and a method for obtaining a microscopic structure of a large-scale seaweed with a section fiber small wire shape.
Background
Microstructural studies, a necessary means to reveal life phenomena and processes, have made many progress since the netherlands scientist hook invention microscope for over 300 years, but the research in this field is often affected by the quality of the tabletting technique during sample processing.
The kelp is a large-scale lower plant in the ocean, mainly species in the order CERAMIALES of Mesona, including Mesona Ceramiun, synechocystis Centroceras, dunaliella Polysiphonia, neurospora Neosiphonia, and Synechocystis Herposiphonia. Microstructure features such as cross section observation of algae, origin of tetrad sporangia, origin of pseudoroot, development of cortex and the like are important features for accurately identifying species, classifying species, carrying out vital activities and the like on the species. However, these species are too fine, with widths ranging from tens of microns to millimeters, and it is difficult to obtain clear and diverse microstructural features. In the past, the scholars in this field have mainly used paraffin sections to obtain their microstructural features. However, the method has obvious defects that 1) paraffin slicing is required to be subjected to multiple procedures of fixing, washing, dehydrating, transparentizing, wax dipping, embedding, slicing, spreading and baking, the operation is complex, time and labor are wasted, 2) paraffin slicing is required to be subjected to instruments such as a dehydrator, an embedding machine and a slicing machine, various reagents are required in the above operation procedures, the input cost is high, 3) certain algae have a width of only forty microns and are very thin, even if the paraffin slicing is difficult to cut out an ideal microstructure, 4) the microstructure obtained by the paraffin slicing is independent cross sections, the integrity is not possessed, the continuous change of a section of algae microstructure cannot be visually embodied, and 5) the paraffin slicing cannot obtain the cortex structural characteristics. There are also methods for dyeing and tabletting which are often used by students, but only cortical structures are often obtained, further clear and complete key microstructures cannot be obtained due to the defects of the formulation, and important features in the structures cannot be highlighted by dyeing, for example, in the prior art, nodes connected with pits and intercellular continuous filaments in seaweed structures cannot be observed by a dyeing method.
How to quickly obtain the microstructure of the kelp with the section fiber and the thread is obviously a scientific problem.
Disclosure of Invention
The first technical problem to be solved by the invention is as follows:
a staining reagent is provided.
The second technical problem to be solved by the invention is as follows:
a method for preparing the staining reagent is provided.
In order to solve the first technical problem, the invention adopts the following technical scheme:
A staining reagent comprising the following components:
P-chlorom-xylenol;
An acidic reagent;
A magenta reagent;
A solvent;
wherein, the volume ratio of the parachlorometaxylenol, the acid reagent and the solvent is 5 (45-80): 55-80;
The acidic reagent comprises glacial acetic acid.
According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:
1. According to the dyeing reagent, on one hand, the sample is dyed through the magenta reagent in the dyeing reagent, certain important characteristics in the structure, such as nodes connected with the pits, are highlighted, on the other hand, various connections, such as intercellular mucilage and intercellular continuous threads, pit connection and the like, exist between the cortical cells and the periaxial cells, between the periaxial cells and between the periaxial cells of the large-scale kelp with the section fiber, so that the connection between the cortical cells and the periaxial cells, between the periaxial cells and between the periaxial cells is required to be dissociated by utilizing the corrosiveness of parachlorometaxylenol and acetic acid, and then the connection between the cells in the cortex is reserved by grasping the volume ratio of parachlorometaxylenol, the acidic reagent and the solvent in the mixed reagent.
2. The dyeing reagent disclosed by the invention can dye certain important features in a salient structure, such as nodes connected by pits, intercellular continuous filaments and the like, by combining glacial acetic acid and a magenta reagent, and the features are favorable for displaying the development of cortex and the development of the ascomycetes of the tetrad more clearly. In addition, compared with phenol in the existing dyeing reagent, the use of the parachlorometaxylenol is more suitable for the large seaweed with the sarcopenia filis, and various clear and complete key microstructures such as the pseudoroot development mode, the tetrasporocyst development mode, the periaxial cell number, the continuous change process of the periaxial cell number, the cortical development process and the like of the large seaweed with the sarcopenia filis further obtained after the treatment of the parachlorometaxylenol.
