CN112120010A - Preparation and observation method of plant nematode lateral line specimen - Google Patents
Preparation and observation method of plant nematode lateral line specimen Download PDFInfo
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- CN112120010A CN112120010A CN202011104844.2A CN202011104844A CN112120010A CN 112120010 A CN112120010 A CN 112120010A CN 202011104844 A CN202011104844 A CN 202011104844A CN 112120010 A CN112120010 A CN 112120010A
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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Abstract
The invention discloses a preparation and observation method of a plant nematode side line specimen, which comprises the steps of adding nematode fixing liquid into the residual liquid containing nematodes; further centrifuging the nematode fixing solution, adding a glycerol aqueous solution, placing at the constant temperature of 35-40 ℃ for 40-50 h, and completing dehydration; under a dissecting mirror, selecting dehydrated adults, and cutting the nematodes into segments with the segment length of 5-10 μm; the cut nematode fragments were then transferred to glycerol and covered with coverslips. The nematode side line production method is simple, convenient and safe, the specimen is clear and complete, the coloring is uniform, the production method is simple and convenient, the storage period is long, and the nematode side line production method is suitable for side line observation of the specimen of the plant nematodes.
Description
Technical Field
The invention relates to the field of biochemistry, in particular to a preparation and observation method of a plant nematode lateral line specimen.
Background
Plant nematodes belong to the phylum of nematodes in invertebrates and can parasitize plant roots, stems, leaves, fruits, seeds and other parts, and the conventional nematode identification methods used in the field of nematode classification and identification mainly comprise morphological identification methods and molecular biological identification methods.
In the research of plant nematode detection and identification, the nematode side line number is one of the important characteristics of nematode species identification. But the number of side lines is difficult to see under a microscope. The general solution is to observe a large number of nematodes as much as possible and search individual side lines to observe clearly for photographing, and to slightly press temporary nematode specimens with fingers to twist the nematodes so as to better show the area where the side lines are located, but the method has high requirements on operator skills.
Therefore, a method for preparing and observing a plant nematode side-line specimen, which can effectively solve the problems, is needed.
Disclosure of Invention
The invention aims to provide a preparation and observation method of a plant nematode lateral line specimen.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention comprises the following steps:
a, adding nematode stationary liquid into the residual liquid containing nematodes;
b, further centrifuging the nematode fixing solution, adding a glycerol aqueous solution (glycerol: distilled water is 1:20), and placing at the constant temperature of 35-40 ℃ for 40-50 h to complete dehydration;
c, under a dissecting mirror, selecting dehydrated adults, and cutting the nematodes to segments, wherein the length of each segment is 5-10 mu m;
transferring the cut nematode fragments into glycerol, placing the nematode fragments on a glass slide provided with a glycerol gel coating, and then covering the glass slide.
Further, the preparation method of the glycerol gel comprises the steps of taking 3g of gelatin, soaking the gelatin in 25mL of distilled water for 2h, adding 22mL of glycerol, and carrying out water bath at 60 ℃ for 1 h.
Further, the aqueous glycerol solution is in accordance with glycerol: mixing distilled water in a volume ratio of 1: 20.
A plant nematode side line specimen observation method is characterized in that a nematode fragment specimen is observed under a low power microscope (5 times objective lens), a cover specimen is slightly pushed by fingers to drive the cut nematode fragments to rotate until the nematode fragments are in a disc shape, a cut faces an observer, and nematode side lines can be observed under a high power microscope (100 times objective lens).
Compared with the prior art, the invention has the following beneficial effects:
the preparation method is simple, convenient and safe, the plant nematode specimen is clear and complete, the coloring is uniform, the preparation method is simple and convenient, the storage period is long, and the preparation method is suitable for the specimen side-line observation of the plant nematode.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be in a manner including, but not limited to, the following examples.
In this embodiment, the present invention includes the following steps:
a. killing and fixing of nematodes: transferring the nematode liquid into a 500-mu L plastic tube, and centrifuging at 2000r/min for 3min to settle the nematodes to the bottom of the plastic tube. The water in the upper layer of the plastic tube was slowly pipetted out to leave only a small amount of nematode solution (about 10. mu.L) in the tube, and then 95 ℃ nematode fixing solution (40% formaldehyde: glycerol: distilled water: 10:2:88) was added to the tube while killing and fixing the nematodes (more than 24h had to be fixed).
b. Centrifuging the plastic tube at 2000r/min for 3min to deposit the nematodes at the bottom of the tube, sucking the upper nematode fixing liquid, transferring the nematodes to a 24-hole cell culture dish by using a liquid transfer device, adding 2mL of glycerol aqueous solution (glycerol: distilled water: 1:20), and placing the dish in a constant temperature incubator at 35-40 ℃ for 40-50 h. At this point, the distilled water in the dish had evaporated completely, and the nematodes had completed the dehydration process and were soaked in glycerol.
