CN104593475A - Fluorescent microscopic counting method for detecting number of bacteria in water body - Google Patents
Fluorescent microscopic counting method for detecting number of bacteria in water body Download PDFInfo
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- CN104593475A CN104593475A CN201510055148.XA CN201510055148A CN104593475A CN 104593475 A CN104593475 A CN 104593475A CN 201510055148 A CN201510055148 A CN 201510055148A CN 104593475 A CN104593475 A CN 104593475A
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- color fading
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Abstract
The invention discloses a fade-resistant agent used in bacteria counting by virtue of a fluorescent microscopy and a fluorescent microscopic counting method for detecting the number of bacteria in a water body and belongs to the field of environmental microbiology. The fade-resistant agent is obtained by dissolving p-phenylenediamine, sodium chloride, disodium hydrogen phosphate, Tris, glycine and glycerol in deionized water and adjusting the pH. The fluorescent microscopic counting method for detecting the number of bacteria in the water body comprises the following steps: adding a fluorescent dye into a water sample, staining, adding a fade-resistant agent working liquid for carrying out fade-resistant treatment, carrying out suction-filtration on the sample subjected to the fade-resistant treatment with a cellulose acetate filtration membrane and observing under the fluorescent microscope. The method for detecting the number of bacteria in the water body is time-saving and effort-saving, low in cost and strong in fade-resistant capability and is free of background fluorescence interference and the number of bacteria in the water sample can be quickly and accurately detected.
Description
Technical field
The invention belongs to environmental microbiology field, be specifically related to the fluorescent microscopic counting method of a kind of anti-color fading agent for fluorescent microscope bacterial count and detection water body bacterial number.
Background technology
Water body total count is one of the index of reflection lake, water quality of river situation.Conventional water body total count detection method is culture method, comprises flat band method, MPN method etc.But and the bacterium of not all can be cultivated, therefore, culture method can cause detected result error larger.For overcoming the shortcoming of culture method, various direct census method is widely used, and wherein result is relatively reliable and comprised Fluorescent microscope counting method and Flow cytometry method by everybody method of extensive accreditation.Because flow cytometer is expensive, operation and maintenance cost is high, applies actually rare on water body total count detects.Fluorescence microscopy direct-counting method is the novel method for bacterial count grown up over nearest 30 years, and it has the features such as quick, accurate, in the environmental samples such as water sample, soil and settling bacterial number mensuration in have a wide range of applications.The fluorescence dye used also develops into SYBR Green line fluorescent dyestuff from AO, DAPI, also comprises the exploitation of the method such as simple stain, two dyeing simultaneously.
Filter membrane conventional in Fluorescent microscope counting is Alumina Inorganic filter membrane.This inorganic filter membrane is without the need to any pre-treatment, easy to use, does not have background fluorescence, but shortcoming to be price more expensive, fertile producer is few, and 2009-2012 has even occurred producer resettlement factory site and caused the situation of whole world supply shortage.Therefore, investigators also attempt using common organic filter membrane (as cellulose acetate filter membrane) to carry out Fluorescent microscope counting.But in the organic filter membrane operation technique reported at present and method, organic filter membrane all will through complicated time-consuming pre-treatment to reduce background fluorescence signal, be unsuitable for rapid detection, as publication number be in the Chinese patent " bacterium viable but non-culturable state fluorescence microscope and method of counting " of 101344476 filter membrane that uses will through hydrophobic treatment with dye dry after could be used for fluorescence microscopy and observe.
Summary of the invention
Object of the present invention is intended to the defect overcoming prior art, there is provided the fluorescent microscopic counting method of a kind of anti-color fading agent for fluorescent microscope bacterial count and detection water body bacterial number, the method is time saving and energy saving, with low cost, the bacterial number that can quick and precisely detect in water body.
Object of the present invention is achieved through the following technical solutions:
For an anti-color fading agent for fluorescent microscope bacterial count, be dissolved in deionized water with Ursol D, sodium-chlor, Sodium phosphate dibasic, Tris, glycine, glycerine, obtain after regulating pH.