3. The staining reagent of the invention can promote the separation of cytoplasmic walls and further make the intercellular continuous filaments clear.
According to one embodiment of the invention, the acidic reagent further comprises lactic acid. By adding an acidic reagent including lactic acid to the acidic reagent, the corrosive effect of the acidic reagent can be promoted.
According to one embodiment of the invention, the volume ratio of parachlorometaxylenol, acidic reagent and solvent is 5 (45-50): 55-60.
According to one embodiment of the present invention, the magenta reagent is added in an excess amount.
In order to solve the second technical problem, the invention adopts the following technical scheme:
A method of obtaining a micro-structure of a kelp having a fibrillar filament shape, comprising the steps of:
S1, configuring the dyeing reagent;
S2, mixing the seaweed sample with a staining reagent to obtain a stained sample;
s3, separating seaweed samples by taking sections as units, wherein each section is a cross section of one sample;
S4, breaking the links of the seaweed separated in the step S3, flattening the broken joints, and observing the microstructure through a microscope.
According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:
1. the method of the invention has the advantages of high tabletting speed, little cost, economy and applicability, and is beneficial to industrial production.
2. The method has good dyeing effect, can obtain various complete and clear microstructures, and is not easy to fade. The dyed sample has sterilizing and preservative effects due to the fact that the dyeing reagent component contains parachlorometaxylenol and an acidic reagent, and can be stored for a long time.
3. The method of the invention not only can obtain various microstructures with clear characteristics of the kelp, but also can intuitively embody continuous change of the microstructure characteristics of a section of kelp, and has important application value in aspects of morphological, anatomical, developmental and evolutionary research of the kelp.
According to one embodiment of the invention, in the step S1, the dyeing reagent is prepared by mixing an acidic reagent in a solvent to obtain a mixed solution, boiling the mixed solution to a boiling point, adding excessive magenta reagent, cooling, filtering, and adding parachlorometaxylenol into the filtrate to obtain the dyeing reagent.
According to one embodiment of the invention, in step S2, the sample and the staining reagent are mixed in a closed tank for a period of 5-6 hours. The mixing time is based on whether the corroded sample can be easily picked up by the acupuncture needle. In the invention, the connection between cells in the cortex is reserved by grasping the content of the ingredients for corrosion in the staining reagent and the time for corrosion, so as to facilitate the subsequent operations, such as opening the algae, the whole cortex or obtaining a cross section.
According to one embodiment of the invention, in step S2, there is further included the step of cleaning the seaweed sample with a brush.
According to one embodiment of the present invention, in step S3, seaweed samples are separated in knots using a separation tool including an acupuncture needle.
According to one embodiment of the invention, the step S3 further comprises the steps of washing the seaweed sample with a solvent to remove excess staining reagent and adding the solvent to suspend the seaweed sample.
According to one embodiment of the present invention, the cross section obtained in step S3 can be photographed directly under 4X and 10X objective lenses without a cover plate.
In step S4, the segments are spread out, including the steps of tiling the segments on a slide and covering the slide with a cover slip.
According to the embodiment of the invention, in step S4, the broken knots are flattened to obtain the cortex structure.
According to one embodiment of the invention, the segments are tiled on a glass slide, and after the cover slip is covered, the steps of adding a moisturizing preservative and a sealing piece are further included.
