CN104789475A - Separation method of pure breed diatom in mixed benthic diatom solution - Google Patents
Separation method of pure breed diatom in mixed benthic diatom solution Download PDFInfo
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Abstract
The invention discloses a method for separating pure breed diatom from a mixed benthic diatom solution, such that all culturable algae pure breeds in the mixed benthic diatom can be obtained in one time, and the nondominant and small-scale pure breed benthic diatom difficultly obtained through other separation methods can be effectively obtained. According to the present invention, based on different sedimentation velocities of the benthic diatoms with different specifications and different forms and the bacteria during centrifugation, the benthic diatoms with different specifications are pre-separated, and the pre-separated algae groups are subjected to centrifugation washing a plurality of times to remove most of bacteria and the algae with the non-target specification, such that it is ensured that the pollution between the bacteria and different algal species are substantially reduced when the single algal community is cultured on the solid flat culture medium by using the flat coating culture so as to effectively and easily obtain the no-pollution pure breed diatom. According to the present invention, the method has characteristics of simple operation, convenience and applicability, and can be directly used for production and research.
Description
Technical field
The present invention relates to the separation method of a kind of purebred benthic diatom, particularly mix the separation method of purebred benthic diatom in benthic diatom solution.
Background technology
Benthic diatom is the open-mouthed bait of Bao Miao during nursery is produced.The benthic diatom basic source that abalone fry-rearing uses in producing is in natural waters, or collect mixed algae kind from sea area algae blade, or collect algae kind at sea area link plate, and then numerous to indoor expansion, but generally algae kind is not selected, after arriving certain density, be directly used in nursery to produce, but the benthic diatom obtained like this, quality can not be guaranteed, and can not ensure the effect of abalone Seedling production.Because benthic diatom is of a great variety in mixed algae kind, the kind of benthic diatom and the content ratio of various benthic diatom can not control, and different sorts benthic diatom, in its body, the content difference of the nutritive ingredient such as unsaturated fatty acids is very large, and the impact that the product confrontation Bao Miao of benthic diatom produces is also very large.So purebred the production Bao Miao of screening and separating high-quality benthic diatom has important practical significance from mixed algae kind.
From mixing benthic diatom, be separated single have several method, 1) micro pipette partition method; 2) sample series isolation by dilution method; 3) water droplet partition method; 4) plate isolation method.No matter be any method, for sociales or the larger kind of individuality, separation is easier to.For less kind individual in the especially non-sociales of non-sociales, be separated very difficult.To be separated in non-sociales individual less single even lucky, to make and to be purebredly also not easy.Because the breeding of the little individual single benthic diatom of non-advantage is often comparatively slow, some mushroom (or on) in algae media can be bred rapidly, floods edominant Micro Algae single, purebred being difficult to is obtained.
Summary of the invention
The present invention is the above-mentioned deficiency in order to solve existing for prior art, a kind of method being separated purebred benthic diatom in mixing benthic diatom solution is proposed, the benthic diatom that it can isolate each classification relatively easily from mixed algae liquid is purebred, especially isolating edominant benthic diatom purebred, providing technical support for screening benthic diatom breeding in abalone fry-rearing production.
