CN102492623A - Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates - Google Patents
Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates Download PDFInfo
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- CN102492623A CN102492623A CN2011104141236A CN201110414123A CN102492623A CN 102492623 A CN102492623 A CN 102492623A CN 2011104141236 A CN2011104141236 A CN 2011104141236A CN 201110414123 A CN201110414123 A CN 201110414123A CN 102492623 A CN102492623 A CN 102492623A
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Abstract
The invention relates to a method for preparing and culturing a culture medium for the supravital culture of seawater scuticociliatida ciliates, and relates to the field of the study of aquatic animal diseases. The method for preparing the in-vitro culture medium comprises the following steps of: mixing fresh brain tissue and muscular tissue of paralichthys olivaceus or turbot fish in a mass ratio of (1:1)-(3:1), and homogenizing to form muddy flesh; adding sterilized seawater into the muddy flesh in a mass/volume ratio of the muddy flesh to the seawater of (1:45)-(1:55), and performing water bath at the temperature of between 65 and 70 DEG C for 2 hours to prepare the in-vitro culture medium, wherein the prepared culture medium is stored at the temperature of 4 DEG C for a long time. The method for preparing the culture medium is simple, and complex components are not needed to be added; and the seawater scuticociliatida ciliates are subjected to the supravital culture by utilizing the culture medium prepared by the method, and the scuticociliatida ciliates can survive for over 30 days, so that the foundation is established for the further study of the pathogenesis and prevention and control technology of the scuticociliatida ciliates.
Description
Technical field:
The invention belongs to the aquatic animal disease research field, relate to a kind of particularly in the method for the external realization seawater of aquatic animal shield ciliate live body long-term cultivation and the preparation of substratum thereof.
Background technology:
The shield ciliate is acknowledged as one type of main pathogenic agent of sea farming, has had the multidigit scholar's research to report its serious harm that sea farming is caused (Azusa Umehara, et al.2003, ESterud et al.2000) both at home and abroad.Generally speaking, shield ciliate battalion free living is a food with the small organic granular (bacterium, protozoon, little algae, organic debris etc.) that suspends in the water.Yet; Under the situation of high-density breeding, the shield ciliate can be changed parasitics into usually, is food with the cell or tissue chips of echinoderms, shellfish, crustaceans and fish; And in host tissue, grow fast, breed; And then cause the degree of depth injury of tissue, and form serious focus, finally cause host's organ failure and death.
Because the serious threat of shield ciliate is to the sound development of sea farming; Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science has successively carried out the sick research of disease popularity comprehensive, system to the shield balantidiasis of China prawn, fish and sea cucumber; But find diversified economy animals such as shield ciliate infection of Chinese prawn, lefteye flounder, turbot, river Puffer, sea cucumber; And deeply carried out the ciliophoran morphological observation (Chinese aquatic science of shield; 2005 12 the 5th phases of volume) with the pathological research that infects these animals (the hydrobiont journal was rolled up for the 5th phase in 20075 31).Simultaneously, carried out Chinese herbal medicine screening (national inventing patent ZL200510044755.2) and prevented and treated Study on Process (national inventing patent ZL200510044757.1) to isolating shield ciliate.These researchs concentrate on aspects such as the histopathology of shield balantidiasis and controls, deeply do not carry out the research of aspects such as shield ciliophoran life history, pathogenesis.
For the control shield balantidiasis of science, except strengthening its Study of Prevention Technology, carry out fundamental researches such as the polypide life history and pathogenesis and also be very necessary, and the research of this respect depends on external and polypide is carried out long live body cultivates.Before the present invention makes; The research report of relevant seawater shield balantidiasis mainly concentrates on aspects such as epidemiology, etiology, taxonomy, control drug screening both at home and abroad; The nutritional needs that has only people such as scholar R.Iglesias to cultivate with regard to shield ciliate polypide supravital carried out research (In vitro growth requirements for the fish pathogen Philasterides dicentrarchi.Veterinary Parasitology; 2003,111:19-30.).They mainly utilize the artificial synthetic medium of containing multiple components external ciliate to be continued to cultivate, and its culture medium prescription is complicated, and the polypide incubation time is also only about 10 days.
