CN102250771A - Quick luminescent bacteria supported based on signal molecule - Google Patents
Quick luminescent bacteria supported based on signal molecule Download PDFInfo
- Publication number
- CN102250771A CN102250771A CN2011101444241A CN201110144424A CN102250771A CN 102250771 A CN102250771 A CN 102250771A CN 2011101444241 A CN2011101444241 A CN 2011101444241A CN 201110144424 A CN201110144424 A CN 201110144424A CN 102250771 A CN102250771 A CN 102250771A
- Authority
- CN
- China
- Prior art keywords
- signaling molecule
- bacterium
- luminous
- microbial inoculum
- quick
- 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.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses quick luminescent bacteria supported based on signal molecule. The quick luminescent bacteria supported based on signal molecule are characterized in that: luminescent bacteria freeze-dried powder is prepared by mixing bacterial mud obtained by centrifuging 10ml of Vibrio fischeri at the concentration of 10<9>/ml and 1ml of cryoprotective agent, and performing freeze drying, and is stored at -20DEG C for 6 months; and the cryoprotective agent is prepared by dissolving 15g of nonfat milk, 5g of glycerol and 12.5g of trehalose in 100ml of signal molecule solution. The invention has the advantages that: by adding signal molecules, the synthesis of luciferase can be promoted, so that the time for the luminescent bacteria freeze-dried powder to restore stable luminescence is shortened from the original 15-30 minutes to 5 minutes; and by adding the composite cryoprotective agent, more bacteria can keep the integrity of cellular structures and the activity of cells during freezing and drying, and can be revived under proper conditions.
Description
Technical field
The present invention relates to detect the microbial inoculum of water quality toxicity, be specifically related to a kind of quick luminous microbial inoculum that supports based on signaling molecule.
Background technology
The Fei Shi vibrios is a kind of special marine bacteria, and it can be survived in seawater and be luminous, and when running into toxic substance, its luminous power can weaken.Change of luminous intensity is relevant with the toxicity size of toxicant, so the change of luminous intensity size is judged the water quality biological acute toxicity when often utilizing polluted water body to contact with photogenic bacterium.Adopt the photogenic bacterium method to detect characteristics such as having time weak point, sensitivity height, and can also remedy the deficiency of conventional physics and chemistry monitoring, therefore be widely adopted.The detection method of national standard method (GT/T15441-1995) luminescent bacteria toxicity inspection is to utilize the relative luminous intensity of luminescent bacteria lyophilized powder to carry out toxicity inspection, the first step of this method is exactly the lyophilized powder recovery that will make photogenic bacterium, even it is luminous that photogenic bacterium recovers, the time of recovering luminous needs 15-30min at least; Because the restriction of these factors can only be carried out this method in the laboratory.Existing both at home and abroad many research institutions utilize the advantage of luminescent bacteria toxicity inspection at monitoring water environment, attempt development photogenic bacterium transmitter be used for on-the-spot continuously, original position, automatic monitoring, below be the main problem that they run into:
1) time that lyophilized powder is recovered is at least 15-30min, and what have is longer, and in the process of measuring, because the serious hysteresis quality of time can not realize the on-line monitoring to water quality;
2) the lyophilized powder stability of photoluminescence after the recovery is bad, influences the toxicity monitoring accuracy.
Studies show that the Fei Shi vibrios utilizes the quorum sensing system to carry out exchanging between cell and the cell, and can regulate the characteristics of luminescence of bacterium.The quorum sensing system is in regulating the luminous process of Fei Shi vibrios; luciferase is by the luxCDABE genes encoding; LuxI and LuxR albumen constitute the modulin of quorum sensing; wherein LuxI is a self-induction thing synthetic enzyme; can synthesize a kind of acyl homoserine lactones compounds (N-(3-oxo-hexanoyl)-homoserine lactone; be AHL), again this compound is become the signaling molecule of quorum sensing.LuxR be activated regulation and control luxICDABE expression of gene after AHL combines.When bacterial density is low, luxI genetic expression is less, can only produce a spot of self-induction thing and free be secreted into the extracellular by cytolemma, make in the cell and the AHL of same concentrations is contained in the extracellular all the time, the quantity not sufficient of luxCDABE genetic expression at this moment is so that thalline is luminous; Along with bacterial number constantly increases, the nectar degree constantly strengthens, and when extracellular self-induction thing reaches capacity concentration (1-10 μ g/ml), just can fully combine with the LuxR albumen in the tenuigenin; The proteic DNA calmodulin binding domain CaM of LuxR that combines the self-induction thing exposes, and the LuxR albumen of this moment can combine with the promotor of luxICDABE gene, thereby activates this expression of gene, finally forms biological luminescence.
