CN102296031B - Double-layer flat plate solid culture method for phaeodactylum tricomutum - Google Patents
Double-layer flat plate solid culture method for phaeodactylum tricomutum Download PDFInfo
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- CN102296031B CN102296031B CN 201110250683 CN201110250683A CN102296031B CN 102296031 B CN102296031 B CN 102296031B CN 201110250683 CN201110250683 CN 201110250683 CN 201110250683 A CN201110250683 A CN 201110250683A CN 102296031 B CN102296031 B CN 102296031B
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Abstract
The invention discloses a double-layer flat plate solid culture method for phaeodactylum tricomutum and relates to the culture of phaeodactylum tricomutum. The method comprises: pouring f/2 culture medium into a flat plate; curing naturally and obtaining lower culture medium; filling f/2 culture medium into a centrifugal pipe, naturally curing, boiling and melting the f/2 culture medium when using the f/2 culture medium, and obtaining upper culture medium; preparing 10 to 20 milliliters of alga solution of phaeodactylum tricomutum growing to a log phase, centrifuging, removing supernate, re-suspending alga cells in f/2 culture medium and obtaining concentrated alga solution; pouring the obtained upper culture medium into concentrated alga solution, placing the mixed on the lower culture medium, and naturally curing; and reversing a flat plate and culturing in light.
Description
Technical field
The present invention relates to the cultivation of Phaeodactylum tricornutum (Phaeodactylum tricornutum, PT), especially relate to a kind of double-layer plate solid culture method of Phaeodactylum tricornutum, dull and stereotyped with the solid algae that obtains Phaeodactylum tricornutum.
Background technology
Molten algae microorganism, such as bacterium, actinomycetes, virus, fungi and the protozoic continuous discovery of molten algae, for wide prospect has been opened up in the microbial control of red tide, and the separation of molten algae microorganism is the basis of microorganisms of red tide control.
The Isolation and screening of molten algae microorganism has two kinds of approach usually: a kind of is the mode that infects by liquid, and another kind is the mode that infects by solid.Whether the method that liquid infects is to be subjected to suppress to judge whether there is molten algae microorganism in the system by the growth of monitoring algae, comprise whether observing frustule with ordinary optical microscope, cracking (1.Takao occurs, Y., K.Nagasaki, K.Mise, T.Okuno, D.Honda, Isolation and characterization of a novel single-stranded RNA virus infectious to a marine fungoid protist, Schizochytrium sp (Thraustochytriaceae, labyrinthulea) .Applied and Environmental Microbiology, 2005.71 (8): 4516-4522.), or by the visual inspection algae liquid color (2.Tomaru that whether changes, Y., Y. Shirai, H.Suzuki, T.Nagumo, K.Nagasaki, Isolation and characterization of a new single-stranded DNA virus infecting the cosmopolitan marine diatom Chaetoceros dehilis.Aquatic Microbial Ecology, 2008.50 (2): 103-112; 3.Imamura, N., I.Motoike, N.Shimada, M.Nishikori, H.Morisaki, H.Fukami, An efficient screening approach for anti-Microcystis compounds based on knowledge of aquatic microbial ecosystem.Journal of Antibiotics, 2001.54 (7): 582-587.), or detect algae fluorescent value whether descend (4.Bettarel, Y., J.Kan, K.Wang, K.E. Williamson, S.Cooney, S.Ribblett, F.Chen, K.E.Wommack, D.W.Coats, Isolation and preliminary characterisation of a small nuclear inclusion virus infecting the diatom Chaetoceros cf.gracilis.Aquatic Microbial Ecology, 2005.40 (2): 103-114; 5.Su, J.Q., X.R.Yang, T.L.Zheng, Y.Tian, N.Z.Jiao, L.Z.Cai, H.S.Hong, Isolation and characterization of a marine algicidal bacterium against the toxic dinoflagellate Alexandrium tamarense.Harmful Algae, 2007.6 (6): 799-810.), judge whether to exist molten algae microorganism, the method