CN114136737B - Microscopic tabletting method for observing lower surface characteristics of mugwort leaves - Google Patents
Microscopic tabletting method for observing lower surface characteristics of mugwort leaves Download PDFInfo
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- CN114136737B CN114136737B CN202111487861.3A CN202111487861A CN114136737B CN 114136737 B CN114136737 B CN 114136737B CN 202111487861 A CN202111487861 A CN 202111487861A CN 114136737 B CN114136737 B CN 114136737B
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- 235000003261 Artemisia vulgaris Nutrition 0.000 title claims abstract description 40
- 240000006891 Artemisia vulgaris Species 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000003292 glue Substances 0.000 claims abstract description 70
- 230000000762 glandular Effects 0.000 claims abstract description 46
- 210000004209 hair Anatomy 0.000 claims abstract description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 39
- 230000003287 optical effect Effects 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 27
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 claims abstract description 21
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 18
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 claims abstract description 18
- 210000001339 epidermal cell Anatomy 0.000 claims abstract description 17
- 239000011259 mixed solution Substances 0.000 claims abstract description 17
- 238000004043 dyeing Methods 0.000 claims abstract description 15
- 238000002791 soaking Methods 0.000 claims abstract description 13
- 239000012153 distilled water Substances 0.000 claims abstract description 10
- 239000011521 glass Substances 0.000 claims abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000010931 gold Substances 0.000 claims abstract description 10
- 229910052737 gold Inorganic materials 0.000 claims abstract description 10
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 9
- 238000004061 bleaching Methods 0.000 claims abstract description 9
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 9
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 9
- 150000002500 ions Chemical class 0.000 claims abstract description 9
- 229940117955 isoamyl acetate Drugs 0.000 claims abstract description 9
- 238000007789 sealing Methods 0.000 claims abstract description 9
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 9
- 235000009518 sodium iodide Nutrition 0.000 claims abstract description 9
- 239000000919 ceramic Substances 0.000 claims description 17
- 230000007935 neutral effect Effects 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 9
- 238000004026 adhesive bonding Methods 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 8
- 238000000576 coating method Methods 0.000 claims description 8
- 239000006059 cover glass Substances 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 8
- 238000000399 optical microscopy Methods 0.000 claims description 8
- 230000018044 dehydration Effects 0.000 claims description 6
- 238000006297 dehydration reaction Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000005507 spraying Methods 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 5
- 238000009826 distribution Methods 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 241000269837 Artemisia dubia Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000035617 depilation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 238000005360 mashing Methods 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Classifications
-
- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to a microscopic flaking method for observing the lower surface characteristics of mugwort leaves, which effectively solves the problem that an optical microscope or a scanning electron microscope can easily observe the distribution of air holes, glandular hairs and non-glandular hairs on the lower surface of mugwort leaves and a vertical wall type sample of epidermal cells, fresh mugwort leaves are glued, the non-glandular hairs on the lower surface are peeled off, small pieces of leaves are taken, distilled water is used for rinsing to remove residual glue, a part of leaves are fixed by methanol, dehydrated by absolute alcohol, soaked by isoamyl acetate, dried by a carbon dioxide critical point and sprayed with gold by an ion meter, and the method is used for scanning electron microscope flaking observation; soaking the other part of small leaves in a mixed solution of sodium hydroxide and sodium hypochlorite, dissociating mesophyll tissues, bleaching, rinsing with clear water, placing on a glass slide, dyeing with toluidine blue O, making sodium iodide solution transparent, and sealing the glass slide to obtain the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescent microscope. The invention has the advantages of simple operation, low cost, small workload, high success rate, clear observation and good effect.
Description
Technical Field
The invention relates to the field of microscopic identification of crude drugs, in particular to a microscopic tabletting method for observing the characteristics of the lower surface of mugwort leaf, which is used for treating the lower surface of mugwort leaf so as to observe the characteristics of leaf epidermis.