Summary of the invention
At the deficiencies in the prior art, the invention provides a kind of photogenic bacterium microbial inoculum of the energy rapid detection water quality toxicity based on quorum sensing effect signal molecule, utilize the signaling molecule that contains in the photogenic bacterium nutrient solution supernatant that the luminous situation of photogenic bacterium lyophilized powder is regulated, make the 15-30min of recovery to the stable time from the commercially available prod of lyophilized powder, shorten to 5min, can can make reaction timely, online, the real-time monitoring of real accomplishing to the toxicity of water ecological environment according to the variation of the light signal strength that stimulation produced of environmental change.
A kind of quick luminous microbial inoculum that supports based on signaling molecule of the present invention, it is characterized in that: the composition of photobacteria lyophilized powder is that concentration is 10
9The Fei Shi vibrios 10ml of individual/ml, bacterium mud and 1ml cryoprotectant after centrifugal mix, and make after lyophilize, heavy altogether 0.5g, and place-20 ℃ condition to preserve 6 months shelf times.
Described a kind of quick luminous microbial inoculum that supports based on signaling molecule is characterized in that: described Fei Shi vibrios obtains the photogenic bacterium bacterium liquid of luminous intensity more than 700mV for cultivating through screening, slant culture and shaking table bacterium liquid.
Described a kind of quick luminous microbial inoculum that supports based on signaling molecule, the composition of its special described cryoprotectant is that skimming milk 15g, glycerine 5g, trehalose 12.5g are dissolved in the 100ml signaling molecule solution formulated.
Described a kind of quick luminous microbial inoculum that supports based on signaling molecule is characterized in that described signaling molecule solution is that the signaling molecule mother liquor is mixed with 3%NaCl solution, and being mixed with volume ratio is the signaling molecule mother liquor: 3%NaCl solution is the mixed solution of 30:70.
Described a kind of quick luminous microbial inoculum that supports based on signaling molecule, its described signaling molecule mother liquor prepares by the following method: photogenic bacterium bacterium liquid centrifugal 5min under 8500rpm that will cultivate 14h, sterilize by the membrane filtration of 0.22 μ m again, every 100ml filtrate adds 3gNaCl, be the signaling molecule mother liquor, place 4 ℃ condition to preserve.
Described a kind of quick luminous microbial inoculum that supports based on signaling molecule is characterized in that it is that photogenic bacterium is inoculated in the liquid nutrient medium that described shaking table bacterium liquid is cultivated, and places shaking table to transfer to 180rpm, and 20 ℃, concussion is cultivated; The liquid culture based component is: 0.5g Yeast diffusion juice, 0.5g Tryptones, 3gNaCl, 0.5gNa
2HPO
4, 0.1gKH
2PO
4, 3ml glycerine, be dissolved in the 100ml distilled water, transfer pH to 6.8.
Described a kind of quick luminous microbial inoculum that supports based on signaling molecule, it is characterized in that, described slant culture is earlier solid medium (to be liquid state when being higher than 40 ℃, condense below 40 ℃ and be solid-state) when high temperature, pour in the 50ml test tube, pouring volume should be test tube volumetrical 1/4-1/3, test tube is tilted to place room temperature, treat that its cooling can form solid-state slant medium, is slant culture with microbionation to solid medium; On the basis of solid medium liquid medium within composition, add the 2%(massfraction again) agar get final product.
