CN108048327A - A kind of new method for efficiently separating diatom - Google Patents
A kind of new method for efficiently separating diatom Download PDFInfo
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- CN108048327A CN108048327A CN201810148737.6A CN201810148737A CN108048327A CN 108048327 A CN108048327 A CN 108048327A CN 201810148737 A CN201810148737 A CN 201810148737A CN 108048327 A CN108048327 A CN 108048327A
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a kind of new methods for efficiently separating diatom, it is main include configuration culture solution and diatom separation and purifying the step of, wherein, the separation and purifying of diatom include gathering the water sample containing diatom, with bolting silk net filtration water sample, water sample sterilization processing, enrichment incubation diatom, count the density of diatom, separate diatom, test tube containing diatom is placed in suitable under conditions of and is cultivated, the new separated diatom kind of microscopy, if without other biological and miscellaneous bacteria, then separate success, the advantage of the invention is that the time used is short, it is easy to operate, it avoids target diatom and there are problems that the probability of success in by bacterium pollution and transfer process to influence work efficiency, microalgae separation is greatly improved on the whole, the work efficiency of purification.Its good separating effect, gained algae density are high, suitable for being isolated and purified from natural water, breeding water body, by the diatom of bacterium pollution or other miscellaneous algae pollutions, therefore possess good application prospect.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of new method for efficiently separating diatom, more particularly, to
The quick separating of diatom, purifying.
Background technology
Culture, the application of microalgae, it is necessary first to algae.Algae is all the fortune from the biota that natural water area mixes at first
It is separated with certain method, obtains monoculture, and preserved, used for culture.Separation, purifying microalgae are its progress
The indispensable technology of scientific research.Diatom be one of main producers of marine organisms and primary productivity of marine ecosystem it
One；Diatom is also the direct or indirect bait of marine animal and its young simultaneously.The annual food of natural marine site Chinese green food
In, diatom accounts for 54%.The juvenile fish such as catfish, sardine are also using diatom as Major Foods.As it can be seen that the height of marine economic animal yield
There is close relationship with the quantity of diatom.With the development of mariculture industry, artificial mass propgation diatom, to solve economic sea
The bait of the animal artificial breeding young is produced, this is to improve the essential key problem in technology of seedling yield.In addition, diatom is being cured at present
Medicine industrially also has been widely used, can be as the filler of filtering agent, adsorbent, papermaking, rubber, cosmetics and coating
Deng and nano material, construction material and thermal insulation material etc..Isolating and purifying for diatom is that it is asked using what must be solved first
Topic.
The prior art mainly has following separation method：Sample series dilution method, water droplet partition method, micro pipette partition method,
Solid medium partition method.
1. isolation by dilution method：A. principle：Under conditions of sterile working, to separated sample using progressively diluted side
Method, side dilution, side microscopy under the microscope when sample is often dripped in the visual field containing only 1 cell, that is, stop dilution, start point
Connect culture.B. method：Algae solution sample to be separated is added in first test tube first, equivalent culture is added in other test tubes
Liquid adds in the volume of culture solution depending on the concrete condition of separation sample (such as 10ml), draws and cultivate from the first test tube
The algae solution sample (10mL) of liquid same volume is added in second test tube, and fully vibration shakes up, and algae solution has been diluted 2 times at this time, then
With the pipette of a new disinfection, the dilution algae solution (10mL) drawn in second test tube is added in the 3rd test tube, as before
Vibration, makes uniformly to dilute, and algae solution has been diluted 4 times at this time, and later test tube is diluted successively using same method, until microscopy
Only 1 cell in dilute sample is often dripped, can be cultivated with such dilution algae solution, final dilution kind is dispensed into not
In same test tube, test tube is stoppered with tampon, test tube is placed on the place of diffusing solar, gently shaken daily several times, find examination
Microscopy is carried out after having algae color in pipe, represents to separate the target for successfully, having reached separation at this time, having purified if microscopy to single,
Culture can be enlarged.C. application scenario：Sample series isolation by dilution method equipment is simple, easy to operate, and workload is small, but this
A technology bears the character of much blindness, and separated sample differs very likely from two or more cells, separated microalgae single
Surely it is target species, but may possibly be separated other kinds or novel species, the initial gross separation of water sample is taken very particularly with from natural water area
It is suitble to, and there is no limit for species, the size for microalgae.
