CN106614520A - Manufacturing method of plant nematode permanent slide - Google Patents
Manufacturing method of plant nematode permanent slide Download PDFInfo
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- CN106614520A CN106614520A CN201611073300.8A CN201611073300A CN106614520A CN 106614520 A CN106614520 A CN 106614520A CN 201611073300 A CN201611073300 A CN 201611073300A CN 106614520 A CN106614520 A CN 106614520A
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- nematode
- glycerine
- slide
- manufacturing
- fixer
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
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- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of manufacturing of plant nematode specimens and discloses a manufacturing method of a plant nematode permanent slide. The manufacturing method of the plant nematode permanent slide comprises the following steps of a, performing warm bath on a nematode, centrifuging, and soaking into fixing liquid at room temperature for more than 24 hours; b, adding a glycerine aqueous solution into the fixed nematode and placing at constant temperature of 35 to 40 DEG C for 40 to 50 hours to obtain the nematode which is completely dehydrated and soaked into glycerine; and c, dropwise adding the glycerine on a glass slide, moving the nematode to a glycerine drop of the glass slide, putting wax blocks on the two sides of the glycerine, adding a cover slip, heating until the wax blocks are melted, cooling and sticking a label. By the manufacturing method of the plant nematode permanent slide, the complex manufacturing procedure of the permanent slide is simplified and the manufacturing difficulty is reduced; and the method is a new, convenient and efficient method for manufacturing a nematode permanent slide.
Description
Technical field
The present invention relates to technical field of plant wireworm specimen manufacture, specially a kind of making side of Plant nematode permanent slide
Method.
Background technology
Plant nematode belongs to the Nematoda in invertebrate, can parasitize the portions such as plant roots, stem, leaf, fruit, seed
Position, its species is various, harm is serious.According to incompletely statistics, the whole world is every year because the farm-forestry crop economy that Plant nematode is caused is damaged
Lose more than hundreds billion of units.In the external plant quarantine harmful organism register that China announces for 2007, there is the line of 20 kinds (category)
Worm is quarantine nematode, its actual up to more than 200 kinds of number of kind.
In Plant nematode Testing and appraisal research work, interim slide typically must be made first, the form of nematode is tied
Structure carries out preliminary observation and photography, and carries out survey meter to various data.Subsequently, need further sample to be made into permanent slide, make
For the sample in kind in laboratory, teaching or scientific research needs can also be available for as the intercepting and capturing evidence of Quarantine Lab, especially to identification
Novel species, must be requested that have type specimen.
General nematode permanent slide manufacturing process is complex, and making step is more, be related to the kill of sample, fixation, dehydration,
The steps such as film-making.And need to take longer, generally require 5～6 weeks.The Chinese patent of Publication No. CN102640742A is disclosed
It is a kind of that traditional improved technical scheme of permanent slide preparation method, but the program are still needed to need 20 with 2 kinds of dehydration liquid, and used time
It or so.
The content of the invention
For problems of the prior art, it is an object of the invention to provide easy nematode permanent slide making side
Method, its method is convenient, workable, effect is good.
Specific technical scheme is as follows：
A kind of preparation method of Plant nematode permanent slide, comprises the following steps：
A, by the aqueous solution containing nematode 55-60 DEG C temperature bath 2-3min after be centrifuged, separate aqueous layer obtains nematode liquid, by institute
State nematode liquid and more than 24h is soaked in fixer；
B, by containing nematode fixer be centrifuged, separate upper strata fixer be fixed after nematode, by the line after fixation
Worm mixes with 1.5～2.5ml glycerine water solutions and is placed in 24 hole Tissue Culture Dish, then by 24 hole Tissue Culture Dish at 35 DEG C
Constant temperature places 40h～50h under the conditions of～40 DEG C, obtains being dehydrated completely the nematode being soaked in glycerine；Wherein, the glycerine is water-soluble
Liquid includes that volume ratio is 1:15～25 glycerine and water；
C, glycerine is added dropwise on slide, the nematode that dehydration is soaked in glycerine described completely is moved into the glycerine of slide
In drop, wax stone, plus cover glass are put on glycerine drop both sides, be heated to being cooled down after wax stone melts.
In the present invention, the nematode fixer includes that volume ratio is 5～15:1～5:65～94 40wt% formaldehyde, glycerine
And water.
