CN103710419A - Method for quickly and simply screening and evaluating marine antifouling compound - Google Patents
Method for quickly and simply screening and evaluating marine antifouling compound Download PDFInfo
- Publication number
- CN103710419A CN103710419A CN201310725873.4A CN201310725873A CN103710419A CN 103710419 A CN103710419 A CN 103710419A CN 201310725873 A CN201310725873 A CN 201310725873A CN 103710419 A CN103710419 A CN 103710419A
- Authority
- CN
- China
- Prior art keywords
- substratum
- value
- compound
- test
- gel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for quickly and simply screening and evaluating a marine antifouling compound. The method comprises the following steps: by using marine bacteria as a tested organism, adding a gelatinizer into a bacterial liquid culture medium, dissolving by heating, cooling, uniformly dispersing the tested compound in the gel solution, uniformly coating the gel solution containing the tested compound on a glass plate to prepare a gel test plate; and putting the gel test plate in seawater in which the tested organism is inoculated, carrying out continuous culture, taking out the gel test plate, rinsing, diluting to the specified volume in a measuring flask, testing the OD (optical density) value or absorbance value, commuting the OD value or absorbance value into antibacterial rate, and comparing the magnitude of the antibacterial rate to evaluate the antifouling property of the tested compound. The evaluation method is simple to operate and easy to implement, has the advantage of short evaluation period, uses the marine bacteria as the tested organism, and can be used for screening the antifouling compounds in the research and development process of marine antifouling paints.
Description
Technical field
The present invention relates to a kind of rapid screening evaluation method, relate in particular to a kind of method of fast simple screening and assessment marine antifoulant.
Background technology
Marine biofouling problem, is restricting the mankind to the development and utilization of marine resources, and brings huge financial loss.At present, preventing and kill off the most frequently used method of marine biofouling is application antifouling paint.Stain control agent is topmost one of the marine biofouling effective ingredient of preventing and kill off of antifouling paint, and screening and assessment stain control agent is the indispensable step of antifouling paint R&D process.Improve the evaluation method of improving antifouling property, significant to antifouling paint exploitation.In the soak test method > > of GB GB/T5370-2007 < < antifouling varnish model shallow sea, provide a kind of antifouling paint evaluation method, stain control agent is made into the antifouling property that also can be used to method evaluation stain control agent in GB GB/T5370-2007 after antifouling paint, but evaluation cycle is long.The China author Tian Bin Master's thesis < < marine anti-pollution coating biology of 2005 is learned in performance study > > and as biological subject, is evaluated the antifouling property of stain control agent with streptococcus aureus, intestinal bacteria, 3 kinds of bacterial strains of distillery yeast, but because biological subject is not typical ocean bacterium, can not represent ocean bacterial classification, evaluation result representativeness is often queried.
Summary of the invention
The methods and applications that the object of this invention is to provide a kind of fast simple screening and assessment marine antifoulant, it take the marine bacteria of separation from ocean is biological subject, gelifying agent is joined in bacterial liquid substratum to the cooling gelating soln of making after heating for dissolving, test-compound is dispersed in gelating soln, then will be containing making gel test panel on the even coated glass plate of test-compound gelating soln, again gel test panel is placed in to the seawater of inoculation biological subject, after cultured continuously, gel test panel is taken out, drip washing, constant volume is in volumetric flask, test OD value (optical density value) or absorbance, OD value or absorbance are converted into bacteriostasis rate, by comparing the size of bacteriostasis rate, evaluate the antifouling property of test-compound.Described evaluation method is simple to operate, easily capable, and evaluation cycle is short, with marine bacteria, is biological subject, can be applicable to the screening of stain control agent in marine antifouling coating R&D process.
Above-mentioned marine bacteria, is characterized in that with selective medium, from seawater, oceanic sediment, relate to extra large facility and soak sea part and isolate.
Above-mentioned selective medium is one or more of synthetic No. 1 substratum of Gao Shi for TCBS substratum, separating payingoff bacteria for Zobell 2216 E substratum, vibrio marinopraesens, humic acid substratum, asparagine substratum, arginine substratum, above-mentioned modified form substratum.
Above-mentioned liquid nutrient medium is one or more of synthetic No. 1 liquid nutrient medium of Gao Shi for TCBS liquid nutrient medium, separating payingoff bacteria for Zobell 2216 E liquid nutrient mediums, vibrio marinopraesens, humic acid liquid substratum, asparagine liquid nutrient medium, arginine liquid nutrient medium, above-mentioned improved liquid substratum.
Above-mentioned gelifying agent is one or more in agar, gelatin, fish glue, carrageenin.
