CN1350180A - Simple and fast light microscope detection technology for blood and lymph of live shrimp and crab - Google Patents
Simple and fast light microscope detection technology for blood and lymph of live shrimp and crab Download PDFInfo
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- CN1350180A CN1350180A CN01137316.4A CN01137316A CN1350180A CN 1350180 A CN1350180 A CN 1350180A CN 01137316 A CN01137316 A CN 01137316A CN 1350180 A CN1350180 A CN 1350180A
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- 210000004369 blood Anatomy 0.000 title claims abstract description 25
- 239000008280 blood Substances 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 title claims description 13
- 210000002751 lymph Anatomy 0.000 title abstract 2
- 238000000034 method Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000010186 staining Methods 0.000 claims abstract description 13
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 244000052769 pathogen Species 0.000 claims abstract description 7
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- 210000000087 hemolymph Anatomy 0.000 claims description 16
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- 238000004040 coloring Methods 0.000 claims description 9
- 238000004043 dyeing Methods 0.000 claims description 6
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- 238000002738 Giemsa staining Methods 0.000 abstract 1
- 239000002253 acid Substances 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 238000007447 staining method Methods 0.000 abstract 1
- 239000012192 staining solution Substances 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 238000010241 blood sampling Methods 0.000 description 15
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Abstract
The prevent invention relates to a simple and quick detectino technique of shrimp-crab intravital blood lymph light microscope, including shrimp and crab living body blood-taking method: cleaning shrimp and crab body, disinfecting by using alcohol, using injector to needle into 7-10 mm of shrimp or crab body to draw blood; blood film preparation and staining method, using 0.05 ml of blood-lymph fluid, dropping on glass slide, adding 4% formaldehyde fixing fluid, drying, staining for 15-20 min. in Giemsa staining solution, washing film with water and making observation by using light microscope, covering with glass plate and making observation by using oil microscope. Said invention adopts meta-acid water flushing scheme to can quickly detect pathogen RLO (Rickettsia-like organism), therefore it possesses high practical value for culturing shrimp and crab.
Description
Technical field
The present invention relates to the detection technique in a kind of aquaculture, particularly a kind of simple and easy, Fast Detection Technique of live shrimp and crab hemolymph light microscopic.
Background technology
In the aquaculture industry, need often to detect the health status of shrimp, crab, so that carry out the control of disease, or with the quality index of testing result as commercial sized prawn, crab.Wherein live shrimp and crab hemolymph light microscopic detection technique is the detection technique of normal employing, comprising blood collecting technology and smear staining technology.Existing blood collecting technology is to bore an aperture in the heart district of the carapace of shrimp crab, inserts pericardial sinus with suction pipe and draws liquid of haemolymph.Another kind of blood sampling mode is intercepting step blood sampling.Preceding a kind of blood sampling mode is that lethal is destroyed; The back is a kind of to be damaging destruction, all can not carry out the real-time detection of live shrimp and crab, and its amount for taking blood all can't accurately be grasped simultaneously.It is technical very strong that pericardial sinus is holed, and the big or small depth of boring is difficult for holding, and is unfamiliar with the anatomical ordinary person of shrimp crab and determines the position of pericardial sinus is difficult.In addition, the external microorganism that adheres to of shrimp crab is a lot, and the blood sampling operation of this dual mode is all brought microorganism in the liquid of haemolymph into easily, pollutes blood sample.Conventional blood film and staining technique that prior art adopts after blood sampling are the film-making decoration method of continuing to use higher mammal basically:
(1) Giemsa dye liquor stoste preparation
Giemsa dye liquor pulvis 0.75g
Glycerine 50mL
Methyl alcohol (AR) 50mL
Pulvis is put into glycerine stir evenly, put into 60 ° of C incubators 24 hours, often shake up.The cold methyl alcohol that adds is again treated in taking-up, is made into stoste and is sealed in the brown bottle and preserves.
The preparation of Giemsa working fluid
Phosphate buffer pH6.8 100mL
Giemsa stoste 5mL
Methyl alcohol (not containing acetone) 5mL
(2) Wright dye liquor preparation
Wright pulvis 0.1g
Methyl alcohol (AR) 60mL
Pulvis is put into the mortar of clean dried, drip methyl alcohol, be ground to moltenly entirely, be sealed in the brown bottle, placed 15～30 days.