2. micro pipette partition method：A principles:Under aseptic technique, with superfine micro pipette, under the microscope target
Algae sample moves on to another slide from a slide, is operated repeatedly using same method, until there was only target list in microscopy water droplet
Until kind.B methods：In the top of micro pipette one medical latex tube for being about 8cm of set, lock out operation, breast is compressed with finger
Sebific duct is to control sucking action.Separated water sample is placed on glass slide, micro pipette mouth is aligned under the microscope to be separated
Frustule, loosens finger, and frustule is inhaled into micro pipette；Then the water droplet of sucking-off is placed on another glass slide, microscope inspection
It looks into water droplet and whether is only drawn onto frustule, by so operating repeatedly again, until reaching the separated target of single.Then separation
The frustule gone out, which pours, to be pre-loaded in the test tube of culture solution, is placed under suitable illumination and is cultivated, and test tube has microscopy after algae color, training
The test tube for single is supported, other are underproof to discard.C is applied：The characteristics of micro pipette partition method is easily to find specific species, institute
Equipment is also relatively simple, but operating technology difficulty is larger, and often drawing a unicellular needs several times several times could be into ten
Work(, and compared suitable for the larger or Filamentous algae of separation individual, such as spirulina, Skeletonema Greville, smaller algae with the method
It is difficult.
3. water droplet partition method:Principle:Under conditions of sterile working, algae sample to be separated is dripped with micro pipette on slide
Into sizeable small water droplet, Tube propagation only can be poured there are one single to be separated in microscopy water droplet.Method:With small
Beaker fills diluted algae sample, by micro pipette be inserted into algae sample in, extract micro pipette, extra algae solution is allowed to ooze, then nozzle with
The glass slide contact sterilized, i.e., stay in there are one small water droplet on glass slide.One 3~4 drop of glass slide drop, is spaced a spacing
From then observing under the microscope.If only there are one separated frustule (no other biological mixes) is needed in a water droplet, i.e.,
The water droplet is poured in test tube with pipette, extract culture solution, test tube mouth tampon beyond the Great Wall is put and cultivated under appropriate conditions.This point
Key from method is that the dilution of algae sample will be suitable for (being diluted to each water droplet, there are about 1~2 frustules), and drop size is suitable, with low
It can see that water droplet is wholly or largely advisable under times mirror, and observe accurate, rapid.Using:Water droplet partition method is easy to operate
It is easy, but it is noted that issue noted above in concrete operations.The method for frustule size there is no limit, be particularly suited for
Separation species dominant in culture solution.