Background
Mugwort leaf is a common traditional Chinese medicine and is produced in most areas of the country. Sun-drying folium Artemisiae Argyi, mashing to obtain "moxa" for moxibustion. Some characteristics of the surface of the mugwort leaf, such as the type and density of the hair quilt, the type of air holes, the air hole index, the vertical peripheral wall pattern of the epidermal cells and the like, have important reference values for identifying the mugwort leaf, and have important effects on researching plant ecology, plant physiology and variety breeding of the mugwort leaf. The upper surface of the mugwort leaf is distributed with glandular hairs and sparse non-glandular hairs, and the upper surface is easy to observe by a scanning electron microscope; the microscopic features of the lower surface are difficult to observe by scanning electron microscopy because the lower surface is densely covered with thick, non-glandular hairs, but not glandular hairs, and covers the glandular hairs, stomata, and epidermal cells that are distributed thereunder. The surface features of the leaves are usually observed by an optical microscope, namely a surface peeling method, a surface peeling method and a transparent surface preparation method, however, as the folium artemisiae argyi is a paper leaf and the leaves are thin, glandular hairs densely distributed on the upper surface and the lower surface of the folium artemisiae argyi are in concave positions, the surface peeling method and the surface peeling method are difficult to implement, and the microscopic features such as pores, epidermal cells and the like are difficult to see because the lower surface of the folium artemisiae argyi is densely covered by thick non-glandular hairs by a simple transparent surface preparation method. Due to the restrictions of the existing tabletting technology, at present, whether a scanning electron microscope or an optical microscope is used, microscopic characteristics such as air holes, glandular hair and non-glandular hair distribution on the lower surface of the mugwort leaf, and the vertical peripheral wall style of the epidermal cells are difficult to observe well. Therefore, a special preparation method is very required to be designed to observe the characteristics of the lower surface of the mugwort leaf, but no disclosure is reported so far.
Disclosure of Invention
Aiming at the situation, the invention aims to overcome the defects of the prior art and provide a microscopic tabletting method for observing the characteristics of the lower surface of the mugwort leaf, which can effectively solve the problem that an optical microscope or a scanning electron microscope can easily observe the distribution of air holes, glandular hairs and non-glandular hairs on the lower surface of the mugwort leaf and the vertical wall type of epidermal cells.
The technical scheme of the invention is that a microscopic tabletting method for observing the lower surface characteristics of mugwort leaves comprises the following steps:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 6-8 mm square small blades at the position where the non-glandular hair is stripped, transferring the small blades into distilled water for rinsing, repeatedly rinsing for 3-5 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, wherein one part is fixed with methanol at room temperature for 6-12 h, the methanol is transferred into absolute alcohol for dehydration for 1h, the absolute alcohol is soaked for 10-20 min by treatment of isoamyl acetate, the critical point of carbon dioxide is dried, an ion instrument sprays gold and is used for slice making and observation by a scanning electron microscope, and the other part is used for slice making by an optical microscope;
(5) Optical microscopy slide: placing the rest other part of small leaves into a mixed solution of sodium hydroxide with the mass concentration of 5% -10% and sodium hypochlorite with the mass concentration of 5% -10% in a volume ratio of 1:1, soaking for 6-12 h at room temperature, dissociating mesophyll tissues, and bleaching simultaneously;
(6) Dyeing: taking out the blades from the mixed solution, rinsing in clear water for 1-3 min, placing the blades on a glass slide, and dripping one drop (about 0.02 ml) of toluidine blue O solution above the blades for dyeing for 1-3 min;
the toluidine blue O solution is prepared by dissolving toluidine blue O in phosphate buffer solution with pH of 4.0, and the mass concentration of the toluidine blue O is 0.1% -0.5%;
(7) And (3) taking out the leaves, using 40-60% sodium iodide solution to be transparent at room temperature for 0.5-1 h, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
The invention has the characteristics of simple operation, low cost, small workload, high success rate, clear observation and good effect, can effectively solve the technical problem of surface observation of the mugwort leaf, has low technical requirements for operators, and is a great innovation in microscopic observation of the characteristic flaking of the lower surface of the mugwort leaf.
Drawings
FIG. 1 is a view of the present invention under a fluorescence microscope, showing the epidermal stomata under mugwort leaf, non-glandular Mao Maoji (white triangle) and glandular wool (white arrow), and under a low power microscope for observation and counting.
