CN112852636B - Collection and culture method and application of sessile ciliates - Google Patents
Collection and culture method and application of sessile ciliates Download PDFInfo
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- CN112852636B CN112852636B CN202110207739.XA CN202110207739A CN112852636B CN 112852636 B CN112852636 B CN 112852636B CN 202110207739 A CN202110207739 A CN 202110207739A CN 112852636 B CN112852636 B CN 112852636B
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- 241000223782 Ciliophora Species 0.000 title claims abstract description 60
- 238000012136 culture method Methods 0.000 title claims abstract description 15
- 241000514719 Mactra quadrangularis Species 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 37
- 239000013535 sea water Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 239000011521 glass Substances 0.000 claims description 28
- 229920000742 Cotton Polymers 0.000 claims description 10
- 239000006059 cover glass Substances 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 229920006395 saturated elastomer Polymers 0.000 claims description 4
- 238000007877 drug screening Methods 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims 1
- 238000005406 washing Methods 0.000 abstract 1
- 230000000384 rearing effect Effects 0.000 description 8
- 238000009395 breeding Methods 0.000 description 6
- 230000001488 breeding effect Effects 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 239000013589 supplement Substances 0.000 description 5
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- 241000238557 Decapoda Species 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000003097 mucus Anatomy 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 241000195493 Cryptophyta Species 0.000 description 2
- 241000514745 Mactra Species 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000007688 edging Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
- 241000250967 Branchia Species 0.000 description 1
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- 241000254173 Coleoptera Species 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 241000283966 Pholidota <mammal> Species 0.000 description 1
- 241000415791 Zoothamnium Species 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- -1 where the retrieved slides are often attached with protozoa Species 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/10—Protozoa; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract
The invention belongs to the technical field of biology, and discloses a collection and culture method of sessile ciliates, which comprises the following steps: culturing Mactra veneriformis in seawater with sessile ciliates, and taking out the Mactra veneriformis when the sessile ciliates attach to the shell edge of the Mactra veneriformis and inlay the edges in black. Taking out collected Mactra veneriformis shell edge black edges, attaching the collected Mactra veneriformis shell edge black edges to a culture device, and culturing while keeping the humidity of the black edges. The method provided by the invention solves the problem that the structure of the micro-community of the sessile ciliates changes after the sessile ciliates leave the original water body, so that observation within 24 hours is not needed; the washing and water changing are convenient; the fixed ciliates growing on the mactra veneriformis edged grow at intervals in order, cannot be piled, and are convenient to observe.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a collection and culture method and application of sessile ciliates.
Background
The sessile ciliate disease is a parasitic disease of shrimps and crabs, and the pathogeny mainly comprises a polypide (Zoothamnium), a coccid (Vortella) and the like in the sessile ciliate disease, which causes little harm to the aquaculture industry. There are many researchers trying to collect sessile ciliates and culture them in laboratory conditions. The existing method for sampling sessile ciliates comprises living body sampling (taking branchia filaments or pangolin scales of shrimps and scales of fishes) and suspension sampling (PFU method, glass slide method and bolting silk concentration method). The living body sampling sample is too thick and is not convenient for observation under a microscope, the water body collected by suspension sampling is changed into unstable environment from stable after artificial treatment, the density of microorganisms in the water body is rapidly increased, the structure of a micro community is changed, and if the micro community is not separated as soon as possible, the ciliates are difficult to separate even if needed species exist. It is necessary to complete species (living body) identification within 24 hours. If the subsequent culture is needed, the swimmers are needed to be collected to culture the adult, the time consumption is long, and the operation requirement is high.
Mactra quadrangularis (Mactra quadrangularis) is a demersal economic shellfish which lives in tidal zones of silt beaches and north and south China and is distributed on the coast, rich in resources, high in nutritional value, tender in meat, delicious in taste and low in price. Mactra veneriformis belongs to genus Mactra of family Mactra of order Lamelliformes of order Lamellibranchiales. Its shell is slightly in the shape of four corners, two shells are symmetrical, and two sides are extremely expanded. The shell is white or yellow, the top of the shell is usually denuded, and the ventral edge is provided with a black edge band. This shell side (shell top web) black band has not been studied.
