CN103175768A - Fluorescent staining kit for rapid detection on biological cell viability, and application of same - Google Patents
Fluorescent staining kit for rapid detection on biological cell viability, and application of same Download PDFInfo
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- CN103175768A CN103175768A CN2013100601403A CN201310060140A CN103175768A CN 103175768 A CN103175768 A CN 103175768A CN 2013100601403 A CN2013100601403 A CN 2013100601403A CN 201310060140 A CN201310060140 A CN 201310060140A CN 103175768 A CN103175768 A CN 103175768A
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- 238000001514 detection method Methods 0.000 title abstract description 10
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Abstract
The invention relates to a fluorescent staining kit for rapid detection on biological cell viability, and application of the same. The kit comprises an anthocyanin dye and a propidium iodide dye. The detection comprises the following steps of: adding the anthocyanin dye and the propidium iodide in the kit are added in cell suspension, observing and counting the cells by a fluorescence microscope or detecting the cells by a microporous fluorescent plate reader under wavelength of emitted light, and reflecting the viability of the cells in biologic sample liquid according to the strength of fluorescence values of different colors. The kit can be used for rapidly observing or detecting the amount of living cells and the amount of dead cells of various cells in the sample in real time, is visual and handy, eliminates the inference of other substances and the defect of long culture cycle, and is not only suitable for animal cells, but also suitable for most gram negative bacteria and gram positive bacteria.
Description
Technical field
The invention belongs to the cell count field, particularly a kind of fast detecting biological cell fluorescent dye kit and application thereof of state anyway.
Background technology
The method of measuring at present viable count has optical microscope reading counting method, dull and stereotyped coating to cultivate colony counting method, photoelectric turbidimetry, maximum most probable number (MPN) method, and membrane filter method etc.
The ascites method: in a small amount the suspending liquid of testing sample is placed on a kind of microslide that has especially definite area and a volume (claiming again meter bacterium device), directly observes counting in microscopically.The method is easy, quick, directly perceived, can be used for the counting of the suspensions such as yeast, bacterium, mycotic spore and zooblast.Shortcoming be for can not accurately identify the life or death state of cell, and measured result is the summation of dead cell and living cells normally.
The method of plate culture count: testing sample is diluted through suitable, make wherein microbe colony or zooblast fully be dispersed into the individual cells state, getting a certain amount of diluted sample liquid is inoculated on plating medium, through cultivating, form macroscopic bacterium colony or population of cells by each unicellular growth and breeding, namely single bacterium colony should represent one in raw sample unicellular.This method is widely used in biological products checks (as active bacteria formulation), and the detection that contains bacterium index or pollution level of food, beverage and water (comprising the water source) etc.shortcoming is, because testing sample often is difficult for being dispersed into fully individual cells, so the single bacterium colony that forms after cultivating may be from the 2-3 in sample or more cells, perhaps because the growth cycle of thalline is different, in the same nutrient culture media due to adaptive difference, some thalline needed 3 days can grow bacterium colony, some thalline need the even longer time in 1 week, therefore the result of plate count is often on the low side, usually use colony-forming units (colony-formingunits, cfu) and do not represent the viable bacteria content of sample with absolute clump count.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of fast detecting biological cell fluorescent dye kit and the application thereof of state anyway, this kit can observe in real time fast or test sample in the living cells of various cells and the quantity of dead cell, intuitively easy, interference and the long shortcoming of cultivation cycle of having got rid of other material, be not only applicable to zooblast, and be applicable to most gramnegative bacterium and gram-positive bacterium.
A kind of fast detecting biological cell of the present invention is the fluorescent dye kit of state anyway, and described kit comprises anthocyanidin dyestuff and propidium iodide dyestuff, and the storage concentration of anthocyanidin dyestuff is 5-6mM, and the storage concentration of propidium iodide dyestuff is 100 μ g/mL.
The dilution buffer liquid that described anthocyanidin dyestuff uses is that 1 * TE buffer[contains 1mM EDTA(ethylenediamine tetraacetic acid) the damping fluid of 10mM Tris-HCl(trishydroxymethylaminomethane-HCl), pH8.0].This dyestuff can be penetrated into the cell under all states, and it tends in conjunction with double-stranded DNA in cell, for single stranded DNA and RNA in conjunction with less, the nucleic acid of cell is dyeed, make cell send green fluorescence.
When described anthocyanidin dyestuff uses, dilution 1000-10000 doubly.
Described anthocyanidin dye solvent is dimethyl sulfoxide (DMSO), and the work ultimate density when using (actual concentrations when working concentration refers to cell dyeing) is 5-6 μ M.