Preferably, the storing solution of the anti-color fading agent described in every 200mL is prepared by the method comprising following steps: be dissolved in 60-80mL deionized water by 1.5-3g Ursol D, 0.5-1g sodium-chlor, 2.5-3.5g Sodium phosphate dibasic, 0.2-0.4g Tris, 3-5g glycine, pH to 7-8 is regulated with hydrochloric acid and sodium hydroxide, add 80-100mL glycerine, be finally settled to 200mL with deionized water again.This storing solution lucifuge 4 DEG C preservation, validity period 3 months.
Preferred, the storing solution of the anti-color fading agent described in every 200mL is prepared by the method comprising following steps: be dissolved in 80mL deionized water by 2g Ursol D, 0.8g sodium-chlor, 3.2g Sodium phosphate dibasic, 0.25g Tris, 4g glycine, with hydrochloric acid or the sodium hydroxide adjustment pH to 7.2 of 0.1M, add 100mL glycerine, be finally settled to 200mL with deionized water.
The working fluid of described anti-color fading agent is for obtaining degerming for storing solution aseptic syringe needle frit with after sterilized water dilution again, and working fluid is now with the current.Preferably, storing solution is diluted 10 times and obtain working fluid.
Above-mentioned anti-color fading agent can be used for, in fluorescent microscope bacterial count, can reducing background fluorescence, obtains good observing effect.
A kind of fluorescent microscopic counting method detecting water body bacterial number, comprise the steps: that in water sample, add fluorescence dye dyes, add above-mentioned anti-color fading agent working fluid again and carry out anti-fade treatment, by the sample of anti-fade treatment after cellulose acetate filter membrane suction filtration at fluorescence microscopy Microscopic observation.
Preferably, the fluorescent microscopic counting method of described detection water body bacterial number, comprises the steps:
(1) sample dyeing: get 1mL water sample in the centrifuge tube of 5mL, adds the SYBR Green I fluorescence dye of 100 μ L aseptic deionized water dilutions, normal temperature lucifuge dyeing 5min.
(2) anti-fade treatment: the anti-color fading agent working fluid adding its 3 times of volumes in the sample that step (1) has dyeed, after mixing, lucifuge leaves standstill 5min.
(3) Fluorescent microscope counting: utilize Vacuum filtration device to filter through the sample of anti-fade treatment step (2), filter membrane used is common cellulose acetate filter membrane, 0.22 μm, aperture.After filtration, being taken out by filter membrane is placed on clean slide glass, attached germy one is made to face up, then on filter membrane, drip an anti-color fading agent working fluid, be covered with cover glass, put on fluorescent microscope Stage microscope, excite with 480nm light wave, 40 times of thing Microscopic observations, the quantity of bacteria particles in random counter 10 visuals field, and calculate mean number.Finally calculate the quantity of bacterium in 1mL sample by formula Y=AB/C, wherein Y represents the quantity of bacterium in 1mL sample, and A represents the mean number of bacterium in a visual field, and B represents membrane retention area, and C represents the area in each visual field.
Major advantage of the present invention is:
(1) the present invention changes anti-fade treatment method, reduces background fluorescence, obtains good observing effect.
(2) the present invention is without the need to loaded down with trivial details organic filter membrane pre-treatment work, improves detection efficiency.
(3) the present invention is without the need to using expensive inorganic filter membrane in testing process, reduces testing cost.
Accompanying drawing explanation
Fig. 1 is the comparative result figure that the inventive method and Anodisk filter method count bacillus coli DH 5 alpha.
Fig. 2 is the design sketch that the water sample after dyeing is observed by 40 times of object lens.
Fig. 3 is 4, East Lake, Wuhan City sub-lake water sample total plate count result figure that the inventive method obtains.
Embodiment
Following examples are used for further illustrating content of the present invention, but should not be construed as limitation of the present invention, and without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement all belong to scope of the present invention.
The preparation of embodiment 1 anti-color fading agent storing solution and anti-color fading agent working fluid
(1) preparation of anti-color fading agent storing solution
2g Ursol D, 0.8g sodium-chlor, 3.2g Sodium phosphate dibasic, 0.25g Tris, 4g glycine are dissolved in 80mL deionized water, with hydrochloric acid or the sodium hydroxide adjustment pH to 7.2 of 0.1M, add 100mL glycerine, finally use deionized water constant volume to 200mL, make anti-color fading agent storing solution.Anti-color fading agent storing solution lucifuge 4 DEG C preservation, validity period 3 months.
