CN103792126A - Staining solution applicable to tea geometrid blood cells and staining method thereof - Google Patents
Staining solution applicable to tea geometrid blood cells and staining method thereof Download PDFInfo
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- CN103792126A CN103792126A CN201310586918.4A CN201310586918A CN103792126A CN 103792126 A CN103792126 A CN 103792126A CN 201310586918 A CN201310586918 A CN 201310586918A CN 103792126 A CN103792126 A CN 103792126A
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Abstract
The invention provides a staining solution applicable to tea geometrid blood cells and a staining method thereof. The staining solution comprises Wright powder and tea polyphenol powder according to a mass ratio of (4-8):1; the staining time of a staining solution prepared from the Wright powder and the tea polyphenol powder lasts for 1-3 minutes; a staining time of a phosphatic buffer solution lasts for 5-10 minutes. The staining solution which is specialty used for identifying tea geometrid blood cells and the staining method thereof have the characteristics of being capable of quickly, accurately and intuitively identifying the types of the tea geometrid blood cells, enriches physiological and biochemical research techniques of the tea geometrids and has a significance in further deep research on invasion rules of biological control measures such as tea geometrid virus and screening of biological control medicines.
Description
Technical field
The present invention relates to dyeing liquor and the colouring method of cell, relate in particular to the dyeing isolation and identification method of the haemocyte in tea geometrid larvae hemolymph.
Background technology
Insect is the widest as organic sphere distribution, a types of populations of One's name is legion, has possessed the adaptive faculty of height and unique immune system in long-term evolutionary process.The immunity of insect comprises humoral immunity and two parts of cellular immunity.Cellular immunity main manifestations is haemocyte mediated immunity reaction, as engulf, tubercle, packing etc., be also responsible in addition removing apoptotic cell and fragment of tissue in body, thereby participate in tissue reconstruction and organ reparation.Humoral immunity effect produces the proteins and peptides of many antibiotic properties, is the important composition composition of defense system.
A lot of about the research of insect blood cell, research contents relates to more than 10 orders more than 100 plant.But insect blood cell form is various, variation is complicated, brings difficulty not only to the classification of haemocyte, but also has affected deep researchs in aspect such as its physiological function, biochemistry.In recent years, obtained new progress in the research of the aspects such as insect blood cell variation of quantity, Observation On The Morphology, physiology, biochemistry, immunology and molecular biology, and its research means has been not quite similar.Research means is mainly the different of colouring method and microexamination technology.Conventional colouring method is to have acridine orange, propidium iodide, dimethyl diaminophenazine chloride, haematine, Giemsa staining, Wright's staining and Ji-Rui union dyeing etc.Microexamination means mainly adopt the technology such as optical microscope, fluorescent microscope, phase microscope, scanning electron microscope and transmission electron microscope.
Though in view of the research of the insect haemocytes such as current Lepidoptera such as bollworm, silkworm, prodenia litura has been reported, the characteristics such as the Physiology and biochemistry of different insects kind all have its singularity.And tea geometrid is as important Pests of Tea-Plants, research in other respects has remarkable progress, the blood cell staining method of tea geometrid is done more deep research by being conducive to its haemocyte, and then to the more deep understanding of the immunization of haemocyte, for the antijamming capability of tea geometrid provides foundation, for the research of control tea geometrid medicine provides reference, finally for the series of problems such as the mechanism of causing a disease of tea geometrid find answer approach.And current conventional decoration method, the problem such as staining cell core, cytoplasmic poor effect simultaneously, exists boundary unclear, and kytoplasm particle Color is poor.
Summary of the invention
The dyeing liquor and the colouring method that the object of the present invention is to provide the Color that a kind of kytoplasm karyon had simultaneously, be specially adapted to the haemocyte in tea geometrid hemolymph.
The object of the invention is to realize by such technical scheme:
The dyeing liquor of tea geometrid haemocyte, comprises Rui Shi powder (Wright) and tea polyphenol powder (TP), and the mass ratio of Rui Shi powder and tea polyphenol powder is 4～8:1, and dyeing time is 1～3min.Preferably, the mass ratio of Rui Shi powder and tea polyphenol powder is 6:1, and dyeing time is 3min.
The dyeing liquor of above-mentioned tea geometrid haemocyte, comprises Rui Shi-Tea Polyphenols dye liquor that Rui Shi powder, tea polyphenol powder, glycerine and methyl alcohol are prepared; Glycerine is 10 times of volumes of Rui Shi powder and tea polyphenol powder general assembly (TW), and methyl alcohol is 500 times of volumes of Rui Shi powder and tea polyphenol powder general assembly (TW).The Color that this dyeing liquor had both had, the term of life that can extend again dyeing liquor, one can be deposited about 1 year.
