CN108318307A - Very thin Euglena measures the algae solution preparation method and application of aquatic particle bio-toxicity - Google Patents
Very thin Euglena measures the algae solution preparation method and application of aquatic particle bio-toxicity Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
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Abstract
The present invention relates to the preparation method and applications for the very thin Euglena algae solution for measuring aquatic particle bio-toxicity, belong to Environmental Health toxicology field, including the culture of algae and the preparation of algae solution, specifically include following steps：Very thin Euglena algae is chosen, algae is seeded in the container equipped with fluid nutrient medium under aseptic condition, inoculation is completed, and it is spare to logarithmic phase to be placed in culture in constant temperature illumination box；By the algae solution of culture to logarithmic phase through secretly postponing, OD is measured with spectrophotometer or microplate reader680nmValue；The OD measured according to step B680nmValue draws cell growth curve, selects cell density range 1 × 105To 1 × 106The very thin Euglena algae solution of the logarithmic phase of a cells/ml, you can for being measured to aquatic particle bio-toxicity.The present invention also provides the very thin Euglena algae solutions in the measurement of aquatic particle bio-toxicity, and the method for the present invention can intuitively react the bio-toxicity of aquatic particle.
Description
Technical field
The invention belongs to Environmental Health toxicology fields, are related to the preparation of very thin Euglena algae solution, and in particular to measure water body
The preparation method and application of the very thin Euglena algae solution of particulate matter bio-toxicity.
Background technology
With the progress of science and technology, the development of industrial and agricultural production, the mankind have synthesized more and more chemicals, therewith
What is come is that the trend that wide variety, quantity take place frequently is presented in the toxic pollutant entered in environment, tends to be complicated to the harm of environment
Change and synthesization, the environmental problem thus caused are increasingly sharpened.The non-speed hair of the heavily contaminateds industry such as plating, process hides, pharmacy, chemical industry
Exhibition, industrial wastewater are largely discharged into municipal sewage plant, and noxious material type also increases therewith in waste water.Pollutant is not only broken up
Water body physical and chemical index, also has an immense impact on to aqueous bio toxicity index, brings to government administration section and greatly choose
War.When there is environmental pollution accident, some conventional physical and chemical indexes for being obtained according to existing chemical measurement method often without
Method judges the severity of pollution, and it is to easily cause masses' fear especially to threaten masses' drinking water safety, and situation is caused to lose
Control.
Aquatic environment is polluted, pollutant often has uncertainty, is also difficult to really to the synthesis toxicity index of water quality
It is fixed.The pollution situation of various complexity can not effectively, be comprehensively coped in view of traditional water quality detection means, there is an urgent need to a kind of spirits
Biological detecting method quick, inexpensive and that biological health indicator can be reacted.And Measurement for Biotechnique, exactly utilize biology by dirt
The variation of the reaction or physiological function that are generated after the harm of dye object or murder by poisoning is conducive to determine poisonous substance peace to evaluate water pollution situation
Full concentration understands pollution and aquatile is directly endangered and influenced, judges water pollution type and extent.Very thin Euglena is acute
Toxicity tries one kind compared with based in the Algal Ecology toxicological experiment of laboratory, has Biological indicators are various, experiment is simple and convenient etc.
Feature, thus this technology is widely used in terms of aqueous bio toxicity.According to current studies have shown that very thin naked
Biological indicators and the water environment biologicals such as algae acute toxicity, Euglena biomass, Contents of Photosynthetic Pigments and superoxide dismutase activity
Toxicity is closely bound up, but nanoparticles and the toxicity and toxicological effect mechanism of plain particles object are unclear in water body, need
It further to study.
Invention content
The present invention is to solve intuitively reflect toxicity performance of the aquatic particle on organism in the prior art, is carried
Preparation method and application for the very thin Euglena algae solution for measuring aquatic particle bio-toxicity in one, can fully understand water
The poisonous effect of body particulate matter, and can deeper into understanding aquatic particle toxicological effect mechanism and approach, can more embody
In practical water body environment particulate matter there are the phenomenon that and information.