FIG. 2 is a diagram showing the observation of the epidermal pores, epidermal cells and non-glands Mao Maoji (arrows) under the mugwort leaf under a bright field microscope according to the present invention.
Fig. 3 is a view showing the lower surface of mugwort leaf observed by scanning electron microscope after depilation in the invention.
Detailed Description
The following describes in detail the embodiments of the present invention with reference to specific cases and examples.
The invention may be embodied by the following examples.
Example 1
The invention discloses a microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which comprises the following steps of:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 7mm square small blades at the position where the non-glandular hair is stripped, transferring the small blades into distilled water for rinsing, repeatedly rinsing for 4 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, fixing one part with methanol at room temperature for 9h, transferring into absolute alcohol for dehydration for 1h, treating and soaking with isoamyl acetate for 15min, drying carbon dioxide critical point, spraying gold by an ion instrument, tabletting and observing by a scanning electron microscope, and tabletting the other part by an optical microscope;
(5) Optical microscopy slide: placing the rest other part of small leaves into mixed solution of sodium hydroxide and sodium hypochlorite with the mass concentration of 7.5% and the volume ratio of 1:1, soaking for 9 hours at room temperature, dissociating mesophyll tissue, and bleaching simultaneously;
(6) Dyeing: taking out the leaves from the mixed solution, rinsing in clear water for 2min, placing on a glass slide, and dropwise adding one drop (about 0.02 ml) of toluidine blue O solution above the leaves for dyeing for 2min, wherein the mass concentration of the toluidine blue O solution is 0.3%;
(7) And taking out the leaves, using 50% sodium iodide solution to be transparent for 0.7h at room temperature, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
Example 2
The invention discloses a microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which comprises the following steps of:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 6mm square small blades at the position where the non-glandular hair is stripped, transferring the small blades into distilled water for rinsing, repeatedly rinsing for 3 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, fixing one part with methanol at room temperature for 6.5h, transferring into absolute alcohol for dehydration for 1h, soaking in isoamyl acetate for 20min, drying at critical point of carbon dioxide, spraying gold by an ion instrument, tabletting and observing by a scanning electron microscope, and tabletting the other part by an optical microscope;
(5) Optical microscopy slide: placing the rest other part of small leaves into mixed solution of sodium hydroxide and sodium hypochlorite with the mass concentration of 6% and the volume ratio of 1:1, soaking at room temperature for 11.5h, dissociating mesophyll tissue, and bleaching at the same time;
(6) Dyeing: taking out the leaves from the mixed solution, rinsing in clear water for 1min, placing on a glass slide, and dropwise adding one drop (about 0.02 ml) of toluidine blue O solution above the leaves for 1min, wherein the mass concentration of the toluidine blue O solution is 0.5%;
(7) And taking out the leaves, using 60% sodium iodide solution to be transparent for 0.5h at room temperature, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
Example 3
The invention discloses a microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which comprises the following steps of:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 8mm square small blades at the position where the non-glandular hair is stripped, transferring into distilled water for rinsing, repeatedly rinsing for 5 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, wherein one part is fixed with methanol at room temperature for 11.5 hours, dehydrated with absolute alcohol for 1 hour, soaked with isoamyl acetate for 10 minutes, dried at a carbon dioxide critical point, sprayed with gold by an ion instrument, used for scanning electron microscope tabletting and observation, and the other part is used for optical microscope tabletting;
(5) Optical microscopy slide: placing the rest other part of small leaves into mixed solution of sodium hydroxide and sodium hypochlorite with the mass concentration of 9.5% and the volume ratio of 1:1, soaking at room temperature for 6 hours, dissociating mesophyll tissue, and bleaching simultaneously;
(6) Dyeing: taking out the leaves from the mixed solution, rinsing in clear water for 3min, placing on a glass slide, and dropwise adding one drop (about 0.02 ml) of toluidine blue O solution above the leaves for 3min, wherein the mass concentration of the toluidine blue O solution is 0.1%;
(7) And (3) taking out the leaves, using 40% sodium iodide solution to be transparent at room temperature for 1h, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
From the above, the method is scientific and reasonable, easy to operate and good in observation effect, the quality and the medicinal value of the mugwort can be effectively ensured, and the result shows that after repeated trial and experiment (taking example 1 as an example), the method is simple and convenient to operate: and (3) firmly adhering the lower surface of the mugwort leaf to paper by using mixed glue, and completely peeling off non-glandular hairs from the lower surface of the mugwort leaf after tearing off the paper strip, and washing off residual mixed glue on the mugwort leaf by using water. A portion of the material was fixed with methanol and observed by a scanning electron microscope. After the other part of the materials are used for transparent tabletting treatment, the distribution of air holes, leaf epidermis and glandular hair and non-glandular hair handles can be clearly seen by a bright field microscope or a fluorescence microscope. After the mugwort leaf is treated by the tabletting method, the type of air holes, the air hole index and the vertical peripheral wall of the epidermal cells can be clearly observed under a scanning electron microscope and an optical microscope (fluorescence or bright field microscope), so that the number of glandular hairs and non-glandular hairs can be conveniently observed and counted, the study and analysis of the lower surface structure of the mugwort leaf can be facilitated, and the quality and the medicinal value of mugwort leaf products can be ensured.