In step a of the present invention or step b, the rotating speed of the centrifugation independently is 2000～4000r/min, and centrifugation time is only
It is on the spot 1～5min.
In step c of the present invention, the nematode for moving to glycerine drop central authorities is that female, the male nematode being dehydrated completely is each some, row
Row are uniform.
In step c, the heating stepses add specifically, the slide containing wax stone is placed in 50～70 DEG C of heating plate
Heat, the time of the heating is 20～40s.
It is labelled on the slide after slide cooling in step c, sample number, host, product are included on the label
Ground, making date.
Compared with prior art, it is an advantage of the current invention that：
This invention simplifies the complicated process that permanent slide makes, only needs to using a kind of dehydration liquid in dehydration,
Its operating process and material requested simplify many than conventional nematode permanent slide flaking method, reduce manufacture difficulty.This
Bright Plant nematode permanent slide simple making method, from the overall process of kill, fixed, dehydration and film-making about 3d, operation letter are only needed
It is single, quick, preferable permanent slide can be obtained to various frequently seen plants nematodes.
Specific embodiment
Below in conjunction with the embodiment in the present invention, the technical scheme in the embodiment of the present invention is carried out clearly and completely
Description, it is clear that described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.Based on this
Embodiment in bright, the every other enforcement that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example, belongs to the scope of protection of the invention.
A kind of preparation method of Plant nematode permanent slide, comprises the following steps：
The kill and fixation of nematode：To be centrifuged after the bath of water-soluble liquid temperature containing nematode, separate aqueous layer obtains micro nematode liquid,
Fixer immersion is added in nematode liquid, is killed and fixed nematode.
Target nematode need to be living in the present invention, preferably just isolate from soil or plant tissue, if isolating
Target nematode place short without the fixed or set time too long, deform can nematode.Water-soluble liquid temperature containing nematode is bathed
To kill nematode.Warm bath temperature is preferably 55-60 DEG C, is further 56-58 DEG C, and the warm bath time is preferably 2-3min.By temperature bath
The centrifugation of the aqueous solution containing nematode afterwards, separates nematode and water.The rotating speed of the centrifugation is 2000～4000r/min, preferably
It is further 3000r/min for 2500～3500r/min；Centrifugation time be 1～5min, preferably 2～4min, more preferably
3min。
Detached water layer is removed, nematode fixer is added in a small amount of nematode liquid for retaining.The nematode fixer is added
Volume can not have the nematode liquid based on, preferably the volume ratio of the nematode liquid and the fixer is 1：10～30,
More preferably 1：15～25, specially 1:20.Wherein, nematode fixer can adopt fixer pair commonly used in the art
Nematode is fixed.The fixer being preferably mixed to form using 40wt% formaldehyde, G ＆ W in the present invention.Wherein,
40wt% formaldehyde, the volume ratio of G ＆ W are 5～15:1～5:65～94, preferably 8～12:2～4:80～90, further
Preferably 10:2:88.There is no special restriction to the temperature of fixer in the present invention, be using the nematode fixer under room temperature
Can.After nematode liquid mixes with fixer, at room temperature soak more than 24 hours, preferably more than 36 hours, further for long in
It is fixed within 48 hours.
After fixation, the fixer containing nematode is centrifuged.The rotating speed of the centrifugation is 2000～4000r/min, preferably
It is further 3000r/min for 2500～3500r/min；Centrifugation time be 1～5min, preferably 2～4min, more preferably
3min。
After centrifugation, fixer is located at upper strata.Separate upper strata fixer be fixed after nematode, by the nematode after fixation with
Glycerine water solution mixes.Upper strata fixer is specially suctioned out, nematode is transferred in container, and add glycerine in the above-described container
Aqueous solution dehydration soaks.The container is preferably with 24 hole Tissue Culture Dish.The glycerine water solution is mixed with water containing glycerine
Solution is closed, glycerine is 1 with the volume ratio of water:10～30, preferably 1:15～25, specially 1:20.Glycerine water solution of the present invention
Volume be 1.5～2.5mL, be further 2mL.
By the container containing nematode-glycerine water solution, constant temperature places 40h～50h under the conditions of 35 DEG C～40 DEG C.Specifically,
The 24 hole Tissue Culture Dish containing glycerine water solution and nematode are placed in constant incubator and are placed, treat the water in glycerine water solution
Evaporating completely, nematode is soaked in glycerine, the nematode being dehydrated completely, for making nematode permanent slide.Wherein, constant temperature training
The temperature setting of foster case is 35 DEG C～40 DEG C, is further 37 DEG C～38 DEG C；The time that nematode is placed in constant incubator is
40h～50h, more preferably 43～47 hours.Now distilled water all evaporates in container, and nematode is soaked in glycerine,
For making nematode permanent slide.
Make nematode permanent slide：Glycerine is added dropwise on slide, the above-mentioned nematode being soaked in glycerine is moved into load glass
In the glycerine drop of piece.Can be selected that the amount of glycerine is added dropwise according to the experience of those skilled in the art, in the present invention, it is preferred to carry
2～4 microlitres of glycerine are added dropwise on slide.Female, male nematode can be placed in each slide each some, be evenly arranged in load fragment.
Wax stone, plus cover glass are put on glycerine drop both sides, natural cooling after wax stone melts is heated to, it is labelled.Those skilled in the art
Conventional mode of heating, such as alcolhol burner heating, it would however also be possible to employ heater plate can be adopted.In embodiments of the present invention, plus
Heat is specially that the slide containing wax stone is placed in heating plate and heats.Heating-up temperature is 50～70 DEG C, preferably 55～65 DEG C, is had
Body is 64 DEG C.After wax stone melts, stop heating.It is preferred that the heat time is 20～40s, it is further 30s.Paste after natural cooling
Upper label.Include the information such as sample number, host, the place of production, making date on label, complete the making of nematode permanent slide.Will system
The nematode sample for performing is put into Riker mount and preserves.Note keeping slide level during preservation, in case nematode slides.
Embodiment 1：
The simple permanent slide making step of pine wood nematode：
A, cleaning slide and cover glass；
B, the kill of pine wood nematode and fixation：Under anatomical lens, with pipettor nematode is drawn from culture dish repeatedly, shifted
To in 500 μ L plastic tubes, plastic tube is taken out after temperature bath 2min at 57 DEG C, 3min is centrifuged with 2500r/min, settle nematode
To plastics bottom of the tube.Slowly the water on plastic tube upper strata is suctioned out with pipettor, makes only to stay a small amount of nematode liquid (about 10 μ L) in pipe,
Then nematode fixer (40% formaldehyde is added in pipe：Glycerine：Distilled water=10:2:88) nematode, is made to soak in fixer
More than 24h, while killing and fixed nematode.
C, by plastic tube 2000r/min be centrifuged 3min, make nematode be sunken to ttom of pipe.After sucking upper strata nematode fixer, with shifting
Nematode is moved in 24 hole Tissue Culture Dish and (to prevent nematode from adhering to tube wall, can draw glycerine water solution with pipettor by liquid device
Draw repeatedly), add glycerine water solution (glycerine：Distilled water=1:20) 2mL, then ware is placed in into placement in 38 DEG C of constant incubators
45h.Now distilled water all evaporates in ware, and nematode has been soaked in glycerine, can be used to make nematode permanent slide.
D, a clean slide is taken, 2 microlitres of glycerine are added dropwise middle, will be dehydrated completely with choosing pin under anatomical lens
Each several of female, male nematode move to the central authorities that glycerine drips, and arrangement is uniform.
E, glycerine drop both sides symmetrically put two pieces of little wax stones, plus cover glass, be placed in 64 DEG C of heating plate heat, after 30s
The natural cooling after wax stone melts.
F, labelling, write sample number, host, the place of production, making date etc. exactly, and nematode permanent slide completes.
Embodiment 2：
The simple permanent slide making step of peanut root-knot nematode：
A, cleaning slide and cover glass；
B, the kill of peanut root-knot nematode and fixation：Under anatomical lens, with pipettor nematode is drawn from culture dish repeatedly,
In being transferred to 500 μ L plastic tubes, plastic tube is taken out after temperature bath 2min at 60 DEG C, 1min is centrifuged with 3000r/min, make nematode
It is deposited to plastics bottom of the tube.Slowly the water on plastic tube upper strata is suctioned out with pipettor, makes only to stay a small amount of nematode liquid (about 10 in pipe
μ L), nematode fixer (40% formaldehyde is then added in pipe：Glycerine：Distilled water=15:5:94) nematode, is made in fixer
Immersion more than 36h, while killing and fixed nematode.
C, by plastic tube 2500r/min be centrifuged 4min, make nematode be sunken to ttom of pipe.After sucking upper strata nematode fixer, with shifting
Nematode is moved in 24 hole Tissue Culture Dish and (to prevent nematode from adhering to tube wall, can draw glycerine water solution with pipettor by liquid device
Draw repeatedly), add glycerine water solution (glycerine：Distilled water=1:30) 1.5mL, then ware is placed in 35 DEG C of constant incubators puts
Put 40h.Now distilled water all evaporates in ware, and nematode has been soaked in glycerine, can be used to make nematode permanent slide.
D, a clean slide is taken, 3 microlitres of glycerine are added dropwise middle, will be dehydrated completely with choosing pin under anatomical lens
Each several of female, male nematode move to the central authorities that glycerine drips, and arrangement is uniform.
E, glycerine drop both sides symmetrically put two pieces of little wax stones, plus cover glass, be placed in 54 DEG C of heating plate heat, after 38s
The natural cooling after wax stone melts.
F, labelling, write sample number, host, the place of production, making date etc. exactly, and nematode permanent slide completes.
Embodiment 3：
The simple permanent slide making step of Aphelenchus avenae：
A, cleaning slide and cover glass；
B, the kill of Aphelenchus avenae and fixation：Under anatomical lens, with the pipettor suction line from culture dish repeatedly
Worm, in being transferred to 500 μ L plastic tubes, plastic tube is taken out after temperature bath 3min at 55 DEG C, and 5min is centrifuged with 2000r/min, is made
Nematode is deposited to plastics bottom of the tube.Slowly the water on plastic tube upper strata is suctioned out with pipettor, makes only to stay a small amount of nematode liquid in pipe
(about 10 μ L), then adds nematode fixer (40% formaldehyde in pipe：Glycerine：Distilled water=7:1:75), while killing and solid
Alignment worm.Make nematode that more than 48h is soaked in fixer；
C, by plastic tube 3500r/min be centrifuged 4min, make nematode be sunken to ttom of pipe.After sucking upper strata nematode fixer, with shifting
Nematode is moved in 24 hole Tissue Culture Dish and (to prevent nematode from adhering to tube wall, can draw glycerine water solution with pipettor by liquid device
Draw repeatedly), add glycerine water solution (glycerine：Distilled water=1:10) 2.5mL, then ware is placed in 40 DEG C of constant incubators puts
Put 40h.Now distilled water all evaporates in ware, and nematode has been soaked in glycerine, can be used to make nematode permanent slide.
D, a clean slide is taken, 4 microlitres of glycerine are added dropwise middle, will be dehydrated completely with choosing pin under anatomical lens
Each several of female, male nematode move to the central authorities that glycerine drips, and arrangement is uniform.
E, glycerine drop both sides symmetrically put two pieces of little wax stones, plus cover glass, be placed in 70 DEG C of heating plate heat, after 24s
The natural cooling after wax stone melts.
F, labelling, write sample number, host, the place of production, making date etc. exactly, and nematode permanent slide completes.
Nematodes species all with the embodiment, can also obtain effect of the present invention.
Above-mentioned Plant nematode permanent slide simple making method, only needs about from the overall process of kill, fixed, dehydration and film-making
3d, it is simple to operate, quick, preferable permanent slide can be obtained to various frequently seen plants nematodes.
It can be that professional and technical personnel in the field realize or use that above-mentioned embodiment is intended to illustrate the present invention, to above-mentioned
Embodiment is modified and will be apparent for those skilled in the art, therefore the present invention is included but is not limited to
Above-mentioned embodiment, it is any to meet the claims or specification description, meet with principles disclosed herein and novelty,
The method of inventive features, technique, product, each fall within protection scope of the present invention.
Claims (7)
1. a kind of preparation method of Plant nematode permanent slide, it is characterised in that comprise the following steps：
A, by the aqueous solution containing nematode 55-60 DEG C temperature bath 2-3min after be centrifuged, separate aqueous layer obtains nematode liquid, by the line
Worm liquid soaks more than 24h in fixer；
B, by containing nematode fixer be centrifuged, separate upper strata fixer be fixed after nematode, by the nematode after fixation and 1
～5ml glycerine water solutions mixing be placed in 24 hole Tissue Culture Dish, then by the culture dish under the conditions of 35 DEG C～40 DEG C constant temperature
40h～50h is placed, obtains being dehydrated completely the nematode being soaked in glycerine；Wherein, the glycerine water solution includes that volume ratio is 1:
15～25 glycerine and water；
C, glycerine is added dropwise on slide, the nematode that dehydration is soaked in glycerine described completely is moved into the glycerine drop of slide
In, wax stone, plus cover glass are put on glycerine drop both sides, it is heated to being cooled down after wax stone melts.