The present invention be realize its purpose the technical solution adopted is that：
Very thin Euglena measures the algae solution preparation method of aquatic particle bio-toxicity, including the culture of algae and the system of algae solution
It is standby, specifically include following steps：
Specifically include following steps：
A, the culture of very thin Euglena：Very thin Euglena algae is chosen, algae is seeded under aseptic condition and is trained equipped with frond
In the conical flask for supporting base, inoculation is completed, and it is spare to logarithmic phase to be placed in culture in constant temperature illumination box；
B, the determination of very thin Euglena cell density：It takes 200ul to cultivate to the frustule culture solution of logarithmic phase, uses microplate reader
Measure absorbance OD at its 680nm680nm, wherein OD680nmThere are following relationships for relationship between cell concentration：
Y=(94.309 × OD680+0.9114)×104(R2=0.999), Y is cell number in above-mentioned conversion relation
(cells/ml), coefficient R2=0.999；
C, the preparation of the very thin Euglena algae solution for measuring aquatic particle bio-toxicity：The algae measured according to step B
Liquid cell content selects cell density range 1 × 105To 1 × 106The very thin Euglena algae solution of logarithmic phase of cells/ml, you can use
It is measured in aquatic particle bio-toxicity.
The condition of culture of the constant temperature illumination box is：Intensity of illumination is 2000lx, and temperature is 25 ± 1 DEG C, and humidity is
75%RH, periodicity of illumination 12h：12h, the initial pH of algae solution are 7.0~8.0, stationary culture, daily shaking flask 2-3 times, while mutually
Replace position.
Preferably, the very thin Euglena algae solution of the biological subject is a concentration of：1×105cells/ml。
The fluid nutrient medium includes following components, in 1000ml fluid nutrient mediums, CH3COONa·3H2O1.0g,
(NH4)2HPO41.0g, KH2PO41.0g, MgSO4·7H2O 0.2g, FeSO4·7H2O 0.003g, CaCl20.02g, EDTA
TriplexIII 0.0637g；Vitamin stock solution 1ml：1,100 121 μ g of μ g, VB of VB；Micro- mother liquor：FeC6H5O75mg,
Na2SiO35mg, ZnSO425 μ g, Na2MoO420 μ g, CuSO4×5H2O 10 μ g, CoCl210 μ g, MnCl2200 μ g, Na2
(EDTA) 5mg, Na2MoO45 μ g, surplus are water.
Measure the very thin Euglena algae solution of aquatic particle bio-toxicity and answering in measurement aquatic particle bio-toxicity
With including the following steps：
A, the culture of very thin Euglena：Very thin Euglena algae is chosen, algae is seeded in aseptic condition and is trained equipped with frond
In the multiple conical flasks for supporting base, inoculation is completed, and it is spare to logarithmic phase to be placed in culture in constant temperature illumination box；
B, the determination of very thin Euglena cell density：It takes 200ul to cultivate to the frustule culture solution of logarithmic phase, measures it
Absorbance OD at 680nm680nm, wherein OD680nmThere are following relationships for relationship between cell concentration：
Y=(94.309 × D680+0.9114)×104(R2=0.999), Y is cell number (cells/ in above-mentioned conversion relation
Ml), coefficient R2=0.999；
C, the preparation of the very thin Euglena algae solution for measuring aquatic particle bio-toxicity：It is measured not according to step b
With the algae solution frustule content of concentration, select cell density range 1 × 105To 1 × 106The logarithmic phase of cells/ml is very thin naked
Algae algae solution, you can measured for aquatic particle bio-toxicity；
D, the acquisition and pretreatment of water body sample：Water sample is acquired, and is retained after filtered through gauze spare；
E, aquatic particle detaches：The water sample of gained in step d is subjected to aquatic particle grain size separation, isolated
Grain object is dissolved in special water, and concentration is consistent with raw water；
F, measurement of the very thin Euglena algae solution to aquatic particle bio-toxicity：Include the following steps,
It takes the very thin Euglena algae solution in logarithmic phase, centrifugation to abandon supernatant, a small amount of PBS is added suspends again and obtain concentrating algae
Liquid takes 10 μ l concentrations algae solutions to be separately added into 50ml each group aquatic particles and measures in sample, mixes and shake up as experimental group, if
It sets special water blank group to be compareed, measures algae growing state, chlorophyll content, superoxide dismutase activity and slender respectively
Born of the same parents' gel electrophoresis takes its stationary value and records, and compares the difference of blank group and experimental group parameter, show that aquatic particle measures sample
This to very thin Euglena each biological indicator damage influence as a result, according to influence result measure aquatic particle bio-toxicity.