The same and similar results were obtained in the same experiment as in example 1, but also in other examples, and are not shown here.
In summary, the microscopic flaking method for observing the lower surface characteristics of the mugwort leaves is characterized in that fresh leaves are taken, a layer of mixture of neutral glue and ceramic glue is coated on the lower surfaces of the leaves, a small paper strip is stuck on the upper side of the mixed glue, lightly pressed and placed on a tray until the paper strip is stuck tightly, and the paper strip is carefully torn. At this point, the non-glandular hairs are stuck on the paper strip and peeled off from the leaves. The leaves were cut, rinsed with distilled water to wash off the remaining mixed gum, fixed with methanol, dehydrated, dried, and sprayed with gold, and then used for observation under a scanning electron microscope. The method has the characteristics of simple operation, low cost, small workload, high success rate, clear observation and good effect, solves the problem that the prior art cannot finish the preparation, has the success rate of one-time preparation of about 99 percent, can easily achieve the aim of satisfactory experiments, provides technical support for researching and analyzing the surface structure of the mugwort leaf, ensures the quality and medicinal value of the mugwort leaf, and is a great innovation for observing the microscopic preparation of the lower surface characteristics of the mugwort leaf.
Claims (4)
1. A microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which is characterized by comprising the following steps:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 6-8 mm square small blades at the position where the non-glandular hair is stripped, transferring the small blades into distilled water for rinsing, repeatedly rinsing for 3-5 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, wherein one part is fixed with methanol at room temperature for 6-12 h, the methanol is transferred into absolute alcohol for dehydration for 1h, the absolute alcohol is soaked for 10-20 min by treatment of isoamyl acetate, the critical point of carbon dioxide is dried, an ion instrument sprays gold and is used for slice making and observation by a scanning electron microscope, and the other part is used for slice making by an optical microscope;
(5) Optical microscopy slide: placing the rest other part of small leaves into a mixed solution of sodium hydroxide with the mass concentration of 5% -10% and sodium hypochlorite with the mass concentration of 5% -10% in a volume ratio of 1:1, soaking for 6-12 h at room temperature, dissociating mesophyll tissues, and bleaching simultaneously;
(6) Dyeing: taking out the blades from the mixed solution, rinsing in clear water for 1-3 min, placing the blades on a glass slide, and dropwise adding a drop of toluidine blue O solution above the blades for dyeing for 1-3 min;
the toluidine blue O solution is prepared by dissolving toluidine blue O in phosphate buffer solution with pH of 4.0, and the mass concentration of the toluidine blue O is 0.1% -0.5%;
(7) And (3) taking out the leaves, using 40-60% sodium iodide solution to be transparent at room temperature for 0.5-1 h, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
2. A microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which is characterized by comprising the following steps:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 7mm square small blades at the position where the non-glandular hair is stripped, transferring the small blades into distilled water for rinsing, repeatedly rinsing for 4 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, fixing one part with methanol at room temperature for 9h, transferring into absolute alcohol for dehydration for 1h, treating and soaking with isoamyl acetate for 15min, drying carbon dioxide critical point, spraying gold by an ion instrument, tabletting and observing by a scanning electron microscope, and tabletting the other part by an optical microscope;
(5) Optical microscopy slide: placing the rest other part of small leaves into mixed solution of sodium hydroxide and sodium hypochlorite with the mass concentration of 7.5% and the volume ratio of 1:1, soaking for 9 hours at room temperature, dissociating mesophyll tissue, and bleaching simultaneously;