In another aspect the invention also relates to the use of said staining reagent in morphology or anatomy or in development or in chemistry. Comprising a staining reagent as described in the example of aspect 1 above. The application adopts all the technical schemes of the dyeing reagent, so that the dyeing reagent has at least all the beneficial effects brought by the technical schemes of the embodiment.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a microstructure of 400 μm of a sample of the kelp of example 1 before treatment;
FIG. 2 is a 20 μm microstructure of the sample of the kelp of example 1 before treatment;
FIG. 3 is a microstructure of the clustering of the kappaphycus alvarezii samples of example 1 at 400 μm prior to treatment;
FIG. 4 is a microstructure of the ascus (T) ring of 20 μm before treatment of the sample of the kelp of example 1;
FIG. 5 is a 100 μm microstructure of a sample of the kelp of example 2 with a section fiber size and a thread shape before treatment;
FIG. 6 is a microstructure of a sample of a kelp with a section fiber and a thread after neutralization treatment in example 1;
FIG. 7 is a microstructure of a section cross section of a sample of a kelp with section fiber and wire;
FIG. 8 is a graph showing the microstructure of the tetraspora (T) ring of a sample of a kelp having a section fiber wire shape after treatment in example 1;
FIG. 9 is a section cross-sectional microstructure of the tetraspora tetradactyla (T) obtained by treatment of the sample of the kelp having section fiber small and wire form;
FIG. 10 is a microstructure of the upper portion of the algal body obtained by treating the sample of the kelp having the Artemisia integrifolia;
FIG. 11 is a microstructure of the lower portion of the algal body obtained by treating a sample of a kelp having a section fiber small wire shape in example 2;
FIG. 12 is a microstructure of a sample of a kelp with a section fiber and a thread after treatment in example 1;
FIG. 13 is a microstructure of a sample of a kelp with a section fiber and a thread after a comparative treatment;
FIG. 14 is a microstructure of the fruit cover cells of the taro hedge prior to treatment in example 3;
FIG. 15 is a microstructure of the fruit cover cells of the taro hedge after the treatment of example 3;
FIG. 16 is a microstructure of a sample of cyst membrane cells of the Heterophylla fence prior to treatment in example 4;
FIG. 17 is a microstructure of a sample of cyst membrane cells of the Heterophylla fence after treatment in example 4.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
The reagents, methods and apparatus employed in the present invention, unless otherwise specified, are all conventional in the art.
Example 1
Example 1 the sample of the kelp with the section fiber is selected as the mesona species Ceramium GARDNERI KYLIN.
A staining reagent comprising the following components:
5ml of parachlorometaxylenol;
45ml of glacial acetic acid;
A magenta reagent;
Distilled water 55ml;
wherein the addition amount of the magenta reagent is excessive.
A method of obtaining a micro-structure of a kelp having a fibrillar filament shape, comprising the steps of:
S1, preparing the dyeing reagent, namely, 45ml of glacial acetic acid and 55ml of distilled water, boiling the mixed solution to a boiling point, adding excessive magenta powder until the mixture cannot be dissolved, cooling the mixture, filtering the mixture, adding 5ml of parachlorometaxylenol into the filtrate, and uniformly mixing the filtrate to obtain the dyeing reagent;
S2, selecting a target sample, cleaning the surface of the algae for 2-3 times by using a brush, removing impurities and miscellaneous algae on the surface of the algae, cutting out algae segments to be observed, putting the selected sample segments into a dyeing tank, adding a dyeing reagent, and performing airtight dyeing and corrosion for about 5 hours;
S3, picking a corroded sample by using a needle tube, placing the sample in a culture dish, dripping clear water to clean the redundant dye solution, adding clear water to enable the sample to suspend lightly, and opening a section of the sample by using an acupuncture needle under a dissecting mirror, wherein each section is a cross section of the sample, and the cross section can be directly photographed by selecting characteristics under 4X and 10X objective lenses without a cover plate;
s4, the section of the link is broken by an acupuncture needle, the broken section is carefully paved on a glass slide, and the cover glass is covered to enable the broken section to be flat, and the flat broken section is a cortical structure.
S5, adding a moisturizing preservative and sealing.
Example 2
Example 2 differs from example 1 in that the species selected is different.
Example 2 the sample of the selected kelp with the section fiber small filiform is a species of the genus medullaria.
A staining reagent comprising the following components:
5ml of parachlorometaxylenol;
45ml of glacial acetic acid;
A magenta reagent;
Distilled water 55ml;
wherein the addition amount of the magenta reagent is excessive.