The very thin Euglena algae solution for measuring aquatic particle bio-toxicity is in measuring in aquatic particle bio-toxicity
Application, step e water sample particulate separation processing further comprises：
The configuration of S1, special water：Measure common zwitterion (Ca in raw water2+,Mg2+,Na+, Cl-,SO4 2-,NO3-,HCO-
Deng) concentration, with laboratory often use salt and pure water configuration containing identical zwitterion concentration special water；
The separation of S2, particulate matter：The raw water for crossing Coarse Mesh Gauze is divided particulate matter in water by grain size through tangential flow filtration system
From the particulate matter of different-grain diameter is dissolved in special water, and concentration is consistent with raw water.
It is described to measure the very thin Euglena algae solution for measuring aquatic particle bio-toxicity in measurement aquatic particle life
The measurement of application in object toxicity, the algae growing state in step f refers to：Each experimental groups of 200 μ l and blank group algae solution are taken, enzyme is used
Mark absorbance OD at instrument or spectrophotometric determination its 680nm680nm, wherein OD680nmRelationship between cell concentration exists such as
Lower relationship：
Y=(94.309 × OD680+0.9114)×104(R2=0.999), Y is cell number in above-mentioned conversion relation
(cells/ml), coefficient R2=0.999.
The very thin Euglena algae solution for measuring aquatic particle bio-toxicity is in measuring in aquatic particle bio-toxicity
Application, the measuring chlorophyll content in step f refers to：It takes the algae solution of each experimental groups of 10ml and blank group to remove supernatant, is added
Quantitative 80% acetone is protected from light extraction chlorophyll for 24 hours；After the completion of extraction, then take supernatant, respectively measure 470nm, 646nm and
Absorbance D at 663nm470、D646And D663, and the content of chlorophyll a, chlorophyll b and carotenoid is calculated as follows：
Ca=12.21D663-2.81D646(mg/L)；
Cb=20.13D646-5.03D663(mg/L)；
CAR=(1000D470-3.27Ca-104Cb)/229(mg/L)。
The superoxide dismutase activity test experiments refer specifically to：Take the experimental group of each particles things exposures of 10ml
With the algae solution centrifugal treating in control group, supernatant is removed, sodium phosphate buffer is then added, is uniformly mixed, then carry out super oxygen
Compound dismutase activity measures.
The SCGE experiment refers specifically to：Take the experimental group and control group of each particles things exposures of 10ml
In algae solution centrifugal treating, remove supernatant, and 1.5% low melting-point agarose mixing takes the 100ul mixed liquors to be added dropwise in pre- place
On glass slide after reason, coated with coverslip, 4 DEG C of standing 10min, condensed offset plate repaves 0.5% low melting-point agarose conduct
Second layer glue, 4 DEG C stand to solidifying, after submerge 20min in the alkaline electrophoresis liquid of precooling, promote DNA to untwist, in 20V and
200mA condition electrophoresis 20min take out offset plate, submerge 15min with neutralizer, upper sem observation is taken pictures, statistical analysis comettail feelings
Condition, in comet, the size that DNA is damaged serious conditions and its tail portion DNA content and back range is proportionate.
