CN101485300A - Method for cultivating Acrobeloides nanus - Google Patents
Method for cultivating Acrobeloides nanus Download PDFInfo
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- CN101485300A CN101485300A CNA2008100101613A CN200810010161A CN101485300A CN 101485300 A CN101485300 A CN 101485300A CN A2008100101613 A CNA2008100101613 A CN A2008100101613A CN 200810010161 A CN200810010161 A CN 200810010161A CN 101485300 A CN101485300 A CN 101485300A
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Abstract
The invention relates to organism cultivation, in particular to a method for cultivating acrobeloides nanus, which comprises the following steps: using an Escherichia coli (E.coli) strain as a growth germ; inoculating the Escherichia coli strain into a liquid beef extract peptone culture medium; performing shake cultivation for 23 to 25 hours at a temperature of between 36.5 and 37.5 DEG C on a shaking table with a speed of between 165 and 175 times per minute to obtain a bacterial solution; adding the bacterial solution to a nematode culture medium; and selecting the aseptically-sterilized acrobeloides nanus which is put into an incubator with constant temperature and constant humidity and is cultivated for 5 to 11 days under the condition of which the humidity is between 50 and 60 percent and the temperature is between 20 and 30 DEG C so as to obtain the cultivated nematode. The method has the advantages of simple operation, low cost and easy implementation; and the acrobeloides nanus obtained by the method can be used as an effective indicator organism for the ecological toxicological diagnosis of environmental pollution so as to solve the problem of biological detection for the environmental pollution.
Description
Technical field
The present invention relates to biological culture, be specially a kind of cultural method of intending beautiful prominent nematode.
Background technology
Nematode is an invertebrate the abundantest in the soil, occupies critical role in soil ecosystem detritus food web.Nematode extensively is present in the soil as the important member in the food chain, because of its form particularity, food selectivity, isolation identification simple relatively, and can make characteristics such as reaction more rapidly to the various variations of environment, can be with the model organism of nematode, for the environmental pollution evaluation provides valuable information as the research of soil pollution toxicological effect.
Intend beautiful prominent nematode (Acrobeloides nanus) heavy metal pollution in soil and the water body is had good biological indicative function, adopt this kind of indoor cultivation nematode to can be used for the biological detection of contaminated environment.Intending beautiful prominent nematode is food bacterium nematode (Laugsch, the M.﹠amp that a kind of apomictic soil is lived; Schierenberg, E.Differences in maternal supply and early development ofclosely related nematode species.International Journal of DevelopmentalBiology, 2004,48:655-662).Intending beautiful prominent nematode becomes about the long 0.5mm of polypide.In the soil of different regions, the world, the chief component that to intend beautiful prominent nematode be nematode group, it has occupied large-scale soil living environment, about 20% (De Goede, R.G.M.﹠amp of the line is busy worm group; Bongers, T.Nematode Communities of Northern Temperate Grassland Ecosystems.Focus, Giessen, Germany.1998).Intending beautiful prominent nematode is food with saprophytic and plant pathogenetic bacteria, by in soil, spread germs and virus indirect act on organic matter decomposition (Ikonen, E.K.Life historystudies of the hermaphrodite nematode Acrobeloides sp.with Pseudomonascepacia as a food source on agar.Nematology, 2001,3:759-766).Intend the cultivation of beautiful prominent nematode and generally adopt adding Escherichia coli OP50 strain bacterial isolates in medium, what the cultivation of this bacterial strain was adopted is the LB medium.But because the successive transfer culture of Escherichia coli OP50 strain bacterial isolates and preservation are difficult, and easily dye assorted bacterium, the LB medium is than other common nutrition bases simultaneously, and cost is higher.Therefore need the bacterial strain of the relatively low and easy cultivation of alternative costs and medium to cultivate and intend beautiful prominent nematode.
