CN108812551A - A kind of hatching of animal gastrointestinal tract nematode larval and collection method - Google Patents
A kind of hatching of animal gastrointestinal tract nematode larval and collection method Download PDFInfo
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- CN108812551A CN108812551A CN201810401665.1A CN201810401665A CN108812551A CN 108812551 A CN108812551 A CN 108812551A CN 201810401665 A CN201810401665 A CN 201810401665A CN 108812551 A CN108812551 A CN 108812551A
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- worm
- ovum
- excrement
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- hatching
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
Abstract
The invention discloses a kind of hatching of animal gastrointestinal tract nematode larval and collection methods, using following steps：The acquisition of excrement sample filters out excrement slag, primary sedimentation, worm's ovum floating, secondary precipitation, three times precipitating, egg hatch, larvae collection.The present invention provides a kind of efficient animal gastrointestinal tract nematode larval hatching and collection method.It avoids the entire hatching process of mould contamination, reduce the removal program and impurity puzzlement of charcoal end and sucrose etc., simplify operating procedure, excrement slag removal rate is high, brooding time is shortened, hatching process can be directly monitored, avoids detection recycling and sample waste, 24 holes, which are individually hatched, can provide scientific experimentation conveniently, be suitable for popularization and application.
Description
Technical field
The present invention relates to a kind of hatching of animal gastrointestinal tract nematode larval and collection methods.
Background technique
Biocontrol of Gastrointestinal Nematodes is a kind of common parasitic zoonoses.Nematode infections have its specific history of life, and one
As for adult colonize in infection host gastrointestinal tract in, generate a large amount of ovum and excreted with excrement, ovum is sent out under optimum conditions
It is bred as infectious worm's ovum or larva, is passed transorally into alimentary canal to infect host.In the related science research of gastrointestinal nematode parasites,
Need to hatch and collect a large amount of each phase larva.However, the still not animal gastrointestinal tract nematode larval hatching of efficient quick at present
With collection method, being mainly obtained using plate hatching method for gastrointestinal nematode parasites larva reapplies soil and plant root-knot nematode
Collection method such as modified Baermann funnel method or sucrose floatation etc..Plate hatching method to the hatching efficiency of animal gastrointestinal tract nematode compared with
Low, mould easy to breed, excrement slag and charcoal end are difficult to remove；Modified Baermann funnel method can not make nematode with it is other thermophilic in excrement
Water worm is separated；The larva amount that sucrose floatation is collected is lower, and is difficult except sugar.For these disadvantages, it is difficult to realize big
Measure the acquisition of larva.Therefore, it is badly in need of a kind of efficient animal gastrointestinal tract nematode larval hatching and collection method.
Summary of the invention
Present invention seek to address that gastrointestinal nematode parasites larvae hatch and collection method lack problem, overcome the incubation rate of the larvaes it is low,
Impurity is not can be removed, mixed infection worm separates, the later period removes sugared, collection difficulty etc., and provides a kind of efficient animal gastrointestinal tract
Nematode larval hatching and collection method.
