CN111238892B - Permanent flaking method of lichen sporophore microstructure - Google Patents
Permanent flaking method of lichen sporophore microstructure Download PDFInfo
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- CN111238892B CN111238892B CN202010059784.0A CN202010059784A CN111238892B CN 111238892 B CN111238892 B CN 111238892B CN 202010059784 A CN202010059784 A CN 202010059784A CN 111238892 B CN111238892 B CN 111238892B
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000000463 material Substances 0.000 claims abstract description 20
- 230000008014 freezing Effects 0.000 claims abstract description 14
- 238000007710 freezing Methods 0.000 claims abstract description 14
- 238000004043 dyeing Methods 0.000 claims abstract description 7
- 238000007789 sealing Methods 0.000 claims abstract description 7
- 239000003292 glue Substances 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- OINQDWUHUPWLHC-UHFFFAOYSA-N 2-hydroxypropanoic acid;phenol Chemical compound CC(O)C(O)=O.OC1=CC=CC=C1 OINQDWUHUPWLHC-UHFFFAOYSA-N 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 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 claims description 3
- 239000005315 stained glass Substances 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims 3
- 238000002360 preparation method Methods 0.000 claims 1
- 210000003484 anatomy Anatomy 0.000 abstract description 8
- 230000006378 damage Effects 0.000 abstract description 3
- 208000005156 Dehydration Diseases 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000018044 dehydration Effects 0.000 abstract description 2
- 238000006297 dehydration reaction Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 235000013399 edible fruits Nutrition 0.000 description 8
- 238000012014 optical coherence tomography Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 5
- 239000000523 sample Substances 0.000 description 3
- 241000235349 Ascomycota Species 0.000 description 2
- 241000512897 Elaeis Species 0.000 description 2
- 235000001950 Elaeis guineensis Nutrition 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000004452 microanalysis Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000005469 synchrotron radiation Effects 0.000 description 1
- 238000004876 x-ray fluorescence Methods 0.000 description 1
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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
-
- 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
- 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
- G01N1/36—Embedding or analogous mounting of samples
-
- 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
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
-
- 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
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- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a method for quickly making and permanently preserving the anatomical structure of lichen fruiting body. The method can solve the problems that qualified microscopic sections can not be obtained quickly and the sections can not be stored permanently in the observation of the microscopic structure of the lichen fruiting body. Firstly, softening and fixing for a certain time, then adopting cheap glue instead of OCT as a freezing embedding agent, controlling the temperature of a box body and a slicing head, sticking, baking and dyeing, finally carrying out a series of dehydration treatment, and then sealing. The whole process has short period, low cost, no deformation of the material, clear microstructure of the sporophore, long storage time and no repeated damage to the researched material. The method is very beneficial to the observation of the anatomical structure of lichen fruiting body and the research of classification and appraisal, and is easy to popularize.
Description
Technical Field
The invention relates to a method for preparing lichen slices, in particular to a method capable of quickly preparing and permanently preserving the anatomical structure of lichen fruiting bodies.
Background
The conventional manual slice making method for researching the structure of the fruiting body in the field of lichen has low slice making rate and uneven thickness, and can not be stored only for temporary observation. Moreover, different researchers may re-sample the same material while conducting the study, resulting in complete destruction of the only reproductive structure of the lichen specimen, compromising the integrity of the lichen specimen. Lichen material is not easily available, so in order to improve the current situation, a method for solving the problems is urgently needed. The adoption of the frozen section has the characteristics of rapidness, simplicity, convenience and easy operation. The technology has wide application in the aspects of animal and human tissues, but the application in the field of lichen is not reported at all. The similar report is applied to a few higher plant tissue slices, but the popularization is not seen.
For example, publication No. CN103926109A, a cryo-sectioning method suitable for rapidly observing the anatomical structure of oil palm leaf. According to the method, an imported embedding medium OCT (optimal cutlingTemperature Compound) is selected for embedding, and a section of an oil palm leaf anatomical structure is obtained. However, the OCT, an imported embedding medium, is expensive and is not easy to purchase.
Publication No. CN108037147A, a method for preparing frozen sections of plant roots for synchrotron radiation X-ray fluorescence microanalysis. The method comprises the steps of cutting plant roots into small blocks, placing the small blocks in an improved OCT (optical coherence tomography) freezing embedding medium, refrigerating and soaking the small blocks for a period of time at 4 ℃, and embedding the small blocks by the improved OCT freezing embedding medium again. The composition of the improved OCT adopted by the method is relatively complex, and the method is inconvenient to popularize.