The principle of luminosity of photogenic bacterium:
The luminescence mechanism of different photogenic bacteriums is identical: be that specific luciferase (LE) is at aerobic (O
2) under the condition that participates in, the flavin nucleotide (FMNH2) and the above long-chain fat aldehyde of eight carbon (RCHO) of reductibility are oxidized to FMN and longer chain fatty acid, discharging maximum emission intensity simultaneously is the process of the blue green light at 450～490nm place at wavelength.Course is as follows:
FMNH2+LE → FMNH2LE+O2 → LEFMNH2O2+RCH → LEFMNH2O2RCHO → LE+FMN+H2O+RCOOH+ light
The present invention compares with prior art has following advantage:
(1) added bacterial population induced signaling molecule in the photogenic bacterium lyophilized powder, this signaling molecule exists can regulate the synthetic of the interior luciferase of cell, and luciferase is the catalyzer that bacterium produces fluorescence.Because the adding of signaling molecule can promote the synthetic of the interior luciferase of cell, photogenic bacterium lyophilized powder recovery to the luminous stable time is shortened, reach stable from traditional 15-30min, shorten to 5min and can reach stable.
(2) owing to need to experience freezing and dry two processes in bacterium lyophilized powder making processes, these two processes can cause great influence to bacterial structure and activity.The protectant adding of composite frozen can make more bacterium keep cyto-architectural complete sum cell activity when freezing and dry, and can recover when condition is suitable.
Embodiment
A kind of quick luminous microbial inoculum that supports based on signaling molecule of the present invention, it is characterized in that: the composition of photobacteria lyophilized powder is that concentration is 10
9Fei Shi vibrios 10ml about individual/ml, bacterium mud and 1ml cryoprotectant after centrifugal mix, and make after lyophilize, heavy altogether 0.5g, and place-20 ℃ condition to preserve 6 months shelf times.
Further, described Fei Shi vibrios is to cultivate through screening, slant culture and shaking table bacterium liquid to obtain the photogenic bacterium bacterium liquid of luminous intensity more than 700mV.
Further, the composition of described cryoprotectant is that skimming milk 15g, glycerine 5g, trehalose 12.5g are dissolved in the 100ml signaling molecule solution formulated.
Further, described signaling molecule solution prepares by the following method: with signaling molecule mother liquor and 6%NaCl(massfraction) solution mixes, and be mixed with the signaling molecule mother liquor: 3%NaCl solution is the mixed solution of 30:70.
Further, described signaling molecule mother liquor prepares by the following method: will cultivate photogenic bacterium bacterium liquid centrifugal 5min under 8500rpm of 14h, by the membrane filtration sterilization of 0.22 μ m, every 100ml filtrate adds 3gNaCl again, be the signaling molecule mother liquor, place 4 ℃ condition to preserve.
Further, it is that photogenic bacterium is inoculated in the liquid nutrient medium that described shaking table bacterium liquid is cultivated, and places shaking table to transfer to 180rpm, and 20 ℃, concussion is cultivated; The liquid culture based component is: 0.5g Yeast diffusion juice, 0.5g Tryptones, 3gNaCl, 0.5gNa
2HPO
4, 0.1gKH
2PO
4, 3ml glycerine is dissolved in the 100ml distilled water, transfers pH to 6.8.
Further, described slant culture is earlier solid medium (to be liquid state when being higher than 40 ℃, condense below 40 ℃ and be solid-state) when high temperature, pour in the 50ml test tube, pouring volume should be test tube volumetrical 1/4-1/3, test tube is tilted to place room temperature, treat that its cooling can form solid-state slant medium, is slant culture with microbionation to solid medium; On the basis of solid medium liquid medium within composition, add the 2%(massfraction again) agar get final product.
The present invention specifically comprises step:
The first step, the cultivation of photogenic bacterium
Detecting all photogenic bacteriums is the Fei Shi vibrios, for independently separating preservation in this laboratory.Before lyophilized powder is made to photogenic bacterium screen, slant culture and liquid culture obtain the fresh bacterium liquid that initial luminous intensity is not less than 700mV, is the good bacterium liquid of activation, and be standby.
The liquid culture based component is: 0.5g Yeast diffusion juice, 0.5g Tryptones, 3gNaCl, 0.5gNa
2HPO
4, 0.1gKH
2PO
4, 3ml glycerine, be dissolved in the 100ml distilled water, transfer pH to 6.8.This is a liquid nutrient medium;
On the basis of solid medium liquid medium within composition, add the 2%(massfraction again) agar get final product.
Second step, the making of photogenic bacterium lyophilized powder
Cultivate 22h in the photogenic bacterium liquid medium within that activation is good; at 8500rpm, 20 ℃ centrifugal down with bacterium liquid, presses 10:1 and concentrate collection (after every 10ml bacterium liquid is centrifugal; pour out supernatant; add the 1ml cryoprotectant), resuspended, be sub-packed in the aseptic ampoul tube; every pipe 0.5ml; place Ultralow Temperature Freezer-70 ℃ pre-freeze 1h earlier, place the dry 10h of freeze drier-50 ℃ low pressure then, Air drying 2h.Lyophilized powder is made to finish and is placed-20 ℃ to preserve down.
The 3rd step, the recovery of photogenic bacterium lyophilized powder
Ampoul tube placed stablize 2-5min under 20 ℃ of constant temperature, every ampoul tube adds 1ml distilled water, and concussion is to dissolving.Recovery 5min promptly can be used for measuring.
The 4th step, the toxicity test of sample
In raw wastewater, add solid NaCl be made into contain the 3%NaCl(massfraction) solution, use the 3%NaCl(massfraction again) solution dilutes the waste water sample by 10 times of stepwise dilution methods, by preliminary experiment judge water sample in, lower bound concentration, be bigness scale one time when bacterium relative luminous intensity during at 1%-100%, which concentration range is the concentration of waste water drop on.Result according to preliminary experiment, to add the raw wastewater concentration of solid NaCl as maximum concentration, the relative luminous intensity that waste water is dropped on bacterium (for example is diluted to 5-6 water sample to be measured by principle such as logarithmic interval such as grade again at the solution of 1%-100% concentration range, if when bacterium relative luminous intensity during at 1%-100%, the concentration of waste water falls between the 1%-10%, then should be diluted to 10%, 6.31%, 3.98%, 2.51%, 1.58,1.00%).The water sample that dilution is good is respectively got 1mL and is added in the testing tube, with the 3%NaCl(massfraction) solution compares, and the every pipe of photogenic bacterium lyophilized powder that recovery is good adds 20 μ L, behind the 10mim, measures luminous intensity, and calculates luminous inhibiting rate.Obtaining luminous relatively inhibiting rate with method of linear interpolation is 50% o'clock pairing waste strength, is this waste water to the luminous inhibiting rate (EC of photogenic bacterium half
50).
The 5th step, the method for expressing of water quality toxicity
Utilize the percentage concentration after former water dilutes to represent its EC
50, judge water quality toxicity by " the percentage ratio grade toxicity criteria for classifying ".
The percentage ratio grade toxicity criteria for classifying
| EC 50 | The toxicity rank | Grade |
| <25% | Strong poison | Ⅰ |
| 25-75% | Poison | Ⅱ |
| 75-100% | Little poison | Ⅲ |
| 100% or can not ask EC 50 | Nontoxic | Ⅳ |
Claims (7)
1. quick luminous microbial inoculum that supports based on signaling molecule, it is characterized in that: the composition of photobacteria lyophilized powder is that concentration is 10
9The Fei Shi vibrios 10ml of individual/ml, bacterium mud and 1ml cryoprotectant after centrifugal mix, and make after lyophilize, and place-20 ℃ condition to preserve 6 months shelf times.
2. a kind of quick luminous microbial inoculum that supports based on signaling molecule according to claim 1 is characterized in that: described Fei Shi vibrios obtains the photogenic bacterium bacterium liquid of luminous intensity more than 700mV for cultivating through screening, slant culture and shaking table bacterium liquid.
3. a kind of quick luminous microbial inoculum that supports based on signaling molecule according to claim 1, it is characterized in that: the composition of described cryoprotectant is that skimming milk 15g, glycerine 5g, trehalose 12.5g are dissolved in the 100ml signaling molecule solution formulated.
4. a kind of quick luminous microbial inoculum that supports based on signaling molecule according to claim 3, it is characterized in that, described signaling molecule solution is to be that 6%NaCl ÷ solution mixes with signaling molecule mother liquor and quality, and being mixed with volume ratio is the signaling molecule mother liquor: 6%NaCl solution is the mixed solution of 30:70.
5. a kind of quick luminous microbial inoculum that supports based on signaling molecule according to claim 4, it is characterized in that, described signaling molecule mother liquor is photogenic bacterium bacterium liquid centrifugal 5min under 8500rpm that will cultivate 14h, sterilize by the membrane filtration of 0.22 μ m again, every 100ml filtrate adds 3gNaCl, be the signaling molecule mother liquor, place 4 ℃ condition to preserve.
6. a kind of quick luminous microbial inoculum that supports based on signaling molecule according to claim 2 is characterized in that it is that photogenic bacterium is inoculated in the liquid nutrient medium that described shaking table bacterium liquid is cultivated, and places shaking table to transfer to 180rpm, and 20 ℃, concussion is cultivated; The liquid culture based component is: 0.5g Yeast diffusion juice, 0.5g Tryptones, 3gNaCl, 0.5gNa
2HPO
4, 0.1gKH
2PO
4, 3ml glycerine, be dissolved in the 100ml distilled water, transfer pH to 6.8.
7. a kind of quick luminous microbial inoculum that supports based on signaling molecule according to claim 2, it is characterized in that, described slant culture is earlier solid medium to be poured in the 50ml test tube when the high temperature, pouring volume should be test tube volumetrical 1/4-1/3, test tube is tilted to place room temperature, treat that its cooling can form solid-state slant medium, is slant culture with microbionation to solid medium; On the basis of solid medium liquid medium within composition, add mass percent again and be 2% agar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101444241A CN102250771A (en) | 2011-05-31 | 2011-05-31 | Quick luminescent bacteria supported based on signal molecule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101444241A CN102250771A (en) | 2011-05-31 | 2011-05-31 | Quick luminescent bacteria supported based on signal molecule |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102250771A true CN102250771A (en) | 2011-11-23 |
Family
ID=44978343
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101444241A Pending CN102250771A (en) | 2011-05-31 | 2011-05-31 | Quick luminescent bacteria supported based on signal molecule |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250771A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468572A (en) * | 2013-09-25 | 2013-12-25 | 北京尚洋东方环境科技股份有限公司 | Freeze-drying protective agent for photogenic bacterium |
| CN103484408A (en) * | 2013-09-25 | 2014-01-01 | 北京尚洋东方环境科技股份有限公司 | Culture medium for light-emitting bacterium |
| CN104419644A (en) * | 2013-09-10 | 2015-03-18 | 上海市刑事科学技术研究院 | Luminous bacteria freeze-dried preparation and preparation method thereof |
| CN106546579A (en) * | 2016-10-28 | 2017-03-29 | 华南理工大学 | A kind of organic solvent improves the method that photobacteria detects toxicant susceptibility |
| CN110747217A (en) * | 2019-11-04 | 2020-02-04 | 南京农业大学 | Iterative gene circuit based on Vibrio fischeri quorum sensing system and T7 expression system and application thereof |
| CN111566199A (en) * | 2019-08-27 | 2020-08-21 | 湖南农业大学 | Luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of luminescent bacterium freeze-drying protective agent and freeze-dried powder in water quality comprehensive toxicity online monitoring |
-
2011
- 2011-05-31 CN CN2011101444241A patent/CN102250771A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104419644A (en) * | 2013-09-10 | 2015-03-18 | 上海市刑事科学技术研究院 | Luminous bacteria freeze-dried preparation and preparation method thereof |
| CN103468572A (en) * | 2013-09-25 | 2013-12-25 | 北京尚洋东方环境科技股份有限公司 | Freeze-drying protective agent for photogenic bacterium |
| CN103484408A (en) * | 2013-09-25 | 2014-01-01 | 北京尚洋东方环境科技股份有限公司 | Culture medium for light-emitting bacterium |
| CN103468572B (en) * | 2013-09-25 | 2016-07-06 | 北京尚洋东方环境科技有限公司 | A kind of freeze drying protectant of luminescent bacteria |