Summary of the invention
The object of the present invention is to provide a kind of cultural method of intending beautiful prominent nematode; With the coli strain is the growth bacterium, and inoculated and cultured obtains bacterium liquid, and bacterium liquid joins nematode culture medium, chooses into the beautiful prominent nematode of plan, and constant temperature culture obtains purebred nematode.The present invention is simple to operate, and is with low cost, implements easily, solves the biological detection problem of environmental pollution.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of cultural method of intending beautiful prominent nematode inserts Escherichia coli E.coli bacterial strain on the beef-protein medium of liquid, obtains bacterium liquid in 23～25 hours 36.5～37.5 ℃ of following 165～175 times/minute shaking table shaken cultivation; Then, bacterium liquid is joined nematode culture medium, choose again, place the constant temperature and humidity incubator, under humidity 50～60%, 20～30 ℃ of conditions of temperature, cultivated 5～11 days, can obtain the nematode of being cultivated into the beautiful prominent nematode of sterile aseptic plan.
Described Escherichia coli E.coli bacterial strain is common wild-type e. coli, and bacterial strain belongs to the Gram-negative brevibacterium, 0.5 * 1～3 microns of sizes, and whole body flagellum can move no gemma.
Described bacterium liquid is with coli strain oese picking, inserts on the beef-protein medium of 40～60mL liquid, and inoculate Escherichia coli quantity is 1 * 10 at every turn
7～3 * 10
7Individual, put shaking table into constant temperature function, to regulate 36.5～37.5 ℃ of shaking table temperature, shake number of times and be set to 165～175 times/minute, shaken cultivation 23～25 hours obtains bacterium liquid.
Described liquid beef-protein medium is by weight: beef extract 5g, peptone 10.0g, NaCl 5g, distilled water 1000mL, regulating pH with 2mol/L NaOH at last is 6.5～7.5.
Described nematode culture medium is by weight: 3g NaCl, 17g agar, 2.5g peptone, be dissolved in the 975mL distilled water, and 120～122 ℃ of sterilizations add 5mg/mL cholesterol stock solution 1mL, 1mol/L CaCl respectively after 29.5～30.5 minutes
21mL, 1mol/L MgSO
41mL, pH are 5.9～6.1 1mol/L phosphate buffer 25mL, shake evenly, promptly get nematode culture medium.
The incubation step of the beautiful prominent nematode of described plan is, get the culture dish that clean diameter is 6cm, nematode culture medium 4～6mL is tiled in the culture dish, leaving standstill 8～10 hours, is 1/50 volume ratio with micropipettor by bacterium liquid/medium, and 80～120 μ L pour culture dish into bacterium liquid, left standstill 3～5 minutes, choose into 10～20 of the beautiful prominent nematodes of aseptic plan with transfer needle, evenly be placed on the medium, cover the culture dish lid; Then, culture dish is placed the constant temperature and humidity incubator, cultivated 5～11 days for 50～60%, 20～30 ℃, can obtain the nematode of being cultivated in humidity.
The present invention has following advantage:
1. the present invention intends the indoor cultivation method of beautiful prominent nematode, at first to the cultivation of Escherichia coli E.coli, cultivates in a large number intending beautiful prominent nematode then, is at last the nematode after cultivating is carried out toxicological experiment, determines the biological indicative function of this nematode.The present invention is simple to operate, and is with low cost, implements easily.
2. the present invention adopts general wild-type e. coli bacterial strain for producing bacterium, and strain cultures is common beef-protein medium.
3. intend beautiful prominent nematode itself among the present invention and cultivate easily, particularly incubation time is shorter.
4. the present invention cultivates the beautiful prominent nematode of plan that obtains, and can be used as a kind of effective indicator organism of the ecological toxicity diagnosis of environmental pollution, solves the biological detection problem of environmental pollution.
