CN107290198A - A kind of method that utilization PIP is dyed to biomaterial - Google Patents
A kind of method that utilization PIP is dyed to biomaterial Download PDFInfo
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- CN107290198A CN107290198A CN201710610328.9A CN201710610328A CN107290198A CN 107290198 A CN107290198 A CN 107290198A CN 201710610328 A CN201710610328 A CN 201710610328A CN 107290198 A CN107290198 A CN 107290198A
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- pip
- biomaterial
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- 239000012620 biological material Substances 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004043 dyeing Methods 0.000 claims abstract description 38
- 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 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 241001465754 Metazoa Species 0.000 claims abstract description 4
- 210000000056 organ Anatomy 0.000 claims abstract description 3
- 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
- 239000000463 material Substances 0.000 claims description 7
- 238000004040 coloring Methods 0.000 claims description 6
- 238000000338 in vitro Methods 0.000 claims description 6
- 239000012128 staining reagent Substances 0.000 claims description 3
- 210000001557 animal structure Anatomy 0.000 claims description 2
- 239000000975 dye Substances 0.000 abstract description 19
- 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
- 241000196324 Embryophyta Species 0.000 abstract 1
- 238000010186 staining Methods 0.000 description 20
- 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
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000000523 sample Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 238000005406 washing Methods 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
- 210000003463 organelle Anatomy 0.000 description 6
- 239000001509 sodium citrate Substances 0.000 description 6
- 235000011083 sodium citrates Nutrition 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
- 230000003612 virological effect 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
- 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
- 238000011017 operating method Methods 0.000 description 3
- 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 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 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
- 210000000170 cell membrane Anatomy 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
- 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
- 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
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant 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
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 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
- 241000894007 species Species 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
Abstract
The invention discloses a kind of method that utilization PIP is dyed to biomaterial.The method that utilization PIP disclosed by the invention is dyed to biomaterial, using PIP or the reagent including PIP dyes the dyeing for completing the biomaterial to biomaterial, PIP chemical structural formula is as shown in formula I；The biomaterial can be microorganism, such as bacteriophage, virus and bacterium, or animal and plant cells, tissue and organ and its section.It is demonstrated experimentally that PIP can be not only used for the dyeing of various biomaterials, and PIP is not dangerous substance, without danger physically or chemically, to health and environment non-hazardous, also easily obtains and is dissolved in water, replaceable uranium acetate is dyed for biomaterial.
Description
Technical field
The present invention relates in biological technical field, a kind of method that utilization PIP is dyed to biomaterial.
Background technology
Transmission electron microscope is to utilize the transmitted electron through sample to be imaged, and most of biological sample is by atoms such as carbon, hydrogen, oxygen
The relatively low atom composition of ordinal number, in order to strengthen the contrast of sample, dyes what is produced after i.e. increase and electron collision using heavy metal
The mode of angle of scattering increases the contrast of sample.The difference for the mode that played a role according to heavy metal to imaging, colouring method is divided into
Negative staining and just two kinds of colouring methods of dyeing.Negative staining is used for 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 in electro-dense grey black background, from
And reach the purpose of enhanced contrast；Complete cell (is cut into 70nm or so section, sees it by positive dyeing mainly for section class
Cell interior structure) sample, using various organelles or the difference of macromolecules adsorption heavy metal quantity or species, so that will
Cell different structure feature shows good contrast.
The double dye methods of uranium acetate-lead citrate are typically passed through for section class sample.Lead citrate is cutting for wide spectrum
Piece dyestuff, has good dyeing to the most cells structure such as film class formation such as cell membrane, cell membrane, mitochondria, endoplasmic reticulum
Effect, but it is poor to nucleic acid genetic class substance stain effect, and uranium acetate has better effect to nuclear targeting.
At present in biological transmission electron microscope observing, uranium acetate is all extensive in negative staining or positive dyeing
The coloring agent used.But because it has a radioactivity, Reagent management and liquid waste processing be always the problem of can not despising, in addition mesh
The preceding reagent is in the strict supervision stage, and most of R＆D institutions are except original stock, it is difficult to buy the reagent.
The content of the invention
Di- μ-iodobis (ethylenediamine) diplatinum (II) is utilized the invention provides one kind
The method that nitrate (abbreviation PIP) is dyed to biomaterial.
The biomaterial colouring method that the present invention is provided, using PIP or the reagent including PIP to biomaterial dye
The dyeing of the biomaterial is completed, PIP chemical structural formula is as shown in formula I：
The active component of the reagent can be PIP, or by PIP and with biomaterial dye function material composition
Composition.
The reagent may also include lead citrate.
The reagent can be specifically made up of PIP with lead citrate.