Technical scheme is as follows：
A kind of hatching of animal gastrointestinal tract nematode larval and collection method, using following steps：
1, excrement sample acquires：The target animal of non-expelling parasite in selection 4 months or more, passes through rectum aseptic collection fresh excreta, is packed into nothing
In bacterium swatch pouches；Collected excrement is transported and saved in 2-8 DEG C, faecal egg collection processing is carried out in 72 h；
2, excrement slag is filtered out：It weighs 20 g fecal samples to be put into clean triangular flask, is smashed to pieces excrement with glass bar, 10 times of weights are added
The sterile purified water of amount is put into bead, repeatedly shaking by swirling triangular flask, keeps excrement sufficiently broken and discharges worm's ovum；It is filtered with 40 meshes
Worm's ovum is sufficiently washed in filtrate from excrement slag except excrement slag, then with sterile purified water, collects filtrate；
3, primary sedimentation：The filtrate of collection is sub-packed in 50 ml centrifuge tubes, 1500 r/min are centrifuged 4 min or standing 20
H makes worm's ovum be deposited to centrifugation bottom of the tube, abandons supernatant；
4, worm's ovum floats：Saturated salt solution is added in precipitating to 50ml scale, whirlpool shakes 15 s, stand 10 min, 1000
R/min is centrifuged 4 min, stands 5 min, is in 45° angle with liquid level immediately with 1000 μ l pipettors, pipette tips mouth half is exposed to
On liquid level, band aspiration takes liquid level surface layer floating material；Every 50 ml draws 5 ml, is transferred in each new centrifuge tube respectively；
5, secondary precipitation：40 ml sterile purified waters are added in each new centrifuge tube, 1000 r/min are centrifuged 4 min, make big
Part worm's ovum is deposited to centrifugation bottom of the tube, draws 25 ml supernatant liquids at leisure from top to bottom with pipettor and discards, and when absorption makes
Pipette tips are just under immersed in liquid level；
6, it precipitates three times：25 ml sterile purified waters are added, 1500 r/min are centrifuged 4 min, worm's ovum is made to be deposited to centrifuge tube bottom
Portion abandons supernatant；
7, egg hatch：According to volume ratio sterile purified water and Earle ' s liquid 1:1 ratio prepares equilibrium liquid, according to practical need
Worm's ovum concentration is adjusted, 5-10 ml equilibrium liquid is added in each new centrifuge tube, mixes and is sub-packed in 24 orifice plates, in 28-32
DEG C, hatch under relative humidity 70-80%；
8, larvae collection：After 48 h are hatched in constant temperature peace and quiet, egg hatch situation is observed under inverted microscope, is observed every 12 h
Once, the most of larvas hatched are deposited in 24 orifice plate bottoms, as needed, can directly collect each phase larva.
The advantage of the invention is that：
The present invention provides a kind of efficient animal gastrointestinal tract nematode larval hatching and collection method.It is entire that it avoids mould contamination
Hatching process reduces the removal program and impurity puzzlement of charcoal end and sucrose etc., simplifies operating procedure, excrement slag removal rate
Height shortens brooding time, can directly monitor hatching process, avoids detection recycling and sample waste, 24 holes are individually hatched can
It is convenient to provide scientific experimentation.
Detailed description of the invention Fig. 1 is process flow chart of the invention；
Fig. 2 is the nematode worm's ovum microscopy figure precipitated in embodiment 1；
Fig. 3 is the nematode larval microscopy figure hatched in embodiment 1.
The present invention will be further explained below with reference to the attached drawings for specific embodiment.
Embodiment 1 is as shown in Figure 1, a kind of animal gastrointestinal tract nematode larval is hatched and collection method, using following steps：
1, excrement sample acquires：Sheep 3 of non-expelling parasite in selection 6 months or more, pass through rectum aseptic collection fresh excreta, and mixing is packed into
In sterile swatch pouches；Collected excrement is transported and be stored in laboratory in 2 DEG C of insulating boxs, carries out excrement worm in 72 h
Ovum collecting processing；
2, excrement slag is filtered out：It weighs 20 g mixing fecal samples to be put into clean triangular flask, is smashed to pieces excrement with glass bar, be added
200 ml sterile purified waters, are put into 50-100 bead, repeatedly shaking by swirling triangular flask, keep excrement sufficiently broken and discharge worm's ovum；
Excrement slag is filtered out with 40 meshes, then is sufficiently washed worm's ovum in filtrate from excrement slag with 100ml sterile purified water, collects filtrate；
3, primary sedimentation：The filtrate of collection is sub-packed in 50 ml centrifuge tubes, 1500 r/min are centrifuged 4 min or standing 20
H makes worm's ovum be deposited to centrifugation bottom of the tube, abandons supernatant；
4, worm's ovum floats：Saturated salt solution is added in precipitating to 50ml scale, whirlpool shakes 15 s, stand 10 min, 1000
R/min is centrifuged 4 min, stands 5 min, is in 45° angle with liquid level immediately with 1000 μ l pipettors, pipette tips mouth half is exposed to
On liquid level, band aspiration takes liquid level surface layer floating material；Every 50 ml draws 5 ml, is transferred in each new centrifuge tube respectively；
5, secondary precipitation：40 ml sterile purified waters are added in each new centrifuge tube, 1000 r/min are centrifuged 4 min, make big
Part worm's ovum is deposited to centrifugation bottom of the tube, draws 25 ml supernatant liquids at leisure from top to bottom with pipettor and discards, and when absorption makes
Pipette tips are just under immersed in liquid level；
6, it precipitates three times：25 ml sterile purified waters are added, 1500 r/min are centrifuged 4 min, worm's ovum is made to be deposited to centrifuge tube bottom
Portion abandons supernatant；
7, egg hatch：According to volume ratio sterile purified water and Earle ' s liquid 1:1 ratio prepares equilibrium liquid, according to practical need
Worm's ovum concentration is adjusted, 5 ml equilibrium liquids are added in each new centrifuge tube, mixes and is sub-packed in 24 orifice plates, in 32 DEG C, phase
It is lower to humidity 70% to hatch；
8, larvae collection：After 48 h are hatched in constant temperature peace and quiet, egg hatch situation is observed under inverted microscope, is observed every 12 h
Once, since the brooding time length of different genera nematode is different, most of worm's ovums can be in hatching in 5-7 days at third-stage larva；
Due to gravity, the most of larvas hatched are deposited in 24 orifice plate bottoms, as needed, can directly collect each phase larva.
Such as Fig. 2, to hatch the preceding nematode worm's ovum microscopy figure precipitated after precipitating three times, worm's ovum is collected more, and rate of recovery height is done
Only, without impurity such as excrement slags, it is convenient for observational study；If Fig. 3 is the nematode larval microscopy figure after hatching.
It is sheep dung with animal wastes in this present embodiment, gastrointestinal nematode parasites Mean abund ance is 3500 EPG
（Egg counts in every gram of excrement）, recovery rate of eggs 71.5%, 120 h the incubation rate of the larvaes are 68.2%.
Embodiment 2 is as shown in Figure 1, a kind of animal gastrointestinal tract nematode larval is hatched and collection method, using following steps：
1, excrement sample acquires：Alpaca 2 of non-expelling parasite in selection 4 months or more, pass through rectum aseptic collection fresh excreta, and mixing is packed into
In sterile swatch pouches；Collected excrement is transported and be stored in laboratory in 8 DEG C of insulating boxs, carries out excrement worm in 72 h
Ovum collecting processing；
2, excrement slag is filtered out：It weighs 20 g mixing fecal samples to be put into clean triangular flask, is smashed to pieces excrement with glass bar, be added
200 ml sterile purified waters are put into 100 beades, repeatedly shaking by swirling triangular flask, keep excrement sufficiently broken and discharge worm's ovum；With 40
Mesh filters out excrement slag, then is sufficiently washed worm's ovum in filtrate from excrement slag with 100ml sterile purified water, collects filtrate；
3, primary sedimentation：The filtrate of collection is sub-packed in 50 ml centrifuge tubes, 1500 r/min are centrifuged 4 min or standing 20
H makes worm's ovum be deposited to centrifugation bottom of the tube, abandons supernatant；
4, worm's ovum floats：Saturated salt solution is added in precipitating to 50ml scale, whirlpool shakes 15 s, stand 10 min, 1000
R/min is centrifuged 4 min, stands 5 min, is in 45° angle with liquid level immediately with 1000 μ l pipettors, pipette tips mouth half is exposed to
On liquid level, band aspiration takes liquid level surface layer floating material；Every 50 ml draws 5 ml, is transferred in each new centrifuge tube respectively；
5, secondary precipitation：40 ml sterile purified waters are added in each new centrifuge tube, 1000 r/min are centrifuged 4 min, make big
Part worm's ovum is deposited to centrifugation bottom of the tube, draws 25 ml supernatant liquids at leisure from top to bottom with pipettor and discards, and when absorption makes
Pipette tips are just under immersed in liquid level；
6, it precipitates three times：25 ml sterile purified waters are added, 1500 r/min are centrifuged 4 min, worm's ovum is made to be deposited to centrifuge tube bottom
Portion abandons supernatant；
7, egg hatch：According to volume ratio sterile purified water and Earle ' s liquid 1:1 ratio prepares equilibrium liquid, according to practical need
Worm's ovum concentration is adjusted, 10 ml equilibrium liquids are added in each new centrifuge tube, mixes and is sub-packed in 24 orifice plates, in 28 DEG C, phase
It is lower to humidity 80% to hatch；
8, larvae collection：After 48 h are hatched in constant temperature peace and quiet, egg hatch situation is observed under inverted microscope, is observed every 12 h
Once, since the brooding time length of different genera nematode is different, most of worm's ovums can be in hatching in 5-7 days at third-stage larva；
Due to gravity, the most of larvas hatched are deposited in 24 orifice plate bottoms, as needed, can directly collect each phase larva.
It is alpaca excrement with animal wastes in this present embodiment, gastrointestinal nematode parasites Mean abund ance is 1500 EPG,
Recovery rate of eggs is 66.7%, and 130 h the incubation rate of the larvaes are 60.1%.
Embodiment 3 is as shown in Figure 1, a kind of animal gastrointestinal tract nematode larval is hatched and collection method, using following steps：
1, excrement sample acquires：Ox 2 of non-expelling parasite in selection 5 months or more, pass through rectum aseptic collection fresh excreta, and mixing is packed into nothing
In bacterium swatch pouches；Collected excrement is transported and be stored in laboratory in 4 DEG C of insulating boxs, carries out faecal egg receipts in 72 h
Collection processing；
2, excrement slag is filtered out：It weighs 20 g mixing fecal samples to be put into clean triangular flask, is smashed to pieces excrement with glass bar, be added
200 ml sterile purified waters are put into 100 beades, repeatedly shaking by swirling triangular flask, keep excrement sufficiently broken and discharge worm's ovum；With 40
Mesh filters out excrement slag, then is sufficiently washed worm's ovum in filtrate from excrement slag with 100ml sterile purified water, collects filtrate；
3, primary sedimentation：The filtrate of collection is sub-packed in 50 ml centrifuge tubes, 1500 r/min are centrifuged 4 min or standing 20
H makes worm's ovum be deposited to centrifugation bottom of the tube, abandons supernatant；
4, worm's ovum floats：Saturated salt solution is added in precipitating to 50ml scale, whirlpool shakes 15 s, stand 10 min, 1000
R/min is centrifuged 4 min, stands 5 min, is in 45° angle with liquid level immediately with 1000 μ l pipettors, pipette tips mouth half is exposed to
On liquid level, band aspiration takes liquid level surface layer floating material；Every 50 ml draws 5 ml, is transferred in each new centrifuge tube respectively；
5, secondary precipitation：40 ml sterile purified waters are added in each new centrifuge tube, 1000 r/min are centrifuged 4 min, make big
Part worm's ovum is deposited to centrifugation bottom of the tube, draws 25 ml supernatant liquids at leisure from top to bottom with pipettor and discards, and when absorption makes
Pipette tips are just under immersed in liquid level；
6, it precipitates three times：25 ml sterile purified waters are added, 1500 r/min are centrifuged 4 min, worm's ovum is made to be deposited to centrifuge tube bottom
Portion abandons supernatant；
7, egg hatch：According to volume ratio sterile purified water and Earle ' s liquid 1:1 ratio prepares equilibrium liquid, according to practical need
Worm's ovum concentration is adjusted, 8 ml equilibrium liquids are added in each new centrifuge tube, mixes and is sub-packed in 24 orifice plates, in 30 DEG C, phase
It is lower to humidity 75% to hatch；
8, larvae collection：After 48 h are hatched in constant temperature peace and quiet, egg hatch situation is observed under inverted microscope, is observed every 12 h
Once, since the brooding time length of different genera nematode is different, most of worm's ovums can be in hatching in 5-7 days at third-stage larva；
Due to gravity, the most of larvas hatched are deposited in 24 orifice plate bottoms, as needed, can directly collect each phase larva.
It is cow dung with animal wastes in this present embodiment, gastrointestinal nematode parasites Mean abund ance is 800 EPG, worm's ovum
The rate of recovery is 62.5%, and 120 h the incubation rate of the larvaes are 78.2%.
Claims (1)
1. a kind of animal gastrointestinal tract nematode larval hatching and collection method, which is characterized in that use following steps：
1, excrement sample acquires：The target animal of non-expelling parasite in selection 4 months or more, passes through rectum aseptic collection fresh excreta, is packed into nothing
In bacterium swatch pouches；Collected excrement is transported and saved in 2-8 DEG C, faecal egg collection processing is carried out in 72 h；
2, excrement slag is filtered out：It weighs 20 g fecal samples to be put into clean triangular flask, is smashed to pieces excrement with glass bar, 10 times of weights are added
The sterile purified water of amount is put into bead, repeatedly shaking by swirling triangular flask, keeps excrement sufficiently broken and discharges worm's ovum；It is filtered with 40 meshes
Worm's ovum is sufficiently washed in filtrate from excrement slag except excrement slag, then with sterile purified water, collects filtrate；
3, primary sedimentation：The filtrate of collection is sub-packed in 50 ml centrifuge tubes, 1500 r/min are centrifuged 4 min or standing 20
H makes worm's ovum be deposited to centrifugation bottom of the tube, abandons supernatant；
4, worm's ovum floats：Saturated salt solution is added in precipitating to 50ml scale, whirlpool shakes 15 s, stand 10 min, 1000
R/min is centrifuged 4 min, stands 5 min, is in 45° angle with liquid level immediately with 1000 μ l pipettors, pipette tips mouth half is exposed to
On liquid level, band aspiration takes liquid level surface layer floating material；Every 50 ml draws 5 ml, is transferred in each new centrifuge tube respectively；
5, secondary precipitation：40 ml sterile purified waters are added in each new centrifuge tube, 1000 r/min are centrifuged 4 min, make big
Part worm's ovum is deposited to centrifugation bottom of the tube, draws 25 ml supernatant liquids at leisure from top to bottom with pipettor and discards, and when absorption makes
Pipette tips are just under immersed in liquid level；
6, it precipitates three times：25 ml sterile purified waters are added, 1500 r/min are centrifuged 4 min, worm's ovum is made to be deposited to centrifuge tube bottom
Portion abandons supernatant；
7, egg hatch：According to volume ratio sterile purified water and Earle ' s liquid 1:1 ratio prepares equilibrium liquid, according to practical need
Worm's ovum concentration is adjusted, 5-10 ml equilibrium liquid is added in each new centrifuge tube, mixes and is sub-packed in 24 orifice plates, in 28-32
DEG C, hatch under relative humidity 70-80%；
8, larvae collection：After 48 h are hatched in constant temperature peace and quiet, egg hatch situation is observed under inverted microscope, is observed every 12 h
Once, the most of larvas hatched are deposited in 24 orifice plate bottoms, as needed, can directly collect each phase larva.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323281A (en) * | 2020-04-01 | 2020-06-23 | 金黑鹰 | Method for extracting fecal cast-off intestinal mucosa sample |
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| CN101424613A (en) * | 2008-04-08 | 2009-05-06 | 廖党金 | Parasite egg detecting method |
| US20130273666A1 (en) * | 2010-11-03 | 2013-10-17 | Teco Diagnostics | Color-Based Reaction Testing of Biological Materials |
| CN106950073A (en) * | 2017-03-17 | 2017-07-14 | 海南医学院 | A kind of method that strongyloides intestinalis larva is collected from watery stools |
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2018
- 2018-04-28 CN CN201810401665.1A patent/CN108812551A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020164675A1 (en) * | 1999-05-28 | 2002-11-07 | My Lien Dao | Methods of propagating and detecting infectious forms of intracellular pathogenic microorganisms |
| CN101424613A (en) * | 2008-04-08 | 2009-05-06 | 廖党金 | Parasite egg detecting method |
| US20130273666A1 (en) * | 2010-11-03 | 2013-10-17 | Teco Diagnostics | Color-Based Reaction Testing of Biological Materials |
| CN106950073A (en) * | 2017-03-17 | 2017-07-14 | 海南医学院 | A kind of method that strongyloides intestinalis larva is collected from watery stools |
Non-Patent Citations (1)
| Title |
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| 夏永高，王权: "虫卵孵化试验对山羊捻转血矛线虫左旋咪唑抗药性检测", 《畜牧与兽医》 * |
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| CN111323281A (en) * | 2020-04-01 | 2020-06-23 | 金黑鹰 | Method for extracting fecal cast-off intestinal mucosa sample |
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