Embodiment
The present invention serves as the growth bacterium with Escherichia coli E.coli bacterial strain, coli strain is inserted on the beef-protein medium of liquid, obtains bacterium liquid in 23～25 hours 36.5～37.5 ℃ of following 165～175 times/minute shaking table shaken cultivation; Then, bacterium liquid is joined nematode culture medium, choose again, cultivated 5～11 days for 50～60%, 20～30 ℃, can gather in the crops the nematode of being cultivated in humidity into through the beautiful prominent nematode of the plan of aseptic sterilization.Details are as follows for concrete grammar:
1) Escherichia coli obtain: the present invention selects common wild-type e. coli for use, and bacterial strain belongs to the Gram-negative brevibacterium, 0.5 * 1～3 microns of sizes, and whole body flagellum can move no gemma.Coli strain can obtain in general microorganism fungus kind laboratory, also can buy from the market.
2) obtaining of bacterium liquid: the coli strain oese picking with step 1) obtains, insert on the beef-protein medium of 40～60mL liquid, inoculate Escherichia coli quantity is 1 * 10 at every turn
7～3 * 10
7Individual, put shaking table into constant temperature function, regulate 36.5～37.5 ℃ of shaking table temperature, shake number of times and be set to 165～175 times/minute, shaken cultivation 23～25 hours, (e. coli concentration is 1 * 10 in the bacterium liquid to obtain bacterium liquid
8～5 * 10
8Individual/mL).Standby.
Described liquid beef-protein medium is by weight: beef extract 5g, peptone 10.0g, NaCl 5g, distilled water 1000mL, the NaOH adjusting pH with 2mol/L is 6.5～7.5 at last.
3) preparation of nematode culture medium: get 1000mL experiment triangular flask, add 975mL distilled water, 3g NaCl, 17g agar, 2.5g peptone, stir with glass bar, insert in the autoclave, under 120～122 ℃ and condition of normal pressure, sterilize after 29.5～30.5 minutes, add cholesterol stock solution 1mL, the 1mol/L CaCl of 5g/L
21mL, 1mol/L MgSO
41mL, pH are 5.9～6.1 1mol/L phosphate buffer 25mL, shake evenly, promptly get nematode culture medium.
4) intend obtaining and surface sterilization of beautiful prominent nematode: soil nematodes is with the graceful funnel method of improved shellfish separation and Extraction, exactly soil specimen is placed in the little screen tray that is covered with 2 layers of face tissue or gauze, then little screen tray is put into the funnel of filling water, after 24 hours, be connected under the emulsion tube with little culture dish or test tube, unclamp tongs, collect nematode suspension.Under anatomical lens, intend beautiful prominent nematode then, it changed in the centrifuge tube of sterilization, handled 20 minutes with thimerosal according to the morphosis picking of nematode, 3000 rev/mins centrifugal 5 minutes, remove supernatant, clean repeatedly 5 times with sterile water again.Fully shake during each the cleaning, 3000 rev/mins centrifugal 5 minutes, abandoning supernatant.The disinfectant percentage by weight is that 0.1% streptomycin sulphate and percentage by weight are the mixed liquor (Li Huixin of 0.002% cycloheximide, Chen Xiaoyun, Hu Feng. separation and the enrichment culture method of soil food bacterium nematode. Agricultural University Of Nanjing's journal, 2002,25 (2): 71-74).
5) cultivation of the beautiful prominent nematode of plan: get the culture dish that clean diameter is 6cm, nematode culture medium 4～6mL that step 3) is obtained is tiled in the culture dish, left standstill 8～10 hours, 's 1/50 volume ratio with micropipettor by bacterium liquid/medium, with step 2) bacterium liquid 80～120 μ L that obtain pour culture dish into, leave standstill 3～5 minutes, choose 10～20 of the beautiful prominent nematodes of the aseptic plan that obtains into step 4) with transfer needle, evenly be placed on the medium, cover the culture dish lid.The aforesaid operations step is all carried out on aseptic superclean bench.Then culture dish is placed the constant temperature and humidity incubator, cultivated 5～11 days for 20～30 ℃, can obtain the nematode of being cultivated in humidity 50～60%, temperature.
Described beef extract is the commercially available prod.Be with the paste of fresh beef, contain the water-soluble substances of creatine, creatine liver, amino acids, mineral matter class and vitamin through a kind of pale brown look of rejecting fat, digestion, filtering, concentrating and obtaining.
