CN110967241A - Dyeing method of Eimeria tenella protoplast - Google Patents
Dyeing method of Eimeria tenella protoplast Download PDFInfo
- Publication number
- CN110967241A CN110967241A CN201911280767.3A CN201911280767A CN110967241A CN 110967241 A CN110967241 A CN 110967241A CN 201911280767 A CN201911280767 A CN 201911280767A CN 110967241 A CN110967241 A CN 110967241A
- Authority
- CN
- China
- Prior art keywords
- eimeria tenella
- oocysts
- freeze
- staining
- hydrochloric acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000223932 Eimeria tenella Species 0.000 title claims abstract description 45
- 210000001938 protoplast Anatomy 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000004043 dyeing Methods 0.000 title claims abstract description 13
- 210000003250 oocyst Anatomy 0.000 claims abstract description 88
- 239000000975 dye Substances 0.000 claims abstract description 17
- 238000010257 thawing Methods 0.000 claims abstract description 12
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000005406 washing Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 238000010186 staining Methods 0.000 claims description 13
- 241000224483 Coccidia Species 0.000 claims description 12
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical group C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 241000287828 Gallus gallus Species 0.000 claims description 6
- 210000004534 cecum Anatomy 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 238000007447 staining method Methods 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 230000005284 excitation Effects 0.000 description 7
- 239000005708 Sodium hypochlorite Substances 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- 230000001351 cycling effect Effects 0.000 description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 235000013330 chicken meat Nutrition 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 208000003495 Coccidiosis Diseases 0.000 description 3
- 206010023076 Isosporiasis Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 102000011782 Keratins Human genes 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
- 238000002594 fluoroscopy Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
- 241000252983 Caecum Species 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- IYVLMMUENZSXFK-UHFFFAOYSA-N ethanol;hydrate;hydrochloride Chemical compound O.Cl.CCO IYVLMMUENZSXFK-UHFFFAOYSA-N 0.000 description 1
- 238000012757 fluorescence staining Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000815 hypotonic solution Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
Images
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
Abstract
The invention relates to a dyeing method of Eimeria tenella protoplasts, which comprises the following steps: (1) taking freshly separated immature eimeria tenella oocysts, and performing freeze-thawing circulation; (2) treating the oocysts subjected to freeze thawing circulation by using hydrochloric acid-ethanol, and after acting, centrifugally washing to remove acting liquid; (3) adding a dye, and carrying out fluorescent staining on the Eimeria tenella protoplast. The invention establishes a wall breaking technology for retaining the complete morphology of the oocyst wall of immature eimeria tenella, reports the dyeing method of the eimeria tenella protoplast for the first time, and has the advantages of simple and convenient procedure, credible method, bright result and easy observation and judgment.
Description
Technical Field
The invention relates to a dyeing method of parasites, in particular to a wall breaking technology for keeping the complete morphology of the oocyst wall of Eimeria tenella, and provides a coccidium protoplast dyeing method with simple dyeing steps and convenient operation.
Background
Immature eimeria tenella (e.tenella) oocyst walls are a strong, dense tissue structure that can withstand certain mechanical insults, and conventional chemicals, digestive enzymes, wash solutions, and hypotonic solutions are difficult to destroy the coccidia oocyst wall structure. The oocyst wall undergoes a significant structural change before and after maturation, and at the moment, the mature sporulated E.tenella oocysts can break the oocyst wall to some extent by adopting a mechanical method of repeated grinding by a grinder to free mature sporangia, but the oocyst wall of the coccidia is cracked into a plurality of small fragments, so that the whole structure does not exist any more. For immature oocysts, no report is found on staining protoplasts in oocysts while maintaining the shape of the oocyst wall. Therefore, it is difficult for scholars at home and abroad to permeate the oocyst wall with conventional dyes after in vitro wall breaking to color the immature E.tenella protoplasts.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the technical defect that the traditional mechanical mode can not keep the complete form of the immature E.tenella oocyst wall, establish a brand-new wall breaking technology and further realize the dyeing of coccidian protoplasts.
The technical scheme is as follows: the purpose of the invention can be realized by the following technical scheme:
a staining method of Eimeria tenella protoplasts comprises the following steps:
(1) taking freshly separated immature eimeria tenella oocysts, and performing freeze-thawing circulation;
(2) treating the oocysts subjected to freeze thawing circulation by using a hydrochloric acid-ethanol aqueous solution, and after acting, centrifugally washing to remove acting liquid;
(3) adding a dye, and carrying out fluorescent staining on the Eimeria tenella protoplast.
Preferably:
the freeze-thaw cycle is performed at 4 deg.C and-20 deg.C for 14-16 min.