2. preparation method according to claim 1, it is characterised in that the fixer includes that volume ratio is 5～15:1～
5:65～94 40wt% formaldehyde, G ＆ W.
3. preparation method according to claim 1, it is characterised in that in step a or step b, the rotating speed of the centrifugation is only
It is on the spot 2000～4000r/min, centrifugation time independently is 1～5min.
4. preparation method according to claim 1, it is characterised in that in step c, moves to the nematode in glycerine drop central authorities for
Female, the male nematode being dehydrated completely is each some, and arrangement is uniform.
5. preparation method according to claim 1, it is characterised in that in step c, the heating stepses will be specifically, will contain
The slide for having wax stone is placed in 50～70 DEG C of heating plate and heats.
6. preparation method according to claim 1 or 5, it is characterised in that in step c, time of the heating is 20～
40s。
7. preparation method according to claim 1, it is characterised in that in step c, also include the slide after the cooling
It is labelled, sample number, host, the place of production, making date are included on the label.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611073300.8A CN106614520A (en) | 2016-11-29 | 2016-11-29 | Manufacturing method of plant nematode permanent slide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611073300.8A CN106614520A (en) | 2016-11-29 | 2016-11-29 | Manufacturing method of plant nematode permanent slide |
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| CN106614520A true CN106614520A (en) | 2017-05-10 |
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ID=58814018
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CN201611073300.8A Pending CN106614520A (en) | 2016-11-29 | 2016-11-29 | Manufacturing method of plant nematode permanent slide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
| CN112120010A (en) * | 2020-10-15 | 2020-12-25 | 宁波检验检疫科学技术研究院 | Preparation and observation method of plant nematode lateral line specimen |
| CN115152738A (en) * | 2022-08-04 | 2022-10-11 | 宁波海关技术中心 | Quick sealing piece propping agent for plant nematodes |
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|---|---|---|---|---|
| CN102640742A (en) * | 2012-04-20 | 2012-08-22 | 杭州市农业科学研究院 | Improved manufacturing method for plant wireworm permanent slide |
| CN103416393A (en) * | 2013-07-30 | 2013-12-04 | 宁波检验检疫科学技术研究院 | Plant nematode temporary glass slide preparation method |
| CN103868774A (en) * | 2014-03-26 | 2014-06-18 | 青岛大学 | Method for manufacturing parasitic ovum permanent slide specimens |
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2016
- 2016-11-29 CN CN201611073300.8A patent/CN106614520A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102640742A (en) * | 2012-04-20 | 2012-08-22 | 杭州市农业科学研究院 | Improved manufacturing method for plant wireworm permanent slide |
| CN103416393A (en) * | 2013-07-30 | 2013-12-04 | 宁波检验检疫科学技术研究院 | Plant nematode temporary glass slide preparation method |
| CN103868774A (en) * | 2014-03-26 | 2014-06-18 | 青岛大学 | Method for manufacturing parasitic ovum permanent slide specimens |
Non-Patent Citations (1)
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109556933A (en) * | 2018-12-12 | 2019-04-02 | 宁波中盛产品检测有限公司 | Root-knot nematode perineal pattern High-speed for preparing Slides |
| CN109556933B (en) * | 2018-12-12 | 2021-08-17 | 宁波中盛产品检测有限公司 | Method for quickly preparing perineal pattern of root-knot nematode |
| CN112120010A (en) * | 2020-10-15 | 2020-12-25 | 宁波检验检疫科学技术研究院 | Preparation and observation method of plant nematode lateral line specimen |
| CN115152738A (en) * | 2022-08-04 | 2022-10-11 | 宁波海关技术中心 | Quick sealing piece propping agent for plant nematodes |
| CN115152738B (en) * | 2022-08-04 | 2023-11-17 | 宁波海关技术中心 | Plant nematode quick-sealing propping agent |
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