CN104593475B - A kind of fluorescent microscopic counting method for detecting water body bacterial number - Google Patents
A kind of fluorescent microscopic counting method for detecting water body bacterial number Download PDFInfo
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- CN104593475B CN104593475B CN201510055148.XA CN201510055148A CN104593475B CN 104593475 B CN104593475 B CN 104593475B CN 201510055148 A CN201510055148 A CN 201510055148A CN 104593475 B CN104593475 B CN 104593475B
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- color fading
- fading agent
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Abstract
The invention discloses a kind of anti-color fading agent for fluorescence microscope count of bacteria and the fluorescent microscopic counting method of detection water body bacterial number, belong to environmental microbiology field.Anti-color fading agent of the invention is to be dissolved in deionized water with p-phenylenediamine, sodium chloride, disodium hydrogen phosphate, Tris, glycine, glycerine, is obtained after regulation pH.Detect that the fluorescent microscopic counting method of water body bacterial number is comprised the following steps：Toward water sample add fluorescent dye dyeed, adding above-mentioned anti-color fading agent working solution carries out anti-fade treatment, by the sample of anti-fade treatment after acetate fiber filter membrane suction filtration in fluorescence microscopy Microscopic observation.The present invention detects that the method for water body bacterial number is time saving and energy saving, with low cost, resistance to discoloration is strong and is disturbed without background fluorescence, can quick and precisely detect the quantity of bacterium in water sample.
Description
Technical field
The invention belongs to environmental microbiology field, and in particular to a kind of anti-colour fading for fluorescence microscope count of bacteria
Agent and the fluorescent microscopic counting method of detection water body bacterial number.
Background technology
Water body total bacteria count is one of reflection lake, the index of water quality of river situation.Conventional water body total bacteria count detection method
It is cultivation, including flat band method, MPN methods etc..But not all of bacterium can be cultivated, therefore, cultivation can cause detection to be tied
Fruit error is larger.To overcome the shortcoming of cultivation, various direct method of counting are widely used, wherein result it is relatively reliable and
The method being widely recognized as by everybody includes Fluorescent microscope counting method and Flow cytometry method.Because flow cytometer price is high
It is expensive, operation and maintenance high cost, using actually rare in the detection of water body total bacteria count.Fluorescence microscopy direct counting method is nearest 30
The new method for count of bacteria that grows up over year, the features such as it has quick, accurate, in water sample, soil and deposit
Had a wide range of applications in measure Deng bacterial number in environmental sample.The fluorescent dye for using also is developed into from AO, DAPI
SYBR Green line fluorescent dyestuffs, while also including the exploitation of the method such as simple stain, double dyeing.
The filter membrane commonly used in Fluorescent microscope counting is Alumina Inorganic filter membrane.This inorganic filter membrane is without any preceding place
Reason, it is easy to use, there is no a background fluorescence, but have the disadvantage expensive, fertile producer is few, and 2009-2012 even occurs
Producer resettlement factory site and cause the situation of whole world supply shortage.Therefore, researchers also attempt using common organic filter membrane（Such as vinegar
Sour fibrous filter membrane）Carry out Fluorescent microscope counting.But the organic filter membrane reported at present is used in technology and method, and organic filter membrane is all
To pass through complicated time-consuming pre-treatment to reduce background fluorescence signal, be unsuitable for quick detection, such as Publication No. 101344476
The filter membrane used in Chinese patent " bacterium viable but non-culturable state fluorescence microscope and method of counting " will be by hydrophobic
Could be used for fluorescence microscopy observation after treatment and dyeing drying.
The content of the invention
The purpose of the present invention is intended to overcome the defect of prior art, there is provided a kind of for the anti-of fluorescence microscope count of bacteria
The fluorescent microscopic counting method of decolourant and detection water body bacterial number, the method is time saving and energy saving, with low cost, can be quick and precisely
Bacterial number in detection water body.
The purpose of the present invention is achieved through the following technical solutions：
A kind of anti-color fading agent for fluorescence microscope count of bacteria, with p-phenylenediamine, sodium chloride, disodium hydrogen phosphate,
Tris, glycine, glycerine are dissolved in deionized water, are obtained after regulation pH.
Preferably, the method that the storing solution of the anti-color fading agent described in every 200mL passes through to comprise the following steps is prepared：Will
1.5-3g p-phenylenediamine, 0.5-1g sodium chloride, 2.5-3.5g disodium hydrogen phosphates, 0.2-0.4g Tris, 3-5g glycine are dissolved in
In 60-80mL deionized waters, pH to 7-8 is adjusted with hydrochloric acid and NaOH, add 80-100mL glycerine, deionization is finally used again
Water is settled to 200mL.4 DEG C of preservations of the storing solution lucifuge, the term of validity 3 months.
It is furthermore preferred that the method that the storing solution of the anti-color fading agent described in per 200mL passes through to comprise the following steps is prepared：
2g p-phenylenediamine, 0.8g sodium chloride, 3.2g disodium hydrogen phosphates, 0.25g Tris, 4g glycine are dissolved in 80mL deionized waters,
PH to 7.2 is adjusted with the hydrochloric acid or NaOH of 0.1M, 100mL glycerine is added, finally 200mL is settled to deionized water.
The working solution of described anti-color fading agent is to dilute storing solution aseptic syringe needle filter filtration sterilization with sterilized water again
After obtain, working solution is now with the current.Preferably, storing solution is diluted 10 times and obtains working solution.
Above-mentioned anti-color fading agent can be used in fluorescence microscope count of bacteria, can reduce background fluorescence, obtain good
Observing effect.
A kind of fluorescent microscopic counting method for detecting water body bacterial number, comprises the following steps：Fluorescence is added toward water sample
Dyestuff is dyeed, and adding above-mentioned anti-color fading agent working solution carries out anti-fade treatment, by the sample of anti-fade treatment through acetic acid
In fluorescence microscopy Microscopic observation after fibrous filter membrane suction filtration.
Preferably, the fluorescent microscopic counting method of described detection water body bacterial number, comprises the following steps：
（1）Sample is dyeed：1mL water samples are taken in the centrifuge tube of 5mL, adding what 100 μ L aseptic deionized waters diluted
SYBR Green I fluorescent dyes, normal temperature lucifuge dyeing 5min.
（2）Anti- fade treatment：To step（1）The anti-color fading agent working solution of its 3 times of volumes is added in the sample for having dyeed, is mixed
Even rear lucifuge stands 5min.
（3）Fluorescent microscope counting：By step（2）Filtered using Vacuum filtration device through the sample of anti-fade treatment,
Filter membrane used is common acetate fiber filter membrane, 0.22 μm of aperture.After filtering is finished, filter membrane is taken out the slide for being placed into cleaning
On, make attached germy one to face up, one is then added dropwise on filter membrane drips anti-color fading agent working solution, is covered with cover glass, puts fluorescence
On microscope carrier, excited with 480nm light waves, 40 times of thing Microscopic observations, the number of bacteria particles in 10 visuals field of random counter
Amount, and calculate average.The quantity of bacterium in 1mL samples is finally calculated by formula Y=AB/C, wherein Y is represented in 1mL samples
The quantity of bacterium, A represents the average of bacterium in a visual field, and B represents membrane retention area, and C represents the area in each visual field.
Main advantages of the present invention are：
（1）The present invention changes anti-fade treatment method, reduces background fluorescence, obtains good observing effect.
（2）The present invention works without cumbersome organic filter membrane pre-treatment, improves detection efficiency.
（3）The present invention, without using expensive inorganic filter membrane, reduces testing cost in detection process.
Brief description of the drawings
Fig. 1 is the comparative result figure that the inventive method counts bacillus coli DH 5 alpha with Anodisk filter method.
Fig. 2 is the design sketch that the water sample after dyeing is observed by 40 times of object lens.
Fig. 3 is the sub- lake water sample total number of bacteria result figure in 4, Wuhan City East Lake that the inventive method is obtained.
Specific embodiment
Following examples are used to further illustrate present disclosure, but should not be construed as limiting the invention, not
In the case of spirit of the invention and essence, the modification or replacement made to the inventive method, step or condition belong to this
The scope of invention.
The preparation of the anti-color fading agent storing solution of embodiment 1 and anti-color fading agent working solution
（1）The preparation of anti-color fading agent storing solution
2g p-phenylenediamine, 0.8g sodium chloride, 3.2g disodium hydrogen phosphates, 0.25g Tris, 4g glycine are dissolved in 80mL and gone
In ionized water, pH to 7.2 is adjusted with the hydrochloric acid or NaOH of 0.1M, add 100mL glycerine, finally arrived with deionized water constant volume
200mL, is made anti-color fading agent storing solution.The 4 DEG C of preservations of anti-color fading agent storing solution lucifuge, the term of validity 3 months.
（2）The preparation of anti-color fading agent working solution
1mL anti-color fading agent storing solutions, aseptic syringe needle filter filtration sterilization are taken, then 10mL is diluted to sterilized water, be made anti-
Decolourant working solution.Anti-color fading agent working solution is now with the current.
The culture of Escherichia coli of embodiment 2 is counted
With the light absorption value at beef extract-peptone fluid nutrient medium culture bacillus coli DH 5 alpha to its 600nm be 0.2 when,
Take 2mL bacterium solutions, 12000g centrifugation 10min give up supernatant, precipitation is resuspended in standby in 2mL SPSSs.
The above-mentioned standby bacteria suspensions of 100 μ L are taken in the centrifuge tube of 5mL, the μ L 10 of addition 10 × SYBR Green I fluorescence
Dyestuff, lucifuge adds 890 μ L SPSSs and 3mL anti-color fading agent working solutions after dyeing 5 minutes, normal temperature lucifuge stands 5 points
Clock, then through 0.22 μm of acetate fiber filter membrane suction filtration in aperture.Filter membrane is placed on the slide of cleaning, is made with large intestine bar
The one of bacterium faces up, and one is then added dropwise on filter membrane drips anti-color fading agent working solution, is covered with cover glass, puts fluorescence microscope objective table
On, excited with 480nm light waves, 40 times of thing Microscopic observations, the bacteria particles number in 5-10 visual field of random counter.Every milliliter of bacterium solution is thin
Bacterium number amount is pressed formula Y=10 × AB/C and is calculated, and wherein Y represents the quantity of bacterium in 1mL samples, and A represents bacterium in a visual field
Average, B represents membrane retention area, and C represents the area in each visual field.Meanwhile, using 0.22 μm of Alumina Inorganic in aperture
Filter membrane carries out same count operation to same sample, compares 2 kinds of differences of filter membrane count results.
3 DH5 α cultures are counted using above-mentioned 2 kinds different filter membranes, acquired results are shown in Fig. 1, as a result confirm this
Invention does not have difference with the result for obtaining the Alumina Inorganic filter membrane fluorescent microscopic counting method being widely recognized as.
The Wuhan East Lake water sample total number of bacteria of embodiment 3 is detected
In October, 2014, with 500mL mineral water bottles respectively from 4 sub- lakes of Wuhan East Lake（Guo Zhenghu, ox Chaohu, soup water chestnut lake,
Guan Qiao lakes）Collection water sample, incubator Refrigerated Transport goes back to laboratory.Every part of water sample takes in the centrifuge tube of 1mL to 5mL respectively, adds
100 μ L 10 × SYBR Green I fluorescent dyes, normal temperature lucifuge dyeing 5min.By anti-color fading agent working solution and dyeing water sample
Volume ratio 3:To anti-color fading agent working solution is added in each water sample for having dyeed, lucifuge stands 5min to 1 ratio after mixing.It is sharp afterwards
Each water sample is filtered with Vacuum filtration device, filter membrane used is common acetate fiber filter membrane, 0.22 μm of aperture.Filtering is finished
Afterwards, filter membrane is taken out and is placed on the slide of cleaning, make attached germy one to face up, a drop is then added dropwise on filter membrane anti-
Decolourant working solution, is covered with cover glass, puts on fluorescence microscope objective table, is excited with 480nm light waves, 40 times of thing Microscopic observations,
Fig. 2 is the design sketch observed by 40 times of object lens after Guo Zheng lake water sample is dyeed.The number of bacteria particles in 10 visuals field of random counter
Amount, and average is calculated, the quantity of bacterium in 1mL samples is finally calculated by formula Y=AB/C, wherein Y is represented in 1mL samples
The quantity of bacterium, A represents the average of bacterium in a visual field, and B represents membrane retention area, and C represents the area in each visual field.
Count results are shown in accompanying drawing 3, and the sub- lake water sample total number of bacteria of as shown by data Wuhan East Lake 4 is all 107Individual/mL ranks,
Wherein Guan Qiao lakes concentration highest, up to 6 × 107Individual/mL, soup water chestnut lake is minimum, is 1.26 × 107Individual/mL.
Claims (6)
1. a kind of anti-color fading agent for fluorescence microscope count of bacteria, it is characterised in that：Per the deposit of the 200mL anti-color fading agent
The method that liquid passes through to comprise the following steps is prepared：By 2g p-phenylenediamine, 0.8g sodium chloride, 3.2g disodium hydrogen phosphates, 0.25g
Tris, 4g glycine are dissolved in 80mL deionized waters, and pH to 7.2 is adjusted with the hydrochloric acid or NaOH of 0.1M, add 100mL sweet
Oil, is finally settled to 200mL with deionized water.
2. the anti-color fading agent for fluorescence microscope count of bacteria according to claim 1, it is characterised in that：The anti-colour fading
The working solution of agent is to be obtained after the filtration sterilization of storing solution aseptic syringe needle filter is diluted with sterilized water again.
3. the anti-color fading agent for fluorescence microscope count of bacteria according to claim 2, it is characterised in that：By storing solution
10 times of dilution obtains working solution.
4. application of the anti-color fading agent described in any one of claim 1-3 in fluorescence microscope count of bacteria.
5. it is a kind of detect water body bacterial number fluorescent microscopic counting method, it is characterised in that comprise the following steps：Toward water sample
Fluorescent dye is added to be dyeed, the working solution for adding the anti-color fading agent described in Claims 2 or 3 carries out anti-fade treatment,
By the sample of anti-fade treatment after acetate fiber filter membrane suction filtration in fluorescence microscopy Microscopic observation.
6. it is according to claim 5 detection water body bacterial number fluorescent microscopic counting method, it is characterised in that including such as
Lower step：
(1) sample dyeing：1mL water samples are taken in the centrifuge tube of 5mL, the SYBR for adding 100 μ L to be diluted with aseptic deionized water
Green I fluorescent dyes, lucifuge dyeing 5min；
(2) anti-fade treatment：It is anti-described in its 3 times of Claims 2 or 3 of volume to being added in the sample that step (1) has been dyeed
The working solution of decolourant, lucifuge stands 5min after mixing；
(3) Fluorescent microscope counting：Step (2) is filtered through the sample of anti-fade treatment using Vacuum filtration device, it is used
Filter membrane is common acetate fiber filter membrane, 0.22 μm of aperture；After filtering is finished, filter membrane is taken out and is placed on slide, make to have
The one of bacterium faces up, and the working solution of the anti-color fading agent described in a drop Claims 2 or 3 is then added dropwise on filter membrane, is covered with lid
Slide, puts on fluorescence microscope objective table, is excited with 480nm light waves, 40 times of thing Microscopic observations, thin in 10 visuals field of random counter
The quantity of bacterium particle, and calculate average；The quantity of bacterium in 1mL samples, wherein Y tables are finally calculated by formula Y=AB/C
Show the quantity of bacterium in 1mL samples, A represents the average of bacterium in a visual field, and B represents membrane retention area, and C represents each
The area in the visual field.
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| CN106153591A (en) * | 2016-08-15 | 2016-11-23 | 吴江华衍水务有限公司 | A kind of measure the method for bacterial density in water sample |
| CN107192655A (en) * | 2017-07-26 | 2017-09-22 | 连云港市质量技术综合检验检测中心 | A kind of household purified water machine Cryptosporidium rejection rate method of inspection |
| CN108387503B (en) * | 2018-01-22 | 2021-06-22 | 北京海岸鸿蒙标准物质技术有限责任公司 | Method for fixing value of particle counting particle standard substance |
| CN109741327A (en) * | 2019-01-15 | 2019-05-10 | 湖北中医药高等专科学校 | A kind of fluorescent microscopic counting method detecting water body bacterial number |
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| CN102321729A (en) * | 2011-07-06 | 2012-01-18 | 华中农业大学 | Fluorescent microscopic counting method for detecting bacterial count in soil and sediment |
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| CN102321729A (en) * | 2011-07-06 | 2012-01-18 | 华中农业大学 | Fluorescent microscopic counting method for detecting bacterial count in soil and sediment |
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| MAKING AND USING AQUEOUS MOUNTING MEDIA;J. A. Kiernan;《Microscopy Today》;19971231;第97卷(第10期);第3页 * |
| molecule localization microscopy.《Histochem Cell Biol》.2014,第567页左栏第1段. * |
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