A method of obtaining a micro-structure of a kelp having a fibrillar filament shape, comprising the steps of:
s1, taking a sample to be observed, cleaning the surface of an alga body for 2-3 times by using a brush, removing impurities and miscellaneous alga on the surface of the alga body, and cutting the sample into alga segments with 3-4 limited branches;
S2, transferring the algae segments into a dyeing tank, adding a dyeing reagent, dyeing and corroding for about 5 hours;
S3, transferring the dyed and dissociated sample into a small beaker, washing the sample for 3-4 times by using distilled water, washing the surface dye liquor of the algae segments, then flatly placing the algae segments on a clean glass slide, dripping a cover plate, and sucking the redundant water.
S4, lightly pressing the algae segments through the cover glass continuously, and simultaneously, placing the sample under a microscope to observe whether the algae segments are scattered or not, so that cell monolayers of the algae segments are paved on the glass slide. Dripping 1-2 drops of sealing liquid, and sealing.
S5, photographing under an imaging microscope to record microscopic features.
Example 3
Example 3 differs from example 1 in that the sample is different. Wherein, example 1 is a selected sample of a large seaweed with a small and filiform node, and example 3 is a selected sample of a cyst membrane cell of a taro hedge.
Example 4
Example 4 differs from example 1 in that the sample is different. Among them, example 1 was a sample of selected kelp with a small and filiform node, and example 4 was a sample of selected cyst cells of a Heterophylla fence.
Comparative example
The comparative example differs from example 1 in that the dyeing reagent composition is different. Among them, the dyeing reagent components of the comparative example were p-chlorom-xylenol, lactic acid, aniline blue and distilled water. The dyeing reagent components of example 1 were p-chlorom-xylenol, glacial acetic acid, magenta reagent and distilled water.
Through testing, the contrast-example dyed seaweed sample with the section fiber small filiform can not clearly observe the development of the ascus tetrandra.
Performance test:
Microstructure photographs were taken of the Mesona species Ceramium GARDNERI KYLIN selected for the Mesona samples of the Mesona-containing filamentous kelp of example 1.
Wherein FIG. 1 is a microstructure of 400 μm of a sample of a kelp with a section fiber thread before treatment in example 1. From FIG. 1, the cortical structure (C) and the formation process of the sample of the kelp with the Artemisia integrifolia shape cannot be seen.
FIG. 2 is a 20 μm microstructure of a sample of a kelp with a section fiber, before treatment in example 1.
Wherein FIG. 3 is a microstructure of a sample of a kelp with a section fiber thread-like kelp clustering of a pseudoroot at 400 μm before treatment in example 1.
FIG. 4 is a microstructure of the ascus (T) ring of the kelp sample of the section fiber-like kelp at 20. Mu.m, before the treatment of example 1.
FIG. 5 is a 100 μm microstructure of a sample of a kelp with a section fiber, before treatment in example 2.
FIG. 6 is a microstructure of a sample of a kelp with a section fiber and a thread after neutralization treatment in example 1. A to I in fig. 6 clearly show the overall process of progressive formation of mature cortical tissue from one pericytes (P) based on intercellular continuous filaments between cortical cells. Wherein A in FIG. 6 is a pericyte, B in FIG. 6 is a pericyte which first divides into 1-stage cortical cells at the left upper corner, C in FIG. 6 is a pericyte which then divides into 1-stage cortical cells at the right upper corner, D in FIG. 6 is a primary cortical cell which then further divides into two-stage cortical cells, E in FIG. 6 is a primary cortical cell which starts dividing into the left lower corner of the pericyte after the occurrence of the secondary cortical cells at the upper end of the pericyte, F in FIG. 6 is a pericyte which starts dividing into 3-stage cortical cells at the upper end of the pericyte, and at the same time, G in FIG. 6 is a secondary cortical cell which also starts dividing into the right lower end of the pericyte, H in FIG. 6 is a pericyte which starts dividing into 4-stage cortical cells at the upper end, and at the same time, 1-stage cortical cell which starts dividing into 1-stage cortical cells directly below the pericyte, and I in FIG. 6 is a mature cortical tissue which comprises a pericyte which starts dividing into 4-stage cortical cells at the two sides, and at the lower-stage cortical cells which develop directly below the 2-stage cortical cells. I.e., a-I in fig. 6 is a complete cortical development process. It follows that the course of sample cortical development can be observed by the stain and method of the present invention.