Lichen is a special fungus, the internal structure of which is completely different from that of higher plants, and the freezing slicing method applied in the field of higher plants is not suitable for the slicing of the microstructure of lichen fruiting bodies, so another set of slicing method which is fast and convenient and is suitable for lichen materials is needed.
Disclosure of Invention
Lichen is a complex composed of fungi and algae (or cyanobacteria), and most of the symbiotic bacteria of lichen are ascomycetes, which produce sexually reproducing fruiting bodies that produce ascomycetes and ascospores. In the classification of lichen, the type of fruit body and the number, shape and structure of spore are all used as very important classification criteria. The invention aims to provide a method for quickly making and permanently preserving the anatomical structure of lichen fruit body. The method can solve the problems that qualified microscopic sections can not be obtained quickly and the sections can not be stored permanently in the observation of the microscopic structure of the lichen fruiting body.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a permanent flaking method of the microscopic structure of the lichen fruiting body is characterized by comprising the following steps:
(1) Softening: soaking lichen material with fruiting body in glycerol solution and vacuumizing;
(2) Fixing: fixing the material obtained in the step (1) in a fixing solution and vacuumizing;
(3) Freezing: placing the material in the step (2) on a cold table, and carrying out freeze embedding by using a freeze embedding solvent;
(4) Slicing: placing the frozen material in the step (3) on a freezing microtome for slicing;
(5) Sticking and baking: uniformly coating a thin layer of sticky tablets on a clean glass slide, sticking the slices in the step (4) on the glass slide, and drying on a 30-50 ℃ exhibition stand;
(6) Dyeing: dyeing the section obtained in the step (5) by adopting lactic acid phenol cotton blue;
(7) And (3) dehydrating: dehydrating the stained glass slide in the step (6) by sequentially using ethanol and n-butanol solutions with different concentration gradient ratios;
(8) And (3) sealing: and (4) sealing the dehydrated lichen fruit body slices in the step (7) by using neutral gum for storage, and drying at room temperature for 24 hours to prepare the microscopic slices of the lichen fruit body capable of being stored permanently.
Further, the glycerol solution in the step (1) is a 1-30% glycerol solution.
Further, the fixing solution in the step (2) comprises 2-3 parts of 1-10% chromic acid aqueous solution, 1-2 parts of glacial acetic acid and 4-5 parts of formalin.
Further, in the step (4), the temperature of the box body is-16 ℃ to-22 ℃, the temperature of the sample head is-16 ℃ to-22 ℃, and the slice thickness is set to be 3-8um.
Aiming at the particularity of the lichen material in the earlier stage, firstly softening and fixing for a certain time, then adopting cheap glue to replace OCT as a freezing embedding agent, controlling the temperature of a box body and a slicing head, sticking, baking and dyeing, and finally sealing after a series of dehydration treatment. The whole process has short period, low cost, no deformation of material, clear microstructure of the fruiting body and long storage time, and the method is very favorable for observing the anatomical structure of lichen fruiting body and researching classification and identification, and is easy to popularize. Compared with the prior art, the invention finds a quick and cheap method suitable for permanent slicing of lichen fruiting bodies. The prepared slice is uniform in thickness and clear in microstructure, and the problems that the slice rate is low, the thickness is not uniform, the structure is unclear, the researched material is repeatedly damaged, and the slice cannot be stored for a long time in the conventional slice making process are solved.
Drawings
FIG. 1 shows a 10X microtome of the fruit body of lichen obtained by the method of the present invention.
FIG. 2 is a 20X microtome of the fruit body of lichen obtained by the method of the present invention.
FIG. 3 is a 40X microtome of the fruit body of lichen obtained by the method of the present invention.
Detailed Description
The following detailed description further describes the present invention for the purpose of illustrating the technical solutions and objects of the present invention.