The anthocyanidin dyestuff, its structural formula is:
The solvent of described propidium iodide dyestuff and thinning agent are ultrapure water.This dyestuff can only be attached on DNA or RNA specifically by the cell membrane of dead cell, makes cell send red fluorescence, therefore can detect dead cell.
The work ultimate density of described propidium iodide dyestuff is 10-20 μ g/mL.
The solvent of described anthocyanidin dyestuff is dimethyl sulfoxide (DMSO), and the solvent of propidium iodide dyestuff is ultrapure water.
A kind of fast detecting biological cell of the present invention application of the fluorescent dye kit of state anyway, detecting step is as follows: add anthocyanidin dyestuff and propidium iodide in kit in cell suspension, utilize the fluorescence microscope counting or utilize porous fluorescent to read the plate device and detect under wavelength of transmitted light, according to the existing state of cell in the power reflection biological sample liquid of different colours fluorescent value.
The described concrete steps of fluorescence microscope counting of utilizing comprise:
(1) draw cell suspension in the centrifuge tube that is surrounded by masking foil;
(2) take the proportioning of volume ratio as 7:3(cell suspension and mixed dye) ratio add volume ratio as the anthocyanidin dyestuff of 1-2:1 and the mixed dye of propidium iodide, fluctuate, be placed in dark 10-30 minute; Perhaps adding volume ratio is the proportioning of 4:1(cell suspension and dyestuff) the anthocyanidin dyestuff, fluctuate, be placed in dark 10-30 minute, survey live body; Perhaps adding volume ratio is the proportioning of 9:1(cell suspension and dyestuff) the PI dyestuff, fluctuate, be placed in dark 10-30 minute, survey dead volume;
(3) will dye the cell sample liquid of look by black filtering membrane (Whatman) filtration of 0.2 μ m, and make cell retention on the black filtering membrane, filtering membrane is transferred on microslide, central authorities drip without fluorescence essential oil, covered at film;
(4) slide is placed under fluorescent microscope, central authorities drip without fluoroscope oil at cover glass, oily sem observation with 100 times, (the suitableeest exciting light of anthocyanidin dyestuff is 488nm at the suitableeest exciting light of anthocyanidin dyestuff and PI dyestuff respectively, the suitableeest exciting light of PI dyestuff is 522nm) under detect, in eyepiece, greeny cell is live body, and the cell that takes on a red color is dead volume.
Describedly utilize porous fluorescent to read the concrete steps that the plate device detects under wavelength of transmitted light to comprise:
(1) will contain the nutrient culture media of cell through the filtering membrane filtration of 0.2 μ m, the cell of collecting is resuspended with physiological saline;
(2) add cell suspension in microwell plate, take the proportioning of volume ratio as 7:3(cell suspension and mixed dye) ratio add volume ratio to be the anthocyanidin dyestuff of 1-2:1 and the mixed dye of propidium iodide in micropore, fluctuate, be placed in dark 10-30 minute; Perhaps adding volume ratio is the proportioning of 4:1(cell suspension and dyestuff) the anthocyanidin dyestuff, fluctuate, be placed in dark 10-30 minute, survey live body; Perhaps adding volume ratio is the proportioning of 9:1(cell suspension and dyestuff) the PI dyestuff, fluctuate, be placed in dark 10-30 minute, survey dead volume; In micropore, cumulative volume is no more than 300 μ L, dyeed 10-30 minute, be 460nm-500nm at exciting light, utilizing emitted light is to detect its fluorescence intensity level under the condition of 500nm-530nm, the power of fluorescence has reflected the quantity of live body in this cell suspension, be 460nm-530nm at exciting light, utilizing emitted light is to detect its fluorescence intensity level under the condition of 600nm-640nm, and the power of fluorescence has reflected the quantity of dead volume in this cell suspension.
Principle of the present invention is to adopt two kinds of fluorescent dyes to carry out fluorescence labeling to the nucleic acid of cell, bioactive difference according to cell in incubation, cell can send different fluorescence, has reflected the ratio of different bioactive cells in nutrient culture media according to the difference of fluorescence intensity.The existing state that is established as the various biological cells of further investigation of this technology provides universal method and technology.Particularly provide a kind of simple and feasible method to the biological characteristics of the less microbial cell of acetobacter xylinum equidimension and the building-up process of bacteria cellulose.
