CN107290198B - A method of biomaterial is dyed using PIP - Google Patents
A method of biomaterial is dyed using PIP Download PDFInfo
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- CN107290198B CN107290198B CN201710610328.9A CN201710610328A CN107290198B CN 107290198 B CN107290198 B CN 107290198B CN 201710610328 A CN201710610328 A CN 201710610328A CN 107290198 B CN107290198 B CN 107290198B
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- 239000012620 biological material Substances 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004043 dyeing Methods 0.000 claims abstract description 46
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 241001515965 unidentified phage Species 0.000 claims abstract description 17
- 241000700605 Viruses Species 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 239000000126 substance Substances 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 210000000056 organ Anatomy 0.000 claims abstract description 4
- 238000000338 in vitro Methods 0.000 claims description 9
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 claims description 8
- 238000004040 coloring Methods 0.000 claims description 6
- 210000001557 animal structure Anatomy 0.000 claims description 3
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 8
- 231100001261 hazardous Toxicity 0.000 abstract description 2
- 238000010186 staining Methods 0.000 description 20
- 239000000975 dye Substances 0.000 description 17
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 16
- 229910052802 copper Inorganic materials 0.000 description 16
- 239000010949 copper Substances 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- SFCVEUVVOJFYSX-UHFFFAOYSA-J [Pb+2].C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].C(C)(=O)[O-].[U+6] Chemical compound [Pb+2].C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].C(C)(=O)[O-].[U+6] SFCVEUVVOJFYSX-UHFFFAOYSA-J 0.000 description 6
- 238000007605 air drying Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 210000003463 organelle Anatomy 0.000 description 6
- 239000001509 sodium citrate Substances 0.000 description 6
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 6
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000005540 biological transmission Effects 0.000 description 4
- 229910001385 heavy metal Inorganic materials 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- NRXMBGQJNZDBOD-UHFFFAOYSA-L ethane-1,2-diamine;iodoplatinum(1+);dinitrate Chemical compound [Pt+]I.[Pt+]I.NCCN.NCCN.[O-][N+]([O-])=O.[O-][N+]([O-])=O NRXMBGQJNZDBOD-UHFFFAOYSA-L 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 238000011017 operating method Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Natural products CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000012128 staining reagent Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- -1 (ethylenediamine) diplatinum (II) dinitrate Chemical compound 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- PMPOMXOPKNLGTQ-UHFFFAOYSA-N [Pt+2].[Pt+2].C(CN)N Chemical compound [Pt+2].[Pt+2].C(CN)N PMPOMXOPKNLGTQ-UHFFFAOYSA-N 0.000 description 1
- 239000012932 acetate dye Substances 0.000 description 1
- ORVXHPPMIWZBCC-UHFFFAOYSA-N acetic acid;dioxouranium Chemical compound O=[U]=O.CC(O)=O ORVXHPPMIWZBCC-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 239000010808 liquid waste Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- HRGDZIGMBDGFTC-UHFFFAOYSA-N platinum(2+) Chemical compound [Pt+2] HRGDZIGMBDGFTC-UHFFFAOYSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000008685 targeting Effects 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
- G01N1/30—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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods dyed using PIP to biomaterial.The method disclosed by the invention dyed using PIP to biomaterial, including the use of PIP or the reagent including PIP completes the dyeing of the biomaterial to biomaterial dyeing, and the chemical structural formula of PIP is as shown in formula I；The biomaterial can be microorganism, can also be animal and plant cells, tissue and organ and its slice such as bacteriophage, virus and bacterium.It is demonstrated experimentally that PIP can be not only used for the dyeing of various biomaterials, and PIP is not dangerous substance, not risk physically or chemically, non-hazardous to health and environment, also easily obtains and be dissolved in water, and replaceable uranium acetate is dyed for biomaterial.
Description
Technical field
The present invention relates in field of biotechnology, a method of biomaterial is dyed using PIP.