(6) Dyeing: taking out the leaves from the mixed solution, rinsing in clear water for 2min, placing on a glass slide, and dropwise adding a drop of toluidine blue O solution above for dyeing for 2min, wherein the mass concentration of the toluidine blue O solution is 0.3%;
(7) And taking out the leaves, using 50% sodium iodide solution to be transparent for 0.7h at room temperature, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
3. A microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which is characterized by comprising the following steps:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 6mm square small blades at the position where the non-glandular hair is stripped, transferring the small blades into distilled water for rinsing, repeatedly rinsing for 3 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, fixing one part with methanol at room temperature for 6.5h, transferring into absolute alcohol for dehydration for 1h, soaking in isoamyl acetate for 20min, drying at critical point of carbon dioxide, spraying gold by an ion instrument, tabletting and observing by a scanning electron microscope, and tabletting the other part by an optical microscope;
(5) Optical microscopy slide: placing the rest other part of small leaves into mixed solution of sodium hydroxide and sodium hypochlorite with the mass concentration of 6% and the volume ratio of 1:1, soaking at room temperature for 11.5h, dissociating mesophyll tissue, and bleaching at the same time;
(6) Dyeing: taking out the leaves from the mixed solution, rinsing in clear water for 1min, placing on a glass slide, and dropwise adding a drop of toluidine blue O solution above for dyeing for 1min, wherein the mass concentration of the toluidine blue O solution is 0.5%;
(7) And taking out the leaves, using 60% sodium iodide solution to be transparent for 0.5h at room temperature, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
4. A microscopic tabletting method for observing the lower surface characteristics of mugwort leaves, which is characterized by comprising the following steps:
(1) Blade gluing: uniformly coating a thin layer of mixture of neutral glue and ceramic glue on the back of the blade, wherein the volume ratio of the neutral glue to the ceramic glue is 2:1, and sticking a paper strip on the mixed glue, so as to ensure firm adhesion;
(2) Stripping non-glandular hairs: after the mixed glue is dried, tearing off the paper strip, and peeling off the non-glandular hairs on the lower surface from the lower surface to be adhered to the paper strip;
(3) Removing residual mixed glue: cutting 8mm square small blades at the position where the non-glandular hair is stripped, transferring into distilled water for rinsing, repeatedly rinsing for 5 times, and removing residual mixed glue on the blades to obtain clean small blades;
(4) Preparing a scanning electron microscope: dividing clean small leaves into two parts, wherein one part is fixed with methanol at room temperature for 11.5 hours, dehydrated with absolute alcohol for 1 hour, soaked with isoamyl acetate for 10 minutes, dried at a carbon dioxide critical point, sprayed with gold by an ion instrument, used for scanning electron microscope tabletting and observation, and the other part is used for optical microscope tabletting;
(5) Optical microscopy slide: placing the rest other part of small leaves into mixed solution of sodium hydroxide and sodium hypochlorite with the mass concentration of 9.5% and the volume ratio of 1:1, soaking at room temperature for 6 hours, dissociating mesophyll tissue, and bleaching simultaneously;
(6) Dyeing: taking out the leaves from the mixed solution, rinsing in clear water for 3min, placing on a glass slide, and dropwise adding a drop of toluidine blue O solution above for dyeing for 3min, wherein the mass concentration of the toluidine blue O solution is 0.1%;
(7) And (3) taking out the leaves, using 40% sodium iodide solution to be transparent at room temperature for 1h, sealing the leaves with a cover glass water, and obtaining the optical microscope lens for observing air holes and epidermal cells by using a common optical microscope or a fluorescence microscope.
Priority Applications (1)
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