Until now, no reports of the mactra veneriformis infecting sessile ciliate insects under natural conditions exist. Also, no ciliate-infected individuals were found in mactra veneriformis purchased in the experiment. In the experiment, the mactra veneriformis shell edge black edging is mainly polypide and beetle.
In taxonomic research, various attempts have been made before for artificial collection and culture of sessile ciliates, mainly by hanging slides, where the retrieved slides are often attached with protozoa, algae and other organisms that interfere with observation, and if washed with sterilized seawater, the slides are easily washed away during operation. Therefore, the swimmer can only be separated by a straw for pure culture, the culture is carried out in a culture dish, water needs to be changed every two to three days, bait needs to be fed, and the operation difficulty is high.
The aquaculture directly takes the pathological materials, however, the pathological materials are too thick to be observed under a microscope, and the swimming body is generally cultured in a culture dish.
Therefore, providing a method for collecting and culturing sessile ciliates, which is convenient for observation, simple and convenient for operation and saves time, is a problem that needs to be solved by the technical personnel in the field.
Disclosure of Invention
The invention provides a method for collecting, culturing and observing sessile ciliates by using mactra veneriformis shell edge rims, and aims to solve the problems that the conventional sessile ciliates collecting and culturing are long in time consumption and difficult to observe.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for collecting sessile ciliates comprises culturing Mactra veneriformis in seawater containing sessile ciliates, and taking out the Mactra veneriformis when the sessile ciliates attach to the shell edge of the Mactra veneriformis and insert the shell edge black.
Preferably, the period of said culturing is one to two weeks.
Preferably, the mactra veneriformis is put into a seawater culture pond or a seawater test area needing sampling for culture.
A culture method of sessile ciliates comprises the following steps: taking out the black rims of the mactra veneriformis shells collected by the method of claim 1, placing the black rims in a culture device, and maintaining the humidity of the black rims for culture.
Preferably, the black border is attached to a slide and placed in the culture device.
Preferably, the slide is a concave slide.
Preferably, sterile seawater is dripped on the black inlaid edge, and the black inlaid edge is subjected to wet culture in the culture device.
Preferably, the moisture preservation method is to put absorbent cotton which is full of moisture into the culture device and supplement the moisture of the glass slide and the moisture of the absorbent cotton in time.
Preferably, the sessile ciliates in the black border are observed during the culture process: finding the fixed position of ciliates under a low power lens, covering a cover glass, and observing under a high power lens.
The invention also provides an application of the culture method of sessile ciliates in drug screening, wherein drugs are contacted with the sessile ciliates in the black inlaid edge, and the effect of the drugs on the sessile ciliates is observed and evaluated.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts mactra veneriformis shell edge rims to collect, culture and observe sessile ciliates. A mucus is formed between shell edge bands of Mactra veneriformis and fixed ciliates, and has protective and nutritional effects, and the fishnet shape of the bands, the fixed ciliates, algae, bacteria and organic matter residues form a small and stable ecological system. The collection and culture method solves the problem of structural change of the micro-community after the sessile ciliates leave the original water body, so that observation within 24 hours is not needed.
The invention solves the problem of difficulty in changing water without fixed flushing, the band is picked off by tweezers and is stuck on the glass slide, and the left end and the right end of the glass slide are fixed on the glass slide after the glass slide is slightly dried due to mucus. Only the middle position is dripped with seawater, and the operations of flushing, water changing and various operations can be carried out.
The invention solves the problem of the microscopic visual field observation caused by the thickness of the pathological material taken by the organism. Although the invention takes the material from the organism directly, the mucilage edging is extremely thin after being attached to the glass slide, is transparent, is suitable for observation under a microscope, and is difficult to have a biological source material to surpass.
The fixed ciliates growing on the selvedges of the invention grow orderly at intervals, do not bundle and are convenient to observe.
Drawings
FIG. 1 is a polypide enveloped by a mesh structure;
FIG. 2 shows the growth of the polypide on the first day of slide culture;
FIG. 3 shows the growth of the polypide on the fourth day of slide culture.