, can be first soluble in water by PIP in specific dyeing, the obtained PIP aqueous solution is contaminated 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 can be adjusted according to specific needs.PIP concentration is in the 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 on the metallic article with conducting function, such as wire netting.It is described
Wire netting can be copper mesh.
Present invention also offers following M1 of PIP or described reagents) or application M2)：
M1) the application in biomaterial dyeing；
M2) the application in biomaterial dyeing product is prepared.
In the present invention, the biomaterial can be microorganism.The microorganism concretely bacteriophage, virus or bacterium.
Any of the biomaterial is alternatively 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 section.
Present invention also offers 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
It is equal to biological sample to play the effect of heavy metal dyeing.In the dyeing to plant sample, PIP can be with nucleic genetic material
The deeper region of electron density is combined to form, illustrates that PIP has 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 animal, the dyeing of microbial cell, illustrates, PIP can be contaminated various cells
Color.In addition, PIP can also carry out negative staining to bacteriophage and virus, show, PIP can substitute uranium acetate to various lifes
Thing material is dyed.It is not dangerous substance or mixture according to the regulation PIP of global coordination system (GHS), current PIP does not have
Danger physically or chemically, to health and environment non-hazardous, PIP easily obtains and is dissolved in water, be easy to apply with popularization, therefore can
Uranium acetate is replaced to dye for biomaterial.
Brief description of the drawings
Fig. 1 is coloration results 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 results 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 results 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 by lead citrate list dye.
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.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified, be
Conventional method.Material, reagent, instrument used etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following examples, is respectively provided with three repetition experiments, results averaged.
In the present invention Di- μ-iodobis (ethylenediamine) diplatinum (II) nitrate (Di- μ-
Iodobis (ethylenediamine) diplatinum (II) dinitrate, abbreviation PIP), the also referred to as (second of two-μ-iodo two
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 products, its chemical structural formula such as formula I
It is shown：
The application of embodiment 1, PIP in negative staining
1st, bacteriophage
Negative staining is carried out to bacteriophage using PIP, and this kind of bacteriophage carried out using no dyeing and uranium acetate
Negative staining is as control, and the concrete operation step that PIP carries out negative staining to bacteriophage is as follows：
After 200 mesh copper mesh glow discharge hydrophilic treateds, take phage suspension drop to be measured on copper mesh, used after 1 minute
After filter paper siphons away residual droplets, after washing is once dyed 1 minute with the PIP aqueous solution of 0.5% (mass percent) afterwards, 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, take phage suspension drop to be measured on copper mesh, used after 1 minute
After filter paper siphons away residual droplets, washing is once dyed 1 minute with the aqueous uranyl acetate of 1% (mass percent) afterwards
Afterwards, surplus liquid is siphoned away with filter paper, upper sem observation after air-drying.
PIP and uranium acetate are distinguished as depicted in figs. 1 and 2 the coloration result of bacteriophage, and undyed bacteriophage is such as
Shown in Fig. 3.
2nd, it is viral
Negative staining is carried out to viral (Beta-fuselloyiridae) using PIP, and utilizes no dyeing and acetic acid dioxygen
Uranium carries out negative staining to this kind virus as control, and the concrete operation step that PIP carries out negative staining to virus is as follows：
After 200 mesh copper mesh glow discharge hydrophilic treateds, viral suspension to be measured is taken to drop in above copper mesh, with filter after 1 minute
After paper siphons away residual droplets, after washing is once dyed 1 minute with the PIP aqueous solution of 0.5% (mass percent) afterwards, 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, viral suspension to be measured is taken to drop in above copper mesh, with filter after 1 minute
After paper siphons away residual droplets, after washing is once dyed 1 minute with the aqueous uranyl acetate of 1% (mass percent) afterwards,
Surplus liquid is siphoned away with filter paper, upper sem observation after air-drying.
To viral coloration result difference as shown in Figure 4 and Figure 5, undyed virus is such as Fig. 6 institutes for PIP and uranium acetate
Show.
3rd, bacterium
Negative staining is carried out to bacterium (Xanthomonas.campestris) using PIP, and utilizes no dyeing and acetic acid
Uranyl carries out negative staining to this kind of bacterium as control, and the concrete operation step that PIP carries out negative staining to bacterium is as follows：
After 200 mesh copper mesh glow discharge hydrophilic treateds, take bacterium solution to be measured to drop in above copper mesh, with filter paper will be many after 1 minute
After extraction raffinate drop is siphoned away, after washing is once dyed 15 seconds with the PIP aqueous solution of 0.01% (mass percent) afterwards, siphoned away with filter paper many
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 to be measured to drop in above copper mesh, with filter paper will be many after 1 minute
After extraction raffinate drop is siphoned away, after washing is once dyed 15 seconds with the aqueous uranyl acetate of 0.02% (mass percent) afterwards, 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 are distinguished the coloration result of bacterium
As shown in Figure 7 and Figure 8, undyed bacterium is as shown in Figure 9.