Technical solution of the present invention is: a kind of separation method mixing purebred diatom in benthic diatom solution, is characterized in that: carry out according to following steps:
First be the particulate debris filtering of in the mixing benthic diatom solution of 0.05-0.15g/ml more than 75 μm by concentration, then mixing benthic diatom solution is divided into three equal parts, put into centrifuge tube respectively, shake up mixing benthic diatom solution in first centrifuge tube, centrifugal treating 4-5min under the condition of 350-450g RCF, is precipitated A and the first supernatant liquor;
To the first supernatant liquor centrifugal treating 4-5min under the condition of 850-950g RCF, be precipitated B;
Shaken up by mixing benthic diatom solution in second centrifuge tube, under the condition of 850-950g RCF, centrifugal treating 4-5min obtains the second supernatant liquor, and to the second supernatant liquor centrifugal treating 4-5min under the condition of 1650-1750g RCF, is precipitated C;
Shaken up by mixing benthic diatom solution in 3rd centrifuge tube, under the condition of 1650-1750g RCF, centrifugal treating 4-5min obtains the 3rd supernatant liquor, and to the 3rd supernatant liquor centrifugal treating 4-5min under the condition of 2650-2750g RCF, is precipitated D;
Get 0.05-0.2g precipitate A, whole precipitate B, whole precipitate C and whole precipitation D, respectively with the antiseptic sea water dilution mixing of 20-40ml, be precipitated A sea water solution, precipitate B sea water solution, precipitate C sea water solution and precipitation D sea water solution respectively;
By precipitate A sea water solution centrifugal treating 5-6min under the condition of 350-450g RCF, be precipitated A
1, to the precipitate A obtained
1carry out the first centrifugal treating 7 times, be precipitated A
2, the first described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 350-450g RCF to this precipitation obtained, obtain precipitation;
By precipitate B sea water solution centrifugal treating 5-6min under the condition of 850-950g RCF, be precipitated B
1, to the precipitate B obtained
1carry out the second centrifugal treating 7 times, be precipitated B
2, the second described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 850-950g RCF to this precipitation obtained;
By precipitate C sea water solution centrifugal treating 5-6min under the condition of 1650-1750g RCF, be precipitated C
1, to the precipitate C obtained
1carry out the 3rd centrifugal treating 7 times, be precipitated C
2, the 3rd described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 1650-1750g RCF to this precipitation obtained, obtain precipitation;
By precipitation D antiseptic sea water solution centrifugal treating 5-6min under the condition of 2650-2750g RCF, be precipitated D
1, to the precipitation D obtained
1carry out the 4th centrifugal treating 7 times, be precipitated D
2, the 4th described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 2650-2750g RCF to this precipitation obtained, obtain precipitation;
By the precipitate A obtained
2, precipitate B
2, precipitate C
2with precipitation D
2transfer in appropriate antiseptic sea water respectively with aseptic method, form A
2, B
2, C
2and D
2seawater mother liquor, then according in seawater total plate count measure time doubling dilution method antiseptic sea water carry out gradient dilution, obtain precipitate A
2, B
2, C
2and D
210 of seawater mother liquor
0, 10
-1, 10
-2, 10
-3, 10
-4each concentration gradient algae liquid;
Use plate isolation method
[1]cultivate A
2, B
2, C
2and D
2benthic diatom in each concentration gradient algae liquid, basis of microscopic observation is also collected the dull and stereotyped upper algae without bacterium and the pollution of other Micro Algae and is fallen, and can obtain various benthic diatom purebred.
Mix a separation method for purebred diatom in benthic diatom solution, it is characterized in that: carry out according to following steps:
First be the particulate debris filtering of in the mixing benthic diatom solution of 0.1g/ml more than 75 μm by concentration, then mixing benthic diatom solution is divided into three equal parts, put into centrifuge tube respectively, shake up mixing benthic diatom solution in first centrifuge tube, centrifugal treating 4min under the condition of 400g RCF, is precipitated A and the first supernatant liquor;
To the first supernatant liquor centrifugal treating 4min under the condition of 900g RCF, be precipitated B;
Shaken up by mixing benthic diatom solution in second centrifuge tube, under the condition of 900g RCF, centrifugal treating 4min obtains the second supernatant liquor, and to the second supernatant liquor centrifugal treating 4min under the condition of 1700g RCF, is precipitated C;