Detailed Description
The invention provides a method for collecting sessile ciliates.
In the collecting method, the mactra veneriformis grows in an intertidal zone and has wide tolerance range on salinity. The collection is preferably to place healthy mactra veneriformis not infected with sessile ciliates into a collection net, place the collecting net into a seawater culture pond or a certain seawater test area to be sampled, and lift the collecting net after one to two weeks. Or putting healthy Mactra veneriformis not infected with sessile ciliates into a temporary rearing box for temporary rearing, collecting the original pond water of the rearing pond, putting the original pond water into the temporary rearing box, and rearing for one week. The source of the Mactra veneriformis is not particularly limited, and commercially available or collected Mactra veneriformis can be adopted.
The invention also provides a culture method of sessile ciliates, which comprises the following steps: taking out collected Mactra veneriformis shell edge black edges, attaching the collected Mactra veneriformis shell edge black edges to a culture device, and culturing while keeping the humidity of the black edges.
In the culture method, the black edged clam shells which are successfully inoculated have mucus, are tough and are not easy to break, can be pulled into a long section which is close to 3 cm, the long section is carefully pasted on a glass slide, and 1 drop of sterile seawater is dripped at the central part of the long section for infiltration. And (4) temporarily breeding mactra veneriformis which is too late to observe in the temporary breeding pond by using the original pond water or the sea water for breeding. The glass slide is preferably a concave glass slide, and the preparation method of the sterilized seawater comprises the steps of boiling and cooling the seawater for cultivation.
In the culture method of the present invention, most of the seawater on the slide glass is sucked with a water-absorbent paper before observation, but the seawater is kept in a wet state. The sessile ciliates growing on the black inlaid edge are orderly arranged at a certain distance from each other, so that the observation is very convenient.
In the culture method, the culture device is preferably a culture dish, and the moisturizing method is to place absorbent cotton saturated with water into the culture dish to maintain the humidity, observe the water evaporation condition and supplement the water of the glass slide and the absorbent cotton in time. 90% of sterilized seawater is dripped into the glass slide to supplement water, and the culture on the glass slide can be in a water-full state after the water is supplemented once every two days generally. The preparation method of the 90% disinfection seawater comprises the following steps: preparing 90% seawater from distilled water and 9 parts seawater for culturing, boiling, and cooling.
In the culture process, the fixed ciliates in the black inlaid edges are observed: finding the fixed position of ciliates under a low power lens, covering a cover glass, and observing under a high power lens.
The invention also provides application of the collection and culture method of sessile ciliates in drug screening, wherein drugs are contacted with the sessile ciliates in the black inlaid edge, and the effect of the drugs on the sessile ciliates is observed and evaluated.
In the present invention, the drug test is carried out by preparing a drug solution with sterilized seawater.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Healthy Mactra veneriformis purchased, and microscopic examination does not infect sessile ciliates. Putting the marine culture pond into a collection net, putting the marine culture pond or a certain seawater test area to be sampled, and lifting the marine culture pond or the certain seawater test area after one week to two weeks;
cutting Mactra veneriformis, removing black rim with tweezers, carefully sticking on concave glass slide, and dripping 1 drop of sterilized seawater on the central part of long section for infiltration. And (4) temporarily breeding mactra veneriformis which is too late to observe in the temporary breeding pond by using the original pond water or the sea water for breeding.
The seawater on the slide was mostly sucked up with a water-absorbent paper before observation, but kept in its wet state. The observation with the high power lens needs to be covered with a cover glass, the position where the ciliates are fixed needs to be found under the low power lens, the position is marked on the glass slide, and the glass slide is covered with the cover glass which is as small as possible. Microscopic observations are shown in FIG. 1, where the polypide is surrounded by a network.