4. solid medium separation method (tablet partition method):It is in routine as the separatory solid medium of microalgae
Fluid nutrient medium in add in a certain amount of agar, and after making its dissolving and autoclave sterilization, by appropriate method,
Algae sample to be separated is seeded on culture medium, is cultivated by the regular hour, and single algae is grown on culture medium and falls and reaches
Separated target.Solid medium prepares and sterilizing:Added in conventional fluid nutrient medium a certain amount of agar (1.0%~
2.0%), after heating for dissolving, it is dispensed into conical flask, is placed in high-pressure steam sterilizing pan device and sterilizes, 121 DEG C of temperature, the time 20
~30min, when culture medium is cooled to 50 DEG C, sterile working is poured into the culture dish of sterilizing for use.The method of inoculation:Inoculation
It is carried out on superclean bench, there are two ways to inoculation.Spray-on process first carries out algae sample to be separated with disinfection seawater
Appropriate dilution is fitted into the laryngeal sprayer sterilized, opens culture dish lid, algae sample is ejected into culture base plane
On, algae sample is made to form finely dispersed one layer of thin droplet in media surface.Water sample is diluted to suitable degree and refers to water sample
More than 1cm must be separated by there are one frustule by being injected on culture base plane, and it is too near that the algae otherwise grown in the future falls distance, is not easy
Separation.Method of scoring after oese is sterilized on alcolhol burner, dips algae sample and line 3 for the first time is gently done on culture base plane
~4, culture dish is rotated about 70 ° of angles, and oese is sterilized on alcolhol burner, second stroke is done by first time drawn area
Line.The the 3rd, the 4th line is done with same method.Since the frustule on first time streak inoculation ring is relatively more, first
Partition frustule intensive may cannot separate, but may possibly be separated out individual algal community by line below.Culture:Training
It supports after being inoculated on base, is placed under suitable illumination and cultivates, through culture after a while, algal community is grown on culture medium,
After microexamination, single target algae is fallen with very thin dissecting needle and is taken out together with a fritter culture medium, immigration is equipped with
It is cultivated in the test tube of culture solution, the microscopy after having algae color in test tube such as mixes without other biological, has reached the separated mesh of single
Mark.The application of solid medium partition method:The separation method of the solid medium made of agar is not appropriate for all microalgaes, greatly
Most green algas can grow on this culture medium, and so as to reach the separated target of single, but the microalgae having is trained in agar
It supports and grows poor on base, or even cannot grow, such as some Freshwater Chrysophytes and Diatomeae, this may be the surface tension of agar medium
Larger reason, can add in the surfactant of 100~500 μ g/L in the medium can promote to grow.General culture
Nitrogen salt in base used in nutritive salt is nitrate nitrogen, and the whips such as sodium nitrate, ball algae, Zhanjiang fork whip algae, Chaetoceros muelleri are replaced with urea
Can be with normal growth on agar medium, these types of single-cell algae has urease, and energy decomposing urea makees nitrogen source, and utilizes nitre
Sour sodium makees nitrogen source, needs nitrate-nitrogen reduction into ammonium nitrogen, and the heterotroph that these types of algae contains is less, and due to sterile
In environment, so algae energy normal growth in ammoniacal nitrogen, and normal growth is unable in nitrate nitrogen.Agar in solid medium
Addition, flexibly to be used according to the quality of agar, culture medium is too hard, influences the sustained release of nutritive salt, and moisture easily loses, unfavorable
The growth that algae falls, culture medium is too soft to be unfavorable for ruling, and is easily scratched culture medium, is influenced to operate.Solid medium partition method works
It is cumbersome, heavy workload, but it is higher to separate the successful probability of single.
In view of the defects of in the presence of the prior art, it is necessary to new thinking is proposed to the separated method of existing diatom.
The content of the invention
In view of the above-mentioned problems, the present invention provides a kind of new methods for efficiently separating diatom.