is applicable to the Isolation and screening of the molten algae microorganism of algae of all size.But there are a lot of shortcomings in the method: for example, sometimes need the whether cracking of the frustule of every day by microscopic examination control group and treatment group, workload is large, and the cell of some algae is less is not easy to judge whether cracking under opticmicroscope; Sometimes some abiotic factors also can cause frustule cracking or growth to be suppressed, and easily produce false positive results; Although sometimes have molten algae microorganism in the system but can not significantly suppress the growth of algae, easily produce false negative result.The method that solid infects is to judge whether there is molten algae microorganism (6.Bellec in the system by whether occurring plaque on the visual inspection algae flat board, L., N.Grimsley, Y.Desdevises, Isolation of Prasinoviruses of the Green Unicellular Algae Ostreococcus spp.on a Worldwide Geographical Scale.Applied and Environmental Microbiology, 2010.76 (1): 96-101; 7.Kim, J.D., J.Y.Kim, J.K.Park, C.G.Lee, Selective Control of the Prorocentrum minimum Harmful Algal Blooms by a Novel Algal-Lytic Bacterium Pseudoalteromonas haloplanktis AFMB-008041.Marine Biotechnology, 2009.11 (4): 463-472.), the Isolation and screening that only is fit to the molten algae microorganism of the algae that can grow at solid medium, but the method that its relative liquid infects is simpler, directly perceived.
Summary of the invention
The double-layer plate solid culture method that the purpose of this invention is to provide a kind of Phaeodactylum tricornutum.
The double-layer plate solid culture method of Phaeodactylum tricornutum of the present invention (Phaeodactylum tricornutum, PT) may further comprise the steps:
1) the f/2 substratum is poured in the flat board, natural coagulation is as lower floor's substratum;
2) the f/2 substratum is packed in the centrifuge tube, natural coagulation is boiled thawing with it during use, as the upper strata substratum;
3) get the Phaeodactylum tricornutum algae liquid 10～20mL that grows to logarithmic phase, centrifugal, abandon supernatant, the resuspended frustule of f/2 substratum must concentrate algae liquid;
4) with step 2) gained upper strata substratum pours step 3 into) in the concentrated algae liquid of gained, repave in step 1) on lower floor's substratum of gained, natural coagulation;
5) flat board is inverted, is cultivated under the illumination.
In step 1) in, the amount of described f/2 substratum can be 20mL, and described f/2 substratum contains 1% agar, and it is that 9cm is dull and stereotyped that described flat board can adopt diameter.
In step 2) in, the amount of described f/2 substratum can be 3.5mL, and described f/2 substratum contains 0.7% agar, and described centrifuge tube can be the 10mL centrifuge tube; During described use it is boiled and melt and place 50 ℃ of water-baths.
In step 3) in, the algae density of described Phaeodactylum tricornutum algae liquid can be 3 * 10
6～10 * 10
6Cells/mL, but the centrifugal 5min of described centrifugal 6000rpm; The resuspended frustule of described f/2 substratum, the resuspended frustule of available 1mL f/2 substratum.
In step 4) in, the temperature of described upper strata substratum can be 50 ℃.
In step 5) in, cultivate under the described illumination and can under 20 ℃ of illumination, cultivate.
The double-layer plate solid culture method of described Phaeodactylum tricornutum can be applicable to the separation of the molten algae microorganism of Phaeodactylum tricornutum.
The double-layer plate solid culture method of described Phaeodactylum tricornutum can be applicable to active detection of molten algae of the molten algae microorganism of Phaeodactylum tricornutum.
The suitableeest algae weight range for the preparation of the Phaeodactylum tricornutum double-layer plate can be 10～20mL.
