CN102175501A - Method for preprocessing plant sample used for microscopic observation - Google Patents
Method for preprocessing plant sample used for microscopic observation Download PDFInfo
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- CN102175501A CN102175501A CN201110003216XA CN201110003216A CN102175501A CN 102175501 A CN102175501 A CN 102175501A CN 201110003216X A CN201110003216X A CN 201110003216XA CN 201110003216 A CN201110003216 A CN 201110003216A CN 102175501 A CN102175501 A CN 102175501A
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Abstract
The invention relates to a method for preprocessing a plant sample used for microscopic observation and belongs to the technical field of microscopic sample preparation. The method comprises the following steps: (1) absorbing a stationary liquid, placing the stationary liquid in a vessel, and cooling, thus acquiring a sample stationary liquid; (2) cutting a plant tissue into tissue blocks, placing the tissue blocks into the sample stationary liquid prepared in the step (1) and sealing the vessel; (3) exhausting the sealed vessel prepared in the step (2), placing the vessel into an ultrasonic cleaner, and cleaning for 2-5 minutes, thus obtaining a cleaned sample; and (4) settling the cleaned sample prepared in the step (3) for 2-4 hours, thereby acquiring the preprocessed plant sample. According to the method, by utilizing the cavitation effect of ultrasonic wave, the motion speed and penetrating power of a medium molecule are increased, the stationary liquid glutaraldehyde and paraformaldehyde molecules are activated, the obstruction of histocyte gap micro-bubbles to the infiltration of the stationary liquid is eliminated, the settling effect of the plant material is efficiently promoted, and simultaneously the effect of cleaning the surface of the material is achieved.
Description
Technical field
The present invention relates to be used for the pre-treating method of fractographic plant sample, particularly the disposal route before a plant material ultra-thin section, half ultra-thin section, the surface scan sample making belongs to the microscope example manufacture technology field.
Technical background
Microtechnic has become indispensable important tool in the modern science and technology research.Transmission and scanning electron microscope be extensive application aspect biology, medical science, materialogy and other subjects, compare with optical microscope, have high resolving power, high-amplification-factor and can do the characteristics of full resolution pricture, utilize it can obtain the meticulous ultrastructure of biological tissue cell, microstructure or spatial structure image, for the research of the fine structure of object tissue surface or section provide light microscope technique the bulk information that can not obtain, for the research microworld has been opened up new field.Provide the wider visual field and optical microscope can be the microstructural observation of biological tissue, the structural information of biological cell microscopic level is provided.The effect of cell ultrastructure, microstructure, configuration of surface structure observation, key are the preparations of sample, and the committed step of specimen preparation is the effective, fixing fast of material.
High crop yield and stable yields are to improve the key factor of China's economic security and grain security.At present, to the observation of crop cell ultrastructure, microstructure and surface characteristics and variation thereof is the importance of estimating crop anti-adversity, adaptability and high-yield character thereof, and high crop yield, high-quality, liquid manure efficiently utilize mechanism etc. to become the research focus of field of biology day by day; Thereby the preparation of electronics and optical microscope sample and observation also become the important means of this area research.Yet the influence of the multifactor and numerous and diverse experimental procedure of audient, the preparation of microscope example often are difficult to successfully with observing.A lot of crops, important crops of China such as wheat, corn, paddy rice for example, its stem, leaf surface are the siliceous cell layer, and be enclosed with cured matter cuticula, hinder immobile liquid and in effective time, enter histocyte, make that material can not be fully fixing, have a strong impact on the observing effect of microscopically, even all that has been achieved is spoiled to cause experiment.In addition, a lot of crops are had a greatly reduced quality owing to the surface contaminants obstacle makes the observing effect of scanning electron microscope.For example, wheat powdery mildew is a fungal disease, their early stage, and blade, leaf sheath and stem stalk, even the wheat head produces the little mildew of white powdery, the surface is covered with one deck white opaque; Stem of plant such as paddy rice, wheat, leaf surface have the hydrophobic white wax cuticula of one deck; Soybean is because longer and intensive glandular hairs and recurrent downy mildew causes blade surface to stick more foreign matters such as assorted dirt; Chinese tamarisk, naringi crenulata, saltbush etc. secrete the salt plant, absorb salinity from soil, and meetings such as part salt ion such as potassium, sodium, calcium, magnesium, chlorine and sulphion are come out by the salt glandular secretion, and the crystalline solid that forms white adheres to the surface of blade or stem.The foreign matter that more than attaches to material surface is difficult to remove with conventional method, and is particularly less and need in time fixing sample to material volume.