The making method of above-mentioned gel test panel, it is characterized in that gelifying agent to join in bacterial liquid substratum the cooling gelating soln of making after heating for dissolving, test-compound is dispersed in gelating soln, then will will be uniformly coated on sheet glass and makes containing test-compound gelating soln.
Above-mentioned sheet glass is long 100 mm, wide 100mm or long 200 mm, wide 200mm or long 250 mm, the ganoid sheet glass of wide 2500mm.
Above-mentioned test OD value or the method for absorbance, is characterized in that it being with microplate reader or visible spectrophotometer, take liquid-based substratum as reference, measures OD value or the absorbance of gel test panel leacheate.
Application when above-mentioned evaluation method is screened stain control agent in research and development marine antifouling coating process.
The advantage of screening and assessment marine anti-pollution agent method of the present invention is to take marine bacteria as biological subject, representative strong, and evaluation method is simple to operate, easily capable, and evaluation cycle is short.
Embodiment
Below in conjunction with attached embodiment, the present invention is described in further detail.
Embodiment:
1, the separation of marine bacteria
Use seawater selective medium, with filter membrane enrichment method or one or more methods in coating method or method of scoring cultivate and separatedly take from seawater, oceanic sediment, relate to the marine bacteria that extra large facility soaks sea part, under low temperature, save backup.
(1) separation of bacterial from seawater:
With the nitrocotton filter membrane of sterilizing, by different volumes seawater amount, carry out enrichment, the filter membrane after enrichment takes off, and is inoculated on the plate culture medium of numbering in advance, cultivates after certain hour, carries out separation, and preserves at low temperatures bacterial classification.
With aseptic sounding bottle from Dongji Island Sea Near three erect-position water samplings.Nitrocotton filter membrane (0.45um) with sterilizing, carries out enrichment to 100mL, 200mL, 300mL seawater sample respectively, takes off filter membrane, is close to respectively on the culture dish that contains Zobell 2216 E solid mediums of above-mentioned three kinds of numbers of finishing in advance.Culture dish is placed after 10min, be inverted and be put in 25 ℃ of biochemical cultivation cases, cultivate 3-5 days, observe its form, record quantity, as impure in bacterium colony, picking colony is separated by method of scoring, until purifying is then inoculated in inclined-plane, glycerine fluid-tight, at-20 ℃, saves backup.
(2) from relating to extra large facility, soak extra large part separation of bacterial:
To soaking the sample that sea part gets and carry out low-speed centrifugal from relating to extra large facility, get supernatant liquor, and be diluted to different multiples, select the diluent of suitable multiple, on the plate culture medium of numbering in advance, inoculate, to cultivate after for some time, separation saves backup under low temperature.The above-mentioned multiple scope that is diluted to is 10
-1～10
-6doubly.
Use aseptic sampling bottle, near deep water mesh cage Dongji Island, select 9 points to sample (with pinching son, gathering different dirt settlings), sample is carried out centrifugal, rotating speed 2000r/min, centrifugal 1 minute, get supernatant liquor, with aseptic seawater, be diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6doubly.Choose 10
-4, 10
-5with 10
-6diluent doubly, is coated with on the culture dish of the Zobell of the number of finishing 2216 E solid mediums with coating method, and each concentration dilution liquid is coated with 3 plates.Culture dish is placed after 10min, be inverted and be put in 25 ℃ of biochemical cultivation cases, cultivate 3-5 days, observe its form, record quantity, as impure in bacterium colony, picking colony is separated by method of scoring, until purifying, then be inoculated in inclined-plane, glycerine fluid-tight, at-20 ℃, saves backup.
(3) separation of bacterial from oceanic sediment:
Get a certain amount of marine sediment samples, be scattered in aseptic seawater, shake into suspension, after dilution different multiples, get the diluent of suitable multiple, be applied in the culture dish that contains substratum, numbering, cultivates after for some time observed and recorded colony growth situation, adopt method of scoring to carry out purifies and separates to bacterium colony, under low temperature, save backup.The above-mentioned multiple scope that is diluted to is 10
-1～10
-6doubly.
Get 10g ooze or sea sand sample, be scattered in the aseptic seawater of 10mL, shake into suspension, with aseptic seawater, be diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6g/mL.Get 10
-4, 10
-5with 10
-6g/mL diluent is coated with on the culture dish of the Zobell of the number of finishing 2216 E solid mediums with coating method, and each concentration dilution liquid is coated with 3 plates.Culture dish is placed after 10min, be inverted and be put in 25 ℃ of biochemical cultivation cases, cultivate 3-5 days, observe its form, record quantity, as impure in bacterium colony, picking colony is separated by method of scoring, until purifying, then be inoculated in inclined-plane, glycerine fluid-tight, at-20 ℃, saves backup.
