CN109042626A - It is located away from the freezen protective and method for resuscitation of the Uronema marinum of Fugu rubripes - Google Patents
It is located away from the freezen protective and method for resuscitation of the Uronema marinum of Fugu rubripes Download PDFInfo
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- CN109042626A CN109042626A CN201810906214.3A CN201810906214A CN109042626A CN 109042626 A CN109042626 A CN 109042626A CN 201810906214 A CN201810906214 A CN 201810906214A CN 109042626 A CN109042626 A CN 109042626A
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- uronema marinum
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- uronema
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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
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Engineering & Computer Science (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses freezing and the method for resuscitation of a kind of Uronema marinum for being located away from Fugu rubripes, i.e. with L-15 complete medium culture Uronema marinum to exponential phase of growth, exponential phase to adult, it is 200,000/mL that its density, which is resuspended, with L-15 complete medium again, Uronema marinum adult liquid is drawn to be added in cryopreservation tube, add the sterile glycerol that volume ratio is 40%, 10% dimethyl sulfoxide, it mixes, cryopreservation tube is quickly put into sequencing gradient cooling cell cryopreservation box, overnight in -80 DEG C, it is then placed in liquid nitrogen, it can long-term preservation.It is quickly put into water-bath from cryopreservation tube is taken out in liquid nitrogen, 37 DEG C of 2 min of water-bath, 4000 rpm are centrifuged 5 min later, abandon supernatant, and L-15 complete medium is added and is resuspended, is transferred in porous plate, cultivates three days, can recover in 20 DEG C of constant incubators.
Description
Technical field
The invention belongs to field of biotechnology, it is related to a kind of ciliophoran preservation of shield and method for resuscitation, especially a kind of point
From in Fugu rubripes Uronema marinum (Uronema marinum) freezen protective and method for resuscitation.
Background technique
Uronema marinum (Uronema marinum) it is under the jurisdiction of shield fibre mesh, Infusoria is a kind of small facultative of individual
Parasitic ciliate.Uronema marinum adaptability is extremely strong, can free living in various breeding water bodies.Once residual bait in breeding water body
Excessively, environmental degradation etc. causes content of organics excessively high, Uronema marinum will mass propagation, parasitize each cultivation phase fish body,
The especially fish body of body-surface rauma, is food with the cell or tissue fragment of host, and secondary bacterial disease when serious causes to cultivate
Fish mortality.Reported Uronema marinum can cause turbot, lefteye flounder, Fugu rubripes and other mariculture fishes
Class morbidity, the death rate are higher.
Marine fish is made currently, having had multidigit scholar to study shield infusorian both at home and abroad and having reported it
At serious harm.However the shield infusorian for being currently used for research mainly passes through continuous passage culture in vitro conservation, there is
In vitro culture passage is cumbersome, passage unsuccessfully leads to worm strain loss, germ contamination, infection ability of damaging by worms decrease, kind cytoplasmic mutation and exempts from
The problems such as epidemic focus is lost.The ciliophoran freezen protective of shield and recovery or blank at present, especially not about ocean tailfiber
The relevant report of worm freezen protective.
Summary of the invention
The present invention is to provide one kind in order to solve technical problem present in the prior art and be located away from Fugu rubripes ocean
The freezen protective and method for resuscitation of Uronema.
The technical solution of the invention is as follows: a kind of freezen protective side for the Uronema marinum being located away from Fugu rubripes
Method, it is characterised in that successively carry out in accordance with the following steps:
A. it is isolated parasitize Fugu rubripes body surface Uronema marinum (Uronema marinum), it is inoculated in broth cultivation
Domestication culture in vitro 15 days in base are supported, Uronema marinum liquid is obtained, the broth bouillon is that every 100 mL sterilizing seawater adds 5g red
10 min are boiled in the homogenate of the fin dongfang globe fish flesh of fish, then the gained after the filtered through gauze of 200 mesh；
It b. by Uronema marinum liquid is by volume again that 1:100 is inoculated in broth bouillon, 20 DEG C of 120 hr of culture are obtained
Uronema marinum liquid in exponential phase of growth；
C. it draws 1 mL to be in the Uronema marinum liquid extremely 1.5mL centrifuge tube of sterilizing of exponential phase of growth, 2000 rpm centrifugation
10 minutes, 1 mL L-15 complete medium is added into centrifuge tube after abandoning supernatant, stands 5 min, Aspirate supernatant is in another
In centrifuge tube, 2000 rpm are centrifuged 10 minutes, are abandoned supernatant, then be resuspended with 1 mL L-15 complete medium, are obtained ocean tailfiber
Worm suspension；The L-15 complete medium is that the mycillin and total matter that gross mass percentage is 1% are added in L-15 culture medium
The fetal calf serum that percentage is 10% is measured, adjusting salinity with sodium chloride is 35 ‰, and adjusting pH is 7.2；
D. use L-15 complete medium serial dilution Uronema marinum suspension, draw that form is complete under the microscope and vigor compared with
Strong single Uronema marinum is added in the porous plate of the L-15 complete medium containing 200 μ L, 20 DEG C of constant temperature incubations 96
Hr, the Uronema marinum liquid purified；
E. the Uronema marinum liquid purified with the dilution of L-15 complete medium is to 1000/mL and 100 μ L of absorption are inoculated in 50
In mL conical flask, the L-15 complete medium of 20 mL, sealed membrane sealing is added, 20 DEG C of 120 hr of constant temperature incubation obtain index
The Uronema marinum liquid of phase；
F. the Uronema marinum liquid of exponential phase is transferred in centrifuge tube, 2000 rpm are centrifuged 10 min, abandon supernatant, are added
L-15 complete medium, 20 DEG C of 5 min of standing, Aspirate supernatant are added in another centrifuge tube, adjust Uronema marinum population
Density is 200,000/mL, obtains Uronema marinum adult liquid；
G. Uronema marinum adult liquid obtained by absorption f step is added in cryopreservation tube and adds sterile glycerol and dimethyl sulfoxide is mixed
Even, the volume ratio of the Uronema marinum adult liquid, sterile glycerol and dimethyl sulfoxide is 5:4:1；
H. cryopreservation tube is put into sequencing gradient cooling cell cryopreservation box, overnight in -80 DEG C, is then placed in liquid nitrogen and protects
It deposits.
A kind of method for resuscitation corresponding with the freezing and storing method of the above-mentioned Uronema marinum for being located away from Fugu rubripes,
It is characterized in that: the cryopreservation tube being stored in liquid nitrogen is put into water-bath, 37 DEG C of water-baths 2 min, 4000rpm are centrifuged 5min, abandon
Supernatant is added L-15 complete medium and is resuspended, is transferred in porous plate, in 20 DEG C of constant incubators, cultivates three days, can recover.
The invention avoids the complicated processes of Uronema marinum serial passages in vitro, while also avoiding thin in succeeding generations
Bacterium pollution can save the strain of original worm, prevent worm strain genetic drift and because of worm strain virulence attenuation of caused by worm strain continuous passage
The advantages that.
Detailed description of the invention
Fig. 1 is 2 polypide recovery number of days of the embodiment of the present invention and quantity schematic diagram.
Fig. 2 is the preceding morphological contrast map with after recovery of polypide of embodiment of the present invention freezing.
Specific embodiment
Embodiment 1:
A. shield infusorian is separated out from the punishment of farm of Daliang City of Liaoning Province illness Fugu rubripes body surface ulcerated tissue, identified
For Uronema marinum (Uronema marinum), obtained Uronema marinum is inoculated in broth bouillon and is tamed in vitro
Culture 15 days, obtains Uronema marinum liquid, and the broth bouillon is that every 100 mL sterilizing seawater adds the 5g Fugu rubripes flesh of fish even
Slurry boils 10 min, then the gained after the filtered through gauze of 200 mesh；
It b. by Uronema marinum liquid is by volume again that 1:100 is inoculated in broth bouillon, 20 DEG C of 120 hr of culture are obtained
Uronema marinum liquid in exponential phase of growth；
C. it draws 1 mL to be in the Uronema marinum liquid extremely 1.5mL centrifuge tube of sterilizing of exponential phase of growth, 2000 rpm centrifugation
10 minutes, be slowly added to 1 mL L-15 complete medium into centrifuge tube after abandoning supernatant, stand 5 min, Aspirate supernatant in
In another centrifuge tube, 2000 rpm are centrifuged 10 minutes, are abandoned supernatant, then be resuspended with 1 mL L-15 complete medium, are obtained ocean
Uronema suspension；The L-15 complete medium is that mycillin and fetal calf serum are added in L-15 culture medium, and salinity is
35 ‰, pH 7.2, the mass ratio of the L-15 culture medium, mycillin and fetal calf serum are 1:0.01:0.1；
D. use L-15 complete medium serial dilution Uronema marinum suspension, draw that form is complete under the microscope and vigor compared with
Strong single Uronema marinum is added in the porous plate of the L-15 complete medium containing 200 μ L, 20 DEG C of constant temperature incubations 96
Hr, the Uronema marinum liquid purified；
E. the Uronema marinum liquid purified with the dilution of L-15 complete medium is to 1000/mL and 100 μ L of absorption are inoculated in 50
In mL conical flask, the L-15 complete medium of 20 mL, sealed membrane sealing is added, 20 DEG C of 120 hr of constant temperature incubation obtain index
The Uronema marinum liquid of phase；
F. the Uronema marinum liquid of exponential phase is transferred in centrifuge tube, 2000 rpm are centrifuged 10 min, abandon supernatant, slowly
L-15 complete medium is added, 20 DEG C of 5 min of standing draw and contain the ciliophoran supernatant of shield, are added in another centrifuge tube,
With plankton counting chamber count population density, adjustings Uronema marinum population density be 200,000/mL, obtain Uronema marinum at
Worm liquid；
G. Uronema marinum adult liquid obtained by absorption f step is added in cryopreservation tube and adds sterile glycerol and dimethyl sulfoxide is mixed
Even, the volume ratio of the Uronema marinum adult liquid, sterile glycerol and dimethyl sulfoxide is 5:4:1；
H. cryopreservation tube is quickly put into sequencing gradient cooling cell cryopreservation box, overnight in -80 DEG C, is then placed in liquid nitrogen
It saves.
Embodiment 2:
Present invention method for resuscitation corresponding with embodiment 1 is as follows: the cryopreservation tube for being stored in 24 hours in liquid nitrogen is quickly put into water
In bath, 37 DEG C of water-baths 2 min, 4000rpm are centrifuged 5min, abandon supernatant, L-15 complete medium is added and is resuspended, is transferred to porous plate
In, in 20 DEG C of constant incubators, cultivate seven days.
Daily microscopically observation simultaneously counts (numerical value is expressed as average value ± standard error), as a result: first day to second day,
In deadtime, the stronger polypide of vigor is not observed；The vigorous polypide of activity is observed in third day, and counting its population density is
1586 ± 82/mL；4th day, counting its population density was 158400 ± 1728/mL；5th day, count its population density
For 590000 ± 20406/ml；6th day, counting its population density was 460133 ± 27280/mL；7th day, count it
Population density is 423066 ± 6130/ml.Concrete outcome is as shown in Figure 1, illustrating that polypide is cultivated three days can recover.
It is as shown in Figure 2 with the form comparison after recovery before the freezing of polypide of the embodiment of the present invention.In Fig. 2, A is real
The form picture of Uronema marinum before applying 1 freezen protective of example (formaldehyde is fixed, 1000 times of visuals field)；B is that embodiment 1 freezes
The form picture (methyl green pyronin staining, 1000 times of visuals field) of Uronema marinum before preservation；C is after embodiment 2 is recovered
The form picture of Uronema marinum (formaldehyde is fixed, 1000 times of visuals field)；D is the form of the Uronema marinum after embodiment 2 is recovered
Picture (methyl green pyronin staining, 1000 times of visuals field).
As seen from Figure 2, the morphological feature of Uronema marinum does not have with the morphological feature for freezing preceding Uronema marinum after recovery
Have apparent difference: polypide is in melon seeds type, and top is slightly round, and naked hair-fields has beak protrusion, all over the body throughout cilium, polypide rear end
There is a dodds.As the result is shown through unna staining: the visible obvious big core of polypide, big core presents it is oval or
Circle is located at polypide front middle part.
To Uronema marinum extracorporeal movement observes result before freezing and after recovery: the Uronema marinum after recovery is with body cilium
Spiral circus movement forward is swung, movement is very fast, and vigor is stronger, does not have notable difference with the Uronema marinum body before freezing.
Claims (2)
1. a kind of freezing and storing method for the Uronema marinum for being located away from Fugu rubripes, it is characterised in that successively according to following step
It is rapid to carry out:
A. it is isolated parasitize Fugu rubripes body surface Uronema marinum (Uronema marinum), it is inoculated in broth cultivation
Domestication culture in vitro 15 days in base are supported, Uronema marinum liquid is obtained, the broth bouillon is that every 100 mL sterilizing seawater adds 5g red
10 min are boiled in the homogenate of the fin dongfang globe fish flesh of fish, then the gained after the filtered through gauze of 200 mesh；
It b. by Uronema marinum liquid is by volume again that 1:100 is inoculated in broth bouillon, 20 DEG C of 120 hr of culture are obtained
Uronema marinum liquid in exponential phase of growth；
C. it draws 1 mL to be in the Uronema marinum liquid extremely 1.5mL centrifuge tube of sterilizing of exponential phase of growth, 2000 rpm centrifugation
10 minutes, 1 mL L-15 complete medium is added into centrifuge tube after abandoning supernatant, stands 5 min, Aspirate supernatant is in another
In centrifuge tube, 2000 rpm are centrifuged 10 minutes, are abandoned supernatant, then be resuspended with 1 mL L-15 complete medium, are obtained ocean tailfiber
Worm suspension；The L-15 complete medium is that the mycillin and total matter that gross mass percentage is 1% are added in L-15 culture medium
The fetal calf serum that percentage is 10% is measured, adjusting salinity with sodium chloride is 35 ‰, and adjusting pH is 7.2；
D. use L-15 complete medium serial dilution Uronema marinum suspension, draw that form is complete under the microscope and vigor compared with
Strong single Uronema marinum is added in the porous plate of the L-15 complete medium containing 200 μ L, 20 DEG C of constant temperature incubations 96
Hr, the Uronema marinum liquid purified；
E. the Uronema marinum liquid purified with the dilution of L-15 complete medium is to 1000/mL and 100 μ L of absorption are inoculated in 50
In mL conical flask, the L-15 complete medium of 20 mL, sealed membrane sealing is added, 20 DEG C of 120 hr of constant temperature incubation obtain index
The Uronema marinum liquid of phase；
F. the Uronema marinum liquid of exponential phase is transferred in centrifuge tube, 2000 rpm are centrifuged 10 min, abandon supernatant, are added
L-15 complete medium, 20 DEG C of 5 min of standing, Aspirate supernatant are added in another centrifuge tube, adjust Uronema marinum population
Density is 200,000/mL, obtains Uronema marinum adult liquid；
G. Uronema marinum adult liquid obtained by absorption f step is added in cryopreservation tube and adds sterile glycerol and dimethyl sulfoxide is mixed
Even, the volume ratio of the Uronema marinum adult liquid, sterile glycerol and dimethyl sulfoxide is 5:4:1；
H. cryopreservation tube is put into sequencing gradient cooling cell cryopreservation box, overnight in -80 DEG C, is then placed in liquid nitrogen and protects
It deposits.
2. a kind of recovery corresponding with the freezing and storing method for the Uronema marinum for being located away from Fugu rubripes described in claim 1
Method, it is characterised in that: the cryopreservation tube being stored in liquid nitrogen is put into water-bath, 37 DEG C of water-bath 2 min, 4000rpm centrifugations
5min abandons supernatant, and L-15 complete medium is added and is resuspended, is transferred in porous plate, in 20 DEG C of constant incubators, culture at least three
It, can recover.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810906214.3A CN109042626B (en) | 2018-08-10 | 2018-08-10 | Cryopreservation and recovery method of sea tail filarial separated from fugu rubripes |
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| CN201810906214.3A CN109042626B (en) | 2018-08-10 | 2018-08-10 | Cryopreservation and recovery method of sea tail filarial separated from fugu rubripes |
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| CN109042626A true CN109042626A (en) | 2018-12-21 |
| CN109042626B CN109042626B (en) | 2023-07-11 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102492623A (en) * | 2011-12-13 | 2012-06-13 | 中国水产科学研究院黄海水产研究所 | Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates |
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2018
- 2018-08-10 CN CN201810906214.3A patent/CN109042626B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492623A (en) * | 2011-12-13 | 2012-06-13 | 中国水产科学研究院黄海水产研究所 | Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates |
Non-Patent Citations (4)
| Title |
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| I. FOLGUEIRAA 等: "Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia)", 《CRYOBIOLOGY》 * |
| R. IGLESIAS 等: "In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida)", 《VETERINARY PARASITOLOGY》 * |
| SALLY A. ANDERSON 等: "In vitro culture and cryopreservation of Uronema marinum isolated from farmed New Zealand groper (Polyprion oxygeneios)", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| 曾红等: "不同培养条件对海洋尾丝虫种群增长的影响", 《福建师范大学学报( 自然科学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
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