As a result show, the looks profile of bacteriophage, virus, bacterium and the attachment by dyeing is not unintelligible, right
In the profile that bacterium is can only see under bacterium and its flagellum, low power, it is impossible to which clear discrimination has atrichous feature, is only put into height
It can just see clearly under times.Its profile can be seen in bacteriophage, virus, the bacterium dyed by PIP and uranium acetate, shows,
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
So that plant leaf blade is cut into slices as an example, effects of the observation PIP in positive dyeing.
Double dyes are carried out to the section of plant leaf blade using PIP-lead citrate, and utilize uranium acetate-lead citrate
Section to the plant leaf blade carries out double dyes and lead citrate to the single dye of section progress of the plant leaf blade as control, using not
The plant leaf blade section of dyeing is used as control of being unstained.
The operating procedure that section of the PIP-lead citrate to plant leaf blade carries out double dyes is as follows：
A clean nitrocellulose filter is paved, 1% (mass percent) PIP aqueous solution is dropped on film, will be loaded with
The copper mesh of section is buckled on drop according to section towards the direction of the PIP aqueous solution, is incubated 25 minutes.After dyeing contained network is pressed from both sides with tweezers
After being cleaned repeatedly in immersion distilled water surplus liquid is blotted with filter paper.Then by the one side for being loaded with section tip upside down on plumbi nitras with
(solution is to dissolve 1.33g plumbi nitras and 1.76g sodium citrates with 30 milliliters of distilled water to the solution that sodium citrate is configured to
And be titrated to sodium hydroxide solution do not precipitate after constant volume to volume to 50 milliliters of obtained solution) in, after dyeing about 5 minutes
Washing is air-dried.
The operating procedure that section of the uranium acetate-lead citrate to plant leaf blade carries out double dyes is as follows：
A clean nitrocellulose filter is paved, 2% (mass percent) aqueous uranyl acetate is dropped on film,
The copper mesh for being loaded with section is buckled on drop according to section towards the direction of aqueous uranyl acetate, is incubated 25 minutes.Dyeing
Immersed afterwards with tweezers folder contained network after being cleaned repeatedly in distilled water and blot surplus liquid with filter paper.To then lurching for section be 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 citrates with 30 in the least
The distilled water risen dissolve and be titrated to sodium hydroxide solution do not precipitate after constant volume to volume to 50 milliliters of obtained solution)
In, dyeing is washed after about 5 minutes and air-dried.
The operating procedure that section of the lead citrate to plant leaf blade carries out single dye is as follows：
The one side that the copper mesh of section will be loaded with tips upside down on the solution that is configured to plumbi nitras and sodium citrate (solution is will
1.33g plumbi nitras and 1.76g sodium citrates, which are dissolved and are titrated to sodium hydroxide solution with 30 milliliters of distilled water, not to be precipitated
Constant volume is to volume to 50 milliliters of obtained solution afterwards) in, dyeing is washed after about 5 minutes and air-dried.
Using each coloration result of transmission electron microscope observation, as a result show, 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
Clearly organelle is not can be only seen it can also be seen that in organelle and nucleus after the double dyes of uranium acetate-lead citrate
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 (8)
1. biomaterial colouring method, using PIP or the reagent including PIP completes the biological material to biomaterial dyeing
The dyeing of material, PIP chemical structural formula is as shown in formula I：
2. according to the method described in claim 1, it is characterised in that：The reagent also includes lead citrate.
3. following M1 of reagent described in PIP described in claim 1 or claim 1 or 2) or application M2)：
M1) the application in biomaterial dyeing；
M2) the application in biomaterial dyeing product is prepared.
4. the application described in method according to claim 1 or 2 or claim 3, it is characterised in that：The biomaterial
For microorganism.
5. method according to claim 4 or application, it is characterised in that：The microorganism is bacteriophage, virus or bacterium.
6. the application described in method according to claim 1 or 2 or claim 3, it is characterised in that：The biomaterial
Any of for 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.
7. method according to claim 6 or application, it is characterised in that：The product is section.
8. biomaterial staining reagent, is the reagent described in claim 1 or 2.
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| CN201710610328.9A CN107290198B (en) | 2017-07-25 | 2017-07-25 | A method of biomaterial is dyed using PIP |
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| CN201710610328.9A CN107290198B (en) | 2017-07-25 | 2017-07-25 | A method of biomaterial is dyed using PIP |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907476A (en) * | 2019-11-14 | 2020-03-24 | 云南省地方病防治所 | Preparation method of phage transmission electron microscope specimen |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060004188A1 (en) * | 2000-09-29 | 2006-01-05 | Wai-Yee Leung | Intermediates and the synthesis of modified carbocyanine dyes and their conjugates |
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