A few clusters of absorbent cotton saturated with water were placed in the petri dish to maintain humidity, and the slide glass was placed therein and the petri dish was covered. Observing the water evaporation condition, supplementing the water of the glass slide and the water of the absorbent cotton in time, dropwise adding 90% of sterilized seawater into the glass slide to supplement the water, and supplementing the water once every two days to ensure that the culture on the glass slide is in a state of full water. The microscopic observation results of the first day of cultivation are shown in FIG. 2, with 12 branches; microscopic observations on the fourth day of culture were shown in FIG. 3, which increased from 12 branches to 15 branches. The fixed position of the cluster of the polycondensed increases the number of branches, which represents the growth and reproduction process of the polycondensed in the period, and the culture condition is suitable for growth.
Example 2
Purchasing healthy Mactra veneriformis in a natural state, and carrying out microscopic examination on the healthy Mactra veneriformis without infecting sessile ciliates. Putting the seeds into a temporary culture box for temporary culture. Collecting the original pond water of the culture pond, putting the original pond water into a temporary culture box, and culturing for one week;
cutting Mactra veneriformis, removing black rim with tweezers, carefully sticking on concave glass slide, and dripping 1 drop of sterilized seawater on the central part of long section for infiltration. Putting mactra veneriformis which cannot be observed in time into a temporary rearing pond, and temporarily rearing the mactra veneriformis in the original pond water or the rearing seawater;
the seawater on the slide was mostly sucked up with a water-absorbent paper before observation, but kept in its wet state. Covering a cover glass when observing with a high power lens, paying attention to the fact that the position where ciliates are fixed needs to be found under a low power lens, marking the position on a glass slide, and covering the position with the cover glass which is as small as possible;
a few clusters of absorbent cotton saturated with water were placed in the petri dish to maintain humidity, and the slide glass was placed therein and the petri dish was covered. Observing the water evaporation condition, supplementing the water of the glass slide and the water of the absorbent cotton in time, dropwise adding 90% of sterilized seawater into the glass slide to supplement the water, and supplementing the water once every two days to ensure that the culture on the glass slide is in a state of full water.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A culture method of sessile ciliates is characterized by comprising the following steps: culturing Mactra veneriformis in seawater with sessile ciliates, taking out the Mactra veneriformis after the sessile ciliates are attached to black edged edges of the shell of the Mactra veneriformis, taking out the collected black edged edges of the shell of the Mactra veneriformis, putting the collected black edged edges of the shell of the Mactra veneriformis in a culture device, and maintaining the humidity of the black edged edges for culturing;
and dripping sterilized seawater on the black inlaid edge, and performing wet culture in the culture device.
2. The method of claim 1, wherein the black border is affixed to a slide and placed in a culture device.
3. The method of claim 2, wherein the slide is a concave slide.
4. The ciliate sesami culture method of claim 1, wherein the moisture retention method is to put absorbent cotton saturated with water into the culture device to replenish the water in the slide glass and the absorbent cotton in time.
5. The sessile ciliate culture method of claim 1, wherein the sessile ciliates in the black border are observed during the culture: finding the fixed position of ciliates under a low power lens, covering a cover glass, and observing under a high power lens.
6. A method for collecting sessile ciliates is characterized in that mactra veneriformis is put into seawater with sessile ciliates for culture, and the mactra veneriformis is taken out after the sessile ciliates are attached to black inlaid edges of shell edges of the mactra veneriformis.
7. The method of claim 6, wherein the culturing is for a period of one to two weeks.
8. The method for collecting sessile ciliates according to claim 6, wherein the mactra veneriformis is cultured in a seawater culture pond or a seawater test area to be sampled.
9. Use of the method according to any one of claims 1 to 8 in drug screening, wherein the drug is contacted with sessile ciliates in the black border, and the effect of the drug on the sessile ciliates is observed and evaluated.
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| 常见原生动物纤毛虫的采集、分离与培养;齐桂兰等;《生物学教学》;20080708(第07期);第38-39页 * |
| 池塘养殖贝类暴发性死亡原因分析;何伟贤等;《水产养殖》;20040830(第04期);第27-28页 * |
| 蛤类寄生性盾纤类纤毛虫的初步研究;王兰萍等;《水利渔业》;20061231;第26卷(第5期);第114页左栏最后1段-第115页左栏最后1段，表1-2，第114页左栏倒数第2段 * |
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