Background technique
Transmission electron microscope is imaged using the transmitted electron through sample, and most of biological sample is by atoms such as carbon, hydrogen, oxygen
The lower atom composition of ordinal number utilizes what is generated after heavy metal dyeing i.e. increase and electron collision to enhance the contrast of sample
The mode of angle of scattering increases the contrast of sample.According to heavy metal to the difference that the mode that plays a role is imaged, colouring method is divided into
Negative staining and just two kinds of colouring methods of dyeing.Negative staining is chiefly used in complete short grained observation, utilizes the weight compared with high electron density
Metallic salt is enclosed in around sample, to set off by contrast the sample of low electron density out in electro-dense grey black background, from
And achieve the purpose that enhanced contrast；Complete cell (is cut into the slice of 70nm or so, sees it by positive dyeing mainly for slice class
Cell interior structure) sample, using various organelles or the difference of macromolecules adsorption heavy metal quantity or type, thus will
Cell different structure feature shows good contrast.
The double dye methods of uranium acetate-lead citrate are typically passed through for slice class sample.Lead citrate is cutting for wide spectrum
Piece dyestuff has good dyeing to the films class formation such as most cells structure such as cell wall, cell membrane, mitochondria, endoplasmic reticulum
Effect, but it is poor to nucleic acid genetic substance dyeing effect, and uranium acetate has better effect to nuclear targeting.
At present in biological transmission electron microscope observing, no matter all extensive in negative staining or positive dyeing uranium acetate is
The coloring agent used.But since it has a radioactivity, Reagent management and liquid waste processing be always the problem of cannot despising, furthermore mesh
The preceding reagent has been in the strict supervision stage, and most of R&D institutions are difficult to buy the reagent in addition to original inventory.
Summary of the invention
Di- μ-iodobis (ethylenediamine) diplatinum (II) is utilized the present invention provides a kind of
The method that nitrate (abbreviation PIP) dyes biomaterial.
Biomaterial colouring method provided by the invention, including the use of PIP or the reagent including PIP dyes biomaterial
The dyeing of the biomaterial is completed, the chemical structural formula of PIP is as shown in formula I:
The active constituent of the reagent can be PIP, can also for by PIP and with biomaterial dye function material composition
Composition.
The reagent may also include lead citrate.
The reagent can be specifically made of PIP and lead citrate.
, can be first soluble in water by PIP in specific dyeing, obtained PIP aqueous solution contaminates the biomaterial
Color.The dyeing, which can directly drop to the PIP aqueous solution, completes dyeing on the biomaterial.PIP in the PIP aqueous solution
Concentration be adjusted according to specific needs.The concentration of PIP is in used PIP aqueous solution in an embodiment of the present invention
0.01% (mass percent) -1% (mass percent), specially 0.01% (mass percent), 0.5% (mass percent)
With 1% (mass percent).
In the above method, the biomaterial can be at having on the metal product of conducting function, such as metal mesh.It is described
Metal mesh can be copper mesh.
The present invention also provides PIP or following M1 of the reagent) or application M2):
M1) the application in biomaterial dyeing；
M2) application in biomaterial dyeing product is being prepared.
In the present invention, the biomaterial can be microorganism.The microorganism concretely bacteriophage, virus or bacterium.
Any one of the biomaterial can also be following A 1)-A6):
A1) plant cell or its product；
A2) plant tissue or its product；
A3) plant organ or its product；
A4) in vitro zooblast or its product；
A5) in vitro animal tissue or its product；
A6) in vitro animal organ or its product.
The product can be slice.
The present invention also provides biomaterial staining reagent, the biomaterial staining reagent concretely examination described above
Agent.
Present invention discover that Di- μ-iodobis (ethylenediamine) diplatinum (II) nitrate is (referred to as
), PIP chemical formula is [Pt2I2(H2NCH2CH2NH2)2](NO3)2Compound either in negative staining or positive colouring method
The effect of heavy metal dyeing can be played to biological sample.In the dyeing to plant sample, PIP can be with nucleic genetic material
In conjunction with the deeper region of electron density is formed, illustrate that PIP has a positive colouring power in the inhereditary material to cell, i.e., it not only can be with
Dyeing for plant cell, it may also be used for the dyeing of animal, microbial cell illustrates that PIP can contaminate various cells
Color.In addition, PIP can also carry out negative staining to bacteriophage and virus, show that PIP can substitute uranium acetate to various lifes
Object material is dyed.Regulation PIP according to global coordination system (GHS) is not dangerous substance or mixture, and PIP does not have at present
Risk physically or chemically, non-hazardous to health and environment, PIP easily obtains and is dissolved in water, convenient for application and popularization, therefore can
Uranium acetate is replaced to dye for biomaterial.