The target algae Phaeodactylum tricornutum that the present invention relates to is the relatively little unicellular algae of a kind of cell, has avette, fusiformis and three and goes out three kinds of different forms of radiation, and these three kinds of forms can change under different envrionment conditionss.As under the condition of normal liquid culture, be mostly that three go out radiation cell and a small amount of spindle cell, these two kinds of forms all do not have siliceous cell walls.Three go out the radiation cell length is about 10～18 μ m (vertical range between two arms), the long 22 μ m of spindle cell, and the wide 5 μ m in middle part, two arm ends are more blunt, the long 8 μ m of avette cell, wide 3 μ m have a crepis face, lack another shell face.(by 10% inoculation, cultivate the fresh algae liquid after 2 weeks, algae density is 3 * 10 to grow to the Phaeodactylum tricornutum algae liquid of logarithmic phase
6～10 * 10
6Cells/mL) mix with the upper strata substratum after concentrated 10～20 times and be down flat plate, can be directly used in subsequent experimental; The double-layer plate for preparing higher algae content can strengthen workload, and nutrient competition and competition for space pressure between the dull and stereotyped upper algae are large, are unfavorable for experiment; And the algae of low concentration can not grow on flat board, perhaps need the long period just can reach the algae density of experiment needs, but algae at this moment is stale, is not suitable for experiment.
Description of drawings
Fig. 1 is the Phaeodactylum tricornutum double-layer plate that contains algae amount 40mL.
Fig. 2 is the Phaeodactylum tricornutum double-layer plate that contains algae amount 30mL.
Fig. 3 is the Phaeodactylum tricornutum double-layer plate that contains algae amount 20mL.
Fig. 4 is the Phaeodactylum tricornutum double-layer plate that contains algae amount 10mL.
Fig. 5 is the Phaeodactylum tricornutum double-layer plate that contains algae amount 5mL.
Fig. 6 is not for containing the double-layer plate blank of Phaeodactylum tricornutum.
Fig. 7 is the plaque result that molten algae microbial lytic Phaeodactylum tricornutum forms.
Fig. 8 is the result that double-layer plate-Maximum probable number method detects molten algae microorganism active.
Embodiment
Phaeodactylum tricornutum double-layer plate of the present invention is referring to Fig. 1～5, and they are respectively algae liquid (the algae density 5.0 * 10 of 40mL, 30mL, 20mL, 10mL and 5mL logarithmic phase
6Cells/mL) be concentrated into the result of the double-layer plate for preparing behind the 1mL.Fig. 1 and Fig. 2 algae flat board are darker tawny, and it is large to contain the algae amount, need to carry out repeatedly with the 10mL centrifuge tube centrifugally, operate more loaded down with trivial details; Fig. 3 and Fig. 4 algae flat board are khaki, with the naked eye can clearly observe the upgrowth situation of algae, and the algae amount are moderate, get final product for centrifugal 1 to 2 time with the 10mL centrifuge tube; The color of Fig. 5 algae flat board is too light, the difficult upgrowth situation of observing algae with the naked eye, and because to contain the algae amount too low, the upgrowth situation of algae is unstable, and therefore algae even can die is not suitable for subsequent experimental sometimes.Phaeodactylum tricornutum can be at double-layer plate survival 1 wheat harvesting period (along with time lengthening, the dull and stereotyped exsiccation, algae be just dead).
The plaque that molten algae microbial lytic Phaeodactylum tricornutum forms is seen Fig. 7, and in Fig. 7, A represents algae, and P represents plaque, and presentation of results can the successful molten algae microorganism that is separated to Phaeodactylum tricornutum by the double-layer plate solid method that infects.
Utilize double-layer plate-Maximum probable number method to detect molten algae microorganism active, the results are shown in Figure 8, stain is for dripping the position of molten algae microorganism, can see that cultivating 2 all rear some Bluepoint positions plaque occurred, namely be positive, the most probable number MPN table is looked in the extent of dilution that record is positive and corresponding repeat number, and the concentration that draws molten algae microorganism is: 4600cells/mL.Here noting will be with the algae dull and stereotyped (within 2 days) of fresh preparation, otherwise experimental result can be poorly.
Experimental result shows: can utilize the dull and stereotyped Phaeodactylum tricornutum of cultivating of solid double-layer, and infect the molten algae microorganism that is separated to Phaeodactylum tricornutum of method success by the double-layer plate solid, and can detect the molten algae microbic activity of Phaeodactylum tricornutum with the algae double-layer plate.
The present invention relates to Double-Medium, it is formulated that they add a certain proportion of agar by the f/2 algae culture medium, and lower floor's substratum is thicker, agar concentration is higher, and for the growth of algae provides nutrition and matrix, the upper strata substratum is very thin one deck, agar concentration is lower, and algae grows in this layer substratum.