c. Under the dissecting mirror, 1 or more of the above-mentioned completely dehydrated adult worms were pricked with nematodes and gently placed in the middle of a lid (right side up) of a clean plastic petri dish.
d. Under a dissecting scope, a razor blade is fixed at one tip, and the nematode is cut into several small pieces (each piece preferably no longer than 10 μm) like sausage.
e. Taking a small amount of glycerin gel (taking 3g of gelatin, soaking in 25mL of distilled water for 2h, adding 22mL of glycerin, and water bathing at 60 ℃ for 1h) with an insect needle, melting under an alcohol lamp, and waiting for 1min, cooling and solidifying the glycerin gel.
g. The nematode fragments were observed under a low power microscope (5 x objective) and the cut nematode fragments were rotated by gently pushing the cover specimen with a finger. Until the segments are in the shape of a disk with the cut facing the viewer. Under a high power microscope (100 times objective lens), nematode lateral lines can be observed.
The preparation method is simple, convenient and safe, the plant nematode specimen is clear and complete, the coloring is uniform, the preparation method is simple and convenient, the storage period is long, and the preparation method is suitable for preparing the plant nematode specimen.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.
Claims (4)
1. The preparation method of the plant nematode side-line specimen is characterized by comprising the following steps:
a, adding nematode stationary liquid into the residual liquid containing nematodes;
b, further centrifuging the nematode fixing solution, adding a glycerol aqueous solution, and placing at the constant temperature of 35-40 ℃ for 40-50 h to complete dehydration;
c, under a dissecting mirror, selecting dehydrated adults, and cutting the nematodes to segments, wherein the length of each segment is 5-10 mu m;
transferring the cut nematode fragments into glycerol, placing the nematode fragments on a glass slide provided with a glycerol gel coating, and then covering the glass slide.
2. The method for preparing the plant nematode side line specimen as claimed in claim 1, wherein the method for preparing the glycerin gel comprises the steps of taking 3g of gelatin, soaking the gelatin in 25mL of distilled water for 2h, adding 22mL of glycerin, and carrying out water bath at 60 ℃ for 1 h.
3. The method for preparing the plant nematode side-line specimen of claim 1 or 2, wherein the glycerin water solution is prepared according to the ratio of glycerin: mixing distilled water in a volume ratio of 1: 20.
4. A plant nematode side-line specimen observing method is characterized in that nematode fragments are observed under a low-power microscope (5-power objective lens), a cover specimen is lightly pushed by fingers to drive the cut nematode fragments to rotate until the nematode fragments are in a disc shape, a cut faces an observer, and nematode side lines can be observed under a high-power microscope (100-power objective lens).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011104844.2A CN112120010A (en) | 2020-10-15 | 2020-10-15 | Preparation and observation method of plant nematode lateral line specimen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011104844.2A CN112120010A (en) | 2020-10-15 | 2020-10-15 | Preparation and observation method of plant nematode lateral line specimen |
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| Publication Number | Publication Date |
|---|---|
| CN112120010A true CN112120010A (en) | 2020-12-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011104844.2A Pending CN112120010A (en) | 2020-10-15 | 2020-10-15 | Preparation and observation method of plant nematode lateral line specimen |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2768162C1 (en) * | 2021-06-09 | 2022-03-23 | Мария Владимировна Видманова | Method for preparation of mature nematodes for identification by maldi tof mass spectrometry |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2625271A2 (en) * | 2010-10-04 | 2013-08-14 | Tree of Knowledge Patents B.V. | Method for extracting dna from nematodes and plant cells |
| 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 |
| CN106614520A (en) * | 2016-11-29 | 2017-05-10 | 宁波检验检疫科学技术研究院 | Manufacturing method of plant nematode permanent slide |
| 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 |
-
2020
- 2020-10-15 CN CN202011104844.2A patent/CN112120010A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2625271A2 (en) * | 2010-10-04 | 2013-08-14 | Tree of Knowledge Patents B.V. | Method for extracting dna from nematodes and plant cells |
| 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 |
| CN106614520A (en) * | 2016-11-29 | 2017-05-10 | 宁波检验检疫科学技术研究院 | Manufacturing method of plant nematode permanent slide |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 王明祖等: "《中国植物线虫研究》", 31 October 1998, 湖北科学技术出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2768162C1 (en) * | 2021-06-09 | 2022-03-23 | Мария Владимировна Видманова | Method for preparation of mature nematodes for identification by maldi tof mass spectrometry |
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Application publication date: 20201225 |
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