Detailed description of the invention
Fig. 1 is coloration result of the PIP to bacteriophage.
Fig. 2 is coloration result of the uranium acetate to bacteriophage.
Fig. 3 is the bacteriophage of no dyeing.
Fig. 4 is coloration result of the PIP to virus.
Fig. 5 is coloration result of the uranium acetate to virus.
Fig. 6 is the virus of no dyeing.
Fig. 7 is coloration result of the PIP to bacterium.
Fig. 8 is coloration result of the uranium acetate to bacterium.
Fig. 9 is the bacterium of no dyeing.
Figure 10 is the blade cell of no dyeing.
Figure 11 is the blade cell contaminated by lead citrate list.
Figure 12 is the blade cell by the double dyes of uranium acetate-lead citrate.
Figure 13 is the blade cell by the double dyes of PIP-lead citrate.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Di- μ-iodobis (ethylenediamine) diplatinum (II) nitrate (Di- μ-in the present invention
Iodobis (ethylenediamine) diplatinum (II) dinitrate, abbreviation PIP), also referred to as two-μ-iodo, two (second
Diamines) two platinum (II) of nitric acid, (Ethylenediamine) iodoplatinum (II) dimer dinitrate, chemical formula is
[Pt2I2(H2NCH2CH2NH2)2](NO3)2(888.17g/mol) is SIGMA-ALDRICH product, chemical structural formula such as formula I
It is shown:
The application of embodiment 1, PIP in negative staining
1, bacteriophage
Negative staining is carried out to bacteriophage using PIP, and this kind of bacteriophage is carried out using no dyeing and uranium acetate
Negative staining as control, PIP to bacteriophage carry out negative staining specific steps are as follows:
After 200 mesh copper mesh glow discharge hydrophilic treateds, takes phage suspension drop to be measured on copper mesh, used after 1 minute
After filter paper siphons away residual droplets, after washing primary latter the dyeing of PIP aqueous solution 1 minute with 0.5% (mass percent), with filter
Paper siphons away surplus liquid, upper sem observation after air-drying.
The step of carrying out negative staining to bacteriophage using uranium acetate is as follows:
After 200 mesh copper mesh glow discharge hydrophilic treateds, takes phage suspension drop to be measured on copper mesh, used after 1 minute
After filter paper siphons away residual droplets, washes the primary rear aqueous uranyl acetate with 1% (mass percent) and dye 1 minute
Afterwards, surplus liquid is siphoned away with filter paper, upper sem observation after air-drying.
PIP and uranium acetate are as depicted in figs. 1 and 2 to the coloration result difference of bacteriophage, and undyed bacteriophage is such as
Shown in Fig. 3.
2, viral
Negative staining is carried out to viral (Beta-fuselloyiridae) using PIP, and utilizes no dyeing and acetic acid dioxygen
Uranium to this kind virus carry out negative staining as control, PIP to virus carry out negative staining specific steps are as follows:
After 200 mesh copper mesh glow discharge hydrophilic treateds, take viral suspension drop to be measured on copper mesh, with filter after 1 minute
After paper siphons away residual droplets, after washing primary latter the dyeing of PIP aqueous solution 1 minute with 0.5% (mass percent), filter paper is used
Surplus liquid is siphoned away, upper sem observation after air-drying.
The step of carrying out negative staining to virus using uranium acetate is as follows:
After 200 mesh copper mesh glow discharge hydrophilic treateds, take viral suspension drop to be measured on copper mesh, with filter after 1 minute
After paper siphons away residual droplets, after washing primary latter aqueous uranyl acetate dyeing 1 minute with 1% (mass percent),
Surplus liquid is siphoned away with filter paper, upper sem observation after air-drying.
