CN105821114B - The Rapid Quantification of sewage treatment plant's polyP bacteria - Google Patents
The Rapid Quantification of sewage treatment plant's polyP bacteria Download PDFInfo
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Abstract
The present invention relates to a kind of Rapid Quantification of sewage treatment plant's polyP bacteria, specific steps include the acquisition of sludge sewage sample and preservation, production sterile gelatin smear, production sample smear, methylene blue staining, DAPI dyeing, micro- sem observation, the determination of polyP bacteria abundance and statistical analysis.According to this method, simple and quick polyP bacteria can be quantified from complicated waste water treatment system mixed microorganism group, operating process is quick, cost is relatively low, it is easy to operate, operator does not need special skills training, place and time to detection is without special requirement, testing result is accurate, and the relevant data of municipal sewage plant technical staff can be quickly provided to, the reason of can be used as monitoring enhanced biological phosphorus removal process exception and foundation, to further adjustment control methods of projects, guarantee that the normal operation of municipal sewage plant's enhanced biological phosphorus removal process has great importance.
Description
Technical field:
The present invention relates to a kind of Rapid Quantification of microorganism, the quick of especially a kind of sewage treatment plant's polyP bacteria is determined
Amount method.
Background technique
With the development of urbanization, water eutrophication problem caused by city and industrial wastewater discharge is more and more prominent,
The main reason is that the excessive emissions of nitrogenous phosphorus sewage, it is therefore necessary to handle sewage.Phosphorus in city and trade effluent
Can be using physics, chemistry and biological treatment removal, compared with traditional physics and chemical method, biological treatment is being removed
There is incomparable advantage in terms of dirty water pollutant, such as: without by high temperature and pressure, can be completed in a mild condition pair
The processing of sewage, therefore processing cost is cheap；The multiplicity of the microbial metabolism type as contained in municipal wastewater, can be to most
The pollutant of number sewage realizes biodegrade；And Sewage Biological Treatment does not generate additional waste, the influence to environment compared with
It is small.Therefore, the sewage disposal process based on activated sludge has become the maximum biotechnology industry of modern society.
Activated sludge process is the processing method of the aerobe of a kind of pair of sewage.Activated sludge is in waste water microbiological treatment
The general designation of the various substances of microbial suspension in water.The essence of activated sludge process is removal dissolution and colloidal from sewage
The organic matter of state and can by the suspended solid of activated sludge absorption and some other substance, while can also remove a part of phosphorus and
Nitrogen.
Activated sludge is made of the flock that bacterium, actinomyces, fungi and protozoan form, the original of activated sludge process
Reason is that air is continuously passed through into waste water, through because aerobic microbiological is bred, and foring sludge-like flocculate after a certain period of time.
Various micropopulations are inhabited thereon, the ability with very strong adsorption capacity and oxidation of organic compounds, for decomposing removal sewage
In organic pollutant.Can thus sludge be made to be separated from water, and most of sludge flows back into aeration tank again, it can be sharp again
With the system is then discharged in redundance.
The important channel of biological phosphate-eliminating is enhanced biological phosphorus removal technology (enhanced biological at present
Phosphorus removal, EBPR).Biological phosphate-eliminating is an important process in biological treatment, and activated sludge is utilized
The phosphorus amount that the phenomenon that a kind of excess phosphorus of middle peculiar microorganism absorbs, the i.e. microorganism absorb is more than needed for itself normal growth
Phosphorus amount, this special microorganism is exactly polyP bacteria.Enhanced biological phosphorus removal mainly passes through polyP bacteria (phosphate
Accumulating organisms, PAOs) phosphorus is discharged under anaerobic, the excessive consumption phosphorus under aerobic condition, and heel row
The excess sludge rich in phosphorus is put, and then achievees the purpose that remove phosphorus from sewage.Enhanced biological dephosphorization mistake is passed through by polyP bacteria
The detailed process for the phosphorus in removal sewage that journey is completed is: by anaerobic zone, amphimicrobian after sewage is mixed with activated sludge
In the process of area and aerobic zone, the polyP bacteria first in activated sludge utilizes internal polyphosphate in anaerobic zone
(polyphosphate, polyP) is the energy, absorbs the short chain fatty acids in sewage and is stored as intracorporal poly- hydroxyl rouge
Fat acid esters (polyhydroxyalkanoates, PHA), and to extracellular release phosphate, it becomes reconciled when into the amphimicrobian phase
Behind oxygen area, polyP bacteria utilizes the phosphorus (PO that intracorporal polyhydroxyalkanoate is in energy absorption sewage4-), it is stored as intracorporal more
Quadrafos is completed at the same time growth course.Latter stage during enhanced biological phosphorus removal in aerobic zone is by discharge rich in phosphorus
Residual active sludge has just achieved the purpose that go dephosphorization from sewage.
The phosphorus of excess in sewage can be sucked itself in vivo in aerobic situation by polyP bacteria, keep intracorporal phosphorus content general
The several times of bacterium phosphorus content, and polyphosphate a part of in anaerobic zone, polyP bacteria meeting decomposer, itself give birth to maintain
It deposits, therefore polyP bacteria is widely used in biological phosphate-eliminating.Microorganism follows the rule of growth curve, in anaerobic zone, poly- phosphorus
Bacterium cell can absorb the phosphate of a large amount of solubilised states from waste water, synthesize polyphosphate in the cell, and store in the cell,
Supply phosphorus element when nucleic acid required for growth period；When the polyP bacteria in activated sludge is raw in environment full of nutrition
When living, into logarithmic growth phase, can largely it be divided in logarithmic growth phase polyP bacteria；PolyP bacteria enters resting stage later, most of
Cell stops breeding, and the synthesis of nucleic acid also stops therewith, and polyP bacteria is very low to the requirement of phosphorus at this time, if but the phosphorus source in environment
Still there is residue, cell has certain energy again when, remains to absorb P elements from the external world, phosphorus is stored in the form of polyphosphate
In intracellular, as storage of cells substance, this summation to phosphorus enables polyP bacteria to assemble to substantially exceed its and grow institute
The phosphorus amount needed；When bacterial cell is in totally unfavorable living condition, polyP bacteria can absorb again acetic acid in sewage, formic acid,
The easily biodegradable organic substance such as propionic acid and ethyl alcohol, storage are used as nutrient source, while the poly- phosphorus that will store in vivo in vivo
Hydrochlorate decomposes, and to obtain energy, needed for maintaining it to survive in adverse environment for bacterium, polyphosphate is just in thallus at this time
It fades away；If such bacterium is again introduced into aerobic environment full of nutrition, it is by the process of repeat body inner product phosphorus.
Many biologies and abiotic factor can all influence the efficiency of enhanced biological phosphorus removal process in sewage disposal process, such as
Sugar source, which accumulates bacterium (glycogen accumulating organisms, GAO) and can compete with polyP bacteria in anaerobic phase, to be absorbed
Same organic substrate results in them aerobic to reduce polyP bacteria in the accumulation of anaerobic phase polyhydroxyalkanoate
The phosphorus of phase and amphimicrobian phase, which absorb, to be reduced；In addition, sewage feature such as C/N also will affect phosphorus than the variation of, pH value and temperature etc.
The efficiency of removal, or even will cause the complete collapse of enhanced biological phosphorus removal process.
In order to sewage be effectively treated and qualified discharge, sewage treatment plant management and operator need monitor and sentence in time
Disconnected the reason of influencing enhanced biological phosphorus removal process exception, to carry out necessary adjustment to activated sludge process and plant facilities,
The normal operation for guaranteeing enhanced biological phosphorus removal process, guarantees the qualified discharge of sewage.
In existing physical chemistry and Biological indicators, the relative abundance and work of polyP bacteria in active sludge microorganism group
Property directly react enhanced biological phosphorus removal process operation situation, be the good index of enhanced biological phosphorus removal process monitoring.But at present
Detection method for polyP bacteria is less, mainly uses pass through quantitatively by the gene probe of target of polyP bacteria 16S rRNA at present
Fluorescence in situ hybridization (quantitative fluorescence in situ hybridization, qFISH) and pass through qPCR
The abundance and the detection of active method of quantitative polyP bacteria.QFISH is mutual according to base using the specific nucleic acid probe of fluorescent marker
Pair principle is mended, hybridizes with intracellular corresponding DNA molecular, forms the double-strandednucleic acid that can be detected, it can by fluorescence microscope
Observe fluorescence signal.QPCR is that fluorogene is added in PCR reaction system, is accumulated using fluorescence signal, monitor PCR into
Journey carries out quantitative analysis to polyP bacteria finally by standard curve.But the method for mesh first two detection is complicated for operation, operator
Member needs certain professional skill, and detection time is too long (7-8 hours), and experimentation material requested and equipment are also more expensive,
It is not suitable as simple and quick detection method.Therefore, a kind of simple, quick, efficiently, inexpensive polyP bacteria detection side is established
Method, suitable for the conventional detection of activated sludge dephosphorization effect and sewage treatment plant's equipment work efficiency, to water conservation environment
There is great meaning with the living standard of the people.
Summary of the invention
The present invention provides a kind of Rapid Quantifications of sewage treatment plant's polyP bacteria, develop a kind of simple and quick from multiple
The means of quantitative polyP bacteria in miscellaneous waste water treatment system mixed microorganism group, methylene blue staining and DAPI dyeing combination can be
PolyP bacteria during 1-2 hours quantification municipal sewage plant activated sludge enhanced biological phosphorus removals, and operating process letter
Single quickly operator does not need professional skill training, and the abundance and activity of polyP bacteria can be used for monitoring sewage treatment plant's reinforcing
The situation of Biological Phosphorus Removal Process operation judges the reason of enhanced biological phosphorus removal process exception, and can take correct engineering in time
Control measure guarantee normal effective operation of municipal sewage plant.
In order to achieve the above objectives, the present invention comprises the steps of:
(1) acquisition of sludge sewage sample and preservation: the aeration zone after aerator is sampled, sample
It is stored in wide mouth glass bottle, and is transported to laboratory in 1 hour.Sample is deposited in centrifuge tube in laboratory, is placed in 4 DEG C of ice
It is saved in case, Coloration experiment is completed in three days.
(2) it makes sterile gelatin smear: under germ-free condition, taking 7 object slide stands, glass slide is filled in object slide stand
In；4-5 drop detergent is added in 3.5L distilled water, is put into object slide stand, boils 10min；Object slide stand is taken out, dry in the air 3min；
Detergent is rinsed well with distilled water, the glass slide state of being kept upright is dried；The ten of 2.5g is added in 3.5L distilled water
The gelatin of sulfate dihydrate potassium chromium and 0.44g is heated to 70 DEG C, and object slide stand, constant temperature 10min are put into after gelatin dissolves substantially；
Object slide stand is taken out, glass slide is tiltedly stood on rack for test tube side and is drying to obtain.
(3) it makes sample smear: under germ-free condition, the centrifuge tube equipped with sample being shaken up in homogenizer, due to sample
In the particle volume that has it is larger, front end is being cut off 2-3 millimeters using preceding by pipette tips, takes about 20ul mixed active sludge with liquid-transfering gun
Sample [8-10g/L mixing liquid (dry weight metering)] is placed in glass slide center, covers the other a piece of glass slide equally handled, uses
The end for pulling two panels glass slide pulls 10-20 times back and forth in the opposite direction, and glass slide is stood and leans against rack for test tube edge, is made
It is air-dried.
(4) methylene blue staining: 10-15ml methylene blue dye liquor drop is taken in air-dried specimen slide, after dyeing 15s
Dye liquor is gone, and with 70% alcohol rinse 30s, it is In Shade to air-dry.
(5) DAPI is dyed: being taken 100-200ul coloring agent D to be covered on air-dried glass slide uniformly spreadable, is placed in dark
Aseptic operation box in dye 10-20min；Coloring agent D is gone, and carefully rinses 2-3 all over (each 1min) with distilled water；It keeps away
Light simultaneously air-dries in aseptic operation box；
(6) micro- sem observation: dried glass slide is placed on fluorescence microscopy endoscope objective lens platform, 100 × object lens under into
Row is observed and takes pictures, and each sample at least acquires 30 sets, and (every sets of data picture includes a methylene blue staining picture and one
DAPI dyes picture) data picture；
(7) determination of polyP bacteria abundance and statistical analysis: same tricks is analyzed respectively using image analysis software ImageJ
The cell number (microbial count) of methylene blue staining positive polyP bacteria cell number and DAPI stained positive in word picture, methylene
Base indigo plant stained positive polyP bacteria cell is counted using the manual count method provided in ImageJ, and DAPI staining cell is using automatic meter
Number method counts:
The abundance of polyP bacteria=methylene blue staining positive polyP bacteria cell number/total number of cells × 100%
T is examined and variance analysis is completed in excel.
Preferably, the preparation method of the methylene blue staining agent are as follows:
A) preparation of A liquid: the KOH solution of mass ratio 10-20%；
B) preparation of B liquid: methylene blue: alcohol=1:33-50 (mass ratio)；
C) preparation of methylene blue staining agent: A liquid is mixed by volume (1:0.5-1) with B liquid.
Preferably, the DAPI dye liquor is the DAPI aqueous solution of concentration 0.0003-0.005mg/ml.
In the present invention, by the way that methylene blue staining method and DAPI (4', 6-diamidino-2-phenylindole) are contaminated
Color method combination, the method for exploring polyP bacteria in a kind of simple and quick qualitative activity sludge microbe group.The principle of method is:
Methylene blue dye is positively charged, and polyphosphoric acids salt particle is negatively charged, therefore the two has very high affinity, the two knot
The light absorption of dyestuff changes after conjunction, and Babes-Ernst bodies and cell can be distinguished by generating different colors.It is contaminated in methylene blue
Chromogenic reaction can occur with the intracorporal polyphosphate of polyP bacteria for color Methylene Blue, chromophore can with conventional microscopy into
Row observation, and their intensity is directly proportional to the amount of polyphosphate, therefore may be used to indicate the activity of potential polyP bacteria.
Therefore the polyP bacteria in sludge sewage microbiologic population can be marked using methylene blue staining method, can be adopted
With all microbial cells in DAPI dye marker active sludge microorganism group, DAPI, can be with as a kind of fluorescent dye
Penetrating cell film plays the effect of label in conjunction with the double-stranded DNA in nucleus, can produce much stronger than DAPI itself 20 times
Fluorescence.It can be seen that the cell of aobvious blue-fluorescence under microscope.It is calculated in conjunction with two methods, and by fluorescence microscope picture
PolyP bacteria accounts for the percentage of all microbial cells in the same visual field out.Up to now, both methods is also never used in combination
It crosses, the successful exploitation of this method will fast and accurately measure the abundance of polyP bacteria in sludge sewage, grasp
With the working condition for judging sewage treatment plant.
The present invention for the first time by methylene blue staining method and 4', 6'- diamidino -2- benzene indole hydrochloride (DAPI) (4', 6 ' -
Diamidino-2-phenylindole) decoration method is combined, and explores a kind of simple and quick qualitative activity sludge microbe group
The method for falling middle polyP bacteria.Methylene blue staining and DAPI dyeing combination can be living 1-2 hours quantification municipal sewage plants
PolyP bacteria during property sludge enhanced biological phosphorus removal, and operating process is simple and quick, and operator does not need professional skill
Training, the abundance and activity of polyP bacteria can be used for monitoring the situation of sewage treatment plant's enhanced biological phosphorus removal process operation, judgement by force
The reason of changing biological phosphate-eliminating process exception, and correct control methods of projects can be taken in time, guarantee municipal sewage plant
Normal effectively operation.
The present invention develops a kind of simple and quick polyP bacteria quantitative from complicated waste water treatment system mixed microorganism group
Means, this means and current molecular biology method (qFISH and qPCR) compare, overcome the deficiency of existing method,
Feature is (can quickly to complete in 1-2 hours), and cost is relatively low, and easy to operate, operator does not need special technical ability training
Instruction, for the place and time to detection without special requirement, testing result is accurate, and can be quickly provided to municipal sewage plant's skill
The relevant data of art personnel can be used as the reason of monitoring enhanced biological phosphorus removal process exception and foundation, to further adjustment engineering
Control measure guarantee that the normal operation of municipal sewage plant's enhanced biological phosphorus removal process has great importance.
Detailed description of the invention
Fig. 1 is the Rapid Quantification flow chart of sewage treatment plant's polyP bacteria；
Fig. 2 is active sludge microorganism group Methylene Blue stained positive polyP bacteria microphoto example, and arrow is signified
Be positive polyP bacteria；
Fig. 3 be DAPI dye sludge microbe group in total microbial cell photographic examples, arrow refer to by
Methylene blue staining positive polyP bacteria is also contaminated by DAPI bright.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
The routine techniques of the molecular biology of domain routine, biochemistry, technique of analytical chemistry and related fields.
The Rapid Quantification of sewage treatment plant's polyP bacteria of the present invention, specific steps:
(1) acquisition of sludge sewage sample and preservation: the aeration zone after aerator is sampled, sample
It is stored in wide mouth glass bottle, and is transported to laboratory in 1 hour.Sample is deposited in centrifuge tube in laboratory, is placed in 4 DEG C of ice
It is saved in case, Coloration experiment is completed in three days.
(2) it makes sterile gelatin smear: under germ-free condition, taking 7 object slide stands, glass slide is filled in object slide stand
In；The daily household washing essence of 4-5 drop is added in 3.5L distilled water, is put into object slide stand, boils 10min；Object slide stand is taken out,
Dry in the air 3min；Detergent is rinsed well with distilled water, the glass slide state of being kept upright is dried；It is added in 3.5L distilled water
The gelatin of the ten sulfate dihydrate potassium chromium and 0.44g of 2.5g, is heated to 70 DEG C, and object slide stand is put into after gelatin dissolves substantially, permanent
Warm 10min；Object slide stand is taken out, glass slide is tiltedly stood on rack for test tube side and is drying to obtain.
(3) it makes sample smear: under germ-free condition, the centrifuge tube equipped with sample being shaken up in homogenizer, due to sample
In the particle volume that has it is larger, front end is being cut off 2-3 millimeters using preceding by pipette tips, takes 20ul mixed active sludge sample with liquid-transfering gun
Product, concentration are 8-10g/L mixing liquid, are placed in glass slide center, cover the other a piece of glass slide equally handled, hand-pull
The end of two panels glass slide pulls 15 times back and forth in the opposite direction, and glass slide is stood and leans against rack for test tube edge, air-dries it.
(4) methylene blue staining: take 10ml methylene blue dye liquor drop will after in air-dried specimen slide, dyeing 15s
Dye liquor is gone, and with 70% alcohol rinse 30s, In Shade to air-dry.
(5) DAPI is dyed: being taken 80ul coloring agent D to be covered on air-dried glass slide uniformly spreadable, is placed in dark nothing
30min is dyed in bacterium control box；Coloring agent D is gone, and is carefully rinsed 2-3 times with distilled water, each 1min；It is protected from light and in nothing
It is air-dried in bacterium control box；
(6) micro- sem observation: dried glass slide is placed on fluorescence microscopy endoscope objective lens platform, 100 × object lens under into
Row observation and take pictures, each sample at least acquire 30 sets (every sets of data picture include green fluorescence starch dye picture with
One DAPI dyes picture) data picture；
(7) determination of polyP bacteria abundance and statistical analysis: same tricks is analyzed respectively using image analysis software ImageJ
The cell number (microbial count) of methylene blue staining positive polyP bacteria cell number and DAPI stained positive in word picture, it is sub-
Methyl blue stained positive polyP bacteria cell is counted using the manual count method provided in ImageJ, and DAPI staining cell is using automatic
Count method:
The abundance of polyP bacteria=methylene blue staining positive polyP bacteria cell number/total number of cells × 100%
T is examined and variance analysis is completed in excel.
Wherein, the preparation method of the methylene blue staining agent are as follows:
(1) preparation of A liquid: the KOH solution of mass ratio 10%；
(2) preparation of B liquid: methylene blue: water: alcohol=1:19.4:12.93 (mass ratio)；
(3) preparation of methylene blue staining agent: A liquid is mixed by volume (1:1) with B liquid.
The DAPI dye liquor is the DAPI aqueous solution of concentration 0.005mg/ml.
By the above experimental program, after the processing and dyeing of the samples different three times of the first sewage treatment plant of Kunming,
Each sample obtains 3 groups of each 15 sets of experimental data pictures, amounts to 135 sets of experiment pictures, and carried out data to these lab diagrams
With statistical analysis.PolyP bacteria ratio shared in each group activated sludge sample and its average value and standard deviation (table 1- table 3),
Table 4 is T inspection result.
Table 1: polyP bacteria ratio shared in each group activated sludge sample and its average value and standard deviation (2012 12
Month sample on the 14th)
Table 2: polyP bacteria ratio shared in each group activated sludge sample and its average value and standard deviation (2013 3
Month sample on the 20th)
Table 3: polyP bacteria ratio shared in each group activated sludge sample and its average value and standard deviation (2013 4
Month sample on the 10th)
Table 4 statisticallys analyze the P value of (T inspection)
(note: sample one is 2012.12.14 sample, and sample two is 2013.3.20 sample, and sample three is 2013.4.10 sample
This.)
It 0.05 is no significant difference that the test stone that T is examined, which is that P value is greater than, less than 0.05 for there were significant differences, by table 2
It is found that without significant difference between sample one and sample two, close to there were significant differences between sample one and sample three, sample two and sample
Without significant difference between sheet three.
Method provided by the invention, can be simple and quick from complicated microbial ecosystem (such as municipal sewage)
Tracer and quantitative polyP bacteria in mixed microorganism group, due to being shown with the method for the molecular biology of some complexity before this
Track and quantitative polyP bacteria pass through quantitative fluorescence in situ hybridization by the gene probe of target of polyP bacteria 16S rRNA including using
(qFISH) and by qPCR the abundance and activity of polyP bacteria are quantified.But the method for qFISH and qPCR detection is complicated for operation, operation
Personnel need certain professional skill training, and detection time is too long (7-8 hours), experimentation material requested and equipment
It is more expensive, it is not suitable for simple and fast detection method.The method of the invention patent and pervious molecular biology method ratio
Compared with feature is can directly to demarcate to fresh activated sludge sample and monitor polyP bacteria group, significantly shortens and reduce labor
Fatigue resistance, cost is relatively low, and operating process is easy, and operator does not need special training, wants to the place of detection without special
Ask, experimental result is accurate, can be quickly provided to the relevant data of waste water treatment plant engineers and technicians (polyP bacteria it is rich
Degree), guarantee that urban sewage treatment system runs well, also can be used as a kind of routine of efficiency for monitoring enhanced biological phosphorus removal process
Detection method, it is ensured that sewage be effectively treated and qualified discharge.
The above is only specific application examples of the invention, are not limited in any way to protection scope of the present invention.All uses
Equivalent transformation or equivalent replacement and the technical solution formed, all fall within rights protection scope of the present invention.
Claims (3)
1. a kind of Rapid Quantification of sewage treatment plant's polyP bacteria, it is characterised in that: the following steps are included:
(1), the acquisition of sludge sewage sample and preservation: the aeration zone after aerator is sampled, and sample is deposited
Enter in wide mouth glass bottle, and be transported to laboratory in 1 hour, sample is deposited in centrifuge tube in laboratory, is placed in 4 DEG C of refrigerators
Middle preservation, Coloration experiment are completed in three days；
(2), it makes sterile gelatin smear: under germ-free condition, taking 7 object slide stands, glass slide is filled in object slide stand；?
The daily house detergent of 4-5 drop is added in 3.5 L distilled water, is put into object slide stand, boils 10 min；Object slide stand is taken out, dries in the air 3
min；Detergent is rinsed well with distilled water, the glass slide state of being kept upright is dried；2.5g is added in 3.5L distilled water
Ten sulfate dihydrate potassium chromium and 0.44g gelatin, be heated to 70 DEG C, object slide stand, constant temperature 10 be put into after gelatin dissolves substantially
min；Object slide stand is taken out, glass slide is tiltedly stood on rack for test tube side and is drying to obtain；
(3), it makes sample smear: under germ-free condition, the centrifuge tube equipped with sample being shaken up on homogenizer, due to having in sample
Particle volume it is larger, front end is being cut off 2-3mm using preceding by liquid-transfering gun pipette tips, takes 20ul mixed active sludge with liquid-transfering gun
Sample is placed in glass slide center, covers the other a piece of glass slide equally handled, hand-pulls the end of two panels glass slide towards phase
Anti- direction pulls 10-20 times back and forth, and glass slide is stood and leans against rack for test tube edge, air-dries it；
(4), it methylene blue staining: takes 10-15 ml methylene blue staining agent drop in air-dried specimen slide, dyes 15 s
Coloring agent is gone afterwards, and with 70% alcohol rinse 30s, it is In Shade to air-dry；
(5), DAPI is dyed: being taken 100-200 ul coloring agent DAPI to be covered on air-dried glass slide uniformly spreadable, is placed in black
10-20 min is dyed in dark aseptic operation box；Coloring agent DAPI is gone, and is carefully rinsed 2-3 times with distilled water, every time 1
min；It is protected from light and is air-dried in aseptic operation box；
(6), micro- sem observation: dried glass slide is placed on fluorescence microscope objective table, 100 × object lens under seen
It examines and takes pictures, each sample at least acquires 30 sets of data pictures, and every sets of data picture includes a processed methylene of step (4)
Base indigo plant dyes picture and the processed DAPI of a step (5) dyes picture；
(7), the determination of polyP bacteria abundance and statistical analysis: same set of datagram is analyzed respectively using image analysis software ImageJ
The cell number of methylene blue staining positive polyP bacteria cell number and DAPI stained positive in piece, the positive poly- phosphorus of methylene blue staining
Bacterium cell is counted using the manual count method provided in ImageJ, and DAPI staining cell uses automatic count method:
The abundance of polyP bacteria=methylene blue staining positive polyP bacteria cell number/total number of cells × 100%
T is examined and variance analysis is completed in excel.
2. the Rapid Quantification of sewage treatment plant's polyP bacteria according to claim 1, it is characterised in that: step (4) institute
State the preparation method of methylene blue staining agent are as follows:
The preparation of A liquid: the KOH solution of mass ratio 10-20%；
The preparation of B liquid: methylene blue in mass ratio: alcohol=1:33-50；
The preparation of methylene blue staining agent: by A liquid, 1:0.5-1 is mixed by volume with B liquid.
3. the Rapid Quantification of sewage treatment plant's polyP bacteria according to claim 1, it is characterised in that: step (5) institute
State the DAPI aqueous solution that coloring agent DAPI is concentration 0.0003-0.005 mg/ml.
Priority Applications (1)
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| 聚磷菌体内多聚物的染色方法;任世英，肖天;《海洋科学》;20051231;第29卷(第1期);59-63 |
| 荧光原位杂交技术检测反应器中微生物实验条件优化的研究;张勇;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20081115(第11期);B027-277 |
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