Following specific embodiment is to further specify of the present invention, but the invention is not restricted to following embodiment.
Embodiment 1: the algae amount for the preparation of the Phaeodactylum tricornutum double-layer plate is determined
(1) 20mL f/2 substratum (containing 1% agar) is down flat plate (diameter is 9cm), and natural coagulation is as lower floor's substratum;
(2) 3.5mL f/2 substratum (containing 0.7% agar) installs in the 10mL centrifuge tube, and natural coagulation is boiled thawing with it during use, and places 50 ℃ of water-baths, as the upper strata substratum;
(3) get Phaeodactylum tricornutum algae liquid (the algae density 5.0 * 10 that grows to logarithmic phase
6Cells/mL) 5～40mL, the centrifugal 5min of 6000rpm, supernatant discarded is with the resuspended frustule of 1mL f/2 substratum;
(4) 50 ℃ upper strata substratum is poured in the above-mentioned concentrated algae liquid, mixing also is laid on lower floor's substratum equably rapidly, natural coagulation, and 1mL f/2 substratum and upper strata substratum are down flat plate as blank;
(5) flat board is inverted, under 20 ℃ of illumination, is cultivated;
(6) for the preparation of the suitableeest algae weight range of Phaeodactylum tricornutum double-layer plate: 10～20mL.The double-layer plate that contains different algae amounts is seen Fig. 1～6.
Embodiment 2: utilize the double-layer plate method to separate the molten algae microorganism of Phaeodactylum tricornutum
(1) gathers the Xiamen sea area surface seawater, be stored in 4 ℃, be used for immediately the separation of molten algae microorganism;
(2) 20mL f/2 substratum (containing 1% agar) is down flat plate (diameter is 9em), and natural coagulation is as lower floor's substratum;
(3) 3.5mL f/2 substratum (containing 0.7% agar) installs in the 10mL centrifuge tube, and natural coagulation is boiled thawing with it during use, and places 50 ℃ of water-baths, as the upper strata substratum;
(4) get Phaeodactylum tricornutum algae liquid (the algae density 6.0 * 10 that grows to logarithmic phase
6Cells/mL) 10mL, the centrifugal 5min of 6000rpm, supernatant discarded, the resuspended frustule of 1mL seawater sample is done 3 repetitions;
(5) 50 ℃ upper strata substratum is poured in the above-mentioned concentrated algae liquid, mixing also is laid on lower floor's substratum natural coagulation equably rapidly;
(6) flat board is inverted, under 20 ℃ of illumination, was cultivated for 2 weeks, observe on the algae plate plaque whether occurs;
(7) experimental result is seen Fig. 7.
Embodiment 3: double-layer plate-Maximum probable number method detects molten algae microorganism active
(1) 20mL f/2 substratum (containing 1% agar) is down flat plate (diameter is 9em), and natural coagulation is as lower floor's substratum;
(2) 3.5mL f/2 substratum (containing 0.7% agar) installs in the 10mL centrifuge tube, and natural coagulation is boiled thawing with it during use, and places 50 ℃ of water-baths, as the upper strata substratum;
(3) get Phaeodactylum tricornutum algae liquid (the algae density 4.8 * 10 that grows to logarithmic phase
6Cells/mL) 10mL, the centrifugal 5min of 6000rpm, supernatant discarded, the resuspended frustule of 1mL f/2 substratum;
(4) 50 ℃ upper strata substratum is poured in the above-mentioned concentrated algae liquid, mixing also is laid on lower floor's substratum natural coagulation equably rapidly;
(5) molten algae microbiological specimens is got respectively 10 μ L stostes and dilution drop on the algae flat board with 10 times and 100 times of f/2 substratum dilutions, and 3 repetitions of each concentration after dull and stereotyped the absorption, are inverted flat board, in 20 ℃ of 2 weeks of illumination cultivation by algae until liquid;
After (6) 2 weeks, extent of dilution and the corresponding repeat number of plaque appears in record, looks into the most probable number MPN table, and the concentration that draws molten algae microorganism is: 4600cells/mL.The results are shown in Figure 8.
