CN107219205A - A kind of pesticide degradation bacteria activity test method based on Fluorescent Staining Observation - Google Patents
A kind of pesticide degradation bacteria activity test method based on Fluorescent Staining Observation Download PDFInfo
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- CN107219205A CN107219205A CN201710411999.2A CN201710411999A CN107219205A CN 107219205 A CN107219205 A CN 107219205A CN 201710411999 A CN201710411999 A CN 201710411999A CN 107219205 A CN107219205 A CN 107219205A
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- degradation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
Proposed by the present invention is a kind of organic agricultural chemicals degradation bacteria activity test method based on Fluorescent Staining Observation, specifically includes following steps：（1）Culture is enlarged for degradation bacteria；（2）Configure the cultivating system of n groups dead bacterium ratio containing different viable bacterias；（3）Calculate viable bacteria fluorescence and dead bacterium fluorescence ratio；（4）The degradation of pesticide activity of n group bacterium solutions is determined by degradation experiment；（5）The correlation curve of fluorescence ratio and degrading activity is drawn, the active calculation formula of degradation bacteria is set up；（6）Being verified with unknown active bacterium solution is used for follow-up test after formula accuracy.Advantage：1）Fluirescence observation can intuitively in reflection system degradation bacteria strains activated state；2）Detect that the used time is short, expense is low, convenient and simple；3）Degradation bacteria activity can real-time be detected, with ageing；4）It is applied widely.
Description
Technical field
The side of organic agricultural chemicals degradation bacteria activity can quickly, conveniently, intuitively, be accurately detected the present invention relates to a kind of
A kind of method, and in particular to pesticide degradation bacteria activity test method based on Fluorescent Staining Observation, belongs to microbial activity detection skill
Art field.
Background technology
Agricultural chemicals is as a kind of important capital goods in the agricultural sector, and to controlling crop diseases and insect pests, boosting agricultural yield has played weight
Act on, long-time, high frequency time, excessively produce a large amount of residuals in the environment using organic agricultural chemicals, seriously threaten ecological environment
Safety and human health, microorganism remediation because with non-toxic efficient, do not produce secondary pollution, it is easy to operate, economical and practical and should
With scope it is wide the advantages of, as remove environment in organic agricultural chemicals pollute a kind of important channel.
Domestic and international researcher by domestication, screening, enrichment and is separated from the environment sample such as sludge, sewage, soil, deposit
A variety of organic agricultural chemicals degradation bacterias have been obtained in product, and have utilized the technologies such as microbial immobilized, degradation bacterial agent preparation by some agricultural chemicals
Degradation bacteria is applied in Polluted area in-situ immobilization, must when carrying out microbial immobilized, degradation bacterial agent preparation and in-situ immobilization
Quantity, ratio and the degrading activity of viable bacteria in palpus guarantee system, thus it is particularly important to the detection of pesticide degradation bacteria activity, to protect
The important prerequisite of the in-situ immobilization effect of pesticide degradation bacteria is demonstrate,proved, the traditional detection method of pesticide degradation bacteria activity is more in a period of time
The residual quantity of target contaminant, calculates degradation efficiency to represent the work of degradation bacteria in system in interior analysis degradation bacteria culture systems
Property, the active monitoring method sampling period length of traditional pesticide degradation bacteria, Monitoring Pesticide Residues are wasted time and energy and spend higher.Meanwhile,
Conventional method is without ageing, when completing in a sampling analysis cycle, the life such as activity, abundance of system Pesticides degradation bacteria
Long status changes already, can not represent time of day during application；Therefore, development is needed badly a kind of quickly, in real time, conveniently, directly
The method seen, accurately detect organic agricultural chemicals degradation bacteria activity.
The content of the invention
Proposed by the present invention is a kind of organic agricultural chemicals degradation bacteria activity test method based on Fluorescent Staining Observation, its purpose
It is intended to make up the deficiency of existing pesticide degradation bacteria activity test method, especially overcoming existing method, time-consuming, costly, ageing
The drawbacks of in terms of difference.
The technical solution of the present invention：Organic agricultural chemicals degradation bacteria activity test method based on Fluorescent Staining Observation, tool
Body comprises the following steps：
（1）Culture is enlarged for degradation bacteria；
（2）Configure the cultivating system of n groups dead bacterium ratio containing different viable bacterias；
（3）Calculate viable bacteria fluorescence and dead bacterium fluorescence ratio；
（4）The degradation of pesticide activity of n group bacterium solutions is determined by degradation experiment；
（5）The correlation curve of fluorescence ratio and degrading activity is drawn, the active calculation formula of degradation bacteria is set up；
（6）Being verified with unknown active bacterium solution is used for follow-up test after formula accuracy.
Advantages of the present invention：
1）Organic agricultural chemicals degradation bacteria activity test method proposed by the present invention based on Fluorescent Staining Observation is by analyzing training system
The different colours fluorescence intensity of viable bacteria and dead bacterium in system, can intuitively reflect the activated state of bacterial strain in cultivating system；
2）Detect that the used time is short, expense is low, be that can evaluate degradation bacteria activity by fluorescence intensity, it is not necessary to which time-consuming, laborious goes inspection
Degradation bacteria is set up to the degradation amount of agricultural chemicals, and this method one in examining system, and follow-up test is very convenient simple, enormously simplify
The step of degradation bacteria Activity determination；
3）Degradation bacteria activity can real-time be detected, with ageing；
4）It is applied widely, it can be used to detect a variety of degradation bacteria activity under difficult environmental conditions, be also applied in in-situ immobilization
The monitoring of degradation bacteria activity.
Brief description of the drawings
Accompanying drawing 1 is the Chlorpyrifos degradation bacteria of embodiment 1SphingomonasSp. Dsp-2 green/reds fluorescence ratio
With the correlation criterion curve of degradation bacteria activity.
Embodiment
Organic agricultural chemicals degradation bacteria activity test method based on Fluorescent Staining Observation, specifically includes following steps：
（1）Culture is enlarged for degradation bacteria；
（2）Configure the cultivating system of n groups dead bacterium ratio containing different viable bacterias；
（3）Calculate viable bacteria fluorescence and dead bacterium fluorescence ratio；
（4）The degradation of pesticide activity of n group bacterium solutions is determined by degradation experiment；
（5）The correlation curve of fluorescence ratio and degrading activity is drawn, the active calculation formula of degradation bacteria is set up；
（6）With unknown active bacterium solution checking formula accuracy；
（7）Degradation bacteria activity in determination sample.
The step（1）Culture is enlarged for degradation bacteria：For certain degradation bacteria, prepare corresponding culture medium, culture
After base sterilizing, inoculation degradation bacteria is enlarged culture, sets wavelength OD600 to detect incubation degradation bacteria using spectrophotometer
Growing state.
The step（2）Configure the cultivating system of n groups dead bacterium ratio containing different viable bacterias：Stationary phase is reached in degraded bacteria growing
Afterwards, the bacterium solution of two parts of equivalent is taken, a copy of it carries out high-temperature inactivation, by the living bacterial liquid after shaking up and inactivated bacterial liquid in varing proportions
Proportioning, the percentage that the viable bacteria formed in n group cultivating systems, each cultivating system accounts for total bacterium is uniformly distributed between 0%-100%, is true
Guarantor's method accuracy, every group at least three repeats, n >=6.
The step（3）Calculate viable bacteria fluorescence and dead bacterium fluorescence ratio：After cultivating system configuration terminates, shake up and take immediately
Bacterium solution in a small amount of difference cultivating system, bacterium solution microscopy under fluorescence microscope, fluorescent dye used after fluorescent dyeing
Viable bacteria and dead bacterium can be distinguished, under different excitation wavelengths, the viable bacteria after dyeing is different with the fluorescence developing of dead bacterium, it is sharp after taking pictures
With different fluorescence optical density values in the image processing software analysis visual field, viable bacteria and dead bacterium fluorescence intensity in different cultivating systems are represented
Ratio.
The step（4）The degradation of pesticide activity of n group bacterium solutions is determined by degradation experiment：First after microscopy, to each culture
The organic agricultural chemicals of same concentration is added in system as the substrate of degradation bacteria, each cultivating system Pesticides of sampling analysis are residual at times
Allowance, the Organic pesticide residues amount that 100% culture group is accounted in viable bacteria terminates degradation experiment when being less than 30%, analyzes each cultivating system
In degradation of pesticide rate.
The step（5）The correlation curve of fluorescence ratio and degrading activity is drawn, the active calculation formula of degradation bacteria is set up：
Using viable bacteria in n group cultivating systems and dead bacterium fluorescence intensity ratio as abscissa, the degradation of pesticide rate of correspondence each group is ordinate, glimmering
Luminous intensity and degradation of pesticide rate are the average value with three repetitions of group, select suitable curve to click through data in two dimensional surface
Row fitting, draws degradation bacteria active testing standard curve, sets up the active calculation formula of degradation bacteria.
The step（6）With unknown active bacterium solution checking formula accuracy：For the degradation bacteria active testing standard of drafting
The accuracy of curve is verified that verification method is that the nutrient solution sampling to unknown viable bacteria percentage carries out Fluorescent Staining Observation,
Simultaneously in test cultures liquid degradation bacteria activity, by compare degradation bacteria activity that degradation bacteria active testing standard curve calculates with
Actually measured activity, verifies the accuracy of degradation bacteria active testing standard curve.
The step（7）Degradation bacteria activity in determination sample：When degradation bacteria in determination sample is active, concussion shakes up bacterium
A small amount of fluorescence intensity ratio for carrying out viable bacteria and dead bacterium in Fluorescent Staining Observation, analysis sample is taken after liquid, it is then strong according to fluorescence
Degree ratio directly finds the activity of degradation bacteria in the sample on standard curve.
Describe the implementation steps of invention in detail below by way of specific embodiment, it is therefore intended that enter those skilled in the art
One step understands the present invention, but the invention is not limited in any way；It should be pointed out that coming to one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.
Embodiment 1
Organophosphorus pesticide chlorpyrifos degrading bacteria based on Fluorescent Staining ObservationSphingomonas sp.Dsp-2 Activity determination.
Using the present invention to pure cultureSphingomonasSp. Dsp-2 chlorpyrifos degradations Activity determination is by following step
Rapid composition：
（1）WillSphingomonasSp. Dsp-2 inoculations are into 4L LB culture mediums（LB culture mediums contain tryptone 10
G/L, the g/L of yeast extract 5, the g/L of sodium chloride 10；pH 7.0）Earthquake shaking table is enlarged culture, shaking speed 200
Rmp, condition of culture is temperature 30oC, the uniform bacterium solutions of 1 ml are taken in superclean bench, spectrophotometric determination is used during culture
Absorbance OD600, detects the growing state of degradation bacteria；
（2）When degradation bacteria grows to stationary phase, 2 L nutrient solutions high-temperature inactivations formation inactivation nutrient solution is taken, remaining 2 L is viable bacteria
Nutrient solution, by the 6 groups of cultivating systems of formation of table 1, every group sets three repetitions：
The culture group 1-6 of table 1 configurations add viable bacteria and inactivation nutrient solution volume
（3）Preparing degraded bacterium suspension after cultivating system configuration terminates, after shaking up is used for fluorescent staining, fluorescent dyeing reagent selection
L7012 LIVE/DEADBacLight Bacterial Viability kits（Invitrogen companies of the U.S.）, the reagent
In box SYTO9 fluorescent dyes combine with it is in heaven or by dead, damaged membrane cell, in the nm/ transmitted waves of excitation wavelength 480
Dead cell aobvious red during long 500 nm；And propidium idodide fluorescent dyes are with reference to intact with cell membrane in kit
Living cells, the aobvious green of these living cells when in 490 nm/ 635 nm of launch wavelength of excitation wavelength, degraded bacterium suspension prepare and
Staining procedure detail is with reference to kit specification；After dyeing terminates, in fluorescence microscopy Microscopic observation, take pictures, utilize
Photoshop softwares obtain visual field Green and red fluorescence OD value, calculate green fluorescence and red fluorescence optical density ratio
Value；
（4）After fluorescence microscope terminates, degradation bacteria is carried out to each culture group to chlorpyrifos degrading Activity determination, to each culture
100 mg/L chlopyrifos are added in group, takes 2 ml to analyze persticide residue every 3 h, 1-6 group chlopyrifos is measured in 48 h
Residual quantity, degradation rate, degradation bacteria activity and fluorescence ratio are as shown in table 2（Display data is three repetition averages）：
The culture group 1-6 fluorescence ratios of table 2 and degrading activity testing result
（5）Using 1-6 groups viable bacteria and dead bacterium fluorescence intensity ratio as abscissa, the degradation of pesticide rate of correspondence each group is ordinate, glimmering
Luminous intensity and degradation bacteria activity are the average value with three repetitions of group, draw degradation bacteria active testing standard curve（It refer to
Accompanying drawing 1）；From standard curve, the culture group Chlorpyrifos degradation bacteria of different viable bacterias and dead bacterium proportioningSphingomonas
Sp. Dsp-2 green/reds fluorescence ratio meets linear relationship with degradation bacteria activity（R2=0.9963）, degradation bacteria activity
Calculation formula for degradation bacteria activity（μg/h）=96.872 × green/red fluorescence ratio -7.504；
（6）The unknown bacterium solution of viable bacteria percentage is taken to carry out Fluorescent Staining Observation, analysis green/red fluorescence ratio is 1.75, will
Ratio substitutes into step（5）It is the μ g of chlorpyrifos degradation 162.0 per hour that middle formula, which calculates degradation bacteria activity in cultivating system, is utilized
The accuracy of degradation experiment verification method, chlopyrifos is added into bacterium solution, final concentration of 100 mg/L, residual in bacterium solution after 48 h
It is 61.20 mg/L to stay chlopyrifos concentration, and degraded total amount is 7.76 mg, and degradation bacteria activity is chlorpyrifos degradation 161.7 per hour
μ g, with the chlorpyrifos degrading bacteria calculated based on fluorescence ratioSphingomonas sp.Dsp-2 degrading activities（162.0 μg/h）
Very little is differed, shows that fluorescence detection method and standard curve have accuracy and reliability.
Embodiment 2
The chlorpyrifos degrading bacteria that charcoal is loaded during pollution waters restorationSphingomonas Sp. Dsp-2 determinations of activity.
By chlorpyrifos degrading bacteriaSphingomonas sp.Dsp-2 is loaded by co-culture method and powdered biological charcoal
Afterwards, degradation bacteria-charcoal complex is fitted into permeable bag, intends rice field water-break system complex ring using ecological water channel mould indoors
Border condition, to ensure high degrading activity, strong adaptability of the complex in actual in-situ immobilization, ecological tank reclaimed water body is derived from
Field paddy rice water-break system, it is determined that being attached to the degradation bacteria on charcoalSphingomonas sp.Dsp-2 growth conditions and
Degrading activity, specifies the evaluation with degeneration system stability to provide theoretical foundation and data supporting for in-situ immobilization strategy.
Detect degradation bacteria-charcoal complex in ecological water using the method proposed by the present invention based on Fluorescent Staining Observation
Chlorpyrifos degrading bacteria after different time is placed in grooveSphingomonas sp.Dsp-2 degrading activity；
With the water body of field rice field water-break system is derived from as culture matrix, by the life of the load degradation bacteria after not inactivating and inactivating
1-6 culture group of the thing charcoal homogenate with different ratio formation as described in example 1 above, takes a small amount of nutrient solution to carry out fluorescent staining sight
Examine, green/red fluorescence ratio is calculated, by the degradation experiment computational load described in embodiment 1 in the degraded on charcoal
Bacterium is to the degrading activity of chlopyrifos, and green/red fluorescence ratio is linearly related to degrading activity, and calculation formula is that degradation bacteria is lived
Property（μg/h）=112.3 × green/red fluorescence ratio+1.023, places in ecological tank and is combined equipped with degradation bacteria-charcoal
The permeable bag of body, before experiment starts（0 h）Fluorescent Staining Observation is carried out first, and calculating obtains degradation bacteria-charcoal and is combined
Body chlorpyrifos degradation activity is 75.3 μ g/h, and degradation bacteria-life is taken from permeable bag after running 8 h, 12 h, 24 h, 48 h, 72 h
Thing charcoal complex carries out Fluorescent Staining Observation, obtains green/red fluorescence ratio, and the poison of different sample times is calculated according to formula
Dead tick degrading activity is as shown in table 3（Display data is three repetition averages）：
Table 3 is attached to fluorescence ratio and degrading activity testing result of the chlorpyrifos degrading bacteria of charcoal in different run times
It is quick, intuitive and accurate using the active quick determination method of the pesticide degradation bacteria proposed by the present invention based on fluorescent staining
The degradation bacteria-charcoal complex is obtained in application different time to the degrading activity of chlopyrifos, so determine the degradation bacteria-
Degrading activity highest of the charcoal complex after 24 h of application to chlopyrifos, degrading activity is less than initial value after 72 h, secretly
Show that the degradation bacteria-charcoal complex highest degrading activity is in 24 h in farmland water-break system pollution by pesticides in situ is repaired, and
Degradation bacteria-charcoal complex in needing to change permeable bag after 72 h of application, to ensure degradation effect.
Claims (7)
1. the organic agricultural chemicals degradation bacteria activity test method based on Fluorescent Staining Observation, it is characterized in that comprising the following steps：
（1）Culture is enlarged for degradation bacteria；
（2）Configure the cultivating system of n groups dead bacterium ratio containing different viable bacterias；
（3）Calculate viable bacteria fluorescence and dead bacterium fluorescence ratio；
（4）The degradation of pesticide activity of n group bacterium solutions is determined by degradation experiment；
（5）The correlation curve of fluorescence ratio and degrading activity is drawn, the active calculation formula of degradation bacteria is set up；
（6）Being verified with unknown active bacterium solution is used for follow-up test after formula accuracy.
2. the organic agricultural chemicals degradation bacteria activity test method according to claim 1 based on Fluorescent Staining Observation, its feature
It is the step（1）Culture is enlarged for degradation bacteria：For certain degradation bacteria, prepare corresponding culture medium, medium sterilization
Afterwards, inoculation degradation bacteria is enlarged culture, and the growth of wavelength OD600 detection incubation degradation bacterias is set using spectrophotometer
Situation.
3. the organic agricultural chemicals degradation bacteria activity test method according to claim 1 based on Fluorescent Staining Observation, its feature
It is the step（2）Configure the cultivating system of n groups dead bacterium ratio containing different viable bacterias：After degraded bacteria growing reaches stationary phase, take
The bacterium solution of two parts of equivalent, a copy of it carries out high-temperature inactivation, and the living bacterial liquid after shaking up is matched in varing proportions with inactivated bacterial liquid,
The percentage that the viable bacteria formed in n group cultivating systems, each cultivating system accounts for total bacterium is uniformly distributed between 0%-100%, for the side of ensuring
Method accuracy, every group at least three repeats, n >=6.
4. the organic agricultural chemicals degradation bacteria activity test method according to claim 1 based on Fluorescent Staining Observation, its feature
It is the step（3）Calculate viable bacteria fluorescence and dead bacterium fluorescence ratio：After cultivating system configuration terminates, shake up and take immediately on a small quantity not
With the bacterium solution in cultivating system, bacterium solution microscopy under fluorescence microscope after fluorescent dyeing, fluorescent dye used being capable of area
Divide viable bacteria and dead bacterium, under different excitation wavelengths, the viable bacteria after dyeing is different with the fluorescence developing of dead bacterium, image is utilized after taking pictures
Different fluorescence optical density values in the software analysis visual field are handled, viable bacteria and dead bacterium fluorescence intensity ratio in different cultivating systems is represented.
5. the organic agricultural chemicals degradation bacteria activity test method according to claim 1 based on Fluorescent Staining Observation, its feature
It is the step（4）The degradation of pesticide activity of n group bacterium solutions is determined by degradation experiment：First after microscopy, into each cultivating system
The organic agricultural chemicals of same concentration is added as the substrate of degradation bacteria, at times each cultivating system Pesticide Residues of sampling analysis,
The Organic pesticide residues amount that 100% culture group is accounted in viable bacteria terminates degradation experiment when being less than 30%, analyzes in each cultivating system
Degradation of pesticide rate.
6. the organic agricultural chemicals degradation bacteria activity test method according to claim 1 based on Fluorescent Staining Observation, its feature
It is the step（5）The correlation curve of fluorescence ratio and degrading activity is drawn, the active calculation formula of degradation bacteria is set up：With n groups
Viable bacteria and dead bacterium fluorescence intensity ratio are abscissa in cultivating system, and the degradation of pesticide rate of correspondence each group is ordinate, and fluorescence is strong
Degree and degradation of pesticide rate are the average value with three repetitions of group, select suitable curve to intend data point in two dimensional surface
Close, draw degradation bacteria active testing standard curve, set up the active calculation formula of degradation bacteria.
7. the organic agricultural chemicals degradation bacteria activity test method according to claim 1 based on Fluorescent Staining Observation, its feature
It is the step（6）It is middle to verify formula accuracy with unknown active bacterium solution：For the degradation bacteria active testing standard curve of drafting
Accuracy verified that verification method is to carry out Fluorescent Staining Observation to the sampling of the nutrient solution of unknown viable bacteria percentage, simultaneously
The activity of degradation bacteria in test cultures liquid, by comparing degradation bacteria activity and reality that degradation bacteria active testing standard curve is calculated
The activity measured, verifies the accuracy of degradation bacteria active testing standard curve.
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| CN109959643A (en) * | 2019-04-10 | 2019-07-02 | 中国环境科学研究院 | A method of assessment toxicant influences activated sludge mass-transfer performance |
| CN110376341A (en) * | 2019-06-24 | 2019-10-25 | 广西大学 | A kind of measuring method of graw mold of tomato protective agents photolysis |
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