The method for permanently preparing the lichen fruit body anatomical structure comprises the following steps: 1. softening: soaking lichen material with fruiting body in 1-30% glycerol solution for 10-60 min, and vacuumizing; 2. fixing: fixing the material in the step 1 in a fixing solution prepared from the following raw materials (2-3 parts of 1-10% chromic acid aqueous solution, 1-2 parts of glacial acetic acid and 4-5 parts of formalin) for 1-12 hours, and vacuumizing; 3. freezing: placing the material in the step 2 on a cold table, and carrying out frozen embedding by using glue (frozen embedding solvent); 4. slicing: placing the frozen material in the step 3 on a freezing microtome, adjusting the frozen sample to a proper position by using a fast forward button, and adjusting the temperature of a box body to be-16 ℃ to-22 ℃ and the temperature of a sample head to be-16 ℃ to-22 ℃. Setting the thickness of the slices to be 3-8um, and slicing; 5. sticking and baking: uniformly coating a thin layer of sticking tablets on a clean glass slide, sticking the slices in the step 4 on the glass slide, and drying on a 30-50 ℃ exhibition stand for 30-60 minutes; 6. dyeing: adopting lactic acid phenol cotton blue to dye for 3-10 minutes; 7. and (3) dehydrating: sequentially subjecting the dyed glass slide to ethanol and n-butanol solutions with different concentration gradient ratios; 8. sealing: the gel is sealed and preserved by neutral gum and dried for 24 hours at room temperature. Making into micro-section of lichen fruiting body capable of being preserved permanently.
As shown in figure 1, the microscopic section obtained by the method of the invention has complete structure, and after the microscopic section is magnified by 20 times, as shown in figure 2, the sporocarpic layer, the sporocarpic group, the algal cell layer, the pith layer and the rhizoid can be seen, the structure of the ascocarp and the lateral filament can be clearly observed under 40 times of oil microscope, the shape of the ascospore in the ascocarp is complete, the structure is clear, and the measurement, the counting and the identification are very easy. The microscopic section obtained by the method has complete structure and does not damage the microscopic structure of the lichen fruiting body. The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (2)
1. A permanent preparation method of a microstructure of a lichen fruiting body is characterized by comprising the following steps:
(1) Softening: soaking lichen material with fruiting body in glycerol solution, and vacuumizing;
(2) Fixing: fixing the material obtained in the step (1) in a fixing solution and vacuumizing;
(3) Freezing: placing the material in the step (2) on a cold table, and carrying out freezing embedding by using a freezing embedding solvent;
(4) Slicing: placing the frozen material in the step (3) on a freezing microtome for slicing;
(5) Sticking and baking: uniformly coating a thin layer of sticky tablets on a clean glass slide, sticking the slices in the step (4) on the glass slide, and drying on a 30-50 ℃ exhibition stand;
(6) Dyeing: dyeing the section obtained in the step (5) by adopting lactic acid phenol cotton blue;
(7) And (3) dehydrating: dehydrating the stained glass slide in the step (6) by sequentially using ethanol and n-butanol solutions with different concentration gradient ratios;
(8) Sealing: sealing and preserving the dehydrated lichen fruiting body in the step (7) by using neutral gum, and drying at room temperature for 24 hours to prepare a micro-section of the lichen fruiting body capable of being preserved permanently;
in the step (2), the fixing solution comprises 2-3 parts of 1-10% chromic acid aqueous solution, 1-2 parts of glacial acetic acid and 4-5 parts of formalin;
in the step (3), the frozen embedding solvent is glue;
in the step (4), the box temperature of the freezing microtome is-16 ℃ to-22 ℃, and the temperature of the sample head is-16 ℃ to-22 ℃; the slice thickness is 3-8um.
2. The method for permanently sectioning the microstructure of a lichen fruiting body according to claim 1, wherein the glycerin solution of the step (1) is a 1-30% glycerin solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010059784.0A CN111238892B (en) | 2020-01-19 | 2020-01-19 | Permanent flaking method of lichen sporophore microstructure |
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| CN111982629B (en) * | 2020-08-25 | 2023-11-17 | 北京市农林科学院 | Ultrathin frozen slicing method for edible fungus tissue cells |
Citations (7)
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| JP2012042414A (en) * | 2010-08-23 | 2012-03-01 | Kyushu Univ | Freezing method and storing method of tissue specimen |
| CN102620960A (en) * | 2012-02-29 | 2012-08-01 | 浙江省农业科学院 | Leaf sheath tissue section manufacturing method for enabling users to observe callose of rice leaf sheath tissues |
| EP2518468A1 (en) * | 2011-04-26 | 2012-10-31 | Milestone S.r.l. | Method, processor and carrier for processing frozen slices of tissue of biospecimens |
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2020
- 2020-01-19 CN CN202010059784.0A patent/CN111238892B/en active Active
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