Shaken up by mixing benthic diatom solution in 3rd centrifuge tube, under the condition of 1700g RCF, centrifugal treating 4min obtains the 3rd supernatant liquor, and to the 3rd supernatant liquor centrifugal treating 4min under the condition of 2700g RCF, is precipitated D;
Get 0.1g precipitate A, whole precipitate B, whole precipitate C and whole precipitation D, respectively with the antiseptic sea water dilution mixing of 30ml, be precipitated A sea water solution, precipitate B sea water solution, precipitate C sea water solution and precipitation D sea water solution respectively;
By precipitate A sea water solution centrifugal treating 6min under the condition of 400g RCF, be precipitated A
1, to the precipitate A obtained
1carry out the first centrifugal treating 7 times, be precipitated A
2, the first described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 400g RCF to this precipitation obtained, obtain precipitation;
By precipitate B sea water solution centrifugal treating 6min under the condition of 900g RCF, be precipitated B
1, to the precipitate B obtained
1carry out the second centrifugal treating 7 times, be precipitated B
2, the second described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 900g RCF to this precipitation obtained;
By precipitate C sea water solution centrifugal treating 6min under the condition of 1700g RCF, be precipitated C
1, to the precipitate C obtained
1carry out the 3rd centrifugal treating 7 times, be precipitated C
2, the 3rd described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 1700g RCF to this precipitation obtained, obtain precipitation;
By precipitation D antiseptic sea water solution centrifugal treating 6min under the condition of 2700g RCF, be precipitated D
1, to the precipitation D obtained
1carry out the 4th centrifugal treating 7 times, be precipitated D
2, the 4th described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 2700g RCF to this precipitation obtained, obtain precipitation;
By the precipitate A obtained
2, precipitate B
2, precipitate C
2with precipitation D
2transfer in appropriate antiseptic sea water respectively with aseptic method, form A
2, B
2, C
2and D
2seawater mother liquor, then according in seawater total plate count measure time doubling dilution method antiseptic sea water carry out gradient dilution, obtain precipitate A
2, B
2, C
2and D
210 of seawater mother liquor
0, 10
-1, 10
-2, 10
-3, 10
-4each concentration gradient algae liquid;
Use plate isolation method
[1]cultivate A
2, B
2, C
2and D
2benthic diatom in each concentration gradient algae liquid, basis of microscopic observation is also collected the dull and stereotyped upper algae without bacterium and the pollution of other Micro Algae and is fallen, and can obtain various benthic diatom purebred.
Compared with the existing technology, tool has the following advantages in the present invention:
The method being separated purebred diatom from mixing benthic diatom solution disclosed in this invention, nearly allly in disposable acquisition mixing benthic diatom can cultivate the purebred of algae kind, and it is purebred effectively can to obtain in other separation method the non-advantage small dimension benthic diatom being difficult to obtain; It based on different size and form benthic diatom and bacterium centrifugal time settling velocity different characteristic, first different size form algae is separated in advance; Then to each group of algae of pre-point through repeatedly centrifuge washing, remove the algae kind of Most bacterial and non-targeted specification; Thus when ensureing that being coated with slat chain conveyor list algae on solid plate substratum falls, the pollution between bacterium and different algae kind reduces greatly, the free of contamination purebred algae of acquisition is become effectively with easy.And present method is simple to operate, convenient applicable, can directly apply to and produce and research.
Accompanying drawing explanation
Fig. 1 to 19 utilizes the purebred microgram of the isolated different benthic diatom of present method.
Figure 20 is the microgram utilizing the isolated green alga of present method purebred.
Embodiment
The specific embodiment of the present invention will be described below:
Mix a separation method for purebred diatom in benthic diatom solution, the method is carried out according to following steps:
First be the particulate debris filtering of in the mixing benthic diatom solution of 0.05-0.15g/ml more than 75 μm by concentration, then mixing benthic diatom solution is divided into three equal parts, put into centrifuge tube respectively, shake up mixing benthic diatom solution in first centrifuge tube, be preferably 400g RCF at 350-450g RCF() condition under centrifugal treating 4-5min(be preferably 4min), be precipitated A and the first supernatant liquor
