CN102321729A - Fluorescent microscopic counting method for detecting bacterial count in soil and sediment - Google Patents
Fluorescent microscopic counting method for detecting bacterial count in soil and sediment Download PDFInfo
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- CN102321729A CN102321729A CN201110187705A CN201110187705A CN102321729A CN 102321729 A CN102321729 A CN 102321729A CN 201110187705 A CN201110187705 A CN 201110187705A CN 201110187705 A CN201110187705 A CN 201110187705A CN 102321729 A CN102321729 A CN 102321729A
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Abstract
The invention relates to a fluorescent microscopic counting method for detecting a bacterial count in soil and sediment, belonging to the field of environmental microbiology. The detection method comprises the following steps: (1) preparing a sample diluent and a fading inhibitor; (2) adding a sample into the sample diluent for processing, thus obtaining a sample counting liquid; and putting the sample counting liquid in a centrifuge tube, adding 10*SYBR Green I fluorescent dye, dyeing while keeping in a dark place, and then adding the fading inhibitor, thus obtaining a dyeing sample; and (3) covering clean cover glass on a counting chamber of a clean blood counting slide, carrying out vortex mixing on the dyeing sample, taking the dyeing sample with a pipette, and loading the sample along the edge of the cover glass to ensure that the dyeing sample fully fills the counting chamber; enabling the blood counting slide loaded with the sample to stand for five minutes, placing onto an objective table of a fluorescent microscope, activating with 480nm light waves, observing under a 40X objective lens, and counting the quantity of bacterial particles in five medium squares; and finally, calculating the bacterial count in 1g of sample in accordance with a formula Y=31.25*A*10<6>, wherein Y represents the bacterial count in 1g of sample, and A represents the average bacterial count in one medium square. The method is time-saving, labor-saving and low in cost, can resist fading, and can quickly and accurately detect the bacterial count in soil and sediment.
Description
Technical field
The invention belongs to the environmental microbiology field, relate to the detection technique of the total bacterial number of a kind of environment, be specifically related to the sample pre-treatments of total plate count fluorescence microscopy counting in a kind of soil and the settling and agent combination and the detection method that fluorescence microscopy is observed.
Background technology
The fluorescent dye direct-counting method is the novel method that is used for bacterial count that grows up over nearest 30 years, and it has characteristics such as quick, accurate, in environmental samples such as water sample, soil and settling, has a wide range of applications in the mensuration of bacterial number.Francisco in 1973 etc. measure to the total plate count in the natural water body, have at first set up the fluorescent microscope dyeing counting method based on the AO dyeing system.Porter in 1980 etc. begin to adopt DAPI that bacterium is dyeed.Velji etc. adopt UW that bacterium in seawater, the settling and marine alga sample have been carried out pre-treatment on the basis of Porter etc., count through the DAPI staining again, obtain better effects.Employing SYBR Green I fluorescent dyeing methods such as Lunau have been measured bacterial number in seawater and the settling, and with the contrast of the dyeing counting of DAPI, AO, find that SYBR Green I and DNA bonded specificity are higher, the better effects if of dyeing counting.
In the total plate count, the pre-treatment of sample is most important in traditional embrane method counting soil or settling.Owing to comprise a large amount of and matrix granule bonded bacterium in soil or the settling; Distributed pole is inhomogeneous; And some part possibly receive opaque mineral microparticle the optical dye absorption and effect is made that the imaging under the fluorescent microscope is not obvious; And (extracellular polymeric substances EPS) has with particulate and combines the formation coacervate closely, thereby possibly not be counted because the extracellular polymeric that microorganism cells produces.In order to obtain accurate measuring result, the key problem in technology that is separated into the pre-treatment of embrane method fluorescence microscope mensuration of bacterium and matrix granule.These separation methods mainly are divided into physics method (comprising centrifugal settling, vibration, ultrasonication, dilution etc.), chemical method (use trisodium phosphate, tensio-active agent, methyl alcohol, etc. dispersion agent) and enzyme facture (EPS enzyme).Current at present way is several kinds of pre-treating process couplings, remedies the deficiency of single treatment process.But the pre-treating process that satisfies embrane method fluorescence microscopy counting is relatively wasted time and energy, and is inappropriate for the rapid detection of great amount of samples in general.The chemical method that trisodium phosphate and polysorbate handle and the physics method processing sample of vibration and ultrasonication combination density gradient centrifugation have been adopted during like bacterium in separating the channel fill deposit thing such as Amalfitano; Though final about 93% bacterial cell is recovered 2.5 hours consuming time of whole pre-treatment.