(2) preparation of anti-color fading agent working fluid
Get 1mL anti-color fading agent storing solution, aseptic syringe needle frit is degerming, then is diluted to 10mL with sterilized water, makes anti-color fading agent working fluid.Anti-color fading agent working fluid is now with the current.
Embodiment 2
culture of Escherichia coli counts
When with extractum carnis-peptone liquid nutrient medium, to cultivate bacillus coli DH 5 alpha to the light absorption value at its 600nm place be 0.2, get 2mL bacterium liquid, the centrifugal 10min of 12000g, gives up supernatant liquor, and precipitation settling flux is for subsequent use in 2mL stroke-physiological saline solution.
Get the above-mentioned bacteria suspension for subsequent use of 100 μ L in the centrifuge tube of 5mL, add 10 μ L 10 × SYBR Green I fluorescence dye, lucifuge adds 890 μ L stroke-physiological saline solution and 3mL anti-color fading agent working fluid after dyeing 5 minutes, normal temperature lucifuge leaves standstill 5 minutes, then through the cellulose acetate filter membrane suction filtration in 0.22 μm of aperture.Filter membrane is placed on clean slide glass, make to face up with colibacillary one, then on filter membrane, an anti-color fading agent working fluid is dripped, be covered with cover glass, put on fluorescent microscope Stage microscope, excite with 480nm light wave, 40 times of thing Microscopic observations, the bacteria particles number in a random counter 5-10 visual field.Every milliliter of bacterium liquid bacterial number is pressed formula Y=10 × AB/C and is calculated, and wherein Y represents the quantity of bacterium in 1mL sample, and A represents the mean number of bacterium in a visual field, and B represents membrane retention area, and C represents the area in each visual field.Meanwhile, utilize the Alumina Inorganic filter membrane in 0.22 μm of aperture to carry out same count operation to same sample, compare the difference of 2 kinds of filter membrane count results.
Utilize above-mentioned 2 kinds of different filter membranes to count 3 DH5 α cultures, acquired results is shown in Fig. 1, and the invention of result proved does not have difference with the result obtaining the Alumina Inorganic filter membrane fluorescent microscopic counting method extensively approved.
Embodiment 3
wuhan East Lake water sample total plate count detects
In October, 2014, gather water sample from Wuhan East Lake 4 sub-lakes (Guo Zhenghu, ox Chaohu, soup water chestnut lake, Guan Qiaohu) respectively with 500mL mineral water bottle, insulation can refrigerated shipment goes back to laboratory.Every part of water sample is got in the centrifuge tube of 1mL to 5mL respectively, add 100 μ L 10 × SYBR Green I fluorescence dye, normal temperature lucifuge dyeing 5min.In the good water sample of each dyeing, add anti-color fading agent working fluid than the ratio of 3:1 in anti-color fading agent working fluid and dyeing volume of water sample, after mixing, lucifuge leaves standstill 5min.Utilize Vacuum filtration device to be filtered by each water sample afterwards, filter membrane used is common cellulose acetate filter membrane, 0.22 μm, aperture.After filtration, being taken out by filter membrane is placed on clean slide glass, attached germy one is made to face up, then on filter membrane, an anti-color fading agent working fluid is dripped, be covered with cover glass, put on fluorescent microscope Stage microscope, excite with 480nm light wave, 40 times of thing Microscopic observations, Fig. 2 is the design sketch observed by 40 times of object lens after the water sample dyeing of Guo Zheng lake.The quantity of bacteria particles in random counter 10 visuals field, and calculate mean number, the quantity of bacterium in 1mL sample is finally calculated by formula Y=AB/C, wherein Y represents the quantity of bacterium in 1mL sample, A represents the mean number of bacterium in a visual field, B represents membrane retention area, and C represents the area in each visual field.
Count results is shown in accompanying drawing 3, and data show that the sub-lake water sample total plate count of Wuhan East Lake 4 is all 10
7individual/mL rank, wherein Guan Qiao lake concentration is the highest, reaches 6 × 10
7individual/mL, soup water chestnut lake is minimum, is 1.26 × 10
7individual/mL.
Claims (8)
1. for an anti-color fading agent for fluorescent microscope bacterial count, it is characterized in that: be dissolved in deionized water with Ursol D, sodium-chlor, Sodium phosphate dibasic, Tris, glycine, glycerine, obtain after regulating pH.
2. the anti-color fading agent for fluorescent microscope bacterial count according to claim 1, it is characterized in that: the storing solution of every this anti-color fading agent of 200mL is prepared by the method comprising following steps: be dissolved in 60-80mL deionized water by 1.5-3g Ursol D, 0.5-1g sodium-chlor, 2.5-3.5g Sodium phosphate dibasic, 0.2-0.4g Tris, 3-5g glycine, pH to 7-8 is regulated with hydrochloric acid and sodium hydroxide, add 80-100mL glycerine, be finally settled to 200mL with deionized water again.
3. the anti-color fading agent for fluorescent microscope bacterial count according to claim 2, it is characterized in that: the storing solution of every this anti-color fading agent of 200mL is prepared by the method comprising following steps: be dissolved in 80mL deionized water by 2g Ursol D, 0.8g sodium-chlor, 3.2g Sodium phosphate dibasic, 0.25g Tris, 4g glycine, with hydrochloric acid or the sodium hydroxide adjustment pH to 7.2 of 0.1M, add 100mL glycerine, be finally settled to 200mL with deionized water.
4. the anti-color fading agent for fluorescent microscope bacterial count according to claim 2, is characterized in that: the working fluid of this anti-color fading agent is for obtaining degerming for storing solution aseptic syringe needle frit with after sterilized water dilution again.
5. the anti-color fading agent for fluorescent microscope bacterial count according to claim 4, is characterized in that: storing solution is diluted 10 times and obtain working fluid.
6. the application of the anti-color fading agent described in any one of claim 1-5 in fluorescent microscope bacterial count.
7. one kind is detected the fluorescent microscopic counting method of water body bacterial number, it is characterized in that comprising the steps: that in water sample, add fluorescence dye dyes, the working fluid adding the anti-color fading agent described in claim 4 or 5 again carries out anti-fade treatment, by the sample of anti-fade treatment after cellulose acetate filter membrane suction filtration at fluorescence microscopy Microscopic observation.
8. the fluorescent microscopic counting method of detection water body bacterial number according to claim 7, is characterized in that comprising the steps:
(1) sample dyeing: get 1mL water sample in the centrifuge tube of 5mL, adds the SYBR Green I fluorescence dye of 100 μ L aseptic deionized water dilutions, lucifuge dyeing 5min;
(2) anti-fade treatment: the working fluid adding the anti-color fading agent described in claim 4 or 5 of its 3 times of volumes in the sample that step (1) has dyeed, after mixing, lucifuge leaves standstill 5min;
(3) Fluorescent microscope counting: utilize Vacuum filtration device to filter through the sample of anti-fade treatment step (2), filter membrane used is common cellulose acetate filter membrane, 0.22 μm, aperture; After filtration, being taken out by filter membrane is placed on slide glass, attached germy one is made to face up, then on filter membrane, drip the working fluid of the anti-color fading agent described in a claim 4 or 5, be covered with cover glass, put on fluorescent microscope Stage microscope, excite with 480nm light wave, 40 times of thing Microscopic observations, the quantity of bacteria particles in random counter 10 visuals field, and calculate mean number; Finally calculate the quantity of bacterium in 1mL sample by formula Y=AB/C, wherein Y represents the quantity of bacterium in 1mL sample, and A represents the mean number of bacterium in a visual field, and B represents membrane retention area, and C represents the area in each visual field.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108387503A (en) * | 2018-01-22 | 2018-08-10 | 北京海岸鸿蒙标准物质技术有限责任公司 | A kind of valued methods of grain count 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| J. A. KIERNAN: "MAKING AND USING AQUEOUS MOUNTING MEDIA", 《MICROSCOPY TODAY》 * |
| M. ONO ET AL: "Quantitative Comparison of Anti-Fading Mounting Media for Confocal Laser Scanning Microscopy", 《THE JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY》 * |
| TERESA KLEIN ET AL: "Eight years of single‑molecule localization microscopy", 《HISTOCHEM CELL BIOL》 * |
| TERESA KLEIN ET AL: "Eight years of single‑molecule localization microscopy", 《HISTOCHEM CELL BIOL》, 5 February 2014 (2014-02-05), pages 567 - 1 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108387503A (en) * | 2018-01-22 | 2018-08-10 | 北京海岸鸿蒙标准物质技术有限责任公司 | A kind of valued methods of grain count particle standard substance |
| 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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