CN105820995A - Method for collecting Chilo suppressalis fat body used for cell culture - Google Patents
Method for collecting Chilo suppressalis fat body used for cell culture Download PDFInfo
- Publication number
- CN105820995A CN105820995A CN201510004977.5A CN201510004977A CN105820995A CN 105820995 A CN105820995 A CN 105820995A CN 201510004977 A CN201510004977 A CN 201510004977A CN 105820995 A CN105820995 A CN 105820995A
- Authority
- CN
- China
- Prior art keywords
- chilo suppressalis
- fat
- fat body
- tweezers
- culture
- 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.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for collecting Chilo suppressalis fat body used for cell culture. The method comprises the followings steps: 1) producing a wax tray; 2) immersing Chilo suppressalis larva in an ethyl alcohol solution with concentration of 75%, performing surface disinfection; 3) cleaning the Chilo suppressalis larva by sterile distilled water; 4) absorbing the Chilo suppressalis larva by sterile filter paper; 5) placing the Chilo suppressalis larva in the wax tray disinfected by ethyl alcohol solution with concentration of 75%, using dissecting needles for fixing a head and a tail; 6) using a disinfected dissecting scissors for shearing the back of Chilo suppressalis from the tail to the head, immediately using a aseptic dissect needle for fixing the sheared skin on the wax tray; 7) taking the fat body attached on the body cavity by a tweezers, placing the fat body in a culture dish containing a HBSS rinse-solution for cleaning; and 8) transferring the cleaned fat body to a cell culture bottle by the tweezers for primary culture. The method can rapidly and massively obtain the fat body required by primary culture of Chilo suppressalis in short time, and can solve the problem of difficult collection of the Chilo suppressalis fat body.
Description
Technical field
The present invention relates to belong to insect anatomy technical field, particularly relate to a kind of collection method cultivating striped rice borer fat-body for cell.
Background technology
Along with the fast development of life sciences, in technical field of bioengineering, cell engineering the most more and more comes into one's own.Cultivate insect cell as research material, always cytobiology, molecular biology and the importance of the scientific research such as biochemistry and insect toxicology.
Fat-body is one of important materials of insecticide physiological and biochemical research, is the important place of insecticide metabolism, is also that in metabolic process, various materials store and the place of exchange.Play an important role at aspects such as the defence of insecticide, freeze proof, immune, damage responses.At present, one of research material that fat-body is the most frequently used in having become insecticide research.Owing to insecticide individuality mostly is less, fat-body content is few, in vitro after be susceptible to dry and hard and blackening reaction, bring certain difficulty to collection work.In recent years, along with going deep into of insecticide fat-body research and increasing of studied caste, the method for collection insecticide fat-body is also in multiformity, and the most quickly, in a large number, the free of contamination fat-body extracting insecticide, is the committed step of subsequent cell cultivation.
Striped rice borerChilosuppressalis(Walker) belonging to Lepidoptera, Pyralidae, is to endanger one of the most serious Occurrence on China Oryza sativa L., be injured in tillering stage and cause withered sheath, withered heart Seedling, being injured in the fringe phase and cause insect bite strain and dead ears, the general time underproduction 3%～5%, time serious, the underproduction is more than 3 one-tenth.In addition to hazard rice, striped rice borer also endangers the crops such as Semen Maydis, Caulis Sacchari sinensis, foxtail millet, Semen Viciae fabae, Caulis Zizaniae caduciflorae, Sorghum vulgare Pers., Brassica campestris L, Semen Tritici aestivi, Herba Astragali Melilotoidis (Herba Astragali Sinici).Striped rice borer has the strongest adaptation ability, can rapidly adapt to adverse environment (such as resistant variety and insecticide), and it causes evil performance and quickly makes a variation.During the preventing and treating of striped rice borer, screening and the qualification of novel biopesticide are increasingly becoming research emphasis.The activity bioassay of biological pesticide needs the striped rice borer examination worm that a large amount of growth is consistent, strict requirements are wanted to raising place, workload is big, if and have the striped rice borer cell of isolated culture, then can study the biological pesticide effect to striped rice borer in vitro, thus contribute to studying the insecticide mechanism of action to striped rice borer at cellular level, contribute to the improvement of current insecticide and the invention of new pesticides.By the invention it is possible to effectively extract striped rice borer fat-body, set up striped rice borer cell line, quantity and the kind of the new insect cell line of China can not only be increased, moreover it is possible to offer reference experience for the foundation of such insect cell line in the future.
Summary of the invention
It is an object of the invention to provide a kind of collection method cultivating striped rice borer fat-body for cell, provide technical support and guarantee for insecticide, particularly striped rice borer physiological and biochemical research, toxicologic study and bioreactor etc..