At 850-950g RCF(, 900g RCF is preferably to the first supernatant liquor) condition under centrifugal treating 4-5min(be preferably 4min), be precipitated B,
Second centrifuge tube is preferably 900g RCF at 850-950g RCF() condition under centrifugal treating 4-5min(be preferably 4min) obtain the second supernatant liquor, and at 1650-1750g RCF(, 1700g RCF is preferably to the second supernatant liquor) condition under centrifugal treating 4-5min(be preferably 4min), be precipitated C
3rd centrifuge tube is preferably 1700g RCF at 1650-1750g RCF() condition under centrifugal treating 4-5min(be preferably 4min) obtain the 3rd supernatant liquor, and at 2650-2750g RCF(, 2700g RCF is preferably to the 3rd supernatant liquor) condition under centrifugal treating 4-5min(be preferably 4min), be precipitated D
Get 0.02-0.2g(and be preferably 0.1g) precipitate A, all precipitate B, all precipitate C and all precipitate D, be preferably 30ml with 20-40ml(respectively) antiseptic sea water dilution mixing, be precipitated A sea water solution, precipitate B sea water solution, precipitate C sea water solution and precipitation D sea water solution respectively;
Precipitate A sea water solution is preferably 400g RCF at 350-450gRCF() condition under centrifugal treating 5-6min(be preferably 6min), be precipitated A
1, to the precipitate A obtained
1carry out the first centrifugal treating 7 times, be precipitated A
2, the first centrifugal treating is here by precipitation appropriate antiseptic sea water dilution mixing, and is preferably 400g RCF to the sea water solution of this precipitation obtained at 350-450g RCF() condition under centrifugal treating 5-6min(be preferably 6min), obtain precipitation;
Precipitate B sea water solution is preferably 900g RCF at 850-950g RCF() condition under centrifugal treating 5-6min(be preferably 6min), be precipitated B
1, to the precipitate B obtained
1carry out the second centrifugal treating 7 times, be precipitated B
2, the second centrifugal treating is here by precipitation appropriate antiseptic sea water dilution mixing, and is preferably 900g RCF to the sea water solution of this precipitation obtained at 850-950g RCF() condition under centrifugal treating 5-6min(be preferably 6min), obtain precipitation;
Precipitate C sea water solution is preferably 1700g RCF at 1650-1750g RCF() condition under centrifugal treating 5-6min(be preferably 6min), be precipitated C
1, to the precipitate C obtained
1carry out the 3rd centrifugal treating 7 times, be precipitated C
2, the 3rd centrifugal treating is here by precipitation appropriate antiseptic sea water dilution mixing, and is preferably 1700g RCF to the sea water solution of this precipitation obtained at 1650-1750g RCF() condition under centrifugal treating 5-6min(be preferably 6min), obtain precipitation;
Precipitation D antiseptic sea water solution is preferably 2700g RCF at 2650-2750g RCF() condition under centrifugal treating 5-6min(be preferably 6min), be precipitated D
1, to the precipitation D obtained
1carry out the 4th centrifugal treating 7 times, be precipitated D
2, the 4th centrifugal treating is here by precipitation appropriate antiseptic sea water dilution mixing, and is preferably 2700g RCF to the sea water solution of this precipitation obtained at 2650-2750g RCF() condition under centrifugal treating 5-6min(be preferably 6min), obtain precipitation;
By the precipitate A obtained
2, precipitate B
2, precipitate C
2with precipitation D
2transfer in appropriate antiseptic sea water respectively with aseptic method, form A
2, B
2, C
2and D
2seawater mother liquor, then according in seawater total plate count measure time doubling dilution method antiseptic sea water carry out gradient dilution, obtain precipitate A
2, B
2, C
2and D
210 of seawater mother liquor
0, 10
-1, 10
-2, 10
-3, 10
-4each concentration gradient algae liquid;
Use plate isolation method
[1]cultivate A
2, B
2, C
2and D
2benthic diatom in each concentration gradient algae liquid.Basis of microscopic observation is also collected the dull and stereotyped upper algae without bacterium and the pollution of other Micro Algae and is fallen, and can obtain various benthic diatom purebred.
Illustrate
Gather the dense thick mixed solution (20ml) of benthic diatom from the waved plate with a large amount of benthic diatom, add seawater, by the above particulate debris of 200 mesh sieve thin,tough silk filtering 75um, obtain 90ml algae liquid, classify in three categories part, and every part is respectively charged in the 45ml centrifuge tube of 1 sterilizing.
Wherein first part shake up after under the condition of 400g RCF centrifugal treating 4min, be precipitated A and the first supernatant liquor, by the first supernatant liquor centrifugal treating 4min under the condition of 900g RCF, be precipitated B.
After second part of algae liquid shakes up, under the condition of 900g RCF, centrifugal treating 4 min obtains the second supernatant liquor, gets the second supernatant liquor centrifugal treating 4min under the condition of 1700g RCF, is precipitated C.
After 3rd part of algae liquid shakes up, under the condition of 1700g RCF, centrifugal treating 4 min obtains the 3rd supernatant liquor, gets the 3rd supernatant liquor centrifugal treating 4 min under the condition of 2700g RCF, is precipitated D.
Get 0.1g precipitate A, all precipitate B, all precipitate C and all precipitate D, respectively with 30ml antiseptic sea water dilution mixing, being precipitated A sea water solution, precipitate B sea water solution, precipitate C sea water solution and precipitation D sea water solution respectively;
By precipitate A sea water solution centrifugal treating 6min under the condition of 400g RCF, be precipitated A
1, to the precipitate A obtained
1carry out the first centrifugal treating 7 times, be precipitated A
2;
By precipitate B sea water solution centrifugal treating 6min under the condition of 900g RCF, be precipitated B
1, to the precipitate B obtained
1carry out the second centrifugal treating 7 times, be precipitated B
2;
By precipitate C sea water solution centrifugal treating 6min under the condition of 1700g RCF, be precipitated C
1, to the precipitate C obtained
1carry out the 3rd centrifugal treating 7 times, be precipitated C
2;
By precipitation D sea water solution centrifugal treating 6min under the condition of 2700g RCF, be precipitated D
1, to the precipitation D obtained
1carry out the 4th centrifugal treating 7 times, be precipitated D
2;
By the precipitate A obtained
2, precipitate B
2, precipitate C
2with precipitation D
2transfer in 0.4ml antiseptic sea water respectively with aseptic method, form A
2, B
2, C
2and D
2seawater mother liquor, then according in seawater total plate count measure time doubling dilution method antiseptic sea water carry out gradient dilution, obtain precipitate A
2, B
2, C
2and D
210 of seawater mother liquor
0, 10
-1, 10
-2, 10
-3, 10
-4each concentration gradient algae liquid;
Use plate isolation method
[1]cultivate A
2, B
2, C
2and D
2benthic diatom in each concentration gradient algae liquid.Basis of microscopic observation is also collected the dull and stereotyped upper algae without bacterium and the pollution of other Micro Algae and is fallen, and isolates purebred 19 of benthic diatom (accompanying drawing 1-19) altogether, also isolates purebred 1 of green alga (accompanying drawing 20) simultaneously, be described in detail as follows:
Fig. 1: about 70um is long, and 5 days algaes fall diameter 1mm, filbert.
Fig. 2: about 55um is long; 5 days algaes fall diameter 1mm, filbert.
Fig. 3: about 45um is long, and 4 days algaes fall diameter 1mm, brown.
Fig. 4: about 40um is long, and 5 days algaes fall diameter 1mm, faint yellow.
Fig. 5: about 40um is long, and 5 days algaes fall diameter 1mm, brown.
Fig. 6: about 35um is long, and 9 days algaes fall diameter 1mm, yellow.
Fig. 7: about 35um is long, and 5 days algaes fall diameter 1mm, light brown.
Fig. 8: about 30um is long, and 7 days algaes fall diameter 1mm, tawny.
Fig. 9: about 30um is long, and fall algae diameter 1mm for 11 days, yellow.
Figure 10: about 30um is long, and 5 days algaes fall diameter 1mm, yellow.
Figure 11: about 30um is long; 6 days algaes fall diameter 1mm, yellow.
Figure 12: about 25um is long, and 5 days algaes fall diameter 1mm, brown.
Figure 13: about 25um is long, and 11 days algaes fall diameter 1mm, light brown.
Figure 14: about 20um is long, and 9 days algaes fall diameter 1mm, faint yellow.
Figure 15: about 20um is long, and 9 days algaes fall diameter 1mm, and faint yellow, one encloses to external expansion.
Figure 16: about 20um is long, and 15 days algaes fall diameter 1mm, faint yellow.
Figure 17: about 15um is long, and 11 days algaes fall diameter 1mm, faint yellow.
Figure 18: about 13um is long, and 11 days algaes fall diameter 1mm; Brown.