Wherein FIG. 7 is a microstructure of a section cross section of a sample of a kelp with section fiber and filiform kelp obtained after the treatment of example 1. As can be seen from FIG. 7, the section cross section obtained after the treatment of example 1 clearly shows the number of periaxial cells (P). In the corresponding FIG. 2, the number of pericytes (P) cannot be shown.
FIG. 8 is a microstructure of the tetraspora (T) ring of a sample of a kelp with a section fiber, obtained after the treatment of example 1. From fig. 8, it can be clearly shown that the pseudoroot is generated from the pericytes (P) and the primary cortical cells (C). While the corresponding FIG. 3 shows a clustered pseudoroot, it is not known whether the pseudoroot is produced by pericytes (P) or cortical cells (C).
FIG. 9 is a microstructure of a section cross section of a tetrasporangium (T) obtained by treating a sample of a kelp with a section fibrillar filament in example 1. From fig. 9, it is clear that two ascus (T) can be produced per periaxial cell (P) based on the node of the ascus to periaxial cell pit connection. While the corresponding FIG. 4 shows that the tetrasporangia (T) is circulating at the node, it is not known from which cells the tetrasporangia (T) develop.
FIG. 10 is a microstructure of the upper part of the alga body obtained by treating the sample of the kelp with the section fiber and the thread type alga in example 2.
FIG. 11 is a microstructure of the lower part of the algal body obtained by treating a sample of a kelp having a Artemisia capillaris strain in example 2.
From fig. 10 and 11, the number of cells around the axis and their change patterns can be clearly seen. In the corresponding FIG. 5, the number of cells around the axis cannot be seen.
FIG. 12 is a microstructure of a sample of a kelp of the section fiber-containing thread-like kelp after treatment in example 1, in which the development of the ascospore is clearly shown, wherein A in FIG. 12 is ascospores, B in FIG. 12 is ascospores, and C in FIG. 12 is ascospores.
FIG. 13 is a microstructure of a sample of a kelp with a section fiber thread after treatment in a comparative example, tested, and the sample of a kelp with a section fiber thread after dyeing in a comparative example, the development of the ascus of the quaternary spore could not be clearly observed. The comparative example is based on the difference from example 1 in that the dyeing reagent composition is different. Among them, the dyeing reagent components of the comparative example were p-chlorom-xylenol, lactic acid, aniline blue and distilled water. The dyeing reagent components of example 1 were p-chlorom-xylenol, glacial acetic acid, magenta reagent and distilled water, and it is known that the process of the development of the tetrasporocyst can be clearly demonstrated using the dyeing reagent of the example of the present invention.
The test was performed on the sample of the cyst membrane cells of the taro hedge of example 3, wherein fig. 14 is a microstructure of the cyst membrane cells of the taro hedge before the treatment of example 3. FIG. 15 is a microstructure of the fruit cover cells of the taro hedge after the treatment of example 3. From fig. 14-15, it can be seen that the intercellular continuous filaments are clearly seen in the microstructure after treatment, thus proving that the staining reagent of the present invention can promote cytoplasmic wall separation and can further make the intercellular continuous filaments clearly visible.
The test was performed on the cyst membrane cell samples of the hedge of example 4, wherein fig. 16 is a microstructure of the cyst membrane cell samples of the hedge of example 4 before treatment. FIG. 17 is a microstructure of a sample of cyst membrane cells of the Heterophylla fence after treatment in example 4. From FIGS. 16 to 17, it is understood that, in the microstructure obtained by the treatment of example 4, the intercellular continuous filaments are clearly seen, thereby proving that the staining reagent of the present invention can promote the separation of cytoplasmic walls and can further make the intercellular continuous filaments clear.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in the relevant art are intended to be included in the scope of the present invention.