The present invention completes by the following technical programs:
(1) cake wax is made；
(2) choose 4-5 Chilo spp larvae in age, Chilo spp larvae is immersed in 75% ethanol solution, carries out surface sterilization 10-15 minute；
(3) Chilo spp larvae is cleaned 3-5 time with sterile distilled water；
(4) polypide is blotted with aseptic filter paper；
(5) polypide is placed in in the 75% dissection cake wax sterilized, will fix end to end with aseptic dissecting needle；
(6) cut off to the end from tail at striped rice borer back with sterilization dissecting scissors, with aseptic dissecting needle, the skin cut off is fixed on cake wax immediately；
(7) will be attached to the fat-body of body cavity and skin inwall with the tweezers sterilized take off, be placed in the culture dish containing HBSS cleanout fluid cleaning 2-3 time；
(8) cleaned fat-body tweezers are transferred to T-25cm2Carrying out original cuiture in Tissue Culture Flask, whole operating process is the most aseptically carried out.
The invention has the beneficial effects as follows: use such scheme, can be a large amount of, rapid extraction striped rice borer fat-body, the fat-body original cuiture for striped rice borer provides material.Striped rice borer is Oryza sativa L. important pests, and the present invention contributes to studying striped rice borer in vitro, provides help for the research of its controlling way and the exploitation of novel biopesticide.
The effect observation that the fat-body that the present invention is extracted is cultivated for cell
1. after aforesaid operations, individual cells the most to the periphery extension (Fig. 1) in a large number bred after 5 days, be can be observed
2., after the 40th day, proliferative cell is paved with Tissue Culture Flask surface (Fig. 2)；Through laboratory observation, cell line major part adherent growth, the shape major part of cell is circular.The cell containing added value, put in new culture bottle together with whole cell culture fluid sucking-offs, according to 1:5(cell volume: last culture fluid volume) carry out Secondary Culture.Chilo spp larvae fat body cells system is tentatively successfully established.
Accompanying drawing explanation
Fig. 1 fat-body cultivates the culture effect of 5 days.
Fig. 2 fat-body cultivates the culture effect of 40 days.
Detailed description of the invention
Below in conjunction with example, the present invention is described in further detail
Embodiment 1
The making of cake wax
(1) hard paraffin is melted in beaker
(2) culture dish of a diameter of 9cm, autoclave sterilization are chosen
(3) in aseptic operating platform, the paraffin that (1) melts is poured in culture dish
(4) culture dish is cooled down, standby.
The extraction of embodiment 2 striped rice borer fat-body
(1) choose 4 age Chilo spp larvae, Chilo spp larvae is immersed in 75% ethanol solution, carries out surface sterilization 10 minutes；
(2) Chilo spp larvae is cleaned 3 times with sterile distilled water；
(3) polypide is blotted with aseptic filter paper；
(4) polypide is placed in in the 75% dissection cake wax sterilized, will fix end to end with aseptic dissecting needle；
(5) cut off to the end from tail at striped rice borer back with sterilization dissecting scissors, with aseptic dissecting needle, the skin cut off is fixed on cake wax immediately；
(6) will be attached to the fat-body of body cavity and skin inwall with the tweezers sterilized take off, be placed in the culture dish containing HBSS cleanout fluid cleaning 2 times；
(7) cleaned fat-body tweezers are transferred to T-25cm2Carrying out original cuiture in Tissue Culture Flask, whole operating process is the most aseptically carried out.
The extraction of embodiment 3 striped rice borer fat-body
(1) choose 5 age Chilo spp larvae, Chilo spp larvae is immersed in 75% ethanol solution, carries out surface sterilization 15 minutes；
(2) Chilo spp larvae is cleaned 5 times with sterile distilled water；
(3) polypide is blotted with aseptic filter paper；
(4) polypide is placed in in the 75% dissection cake wax sterilized, will fix end to end with aseptic dissecting needle；
(5) cut off to the end from tail at striped rice borer back with sterilization dissecting scissors, with aseptic dissecting needle, the skin cut off is fixed on cake wax immediately；
(6) will be attached to the fat-body of body cavity and skin inwall with the tweezers sterilized take off, be placed in the culture dish containing HBSS cleanout fluid cleaning 3 times；
(7) cleaned fat-body tweezers are transferred to T-25cm2Carrying out original cuiture in Tissue Culture Flask, whole operating process is the most aseptically carried out.
The original cuiture of embodiment 4 striped rice borer fat body cells
(1) cleaned fat-body is placed in fills the T-25cm of 1ml cell culture fluid in advance2In Tissue Culture Flask, put in the cell culture incubator of 27 DEG C of unglazed photographs and cultivate；
(2) after overnight, after fat-body is adherent, add 2ml cell culture fluid, make fat-body be mostly submerged in this culture fluid.Note not making tissue suspension liquid feeding when in cell culture fluid；
(3) every sucking-off in 7 days and the passage culture fluid that changes to 70% amount, until extending and the cell bred is full of culture bottle.