PIP and uranium acetate are as shown in Figure 4 and Figure 5 to the coloration result difference of virus, and undyed virus is such as Fig. 6 institute
Show.
3, bacterium
Negative staining is carried out to bacterium (Xanthomonas.campestris) using PIP, and utilizes no dyeing and acetic acid
Uranyl carries out negative staining as control to this kind of bacterium, PIP to bacterium carry out negative staining specific steps are as follows:
After 200 mesh copper mesh glow discharge hydrophilic treateds, take bacterium solution drop to be measured on copper mesh, it will be more with filter paper after 1 minute
After extraction raffinate drop siphons away, after washing primary latter the dyeing of PIP aqueous solution 15 seconds with 0.01% (mass percent), siphoned away with filter paper more
Extraction raffinate body, upper sem observation after air-drying.
The step of carrying out negative staining to bacterium using uranium acetate is as follows:
After 200 mesh copper mesh glow discharge hydrophilic treateds, take bacterium solution drop to be measured on copper mesh, it will be more with filter paper after 1 minute
After extraction raffinate drop siphons away, after washing primary latter aqueous uranyl acetate dyeing 15 seconds with 0.02% (mass percent), with filter
Paper siphons away surplus liquid, upper sem observation after air-drying.
Using each coloration result of transmission electron microscope observation, PIP and uranium acetate distinguish the coloration result of bacterium
As shown in Figure 7 and Figure 8, undyed bacterium is as shown in Figure 9.
The results show that the looks profile of bacteriophage, virus, bacterium and the attachment by dyeing is not unintelligible, it is right
It can only see the profile of bacterium under bacterium and its flagellum, low power, clearly discrimination can not have atrichous feature, only be put into height
Can just it see clearly under times.Its profile can be seen by bacteriophage, virus, bacterium that PIP and uranium acetate dye, show
In negative staining, PIP can substitute uranium acetate and be dyed, and illustrate that PIP can be used for the negative staining to bacteriophage, virus and bacterium
Color.
The application of embodiment 2, PIP in positive dyeing
By taking plant leaf blade is sliced as an example, effect of the observation PIP in positive dyeing.
Double dyes are carried out using slice of the PIP-lead citrate to plant leaf blade, and utilize uranium acetate-lead citrate
Double dyes and lead citrate are carried out to the slice of the plant leaf blade, single dye is carried out as control, using not to the slice of the plant leaf blade
The plant leaf blade slice of dyeing is as control of being unstained.
The operating procedure that PIP-lead citrate carries out double dyes to the slice of plant leaf blade is as follows:
A clean nitrocellulose filter is paved, by the 1% water-soluble drop of (mass percent) PIP on film, will be loaded with
The copper mesh of slice is buckled on drop according to slice towards the direction of PIP aqueous solution, is incubated for 25 minutes.Support grid is pressed from both sides with tweezers after dyeing
It immerses in distilled water after cleaning repeatedly and blots surplus liquid with filter paper.Then by be loaded with slice tip upside down on one side with plumbi nitras with
(solution is to dissolve 30 milliliters of distilled water of 1.33g plumbi nitras and 1.76g sodium citrate to the solution that sodium citrate is configured to
And be titrated to sodium hydroxide solution do not precipitate after the solution that is obtained to volume to 50 milliliters of constant volume) in, dyeing is about after five minutes
Washing air-dries.
The operating procedure that uranium acetate-lead citrate carries out double dyes to the slice of plant leaf blade is as follows:
A clean nitrocellulose filter is paved, by 2% (mass percent) aqueous uranyl acetate drop on film,
The copper mesh for being loaded with slice is buckled on drop according to slice towards the direction of aqueous uranyl acetate, is incubated for 25 minutes.Dyeing
It is immersed in distilled water with tweezers folder support grid after cleaning repeatedly afterwards and blots surplus liquid with filter paper.Lurching for slice will be then loaded with
Be buckled in be configured to plumbi nitras and sodium citrate solution (solution be by 1.33g plumbi nitras and 1.76g sodium citrate with 30 in the least
The distilled water risen dissolve and be titrated to sodium hydroxide solution do not precipitate after the solution that is obtained to volume to 50 milliliters of constant volume)
In, about washing air-dries after five minutes for dyeing.
