CN102087277A - Method for monitoring benzo(a)pyrene pollution of seawater by utilizing fish blood cells - Google Patents
Method for monitoring benzo(a)pyrene pollution of seawater by utilizing fish blood cells Download PDFInfo
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- CN102087277A CN102087277A CN2010105878029A CN201010587802A CN102087277A CN 102087277 A CN102087277 A CN 102087277A CN 2010105878029 A CN2010105878029 A CN 2010105878029A CN 201010587802 A CN201010587802 A CN 201010587802A CN 102087277 A CN102087277 A CN 102087277A
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- pyrene
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Abstract
The invention provides a method for monitoring benzo(a)pyrene in seawater by utilizing fish blood cells. The method comprises the following steps: taking 50mu L of staphylococcus aureus suspension obtained through treatment of buffer solution on ice and mixing the staphylococcus aureus suspension with isovolumetric paralichthys olivaceus erythrocyte suspension exposed in the seawater containing benzo(a)pyrene and incubating the mixture at the constant temperature of 20 DEG C for 30 minutes, wherein the ratio of the cell number is 20:1; shaking the mixture to mix the mixture uniformly every 10 minutes to prevent precipitates; terminating reaction after completion of incubation, adding 20mu L of 0.25% of precooled glutaraldehyde solution to each tube, immobilizing the tube in a refrigerator at the temperature of 4 DEG C for 15 minutes, taking incubation solution to prepare smears and immobilizing the smears with methanol after airing the smears; dying each smear with Wright's dye solution for 3 minutes, washing the smear with distilled water until the smear becomes colorless, carrying out observation under an oil lens after airing the smear and computing the erythrocyte C3b receptor rosette rate; and forming a rosette by adhering two or more staphylococcus aurei to each erythrocyte. The detection method is completed in one hour and whether the seawater is polluted by benzo(a)pyrene can be better monitored and analyzed by the method.
Description
Technical field
The invention belongs to the detection method that the present invention relates to based on the seawater toxic organic pollutant of fish blood cell immunity adhesion activity variation.
Background technology
In recent years, development along with progress of science and technology and industrial and agricultural production, the toxic organic pollutant (comprising petroleum hydrocarbon, polychlorinated biphenyl, organo-chlorine pesticide, palycyclic aromatic, tributyl tin etc.) of more and more synthetic generally detects in coastal seawater, sediment and marine organism, the organic contamination problem is outstanding day by day, has become the new focus that countries in the world scientific circles and government pay close attention to.Studies show that, toxic organic pollutant toxicity is big, be dissolved in easily in the biological organic phase, in the enrichment of marine organism inner height, destroy halobiontic inhereditary material, influence halobiontic fertility, the halobiontic population that changes contaminated waters is formed, cause the marine ecosystems imbalance, and can enter human body, jeopardize human health by fish, shellfish and other marine product.Therefore, domestic and international researcher pays close attention to the pollution monitoring and the potential hazard thereof of toxic organics in the ocean all the more.
Monitoring to toxic organics in the Yu Haiyang, mainly contain two kinds of methods of chemical monitoring and biological monitoring, wherein chemical monitoring method is mainly by the liquid-liquid extraction method, toxic organics in the enriching seawater, reflect their pollution levels by the amount of measuring organic compound in the extract, comprise fluorescence spectrophotometry marine environment
1, ultraviolet spectrophotometry, gas chromatography-mass spectrography (GC-MS) coupling technique etc.
2The biological monitoring method mainly is to utilize the residual level of organic contaminant in the filter-feeding shellfish bodies such as marine benthos mussel, clam, oyster to reflect various toxic organic pollutant matter content in the background water body
3But at actual ocean toxic organic pollutant observation process, the chemical monitoring method is loaded down with trivial details relatively, and the high more required instrument price of detection sensitivity is just expensive more, therefore is not easy to promote in marine environmental monitoring unit of local basic unit.The biological monitoring method then mainly with benthic mollusca as indicator species, can not truly reflect the organic contamination situation in the surface seawater.Therefore with the indicator species of fish as toxic organic pollutant, set up a kind of sensitive relatively and check and analysis method easily, for enrich toxic organic pollutant detection method in the seawater, to improve the seawater quality examination criteria significant.
List of references
1. Wei is climing new, He Benmao. and the nearly 20a of Qinzhou Wan comes the distribution characteristics and the pollution situation thereof of water environment index variation tendency II oils. marine environment science, 2003,22 (2): 49-52.
2. osmanthus, river is refined, Xu Fuzheng, He Bin etc. organo-tin compound study on determination method progress. and marine environmental chemistry, 1999,18 (3): 61-68.
3. Liu Ren edge, Wu Shipei, Wang Bin. the distribution and the evaluation of organo-chlorine pesticide and polychlorinated biphenyl in the coastal main economic shellfish to the north of the entrance of Changjiang River. marine environment science, 1996,15 (3): 29-35.
Summary of the invention
The present invention is intended to set up a kind of method of polluting based on benzo a pyrene in the fast monitored seawater of lefteye flounder blood cell immunity adhesion activity variation under the different briny environment conditions.By at normal seawater with contain the variation of the haemocyte of lefteye flounder in the benzo a pyrene seawater to the immune adherence activity of staphylococcus aureus, preliminary foundation researched and analysed the pollution condition of benzo a pyrene in the seawater and the experimental technique of poisonous effect thereof.At 0.1mol/L PBS
++In the buffer solution system, gather respectively and culture at normal seawater and the lefteye flounder haemocyte that contains in the benzo a pyrene seawater, hatch with golden yellow grape ball, observation is calculated the fish blood cell immunity and is adhered to the percent that staphylococcus aureus forms the C3b receptor garland.Each two of haemocyte adhesion and two above staphylococcus aureuses are designated as a garland, count 200 haemocytes, calculate the garland positive percentage.Haemocyte C3b receptor rosette rate=one-tenth garland cell number/200 * 100%.
Concrete grammar and step are:
1. the preparation of lefteye flounder serum and bacteria suspension:
Respectively from the tail vein blood of experiment lefteye flounder, inject the 5mL centrifuge tube with asepsis injector, lie against on the desktop, room temperature is placed 30min.After treating blood clotting, be put in 4 ℃ of refrigerator overnight.The serum that sucking-off is separated out is sub-packed in the 1.5mL sterilization centrifuge tube, and-80 ℃ of preservations are standby.Get the staphylococcus aureus suspension 300 μ L that handle well in 2mL sterilization centrifuge tube, add equal-volume lefteye flounder serum, fully behind the mixing, hatch 30min on ice in 20 ℃.Every 10min gently mixing once prevent bacterial sediment.Hatch finish after, the centrifugal 5min of 10000rpm under 4 ℃ of conditions, bacterial sediment is resuspended with the corresponding damping fluid of 300 μ L, returns to original bacteria suspension concentration, 4 ℃ of preservations are standby.
2. the preparation of flounder erythrocyte:
Get blood from the tail vein of experiment lefteye flounder, adopt the A Shi anti-coagulants to mix at 1: 1 with the lefteye flounder peripheral blood.Utilizing proportion is that 1.083 lymphocyte separation medium (TBD company) separates the preparation flounder erythrocyte.Use 0.1M PBS in 4 ℃ of following centrifuge washings twice erythroprecipitin of the bottom, adjust concentration to 1 * 10 with blood counting chamber counting back
7/ mL.Get an amount of red blood cell then and use damping fluid, return to 1 * 10 in 4 ℃ of centrifuge washings twice
7/ mL.
3. the detection of erythrocyte immune adhesion activity:
Get the bacteria suspension that 50 μ L handle with damping fluid respectively and mix with the red cell suspension equal-volume, the ratio of cell number is about 20: 1 (aforesaid operations carries out on ice), hatches 30min in 20 ℃ of constant temperature.Hatch in the process and shake mixing gently, prevent red blood cell and somatic cells precipitation every 10min.Hatch finish after, in cessation reaction on ice.Every pipe adds 0.25% glutaraldehyde solution of 20 μ L precoolings, fixing 15min in 4 ℃ of refrigerators.Get an amount of Incubating Solution and make smear, dry fixedly 2min of back methyl alcohol.Every smear is through Wright ' s dye liquor dyeing 3min, with distilled water flushing to current colourless till.Dry the oily mirror in back and observe calculating red blood cell C3b receptor rosette rate down.In the experiment two of each red blood cell adhesions and two above staphylococcus aureuses are designated as a garland.Each sample is coated with 3 parallel plates and observes counting.Red blood cell C3b receptor rosette rate=one-tenth garland cell number/200 * 100%.
The statistical procedures of experimental result adopts SPSS 13.0 statistical softwares to carry out the t check analysis.
This detection method mainly is the organic contamination situation of doing in the qualitative detection marine environment, but when the needs accurate quantification detects, does not advise adopting this method.
Testing process of the present invention is simple to operate, and required reagent preparation is convenient, and does not need to use complicated instrument and equipment.
Description of drawings
Fig. 1: the coherent condition that is subjected to lefteye flounder erythrocyte immune adhesion staphylococcus aureus in the organic contamination seawater for normal nature seawater neutralization.
Fig. 2: the lefteye flounder erythrocyte immune adheres to the coherent condition of staphylococcus aureus in the benzo a pyrene mark-on seawater
Embodiment
Embodiment 1
With staphylococcus aureus with 0.1mol/L PBS damping fluid centrifuge washing 3 times, and to be adjusted to concentration be 2 * 10
8The bacteria suspension of CFU/mL, 4 ℃ of preservations are standby.
2. get 50 μ L staphylococcus aureus suspensions and mix respectively with at normal seawater and the red cell suspension equal-volume that contains the lefteye flounder of benzo a pyrene seawater breeding, the cell number ratio is about 20: 1, behind the mixing, hatches 30min in 20 ℃ of constant temperature gently.
3. hatch in the process and shake mixing gently, prevent red blood cell and somatic cells precipitation every 10min.
4. hatch finish after, in cessation reaction on ice.Every pipe adds 0.25% glutaraldehyde solution of 20 μ L precoolings, 4 ℃ of fixing 15min.Get an amount of Incubating Solution and make smear, dry fixedly 2min of back methyl alcohol.
5. every smear adds an amount of Wright ' s dye liquor dyeing 3min, and is colourless to current with distilled water flushing.Dry the oily mirror in back and observe the immune adherence of red blood cell down, calculate red blood cell C3b receptor rosette rate antigen.
6. each sample is coated with 3 sheets and observes counting and average.Two of each red blood cell adhesions and two above staphylococcus aureuses are designated as a garland.
7.0.1mol/L PBS
++The garland number scale that forms in 200 red blood cells in the experimental group is x
1, the garland number scale that forms in 200 red blood cells in the 0.1mol/LPBS-EDTA negative control group is x
2
8. red blood cell C3b receptor rosette rate=(x
1-x
2)/200 * 100%.Experimental result adopts SPSS 13.0 softwares to carry out statistical analysis.
Embodiment 2
The glass beaker of selecting 3L for use is as experiment container, and adding cumulative volume is the mark-on seawater of 2L, and each beaker is put experiment fish 5 tails, and 2 of each mark-on concentration are parallel.Benzo a pyrene concentration gradient: 0.49 μ g/L, 0.98 μ g/L, 1.96 μ g/L, 3.92 μ g/L, 7.84 μ g/L.Experimental period 14d, temperature (20 ± 2) ℃, 24h changes mark-on liquid, measures the situation of change of blood cell immunity adhesion rate.
1. use the 2.5mL asepsis injector from experiment fish tail venous blood collection, adopt the A Shi anti-coagulants to mix at 1: 1, be put in 4 ℃ of refrigerators and preserve standby with the lefteye flounder peripheral blood
With staphylococcus aureus with 0.1mol/L PBS damping fluid centrifuge washing 3 times, and to be adjusted to concentration be 2 * 10
8The bacteria suspension of CFU/mL, 4 ℃ of preservations are standby
3. get 50 μ L staphylococcus aureus suspensions and mix with red cell suspension equal-volume at the lefteye flounder of normal seawater and mark-on seawater breeding respectively, the cell number ratio is about 20: 1, behind the mixing, hatches 30min in 20 ℃ of constant temperature gently.
4. hatch finish after, in cessation reaction on ice.Every pipe adds 0.25% glutaraldehyde solution of 20 μ L precoolings, 4 ℃ of fixing 15min.Get an amount of Incubating Solution and make smear, dry fixedly 2min of back methyl alcohol.
5. every smear adds an amount of Wright ' s dye liquor dyeing 3min, and is colourless to current with distilled water flushing.Dry the oily mirror in back and observe the immune adherence of red blood cell down, calculate red blood cell C3b receptor rosette rate antigen.
6. each sample is coated with 3 sheets and observes counting and average.Two of each red blood cell adhesions and two above staphylococcus aureuses are designated as a garland.
7.0.1mol/L PBS
++The garland number scale that forms in 200 red blood cells in the experimental group is x
1, the garland number scale that forms in 200 red blood cells in the 0.1mol/LPBS-EDTA negative control group is x
2
Red blood cell C3b receptor rosette rate=(x
1-x
2)/200 * 100%.Experimental result adopts SPSS 13.0 softwares to carry out statistical analysis.
Claims (2)
1. the method for utilizing fish haemocyte monitoring seawater benzo a pyrene to pollute is characterized in that concrete check and analysis method:
(1) with staphylococcus aureus with 0.1mol/L PBS damping fluid centrifuge washing 3 times, and to be adjusted to concentration be 2 * 10
8The bacteria suspension of CFU/mL, 4 ℃ of preservations are standby;
(2) get 50 μ L staphylococcus aureus suspensions and mix respectively with at normal seawater and the red cell suspension equal-volume that contains the lefteye flounder of benzo a pyrene sea-farming, the cell number ratio is about 20: 1, behind the mixing, hatches 30min in 20 ℃ of constant temperature gently;
(3) hatch in the process and shake mixing gently, prevent red blood cell and somatic cells precipitation every 10min;
(4) hatch finish after, in cessation reaction on ice.Every pipe adds 0.25% glutaraldehyde solution of 20 μ L precoolings, 4 ℃ of fixing 15min.Get an amount of Incubating Solution and make smear, dry fixedly 2min of back methyl alcohol;
(5) every smear adds an amount of Wright ' s dye liquor dyeing 3min, and is colourless to current with distilled water flushing.Dry the oily mirror in back and observe the immune adherence of red blood cell down, calculate red blood cell C3b receptor rosette rate antigen;
(6) each sample is coated with 3 sheets and observes counting and average.Two of each red blood cell adhesions and two above staphylococcus aureuses are designated as a garland;
(7) 0.1mol/L PBS
++The garland number scale that forms in 200 red blood cells in the experimental group is x
1, the garland number scale that forms in 200 red blood cells in the 0.1mol/LPBS-EDTA negative control group is x
2
(8) red blood cell C3b receptor rosette rate=(x
1-x
2)/200 * 100%.Experimental result adopts SPSS 13.0 softwares to carry out statistical analysis;
The preparation method of described staphylococcus aureus suspension is: in the LB fluid nutrient medium, 37 ℃ of overnight shakings are cultivated with the single bacterium colony of staphylococcus aureus.With 0.1M PBS damping fluid centrifuge washing twice, the 65 ℃ of water-bath 40min of formalin solution that use final concentration 1% are with its deactivation.Get staphylococcus aureus suspension 300 μ L in 2mL sterilization centrifuge tube, add equal-volume lefteye flounder serum, behind the abundant mixing, hatch 30min on ice in 20 ℃.Hatch finish after, the centrifugal 5min of 10000rpm under 4 ℃ of conditions, bacterial sediment is resuspended with the corresponding damping fluid of 300 μ L, returns to original bacteria suspension concentration, 4 ℃ of preservations are standby;
Used damping fluid and other liquid are in the described reaction:
PBS damping fluid: NaCl 8g, KCl 0.2g, KH
2PO
40.24g, Na
2HPO
4-12H
2O 2.9g, distilled water is settled to 1000ml, pH7.4; 10 times of lefteye flounder serum dilutions.
2. according to the described method of utilizing fish haemocyte monitoring seawater benzo a pyrene to pollute of claim 1, it is characterized in that in the step (2), the fish haemocyte and the staphylococcus aureus suspension mixing that will be exposed in the benzo a pyrene polluted seawater are hatched, and hatch 30min in 20 ℃ of constant temperature, slightly shake.
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| CN111487399A (en) * | 2020-03-26 | 2020-08-04 | 湖南师范大学 | Application of protein molecular marker in research on fish germ cell development |
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Application publication date: 20110608 |