The filter membrane that uses during the tradition embrane method detects divides two types of organic and inorganic filter membranes.Organic filter membrane uses early; Cheap relatively; But filter membrane will pass through dyeing and handle; More time-consuming, be inappropriate for rapid detection, to pass through like the filter membrane that uses in the patent " but non-cultivation conditions fluorescence microscope and method of counting of bacteria in viable " of Li Ying etc. and just can be used for fluorescence microscopy after hydrophobic treatment and the dyeing oven dry and observe.Filter membrane commonly used now is the inorganic filter membrane of aluminum oxide.Inorganic filter membrane dispense with dyeing, easy to use, but price is more expensive.In addition, the anti-fade treatment of embrane method is carried out on film, and the topmost shortcoming of this treating processes is that anti-decolourant adhesion amount is few on the film, and anti-fading effect is relatively poor, and the dye fluorescence cancellation is very fast, is unfavorable for observing counting.
Summary of the invention
The present invention is intended to overcome the defective of prior art, provides a kind of time saving and energy saving, with low cost, anti-fading effect better, and can quick and precisely detect the method for bacterial number in earth and the settling.
The present invention realizes through following technical scheme:
(1) damping fluid preparation: take by weighing 0.446g trisodium phosphate and 0.15g EDTA, be dissolved in the 100ml sterile distilled water, process sample diluting liquid; Take by weighing the 0.1g Ursol D, be dissolved in the 1ml PBS-glycerine damping fluid, process anti-decolourant.
(2) sample pre-treatments: take by weighing 0.1g mud appearance in aseptic Eppendolf pipe, add the 1ml sample diluting liquid, vortex vibration 5min processes even suspension liquid.Get the 0.1ml suspension liquid in the 0.9ml sample diluting liquid, vortex vibration 5min processes sample counting liquid.Get 80 μ l samples countings liquid in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I optical dye, add the anti-decolourant of 10 μ l behind the lucifuge dyeing 1min, process stained specimens.
(3) fluorescence microscopy counting: the deckglass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor, and the application of sample along the deckglass edge makes stained specimens be full of nucleonics.The blood counting chamber of application of sample is left standstill 5min, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, 40 times of object lens are observed down, count the quantity of bacteria particles in 5 medium squares.Y=31.25 * A * 10 by formula at last
6Calculate number of bacteria in the 1g sample, wherein Y representes number of bacteria in the 1g sample, and A representes the mean number of bacterium in the medium square.
Major advantage of the present invention is:
1, the present invention need not loaded down with trivial details sample pre-treatments work, has shortened time for sample pretreatment greatly, has improved detection efficiency, and is more time saving and energy saving than conventional fluorescent microscopic method, can quick and precisely detect number of bacteria in earth and the settling.
2, the present invention need not to use expensive inorganic filter membrane in testing process, has reduced the detection cost.
3, the present invention has changed anti-fade treatment method, has significantly improved anti-fading effect.
Description of drawings
Fig. 1: be the design sketch that the settling bacterium after the dyeing is observed through 40 times of object lens in blood counting chamber.
Fig. 2: be the pond settling total plate count that blood counting chamber fluorescence microscopy counting process obtains.
Embodiment
Below in conjunction with Figure of description and embodiment the present invention is done further explanation, but be not restriction the present invention.
The comparison of 1 three kinds of method of counting of embodiment
The light absorption value of cultivating bacillus coli DH 5 alpha (teacher Yang Jiaoyan of Central China Normal University is so kind as to give) to its 600nm place with the LB liquid nutrient medium is 0.2～0.3 o'clock, and the saline water with 0.9% suitably dilutes.
Colony counting method: select suitable dilution gradient, draw 0.2 milliliter of spread plate, the bacterium colony number that makes each dull and stereotyped top growth is between 30～300.The counting flat board is gone up colony count behind 37 ℃ of cultivation 36h, calculates the concentration of original bacterium liquid according to the bacterium colony number.Every milliliter of bacterium liquid bacterial count=3 multiple bacterium colonies of same extent of dilution mean number * extension rate.
Blood counting chamber fluorescence microscopy counting process: get sample 80 μ l after the dilution in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I optical dye, dyeing adds the anti-decolourant of 10 μ l after 1 minute.The deckglass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor, and the application of sample along the deckglass edge makes stained specimens be full of nucleonics.The blood counting chamber of application of sample is left standstill 5min, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, 40 times of object lens are observed down.With the tally of 25 medium squares, press the diagonal lines orientation during counting, get upper left, following, upper right, bottom right, a left side and 5 medium squares of intermediary and count.Every milliliter of bacterium liquid bacterial count is Y=31.25 * A * C * 10 by formula
4, wherein Y is every milliliter of bacterium liquid bacterial count, and A is every medium square bacterium mean number, and C is an extension rate.
Filter membrane fluorescence microscopy counting process: get the suitably sample of dilution of 80 μ l, bacterium is dyeed and anti-fade treatment, be diluted to 25ml with SPSS then, after the inorganic membrane filtration of 0.22 μ m according to the description in the blood counting chamber method.Filter membrane is put on the slide glass, excited with the 480nm light wave under the fluorescent microscope, 40 times of object lens are observed down, count the bacteria particles number in 5 visuals field at random.Every milliliter of bacterium liquid bacterial count Y=1.25 * P * K * C calculating by formula, wherein Y is every milliliter of bacterium liquid bacterial count, and P is the mean number of bacterium in each visual field, and K is the visual field number of filter membrane, and C is an extension rate.
Utilize above-mentioned 3 kinds of methods that 3 DH5 α cultures are counted, the gained result sees table 1, and the result proves that blood counting chamber fluorescence microscopy counting process recall rate will be higher than other 2 kinds of methods.
Table 1.3 kind of method of counting counting bacillus coli DH 5 alpha result relatively
Total plate count investigation in the settling of pond, embodiment 2 Chong Hu fishing ground
Take surface deposit 200g from center, 21 mouthfuls of ponds, Chong Hu fishing ground in October, 2010, the aseptic valve bag of packing into, and the insulation can refrigerated shipment is gone back to the laboratory.Take by weighing different sediment sample 0.1g with the 1.5ml centrifuge tube, add 1ml trisodium phosphate-edta buffer liquid, vortex vibration 5min gets the 0.1ml suspension liquid, joins in 0.9ml trisodium phosphate-edta buffer liquid, vortex vibration 5min again.Get the good suspension liquid of 80 μ l dilution in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I dyestuff, lucifuge dyeing 1min adds 10 μ l antagonism decolourant then.The deckglass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor, and the application of sample along the deckglass edge makes stained specimens be full of nucleonics.The blood counting chamber of application of sample is left standstill 5min, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, 40 times of object lens are observed counting down.With the tally of 25 medium squares, press the diagonal lines orientation during counting, get upper left, following, upper right, bottom right, a left side and 5 medium squares of intermediary and count.Every gram sample bacterial count is Y=31.25 * A * 10 by formula
6, wherein Y is every gram sample bacterial count, A is every medium square bacterium mean number.Effect such as Fig. 1 that settling bacterium after the dyeing is observed through 40 times of object lens in blood counting chamber.Count results such as Fig. 2,24 mouthfuls of sedimental bacterial numbers in fish pond are basically all 10
8The cell/g order of magnitude, but still there is significant difference in the quantity between different pond.
Claims (4)
1. fluorescence microscopy method of counting that detects bacterial number in soil and the settling is characterized in that may further comprise the steps:
(1) preparation of damping fluid: be dissolved in the sterile distilled water with trisodium phosphate and EDTA, be mixed with sample diluting liquid; Be dissolved in the PBS-glycerine damping fluid with Ursol D, be mixed with anti-decolourant;
(2) sample pre-treatments: take by weighing 0.1g mud appearance in aseptic Eppendolf pipe, add the 1ml sample diluting liquid, vortex vibration 5min; Process even suspension liquid, get the 0.1ml suspension liquid in the 0.9ml sample diluting liquid, vortex vibration 5min; Process sample counting liquid, get 80 μ l samples counting liquid in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I optical dye; Add the anti-decolourant of 10 μ l behind the lucifuge dyeing 1min, process stained specimens;
(3) fluorescence microscopy counting: the deckglass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor; The application of sample along the deckglass edge makes stained specimens be full of nucleonics, and the blood counting chamber of application of sample is left standstill 5min; Put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, 40 times of object lens are observed down; Count the quantity of bacteria particles in 5 medium squares, at last Y=31.25 * A * 10 by formula
6Calculate number of bacteria in the 1g sample, wherein Y representes number of bacteria in the 1g sample, and A representes the mean number of bacterium in the medium square.