(3) aqueous acetic acid
Glacial acetic acid 1mL
Distilled water 400ml
(4) dyeing course
Drop of blood on clean slide, is coated with straticulation, the flame drying;
Keep flat section, dripped full Wright dye liquor 5 minutes;
Add equivalent distilled water, metallic luster occurs, 5 minutes to liquid level;
Outwell dye liquor,, immerse in the Giemsa working fluid in the staining jar 45 minutes without washing;
Color separation and dehydration
Aqueous acetic acid is rinsed once, changes aqueous acetic acid and changes and rinse twice; Distilled water changes rinses twice;
95% alcohol changes rinses three times, and anhydrous alcohol changes rinses three times; 1: 1 dimethylbenzene-no watery wine
Essence is changed and is rinsed three times; Dimethylbenzene changes rinses twice; The synthetic resin mounting adds cover glass.
The liquid of haemolymph of shrimp crab than the blood of higher mammal denseer thick, easily solidify, adopt the colouring method of above higher mammal blood, often poor effect; Conventional Giemsa decoration method complexity, loaded down with trivial details, time-consuming, and technical very strong, facility needs more than 10 of staining jars, and 5～8 kinds of medicines are difficult for promoting the use of in basic unit.Its used dimethylbenzene is poisoned bigger to human body simultaneously.
Summary of the invention
Above deficiency at prior art, the present invention will provide a kind of simple and easy, Fast Detection Technique of new live shrimp and crab hemolymph light microscopic, this technology comprises blood collecting technology and staining technique, on the blood sampling mode, will abandon the lethal of prawn crab or than the method for macrolesion, adopt a kind of animal that neither damages, reduce the blood sampling mode of blood contamination chance again; At shrimp crab liquid of haemolymph dense thick, easily characteristic such as solidify, provide a kind of simple and easy, light microscopic colouring method fast, so that the health status of prawn crab is carried out the fast detecting of real-time physiological detection and pathogen.Blood-sampling method of the present invention and colouring method use separately respectively all can reach purpose of the present invention, and both complement one another, and are more superior when both use simultaneously.Prioritization scheme of the present invention also can carry out fast detecting to the pathogen RLO (rickettsia-like organism) that classic method can't dye.
The scheme of finishing the foregoing invention task comprises blood collecting technology and staining technique respectively:
The live shrimp and crab blood extracting method:
Shrimp and crab body is cleaned up outward
At the uromere interface of shrimp or 3～4 pairs of step base portion alcohol disinfectings of crab
Thrust 7～10mm in the shrimp and crab body with sterile syringe at the position of sterilizing, extract blood.
The film-making of blood film and colouring method
Get the liquid of haemolymph about 0.05mL, drop on the clean microslide, on drop, add the formaldehyde fixed liquid of equivalent 4% immediately
Wait to drip and put into the Giemsa dyeing liquor after the sheet air dry and dyeed 15～20 minutes
Promptly can be used for om observation behind the water flushing staining section.
Prioritization scheme of the present invention is: add a cover cover glass and get final product the fuel feeding sem observation; When needing to preserve section,
Cover glass is removed, put into box after the air dry and preserve, can supply after the water flushing when observing once more
Om observation; Equally, but covered fuel feeding sem observation once more.
The present invention program's further optimization, can carry out the fast detecting of pathogen RLO (rickettsia-like organism): promptly, dyeing back washes with the water (about pH=5.6) of slant acidity that (potential of hydrogen of certain areas tap water is pH=5.6, but the pH value of well water or mineral water is too high), can demonstrate peach RLO inclusion body in the haemocyte matter.
The present invention has remedied the deficiencies in the prior art, simple and easy, the Fast Detection Technique of this new live shrimp and crab hemolymph light microscopic avoided the lethal of prawn crab or than the blood-sampling method of macrolesion on the blood sampling mode, new blood sampling mode is neither damaged animal, can reduce the blood contamination chance again, also be easy to control blood sampling volume.New colouring method at shrimp crab liquid of haemolymph dense thick, easily characteristics design such as solidify, be a kind of simple and easy, colouring method fast.The health status of being convenient to the prawn crab is carried out the fast detecting of real-time physiological detection and pathogen.When using simultaneously, blood-sampling method of the present invention and colouring method be optimization scheme.All can reach purpose of the present invention when two parts use separately respectively, both complement one another.The prioritization scheme that adopts slant acidity water to wash can carry out the fast detecting of pathogen RLO (rickettsia-like organism), and the practical value of prawn crab aquaculture is very high.
Description of drawings
Fig. 1 is shrimp body blood sampling position view;
Fig. 2 is crab body blood sampling position view.
Embodiment
Embodiment 1, with reference to Fig. 1, Fig. 2: shrimp and crab body is cleaned up outward, with 70% cotton ball soaked in alcohol in the uromere interface of shrimp or twice sterilization of 3～4 pairs of step base portions wiping of crab.With the alcohol 0.2～0.3mL in the 1mL syringe absorption cotton ball soaked in alcohol, the inner sterilization of syringe is carried out in suction back and forth in syringe.Alcohol is released in will managing, and with cotton ball soaked in alcohol wiping syringe needle, thrusts 7～10mm in the shrimp and crab body at the already sterilised position of shrimp crab, extracts the blood of aequum.
The film-making of blood film and colouring method
Get the liquid of haemolymph about 0.05mL, drop on the clean microslide, on drop, add the formaldehyde fixed liquid of equivalent 4% immediately
Wait to drip and put into the Giemsa dyeing liquor after the sheet air dry and dyeed 15～20 minutes
Promptly can be used for om observation behind the water flushing staining section.
Staining section needs to observe having under water (about the pH5.6) condition, and dry back blood cell shape and color all change, and it is unintelligible to become under light microscopic, but recovers normal again after adding entry, so an observing time can not be oversize, long-time observation need constantly add water.Because mounting not,, to pay special attention to during preservation so fall to going up behind the dust not easy cleaning on the staining section.
Claims (3)
1, a kind of simple and easy, Fast Detection Technique of live shrimp and crab hemolymph light microscopic, its blood collecting technology is:
The live shrimp and crab blood extracting method:
Shrimp and crab body is cleaned up outward
At the uromere interface of shrimp or 3～4 pairs of step base portion alcohol disinfectings of crab
Thrust 7～10mm in the shrimp and crab body with sterile syringe at the position of sterilizing, extract blood
Its staining technique is:
The film-making of blood film and colouring method:
Get the liquid of haemolymph about 0.05mL, drop on the clean microslide, on drop, add the formaldehyde fixed liquid of equivalent 4% immediately
Wait to drip and put into the Giemsa dyeing liquor after the sheet air dry and dyeed 15～20 minutes
Promptly be used for om observation behind the water flushing staining section.
2, according to simple and easy, the Fast Detection Technique of the described live shrimp and crab hemolymph of claim 1 light microscopic, it is characterized in that further comprising the steps of:
Add a cover cover glass fuel feeding sem observation
When needing to preserve section, cover glass is removed, put into box after the air dry and preserve.
3, according to simple and easy, the Fast Detection Technique of claim 1 or 2 described live shrimp and crab hemolymph light microscopics, it is characterized in that may further comprise the steps:
When carrying out the fast detecting of pathogen rickettsia-like organism: the used wash-down water in dyeing back is the slant acidity water about pH=5.6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01137316.4A CN1128364C (en) | 2001-11-28 | 2001-11-28 | Simple and fast light microscope detection technology for blood and lymph of live shrimp and crab |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01137316.4A CN1128364C (en) | 2001-11-28 | 2001-11-28 | Simple and fast light microscope detection technology for blood and lymph of live shrimp and crab |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1350180A true CN1350180A (en) | 2002-05-22 |
| CN1128364C CN1128364C (en) | 2003-11-19 |
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808550A (en) * | 2014-02-19 | 2014-05-21 | 上海海洋大学 | Myxosporean dyeing and sealed-storage method convenient for morphological observation |
| CN109644912A (en) * | 2019-01-29 | 2019-04-19 | 中国水产科学研究院黄海水产研究所 | A kind of Portunus trituberculatus Miers specific pathogen free offspring seed cultivation method |
| CN112237160A (en) * | 2020-10-22 | 2021-01-19 | 宁波大学 | Method for reducing attack fighting behavior of portunus trituberculatus |
-
2001
- 2001-11-28 CN CN01137316.4A patent/CN1128364C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808550A (en) * | 2014-02-19 | 2014-05-21 | 上海海洋大学 | Myxosporean dyeing and sealed-storage method convenient for morphological observation |
| CN103808550B (en) * | 2014-02-19 | 2016-08-17 | 上海海洋大学 | A kind of Myxosporean dyeing method of seal being easy to morphological observation |
| CN109644912A (en) * | 2019-01-29 | 2019-04-19 | 中国水产科学研究院黄海水产研究所 | A kind of Portunus trituberculatus Miers specific pathogen free offspring seed cultivation method |
| CN109644912B (en) * | 2019-01-29 | 2020-06-02 | 中国水产科学研究院黄海水产研究所 | Specific pathogen-free seedling cultivation method for portunus trituberculatus |
| CN112237160A (en) * | 2020-10-22 | 2021-01-19 | 宁波大学 | Method for reducing attack fighting behavior of portunus trituberculatus |
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| Publication number | Publication date |
|---|---|
| CN1128364C (en) | 2003-11-19 |
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