The hydrochloric acid is concentrated hydrochloric acid with the concentration of 36-38%, the ethanol water solution is 93-97% ethanol water solution, and the volume ratio of the hydrochloric acid to the ethanol water solution is 1 (0.5-1.5).
The dye is Hoechst 33258 dye, and the final concentration of the dye is 0.5-1.5 mu g/ml.
The number of freeze-thaw cycles is three, and the number of centrifugal washes is three.
The oocysts after freeze thawing circulation are treated by using a hydrochloric acid-ethanol aqueous solution, the action time is 3-5h, and the action temperature is 4 ℃.
The oocysts were fresh immature eimeria tenella oocysts isolated from the cecum after coccidia infection of the chickens.
More preferred methods are as follows:
(1) taking fresh immature Eimeria tenella oocysts separated from caecum after coccidium infects chickens, and respectively placing the fresh immature Eimeria tenella oocysts under the conditions of 4 ℃ and-20 ℃ for freeze thawing cycle for three times, wherein the action time is 15min each time;
(2) treating oocysts subjected to freeze thawing circulation by using 37% concentrated hydrochloric acid-95% ethanol aqueous solution (volume ratio of 1:1), acting for 4h, and centrifuging and washing for three times to remove acting liquid;
(3) adjusting the concentration of oocysts to 1 × 103Adding Hoechst 33258 dye into the mixture per mu l, and adjusting the final concentration of the dye to be 1 mu g/ml;
(4) and (3) observing the fluorescence characteristics of the protoplast in the oocyst under the dark field of a fluorescence inverted microscope at an excitation wavelength of 532 nm.
The invention adopts a freeze-thaw cycle technology and hydrochloric acid-ethanol pretreatment to establish a wall breaking technology and a coccidian protoplast dyeing method which can perfectly preserve the whole morphology of immature E.tenella oocyst walls. The fluorescence microscope dark field condenser is adopted to enhance the brightness and contrast of the stained oocysts, improve the image quality and facilitate observation of the fluorescence stained protoplasts.
The freeze-thaw cycling technique refers to the alternating occurrence of freezing (below 0 ℃) and thawing (above 0 ℃) of moisture outside and inside the structure of the material. Research shows that the freeze thawing technology can rearrange the structure of soil grains and affect the physical and mechanical properties of soil. The technology is applied to destroy the oocyst wall of immature E.tenella, the temperature of the external structure of the oocyst wall is reduced to be below 0 ℃ so that water is frozen into ice, the temperature is maintained to be above 0 ℃ for a long time, the ice on the surface of the oocyst wall is melted into water, nonporous uninformed water drops penetrate inwards along gaps of the oocyst wall, and large expansion force is generated by repeating three times of freeze-thaw cycles so as to destroy the physical structure of the oocyst wall.
The immature e.tenella oocyst wall has two layers, the outer layer is a protective structure with chemical components similar to keratin; the inner layer is composed of small particles formed by zygotes in the development process, and the chemical components belong to lipid. The concentrated hydrochloric acid treatment can partially hydrolyze keratin on the outer layer, and the ethanol treatment can dissolve part of lipid on the inner layer, so that the chemical structure of the oocyst wall is damaged, but the appearance of the oocyst wall is not influenced. The three-time freeze-thaw cycle and the concentrated hydrochloric acid-ethanol water solution combined treatment method used in the invention can achieve 45-50% of damage rate to the oocyst wall, increase the permeability of the oocyst wall and lay a key foundation for the next coccidian protoplast staining.
Hoechst 33258 is a blue fluorescent dye which can penetrate cell membranes, has low cytotoxicity, is usually used for dyeing common cell nuclei, and is detected by a fluorescence microscope or a flow cytometer after dyeing. Fluorescence staining results show that the oocysts of the combined treatment group can obviously see the complete outline of cell membranes and oocyst walls, and cell nuclei are specifically stained to be bright blue (see figure 1), while the normal control group and the freezing-thawing cycle treatment group only do not see the nuclear deep staining (figures 2 and 3). The invention reports a staining method of E.tenella protoplast for the first time, and can capture a fluorescence microscopic image with clear staining effect.
Coccidian protoplast is not easy to stain, but after the combined treatment of freeze-thaw cycle and hydrochloric acid-ethanol, certain components in the oocyst wall structure are destroyed to allow dye to enter the oocyst and stain the protoplast, the method can effectively destroy the compact tissue structure of the oocyst wall and can completely preserve the shape of the oocyst wall, so that Hoechst 33258 dye can more easily permeate into the oocyst through the structural gaps of the destroyed oocyst wall and penetrate through cell membranes, thereby leading to the dense staining of the protoplast. Therefore, the writer adopts the wall breaking technology to realize the fluorescent staining of the protoplast in the coccidian oocyst.
The technical effects are as follows: compared with the prior art, the traditional mechanical grinding wall-breaking method can completely collapse the whole structure of the oocyst wall and release the sporangium, and is mainly applied to wall-breaking treatment of mature E.tenella sporulated oocysts. The research successfully establishes the wall breaking technology of the immature E.tenella oocysts by carrying out freeze-thaw cycle and hydrochloric acid-ethanol combined treatment on the immature E.tenella oocysts, well retains the overall morphological structure of the oocyst wall, realizes the fluorescent dyeing of coccidia protoplasts, and has the advantages of simple operation, credible method, clear result and easy observation and judgment.
Drawings
FIG. 1 is a fluorescence plot (excitation wavelength 532nm) of freeze-thaw cycles + HCl-ethanol treatment oocysts;
FIG. 2 is a fluorescence image (excitation wavelength 532nm) of oocysts treated with freeze-thaw cycles only;
FIG. 3 is a fluorescence image (excitation wavelength 532nm) of normal coccidia oocysts;
Detailed Description
The present invention will be described in further detail with reference to specific examples below:
example 1
(1) Fresh immature Eimeria tenella oocysts isolated from ceca after coccidiosis infection in chickens were taken, treated with 20% sodium hypochlorite solution in a refrigerator at 4 ℃ for 10min, and centrifuged and washed 3 times with sterile water to remove sodium hypochlorite. Adding 1.1mol/L sucrose solution into the final precipitate, centrifuging at 2000rpm for 10min, floating oocysts, sucking the oocysts into another centrifuge tube, and washing with sterilized water for 3 times to remove sucrose.
(2) Respectively placing the purified immature Eimeria tenella oocysts at 4 ℃ and-20 ℃ for 15min, and performing freeze-thaw cycling for three times;
(3) treating immature Eimeria tenella oocysts subjected to freeze-thaw cycle with 37% concentrated hydrochloric acid-95% ethanol water solution (volume ratio of 1:1), acting at 4 deg.C for 4 hr, centrifuging to remove acting solution, washing with distilled water for 3 times, and adjusting the concentration of oocysts to 1 × 103Mu/l;
(4) adding Hoechst 33258 dye into Eppendorf tube containing the processed oocysts, and adjusting the final concentration to be 1 μ g/ml;
(5) and (3) observing the staining characteristic of the protoplast in the oocyst in a dark field by adopting a dark field condenser of a fluorescence inverted microscope at an excitation wavelength of 532 nm. Fluorescence results show that protoplasts in normal coccidia and only freeze-thaw cycle group oocysts are not stained (see fig. 2 and 3), and the freeze-thaw cycle and hydrochloric acid-ethanol treatment group oocysts can obviously see that the protoplasts are specifically stained into brilliant blue (as shown in fig. 1), which indicates that the freeze-thaw cycle and hydrochloric acid-ethanol combined treatment can destroy compact tissue structures of oocyst walls, increase permeability of oocyst walls, enable Hoechst 33258 dye to easily permeate into the oocysts and penetrate cell membranes, so that the protoplasts are heavily stained, and excellent fluorescence microscopic observation results are obtained.
Example 2
(1) Fresh immature Eimeria tenella oocysts isolated from ceca after coccidiosis infection in chickens were taken, treated with 20% sodium hypochlorite solution in a refrigerator at 4 ℃ for 10min, and centrifuged and washed 3 times with sterile water to remove sodium hypochlorite. Adding 1.1mol/L sucrose solution into the final precipitate, centrifuging at 2000rpm for 10min, floating oocysts, sucking the oocysts into another centrifuge tube, and washing with sterilized water for 3 times to remove sucrose.
(2) Placing the purified immature Eimeria tenella oocysts at 4 ℃ and-20 ℃ for 14min respectively, and performing freeze-thaw cycling for three times;
(3) treating immature Eimeria tenella oocysts subjected to freeze-thaw cycling by using 36% concentrated hydrochloric acid-93% ethanol aqueous solution (volume ratio of 1:0.5), acting at 4 ℃ for 3h, centrifuging to remove acting liquid, washing with distilled water for 3 times, and adjusting the concentration of oocysts to 1 × 103Mu/l;
(4) adding Hoechst 33258 dye into Eppendorf tube containing the processed oocysts, and adjusting the final concentration to 0.5 mug/ml;
(5) and (3) observing the staining characteristic of the protoplast in the oocyst in a dark field by adopting a dark field condenser of a fluorescence inverted microscope at an excitation wavelength of 532 nm. The results of the fluoroscopy were substantially the same as in example 1.