Detect principle: when add simultaneously two kinds of dyestuffs in cell suspension, the anthocyanidin dyestuff can enter all cells, makes cell send green fluorescence.When cell is in existing state, cell membrane only allows the anthocyanidin dyestuff to enter cell, and the PI dyestuff can not enter cell, and the cell of existing state only shows green fluorescence.When cell is in moribund condition, membrane passage increases, and PI can partly enter into cell, be embedded into the nucleic acid moiety that dyes the cell that green fluorescence is arranged, override the part green fluorescence, make cell send the red fluorescence of part, what detect at microscopically is that cell sends fluorescent orange.When cell is in dead state, PI can pass through cell membrane in large quantities, is embedded on the nucleic acid of cell, overrides the green fluorescence of anthocyanidin, and dead cell can wholely show red fluorescence.
Beneficial effect
The present invention can observe in real time fast or test sample in the living cells of various cells and the quantity of dead cell, intuitively easy.And the anthocyanidin dyestuff derives from nature, and toxicity is low, and is easy to use.Send fluorescence on double-strandednucleic acid because dyestuff can only be combined in specific manner, got rid of interference and the long shortcoming of cultivation cycle of other material.This colouring method is not only applicable to zooblast, and is applicable to most gramnegative bacterium and gram-positive bacterium.
Description of drawings
Fig. 1 is with the fluorescence microscopy microscopy and is coated with the acetobacter xylinum that flat band method detects same system, the contrast of testing result;
Fig. 2 is the optimum dye concentration of anthocyanidin dyeing thalline, the fluorescence of the RFU=dyeing thalline total fluorescence intensity-blank fluorescence of thalline-dyestuff blank.
Fig. 3 is the optimum dye concentration of PI dyeing thalline, the fluorescence of the RFU=dyeing thalline total fluorescence intensity-blank fluorescence of thalline-dyestuff blank;
Fig. 4 detects with kit the staphylococcus aureus suspension that mixes in the ratio of different dead bacterium and viable bacteria, and the ratio of its RFU accumulative total intensity changes, the fluorescence of the RFU=dyeing thalline total fluorescence intensity-blank fluorescence of thalline-dyestuff blank;
Fig. 5 detects with kit the Escherichia coli suspension that mixes in the ratio of different dead bacterium and viable bacteria, and the ratio of its RFU accumulative total intensity changes, the fluorescence of the RFU=dyeing thalline total fluorescence intensity-blank fluorescence of thalline-dyestuff blank;
Fig. 6 detects with kit the acetobacter xylinum suspension that mixes in the ratio of different dead bacterium and viable bacteria, and the ratio of its RFU accumulative total intensity changes, the fluorescence of the RFU=dyeing thalline total fluorescence intensity-blank fluorescence of thalline-dyestuff blank;
Fig. 7 detects the typical curve of acetobacter xylinum viable bacteria with kit;
Fig. 8 detects the typical curve of the dead bacterium of acetobacter xylinum with kit.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
(1) the anthocyanidin dyestuff is dissolved to 5-6mM concentration with dimethyl sulfoxide (DMSO), and this anthocyanidin dyestuff for preparation stores strong solution.
(2) the PI dyestuff is dissolved to 100 μ g/mL with ultrapure water, and lucifuge stores
(3) draw the acetobacter xylinum bacterium liquid of 1mL in the 2mL centrifuge tube that is surrounded by masking foil.
(4) add 300 μ L after 1000 times of damping fluid dilutions the anthocyanidin dyestuff and the 100 μ g/mL PI dyestuffs of 200 μ L, fluctuate repeatedly, be placed in dark 10-30 minute, dilution buffer liquid be 1 * TE buffer (the 10mM Tris-HCl damping fluid that contains 1mM EDTA, pH8.0).
(5) will dye the bacterium liquid of look by black filtering membrane (Whatman) filtration of 0.2 μ m, thalline is trapped on the black filtering membrane, filtering membrane is transferred on microslide, central authorities drip without fluorescence essential oil, covered at film.
(6) slide is placed under 100 times of object lens of fluorescent microscope and observes, because different dyestuffs has the suitableeest different wave band that excites, therefore need to detect under the suitableeest exciting light of anthocyanidin dyestuff and PI dyestuff, in eyepiece, greeny thalline is the viable bacteria body, and the thalline that takes on a red color is dead thalline.Take out the acetobacter xylinum of 1mL from same system, with 10 times of its dilutions, 100 times, 1000 times, 10000 times, the thalline that dilution is good is coated with respectively flat board, bacterium colony to be grown compares (experimental result such as Fig. 1) with calculating the colony counts of gained and the experimental result of fluorescent microscope.Experimental result shows, use the measured thalline quantity result of fluorescent microscope on average to exceed 1-2 the order of magnitude than the testing result with the method for plate culture count gained, this with document in report identical, during due to the use colony counting method, testing sample often is difficult for being dispersed into fully individual cells, so the single bacterium colony that forms after cultivating may be from the 2-3 in sample or more cells, so the result of plate count is often on the low side.But the linear relationship as a result that itself and fluorescence microscopy detect is good, R
2=0.9872.
(1) the anthocyanidin dyestuff is stored 1000 times to 20000 times of strong solution dilutions, with volume is identical but the dyestuff that extension rate is different by the thalline that dyes of the step 1-3 in embodiment 1, use Molecular Devices Flexstation II type calcium current luminoscope to detect, the fluorescence intensity of discovery thalline descends with the increase of the extension rate of dyestuff, when diluting 5000 times, its Color is better and dye dosage is relatively low, and is both economical.If extension rate increases, detected fluorescent value is near limits of error in-scope, and the error of detected value increases (experimental result such as Fig. 2).
(2) concentration with dyestuff PI is mixed with from 1 μ g/mL to 100 μ g/mL, with identical but the dyeing thalline that extension rate is different of volume, and its Color best (experimental result such as Fig. 3) when finding that concentration is 10 μ g/mL.
(1) staphylococcus aureus being cultivated the dense turbidity OD value of bacterium is 0.7, gets 25mL bacterium liquid under the 12000r/min condition centrifugal 10-15 minute;
(2) collecting thalline is resuspended in the physiological saline of 2mL;
(3) get 1mL bacterium liquid and be dissolved in 20mL physiological saline, separately get 1mL and be dissolved in the 20mL70% isopropyl alcohol;
(4) stirred once incubated at room 1 hour in every 15 minutes;
(5) under the 12000r/min condition centrifugal 10-15 minute;
(6) be resuspended in respectively the physiological saline of 20mL, then centrifuge washing, operation steps is the same;
(7) use respectively the physiological saline of 10mL resuspended, viable bacteria is mixed according to the ratio of table one with dead bacterium;
Table one
| The ratio of viable bacteria and dead bacterium | The volume of viable bacteria (mL) | The volume (mL) of dead bacterium |
| 0:100 | 0 | 2 |
| 10:90 | 0.2 | 1.8 |
| 50:50 | 1 | 1 |
| 100:0 | 2 | 0 |
(8) the bacterium liquid that adds 140 μ L to mix in microwell plate, add the 5mM anthocyanidin dyestuff of 40 μ L and the 100 μ g/mL PI dyestuffs of 20 μ L under dark room conditions, be to excite under 488nm at exciting light, detect under the detection light of 522nm and 622nm respectively, the fluorescence intensity that records under 522nm divided by the fluorescence intensity that records under 622nm, is obtained the ratio of RFU.Then with the ratio of viable bacteria, RFU ratio is mapped, return and obtain regression equation and R
2Value, experimental result such as Fig. 4.Result shows the linear fine of regression equation, R
2=0.9599, after the dyeing cell in this kit was described, the fluorescence intensity that excites under relevant excitation wavelength can be corresponding well with the life or death state of cell.
Embodiment 4
(1) Escherichia coli being cultivated the dense turbidity OD value of bacterium is 1.2, gets 25mL bacterium liquid under the 12000r/min condition centrifugal 10-15 minute;
(2) collecting thalline is resuspended in the physiological saline of 2mL;
(3) get 1mL bacterium liquid and be dissolved in 20mL physiological saline, separately get 1mL and be dissolved in the 20mL70% isopropyl alcohol;
(4) stirred once in every 15 minutes, the room temperature insulation was hatched 1 hour;
(5) under the 12000r/min condition centrifugal 10-15 minute;
(6) be resuspended in respectively the physiological saline of 20mL, then centrifuge washing, operation steps is the same;
(7) use respectively the physiological saline of 10mL resuspended, viable bacteria is mixed according to the ratio of table one with dead bacterium;
(8) the bacterium liquid that adds 140 μ L to mix in microwell plate, add the 5mM anthocyanidin dyestuff of 40 μ L and the 100 μ g/mL PI dyestuffs of 20 μ L under dark room conditions, be to excite under 488nm at exciting light, detect under the detection light of 522nm and 622nm, the fluorescence intensity that records under 522nm divided by the fluorescence intensity that records under 622nm, is obtained the ratio of RFU.Then with the ratio of viable bacteria, RFU ratio is mapped, return and obtain regression equation and R
2Value, experimental result such as Fig. 5.Result shows the linear fine of regression equation, R
2=0.9804, after the dyeing cell in this kit was described, the fluorescence intensity that excites under relevant excitation wavelength can be corresponding well with the life or death state of cell.