Figure 19: about 12um is long, and 4 days algaes fall diameter 1mm, light brown.
Figure 20: about 30um is long, and 5 days algaes fall diameter 1mm, blackish green (green alga).
【1】Matthews I，Cook P A. Short Commutication: Diatom diet of abalone post-larvate(Haliotis midae) and the effect of pre-grazing the diatom overstory. Mar.freshwat. Res. 1995, 46:545-548.
Claims (2)
1. mix a separation method for purebred diatom in benthic diatom solution, it is characterized in that: carry out according to following steps:
First be the particulate debris filtering of in the mixing benthic diatom solution of 0.05-0.15g/ml more than 75 μm by concentration, then mixing benthic diatom solution is divided into three equal parts, put into centrifuge tube respectively, shake up mixing benthic diatom solution in first centrifuge tube, centrifugal treating 4-5min under the condition of 350-450g RCF, is precipitated A and the first supernatant liquor;
To the first supernatant liquor centrifugal treating 4-5min under the condition of 850-950g RCF, be precipitated B;
Shaken up by mixing benthic diatom solution in second centrifuge tube, under the condition of 850-950g RCF, centrifugal treating 4-5min obtains the second supernatant liquor, and to the second supernatant liquor centrifugal treating 4-5min under the condition of 1650-1750g RCF, is precipitated C;
Shaken up by mixing benthic diatom solution in 3rd centrifuge tube, under the condition of 1650-1750g RCF, centrifugal treating 4-5min obtains the 3rd supernatant liquor, and to the 3rd supernatant liquor centrifugal treating 4-5min under the condition of 2650-2750g RCF, is precipitated D;
Get 0.05-0.2g precipitate A, whole precipitate B, whole precipitate C and whole precipitation D, respectively with the antiseptic sea water dilution mixing of 20-40ml, be precipitated A sea water solution, precipitate B sea water solution, precipitate C sea water solution and precipitation D sea water solution respectively;
By precipitate A sea water solution centrifugal treating 5-6min under the condition of 350-450g RCF, be precipitated A
1, to the precipitate A obtained
1carry out the first centrifugal treating 7 times, be precipitated A
2, the first described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 350-450g RCF to this precipitation obtained, obtain precipitation;
By precipitate B sea water solution centrifugal treating 5-6min under the condition of 850-950g RCF, be precipitated B
1, to the precipitate B obtained
1carry out the second centrifugal treating 7 times, be precipitated B
2, the second described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 850-950g RCF to this precipitation obtained;
By precipitate C sea water solution centrifugal treating 5-6min under the condition of 1650-1750g RCF, be precipitated C
1, to the precipitate C obtained
1carry out the 3rd centrifugal treating 7 times, be precipitated C
2, the 3rd described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 1650-1750g RCF to this precipitation obtained, obtain precipitation;
By precipitation D antiseptic sea water solution centrifugal treating 5-6min under the condition of 2650-2750g RCF, be precipitated D
1, to the precipitation D obtained
1carry out the 4th centrifugal treating 7 times, be precipitated D
2, the 4th described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 5-6min under the condition of 2650-2750g RCF to this precipitation obtained, obtain precipitation;
By the precipitate A obtained
2, precipitate B
2, precipitate C
2with precipitation D
2transfer in appropriate antiseptic sea water respectively with aseptic method, form A
2, B
2, C
2and D
2seawater mother liquor, then according in seawater total plate count measure time doubling dilution method antiseptic sea water carry out gradient dilution, obtain precipitate A
2, B
2, C
2and D
210 of seawater mother liquor
0, 10
-1, 10
-2, 10
-3, 10
-4each concentration gradient algae liquid;
Use plate isolation method
[1]cultivate A
2, B
2, C
2and D
2benthic diatom in each concentration gradient algae liquid, basis of microscopic observation is also collected the dull and stereotyped upper algae without bacterium and the pollution of other Micro Algae and is fallen, and can obtain various benthic diatom purebred.