Example 3
(1) Fresh immature Eimeria tenella oocysts isolated from ceca after coccidiosis infection in chickens were taken, treated with 20% sodium hypochlorite solution in a refrigerator at 4 ℃ for 10min, and centrifuged and washed 3 times with sterile water to remove sodium hypochlorite. Adding 1.1mol/L sucrose solution into the final precipitate, centrifuging at 2000rpm for 10min, floating oocysts, sucking the oocysts into another centrifuge tube, and washing with sterilized water for 3 times to remove sucrose.
(2) Respectively placing the purified immature Eimeria tenella oocysts at 4 ℃ and-20 ℃ for 16min, and performing freeze-thaw cycling for three times;
(3) treating immature Eimeria tenella oocysts subjected to freeze-thaw cycling by using 38% concentrated hydrochloric acid-97% ethanol aqueous solution (volume ratio of 1:1.5), acting at 4 ℃ for 5h, centrifuging to remove acting liquid, washing with distilled water for 3 times, and adjusting the concentration of oocysts to 1 × 103Mu/l;
(4) adding Hoechst 33258 dye into Eppendorf tube containing the processed oocysts, and adjusting the final concentration to be 1.5 mu g/ml;
(5) and (3) observing the staining characteristic of the protoplast in the oocyst in a dark field by adopting a dark field condenser of a fluorescence inverted microscope at an excitation wavelength of 532 nm. The results of the fluoroscopy were substantially the same as in example 1.
Claims (7)
1. A staining method of Eimeria tenella protoplasts is characterized by comprising the following steps:
(1) taking freshly separated immature eimeria tenella oocysts, and performing freeze-thawing circulation;
(2) treating the oocysts subjected to freeze thawing circulation by using a hydrochloric acid-ethanol aqueous solution, and after acting, centrifugally washing to remove acting liquid;
(3) adding a dye, and carrying out fluorescent staining on the Eimeria tenella protoplast.
2. The method for staining Eimeria tenella protoplasts according to claim 1, wherein the freeze-thaw cycle is performed at 4 ℃ and-20 ℃ for 14-16min each time.
3. The method for dyeing Eimeria tenella protoplasts according to claim 1, wherein the hydrochloric acid is concentrated hydrochloric acid with a concentration of 36-38%, the ethanol is 93-97% ethanol aqueous solution, and the volume ratio of the hydrochloric acid to the ethanol aqueous solution is 1 (0.5-1.5).
4. The method for staining Eimeria tenella protoplasts according to claim 1, wherein the dye is Hoechst 33258 dye at a final concentration of 0.5 to 1.5 μ g/ml.
5. The method for staining Eimeria tenella protoplasts according to claim 1, wherein the number of freeze-thaw cycles is three, and the number of centrifugal washes is three.
6. The method for staining Eimeria tenella protoplasts according to claim 1, wherein the oocysts after freeze-thaw cycle are treated with hydrochloric acid-ethanol for 3-5h at 4 ℃.
7. The method for staining Eimeria tenella protoplasts according to claim 1, wherein the oocysts are fresh immature Eimeria tenella oocysts isolated from the cecum after coccidia infection of the chicken.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911280767.3A CN110967241B (en) | 2019-12-13 | 2019-12-13 | Dyeing method of Eimeria tenella protoplast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911280767.3A CN110967241B (en) | 2019-12-13 | 2019-12-13 | Dyeing method of Eimeria tenella protoplast |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110967241A true CN110967241A (en) | 2020-04-07 |
| CN110967241B CN110967241B (en) | 2022-07-29 |
Family
ID=70034293
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911280767.3A Active CN110967241B (en) | 2019-12-13 | 2019-12-13 | Dyeing method of Eimeria tenella protoplast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110967241B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995031481A1 (en) * | 1994-05-18 | 1995-11-23 | The Research And Development Institute, Inc. | Simple, rapid method for the detection, identification and enumeration of specific viable microorganisms |
| WO2005124351A2 (en) * | 2004-06-09 | 2005-12-29 | The United States Of America, As Represented By The Secretary Of Agriculture | A sensitive antibody-based method for detecting cryptosporidium parvum oocysts in water |
| CN101928771A (en) * | 2009-11-26 | 2010-12-29 | 扬州大学 | Multiplex PCR method for quickly identifying species of chicken coccidia and kit |
