CN102321729B - 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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- CN102321729B CN102321729B CN 201110187705 CN201110187705A CN102321729B CN 102321729 B CN102321729 B CN 102321729B CN 201110187705 CN201110187705 CN 201110187705 CN 201110187705 A CN201110187705 A CN 201110187705A CN 102321729 B CN102321729 B CN 102321729B
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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 a kind of detection technique of environment total bacteria amount, be specifically related to the sample pre-treatments of total plate count Fluorescent microscope counting in a kind of soils and sediments 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 the characteristics such as quick, accurate, has a wide range of applications in the mensuration of bacterial number in the environmental samples such as water sample, soil and settling.Francisco in 1973 etc. measure for the total plate count in natural water body, model based on the fluorescent microscope dyeing counting method of AO dyeing system.Porter in 1980 etc. begin to adopt DAPI that bacterium is dyeed.Velji etc. adopt ultrasonic wave to carry out pre-treatment to the bacterium in seawater, settling and marine alga sample on the basis of Porter etc., then count through the DAPI staining, obtain better effects.The employing SYBR Green I fluorescent dyeing methods such as Lunau have been measured bacterial number in seawater and settling, and with the contrast of the dyeing counting of DAPI, AO, find that the specificity that SYBR Green I is combined with DNA is higher, the better effects if of dyeing counting.
In total plate count, determination is most important in traditional embrane method counting soil or settling.Owing to comprising the bacterium of being combined with matrix granule in a large number in soil or settling, distributed pole is inhomogeneous, and some part may be subject to opaque mineral microparticle and make the imaging under fluorescent microscope not obvious on the impact that fluorescence dye absorbs, and extracellular polymeric (the extracellular polymeric substances due to the microorganism cells generation, EPS) have to be combined closely with particulate and form coacervate, thereby may not be counted.In order to obtain the Measurement accuracy 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 Physical (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 way is several pre-treating process couplings at present, makes up the deficiency of single treatment process.But in general, the pre-treating process that satisfies the embrane method Fluorescent microscope counting is relatively wasted time and energy, and is unsuitable for the rapid detection of great amount of samples.Adopted chemical method that trisodium phosphate and polysorbate process and vibration and ultrasonication to process sample in conjunction with the Physical of density gradient centrifugation during bacterium as Amalfitano etc. in separating the channel fill deposit thing, although final approximately 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 organic and inorganic filter membrane two classes.Organic filter membrane uses early, relatively cheap, but filter membrane will be processed through dyeing, more time-consuming, be unsuitable for rapid detection, will be through could be for the fluorescence microscopy observation after hydrophobic treatment and dyeing oven dry as the filter membrane that uses in the patent " non-culmrable sate fluorescence microscope and the method for counting of bacteria in viable " of Li Ying etc.Filter membrane commonly used is the inorganic filter membrane of aluminum oxide now.Inorganic filter membrane need not dyeing, and is 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 on film, anti-decolourant adhesion amount is few, 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, provide a kind of time saving and energy saving, with low cost, anti-fading effect is better, and can quick and precisely detect the method for bacterial number in earth and settling.
The present invention is achieved through the following technical solutions:
(1) damping fluid preparation: take 0.446g trisodium phosphate and 0.15g EDTA, be dissolved in the 100ml sterile distilled water, make sample diluting liquid; Take the 0.1g Ursol D, be dissolved in 1ml PBS-glycerine damping fluid, make anti-decolourant.
(2) sample pre-treatments: take 0.1g mud sample in aseptic Eppendolf pipe, add the 1ml sample diluting liquid, vortex vibration 5min makes even suspension liquid.Get the 0.1ml suspension liquid in the 0.9ml sample diluting liquid, vortex vibration 5min makes 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 fluorescence dye, add the 10 anti-decolourants of μ l after lucifuge dyeing 1min, make stained specimens.
(3) Fluorescent microscope counting: the cover glass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor, along cover glass edge application of sample, makes stained specimens be full of nucleonics.With the standing 5min of the blood counting chamber of application of sample, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, observe under 40 times of object lens, count the quantity of bacteria particles in 5 medium squares.Y=31.25 * A * 10 by formula at last
6Calculate the quantity of bacterium in the 1g sample, wherein Y represents the quantity of bacterium in the 1g sample, and A represents the mean number of bacterium in a 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 greatly shortened time for sample pretreatment, has improved detection efficiency, and is more time saving and energy saving than conventional fluorescent microscopic method, can quick and precisely detect the quantity of bacterium in earth and settling.
2, the present invention need not to use expensive inorganic filter membrane in testing process, has reduced testing 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 dyeing is observed by 40 times of object lens in blood counting chamber.
Fig. 2: be the pond settling total plate count that blood counting chamber Fluorescent microscope counting method obtains.
Embodiment
The present invention is described further below in conjunction with Figure of description and embodiment, but be not restriction the present invention.
The comparison of 1 three kinds of method of counting of embodiment
Cultivating bacillus coli DH 5 alpha (teacher Yang Jiaoyan of Central China Normal University is so kind as to give) to the light absorption value at its 600nm place with the LB liquid nutrient medium is 0.2～0.3 o'clock, and the physiological saline with 0.9% suitably dilutes.