Therefore, the microscopic examination plant sample fast, cleaning effectively fixing and the surface adhesion object is the committed step that guarantees that vegetable cell ultrastructure, microstructure, tissue surface morphosis are observed.
Publication number is the Chinese patent of ℃ N1673722A (application number is 200510067183.x), discloses a kind of cell agglutinin probe.It is provided with component I, II, III, wherein; III is the sample fixed system, be selected from a kind of in Lu Geshi iodine liquid, paraformaldehyde and the glutaraldehyde, the concentration of Lu Geshi iodine liquid is 10%～20%, the final concentration of Lu Geshi iodine liquid is 0.5%～1.5%, the concentration of paraformaldehyde is 10%～20%, the final concentration of paraformaldehyde is 0.5%～1.5%, and the concentration of glutaraldehyde is 20%～25%, and the final concentration of glutaraldehyde is 1.0%～2.5%.Fixed system in this application is to be used for fixing red tide plankton, owing to do not have the siliceous cell layer and the wax cuticula of crop surface in the red tide plankton, so this immobile liquid can't enter histocyte, fully immobilization material in effective time.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of pre-treating method that is used for fractographic plant sample is provided, this method can improve the cleaning on the fixing and surface of vegetable cell ultrastructure, microstructure, configuration of surface structure effectively, improves success ratio and observing effect that plant sample is made.
The technical scheme that technical solution problem of the present invention is taked is:
Be used for the pre-treating method of fractographic plant sample, step is as follows:
(1) draw immobile liquid 2-3ml and insert in the vessel of volume 5-10ml, cooling then gets the sample immobile liquid;
(2) plant tissue is cut into 1-3m
3Piece of tissue, insert in the sample immobile liquid that step (1) makes the sealing vessel then;
(3) vessel after the sealing that step (2) is made are bled, and make the plant tissue piece all sink to the vessel bottom; Then vessel are put into ultrasonic cleaner, adjust water temperature, frequency of operation 40-60kHz, power 0.3-0.6w/cm
2, cleaned 2-5 minute, must clean sample;
(4) the cleaning sample that step (3) is made descended fixedly 2-4 hour at 4 ℃, must be through the plant sample of pre-treatment.
Immobile liquid in the described step (1) is prepared by the following method:
Glutaraldehyde and paraformaldehyde are added to respectively in the phosphate buffer, and the final concentration that makes glutaraldehyde is 2.2-2.8% (percent by volume), and the final concentration of paraformaldehyde is 2.8-3.2% (percent weight in volume), and the phosphate buffer final concentration is 100mM.
Preferably, the final concentration of described glutaraldehyde is 2.5% (percent by volume), and the final concentration of paraformaldehyde is 3% (percent weight in volume).
Preferably, described percent weight in volume unit is: g/ml.
Preferably, the pH=7.2 of described phosphate buffer.
Chilling temperature in the described step (1) is 4-8 ℃.
Described step (3) is put into ultrasonic cleaner with vessel, and 80% of vessel are submerged in the water of rinse bath.
Adjust water temperature in the described step (3) to 4-8 ℃.
Through the plant sample processing subsequently of pre-treatment routinely prior aries such as ultra-thin section, semithin section, sem observation sample making method handle.
Beneficial effect
1, employing ultrasonic cleaner of the present invention is handled the plant tissue piece, by the cavitation effect that ultrasound wave had, increase the movement velocity and the penetration power of medium molecule, activate immobile liquid glutaraldehyde and paraformaldehyde molecule, eliminate the obstruction that histocyte gap micro-bubble infiltrates to immobile liquid, effectively promote the fixed effect of vegetable material, play the effect of material surface cleaning simultaneously;
2, ultrasonic cleaner must be handled indirectly for the inevitable material that needs to isolate, promptly be placed on to place again in the rinse bath in the container and carry out, specific ultrasonic frequency of the present invention and power make the immobile liquid molecule have very strong penetration power, and making to handle indirectly with direct the processing has same effect;
3, operation steps of the present invention all is to carry out under 4-8 ℃, enzyme of proteolysis in the cell, lipase, amylases etc. are in passive state, avoid the degraded of above enzyme pair cell structure and organic macromolecule, help stabilized cell ultrastructure, microstructure and configuration of surface structure, make observing effect reach optimum condition;
4, add 3% paraformaldehyde in the sample immobile liquid, paraformaldehyde is dissolved in that depolymerization is less monomer of molecular weight or oligomer behind the water, can be penetrated into cell interior by cell membrane, cytoplasma membrane fast, plays fixing fast effect;
5, the present invention can improve the Ultrastructural fixing and surface cleaning effect of vegetable material histocyte effectively, and film system definitions such as the interior chloroplast of cell, mitochondria, nucleus are significantly increased; More remarkable in routine operation, be difficult to its fixed effect of observed cellular component such as plasmodesmus etc.; Simultaneously, because material surface has been carried out effective cleaning, thereby more clear to the observation of vegetable material configuration of surface structures such as the arrangement of organization table chrotoplast, gas cell distribution, glandular hairs, salt-secreting gland under the surface sweeping electron microscope;
6, the present invention is simple to operate, and used ultrasonic cleaner is the conventional instrument in the laboratory, and cost is low, is convenient to use;
7, the present invention is applicable to the making of the plant sample that all need be fixed, and particularly there are thicker wax cuticula, top layer in the surface is the vegetable material of siliceous cell, for example stem, leaf textures such as wheat, corn, paddy rice, and effect is better.