Summary of the invention:
Technical problem to be solved by this invention provides a kind of preparation of seawater shield ciliate supravital culture medium and utilizes this substratum to carry out seawater shield ciliate supravital cultured method; The medium preparation method is simple; Can realize shield ciliate live body in the external long-term cultivation of aquatic animal, for fundamental researches such as further investigation shield ciliophoran life history, pathogenesis provide indispensable technical support.
The present invention adopts following technical scheme to be achieved:
A kind of preparation method of seawater shield ciliate supravital culture medium; May further comprise the steps: the cerebral tissue of clip healthy Paralichthys olivaceus fish or turbot and muscle tissue; With both according to 1: 1～3: 1 mixed of mass ratio after, the sterilization seawater volume that adds behind membrane filtration is V
1, be prepared into muddy flesh with tissue refiner; The sterilization seawater volume that in muddy flesh, adds behind membrane filtration is V
2The mass volume ratio of the seawater TV of muddy flesh and twice adding is 1: 45～1: 55; Vibration evenly is placed in sealing, the aseptic Glass Containers; 65～70 ℃ of water-baths are prepared into shield ciliate live body substratum after 2～3 hours, the shield ciliate live body substratum for preparing is placed on prolonged preservation under 4 ℃ of conditions.
Further, the described filter membrane that is used for filtering sea is preferably the cellulose mixture filter membrane of aperture 0.22 μ m.
The present invention also provides a kind of substratum that utilizes the seawater shield ciliate supravital cultivation of said method preparation.
The present invention also provides a kind of seawater shield ciliate supravital cultured method.
The present invention realizes by following technical scheme:
A kind of seawater shield ciliate supravital cultural method; Concrete steps are: clip infects the cultivated animals part lesion tissue of shield balantidiasis; After aseptic technique shreds in glass culture dish; Splash into the sterilization seawater behind membrane filtration of 15～20 times of volumes, place in 18～20 ℃ of constant incubators and left standstill 2～4 hours; Under anatomical lens, draw in the petridish after leaving standstill free polypide in the liquid, splash in the glass culture dish that the shield ciliate substratum that 15～30ml prepares is housed, place 18～20 ℃ of constant temperature culture with aseptic glue head dropper; Every at a distance from 20～24 hours; Drawing 5～10ml nutrient solution is transferred in another glass culture dish that fresh shield ciliate substratum of 15～30mL is housed; Rock gently mix for several times after; Be placed on 18～20 ℃ of constant incubators again and continue to cultivate, realize the purpose of living worm body long-term cultivation with this, remaining nutrient solution is used for the polypide counting in the former petridish.
Further, the described filter membrane that is used for filtering sea is preferably the cellulose mixture filter membrane of aperture 0.22 μ m.
The correlated beneficial effect of the present invention and prior art:
1. the making method of shield ciliate live body substratum is simple, need not to add multiple complicated composition, makes things convenient in the laboratory in a large number, prepares fast.
2. cultural method is simple, and required plant and instrument is few, under general laboratory condition, all can cultivate.
3. the incubation time of external shield ciliate live body is long, but cultured continuously more than 30 days, considerably beyond about 10 days of similar information report.
Description of drawings:
The live body shield ciliate that Fig. 1 vitro culture was examined under a microscope in 15 days.
Fig. 2 adds the liquid-solid shield ciliate polypide after fixed of Lu Geshi iodine.
Embodiment:
Combine accompanying drawing that technical scheme of the present invention is elaborated through embodiment below:
Embodiment 1
In June, 2011, Yantai, Shandong turbot is cultured the turbot generation shield balantidiasis that factory cultures, and carries out pathogen separation and cultivation with taking back Qingdao Huanghai Sea aquatic products institute laboratory after the ill turbot packing oxygenation.
In the laboratory, at first prepare shield ciliate live body substratum.Select 20 from culturing the healthy turbot that factory takes back; About 1g of its cerebral tissue of clip and the about 1g of its back peeling muscle pack both mixing in the big centrifuge tube into, add 20ml and sterilize seawater with tissue refiner's homogenate after 10 minutes; Add 70ml sterilization seawater again and fully vibrate, mix.The 90ml mixture that mixes is transferred in the 200ml vial that has sealing cover, was placed in 65 ℃ of water-baths water-bath behind the good seal 2 hours, during whenever jiggled once at a distance from 15 minutes.After water-bath finishes, put into 4 ℃ of preservations after vial naturally cooled to room temperature.
Select the ciliophoran ill turbot of severe infections shield and put into Dissecting tray, with its lesions position of aseptic seawater flushing 3 times.Put into the glass culture dish of an aseptic diameter 9cm then with the about 5g lesion tissue of Dissecting scissors clip; Aseptic technique shreds this lesion tissue; And the about 75ml via hole diameter of adding is the sterilization seawater behind the 0.22 μ m cellulose mixture membrane filtration in petridish; After rocking petridish gently homogeneous microstructure being scattered, cover the petridish lid and put into 18 ℃ of constant incubators and left standstill 2 hours.Petridish after will leaving standstill then is placed under the anatomical lens; Draw free shield ciliate polypide in the some amount liquid with aseptic glue head dropper; Be transferred in another new glass culture dish that 15ml shield ciliate live body substratum is housed behind high-temperature sterilization; Cover the petridish lid and rock gently for several times, put into 18 ℃ of constant incubators and cultivate.
Cultivate after 24 hours; Taking out petridish rocks for several times gently; Nutrient solution with aseptic glue head dropper absorption 5ml is transferred in another new glass culture dish that the fresh shield ciliate live body substratum of 15ml is housed behind high-temperature sterilization then; Cover after the petridish lid rocks for several times gently, continue to put into 18 ℃ of constant incubators and cultivate.Remaining nutrient solution is used for the polypide counting.After this every this step that repeated at a distance from 24 hours finishes until experiment.Experimental session in time prepares and additional fresh shield ciliate live body substratum.
The method of polypide counting is in the culturing process: remaining nutrient solution exhausts with aseptic glue head dropper fully after will drawing 5ml, splashes in the funnel that filter paper is housed and filters.During filtration, nutrient solution is dripped in the filter paper centre bottom as much as possible, make filtering cross-sectional area as much as possible little with glue head dropper.After filtering completion, use 10ml via hole diameter is the filtration position that the sterilization seawater behind the 0.22 μ m cellulose mixture membrane filtration oppositely drips filter wash paper, drips a washing lotion and is collected in the centrifuge tube of a sterilization, and the polypide that obtains 10ml is collected liquid.The Lu Geshi iodine liquid of adding 1% in collecting liquid (Lugol ' s solution) fixing; Fully absorption 0.1ml collects liquid and puts into plankton counting chamber at the microscopically counting after the vibration evenly, and the measurement of counting volume is a standard with final 10ml polypide collection liquid.
This external shield ciliate live body culture experiment has been carried out 37 days altogether, and polypide is counted per 3 days once, and count results is seen table 1.In the experimentation, the shield ciliate polypide in the nutrient solution is remaining vigorous vitality and reproductivity (see figure 1).
The count results that table 1 shield ciliate live body is cultivated
Embodiment 2
In August, 2011, the shield balantidiasis also takes place in the lefteye flounder that Jiaonan City, Qingdao lefteye flounder is cultured factory's breed, carries out pathogen separation and cultivation with taking back Qingdao Huanghai Sea aquatic products institute laboratory after the ill lefteye flounder packing oxygenation.
In the laboratory, at first prepare shield ciliate live body substratum.Select 30 from culturing the healthy Paralichthys olivaceus that factory takes back; About 1.5g of its cerebral tissue of clip and the about 0.5g of its back peeling muscle pack both mixing in the big centrifuge tube into, add 20ml and sterilize seawater with tissue refiner's homogenate after 10 minutes; Add 90ml sterilization seawater again and fully vibrate, mix.The 110ml mixture that mixes is transferred in the 200ml vial that has sealing cover, was placed in 70 ℃ of water-baths water-bath behind the good seal 3 hours, whenever rocked once in the water-bath process at a distance from 15 minutes.After water-bath finishes, vial naturally cooled to put into 4 ℃ of refrigerators after the room temperature and preserve.
Select and infect the ciliophoran ill lefteye flounder of shield and put into Dissecting tray, with its lesions position of aseptic seawater flushing 3 times.Put into the glass culture dish of an aseptic diameter 9cm then with the about 5g lesion tissue of Dissecting scissors clip; Aseptic technique shreds this lesion tissue; And the about 100ml via hole diameter of adding is the sterilization seawater behind the 0.22 μ m cellulose mixture membrane filtration in petridish; After rocking petridish gently homogeneous microstructure being scattered, cover the petridish lid and put into 20 ℃ of constant incubators and left standstill 4 hours.Petridish after will leaving standstill then is placed under the anatomical lens; Draw free shield ciliate polypide in the some amount liquid with aseptic glue head dropper; Be transferred in another new glass culture dish that the fresh shield ciliate live body substratum of 30ml is housed behind high-temperature sterilization; Cover the petridish lid and rock gently for several times, put into 20 ℃ of constant incubators and cultivate.
Cultivate after 20 hours; Taking out petridish rocks for several times gently; Nutrient solution with aseptic glue head dropper absorption 10ml is transferred in another new glass culture dish that 30ml shield ciliate live body substratum is housed behind high-temperature sterilization then; Cover after the petridish lid rocks for several times gently, continue to put into 20 ℃ of constant incubators and cultivate.Remaining nutrient solution is used for the polypide counting.After this every this step that repeated at a distance from 20 hours finishes until experiment.Experimental session, preparation in time replenishes fresh shield ciliate live body substratum.
The method of polypide counting is in the culturing process: remaining nutrient solution exhausts with aseptic glue head dropper fully after will drawing 10ml, splashes in the funnel that filter paper is housed and filters.During filtration, nutrient solution is dripped in the filter paper centre bottom as much as possible, make filtering cross-sectional area as much as possible little with glue head dropper.After filtering completion, use 10ml via hole diameter is the filtration position that the sterilization seawater behind the 0.22 μ m cellulose mixture membrane filtration oppositely drips filter wash paper, drips a washing lotion and is collected in the centrifuge tube of a sterilization, and the polypide that obtains 10ml is collected liquid.The Lu Geshi iodine liquid of adding 1% in collecting liquid (Lugol ' s solution) fixing; Fully absorption 0.1ml collects liquid and puts into plankton counting chamber in microscopically counting (see figure 2) after the vibration evenly, and the measurement of counting volume is a standard with final 10ml polypide collection liquid.
This external shield ciliate live body culture experiment has been carried out 920 hours (about 38 days) altogether, and polypide is counted per 60 hours once, and count results is seen table 2.
The count results that table 2 shield ciliate live body is cultivated
Through repeatedly experiment, prove that the present invention can provide indispensable technical guarantee for carrying out fundamental researches such as the ciliophoran life history of shield and pathogenesis in the long-term cultivation of external realization shield ciliate live body.