The operating procedure that lead citrate singly contaminates the slice of plant leaf blade is as follows:
By the solution that is configured to plumbi nitras and sodium citrate of tipping upside down on one side for the copper mesh for being loaded with slice, (solution is will
1.33g plumbi nitras and 1.76g sodium citrate, which are dissolved and are titrated to sodium hydroxide solution with 30 milliliters of distilled water, not to be precipitated
The solution that constant volume is obtained to volume to 50 milliliters afterwards) in, about washing air-dries after five minutes for dyeing.
Using each coloration result of transmission electron microscope observation, the results show that the plant cell of no dyeing can only be seen
To the general outline (Figure 10) of cell, it can further be seen that clearly organelle (Figure 11) after single dye of lead citrate, and pass through
Not can be only seen clearly organelle after the double dyes of uranium acetate-lead citrate can also be seen that in organelle and nucleus
Inhereditary material (Figure 12), it can also be seen that clearly organelle and organelle and nucleus after the double dyes of PIP-lead citrate
In inhereditary material (Figure 13), show PIP-lead citrate also replaceable uranium acetate-lead citrate be used for biologic slice
Dyeing.
Claims (10)
1. biomaterial colouring method completes the biomaterial to biomaterial dyeing including the use of the reagent containing PIP
Dyeing, the chemical structural formula of PIP is as shown in formula I:
2. according to the method described in claim 1, it is characterized by: the reagent further includes lead citrate.
3. method according to claim 1 or 2, it is characterised in that: the biomaterial is microorganism.
4. method according to claim 1 or 2, it is characterised in that: the biomaterial be following A 1)-A6) and in any
Kind:
A1) plant cell or its product；
A2) plant tissue or its product；
A3) plant organ or its product；
A4) in vitro zooblast or its product；
A5) in vitro animal tissue or its product；
A6) in vitro animal organ or its product.
5. according to the method described in claim 4, it is characterized by: the product is slice.
6. following M1 of reagent as claimed in claim 1 or 2) or application M2):
M1) the application in biomaterial dyeing；
M2) application in biomaterial dyeing product is being prepared.
7. application according to claim 6, it is characterised in that: the biomaterial is microorganism.
8. application according to claim 7, it is characterised in that: the microorganism is bacteriophage, virus or bacterium.
Any one of 9. application according to claim 6, it is characterised in that: the biomaterial is following A 1)-A6):
A1) plant cell or its product；
A2) plant tissue or its product；
A3) plant organ or its product；
A4) in vitro zooblast or its product；
A5) in vitro animal tissue or its product；
A6) in vitro animal organ or its product.
10. application according to claim 9, it is characterised in that: the product is slice.
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| US7020563B1 (en) * | 1997-11-27 | 2006-03-28 | Commonwealth Scientific And Industrial Research Organisation | Method of designing agonists and antagonists to IGF receptor |
| CN101936837A (en) * | 2009-06-26 | 2011-01-05 | 南京金斯瑞生物科技有限公司 | Rapid staining method for biopolymers |
| CN104458371A (en) * | 2014-11-26 | 2015-03-25 | 上海华力微电子有限公司 | Preparation method of TEM sample |
| CN106338420A (en) * | 2016-08-19 | 2017-01-18 | 上海华力微电子有限公司 | Transmission electron microscope sample junction staining method |
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| US7020563B1 (en) * | 1997-11-27 | 2006-03-28 | Commonwealth Scientific And Industrial Research Organisation | Method of designing agonists and antagonists to IGF receptor |
| CN101936837A (en) * | 2009-06-26 | 2011-01-05 | 南京金斯瑞生物科技有限公司 | Rapid staining method for biopolymers |
| CN104458371A (en) * | 2014-11-26 | 2015-03-25 | 上海华力微电子有限公司 | Preparation method of TEM sample |
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