Description of drawings
Fig. 1 is the photo that sample that the method according to embodiment 1 makes is observed under transmission electron microscope;
Fig. 2 is the photo that sample that the method according to the reference examples among the embodiment 1 makes is observed under transmission electron microscope;
Fig. 3 is the photo that sample that the method according to embodiment 2 makes is observed under optical microscope;
Fig. 4 is the photo that sample that the method according to the reference examples among the embodiment 2 makes is observed under optical microscope;
Fig. 5 is the photo that sample that the method according to embodiment 3 makes is observed under scanning electron microscope;
Fig. 6 is the photo that sample that the method according to the reference examples among the embodiment 3 makes is observed under scanning electron microscope.
Embodiment
Below in conjunction with embodiment the present invention is further elaborated, but institute of the present invention protection domain is not limited thereto.
Glutaraldehyde is available from Sigma company, and paraformaldehyde tries a chemical reagent company limited available from Shanghai.
Embodiment 1, be used under the wheatear that the stem histocyte is fixed, under the transmission electron microscope to Ultrastructural observation
Be used for the pre-treating method of fractographic plant sample, step is as follows:
(1) draw immobile liquid 2-3ml and insert in the vial of volume 5ml, be cooled to 4 ℃ then, get the sample immobile liquid, immobile liquid is prepared by the following method:
Glutaraldehyde and paraformaldehyde are added to respectively in the phosphate buffer of pH7.2, and making glutaraldehyde get final concentration is 2.5% (percent by volume), the final concentration of paraformaldehyde be 3% (percent weight in volume, g/ml), the phosphate buffer final concentration is 100mM;
(2) stem under the fringe is cut into 1m
3Piece of tissue, insert then in the sample immobile liquid that step (1) makes, bottle is tight with rubber bottle cap lid;
(3) injector for medical purpose syringe needle inserting step (2) is made with in the tight bottle of rubber bottle cap lid, bled 3-5 minute, be as the criterion at the bottom of all sinking to bottle with sample; Then vessel are put into ultrasonic cleaner, 80% of vessel are submerged in the water of rinse bath, adjust water temperature to 4-8 ℃, frequency of operation 40-60kHz, power 0.3-0.6w/cm
2, cleaned 2-5 minute, must clean sample;
(4) the cleaning sample that step (3) is made descended fixedly 2-4 hour at 4 ℃, must be through the plant sample of pre-treatment.
(5) according to the record of " practical Electron Microscopy " (ISBN:978-7-206-05491-4, publish in Dec, 2007) 49-66 page or leaf, operation steps subsequently is identical with the making of conventional ultra-thin section.
Stem stalk histocyte ultra-thin section under the wheatear of finally making is observed under transmission electron microscope, and observations is (scale=1 μ m) as shown in Figure 1.
Reference examples
According to " practical Electron Microscopy " (ISBN:978-7-206-05491-4, in Dec, 2007 publication) record of 49-66 page or leaf is operated, stem stalk histocyte ultra-thin section under the wheatear of finally making is observed under transmission electron microscope, and observations is (scale=1 μ m) as shown in Figure 2.
By Fig. 1 as a result of embodiment 1 and Fig. 2 as a result of reference examples are compared, can draw, observed its Ultrastructural observation effect of wheat stalk stalk histocyte of ultra-thin section of making of the present invention significantly improves, even the thylakoid lamella is also very clear in old and feeble its chloroplast of stalk cell, illustrate that the present invention can effectively have the wheat stalk histocyte ultrastructure of thicker wax cuticula, siliceous cell layer fixing fast to the surface.