Described peptone is the commercially available prod.Be to mix with fresh ox bone and beef to extract, through digestion, filter, concentrate, atomized drying and a kind of light yellow dried powder that obtains to off-white color.Soluble in water, it is faint yellow that the aqueous solution is.
Described NaCl is that the laboratory is with analyzing pure NaCl.
The preparation of the NaOH of described 2mol/L: get the 100mL beaker, add the 8g laboratory with analyzing pure NaOH, add 50mL distilled water, stir with glass bar, make it abundant dissolving, solution is transferred to the 100mL volumetric flask, clean beaker 4～5 times with 6～8mL distilled water at every turn, cleaning fluid is transferred to volumetric flask, is settled to 100mL at last, shakes evenly.Standby.
Described shaking table is the experiment shaking table with temperature control functional device, as the HZQ-R type shaking table of east connection electronic technology development corporation, Ltd. product.
Described agar is the commercially available prod, is the polysaccharide of obtaining through refining from rhodophytas such as agar, fragrant plants mentioned in ancient texts.Its main component is the sulfuric acid ester of poly galactose.What the commodity of making had is strip, and what have is powdery.The useful properties of agar is that its condensation point and the temperature between the fusing point differ greatly.It just begins fusing in the time of need being heated to 95 ℃ in water, just begin when the solution temperature after the fusing need drop to 40 ℃ to solidify, so it is the best coagulating agent of preparation solid culture medium.
Described cholesterol stock solution preparation: get 1000mL experiment volumetric flask, add 800～900mL ethanol, the 5g cholesterol is dissolved in the ethanol, shake volumetric flask and make the cholesterol dissolving, be settled to 1000mL, shake evenly with ethanol.Standby.
Described 1mol/L CaCl
2Preparation: get 1000mL experiment volumetric flask, adding 800～900mL distilled water, weighing 111g analyzes pure anhydrous CaCl
2, shake volumetric flask and make CaCl
2Dissolving is settled to 1000mL with distilled water, shakes evenly.Standby.
Described 1mol/L MgSO
4Be equipped with: get 1000mL experiment volumetric flask, add 800～900mL distilled water, weighing 120g analyzes pure anhydrous MgSO
4, shake volumetric flask and make MgSO
4Dissolving is settled to 1000mL with distilled water, shakes evenly.Standby.
The preparation of described 1mol/L phosphate buffer: get 1000mL experiment volumetric flask, add 800～900mL distilled water, add 136g and analyze pure KH
2PO
4, shake volumetric flask and make KH
2PO
4Dissolving is settled to 1000mL with distilled water, shakes evenly.Standby.
Described 0.1% streptomycin sulphate and the preparation of 0.002% cycloheximide mixed liquor: get 100mL experiment volumetric flask, add 80～90mL distilled water, add the medical white powder streptomycin sulphate of 10g (being accurate to 0.01g), shake volumetric flask and make the streptomycin sulphate dissolving, be settled to 100mL with distilled water, shake evenly.Getting weight ratio is 10% streptomycin sulphate.Get 100mL experiment volumetric flask, add 80～90mL distilled water, add 0.5g (being accurate to 0.001g) cycloheximide, shake volumetric flask and make the cycloheximide dissolving, be settled to 100mL with distilled water, shake evenly, being mixed with weight ratio is 0.5% cycloheximide solution.Get the 1000mL volumetric flask, add 800～900mL distilled water, adding weight ratio is that 10% streptomycin sulphate solution 10mL, weight ratio are 0.5% cycloheximide solution 4mL, be settled to 1000mL with distilled water, shake evenly, weight ratio is that 0.1% streptomycin sulphate and weight ratio are 0.002% cycloheximide mixed liquor.
Described cycloheximide chemical name is 3-[2-(3,5-dimethyl-2-oxo cyclohexyl)-2-carboxy ethyl] glutaramide, molecular formula C
15H
23NO
4, molecular weight 281.35.