Claims (4)
1. the collection method cultivating striped rice borer fat-body for cell, it is characterised in that the method comprises the following steps:
(1) cake wax is made；
(2) choose 4-5 Chilo spp larvae in age, Chilo spp larvae is immersed in 75% ethanol solution, carries out surface sterilization 10-15 minute；
(3) Chilo spp larvae is cleaned 3-5 time with sterile distilled water；
(4) polypide is blotted with aseptic filter paper；
(5) polypide is placed in in the 75% dissection cake wax sterilized, will fix end to end with aseptic dissecting needle；
(6) cut off to the end from tail at striped rice borer back with sterilization dissecting scissors, with aseptic dissecting needle, the skin cut off is fixed on cake wax immediately；
(7) will be attached to the fat-body of body cavity and skin inwall with the tweezers sterilized take off, be placed in the culture dish containing HBSS cleanout fluid cleaning 2-3 time；
(8) cleaned fat-body tweezers are transferred to T-25cm2Tissue Culture Flask carries out original cuiture;
Whole operating process is the most aseptically carried out.
Method the most according to claim 1, it is characterised in that described cake wax be hard paraffin is dissolved after, pour in the 9cm culture dish that autoclave sterilization processed, in super-clean bench, treat that its solidification is standby.
Method the most according to claim 2, it is characterised in that described HBSS cleanout fluid is HBSS solution and the mixed liquor of penicillin, content of streptomycin and amphotericin B.
Method the most according to claim 3, it is characterised in that Penicillin Content is 200U/ml, content of streptomycin is 0.2 μ g/ml, and amphotericin B content is 0.5 μ g/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510004977.5A CN105820995A (en) | 2015-01-07 | 2015-01-07 | Method for collecting Chilo suppressalis fat body used for cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510004977.5A CN105820995A (en) | 2015-01-07 | 2015-01-07 | Method for collecting Chilo suppressalis fat body used for cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105820995A true CN105820995A (en) | 2016-08-03 |
Family
ID=56513654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510004977.5A Pending CN105820995A (en) | 2015-01-07 | 2015-01-07 | Method for collecting Chilo suppressalis fat body used for cell culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105820995A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820380A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis blood lymphocytes |
| CN103820382A (en) * | 2014-02-10 | 2014-05-28 | 中国计量学院 | Construction method for fat body cell line of chilo suppressalis larva |
| CN103820381A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis cells |
-
2015
- 2015-01-07 CN CN201510004977.5A patent/CN105820995A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820380A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis blood lymphocytes |
| CN103820381A (en) * | 2014-01-23 | 2014-05-28 | 中国计量学院 | Primary culture method for chilo suppressalis cells |
| CN103820382A (en) * | 2014-02-10 | 2014-05-28 | 中国计量学院 | Construction method for fat body cell line of chilo suppressalis larva |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mandawala et al. | Cryopreservation of animal oocytes and embryos: Current progress and future prospects | |
| Vajta et al. | Improving cryopreservation systems | |
| Lal et al. | Commercial scale micropropagation of sugarcane: constraints and remedies | |
| CN105713871A (en) | Human chorion mesenchymal stem cell isolated culture method | |
| CN101984049B (en) | Method for separating mesenchymal stem cells from dispose tissues | |
| CN103820381B (en) | A kind of primary culture method of striped rice borer cell | |
| US20150313211A1 (en) | A Method of Vitrification | |
| CN107372361A (en) | A kind of honeybee ovum superfreeze method and its kit | |
| Wang et al. | Cryopreservation of Musca domestica (Diptera: Muscidae) embryos | |
| Bové et al. | Culture techniques for spiroplasmas from plants | |
| CN109706115A (en) | A kind of construction method of Marrow Mesenchymal Stem Cells cell line | |
| CN107937444A (en) | The method of somatic cell clone dog | |
| CN103820382B (en) | A kind of construction method of Chilo spp larvae fat body cells system | |
| CN105265320A (en) | Tissue culture propagation method for herba aristolochia mollissima | |
| CN104686347A (en) | Ultralow-temperature preservation technique of callus of divaricate saposhnikovia root | |
| CN107129962B (en) | A kind of primary culture method of Hirudo japonica salivary gland cell | |
| CN105820995A (en) | Method for collecting Chilo suppressalis fat body used for cell culture | |
| CN103820380B (en) | A kind of primary culture method of striped rice borer blood lymphocyte | |
| Souza et al. | Cryopreservation of pineapple shoot tips by the droplet vitrification technique | |
| CN100463960C (en) | Separation technology of orchidaceal plant mycorrhizal fungi monobyssisede | |
| Neumann et al. | Callus cultures | |
| CN103461323B (en) | One can be used for external time delay and preserves the bioactive method of egg mother cell | |
| Yuliani et al. | The Morphological, Anatomical, And Physiological Characteristics of Elephantopus scaber As Explant Source For Tissue Culture | |
| CN205803502U (en) | A kind of tumor stem cell culture dish | |
| Boisseau et al. | In vitro rearing of Pratylenchidae nematodes on carrot discs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160803 |
|
| WD01 | Invention patent application deemed withdrawn after publication |