2. the separation method mixing purebred diatom in benthic diatom solution according to claim 1, is characterized in that:
First be the particulate debris filtering of in the mixing benthic diatom solution of 0.1g/ml more than 75 μm by concentration, then mixing benthic diatom solution is divided into three equal parts, put into centrifuge tube respectively, shake up mixing benthic diatom solution in first centrifuge tube, centrifugal treating 4min under the condition of 400g RCF, is precipitated A and the first supernatant liquor;
To the first supernatant liquor centrifugal treating 4min under the condition of 900g RCF, be precipitated B;
Shaken up by mixing benthic diatom solution in second centrifuge tube, under the condition of 900g RCF, centrifugal treating 4min obtains the second supernatant liquor, and to the second supernatant liquor centrifugal treating 4min under the condition of 1700g RCF, is precipitated C;
Shaken up by mixing benthic diatom solution in 3rd centrifuge tube, under the condition of 1700g RCF, centrifugal treating 4min obtains the 3rd supernatant liquor, and to the 3rd supernatant liquor centrifugal treating 4min under the condition of 2700g RCF, is precipitated D;
Get 0.1g precipitate A, whole precipitate B, whole precipitate C and whole precipitation D, respectively with the antiseptic sea water dilution mixing of 30ml, be precipitated A sea water solution, precipitate B sea water solution, precipitate C sea water solution and precipitation D sea water solution respectively;
By precipitate A sea water solution centrifugal treating 6min under the condition of 400g RCF, be precipitated A
1, to the precipitate A obtained
1carry out the first centrifugal treating 7 times, be precipitated A
2, the first described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 400g RCF to this precipitation obtained, obtain precipitation;
By precipitate B sea water solution centrifugal treating 6min under the condition of 900g RCF, be precipitated B
1, to the precipitate B obtained
1carry out the second centrifugal treating 7 times, be precipitated B
2, the second described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 900g RCF to this precipitation obtained;
By precipitate C sea water solution centrifugal treating 6min under the condition of 1700g RCF, be precipitated C
1, to the precipitate C obtained
1carry out the 3rd centrifugal treating 7 times, be precipitated C
2, the 3rd described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 1700g RCF to this precipitation obtained, obtain precipitation;
By precipitation D antiseptic sea water solution centrifugal treating 6min under the condition of 2700g RCF, be precipitated D
1, to the precipitation D obtained
1carry out the 4th centrifugal treating 7 times, be precipitated D
2, the 4th described centrifugal treating is by the appropriate antiseptic sea water dilution mixing of precipitation, and the sea water solution centrifugal treating 6min under the condition of 2700g RCF to this precipitation obtained, obtain precipitation;
By the precipitate A obtained
2, precipitate B
2, precipitate C
2with precipitation D
2transfer in appropriate antiseptic sea water respectively with aseptic method, form A
2, B
2, C
2and D
2seawater mother liquor, then according in seawater total plate count measure time doubling dilution method antiseptic sea water carry out gradient dilution, obtain precipitate A
2, B
2, C
2and D
210 of seawater mother liquor
0, 10
-1, 10
-2, 10
-3, 10
-4each concentration gradient algae liquid;
Use plate isolation method
[1]cultivate A
2, B
2, C
2and D
2benthic diatom in each concentration gradient algae liquid, basis of microscopic observation is also collected the dull and stereotyped upper algae without bacterium and the pollution of other Micro Algae and is fallen, and can obtain various benthic diatom purebred.
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| CN108048327A (en) * | 2018-02-13 | 2018-05-18 | 海南大学 | A kind of new method for efficiently separating diatom |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280271A (en) * | 2008-05-27 | 2008-10-08 | 蔡志武 | Production unit for microalgae industrialization and method for producing microalgae |
| CN102643751A (en) * | 2012-04-25 | 2012-08-22 | 同济大学 | Method for quickly separating and purifying chlorella |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101280271A (en) * | 2008-05-27 | 2008-10-08 | 蔡志武 | Production unit for microalgae industrialization and method for producing microalgae |
| CN102643751A (en) * | 2012-04-25 | 2012-08-22 | 同济大学 | Method for quickly separating and purifying chlorella |
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| Title |
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| 李洪武，宋培学: "《海洋浮游生物学》", 31 August 2012 * |
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