Claims (8)
1. A method for obtaining a micro-structure of a kelp with a section fiber thread using a staining reagent, characterized by:
the dyeing reagent comprises the following components:
P-chlorom-xylenol;
An acidic reagent;
A magenta reagent;
A solvent;
The solvent is distilled water;
wherein the volume ratio of the parachlorometaxylenol to the acidic reagent to the solvent is 5:45-80:55-80;
The acidic reagent comprises glacial acetic acid;
the preparation method of the dyeing reagent comprises the following steps:
mixing an acidic reagent in a solvent to obtain a mixed solution, boiling the mixed solution to a boiling point, adding excessive magenta reagent, cooling, filtering, and adding parachlorometaxylenol into the filtrate to obtain a dyeing reagent;
a method for obtaining a micro-structure of a large seaweed with a section fiber thread using the dyeing agent, comprising the steps of:
S1, mixing a seaweed sample with the dyeing reagent to obtain a dyed sample;
S2, separating seaweed samples by taking sections as units, wherein each section is a cross section of one sample;
s3, breaking the links of the seaweed separated in the step S2, flattening the broken joints, and observing the microstructure through a microscope.
2. The method of claim 1, wherein the acidic reagent further comprises lactic acid.
3. The method according to claim 1, wherein the volume ratio of parachlorometaxylenol, acidic reagent and solvent is 5:45-50:55-60.
4. The method according to claim 1, wherein in step S1, the sample and the staining reagent are mixed in a closed tank for a period of 5 to 6 hours.
5. The method according to claim 1, wherein in step S1, the seaweed sample is cleaned with a brush.
6. The method according to claim 1, wherein in step S2, the seaweed sample is separated in knots using a separation tool comprising an acupuncture needle.
7. The method according to claim 1, wherein in step S2, the method further comprises the steps of washing the seaweed sample with a solvent to remove excess staining reagent, and adding the solvent to suspend the seaweed sample.
8. The method of claim 1, wherein the step of flattening the segments in step S3 comprises the step of tiling the segments on a slide and covering the slide with a cover slip.
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| JP2003329670A (en) * | 2002-05-15 | 2003-11-19 | Kyokuto Seiyaku Kogyo Kk | Reagent for urinary sediment examination and method of urinary sediment examination |
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| GB1151740A (en) * | 1965-10-14 | 1969-05-14 | Sumitomo Chemical Co | Process for Dyeing Polyolefinic Materials |
| JPS58107198A (en) * | 1981-12-18 | 1983-06-25 | Hitachi Ltd | Enzymatic dyeing of separated protein |
| KR100578034B1 (en) * | 2003-09-23 | 2006-05-11 | 주식회사 정우인터내셔날 | Dye composition for freeze section staining and freeze section staining method |
| WO2005105984A2 (en) * | 2004-04-12 | 2005-11-10 | The Trustees Of The University Of Pennsylvania | Culture conditions and growth factors affecting fate determination, self-renewal and expansion of mouse spermatogonial stem cells |
| US20070218278A1 (en) * | 2004-08-04 | 2007-09-20 | Sunghoon Choi | Method for Dyeing Microfibers and Fabric Woven with Microfibers Dyed Thereby |
| CN104215485A (en) * | 2014-09-05 | 2014-12-17 | 江苏农林职业技术学院 | Flaking method of petunia chromosome |
| WO2019118591A1 (en) * | 2017-12-12 | 2019-06-20 | Cbd Sciences Group Llc | Burn treatment formulation and dressing and method for treating burns |
| JP2021522938A (en) * | 2018-05-09 | 2021-09-02 | ザ ジョンズ ホプキンス ユニバーシティ | Nanofiber-hydrogel complex for cell and tissue delivery |
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