Embodiment 2, be used under the wheatear under stem fixation of tissue, the optical microscope microstructural observation
Be used for the pre-treating method of fractographic plant sample, step is as follows:
(1) draw immobile liquid 2-3ml and insert in the vial of volume 8ml, be cooled to 4 ℃ then, get the sample immobile liquid, immobile liquid is prepared by the following method:
Glutaraldehyde and paraformaldehyde are added to respectively in the phosphate buffer of pH7.2, and making glutaraldehyde get final concentration is 2.5% (percent by volume), the final concentration of paraformaldehyde be 3% (percent weight in volume, g/ml), the phosphate buffer final concentration is 100mM;
(2) stem under the fringe is cut into 3m
3Piece of tissue, insert then in the sample immobile liquid that step (1) makes, bottle is tight with rubber bottle cap lid;
(3) injector for medical purpose syringe needle inserting step (2) is made with in the tight bottle of rubber bottle cap lid, bled 5-8 minute, be as the criterion at the bottom of all sinking to bottle with sample; Then vessel are put into ultrasonic cleaner, 80% of vessel are submerged in the water of rinse bath, adjust water temperature to 4-8 ℃, frequency of operation 40-60kHz, power 0.3-0.6w/cm
2, cleaned 3-5 minute, must clean sample;
(4) the cleaning sample that step (3) is made descended fixedly 2-4 hour at 4 ℃, must be through the plant sample of pre-treatment.
(5) according to the record of " practical Electron Microscopy " (ISBN:978-7-206-05491-4, in Dec, 2007 publish) 49-66 page or leaf and 68 pages, operation steps subsequently is identical with the making of conventional ultra-thin section.
Stem stalk histocyte ultra-thin section under the wheatear of finally making is observed under optical microscope, and observations is (scale=200 μ m) as shown in Figure 3.
Reference examples
According to " practical Electron Microscopy " (ISBN:978-7-206-05491-4, publish in Dec, 2007) record of 49-66 page or leaf and 68 pages operates, stem stalk histocyte ultra-thin section under the wheatear of finally making is observed under optical microscope, and observations is (scale=200 μ m) as shown in Figure 4.
By Fig. 3 as a result of embodiment 2 and Fig. 4 as a result of reference examples are compared, can draw, observed its displaing microstructure observing effect of wheat stalk stalk histocyte of half ultra-thin section of making of the present invention significantly improves, number of chloroplast illustrates that obviously more than the number of chloroplast in the reference examples the present invention can be effectively fixing fast to the wheat stalk histocyte microstructure that material volume is big, there are thicker wax cuticula, siliceous cell layer in the surface in its parenchyma cell.
Embodiment 3, be used under fixing, the scanning electron microscope of stem tissue surface under the wheatear surperficial morphology and structure
Be used for the pre-treating method of fractographic plant sample, step is as follows:
(1) draw immobile liquid 2-3ml and insert in the vial of volume 10ml, be cooled to 4 ℃ then, get the sample immobile liquid, immobile liquid is prepared by the following method:
Glutaraldehyde and paraformaldehyde are added to respectively in the phosphate buffer of pH7.2, and making glutaraldehyde get final concentration is 2.5% (percent by volume), the final concentration of paraformaldehyde be 3% (percent weight in volume, g/ml), the phosphate buffer final concentration is 100mM;
(2) the wheat leaf blade tissue is cut into 3m
3Piece of tissue, insert then in the sample immobile liquid that step (1) makes, bottle is tight with rubber bottle cap lid;
(3) injector for medical purpose syringe needle inserting step (2) is made with in the tight bottle of rubber bottle cap lid, bled 5-8 minute, be as the criterion at the bottom of all sinking to bottle with sample; Then vessel are put into ultrasonic cleaner, 80% of vessel are submerged in the water of rinse bath, adjust water temperature to 4-8 ℃, frequency of operation 40-60kHz, power 0.3-0.6w/cm
2, cleaned 3-5 minute, must clean sample;
(4) the cleaning sample that step (3) is made descended fixedly 2-4 hour at 4 ℃, must be through the plant sample of pre-treatment.
(5) according to the record of " practical Electron Microscopy " (ISBN:978-7-206-05491-4, publish in Dec, 2007) 89-99 page or leaf, operation steps subsequently is identical with the making of conventional ultra-thin section.
Stem tissue surface under the wheatear of finally making is observed under scanning electron microscope, and observations is (scale=30 μ m) as shown in Figure 5.