Use the colouring method of above-mentioned tea geometrid blood cell staining liquid, the dyeing liquor dyeing time of Rui Shi powder and tea polyphenol powder preparation is 1～3min, and phosphate buffer dyeing time is 5～10min.
The colouring method of above-mentioned tea geometrid haemocyte, comprises the following steps:
Reagent preparation:
Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder, according to the mass ratio of 4～8:1, put into mortar, then add the glycerine of Rui Shi and 10 times of volumes of tea polyphenol powder general assembly (TW), grind until without particle, dissolve in the methyl alcohol of Rui Shi and 500 times of volumes of tea polyphenol powder general assembly (TW);
The potassium dihydrogen phosphate of phosphate buffer: 10g/L, the sodium hydrogen phosphate of 10g/L, PH6.8～7.4;
Sampling: tea geometrid larvae ice bath 10min, gets the liquid of haemolymph of abdominal foot;
Dyeing: will dye drop on the liquid of haemolymph of step (1), and after dyeing 1-3min, add phosphate buffer in the dyeing liquor of liquid of haemolymph, dyeing 5～10min, rinses, and dries.
Particularly, the colouring method of above-mentioned tea geometrid haemocyte, comprises the following steps
Reagent preparation:
Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder according to the mass ratio of 4～8:1, put into mortar, add again the glycerine of Rui Shi and 10 times of volumes of tea polyphenol powder general assembly (TW), grind until without particle, dissolve in the methyl alcohol of Rui Shi and 500 times of volumes of tea polyphenol powder general assembly (TW);
The potassium dihydrogen phosphate of phosphate buffer: 10g/L, the sodium hydrogen phosphate of 10g/L, PH6.8～7.4;
Sampling: after tea geometrid larvae cleans up with distilled water, with 75% alcohol disinfecting, after ice bath 10min, cut off an abdominal foot, drip one and bleed lymph on the clean microslide of ethanol, for subsequent use.
Dyeing: after tea geometrid larvae liquid of haemolymph is natural drying, drips Rui Shi-Tea Polyphenols dyeing liquor number and drip, dyeing 1-3min, then adds above-mentioned phosphate buffer in dyeing liquor, mixes, dyeing 5-10min, and tap water rinses, and dries, and envelope is hidden.
Beneficial effect
1. dyeing liquor and the colouring method of differentiating tea geometrid haemocyte of being exclusively used in provided by the invention, to differentiating the kind of tea geometrid haemocyte, have fast, accurately, feature intuitively, enriched tea geometrid physiological and biochemical research technology.Wait invasion rule, the biological control drug screening etc. of bio-control method significant to further further investigation tea geometrid virus.
2. the tea geometrid blood cell staining of the present invention's dyeing is clear, even, and obviously, cell outline is clear for nucleus and tenuigenin contrast, is convenient to observe and identify.
3. the present invention had both had the hemolymph of preventing melanism function, had kept again the activity of tea geometrid haemocyte, maintained as much as possible the original life state of cell, so that coloration result can reflect the immunity of cell, the physiological biochemical property such as anti-interference intuitively, really.
Accompanying drawing explanation
Fig. 1-2 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 6:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 5min;
Fig. 3 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 6:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 10min;
Fig. 4 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 8:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 5min;
Fig. 5 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 8:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 10min;
Fig. 6 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 5:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 5min;
Fig. 7 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 5:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 10min;
Fig. 8 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 4:1, after dyeing time is 3min, drips dye the respectively coloration result of 5～10min of PBS.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated, but not only for so.For a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Embodiment 1
The preparation of Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder 0.5g altogether according to the ratio of 4:1, put into mortar, then add 5mL glycerine, be ground to without till particle, dissolve in 250mL methyl alcohol, after filtering with absorbent cotton, place shady place, fully dissolve, within one 1 month, be advisable above.
Get tea geometrid 4～5 instar larvaes, after cleaning up with distilled water, use 75% alcohol disinfecting, ice bath 10min again, operating scissors cut off a larva abdominal foot, drip one and bleed lymph liquid on the microslide after ethanol is clean, with one side contact drop of blood of cover glass, then with 45.Angle is close to microslide and is moved right and pull into uniform one deck blood film.On the blood film drying, drip above-mentioned Rui Shi-Tea Polyphenols dye liquor, to covering whole blood film, unnecessary dye liquor sucks with filter paper, dyeing 1～3min.Then add and be overlying on the phosphate buffer (PH6.8～7.4) doubly of Rui Shi-Tea Polyphenols dye liquor volume 2-4 on blood film in mixing on dye liquor, mix gently (not encountering microslide is advisable) with liquid transfer gun head, continue after dyeing 5～10min, control tap water current, become shallow bid, slowly rinse (thereby in order to avoid a large amount of haemocytes is fallen and affects later observation from microslide upper punch), no longer include dyeing liquor to slide surface.After being put into shady and cool place and drying, use neutral gum mounting.In 1000 times of optical microphotograph Microscopic observations.
Embodiment 2
The preparation of Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder 0.3g altogether, according to the ratio of 5:1, put into mortar respectively, then add 3mL glycerine, grind until without after particle, dissolve in 150mL methyl alcohol, after filtering with absorbent cotton, place shady place, fully dissolve, within one 1 month, be advisable above.
Concrete dyeing course is with embodiment 1.
Embodiment 3
The preparation of Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder 0.35g altogether according to the ratio of 6:1, put into mortar, then add 3.5mL glycerine, be ground to without after particle, dissolve in 175mL methyl alcohol, after filtering with absorbent cotton, place shady place, fully dissolve, within one 1 month, be advisable above.
Concrete dyeing course is with embodiment 1.
Embodiment 4
The preparation of Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder 0.36g altogether according to the ratio of 8:1, put into mortar, then add 3.6mL glycerine, grind until without after particle, dissolve in 180mL methyl alcohol, after filtering with absorbent cotton, place shady place, fully dissolve, within one 1 month, be advisable above.
Concrete dyeing course is with embodiment 1.
Table 1 be according to embodiment to contrast situation to haemocyte (take plasmatocytes as example) Color of the different proportionings of Rui Shi-Tea Polyphenols dyeing liquor (W-G), dyeing time and phosphate buffer (PBS) coloured differently time.
Table 1
Note: in table, W-T is Rui Shi and Tea Polyphenols mixing dye liquor; PBS is phosphate buffer
Fig. 1-2 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 6:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 5min, the blood cell staining of tea geometrid is clear, even, and obviously, cell outline is clear for nucleus and tenuigenin contrast, be convenient to observe and identify, cytoactive is high;
Fig. 3 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 6:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 10min, and the haemocyte tenuigenin dyeing of tea geometrid is slightly partially light, but nucleus and tenuigenin contrast are obviously, and whole cell outline is clear;
Fig. 4 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 8:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 5min, and the nucleus of tea geometrid haemocyte and tenuigenin dyeing are all excessively dark, are difficult to distinguish nuclear profile;
Fig. 5 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 8:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 10min, and the tenuigenin of tea geometrid haemocyte and nucleus dyeing are all partially dark, be dark blue purple, but karyon border are more clear;
Fig. 6 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 5:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 5min, and nucleus dyeing is clear, and tenuigenin dyeing is too light, and cell boundaries only has part clear;
Fig. 7 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 5:1, after dyeing time is 3min, drips the coloration result of PBS dyeing 10min, and the tenuigenin dyeing of tea geometrid haemocyte is partially light, and karyon border is more complete;
Fig. 8 is that Rui Shi-Tea Polyphenols dye liquor proportioning is 4:1, after dyeing time is 3min, drips dye the respectively coloration result of 5～10min of PBS, and the tenuigenin dyeing that all shows as tea geometrid haemocyte is too light, almost illegible cell outline.
Can find out by the proportionings different from tea polyphenol powder of the Rui Shi powder in embodiment and the Color of coloured differently time, Rui Shi powder and tea polyphenol powder are mixed comparatively desirable with the ratio of 6:1, and its dyeing time Rui Shi-Tea Polyphenols dye liquor is 3min, and PBS dyeing time is that 5～10min all can.The Color of other proportionings and dyeing time mainly contains following several situation: the nucleus of haemocyte and tenuigenin dyeing are partially dark, but karyon border is also more clear, is even difficult to distinguish nuclear profile; It is all partially shallow that another is exactly the tenuigenin of haemocyte and nucleus dyeing, but also more complete or almost illegible cell outline of karyon border; Again one be exactly the nucleus of haemocyte and tenuigenin one of them is partially light, another is but partially dark, most tenuigenin does not dye easily, and easy dyeing is partially light.
Claims (6)
1. a dyeing liquor for tea geometrid haemocyte, is characterized in that: comprise Rui Shi powder and tea polyphenol powder, the mass ratio of Rui Shi powder and tea polyphenol powder is 4 ~ 8:1, and dyeing time is 1-3min.
2. the dyeing liquor of tea geometrid haemocyte as claimed in claim 1, is characterized in that: the mass ratio of Rui Shi powder and tea polyphenol powder is 6:1, and dyeing time is 3min.