| CN101955956A (en) * | 2010-08-13 | 2011-01-26 | 中国农业科学院上海兽医研究所 | Eimeria tenella calcium-dependent protein kinase gene and application thereof |
| CN102154496A (en) * | 2011-03-19 | 2011-08-17 | 吉林大学 | Various important aquagenic zoonoses protozoa simultaneous assay kit and preparation method thereof |
| CN110484450A (en) * | 2019-07-25 | 2019-11-22 | 扬州大学 | A kind of preparation method of Eimeria unsporulated oocysts wall |
-
2019
- 2019-12-13 CN CN201911280767.3A patent/CN110967241B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995031481A1 (en) * | 1994-05-18 | 1995-11-23 | The Research And Development Institute, Inc. | Simple, rapid method for the detection, identification and enumeration of specific viable microorganisms |
| WO2005124351A2 (en) * | 2004-06-09 | 2005-12-29 | The United States Of America, As Represented By The Secretary Of Agriculture | A sensitive antibody-based method for detecting cryptosporidium parvum oocysts in water |
| CN101928771A (en) * | 2009-11-26 | 2010-12-29 | 扬州大学 | Multiplex PCR method for quickly identifying species of chicken coccidia and kit |
| CN101955956A (en) * | 2010-08-13 | 2011-01-26 | 中国农业科学院上海兽医研究所 | Eimeria tenella calcium-dependent protein kinase gene and application thereof |
| CN102154496A (en) * | 2011-03-19 | 2011-08-17 | 吉林大学 | Various important aquagenic zoonoses protozoa simultaneous assay kit and preparation method thereof |
| CN110484450A (en) * | 2019-07-25 | 2019-11-22 | 扬州大学 | A kind of preparation method of Eimeria unsporulated oocysts wall |
Non-Patent Citations (3)
| Title |
|---|
| SASAI, K: "A chicken anti-conoid monoclonal antibody identifies a common epitope which is present on motile stages of Eimeria, Neospora, and Toxoplasma", 《JOURNAL OF PARASITOLOGY》 * |
| 郭海宁: "鸡艾美耳球虫卵囊染色试验初报", 《中国家禽》 * |
| 黄杰: "青蒿素与癸氧喹酯联用对鸡柔嫩艾美耳球虫感染的预防作用", 《畜牧与兽医》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110967241B (en) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103835003B (en) | A kind of preparation method of bluish dogbane degummed ramie | |
| CN105001971B (en) | A kind of method of two-step method production microalgae bio oil | |
| CN107389641A (en) | A kind of method based on immune vestige method detection ancient times argillization silk goods | |
| JPWO2011115039A1 (en) | Production method and apparatus for sugar solution | |
| CN110967241B (en) | Dyeing method of Eimeria tenella protoplast | |
| CN105385652A (en) | High-purity cardiac muscle cell primary culture method | |
| CN104746348A (en) | A cotton yarn scouring method by applying pectinase and neutral cellulase | |
| CN101607933B (en) | Technology for preparing astaxanthin using microwave-assisted dimethyl sulfoxide method | |
| CN106554984A (en) | The method for extracting bata-carotene | |
| CN109322161A (en) | A kind of cotton-polyester blend fabric scouring agent and its method for refining | |
| Ren et al. | Preparation and regeneration of protoplasts from the ethyl vincamine producing fungus CH1 (Geomyces sp.) | |
| CN111448298B (en) | Method for separating microbial oil | |
| CN112480241A (en) | Method for extracting phycocyanin from fresh spirulina harvested in high-salt environment | |
| CN102835652B (en) | Method for extracting cordyceps hirsutella sinensis mycelia on basis of membrane technology | |
| CN104651948B (en) | A kind of lithographic method of c surface sapphires | |
| CN102517872B (en) | Oxygen bleaching-alkali decrement-bleaching one-bath treatment method for T/C and CVC fabrics | |
| CN113862312A (en) | Preparation method of schizochytrium oil containing EPA and DHA | |
| CN104711252A (en) | Extraction method for fresh water red algae DNA | |
| Wisher et al. | The large-scale preparation of purified sporozoites of Eimeria spp. by metrizamide density-gradient centrifugation | |
| CN105507003A (en) | Textile | |
| KR20020093456A (en) | In situ extraction of hydrocarbon from the culture of microalgae | |
| CN106987534B (en) | A kind of method of pepper fresh fruit biology decortication preparation white pepper | |
| CN107141365A (en) | A kind of method that pressure of plus-minus repeatedly efficiently purifies Phellinus polysaccharide | |
| Katoh et al. | [37] Xanthosomes: Supramolecular assemblies of xanthophyll-chlorophyll a/c protein complexes | |
| CN114023638B (en) | Method for removing inversion layer of silicon wafer after phosphorus diffusion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |