CN103940789B - The specification of photobacteria Fluorescence behaviour and numeric value analysis in water toxicity detection - Google Patents
The specification of photobacteria Fluorescence behaviour and numeric value analysis in water toxicity detection Download PDFInfo
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Abstract
Although photobacteria contact poison can be good at quantifying toxicity, but due to bio-diversity, its toxicity quantized values is the most difficult for scientific communication, it is impossible to for the detection of actual water environment and monitoring.The present invention greatly improves photobacteria for the reproducibility that toxicity detects, if detecting used same normaltoxin and photobacteria dosage form, result of the test error is less than 10%.Such as, the specific Fermi operator of lyophilizing (Aliivibrio Fischeri) strain is used to detect the toxicity of phenol (analytical pure), its toxicity detection result has high reproducibility, does not changes because of operator or geographical diversity, and result of the test error is less than 10%.Being used as the assessment of water body toxicity completely, its result may be used for scientific communication.By the laboratory observation long-term to photobacteria with repeat test, sum up and a set of be greatly improved the photobacteria method for reproducibility that toxicity detects, mainly included following two inventions: 1) way of " photobacteria Fluorescence behaviour specification "；2) on numeric value analysis, improvement reduces error.
Description
The specification of photobacteria Fluorescence behaviour and numeric value analysis in water toxicity detection
1. technical field
The present invention can in the short time, (one little to the eco-toxicity (ecotoxicity) of the most unknown soluble toxins
Time) quantification, these toxin include inorganic salt, organic compound and the water Han the colorful one；Can there is no any analysisization
In the case of background of gaining knowledge, the toxicity to water makes early warning.Briefly, it is not known that, being also indifferent to is any water-soluble material,
But it is much to wonder that toxicity has on earth？What biological (ecological) consequence is？
The present invention is not suitable for chemical substance carcinogenecity or the assessment of pathogen danger and judgement；It is also not suitable for eucaryon thin
The toxic reaction of born of the same parents' receptor judges, also referred to as pharmacological toxicology.
The present invention is likely to be of potential misleading, and such as ethanol result in this detection is poisonous, and actual ethanol is at ring
There is no directly injury in border, this is that attribution ethanol has albuminous degeneration function, also poisonous to human organ.
2. background technology
The toxic contaminants source of water body environment can be many, the discharge of such as industrial wastewater containing toxic, industry or
Person's vehicle accident causes chemical substance river pollution, even sewage itself not to have toxicity, but due to broken in biodegradation process
After the molecular polarity of bad organic compound makes sewage enter in environment, toxicity increases suddenly.In the case of these, it is difficult in a short time to dirt
The chemical property of water carefully analyzes, it is desirable to do not have the poison to water in the case of any analytical chemistry knowledge to given sewage sample
Property judges, and action will be directly instructed in these judgements.This process, time is precious.
During water body toxicity in the past is assessed, rainbow Squaliobarbus ourriculus (Oncorhynchus mikiss) or water was once used to jump
The flea (Daphnia magna) mortality rate in water quantifies the toxicity of water.Rainbow Squaliobarbus ourriculus and the activity criteria of water flea
It is very difficult to define, and the aquatic animal of business-like standard is unusual costliness, it is impossible to use in practice.
Photobacteria or fluorescent bacteria (bioluminescent bacteria or luminescent bacteria)
Luminescent behavior and its respiratory chain chemical reaction be closely related.When, after photobacteria contact noxious substance, noxious substance destroys
The physiological metabolism of photobacteria, thus the strength retrogression of luminescence.The luminous intensity of photobacteria is expressed as " Relative fluorescence units "
The pad value of (Relative Luminescent Unit, RLU) comes toxicity evaluation and quantization.
Utilizing photobacteria contact poison is a technology the most charming to quantify toxicity, and it can be in the shortest time
Inside provide toxicity quantized data.Laboratory research for many years and domestic and foreign literature confirm the photobacteria Fluorescence behaviour of antibacterial
(bioluminescent behavior) is a behavior the most changeable, has extremely complex bio-diversity, such as same
The RLU value of the photobacteria of a collection of cultivation can be tens times even difference of hundreds of times, can describe carefully with being happy and angry uncertainly
Bacterium luminescent behavior.
Additionally, due to the international standard that Relative fluorescence units RLU neither one is unified, the luminous detection that different manufacturers produces
Instrument (Luminometer) employs different photomultiplier tube (photomultiplier) and signal amplification circuit, thus is
Making luminous intensity consistent, the RLU reading of the luminometer of different manufacturers also varies.
Although photobacteria contact poison can be good at quantifying toxicity, but owing to the reproducibility of RLU is extremely low, its poison
Property quantized values and unit results are the most difficult for scientific communication, thus accuracy and reproducibility are had a greatly reduced quality.It is not used to
The detection of actual water environment and monitoring.
The photobacteria that nature is selected at present is all facultative psychrophile (psychrotrophic bacterium), generally
Live in 10 °-20 DEG C；Marine bacteria (Fermi operator strain Aliivibrio fischeri) and light it is divided into from the point of view of ecological branch
Water bacteria (Qinghai Vibrion vibrio Qinghaiensis).These photobacterias all have been used for water toxicity detection.
Record the Fluorescence behaviour of how these photobacterias of specification currently without document thus obtain one and how can believe
Toxicity detection data.
3. summary of the invention
The present invention greatly improves photobacteria for the data reproducibility that toxicity detects, if detection is used same
Planting normaltoxin and photobacteria dosage form, the usual error of result of the test is less than 10%.Such as, lyophilizing specific Fermi operator strain is used
(Aliivibrio fischeri ATCC700601) detects the toxicity of phenol (analytical pure), and its toxicity detection result has pole
High reproducibility, does not changes because of operator or geographical diversity, and result of the test error is less than 10%.Completely can by with
Making the assessment of water body toxicity, its result may be used for scientific communication.
One, the method for photobacteria Fluorescence behaviour specification
The present invention compared for two groups of different classes of antibacterials: marine bacteria (Fermi operator strain Aliivibrio
And fresh water bacteria (Qinghai Vibrion Vibrio qinghaiensis) Fischeri).By the Fluorescence behaviour of two groups of antibacterials is seen
Examining, induction and conclusion has gone out the way of a set of specification " photobacteria Fluorescence behaviour ".
These ways include that strictly controlling cell cultivates, results, environment when preservation and detection and process.During concrete
Control the age of antibacterial, population effect (Quorum sensing), physiological status, cell density.During detection, strictly control
Liquid oxygen concentration processed, brooding time and temperature carry out the luminescent behavior of specification antibacterial.
Two, the numeric value analysis of bacterial fluorescence of giving out light behavior
Use modifying factor to revise check sample reading, reduce the error caused due to bio-diversity；
Set up concentration of specimens, time and the linear relationship of poisonous substance gamma value.For coloured sewage, use doing of color correction
Method removes the error that optical interference causes.
Three, contrast Marine Luminous Bacteria (Fermi operator strain Aliivibrio Fischeri) and fresh water photobacteria (Qinghai
Vibrio vibrio Qinghaiensis) luminescent behavior after, sum up the suitability of this method.
Four, the luminometer (Luminometer) that detection different manufacturers produces.
In photobacteria Fluorescence behaviour numerical value, have employed ratio mode, cancel all different manufacturers illumination instrument
Any difference of detector.When specifically Control of Fluorescence effect being deducted a certain contact equal to the fluorescence intensity of zero time point
Between fluorescence intensity (drawing the fluorescence intensity of decay) in the fluorescence intensity of division by 0 time point.By such mode, by instrument
The different number of degrees between device are all united.
Detailed description of the invention
The most supporting culture medium of strain and reagent
1. Marine Luminous Bacteria Fermi operator (Aliivibrio fischeri ATCC700601)
(American Type Culture Collection) is buied by American Type Culture Collecti
Culture medium: every 1 liter of culture medium contains: 5 grams of yeast extracts, 5 grams of tryptones, 0.5 gram of magnesium sulfate, 1 gram of calcium carbonate, 3 grams of phosphorus
Potassium hydrogen phthalate, 30 grams of sodium chloride, agar culture medium separately adds 20 grams of agar if necessary.After 121 ° of high pressure steam sterilizations, room temperature preserves.
Frost protection liquid: every 100 milliliters of frost culture fluid contain: 60 grams of glucoses, 4 grams of sodium chloride, 2 grams of L-Histidines,
0.5 gram of bovine serum albumin, it is 7 standby for adjusting pH value after fully dissolving.
Frozen-dried protective liquid: every 100 milliliters of lyophilizing liquid contain: 8 grams of defatted milk powder, 8 grams of glucoses, 0.5 gram of arginine and 3 grams
Sodium chloride, it is 7 standby for adjusting pH value after fully dissolving.
Antibacterial is brought back to life and detection buffer: every 100 milliliter buffer contain: 7.3 grams of glucoses, 2.3 gram of 6 aqueous magnesium chloride,
0.3 gram of potassium chloride, 12 grams of HEPES (N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic acid),
Sigma company .) 20 grams of sodium chloride；PH is adjusted to 7.0.
2. fresh water photobacteria: Qinghai Vibrion vibrio Qinghaiensis CS235
By shore, Beijing, pine photonic propulsion commerce and trade (Chinese) company limited buys
Culture medium:Every 1 liter of culture medium contains 2.5 grams of magnesium sulfate, 0.5 gram of magnesium carbonate, 0.5 gram of calcium carbonate, 0.15 gram of chlorination
Potassium, 8 grams of sodium chloride, 5 grams of yeast extracts, 5 grams of tryptones, agar culture medium separately adds 20 grams of agar if necessary.121.High steam goes out
After bacterium, room temperature preserves.
Frost protection liquid:Every 100 milliliters of frost culture fluid contain: 60 grams of glucoses, 0.8 gram of sodium chloride, 2 grams of L-group ammonia
Acid, 0.5 gram of bovine serum albumin.It is 7 standby for adjusting pH value after fully dissolving.
Frozen-dried protective liquid:Every 100 milliliter lyophilizing liquid contain: 8 grams of defatted milk powder, 8 grams of glucoses, 0.5 gram of arginine, fully
It is 7 standby for adjusting pH value after dissolving.
Antibacterial is brought back to life and detection buffer: every 100 milliliters of buffer contain: 7.3 grams of glucoses, 23 gram of 6 aqueous magnesium chloride,
0.3 gram of potassium chloride, 12 grams of HEPES (N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic
Acid), Sigma company .) 3 grams of sodium chloride；PH is adjusted to 7.0.
Test operation step
A kind of method utilizing photobacteria after contacting water solublity poisonous substance to quantify poisonous substance toxicity is standardized behaviour
Make.Make the detection toxicity of standard poisonous substance less than 10%.The effectively Fluorescence behaviour of specification antibacterial reaches standardization purpose；
1) cultivation of photobacteria and results:
The first step: single bacterium colony photobacteria is seeded in the aseptic liquid nutrient medium liquid conical flask (capacity of 10 milliliters
50 milliliters) in, shaking table 180 rpms, it is incubated 20 DEG C, takes 1 milliliter of bacterium solution after cultivating 24 hours and carry out secondary inoculation；
Second step: 1 milliliter of bacterium solution of giving out light is inoculated into the aseptic liquid nutrient medium conical flask (capacity 250 of 50 milliliters again
Milliliter) in, shaking table 180 rpms, it is incubated 20 DEG C, after cultivating 20 hours, detects cell density rear (OD600=0.8-up to standard
1.0) at once bacterium solution is placed on ice bath 15 minutes on Sorbet；
3rd step: bacterium solution after ice bath is carried out 4 DEG C of frozen centrifugations (6000g) and removes supernatant harvesting after 15 minutes.
2) antibacterial gathered in the crops preserves:
● stored frozen: by harvesting ice bath 15 points in 100 milliliters of stored frozen liquid (pre-cooling 15 minutes in ice bath)
Clock stirs evenly, 100 microlitre subpackages, puts in liquid nitrogen and solidifies 1 minute, puts into-70 DEG C of Refrigerator stores 2 years until using；
● lyophilization preserves: by harvesting ice bath in 100 milliliters of lyophilization liquid (pre-cooling 15 minutes in ice bath)
Within 15 minutes, stirring evenly, 100 microlitre subpackages, put in liquid nitrogen and solidify 1 minute, put into freezer dryer dried, room temperature darkroom preserves 2
Year is until using.
3) resurrection of antibacterial is prepared with test bacterium solution
Portion stored frozen or cryodesiccated photobacteria are dissolved in 100 milliliters of resurrection liquids, will examination during dissolving
Pipe is jiggled, it is to avoid produce bubble.The cell being completely dissolved is placed in 20 DEG C of aluminium ingot temperature controls silently hatch 15 minutes-30 points
Clock.Prepare complete
4) prepared by test poison sewage
By sewage with arbitrary proportion serial dilution 10 times (generally advising 10%), if using Marine Luminous Bacteria detection
Final sodium chloride concentration is controlled 2% by sample.Finally poison sewage is placed in 15 DEG C of aluminium ingot temperature controls and silently hatches 15 minutes.
5) toxicity detection reading
Poison sewage is added in the photobacteria solution of previously preparation with same capacity ratio, when recording zero after record contact
Between, 5 minutes, light intensity (RLU) value of 15 minutes and 30 minutes.Use the luminometer that different manufacturers produces
(Luminometer) including: CleanTrace (3M company), (shore, Beijing pine photon has shore pine BHP9514 type drinking water detector
Limit company).All processes is temperature control in 15 DEG C of aluminium ingot temperature controls.
Data parsing:
In order to reduce the error that bio-diversity causes, it is firstly introduced into fluorescence intensity correction factor, as formula 1 describes, one
Individual matched group antibacterial, in the case of not having any toxicant exposure, fluorescence intensity also can decay sometimes, and the ratio of its decay is referred to as
Fluorescence intensity correction factor:
Formula 1: the correction factor of fluorescence intensity:
fkt=Ikt/I0
fktThe correction factor (time be often registered as minute) of contact T time
I0The fluorescence intensity (sample in matched group, unit is RLU) of contact zero-time
IktThe fluorescence intensity (sample in matched group, unit is RLU) of contact T time
In poisonous substance detection sample, its fluorescence radiation intensity is amassed as the fluorescence intensity after correcting with correction factor.Such as public affairs
Formula 2:
Formula 2: the fluorescence intensity after correction
Ict=I0fkt
fktIt is fktAverage；
I0It it is the fluorescence intensity (RLU) of contact zero-time
IctI after correction0(RLU)
Antibacterial is after contact poison T time, and its fluorescence intensity has decayed, and its Control of Fluorescence effect is expressed as formula 3:
Formula 3:(antibacterial) Control of Fluorescence effect
Ht=(Ict-ITt)/Ict×100
HtIt it is given sample Control of Fluorescence effect (as a percentage) within fixing time of contact
IctI after correction0；
ITtIt it is given sample fluorescence intensity (RLU) within fixing time of contact
In order to assess the relation of concentration and toxicity, introduce gamma value and describe each diluted concentration under a certain special time
Inhibition.
Formula 4: the gamma value of toxicant
Γt=Ht/(100-Ht)
ГtIs measures the gamma value after the sample contact time
HtIt it is given sample Control of Fluorescence effect (as a percentage) within fixing time of contact
With TC, and the gamma value (toxicological characteristics of poisonous substance itself) of certain fixing time of contact set up one linear
Relation such as formula 5:
Formula 5: toxicity and the relation of concentration under given time of contact
Lgc=blg Γ t+1ga
ΓtMeasure the gamma value after the sample contact time；
CtThe dilution ratio (the most as a percentage) of test sample；
Such as Гt=0.25, then Ct=IC20, or such as Γ t=1, then Ct=IC50；
ΓtIt it is the gamma value after measuring the sample contact time；
B: slope
Inhibition concentration (Inhibition Concentration, IC) is used for expressing the concentration of a given sewage sample and presses down
The ratio of bacterial luminescence processed.Such as IC20Represent the sewage of certain concentration inhibit within a time of contact 20% antibacterial maximum
Luminous strong, or IC50Represent the sewage of certain concentration inhibit within a time of contact 50% antibacterial maximum luminous strong.IC value
The least expression sewage toxicity is the biggest.And time of contact, the shortest (such as 5 minutes) just demonstrated that toxicity is acute toxicity.
Chromatic aberration correction:
The usual emission wavelength of photobacteria in 490 nanometers (yellow green), but some detection samples may containing color can
Interference can be brought, it is therefore necessary to carried out chromatic aberration correction before toxotest.
Use spectrophotometer (Shanghai Ao Xi scientific instrument company limited AX6001) that sewage reads the absorption of 490 nanometers
Value (contrast matched group deionized water):
Formula 6:490 nanometer absorbs contribution margin
Ax=2.303 × ABSx
ABSxIt is 490 nanometer absorption values
AxIt is that 490 nanometers absorb contribution margin
Formula 7: color transmittance (Transmittance)
Tx=(1-e-Ax)/Ax
TxIt it is transmittance
AxIt is that 490 nanometers absorb contribution margin
Formula 8: the luminous intensity after color correction
ACI0=I0×Tx
ACI0It it is the luminous intensity after color correction
I0It it is the fluorescence intensity (RLU, as formula 2 describes) of contact zero-time
TxIt it is transmittance
For the toxicity detection after color correction, by formula 8 numerical value ACI0Replace formula I0, ACITtReplace ITtContinue fortune
Calculate.
The half-inhibition concentration (IC50) of common Toxic
Inorganic matter sample lyophilizing ocean and fresh water photobacteria half-inhibition concentration (IC50)
Organic sample freezing and lyophilizing Marine Luminous Bacteria half-inhibition concentration (IC50)
Organic sample lyophilizing ocean and fresh water photobacteria half-inhibition concentration (IC50)
Half-inhibition concentration (IC50) after toxicant lyophilizing Marine Luminous Bacteria and pigment interference
Claims (3)
1. the method for photobacteria Fluorescence behaviour specification in toxicity detection, comprises the following steps:
A) resurrection of antibacterial is prepared with test bacterium solution
During portion stored frozen or cryodesiccated photobacteria are dissolved resurrection liquid, after needing to be completely dissolved antibacterial
It is placed in 15-25 DEG C of aluminium ingot temperature control and silently hatches 15 minutes-45 minutes；
B) prepared by test poison sewage
Sewage in 10-20 DEG C of aluminium ingot temperature control, is hatched 10-25 silently divide must be placed on by sewage after arbitrary proportion serial dilution
Clock；
C) toxicity detection reading
Poison sewage is added in the photobacteria solution of preparation with same capacity ratio, the light intensity of each time point after record contact
(Relative Luminescent Unit) value；
D) numeric value analysis
In order to reduce the error that bio-diversity causes, being firstly introduced into fluorescence intensity correction factor, as formula 1 describes, one right
According to group antibacterial, in the case of not having any toxicant exposure, fluorescence intensity also can decay sometimes, and the ratio of its decay is referred to as fluorescence
Intensity correction factor:
Formula 1: fluorescence intensity correction factor:
fkt=Ikt/I0
fktThe correction factor of contact T time, the time is recorded as minute
I0The fluorescence intensity of contact zero-time, the sample in matched group, unit is RLU
IktThe fluorescence intensity of contact T time, the sample in matched group, unit is RLU
In poisonous substance detection sample, its fluorescence radiation intensity and correction factor amass as the fluorescence intensity after correcting, such as formula 2:
Formula 2: the fluorescence intensity after correction
It is fktAverage；
I0It it is the fluorescence intensity (RLU) of contact zero-time
IctI after correction0(RLU)
Antibacterial is after contact poison T time, and its fluorescence intensity has decayed, and its Control of Fluorescence effect is expressed as formula 3:
Formula 3: the Control of Fluorescence effect of antibacterial
Ht=(Ict-ITt)/Ict×100
HtIt is given sample Control of Fluorescence effect within fixing time of contact, as a percentage
IctI after correction0；
ITtIt it is given sample fluorescence intensity (RLU) within fixing time of contact
In order to assess the relation of concentration and toxicity, introduce gamma value and describe pressing down of each diluted concentration under a certain special time
Effect processed, such as formula 4:
Formula 4: the gamma value of toxicant
Гt=Ht/(100-Ht)
ГtIt it is the gamma value after measuring the sample contact time
HtIt it is given sample Control of Fluorescence effect within fixing time of contact
With TC, and the gamma value of certain fixing time of contact sets up a linear relationship such as formula 5:
Formula 5: toxicity and the relation of concentration under given time of contact
LgCt=blg Г t+lga
ГtMeasure the gamma value after the sample contact time；
CtThe dilution ratio of test sample, as a percentage；
ГtIt it is the gamma value after measuring the sample contact time；
B: slope.
2. the method for claim 1, also includes chromatic aberration correction:
Use spectrophotometer that sewage reads the absorption value of 490 nanometers, contrast matched group deionized water:
Formula 6:490 nanometer absorbs contribution margin
Ax=2.303 × ABSx
ABSxIt is 490 nanometer absorption values
AxIt is that 490 nanometers absorb contribution margin
Formula 7: color transmittance (Transmittance)
Tx=(1-e-Ax)/Ax
TxIt it is transmittance
AxIt is that 490 nanometers absorb contribution margin
Formula 8: the luminous intensity after color correction
ACI0=I0×Tx
ACI0It it is the luminous intensity after color correction.
3. the method for claim 1, contrast Marine Luminous Bacteria Fermi operator strain Aliivibrio Fischeri and light
After the luminescent behavior of water photobacteria Qinghai Vibrion vibrio Qinghaiensis, sum up the method and be applicable to fresh water and sea
The toxicity detection of ocean photobacteria.
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| CN102213721A (en) * | 2011-04-11 | 2011-10-12 | 同济大学 | Method for detecting toxicity of luminescent bacteria |
| CN102465167A (en) * | 2010-11-10 | 2012-05-23 | 中国科学院生态环境研究中心 | Rapid and high-flux acute toxicity test method for luminous bacteria |
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| CN102465167A (en) * | 2010-11-10 | 2012-05-23 | 中国科学院生态环境研究中心 | Rapid and high-flux acute toxicity test method for luminous bacteria |
| CN102213721A (en) * | 2011-04-11 | 2011-10-12 | 同济大学 | Method for detecting toxicity of luminescent bacteria |
Non-Patent Citations (1)
| Title |
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| Toxicity Evaluation of Antibiotics in Piggery Wastewater by Luminescent Bacteria;Jianping Sun 等;《Pol. J. Environ. Stud.》;20121231;第21卷(第3期);第741-747页 * |
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