3. the dyeing liquor of tea geometrid haemocyte as claimed in claim 1 or 2, is characterized in that: comprise Rui Shi-Tea Polyphenols dye liquor of Rui Shi powder, tea polyphenol powder, glycerine and methyl alcohol preparation, and phosphate buffer; Glycerine is 10 times of volumes of Rui Shi powder and tea polyphenol powder general assembly (TW), and methyl alcohol is 500 times of volumes of Rui Shi powder and tea polyphenol powder general assembly (TW), and phosphate buffer is 2 ~ 4 times of Rui Shi-Tea Polyphenols dye liquor volume; Described phosphate buffer is 10g/L potassium dihydrogen phosphate, 10g/L sodium hydrogen phosphate, PH6.8 ~ 7.4.
4. right to use requires the colouring method of the dyeing liquor of the tea geometrid haemocyte described in 3, it is characterized in that: Rui Shi-Tea Polyphenols dye liquor dyeing time is 1-3min, and phosphate buffer dyeing time is 5-10min.
5. the colouring method of the dyeing liquor of the tea geometrid haemocyte as described in claim 4 or 5, comprises the following steps:
● reagent preparation:
Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder, according to the mass ratio of 4 ~ 8:1, put into mortar, then add the glycerine of Rui Shi and 10 times of volumes of tea polyphenol powder general assembly (TW), grind until without particle, dissolve in the methyl alcohol of Rui Shi and 500 times of volumes of tea polyphenol powder general assembly (TW);
The potassium dihydrogen phosphate of phosphate buffer: 10g/L, the sodium hydrogen phosphate of 10g/L, PH6.8 ~ 7.4;
● sampling: tea geometrid larvae ice bath 10min, gets the liquid of haemolymph of abdominal foot;
● dyeing: will dye drop on liquid of haemolymph, and after dyeing 1-3min, add phosphate buffer in the dyeing liquor of liquid of haemolymph, dyeing 5-10min, rinses, and dries.
6. the colouring method of the dyeing liquor of tea geometrid haemocyte as claimed in claim 6, comprises the following steps:
● reagent preparation:
Rui Shi-Tea Polyphenols dye liquor: get Rui Shi powder and tea polyphenol powder according to the mass ratio of 4 ~ 8:1, put into mortar, add again the glycerine of Rui Shi and 10 times of volumes of tea polyphenol powder general assembly (TW), grind until without particle, dissolve in the methyl alcohol of Rui Shi and 500 times of volumes of tea polyphenol powder general assembly (TW);
The potassium dihydrogen phosphate of phosphate buffer: 10g/L, the sodium hydrogen phosphate of 10g/L, PH6.8 ~ 7.4;
● sampling: after tea geometrid larvae cleans up with distilled water, with 75% alcohol disinfecting, after ice bath 10min, cut off an abdominal foot, drip one and bleed lymph on the clean microslide of ethanol, for subsequent use;
● dyeing: after tea geometrid larvae liquid of haemolymph is natural drying, drip Rui Shi-Tea Polyphenols dyeing liquor dyeing 1-3min, then add above-mentioned phosphate buffer in dyeing liquor, mix, dyeing 5-10min, tap water rinses, and dries, and envelope is hidden.
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Citations (4)
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| CN1434127A (en) * | 2002-01-22 | 2003-08-06 | 裴芳君 | Staining kit and preparation method, staining method, and use thereof |
| CN101122601A (en) * | 2007-09-24 | 2008-02-13 | 孙艳萍 | Method for separating and authenticating erythroblast of blood |
| US20110171214A1 (en) * | 2008-05-22 | 2011-07-14 | Larry Mark Weisenthal | Efficient well being assessment and improved treatment protocol |
| CN103323313A (en) * | 2012-03-23 | 2013-09-25 | 黄伏生 | Liquid-based cell analyzer staining method of cells exfoliated from serous cavity by using Wright staining method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1434127A (en) * | 2002-01-22 | 2003-08-06 | 裴芳君 | Staining kit and preparation method, staining method, and use thereof |
| CN101122601A (en) * | 2007-09-24 | 2008-02-13 | 孙艳萍 | Method for separating and authenticating erythroblast of blood |
| US20110171214A1 (en) * | 2008-05-22 | 2011-07-14 | Larry Mark Weisenthal | Efficient well being assessment and improved treatment protocol |
| CN103323313A (en) * | 2012-03-23 | 2013-09-25 | 黄伏生 | Liquid-based cell analyzer staining method of cells exfoliated from serous cavity by using Wright staining method |
Non-Patent Citations (1)
| Title |
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| 杨祚升等: "血细胞快速染色法及临床应用", 《衡阳医学院学报》 * |
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