The beneficial effects of the invention are as follows：Particulate matter component is complicated in water body, and each pollutant of single evaluation can not evaluate water body
The comprehensive toxicity of particulate matter, the present invention using the growth of very thin Euglena, chlorophyll content, superoxide dismutase activity index and
The situation of change of DNA loss parameters indicates the bio-toxicity of aquatic particle, realize comprehensively, intuitively react aquatic particle
Synthetic biological toxicity, accomplish early warning, ensure water quality safety.The present invention is using very thin Euglena as instruction biological detection water
The bio-toxicity of body, growth is very fast, culture is relatively easy, cost is smaller, sensitive to water environment toxic reaction, with chemical detection
Method is compared, and more can really reflect the bio-toxicity of water environment.It is tried using very thin algae acute toxicity in addition, the present invention is also demonstrated
The reliability as particulate matter bio-toxicity pollution level index in water environment is tested, is a kind of effective and convenient evaluation method.
Description of the drawings
Fig. 1 is the Gong Hu (A) and Ecological Restoration Area (B) water sample particle size distribution map of the present invention；
Fig. 2 is derived from the very thin Euglena growth curve of tribute lake water sample (A) and Ecological Restoration Area water sample (B)；
Fig. 3 is derived from the very thin Euglena green cellulose content of tribute lake water sample (A) and Ecological Restoration Area water sample (B)；
Fig. 4 is derived from the superoxide dismutase activity of tribute lake water sample (A) and Ecological Restoration Area water sample (B)；
Fig. 5 is derived from the detection of the very thin Euglena DNA damage of tribute lake water sample (A) and Ecological Restoration Area water sample (B).
Diagram：Grain size ＜ 1 indicates that water sample particle size is less than 1um；The expression water sample particle size of grain size >=1 be more than or
Equal to 1um；Chl-a indicates chlorophyll a；Chl-b indicates chlorophyll b；CAR indicates carotenoid
Specific implementation mode
It is measured by very thin Euglena and finds that aquatic particle influences the algae growing state of very thin Euglena, chlorophyll content, surpasses
Superoxide dismutase activity and DNA parameters, therefore pass through algae growing state, chlorophyll content, the superoxides to very thin Euglena
The measurement of dismutase activity and DNA parameters can intuitively reflect toxicity performance of the aquatic particle on organism, below by tool
The present invention will be further described for body embodiment.
1, material
Algae：The very thin Euglena of algae used in the present invention, algae are cultivated by Inst. of Hydrobiology, Chinese Academy of Sciences typical case
Object preservation committee fresh water algae algae library (FACHB) provides.
Instrument：Illumination box, spectrophotometer or microplate reader, Zata- potentiometers
Water sample sampling：
By on-the-spot investigation, investigation, on the basis of considering natural conditions, various human factors, we are formed in
Tribute arm of lake waters sets the scheme of sampled point.In December, 2016, Taihu Lake is divided into Gong Hu and its Ecological Restoration Area by us, is divided
Each 4 points are had chosen respectively from Gong Hu and its Ecological Restoration Area, amount to 8 sampled points, and water sample is carried out at the 0.5m of underwater
Acquisition.Each sample is all accurately positioned with GPS in gatherer process, and investigated the environmental condition near each sampled point,
Reason situation.Plant residue, the stone etc. in water sample are removed, is then individually positioned in collection vessel.Sampling point number, longitude and latitude
It is shown in Table 1.