2, the preparation of gel test panel
In liquid nutrient medium, add respectively gelifying agent, heating for dissolving is prepared certain density gelifying agent solution, cooling after, add respectively wherein test-compound stain control agent, make the gelating soln containing test-compound, be uniformly coated on sheet glass.Above-mentioned certain density gelifying agent solution refers to that gelifying agent concentration range is 1%～30% solution (weight percentage).The above-mentioned gelating soln containing test-compound refers to the gelating soln of concentration range 1 mg/mL～400 mg/mL test-compound.
In Zobell 2216 E liquid nutrient mediums, add agar powder, it is 1% agar-gel solution that heating for dissolving is mixed with agar concentration, is cooled to 50～60 ℃, stand-by.Get 5 parts of above-mentioned agar-gel solutions and add respectively TBTO, CPT, ZPT, DCDMA, five kinds of test-compounds of Diuron, making test-compound concentration is 5 kinds of agar-gel solutions of 1 mg/mL, being uniformly coated on specification is on 100 * 100mm sheet glass, dry, form smooth gel surface.Using and do not add gel test panel that the agar-gel solution of test-compound manufactures as blank.
3, the cultivation of gel test panel in inoculation biological subject seawater
In specification, be in 630 * 370 * 500mm glass jar, add the aseptic seawater of 10L, and cultivate each 1mL of tested bacteria suspension after 10 hours, keep 25 ℃ of temperature, be cultured to exponential growth after date, agar gel test panel inserted in glass jar seawater to cultured continuously 24h.
4, the mensuration of OD value and anti-fouling effect evaluation
Agar gel test panel is taken out, use liquid nutrient medium drip washing, with liquid nutrient medium, be settled in 50 mL volumetric flasks, measure OD value or absorbance.
The calculating of 24h bacteriostasis rate: R/%=
,
Wherein, R-bacteriostasis rate;
Average OD value or the mean light absorbency value of B-blank plate leacheate;
C-containing average OD value or the mean light absorbency value of different test-compound gel test panel leacheates.
OD value or absorbance are converted into bacteriostasis rate in Table 1.
The indoor link plate result of the different stain control agent 24h of table 1
From table 1, the biocidal property size order of 5 kinds of biological subjects is TBTO > CPT > ZPT > DCDMA > Diuron.Biocidal property size order is with consistent by the antifouling property result of GB GB/T5370-2007 < < antifouling varnish model shallow sea soak test method > > test.
Although described the present invention in conjunction with preferred embodiment; so it is not in order to limit the present invention; any those skilled in the art; without departing from the spirit and scope of the present invention; can implement to the theme of listing displacement and the modification of various changes, coordinator here, so protection scope of the present invention is as the criterion when the scope of the claim restriction depending on proposed.
Claims (4)
1. the method for a fast simple screening and assessment marine antifoulant, it is characterized in that: the marine bacteria of separation from ocean of take is biological subject, by gelifying agent join heating for dissolving in bacterial liquid substratum, cooling after, test-compound is dispersed in gelating soln, then will be containing making gel test panel on the even coated glass plate of test-compound gelating soln; Gel test panel is placed in to the seawater of inoculation biological subject, after cultured continuously, by the taking-up of gel test panel, drip washing, constant volume is in volumetric flask, test OD value or absorbance, OD value or absorbance are converted into bacteriostasis rate, by comparing the size of bacteriostasis rate, evaluate the antifouling property of test-compound;
Described comparison bacteriostasis rate be R/%=
,
Wherein, R-bacteriostasis rate;
Average OD value or the mean light absorbency value of B-blank plate leacheate;
C-containing average OD value or the mean light absorbency value of different test-compound gel test panel leacheates.
2. the method for a kind of fast simple screening and assessment marine antifoulant according to claim 1, is characterized in that: described substratum is selective medium, and this substratum separation is from seawater or oceanic sediment or soak extra large facility and soak extra large part.
3. the method for a kind of fast simple screening and assessment marine antifoulant according to claim 2, is characterized in that: described liquid nutrient medium is selected from Zobell 2216E substratum, synthetic No. 1 substratum of Gao Shi for TCBS substratum, separating payingoff bacteria for vibrio marinopraesens, humic acid substratum, asparagine substratum, arginine substratum, above-mentioned modified form substratum one or more.