| CN106546579A (en) * | 2016-10-28 | 2017-03-29 | 华南理工大学 | A kind of organic solvent improves the method that photobacteria detects toxicant susceptibility |
| CN111566199A (en) * | 2019-08-27 | 2020-08-21 | 湖南农业大学 | Luminescent bacterium freeze-drying protective agent, freeze-dried powder and application of luminescent bacterium freeze-drying protective agent and freeze-dried powder in water quality comprehensive toxicity online monitoring |
| CN110747217A (en) * | 2019-11-04 | 2020-02-04 | 南京农业大学 | Iterative gene circuit based on Vibrio fischeri quorum sensing system and T7 expression system and application thereof |
| CN110747217B (en) * | 2019-11-04 | 2022-12-20 | 南京农业大学 | Iterative gene circuit based on Vibrio fischeri quorum sensing system and T7 expression system and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102250771A (en) | Quick luminescent bacteria supported based on signal molecule | |
| Wang et al. | Enhanced removal of antibiotic resistance genes and mobile genetic elements during swine manure composting inoculated with mature compost | |
| Min et al. | Cultivating Chlorella sp. in a pilot-scale photobioreactor using centrate wastewater for microalgae biomass production and wastewater nutrient removal | |
| Chang et al. | Cultivation of Spirulina platensis for biomass production and nutrient removal from synthetic human urine | |
| Abdelaziz et al. | Screening microalgae native to Quebec for wastewater treatment and biodiesel production | |
| Mierzwa-Hersztek et al. | Effect of coapplication of biochar and nutrients on microbiocenotic composition, dehydrogenase activity index and chemical properties of sandy soil | |
| Maitra et al. | Ecological significance and phosphorus release potential of phosphate solubilizing bacteria in freshwater ecosystems | |
| CN104212431A (en) | Petroleum endogenous microbe activating system, and screening method and application thereof | |
| CN102391961B (en) | Raoultella ornithinolytica N-4 bacterial strains and preparation and application thereof | |
| Zhang et al. | Revealing the characteristics and formation mechanisms of partial denitrification granular sludge for efficient nitrite accumulation driven by glycerol | |
| CN102864102B (en) | Phthalate degrading strain and application thereof | |
| Oscar et al. | Deciphering the diversity of microalgal bloom in wastewater-an attempt to construct potential consortia for bioremediation | |
| CN114057284B (en) | Device and method for improving salt impact resistance of anaerobic ammonia oxidation particle system based on quorum sensing | |
| Aravinthan et al. | Coal seam gas water as a medium to grow Dunalliella tertiolecta microalgae for lipid extraction | |
| Zafar et al. | Performance of immobilized microalgal strains for biodesalination of real seawater | |
| Zhang et al. | Development, performance and microbial community analysis of a continuous-flow microalgal-bacterial biofilm photoreactor for municipal wastewater treatment | |
| CN105645596B (en) | A kind of activated sludge pre-treatment medicaments and its preparation method and application method | |
| Sládečková et al. | The utilization of periphyton in waterworks pre-treatment for nutrient removal from enriched influents | |
| CN103468646A (en) | Method for efficiently screening low-temperature bacteriophages | |
| Cervetto et al. | Feeding behaviour and migrations in a natural population of the copepod Acartia tonsa | |
| Chintala et al. | Examining biochar impacts on soil abiotic and biotic processes and exploring the potential for pyrosequencing analysis | |
| Zhang et al. | Extracellular polymeric substances extraction induced the increased purification performance of percoll density gradient centrifugation for anammox bacteria | |
| CN108690863A (en) | A method of measuring sludge heavy-metal Ecotoxicology using portable glucose meter | |
| KR20140022211A (en) | Culturing method of microalgae for producing biodiesel by using wastewater and abandoned mine drainage | |
| CN101386828B (en) | Reporting bacterial strain sensitive to oxidation-reduction cycle reactant and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20111123 |