1 sampling point distributions table of table
2, the preparation of very thin Euglena algae solution
A, the culture of very thin Euglena algae solution：It chooses：A concentration of 1 × 105The very thin Euglena algae of cells/ml, in sterile item
Algae is seeded in the conical flask equipped with fluid nutrient medium under part, inoculation is completed, and is placed in constant temperature illumination box and is cultivated, and is trained
Foster condition is：Intensity of illumination is 2000lx, and temperature is 25 ± 1 DEG C, humidity 75%RH, periodicity of illumination 12h：12h, at the beginning of algae solution
Beginning pH is 7.0~8.0, stationary culture, daily shaking flask 2-3 times, while mutually replacing position, is cultivated to logarithmic phase, spare；
Aforesaid liquid culture medium prescription is：Euglena uses Checcucci medium cultures, specific formula as follows：1000ml
Fluid nutrient medium contains CH3COONa·3H2O 1.0g, (NH4)2HPO41.0g, KH2PO41.0g, MgSO4·7H2O 0.2g,
FeSO4·7H2O 0.003g, CaCl20.02g, EDTA TriplexIII0.0637g；Vitamin stock solution 1ml：1100 μ g of VB,
VB 121μg；Micro- mother liquor：FeC6H5O75mg, Na2SiO35mg, ZnSO425 μ g, Na2MoO420 μ g, CuSO4×5H2O
10 μ g, CoCl210 μ g, MnCl2200 μ g, Na2(EDTA) 5mg, Na2MoO45 μ g, surplus are water, and culture medium is sequentially weighed and is matched
After placing a night after system or so that solid is completely dissolved using magnetic stirring apparatus, 121 DEG C, 20min high-temperature sterilizations are added after cooling
The vitamin of filtration sterilization and micro- mother liquor.
B, the determination of very thin Euglena cell density：It takes 200ul to cultivate to the frustule culture solution of logarithmic phase, uses microplate reader
Measure absorbance OD at its 680nm680nm, wherein OD680nmThere are following relationships for relationship between cell concentration：
Y=(94.309 × D680+0.9114)×104(R2=0.999), Y is cell number (cells/ in above-mentioned conversion relation
Ml), coefficient R2=0.999；
C, the preparation of the very thin Euglena algae solution for measuring aquatic particle bio-toxicity：The algae measured according to step B
Liquid cell content selects cell density range 1 × 105To 1 × 106The very thin Euglena algae solution of logarithmic phase of cells/ml, you can use
It is measured in aquatic particle bio-toxicity.
3, the application in aquatic particle bio-toxicity is measured
A, the very thin Euglena algae solution for measuring aquatic particle bio-toxicity of the preparation in selecting step 2；
D, the acquisition and pretreatment of water body sample：The water sample acquired in step 1 is taken, and is retained after filtered through gauze spare；
C, the preparation of special water and aquatic particle separation：Common zwitterion (Ca in determination step d water samples2+,Mg2+,
Na+, Cl-,SO4 2-,NO3-,HCO-Deng) concentration, with laboratory often use salt and pure water configuration contain identical zwitterion concentration
Special water；Again particulate matter in water is detached by the raw water for crossing Coarse Mesh Gauze by grain size through tangential flow filtration system, it is small to be divided into grain size
The water sample particulate matter of sample, grain size more than or equal to 1um, which is measured, in the water sample particulate matter of 1uL measures sample, of different-grain diameter
Grain object is dissolved in special water, and concentration is consistent with raw water.As shown in Figure 1, derived from the water sample of Gong Hu and Ecological Restoration Area
Suspended particle size size is differed from 1nm to 22.9um, and the particle size of the two is distributed without marked difference, is all shown as
Nanoparticles total amount is about 75%, and plain particles object total amount is about 25%.
D, measurement of the very thin Euglena algae solution to aquatic particle bio-toxicity：Include the following steps；
Take the very thin Euglena algae solution of step a, supernatant is abandoned in centrifugation, a small amount of PBS is added suspends again and obtain concentrating algae solution, is taken
The aquatic particle that 10 μ l concentrations algae solutions are separately added into 50ml different-grain diameters measures in sample, mixes and shakes up as experimental group,
Special water blank group is arranged to be compareed, measures algae growing state, chlorophyll content, superoxide dismutase activity and list respectively
Cellular gels electrophoresis takes its stationary value and records, and compares the difference of blank group and experimental group parameter, show that aquatic particle measures
The influence that sample damages each biological indicator of very thin Euglena according to result is influenced as a result, measure aquatic particle biology poison
Property.
The algae growing state for determining each experimental group and blank group in the present embodiment respectively takes each experimental groups of 200ul and sky
White group frustule culture solution, absorbance OD at its 680nm is measured using microplate reader680nm, wherein OD680nmBetween cell concentration
Relationship there are following relationships：
Y=(94.309 × D680+0.9114)×104(R2=0.999), Y is cell number (cells/ in above-mentioned conversion relation
Ml), coefficient R2=0.999, obtain algae cell density.As shown in Fig. 2, the particle isolated for same volume water sample
Object, grain size in tribute arm of lake water sample<The inhibition that 1 μm of particulate matter grows very thin Euglena is significantly higher than>1 μm of particulate matter, and it is ecological
It repairs area's water sample and shows that the particulate matter of different-grain diameter has no the apparent inhibition of performance to the growth of very thin Euglena.
The present embodiment Determination of Chlorophyll assay carries out by the following method：Take the algae solution of 10ml each experimental group and blank group
Supernatant is removed, quantitative 80% acetone is added, is protected from light extraction chlorophyll for 24 hours；After the completion of extraction, then supernatant is taken, measured respectively
Absorbance D at 470nm, 646nm and 663nm470、D646And D663, and chlorophyll a, chlorophyll b and class is calculated as follows
The content of carrotene：
Ca=12.21D663-2.81D646(mg/L)；
Cb=20.13D646-5.03D663(mg/L)；
CAR=(1000D470-3.27Ca-104Cb)/229(mg/L).As shown in figure 3, compared with blank group, it is exposed to water
The very thin Euglena chlorophyll content of body particulate matter shows notable raising, this may with tribute arm of lake water nutrition degree is higher has
It closes.
Superoxide dismutase activity test experiments carry out as follows in the present embodiment：Take each grain sizes of 10ml
Algae solution centrifugal treating in the experimental group and control group of grain object exposure, removes supernatant, sodium phosphate buffer is then added, mix
Uniformly, then Determination of erythrocyte superoxide dismutase activity is carried out.As shown in figure 4, compared with blank group, tribute arm of lake water body grain size<1 μm
Under particle and total particle exposure, the superoxide dismutase activity in very thin Euglena body shows significantly to increase.And it is repaiied in ecology
In the particulate matter of multiple area's water body separation, only total particulate processing group shows to promote superoxide dismutase activity raised
Phenomenon.
DNA damage detection is carried out by SCGE experiment in the present embodiment, is as follows：Take 10ml each
Algae solution centrifugal treating in the experimental group and control group of a particles things exposure, removes supernatant, with 1.5% low melting point agar
Sugared mixing takes the 100ul mixed liquors to be added dropwise on pretreated glass slide, and coated with coverslip, 4 DEG C stand 10min, after condensation
Offset plate repave 0.5% low melting-point agarose as second layer glue, 4 DEG C stand to solidifying, after in the alkaline electrophoresis liquid of precooling
20min is submerged, DNA is promoted to untwist, in 20V and 200mA condition electrophoresis 20min, takes out offset plate, 15min is submerged with neutralizer, on
Sem observation is taken pictures, statistical analysis comettail situation, in comet, DNA be damaged serious conditions and its tail portion DNA content and
The size of back range is proportionate.As shown in figure 5, the very thin Euglena DNA for being exposed to aquatic particle show it is different degrees of
Damage, wherein for the particulate matter that same volume is isolated, grain size in tribute Lake Water Body<1 μm of particulate matter is to very thin Euglena DNA
Damage is significantly higher than other experimental groups.