4. the method for a kind of fast simple screening and assessment marine antifoulant according to claim 1, is characterized in that: described gelifying agent is one or more in agar, gelatin, fish glue, carrageenin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310725873.4A CN103710419B (en) | 2013-12-25 | 2013-12-25 | Method for quickly and simply screening and evaluating marine antifouling compound |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310725873.4A CN103710419B (en) | 2013-12-25 | 2013-12-25 | Method for quickly and simply screening and evaluating marine antifouling compound |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103710419A true CN103710419A (en) | 2014-04-09 |
| CN103710419B CN103710419B (en) | 2015-07-22 |
Family
ID=50403773
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310725873.4A Active CN103710419B (en) | 2013-12-25 | 2013-12-25 | Method for quickly and simply screening and evaluating marine antifouling compound |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103710419B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316475B (en) * | 2014-09-10 | 2017-02-15 | 深圳大学 | Living spore content high throughput detection method based on spectrophotometry method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624056A (en) * | 2004-11-05 | 2005-06-08 | 叶德佳 | Harmless antifouling paint |
-
2013
- 2013-12-25 CN CN201310725873.4A patent/CN103710419B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624056A (en) * | 2004-11-05 | 2005-06-08 | 叶德佳 | Harmless antifouling paint |
Non-Patent Citations (3)
| Title |
|---|
| 易定和等: "海洋污损生物防除的方法概述及发展趋势", 《腐蚀科学与防护技术》 * |
| 沈明等: "针对海洋细菌防污剂的快速评价方法及其应用", 《涂料工业》 * |
| 田斌: "海洋防污涂层生物学性能研究", 《优秀硕士论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316475B (en) * | 2014-09-10 | 2017-02-15 | 深圳大学 | Living spore content high throughput detection method based on spectrophotometry method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103710419B (en) | 2015-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Leynaert et al. | Diatom succession, silicification and silicic acid availability in Belgian coastal waters (Southern North Sea) | |
| CN102286405A (en) | Pseudomonas, use thereof and method for removing cadmium pollution to environment | |
| Asadishad et al. | Method for the direct observation and quantification of survival of bacteria attached to negatively or positively charged surfaces in an aqueous medium | |
| US20150337351A1 (en) | Methods of microorganism immobilization | |
| Handy et al. | Evaluating vertical migration behavior of harmful raphidophytes in the Delaware Inland Bays utilizing quantitative real-time PCR | |
| Qu et al. | Responses of the large centric diatom Coscinodiscus sp. to interactions between warming, elevated CO2, and nitrate availability | |
| Chen et al. | Transparent exopolymer particle production and aggregation by a marine planktonic diatom (Thalassiosira weissflogii) at different growth rates | |
| Rodríguez et al. | Macronutrients requirements of the dinoflagellate Protoceratium reticulatum | |
| CN104390920A (en) | Microplate analysis method for time toxicity of environmental pollutants on basis of chlorella pyrenoidosa | |
| Papkovsky et al. | Oxygen sensor-based respirometry and the landscape of microbial testing methods as applicable to food and beverage matrices | |
| Wang et al. | Distribution of microbial populations and their relationship with environmental parameters in the coastal waters of Qingdao, China | |
| CN103710419B (en) | Method for quickly and simply screening and evaluating marine antifouling compound | |
| CN103710422A (en) | Reagent combination for screening and evaluating marine antifouling compounds | |
| Villareal et al. | Occurrence of the toxic diatom Nitzschia pungens f. multiseries in Massachusetts Bay, Massachusetts, USA | |
| Mitbavkar et al. | Picophytoplankton community from tropical marine biofilms | |
| Znachor et al. | APPLICATION OF THE PDMPO TECHNIQUE IN STUDYING SILICA DEPOSITION IN NATURAL POPULATIONS OF FRAGILARIA CROTONENSIS (BACILLARIOPHYCEAE) AT DIFFERENT DEPTHS IN A EUTROPHIC RESERVOIR 1 | |
| Komar et al. | Development of a Method for Assessing the Resistance of Building Coatings to Phoatoautotrophic Biofouling | |
| Uribe et al. | Early succession of micro‐periphyton communities in kelp bed and barren ground ecological systems | |
| CN110726804B (en) | Quick evaluation method for antifouling performance of bionic and low-surface-energy marine antifouling coating | |
| Béchet et al. | The colorimetric assay of viability for algae (CAVA): a fast and accurate technique | |
| Ma et al. | Seasonal and spatial variability of virioplanktonic abundance in Haihe River, China | |
| Manti et al. | What flow cytometry can tell us about marine micro-organisms—current status and future applications | |
| Wang et al. | Biological characteristics of biofilms formed on different substrata in a shallow lake in Haihe basin (China) | |
| CN1793910A (en) | Method for evaluating antifouling performance of antifouling coating layer coated on plate in room | |
| Bober | Quantification of single-species marine biofilm with alcian blue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |