CN102492623B - 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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- CN102492623B CN102492623B CN2011104141236A CN201110414123A CN102492623B CN 102492623 B CN102492623 B CN 102492623B CN 2011104141236 A CN2011104141236 A CN 2011104141236A CN 201110414123 A CN201110414123 A CN 201110414123A CN 102492623 B CN102492623 B CN 102492623B
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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 particularly and a kind ofly realize the method for seawater shield ciliate live body long-term cultivation and the preparation of substratum thereof aquatic animal is external.
Background technology:
The shield ciliate is acknowledged as the main class 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, shield ciliate battalion free living, the small organic granular (bacterium, protozoon, little algae, organic debris etc.) that suspends in the water is for eating.Yet, in the situation that high-density breeding, the shield ciliate can be changed parasitics into usually, take the cell or tissue chips of echinoderms, shellfish, crustaceans and fish as food, and in host tissue Fast Growth, propagation, and then cause the degree of depth injury of tissue, and form serious focus, finally cause host's organ failure and death.
due to the sound development of serious threat of shield ciliate to sea farming, Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science is successively to China prawn, the shield balantidiasis of fish and sea cucumber has carried out comprehensively, the sick learning of the disease popularity of system studied, but find shield ciliate infection of Chinese prawn, lefteye flounder, turbot, the river Puffer, the diversified economy animals such as sea cucumber, and deeply carried out the ciliophoran morphological observation (Chinese aquatic science of shield, 12 the 5th phases of volume in 2005) and infect pathological research (the hydrobiont journals of these animals, 31 the 5th phases of volume in 20075).Simultaneously, carried out Chinese herbal medicine screening (national inventing patent ZL200510044755.2) and prevented and treated Study on Process (national inventing patent ZL200510044757.1) for the shield ciliate that separates.These researchs concentrate on aspects such as the histopathology of shield balantidiasis and controls, deeply do not carry out the research of the aspects such as shield ciliophoran life history, pathogenesis.
For the control shield balantidiasis of science, except strengthening its Study of Prevention Technology, it is also very necessary carrying out the fundamental researches such as the polypide life history and pathogenesis, 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 the aspects such as epidemiology, etiology, taxonomy, Fungicide screning both at home and abroad, the nutritional needs that only has the 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 is to provide a kind of preparation of seawater shield ciliate supravital culture medium and utilizes this substratum to carry out the method that seawater shield ciliate supravital is cultivated, the substratum preparation method is simple, can realize shield ciliate live body in the external long-term cultivation of aquatic animal, for the 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, comprise the following steps: the cerebral tissue of clip healthy Paralichthys olivaceus fish or turbot and muscle tissue, after both were mixed according to the mass ratio ratio of 1: 1～3: 1, adding the sterilization seawater volume after membrane filtration was V
1, be prepared into muddy flesh with tissue refiner; Adding the sterilization seawater volume after membrane filtration in muddy flesh is V
2The mass volume ratio of muddy flesh and the seawater cumulative volume that adds for twice is 1: 45～1: 55, vibration evenly is placed in sealing, 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, described filter membrane 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 described method preparation.
A kind of method that the present invention also provides seawater shield ciliate supravital to cultivate.
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 after membrane filtration of 15～20 times of volumes, be placed in 18～20 ℃ of constant incubators interior standing 2～4 hours; Draw in culture dish after standing free polypide in liquid with aseptic glue head dropper under anatomical lens, splash in the glass culture dish that the shield ciliate substratum that 15～30ml prepares is housed, be placed in 18～20 ℃ of constant temperature culture; Every 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, being placed on 18～20 ℃ of constant incubators continues to cultivate again, realize the purpose of living worm body long-term cultivation with this, in former culture dish, remaining nutrient solution is used for the polypide counting.
Further, described filter membrane for filtering sea is preferably the cellulose mixture filter membrane of aperture 0.22 μ m.
The beneficial effect that the present invention is compared with the prior art:
1. the making method of shield ciliate live body substratum is simple, need not to add the Various Complex composition, facilitates in the laboratory in a large number, prepares fast.
2. cultural method is simple, and required plant and instrument is few, all can cultivate under general laboratory condition.
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:
By reference to the accompanying drawings technical scheme of the present invention is elaborated below by embodiment:
Embodiment 1
In June, 2011, the turbot generation shield balantidiasis of Yantai, Shandong turbot cultivation factory cultivation carries out pathogen separation and cultivation with taking back Qingdao Huanghai Sea aquatic products institute laboratory after ill turbot packing oxygenation.
At first prepare shield ciliate live body substratum in the laboratory.Select 20 healthy turbot of taking back from cultivating factory, its cerebral tissue of clip is the about 1g of 1g and its back peeling muscle approximately, and both mixing are packed in a large centrifuge tube, adds 20ml sterilization seawater with tissue refiner's homogenate after 10 minutes, add again 70ml sterilization seawater fully to vibrate, mix.The 90ml mixture that mixes is transferred in 200ml vial with sealing cover, was placed in 65 ℃ of water-baths water-bath after good seal 2 hours, during jiggled once every 15 minutes.After water-bath is complete, after being naturally cooled to room temperature, vial puts into 4 ℃ of preservations.
Select the ciliophoran ill turbot of severe infections shield and put into Dissecting tray, with its lesions position of aseptic seawater flushing 3 times.Then with the Dissecting scissors clip approximately the 5g lesion tissue put into the glass culture dish of an aseptic diameter 9cm, aseptic technique shreds this lesion tissue, and to add about 75ml via hole diameter in the culture dish be sterilization seawater after 0.22 μ m cellulose mixture membrane filtration, after rocking gently culture dish homogeneous microstructure being scattered, cover the culture dish lid and put into 18 ℃ of constant incubators standing 2 hours.Then the culture dish after standing is placed under anatomical lens, draw shield ciliate polypide free in 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 after high-temperature sterilization, cover the culture dish lid and rock gently for several times, put into 18 ℃ of constant incubators and cultivate.
Cultivate after 24 hours, taking out culture dish rocks for several times gently, then the 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 after high-temperature sterilization, after covering the culture dish lid and rocking gently for several times, continue to put into 18 ℃ of constant incubators and cultivate.Remaining nutrient solution is used for the polypide counting.After this repeated this step every 24 hours, until experiment finishes.Experimental session in time prepares and replenishes fresh shield ciliate live body substratum.
In culturing process, the method for polypide counting is: after drawing 5ml, remaining nutrient solution exhausts fully with aseptic glue head dropper, splashes in the funnel that filter paper is housed and filters.During filtration, with glue head dropper, nutrient solution is dripped as much as possible in the filter paper centre bottom, make the cross-sectional area of filtration as much as possible little.After filtration is completed, be the filtration position that sterilization seawater after 0.22 μ m cellulose mixture membrane filtration oppositely drips filter wash paper with the 10ml via hole diameter, drip a washing lotion and be collected in the centrifuge tube of a sterilization, the polypide that obtains 10ml is collected liquid.Add 1% Lu Geshi iodine liquid (Lugol ' s solution) fixing in liquid to collecting, fully after vibration evenly, absorption 0.1ml collects liquid and puts into plankton counting chamber at the microscopically counting, and the measurement of counting volume is collected liquid as standard take final 10ml polypide.
This external shield ciliate live body culture experiment has been carried out 37 days altogether, and once count results saw Table 1 to the polypide counting in every 3 days.In experimentation, the shield ciliate polypide in nutrient solution is remaining vigorous vitality and reproductivity (seeing Fig. 1).
The count results that table 1 shield ciliate live body is cultivated
Embodiment 2
In August, 2011, the shield balantidiasis also occurs in the lefteye flounder of Jiaonan City, Qingdao lefteye flounder cultivation factory cultivation, carries out pathogen separation and cultivation with taking back Qingdao Huanghai Sea aquatic products institute laboratory after ill lefteye flounder packing oxygenation.
At first prepare shield ciliate live body substratum in the laboratory.Select 30 healthy Paralichthys olivaceus of taking back from cultivating factory, its cerebral tissue of clip is the about 0.5g of 1.5g and its back peeling muscle approximately, and both mixing are packed in a large centrifuge tube, adds 20ml sterilization seawater with tissue refiner's homogenate after 10 minutes, add again 90ml sterilization seawater fully to vibrate, mix.The 110ml mixture that mixes is transferred in 200ml vial with sealing cover, was placed in 70 ℃ of water-baths water-bath after good seal 3 hours, rocked once every 15 minutes in the water-bath process.After water-bath is complete, after being naturally cooled to room temperature, vial puts into 4 ℃ of Refrigerator stores.
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.Then with the Dissecting scissors clip approximately the 5g lesion tissue put into the glass culture dish of an aseptic diameter 9cm, aseptic technique shreds this lesion tissue, and to add about 100ml via hole diameter in the culture dish be sterilization seawater after 0.22 μ m cellulose mixture membrane filtration, after rocking gently culture dish homogeneous microstructure being scattered, cover the culture dish lid and put into 20 ℃ of constant incubators standing 4 hours.Then the culture dish after standing is placed under anatomical lens, draw shield ciliate polypide free in 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 after high-temperature sterilization, cover the culture dish lid and rock gently for several times, put into 20 ℃ of constant incubators and cultivate.
Cultivate after 20 hours, taking out culture dish rocks for several times gently, then the 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 after high-temperature sterilization, after covering the culture dish lid and rocking gently for several times, continue to put into 20 ℃ of constant incubators and cultivate.Remaining nutrient solution is used for the polypide counting.After this repeated this step every 20 hours, until experiment finishes.Experimental session, in time preparation replenishes fresh shield ciliate live body substratum.
In culturing process, the method for polypide counting is: after drawing 10ml, remaining nutrient solution exhausts fully with aseptic glue head dropper, splashes in the funnel that filter paper is housed and filters.During filtration, with glue head dropper, nutrient solution is dripped as much as possible in the filter paper centre bottom, make the cross-sectional area of filtration as much as possible little.After filtration is completed, be the filtration position that sterilization seawater after 0.22 μ m cellulose mixture membrane filtration oppositely drips filter wash paper with the 10ml via hole diameter, drip a washing lotion and be collected in the centrifuge tube of a sterilization, the polypide that obtains 10ml is collected liquid.Add 1% Lu Geshi iodine liquid (Lugol ' s solution) fixing in liquid to collecting, fully after vibration evenly, absorption 0.1ml collects liquid and puts into plankton counting chamber at microscopically counting (seeing Fig. 2), and the measurement of counting volume is collected liquid as standard take final 10ml polypide.
This external shield ciliate live body culture experiment has been carried out 920 hours (approximately 38 days) altogether, and once count results saw Table 2 to the polypide counting in every 60 hours.
The count results that table 2 shield ciliate live body is cultivated
By many experiments, prove that the present invention can in the external long-term cultivation that realizes shield ciliate live body, provide indispensable technical guarantee for carrying out the fundamental researches such as the ciliophoran life history of shield and pathogenesis.
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, after both were mixed according to the ratio of mass ratio 1:1～3:1, adding the sterilization seawater volume after membrane filtration was V
1, be prepared into muddy flesh with tissue refiner;
B. adding the sterilization seawater volume after membrane filtration in muddy flesh is V
2The mass volume ratio of muddy flesh and the seawater cumulative volume that adds for twice is 1:45～1:55, vibration evenly is placed in sealing, aseptic Glass Containers, 65～70 ℃ of water-baths are prepared into seawater shield ciliate live body substratum after 2～3 hours, the seawater shield ciliate live body substratum for preparing is placed on prolonged preservation under 4 ℃ of conditions.
2. method according to claim 1, is characterized in that described filter membrane for filtering sea is the cellulose mixture filter membrane of aperture 0.22 μ m.
3. a kind of seawater shield ciliate live body substratum of described method preparation according to claim 1 and 2.
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 after membrane filtration of 15～20 times of volumes, is placed in 18～20 ℃ of constant incubators interior standing 2～4 hours;
B. draw in culture dish after standing free polypide in liquid with aseptic glue head dropper under anatomical lens, splash in the glass culture dish that the seawater shield ciliate live body substratum claimed in claim 3 that 15～30ml prepares is housed, be placed in 18～20 ℃ of constant temperature culture;
C. every 20～24 hours, drawing 5～10ml nutrient solution is transferred in another glass culture dish that fresh seawater shield ciliate live body substratum claimed in claim 3 of 15～30mL is housed, rock gently mix for several times after, be placed on again 18～20 ℃ of constant incubators and continue to cultivate, realize the purpose of living worm body long-term cultivation with this.
5. method according to claim 4, is characterized in that described filter membrane for filtering sea is the cellulose mixture filter membrane of aperture 0.22 μ m.
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| 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 |
| CN108056320B (en) * | 2017-11-29 | 2021-09-14 | 天津农学院 | Culture medium and method for protecting seeds of broad bean worms with salt |
| CN108633843B (en) * | 2018-04-18 | 2020-10-27 | 中国科学院水生生物研究所 | In-vitro culture method of grass carp intestinal bagworms and preparation method of used culture medium |
| CN109042626B (en) * | 2018-08-10 | 2023-07-11 | 大连海洋大学 | Cryopreservation and recovery method of sea tail filarial separated from fugu rubripes |
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