Very thin Euglena acute toxicity test by aquatic particle to the algae growing state of very thin Euglena, chlorophyll content,
The influence of superoxide dismutase activity and DNA are as a result, obtain either grain size<1 μm of particulate matter, the particle of still >=1um
Object obviously embodies the inhibiting effect to very thin Euglena superoxide dismutase activity, to DNA loss facilitation, from
And realize bio-toxicity that is comprehensive, getting information about aquatic particle.
In conclusion very thin Euglena acute toxicity test accurately and can be detected delicately in water-outlet body actual environment sample
Pollutant provides data and theories integration to the genetoxic of biology to evaluate its potential health and ecological risk.
Above-mentioned combination specific example elaborates embodiments of the present invention, but the present invention is not limited to above-mentioned realities
Apply mode, the technical field those of ordinary skill within the scope of knowledge, present inventive concept can also not departed from
Under the premise of make a variety of changes.
Claims (8)
1. a kind of very thin Euglena measures the algae solution preparation method of aquatic particle bio-toxicity, include culture and the algae solution of algae
It prepares, which is characterized in that specifically include following steps：
A, the culture of very thin Euglena：Very thin Euglena algae is chosen, very thin Euglena algae is seeded to equipped with algae under aseptic condition
In the conical flask of body culture medium, inoculation is completed, it is standby to logarithmic phase that conical flask is then placed in culture in constant temperature illumination box
With；
B, the determination of very thin Euglena cell density：Culture is measured to absorbance at the frustule culture solution 680nm of logarithmic phase
OD680nm, wherein OD680nmThere are following relationships for relationship between cell concentration：
Y=(94.309 × D680+0.9114)×104(R2=0.999), wherein Y is cell number (cells/ml), coefficient R2
=0.999；
C, the preparation of the very thin Euglena algae solution for measuring aquatic particle bio-toxicity：The frustule measured according to step B
Culture solution cell content selects cell density range 1 × 105To 1 × 106The very thin Euglena algae solution of logarithmic phase of cells/ml, i.e.,
It can be used for aquatic particle bio-toxicity measurement.
2. very thin Euglena according to claim 1 measures the algae solution preparation method of aquatic particle bio-toxicity, feature
It is：The condition of culture of the constant temperature illumination box is：Intensity of illumination is 2000lx, and temperature is 25 ± 1 DEG C, humidity 75%
RH, periodicity of illumination 12h：12h, the initial pH of algae solution are 7.0~8.0, stationary culture, daily shaking flask 2-3 times, while mutually being replaced
Position.
3. very thin Euglena according to claim 1 measures the algae solution preparation method of aquatic particle bio-toxicity, feature
It is：A concentration of the 1 × 10 of the very thin Euglena algae of biological subject5cells/ml。
4. measure the very thin Euglena algae solution of aquatic particle bio-toxicity in measuring the application in aquatic particle bio-toxicity,
Characterized by the following steps：
A, the culture of very thin Euglena：Very thin Euglena algae is chosen, very thin Euglena algae is seeded to equipped with algae in aseptic condition
1-2 conical flask of body culture medium completes inoculation, and then conical flask is placed in constant temperature illumination box and is cultivated to logarithmic phase,
It is spare；
B, the determination of very thin Euglena cell density：Measure culture to logarithmic phase frustule culture solution at 680nm absorbance
D680nm, wherein D680There are following relationships for relationship between cell concentration：
Y=(94.309 × D680+0.9114)×104(R2=0.999)
Wherein, Y is cell number (cells/ml), coefficient R2=0.999；
C, the preparation of the very thin Euglena algae solution for measuring aquatic particle bio-toxicity：The difference measured according to step b is dense
The algae solution frustule content of degree selects cell density range 1 × 105To 1 × 106The very thin Euglena algae of logarithmic phase of cells/ml
Liquid, you can measured for aquatic particle bio-toxicity；
D, the acquisition and pretreatment of water body sample：Water sample is acquired, and is retained after filtered through gauze spare；
E, aquatic particle detaches：The water sample of gained in step d is subjected to aquatic particle grain size separation, the particulate matter isolated
It is dissolved in special water, concentration is consistent with raw water；
F, measurement of the very thin Euglena algae solution to aquatic particle bio-toxicity：Include the following steps,
It takes the very thin Euglena algae solution in logarithmic phase, centrifugation to abandon supernatant, a small amount of PBS is added suspends again and obtain concentrating algae solution,
It takes 10 μ l concentrations algae solutions to be separately added into 50ml each group aquatic particles to measure in sample, mixes and shake up as experimental group, setting
Special water blank group is compareed, and measures algae growing state, chlorophyll content, superoxide dismutase activity and unicellular respectively
Gel electrophoresis takes its stationary value and records, and compares the difference of blank group and experimental group parameter, show that aquatic particle measures sample
Influence to the damage of very thin Euglena each biological indicator according to result is influenced as a result, measure aquatic particle bio-toxicity.