Claims (5)
1. the preparation method of a seawater shield ciliate live body substratum is characterized in that its preparation process is:
A. the cerebral tissue of clip healthy Paralichthys olivaceus fish or turbot and muscle tissue, with both according to 1: 1～3: 1 mixed of mass ratio after, the sterilization seawater volume that adds behind membrane filtration is V
1, be prepared into muddy flesh with tissue refiner;
B. the sterilization seawater volume that in muddy flesh, adds behind membrane filtration is V
2The mass volume ratio of the seawater TV of muddy flesh and twice adding is 1: 45～1: 55; Vibration evenly is placed in sealing, the aseptic Glass Containers; 65～70 ℃ of water-baths are prepared into shield ciliate live body substratum after 2～3 hours, the shield ciliate live body substratum for preparing is placed on prolonged preservation under 4 ℃ of conditions.
2. according to the said method of claim 1, it is characterized in that the described filter membrane that is used for filtering sea is the cellulose mixture filter membrane of aperture 0.22 μ m.
3. according to a kind of seawater shield ciliate live body substratum of claim 1 or the preparation of 2 said methods.
4. ciliophoran supravital cultural method of seawater shield is characterized in that its method steps is:
A. clip infects the cultivated animals part lesion tissue of shield balantidiasis, after aseptic technique shreds in glass culture dish, splashes into the sterilization seawater behind membrane filtration of 15～20 times of volumes, places in 18～20 ℃ of constant incubators and leaves standstill 2～4 hours;
B. under anatomical lens, draw in the petridish after leaving standstill free polypide in the liquid, splash in the glass culture dish that the shield ciliate substratum that 15～30ml prepares is housed, place 18～20 ℃ of constant temperature culture with aseptic glue head dropper;
C. every at a distance from 20～24 hours; Drawing 5～10ml nutrient solution is transferred in another glass culture dish that fresh shield ciliate substratum of 15～30mL is housed; Rock gently mix for several times after; Be placed on 18～20 ℃ of constant incubators again and continue to cultivate, realize the purpose of living worm body long-term cultivation with this.
5. according to the said method of claim 4, it is characterized in that the described filter membrane that is used for filtering sea is the cellulose mixture filter membrane of aperture 0.22 μ m.
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| CN102907377A (en) * | 2012-10-25 | 2013-02-06 | 中国水产科学研究院黄海水产研究所 | Preparation and cultivation methods of fluid nutrient medium of stichopus japonicus in-vivo parasitic deuterostomia |
| CN102928274A (en) * | 2012-10-25 | 2013-02-13 | 中国水产科学研究院黄海水产研究所 | Method for depilating cilium on body surface of seawater infusorians |
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| CN102928274A (en) * | 2012-10-25 | 2013-02-13 | 中国水产科学研究院黄海水产研究所 | Method for depilating cilium on body surface of seawater infusorians |
| CN102907377B (en) * | 2012-10-25 | 2013-12-18 | 中国水产科学研究院黄海水产研究所 | Preparation and cultivation methods of fluid nutrient medium of stichopus japonicus in-vivo parasitic deuterostomia |
| CN102928274B (en) * | 2012-10-25 | 2014-09-17 | 中国水产科学研究院黄海水产研究所 | Method for depilating cilium on body surface of seawater infusorians |
| CN105132287A (en) * | 2015-10-19 | 2015-12-09 | 吉林省水产科学研究院 | Method for culturing myxobolus in vitro |
| CN105675882A (en) * | 2016-02-04 | 2016-06-15 | 中国水产科学研究院黄海水产研究所 | Method for evaluating inhibition of marine fish mucus lectin for paralembus digitiformis |
| CN108056320A (en) * | 2017-11-29 | 2018-05-22 | 天津农学院 | A kind of culture medium and protecting method of salt Bruchus rufimanus conservation |
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| CN108633843A (en) * | 2018-04-18 | 2018-10-12 | 中国科学院水生生物研究所 | A kind of culture medium, preparation method and the extracorporeal culturing method of grass carp bowel bag worm applied to grass carp bowel bag worm in vitro culture |
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