Claims (7)
1. the double-layer plate solid culture method of Phaeodactylum tricornutum is characterized in that may further comprise the steps:
1) the f/2 substratum is poured in the flat board, natural coagulation is as lower floor's substratum; Described f/2 substratum contains 1% agar;
2) the f/2 substratum is packed in the centrifuge tube, natural coagulation is boiled thawing with it during use, as the upper strata substratum; Described f/2 substratum contains 0.7% agar;
3) get the Phaeodactylum tricornutum algae liquid 10～20mL that grows to logarithmic phase, centrifugal, abandon supernatant, the resuspended frustule of f/2 substratum must concentrate algae liquid; The algae density of described Phaeodactylum tricornutum algae liquid is 3 * 10
6～10 * 10
6Cells/mL, described centrifugal be the centrifugal 5min of 6000rpm; The resuspended frustule of described f/2 substratum is with the resuspended frustule of 1mL f/2 substratum;
4) with step 2) gained upper strata substratum pours in the concentrated algae liquid of step 3) gained, repaves on lower floor's substratum of step 1) gained natural coagulation;
5) flat board is inverted, is cultivated under the illumination.
2. the double-layer plate solid culture method of Phaeodactylum tricornutum as claimed in claim 1 is characterized in that in step 1), and the amount of described f/2 substratum is 20mL.
3. the double-layer plate solid culture method of Phaeodactylum tricornutum as claimed in claim 1 is characterized in that in step 1), and the described dull and stereotyped diameter that adopts is that 9cm is dull and stereotyped.
4. the double-layer plate solid culture method of Phaeodactylum tricornutum as claimed in claim 1 is characterized in that in step 2) in, the amount of described f/2 substratum is 3.5mL.
5. the double-layer plate solid culture method of Phaeodactylum tricornutum as claimed in claim 1 is characterized in that in step 2) in, during described use it is boiled and melt and place 50 ℃ of water-baths.
6. the double-layer plate solid culture method of Phaeodactylum tricornutum as claimed in claim 1 is characterized in that in step 4), and the temperature of described upper strata substratum is 50 ℃.
7. the double-layer plate solid culture method of Phaeodactylum tricornutum as claimed in claim 1 is characterized in that in step 5), is incubated under the described illumination under 20 ℃ of illumination to cultivate.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100400642C (en) * | 2004-06-18 | 2008-07-09 | 徐州师范大学 | Traingular brown algae open culture method and its special culture meidum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100400642C (en) * | 2004-06-18 | 2008-07-09 | 徐州师范大学 | Traingular brown algae open culture method and its special culture meidum |
Non-Patent Citations (6)
| Title |
|---|
| GUTENBRUNNER, SA.PROTEINACEOUS AND IMMUNOCHEMICAL DISTINCTIONS BETWEEN THE OVAL AND FUSIFORM MORPHOTYPES OF PHAEODACTYLUM-TRICORNUTUM (BACILLARIOPHYCEAE).《JOURNAL OF PHYCOLOGY》.1994,第30卷(第1期),129-136. |
| PROTEINACEOUS AND IMMUNOCHEMICAL DISTINCTIONS BETWEEN THE OVAL AND FUSIFORM MORPHOTYPES OF PHAEODACTYLUM-TRICORNUTUM (BACILLARIOPHYCEAE);GUTENBRUNNER, SA;《JOURNAL OF PHYCOLOGY》;19940228;第30卷(第1期);129-136 * |
| 一种筛选溶藻放线菌的新方法;喻融;《华中师范大学研究生学报》;20051231;第12卷(第4期);142-144 * |
| 单胞藻软琼脂克隆固体培养基保藏技术;陆德祥;《河北渔业》;20051231(第5期);49-51 * |
| 喻融.一种筛选溶藻放线菌的新方法.《华中师范大学研究生学报》.2005,第12卷(第4期),142-144. |
| 陆德祥.单胞藻软琼脂克隆固体培养基保藏技术.《河北渔业》.2005,(第5期),49-51. |
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