2. a kind of fluorescence microscopy method of counting that detects bacterial number in soil and the settling as claimed in claim 1 is characterized in that said sample diluting liquid is with 0.446g trisodium phosphate and 0.15g EDTA, is dissolved in the 100ml sterile distilled water formulated.
3. a kind of fluorescence microscopy method of counting that detects bacterial number in soil and the settling as claimed in claim 1 is characterized in that said anti-decolourant is dissolved in the 1ml PBS-glycerine damping fluid 0.1g Ursol D formulated.
4. a kind of fluorescence microscopy method of counting that detects bacterial number in soil and the settling as claimed in claim 1, the application of its bacterial number in detecting soil and settling.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593475A (en) * | 2015-02-03 | 2015-05-06 | 武汉市环境保护科学研究院 | Fluorescent microscopic counting method for detecting number of bacteria in water body |
| CN106636299A (en) * | 2016-12-27 | 2017-05-10 | 扬州大学 | Bacterial pollution detection method for edible fungus liquid strain |
| CN106893673A (en) * | 2017-04-11 | 2017-06-27 | 尹康康 | A kind of soil bacteria separates counting experimental provision |
| CN107326063A (en) * | 2017-07-31 | 2017-11-07 | 农业部沼气科学研究所 | A kind of method that fixation of bacteria observation is counted |
| CN108034691A (en) * | 2017-12-01 | 2018-05-15 | 汕头大学 | A kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph |
| CN109696393A (en) * | 2019-01-17 | 2019-04-30 | 汕尾市海洋产业研究院 | A kind of method that determination of the environment tests polystyrene microsphere content under simulated conditions |
| CN114004851A (en) * | 2021-11-26 | 2022-02-01 | 广州市艾贝泰生物科技有限公司 | Cell image segmentation method and device and cell counting method |
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| CN2377532Y (en) * | 1999-06-10 | 2000-05-10 | 中国人民解放军第二军医大学 | Microfluorecyte counting plate |
| CN101482512A (en) * | 2009-02-10 | 2009-07-15 | 中国农业大学 | Total bacteria count measuring method based on ultrasonic and fluorescent observation |
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Patent Citations (2)
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| CN2377532Y (en) * | 1999-06-10 | 2000-05-10 | 中国人民解放军第二军医大学 | Microfluorecyte counting plate |
| CN101482512A (en) * | 2009-02-10 | 2009-07-15 | 中国农业大学 | Total bacteria count measuring method based on ultrasonic and fluorescent observation |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593475A (en) * | 2015-02-03 | 2015-05-06 | 武汉市环境保护科学研究院 | Fluorescent microscopic counting method for detecting number of bacteria in water body |
| CN104593475B (en) * | 2015-02-03 | 2017-06-09 | 武汉市环境保护科学研究院 | A kind of fluorescent microscopic counting method for detecting water body bacterial number |
| CN106636299A (en) * | 2016-12-27 | 2017-05-10 | 扬州大学 | Bacterial pollution detection method for edible fungus liquid strain |
| CN106636299B (en) * | 2016-12-27 | 2021-04-27 | 扬州大学 | Method for detecting bacterial contamination of edible fungus liquid strain |
| CN106893673A (en) * | 2017-04-11 | 2017-06-27 | 尹康康 | A kind of soil bacteria separates counting experimental provision |
| CN107326063A (en) * | 2017-07-31 | 2017-11-07 | 农业部沼气科学研究所 | A kind of method that fixation of bacteria observation is counted |
| CN108034691A (en) * | 2017-12-01 | 2018-05-15 | 汕头大学 | A kind of method of the abundance of microorganism in accurate counting invertebrate hemolymph |
| CN108034691B (en) * | 2017-12-01 | 2021-08-06 | 汕头大学 | Method for accurately counting abundance of microorganisms in invertebrate haemolymph |
| CN109696393A (en) * | 2019-01-17 | 2019-04-30 | 汕尾市海洋产业研究院 | A kind of method that determination of the environment tests polystyrene microsphere content under simulated conditions |
| CN114004851A (en) * | 2021-11-26 | 2022-02-01 | 广州市艾贝泰生物科技有限公司 | Cell image segmentation method and device and cell counting method |
| CN114004851B (en) * | 2021-11-26 | 2022-11-29 | 广州市艾贝泰生物科技有限公司 | Cell image segmentation method and device and cell counting method |
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