Described micropipettor scope is 100～500 μ L, as 100～500 μ L micropipettors of Qiujing Bio-Chemical Reagent Instrument Co., Ltd., Shanghai's production.
In the incubation step of the beautiful prominent nematode of described plan, put into each preceding operating procedure of incubator and need carry out on aseptic workbench, described aseptic workbench is that the used conventional aseptic superclean bench of Experiment on Microbiology is carried out in the laboratory.
Embodiment 1:
The preparation of bacterium liquid: insert on the beef-protein medium of 50mL liquid with oese picking coli strain, the oese diameter that adopts is 2.5mm, and inoculate Escherichia coli quantity is 2 * 10 at every turn
7Individual, (e. coli concentration is 3 * 10 in the bacterium liquid to obtain bacterium liquid in 24 hours 37 ℃ of following 170 rev/mins of shaking table shaken cultivation
8Individual/mL), standby.Culture medium prescription is: beef extract 5g, peptone 10.0g, NaCl 5g, distilled water 1000ml, regulating pH at last is 7.
Intend the cultivation of beautiful prominent nematode: the culture dish that to get 18 clean diameters be 6cm, the nematode culture medium 5mL that step 3) is obtained is tiled in the culture dish, left standstill 10 hours, it by bacterium liquid/medium 1/50 volume ratio, pour the above-mentioned bacterium liquid that obtains 100 μ L into culture dish, left standstill 5 minutes, in per 6 culture dishes, choose 10,15 and 20 of the beautiful prominent nematodes of the aseptic plan that obtains into step 4) with transfer needle respectively, evenly be placed on the medium, cover the culture dish lid.The aforesaid operations step is all carried out on aseptic workbench.Then culture dish is placed the constant temperature and humidity incubator, cultivated 11 days, can obtain the nematode of being cultivated humidity 50%, 20 ℃.
Embodiment 2
The preparation of bacterium liquid: insert on the beef-protein medium of 40mL liquid with oese picking coli strain, the oese diameter that adopts is 2.5mm, and inoculate Escherichia coli quantity is 1 * 10 at every turn
7Individual, (e. coli concentration is 1 * 10 in the bacterium liquid to obtain bacterium liquid in 25 hours 36.5 ℃ of following 175 rev/mins of shaking table shaken cultivation
8Individual/mL), standby.Culture medium prescription is: beef extract 5g, peptone 10.0g, NaCl 5g, distilled water 1000ml, regulating pH at last is 6.5.
Intend the cultivation of beautiful prominent nematode: the culture dish that to get 18 clean diameters be 6cm, the nematode culture medium 4mL that step 3) is obtained is tiled in the culture dish, left standstill 8 hours, it by bacterium liquid/medium 1/50 volume ratio, pour the above-mentioned bacterium liquid that obtains 80 μ L into culture dish, left standstill 3 minutes, in per 6 culture dishes, choose 10,15 and 20 of the beautiful prominent nematodes of the aseptic plan that obtains into step 4) with transfer needle respectively, evenly be placed on the medium, cover the culture dish lid.The aforesaid operations step is all carried out on aseptic workbench.Then culture dish is placed the constant temperature and humidity incubator, humidity 55%, 21 ℃ were cultivated 10 days, can obtain the nematode of being cultivated.
Embodiment 3
The preparation of bacterium liquid: insert on the beef-protein medium of 60mL liquid with oese picking coli strain, the oese diameter that adopts is 2.5mm, and inoculate Escherichia coli quantity is 3 * 10 at every turn
7Individual, (e. coli concentration is 5 * 10 in the bacterium liquid to obtain bacterium liquid in 23 hours 37.5 ℃ of following 165 rev/mins of shaking table shaken cultivation
8Individual/mL), standby.Culture medium prescription is: beef extract 5g, peptone 10.0g, NaCl 5g, distilled water 1000ml, regulating pH at last is 7.5.