The present invention is that technical solution is used by solving its technical problem：
A kind of new method for efficiently separating diatom, it is characterised in that including following preparation steps：
Step 1：Culture solution is configured, wherein, the culture solution includes I liquid and II liquid；
1.1 configuration I liquid：Sodium nitrate 4-6g, urea 1-3 g, potassium dihydrogen phosphate 0.5-1.5g, ironic citrate 0.2-0.4g, sulfuric acid
Manganese 0.01-0.03g, EDTA-Na20.5-1.5g, vitamin B110-30 mg, vitamin B120.05-0.15mg, pure water
50-150mL；
1.2 configuration II liquid：Sodium metasilicate 5-7g, pure water 50-150mL；
1.3 are separately added into I liquid and II liquid according to 1/1000 ratio, and shake is mixed nutritive salt and supplement boils the O of consumption2And CO2,
To obtain culture solution；
Step 2：The separation and purifying of diatom；
2.1 take the shrimp-cultivation pond water sample containing diatom of brown with hydrophore；
2.2 filter water sample through 300-500 mesh sieve tulles, remove the enemy zooplankter of diatom；
2.3 water sample sterilization processings proportionally add in penicillin 200-300mg/L, kanamycins 100-140mg/ into water sample
L, streptomysin 100-140mg/L shakes up, and removes the bacterium in water sample；
2.4 are contained in water sample in conical flask, diatom culture solution are added in, to the nutritive salt of diatom abundance, i.e. enrichment incubation diatom
3-5 days；
2.5 count the density of microalgae cell with blood counting chamber, and are diluted to algae cell density as 300-500/ml；
2.6 microscopies separate diatom, and the water sample containing diatom is shaken up, and 8-12 microlitres of water sample is drawn with liquid-transfering gun, divide 3-5 etc. points of drops
On the special glass slide on conventional slide, biology microscope Microscopic observation is placed in, not lid coverslip：
If the water droplet only has 1 target frustule without other biological, which is put into equipped with 4-6 milliliters
In the test tube of diatom culture solution；
If containing target diatom simultaneously also containing miscellaneous biology n in the water droplet, n 2.5 μ l disinfections are added on the water droplet
Water or culture solution, then be mixed water sample with another disinfection pipette tips agitation and draw the water sample, the water droplet for being divided into 2.5 μ l of every drop drops in load
On special glass slide on slide；
Microscopy again puts the special glass slide only containing target diatom in the test tube for filling culture solution into, repetitive operation 20
It is more than sample；
2.7 are placed in test tube in illumination box, and it is 60-80 μm of ol/m to control 24-28 DEG C of temperature, illuminance2·S-1, cultivate 5-
10 days；
There is brown in 2.8 test tube bottoms, are placed in disinfection suction pipe absorption water sample on the glass slide of disinfection and cover sterilized lid
Slide is placed in micro- Microscopic observation, if only a kind of diatom, without other biologicals such as miscellaneous algae, bacterium and fungies, shows to be separated into
Work(.
Further, in step 1, I liquid is configured：Sodium nitrate 5g, urea 2g, potassium dihydrogen phosphate 1g, ironic citrate 0.3g, sulphur
Sour manganese 0.02g, EDTA-Na21g, vitamin B120mg, vitamin B120.1mg, pure water 100mL configure II liquid：Sodium metasilicate
6g, pure water 100mL, the configuration of culture solution should be weighed, dissolved one by one one by one, and water temperature could add in dimension life after should being less than 60 DEG C
Element.
Further, in step 2 2.2, water sample is filtered through 400 mesh sieve tulles, removes the enemy zooplankter of diatom.
Further, in step 2 2.3, configuration penicillin 250mg/L, kanamycins 120mg/L, streptomysin 120mg/L.
Further, in step 2 2.4, volume of water sample is no more than the 2/3 of container, is placed on 24-28 DEG C of temperature, 80 μm of ol/
m2·S-1Illumination under preculture 3-5 days, increase the diatom density in water sample, convenient for separation screening.
Further, in step 2 2.5, algae cell density is 400/ml.
Further, in step 2 2.6, special glass slide is that the coverslip for being 0.13-0.17mm with thickness is cut into 12*24mm
Specification.
In addition, in step 2 2.4, water sample is contained in 500mL or 1000mL conical flasks, be placed in illumination box or
In the air-conditioned room for having illumination cultivation frame.
The beneficial effects of the invention are as follows：The present invention is main to include configuration training as a kind of new method for efficiently separating diatom
The step of separation and purifying of nutrient solution and diatom, the advantage of the invention is that the time used it is short, it is easy to operate, avoid target
Diatom is greatly improved micro- on the whole without there is the probability of success in by bacterium pollution and transfer process to influence work efficiency
Algae separation, the work efficiency of purification.Its good separating effect, gained algae density are high, suitable for from natural water, breeding water body, by
The diatom of bacterium pollution or other miscellaneous algae pollutions isolates and purifies, therefore possesses good application prospect.
Specific embodiment
The method of the present invention for efficiently separating beneficial diatoms is carried out below by specific embodiment further
Explanation：
By the use of diatom as the seed of prawn seed-rearing open-mouthed bait, not only survival rate of seedling is high, and shrimp seedling health is healthy and strong, beneficial to the later stage into
The success of shrimp aquaculture, in addition in the shrimp-cultivation pond of beneficial diatoms rich content, the disease-free and speed of growth of prawn health is fast.For
Beneficial target diatom is obtained, it is necessary to carry out single separation, selection and breeding excellent mesh to the biological group mixed in pond waters
Diatom is marked, it is generated after frustule division 2 new thin in addition, cell division is a kind of most common modes of reproduction of diatom
In born of the same parents, one is identical with mother cell size, another is then small compared with mother cell, and so continuous division is gone down, and individual will be less and less,
Therefore laboratory cultures diatom, it is necessary to constantly separation, screening high-quality algae.Thus, the isolation technics of microalgae is very heavy
Will, it is microdisk electrode and the basis further applied and key link.Different microalgaes selects suitable separation method, right
Single separation success or not is critically important.
Separation, purifying the invention solves diatom in cultivating pool, in order to train and be applied to prawn for a certain area later
Nursery, cultivating system realize healthy breeding, the cultivation of prawn.
It is as follows：
Step 1: configuration culture solution
Cultivate mother liquor（1000 times of concentration）Formula：I liquid：Sodium nitrate 5g, urea 2g, potassium dihydrogen phosphate 1g, ironic citrate 0.3g, sulphur
Sour manganese 0.02g, EDTA-Na21g, vitamin B1 20mg, vitamin B12 0.1mg, pure water 100mL.II liquid：Sodium metasilicate
6g, pure water 100mL.The configuration of culture solution should weigh one by one, dissolve one by one, and water temperature could add in dimension life after should being less than 60 DEG C
Element.The preparation method of liquid medium is：Nature seawater 2000mL is added in the conical flask of 3000mL, scalding is cold
But after, on superclean bench, I liquid and II liquid are separately added into according to 1/1000 ratio, is shaken, nutritive salt is mixed and supplement is boiled
The O of consumption2And CO2。
The fast-growth breeding of the particularly suitable diatom of culture formula of liquid such as adds in the existing diatom culture medium of amount of sodium metasilicate
3 times, silicon concentration is the optimised quantity needed for the breeding of most of growth of diatom algae, because frustule wall contains silicon, is contained in environment
There is element silicon abundant in right amount, growth of diatom algae rate can be made up to maximum.The addition of vitamin is also needed for most of diatom
Optimised quantity.
Step 2: the separation of diatom, purification step
The shrimp-cultivation pond water sample containing diatom of brown is taken with hydrophore.Water sample is filtered through 400 mesh sieve tulles, is removed
The enemy zooplankter of diatom（The filtering of 400 mesh sieve tulles can remove Copepods, cladocera, wheel animalcule and trip and flutter the life of the enemies such as worm
Object avoids diatom from being consumed）.Water sample sterilization processing proportionally adds in penicillin 250mg/L, kanamycins into water sample
120mg/L, streptomysin 120mg/L, shake up, and remove the bacterium in water sample（Penicillin is to gram-positive bacteria, Grain-negative ball
Bacterium, conveyor screw and actinomyces have good bactericidal effect and therapeutic effect；Streptomysin is used to kill except gram-negative bacteria；Kanamycins
There is killing or inhibiting effect to gram-positive bacteria and negative bacterium and mycoplasma, three kinds of antibiotic add together, and bactericidal effect is more
It is good）.Water sample is contained in 500mL or 1000mL conical flasks, adds in diatom culture solution, to the nutritive salt of diatom abundance, i.e.,
Enrichment incubation diatom 3-5 days.Volume of water sample is no more than the 2/3 of container, is placed on 24-28 DEG C, 60-80 μm ol/m of temperature2·S-1
Illumination under preculture 3-5 days（It is placed in illumination box or has in the air-conditioned room of illumination cultivation frame）, increase the diatom in water sample
Density, convenient for separation screening.（If the density in water sample is sufficiently large, this step can be removed）It is counted with blood counting chamber micro-
The density of frustule, and be diluted to algae cell density as 400/ml, i.e. 10 μ l contain 4 frustules.Microscopy divided silicon
Algae：Water sample containing diatom is shaken up, draws 10 microlitres of water sample with liquid-transfering gun, point 4 deciles drop in the spy on conventional slide
On glass slide processed（Special glass slide is cut into 12*24 mm specifications with the coverslip that thickness is 0.13-0.17mm, this specification is most suitable
It closes）.（It is required that：Pipette tips, glass slide, special glass slide and culture solution all sterilize by strict sterilization；Often drip 2.5 μ l of sample, this
Amount can see the water droplet largely or entirely under 10 times of eyepieces, and water droplet is too small to be easy to kill, and water droplet is unfavorable for greatly very much one
Secondary property observe and determine this in dripping whether only there are one frustule, the water size of this water droplet is critically important.）.It is placed in biology
Micro- Microscopic observation（Not lid coverslip）If only there are one target frustules without other biological for the water droplet, this is special
Glass slide is put into the test tube equipped with 5 milliliters of diatom culture solutions.If also contain miscellaneous life simultaneously containing target diatom in the water droplet
Object n then adds in n 2.5 μ l on the water droplet（Or write as 2.5n μ l）Disinfectant or culture solution, then stirred with another disinfection pipette tips
Dynamic to be mixed water sample and draw the water sample, the water droplet for being divided into 2.5 μ l of every drop is dropped on the special glass slide on glass slide（This place
Theoretically the water droplet of every 2.5 μ l is biological containing there are one after reason）, microscopy, the special glass slide only containing target diatom is put again
Into in the test tube for filling culture solution.（Entire special glass slide containing target diatom is put into test tube and is cultivated, before avoiding
Operating method target microalgae is rushed in the loss occurred in test tube, improve the separated success rate of target diatom）.Repetitive operation
It is more than 20 samples.Test tube is placed in illumination box（It is 60-80 μm of ol/m to control 24-28 DEG C of temperature, illuminance2·
S-1）Culture 1 week.There is brown in test tube bottom, is placed on the glass slide of disinfection and is covered through disappearing with disinfection suction pipe absorption water sample
The coverslip of poison, is placed in micro- Microscopic observation, if only a kind of diatom, without other biologicals such as miscellaneous algae, bacterium and fungies, shows
It separates successfully.
Above the preferable of the present invention is implemented to be illustrated, certainly, the present invention can also use and above-mentioned implementation
The different form of mode and similar field, these do not form any restrictions to present embodiment, are familiar with this field
The equivalent conversion or corresponding change that technical staff is made on the premise of without prejudice to spirit of the invention, should all belong to this hair
In bright protection domain.
Claims (8)
1. a kind of new method for efficiently separating diatom, it is characterised in that including following preparation steps：
Step 1：Culture solution is configured, wherein, the culture solution includes I liquid and II liquid；
1.1 configuration I liquid：Sodium nitrate 4-6g, urea 1-3 g, potassium dihydrogen phosphate 0.5-1.5g, ironic citrate 0.2-0.4g, sulfuric acid
Manganese 0.01-0.03g, EDTA-Na20.5-1.5g, vitamin B110-30 mg, vitamin B120.05-0.15mg, pure water
50-150mL；
1.2 configuration II liquid：Sodium metasilicate 5-7g, pure water 50-150mL；
1.3 are separately added into I liquid and II liquid according to 1/1000 ratio, and shake is mixed nutritive salt and supplement boils the O of consumption2And CO2,
To obtain culture solution；
Step 2：The separation and purifying of diatom；
2.1 take the shrimp-cultivation pond water sample containing diatom of brown with hydrophore；
2.2 filter water sample through 300-500 mesh sieve tulles, remove the enemy zooplankter of diatom；
2.3 water sample sterilization processings proportionally add in penicillin 200-300mg/L, kanamycins 100-140mg/ into water sample
L, streptomysin 100-140mg/L shakes up, and when placement 12 is small, removes the bacterium in water sample；
2.4 are contained in water sample in conical flask, diatom culture solution are added in, to the nutritive salt of diatom abundance, i.e. enrichment incubation diatom
3-5 days；
2.5 count the density of microalgae cell with blood counting chamber, and are diluted to algae cell density as 300-500/ml；
2.6 microscopies separate diatom, and the water sample containing diatom is shaken up, and 8-12 microlitres of water sample is drawn with liquid-transfering gun, divide 3-5 etc. points of drops
On the special glass slide on conventional slide, 10 times of biology microscope Microscopic observations are placed in, not lid coverslip：
If the water droplet only has 1 target frustule without other biological, which is put into dress together with water droplet
In the test tube for having 4-6 milliliters of diatom culture solutions；
If containing target diatom simultaneously also containing miscellaneous biology n in the water droplet, n 2.5 μ l disinfections are added on the water droplet
Water or culture solution, then be mixed water sample with another disinfection pipette tips agitation and draw the water sample, the water droplet for being divided into 2.5 μ l of every drop drops in load
On special glass slide on slide；
Microscopy again puts the special glass slide only containing target diatom in the test tube for filling culture solution together with water droplet into, repeats
It operates more than 20 samples；
2.7 are placed in test tube in illumination box, and it is 60-80 μm of ol/m to control 24-28 DEG C of temperature, illuminance2·S-1, cultivate 5-
10 days；
There is brown in 2.8 test tube bottoms, are placed in disinfection suction pipe absorption water sample on the glass slide of disinfection and cover sterilized lid
Slide is placed in micro- Microscopic observation, if only a kind of diatom, without other biologicals such as miscellaneous algae, bacterium and fungies, shows to be separated into
Work(.
2. a kind of new method for efficiently separating diatom according to claim 1, it is characterised in that：In step 1, I is configured
Liquid：Sodium nitrate 5g, urea 2g, potassium dihydrogen phosphate 1g, ironic citrate 0.3g, manganese sulfate 0.02g, EDTA-Na21g, vitamin B1
20mg, vitamin B120.1mg, pure water 100mL configure II liquid：Sodium metasilicate 6g, pure water 100mL, the configuration of culture solution
It should weigh, dissolve one by one one by one, water temperature could add in vitamin after should being less than 60 DEG C.
3. a kind of new method for efficiently separating diatom according to claim 2, it is characterised in that：In step 2 2.2, warp
400 mesh sieve tulles filter water sample, remove the enemy zooplankter of diatom.
4. a kind of new method for efficiently separating diatom according to claim 1 or 3, it is characterised in that：In step 2 2.3,
Configure penicillin 250mg/L, kanamycins 120mg/L, streptomysin 120mg/L.
5. a kind of new method for efficiently separating diatom according to claim 1 or 3, it is characterised in that：In step 2 2.4,
Volume of water sample is no more than the 2/3 of container, is placed on 24-28 DEG C of temperature, 80 μm of ol/m2·S-1Illumination under preculture 3-5 days,
Increase the diatom density in water sample, convenient for separation screening.
6. a kind of new method for efficiently separating diatom according to claim 1 or 3, it is characterised in that：In step 2 2.5,
Algae cell density is 400/ml.
7. a kind of new method for efficiently separating diatom according to claim 1 or 3, it is characterised in that：In step 2 2.6,
Special glass slide is that the coverslip for being 0.13-0.17mm with thickness is cut into 12*24mm specifications.
8. a kind of new method for efficiently separating diatom according to claim 5, it is characterised in that：In step 2 2.4, by water
Sample is contained in 500mL or 1000mL conical flasks, is placed in illumination box or has in the air-conditioned room of illumination cultivation frame.
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| CN113881568A (en) * | 2021-10-15 | 2022-01-04 | 内蒙古医科大学 | Microalgae separation method by means of cell climbing sheet |
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