5. measuring the very thin Euglena algae solution of aquatic particle bio-toxicity as claimed in claim 4 in measurement aquatic particle
Application in bio-toxicity, it is characterised in that：The water sample particulate separation processing of step e further comprises：
The configuration of S1, special water：Measure common zwitterion (Ca in raw water2+,Mg2+,Na+, Cl-,SO4 2-,NO3 -,HCO-Deng)
Concentration often uses the special water of salt and pure water configuration containing identical zwitterion concentration with laboratory；
The separation of S2, particulate matter：The raw water for crossing Coarse Mesh Gauze is detached particulate matter in water by grain size through tangential flow filtration system, no
Particulate matter with grain size is dissolved in special water, and concentration is consistent with raw water.
6. measuring the very thin Euglena algae solution of aquatic particle bio-toxicity as claimed in claim 4 in measurement aquatic particle
Application in bio-toxicity, it is characterised in that：The measurement of step f Determination of Chlorophyll contents refers to：Take each experimental groups of 10ml and blank group
Algae solution remove supernatant, quantitative 80% acetone is added, is protected from light extraction chlorophyll for 24 hours；After the completion of extraction, then supernatant is taken, point
Absorbance D that Ce Ding be at 470nm, 646nm and 663nm470、D646And D663, and chlorophyll a, chlorophyll is calculated as follows
The content of b and carotenoid：
Ca=12.21D663-2.81D646(mg/L)；
Cb=20.13D646-5.03D663(mg/L)；
CAR=(1000D470-3.27Ca-104Cb)/229(mg/L)。
7. measuring the very thin Euglena algae solution of aquatic particle bio-toxicity as claimed in claim 4 in measurement aquatic particle
Application in bio-toxicity, it is characterised in that：The measurement single cell gel electrophoresis refers to：It takes at the algae solution centrifugation in 10ml c
Reason removes supernatant, with 1.5% low melting-point agarose mixing, the 100ul mixed liquors is taken to be added dropwise in pretreated glass slide
On, coated with coverslip, 4 DEG C of standing 10min, condensed offset plate repaves 0.5% low melting-point agarose as second layer glue, 4 DEG C
Stand to solidify, after submerge 20min in the alkaline electrophoresis liquid of precooling, promote DNA to untwist, in 20V and 200mA condition electrophoresis
20min takes out offset plate, submerges 15min with neutralizer, upper sem observation is taken pictures, statistical analysis comettail situation.
8. measuring the very thin Euglena algae solution of aquatic particle bio-toxicity as claimed in claim 4 in measurement aquatic particle
Application in bio-toxicity, it is characterised in that：The measurement superoxide dismutase activity method is：Take each grain sizes of 10ml
Algae solution centrifugal treating in the experimental group and control group of grain object exposure, removes supernatant, sodium phosphate buffer is then added, mix
Uniformly, then Determination of erythrocyte superoxide dismutase activity is carried out.
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