Intend the cultivation of beautiful prominent nematode: the culture dish that to get 18 clean diameters be 6cm, the nematode culture medium 6mL that step 3) is obtained is tiled in the culture dish, left standstill 9 hours, it by bacterium liquid/medium 1/50 volume ratio, pour the above-mentioned bacterium liquid that obtains 120 μ L into culture dish, left standstill 4 minutes, in per 6 culture dishes, choose 10,15 and 20 of the beautiful prominent nematodes of the aseptic plan that obtains into step 4) with transfer needle respectively, evenly be placed on the medium, cover the culture dish lid.The aforesaid operations step is all carried out on aseptic workbench.Then culture dish is placed the constant temperature and humidity incubator, humidity 60%, 22 ℃ were cultivated 10 days, can obtain the nematode of being cultivated.
Embodiment 4～11:
Embodiment is with embodiment 1, and difference is culture of nematodes temp. and humidity and incubation time difference (seeing Table 1) to some extent.
Table 1
| Numbering | Cultivate humidity (%) | Cultivation temperature (℃) | Incubation time (my god) |
| 4 | 51 | 23 | 9 |
| 5 | 52 | 24 | 9 |
| 6 | 53 | 25 | 8 |
| 7 | 54 | 26 | 8 |
| 8 | 56 | 27 | 7 |
| 9 | 57 | 28 | 7 |
| 10 | 58 | 29 | 6 |
| 11 | 59 | 30 | 5 |
Application examples 1:
Should use-case adopt the cultural method of embodiment 1 (20 ℃), (data are for getting 1cm in the table to obtain nematode population such as table 2 after the cultivation
2The plane, the observation nematode population according to the culture dish area, multiply by 25 with observation data and obtains, down together).
Table 2
| Add 10 nematodes | Add 15 nematodes | Add 20 nematodes | |
| Culture dish 1 | 3100 | 3400 | 2600 |
| Culture dish 2 | 2175 | 3825 | 3425 |
| Culture dish 3 | 2325 | 2350 | 1950 |
| Culture dish 4 | 2900 | 3275 | 3025 |
| Culture dish 5 | 2675 | 2875 | 2725 |
| Culture dish 6 | 1975 | 1600 | 3100 |
| On average | 2525 | 2888 | 2804 |
According to table 2 result, add before cultivating and intend 10,15 of beautiful prominent nematodes and 20 processing, after cultivating in 11 days, on average reach 2525,2888 and 2804 respectively.
Application examples 2:
The cultural method of embodiment 6 (25 ℃) be should use-case adopt, nematode population such as table 3 obtained after the cultivation.
Table 3
| Add 10 nematodes | Add 15 nematodes | Add 20 nematodes | |
| Culture dish 1 | 2950 | 3575 | 3875 |
| Culture dish 2 | 4275 | 3300 | 4050 |
| Culture dish 3 | 3375 | 4475 | 3825 |
| Culture dish 4 | 3225 | 4625 | 4125 |
| Culture dish 5 | 4075 | 3025 | 3450 |
| Culture dish 6 | 3725 | 3425 | 4400 |
| On average | 3604 | 3738 | 3954 |
According to table 3 result, add before cultivating and intend 10,15 of beautiful prominent nematodes and 20 processing, after cultivating in 5 days, on average reach 3604,3738 and 3954 respectively.
Application examples 3:
The cultural method that should use-case adopts embodiment 11 (30 ℃), the nematode population such as the table 4 that obtain after the cultivation.
Table 4
| Add 10 nematodes | Add 15 nematodes | Add 20 nematodes | |
| Culture dish 1 | 4175 | 3950 | 4775 |
| Culture dish 2 | 2525 | 2425 | 2725 |
| Culture dish 3 | 3675 | 4600 | 3025 |
| Culture dish 4 | 3125 | 3475 | 3500 |
| Culture dish 5 | 4300 | 3275 | 4225 |
| Culture dish 6 | 4550 | 3825 | 3675 |
| On average | 3725 | 3592 | 3654 |
According to table 4 result, add before cultivating and intend 10,15 of beautiful prominent nematodes and 20 processing, after cultivating in 5 days, on average reach 3725,3592 and 3654 respectively.
Claims (6)
1. a cultural method of intending beautiful prominent nematode is characterized in that: Escherichia coli E.coli bacterial strain is inserted on the beef-protein medium of liquid, obtained bacterium liquid in 23～25 hours 36.5～37.5 ℃ of following 165～175 times/minute shaking table shaken cultivation; Then, bacterium liquid is joined nematode culture medium, choose again, place the constant temperature and humidity incubator, under humidity 50～60%, 20～30 ℃ of conditions of temperature, cultivated 5～11 days, can obtain the nematode of being cultivated into the beautiful prominent nematode of sterile aseptic plan.
2. the cultural method of the beautiful prominent nematode of plan according to claim 1 is characterized in that: described Escherichia coli E.coli bacterial strain is common wild-type e. coli, and bacterial strain belongs to the Gram-negative brevibacterium, 0.5 * 1～3 microns of sizes, whole body flagellum can move no gemma.
3. the cultural method of the beautiful prominent nematode of plan according to claim 1, it is characterized in that: described bacterium liquid is with coli strain oese picking, insert on the beef-protein medium of 40～60mL liquid, inoculate Escherichia coli quantity is 1 * 10 at every turn
7～3 * 10
7Individual, put shaking table into constant temperature function, to regulate 36.5～37.5 ℃ of shaking table temperature, shake number of times and be set to 165～175 times/minute, shaken cultivation 23～25 hours obtains bacterium liquid.
4. the cultural method of the beautiful prominent nematode of plan according to claim 1, it is characterized in that: described liquid beef-protein medium is by weight: beef extract 5g, peptone 10.0g, NaCl 5g, distilled water 1000mL, regulating pH with 2mol/LNaOH at last is 6.5～7.5.
5. the cultural method of the beautiful prominent nematode of plan according to claim 1, it is characterized in that: described nematode culture medium is by weight: 3g NaCl, 17g agar, 2.5g peptone, be dissolved in the 975mL distilled water, 120～122 ℃ of sterilizations add 5mg/mL cholesterol stock solution 1mL, 1mol/L CaCl respectively after 29.5～30.5 minutes
21mL, 1mol/L MgSO
41mL, pH are 5.9～6.1 1mol/L phosphate buffer 25mL, shake evenly, promptly get nematode culture medium.
6. the cultural method of the beautiful prominent nematode of plan according to claim 1, it is characterized in that: the incubation step of the beautiful prominent nematode of described plan is, get the culture dish that clean diameter is 6cm, nematode culture medium 4～6mL is tiled in the culture dish, left standstill 8～10 hours, 's 1/50 volume ratio with micropipettor by bacterium liquid/medium, 80～120 μ L pour culture dish into bacterium liquid, left standstill 3～5 minutes, choose into 10～20 of the beautiful prominent nematodes of aseptic plan with transfer needle, evenly be placed on the medium, cover the culture dish lid; Then, culture dish is placed the constant temperature and humidity incubator, cultivated 5～11 days for 50～60%, 20～30 ℃, can obtain the nematode of being cultivated in humidity.
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| CN104542505A (en) * | 2015-01-23 | 2015-04-29 | 温州医科大学 | In vitro culturing method for parasitic nematodes in parasitic stage |
| CN105536367A (en) * | 2016-02-19 | 2016-05-04 | 山东省计量科学研究院 | Air dedusting and purifying system for constant temperature and humidity chamber |
| CN105891177A (en) * | 2016-04-13 | 2016-08-24 | 中国科学院东北地理与农业生态研究所 | Method for detecting trace BPA (bisphenol A) through nematode dyeing with Nile red |
| CN105891177B (en) * | 2016-04-13 | 2019-02-19 | 中国科学院东北地理与农业生态研究所 | Nematode dyeing detection trace BPA method is carried out using Nile red |
| CN116326545A (en) * | 2022-09-16 | 2023-06-27 | 黑龙江省农业科学院园艺分院 | Culture method of Zhongzhen nematodes |
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