Reference examples
According to " practical Electron Microscopy " (ISBN:978-7-206-05491-4, in Dec, 2007 publication) record of 89-99 page or leaf is operated, stem tissue surface under the wheatear of finally making is observed under scanning electron microscope, and observations is (scale=30 μ m) as shown in Figure 6.
By Fig. 5 as a result of embodiment 3 and Fig. 6 as a result of reference examples are compared, can draw, very clean with stem tissue surface under the observed wheatear of the ultra-thin section of the present invention's making, configuration of surface structure observation effects such as its pore significantly improve, and illustrate that the present invention can be effectively effectively cleans the wheat stalk tissue surface of surperficial adularescent wax coat.
Claims (8)
1. be used for the pre-treating method of fractographic plant sample, it is characterized in that, step is as follows:
(1) draw immobile liquid 2-3ml and insert in the vessel of volume 5-10ml, cooling then gets the sample immobile liquid;
(2) plant tissue is cut into 1-3m
3Piece of tissue, insert in the sample immobile liquid that step (1) makes the sealing vessel then;
(3) vessel after the sealing that step (2) is made are bled, and make the plant tissue piece all sink to the vessel bottom; Then vessel are put into ultrasonic cleaner, adjust water temperature, frequency of operation 40-60kHz, power 0.3-0.6w/cm
2, cleaned 2-5 minute, must clean sample;
(4) the cleaning sample that step (3) is made descended fixedly 2-4 hour at 4 ℃, must be through the plant sample of pre-treatment.
2. pre-treating method as claimed in claim 1 is characterized in that, the immobile liquid in the described step (1) is prepared by the following method:
Glutaraldehyde and paraformaldehyde are added to respectively in the phosphate buffer, and the final concentration that makes glutaraldehyde is 2.2-2.8% (percent by volume), and the final concentration of paraformaldehyde is 2.8-3.2% (percent weight in volume), and the phosphate buffer final concentration is 100mM.
3. pre-treating method as claimed in claim 2 is characterized in that, the final concentration of described glutaraldehyde is 2.5% (percent by volume), and the final concentration of paraformaldehyde is 3% (percent weight in volume).
4. pre-treating method as claimed in claim 2 is characterized in that described percent weight in volume unit is: g/ml.
5. pre-treating method as claimed in claim 2 is characterized in that, the pH=7.2 of described phosphate buffer.
6. pre-treating method as claimed in claim 1 is characterized in that, the chilling temperature in the described step (1) is 4-8 ℃.
7. pre-treating method as claimed in claim 1 is characterized in that, described step (3) is put into ultrasonic cleaner with vessel, and 80% of vessel are submerged in the water of rinse bath.
8. pre-treating method as claimed in claim 1 is characterized in that, adjusts water temperature in the described step (3) to 4-8 ℃.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033401A (en) * | 2011-09-30 | 2013-04-10 | 汕头大学 | Rapid tableting method of large-sized alga in-vitro dyeing epidermal lamella |
| CN104949869A (en) * | 2015-05-29 | 2015-09-30 | 浙江大学 | Method for rapid acquisition of efficient formaldehyde stationary liquid from paraformaldehyde solid reagent |
| CN107132097A (en) * | 2017-06-29 | 2017-09-05 | 中国热带农业科学院橡胶研究所 | A kind of dicing method of suitable observation rubber tree floral organ anatomical structure |
| CN111982629A (en) * | 2020-08-25 | 2020-11-24 | 北京市农林科学院 | Ultrathin freezing slicing method for edible mushroom tissue cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033401A (en) * | 2011-09-30 | 2013-04-10 | 汕头大学 | Rapid tableting method of large-sized alga in-vitro dyeing epidermal lamella |
| CN103033401B (en) * | 2011-09-30 | 2015-11-18 | 汕头大学 | A kind of tangleweed dyes the High-speed for preparing Slides of epidermis lamella in vitro |
| CN104949869A (en) * | 2015-05-29 | 2015-09-30 | 浙江大学 | Method for rapid acquisition of efficient formaldehyde stationary liquid from paraformaldehyde solid reagent |
| CN107132097A (en) * | 2017-06-29 | 2017-09-05 | 中国热带农业科学院橡胶研究所 | A kind of dicing method of suitable observation rubber tree floral organ anatomical structure |
| CN107132097B (en) * | 2017-06-29 | 2020-05-05 | 中国热带农业科学院橡胶研究所 | Slicing method suitable for observing anatomical structures of flower organs of rubber trees |
| CN111982629A (en) * | 2020-08-25 | 2020-11-24 | 北京市农林科学院 | Ultrathin freezing slicing method for edible mushroom tissue cells |
| 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 |
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Application publication date: 20110907 |