CN111238892B - Permanent flaking method of lichen sporophore microstructure - Google Patents

Permanent flaking method of lichen sporophore microstructure Download PDF

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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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lichen
fruiting body
freezing
fixing
microstructure
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CN111238892A (en
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牛东玲
韩晓鹏
余德菊
苗申奥
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Ningxia University
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36Embedding or analogous mounting of samples
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42Low-temperature sample treatment, e.g. cryofixation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84Systems specially adapted for particular applications

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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

Permanent flaking method of lichen sporophore microstructure
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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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
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CN103776657A (en) * 2014-01-26 2014-05-07 中国热带农业科学院椰子研究所 Manufacturing method of coconut blade freezing slice
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EP2518468A1 (en) * 2011-04-26 2012-10-31 Milestone S.r.l. Method, processor and carrier for processing frozen slices of tissue of biospecimens
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
CN103776657A (en) * 2014-01-26 2014-05-07 中国热带农业科学院椰子研究所 Manufacturing method of coconut blade freezing slice
CN106053177A (en) * 2016-07-28 2016-10-26 江苏农林职业技术学院 Specimen making method for observing anatomic structure of lamina of tillandsia
CN106525530A (en) * 2016-11-04 2017-03-22 河南科技大学 Paraffin slicing method of tree stem tissue
CN110132673A (en) * 2019-05-22 2019-08-16 上海市农业科学院 A method of preparing acupuncture needle massee fruiting bodies histotomy

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