Colony counting method: select suitable dilution gradient, draw 0.2 milliliter of spread plate, make bacterium colony number that each flat board grows above between 30～300.The dull and stereotyped upper colony number of counting after 37 ℃ of cultivation 36h is according to the concentration of the original bacterium liquid of bacterium colony number calculating.Every milliliter of bacterium colony mean number * extension rate that bacterium liquid bacterial count=same extent of dilution 3 times repeats.
Blood counting chamber Fluorescent microscope counting method: get sample 80 μ l after dilution in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I fluorescence dye, dyeing adds the 10 anti-decolourants of μ l after 1 minute.The cover glass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor, along cover glass edge application of sample, makes stained specimens be full of nucleonics.With the standing 5min of the blood counting chamber of application of sample, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, observe under 40 times of object lens.With the tally of 25 medium squares, press the diagonal lines orientation during counting, get upper left, lower-left, upper right, bottom right and 5 middle medium squares 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 extension rate.
Filter membrane Fluorescent microscope counting method: get the suitably sample of dilution of 80 μ l, bacterium is dyeed and anti-fade treatment according to the description in the blood counting chamber method, then be diluted to 25ml with stroke-physiological saline solution, finally by the 0.22 inorganic membrane filtration of μ m.Filter membrane is put on slide glass, excited with the 480nm light wave under fluorescent microscope, observe under 40 times of object lens, count at random the bacteria particles number in 5 visuals field.Every milliliter of by formula Y=1.25 * P * K * C calculating of bacterium liquid bacterial count, 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 extension rate.
Utilize above-mentioned 3 kinds of methods that 3 DH5 α cultures are counted, acquired results sees Table 1, and result proof blood counting chamber Fluorescent microscope counting method 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
Investigation On The Total Count of Bacteria in embodiment 2 pond, Chong Hu fishing ground settlings
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 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, join in 0.9ml trisodium phosphate-edta buffer liquid, then vortex vibration 5min.Get 80 μ l and dilute good suspension liquid in the centrifuge tube of 200 μ l, add 10 μ l 10 * SYBR Green I dyestuff, then lucifuge dyeing 1min adds 10 μ l antagonism decolourants.The cover glass of cleaning is covered on clean blood counting chamber nucleonics, and vortex mixing stained specimens is got 20 μ l stained specimens with pipettor, along cover glass edge application of sample, makes stained specimens be full of nucleonics.With the standing 5min of the blood counting chamber of application of sample, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, observe counting under 40 times of object lens.With the tally of 25 medium squares, press the diagonal lines orientation during counting, get upper left, lower-left, upper right, bottom right and 5 middle medium squares 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 dyeing is observed by 40 times of object lens in blood counting chamber.Count results such as Fig. 2, the bacterial number of 24 mouthfuls of Sediment of Aquaculture Ponds is substantially all 10
8The cell/g order of magnitude, but still there is significant difference in the quantity between different pond.
Claims (2)
1. fluorescent microscopic counting method that detects bacterial number in soils and sediments is characterized in that comprising the following steps:
(1) preparation of damping fluid: the preparation of damping fluid: be dissolved in the 100ml sterile distilled water with 0.446g trisodium phosphate and 0.15g EDTA, be mixed with sample diluting liquid; Be dissolved in the PBS-glycerine damping fluid that the 1ml volume ratio is 1:1 with the 0.1g Ursol D, be mixed with anti-decolourant;
(2) sample pre-treatments: take 0.1g mud sample in aseptic Eppendolf pipe, add the 1ml sample diluting liquid, vortex vibration 5min makes even suspension liquid, gets the 0.1ml suspension liquid in the 0.9ml sample diluting liquid, vortex vibration 5min, make sample counting liquid, get 80 μ l samples counting liquid in the centrifuge tube of 200 μ l, add 10 μ l10 * SYBR Green I fluorescence dye, add the 10 anti-decolourants of μ l after lucifuge dyeing 1min, make stained specimens;
(3) Fluorescent microscope counting: the cover glass of cleaning is covered on clean blood counting chamber nucleonics, vortex mixing stained specimens, get 20 μ l stained specimens with pipettor, along cover glass edge application of sample, make stained specimens be full of nucleonics, with the standing 5min of the blood counting chamber of application of sample, put on the fluorescent microscope Stage microscope, excite with the 480nm light wave, observe under 40 times of object lens, count the quantity of bacteria particles in 5 medium squares, at last Y=31.25 * A * 10 by formula
6Calculate the quantity of bacterium in the 1g sample, wherein Y represents the quantity of bacterium in the 1g sample, and A represents the mean number of bacterium in a medium square.
2. a kind of fluorescent microscopic counting method that detects bacterial number in soils and sediments as claimed in claim 1, the application of its bacterial number in detecting soils and sediments.
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| 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 |
| CN106636299B (en) * | 2016-12-27 | 2021-04-27 | 扬州大学 | Method for detecting bacterial contamination of edible fungus liquid strain |
| CN106893673B (en) * | 2017-04-11 | 2019-09-06 | 宿州市鑫尧健康科技有限公司 | A kind of soil bacteria separation 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 |
| 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 |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| 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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