(1) filtering membrane of 25mL acetobacter xylinum with 0.22 μ m filtered, resuspended with 2mL physiological saline;
(2) get the physiological saline that 1mL bacterium liquid is dissolved in 1mL, separately get 1mL bacterium liquid and fix with the 1mL5% glutaraldehyde;
(3) stirred once in every 15 minutes, placed 1 hour under room temperature;
(4) filtering membrane with 0.22 μ m filters, with the thalline physiological saline washed twice that is retained on filtering membrane.
(5) washed viable bacteria body and dead thalline are resuspended in respectively in the physiological saline of 10mL, according to the ratio of table two, viable bacteria are mixed with dead bacterium.
(6) the bacterium liquid that adds 140 μ L to mix in microwell plate, the 100 μ g/mL PI dyestuffs that add 5mM anthocyanidin dyestuff and 20 μ L under dark room conditions, be to excite under 488nm at exciting light, detect under the detection light of 522nm and 622nm, the fluorescence intensity that records under 522nm divided by the fluorescence intensity that records under 622nm, is obtained the ratio of RFU.Then with the ratio of viable bacteria, RFU ratio is mapped, return and obtain equation and R
2Value, experimental result such as Fig. 6.Result shows the linear fine of regression equation, R
2=0.977, after the dyeing cell in this kit was described, the fluorescence intensity that excites under relevant excitation wavelength can be corresponding well with the life or death state of cell.
Table two
| The ratio of viable bacteria and dead bacterium | The volume of viable bacteria (mL) | The volume (mL) of dead bacterium |
| 0:100 | 0 | 2 |
| 20:80 | 0.4 | 1.6 |
| 50:50 | 1 | 1 |
| 80:20 | 1.6 | 0.4 |
(1) filtering membrane of acetobacter xylinum with 0.22 μ m filtered, with the resuspended bacteria suspension of making of physiological saline.Add anthocyanidin dyestuff and PI dyestuff in bacteria suspension, take out a part and be used for the fluorescent microscope detection from same system, a part is used for fluorescence and reads the detection of plate device, figure is in Bacterial stain number in fluorescent microscope and the fluorescence intensity level comparison of reading to read in the plate device, obtain the corresponding relation (experimental result such as Fig. 7) of acetobacter xylinum viable count and fluorescence intensity, and the corresponding relation (see figure 8) of the fluorescence intensity of the dead bacterium number of acetobacter xylinum and PI dyeing acquisition.
Fig. 7 shows the dense coefficient R with fluorescence intensity RFU of the bacterium of anthocyanidin dyeing acetobacter xylinum
2=0.9379, illustrate linear fine.
Fig. 8 shows the coefficient R of dead bacteria concentration and the fluorescence intensity RFU of PI dyeing acetobacter xylinum
2=0.9723, illustrate that the fluorescence intensity and the linear relationship between dead bacteria concentration that generate after PI dyeing are also fine.
(2) anthocyanidin dyestuff and PI dyeing Sensitivity be bacterium dense be 10
5, highest detection is limited to bacterium dense 10
9
(3) there is error in the fluorescence detection of reading the plate device, and the registration of fluorescent microscope is in the situation that the bacterium amount is too much, and can there be error in count results, and also can there be error in the dilution of bacterium liquid, often can not be exactly according to order of magnitude stepwise dilution.
Claims (9)
1. the fast detecting biological cell fluorescent dye kit of state anyway, it is characterized in that: described kit comprises anthocyanidin dyestuff and propidium iodide dyestuff, the storage concentration of anthocyanidin dyestuff is 5-6mM, and the storage concentration of propidium iodide dyestuff is 100 μ g/mL.
2. a kind of fast detecting biological cell according to claim 1 fluorescent dye kit of state anyway, it is characterized in that: the dilution buffer liquid that described anthocyanidin dyestuff uses is 1 * TE buffer, and the thinning agent that the propidium iodide dyestuff uses is ultrapure water.
3. the fluorescent dye kit of a kind of fast detecting biological cell life or death state according to claim 1, is characterized in that: dilute 1000-10000 when described anthocyanidin dyestuff uses doubly.
4. a kind of fast detecting biological cell according to claim 1 fluorescent dye kit of state anyway, it is characterized in that: the work ultimate density when described anthocyanidin dyestuff uses is 5-6 μ M.
5. a kind of fast detecting biological cell according to claim 1 fluorescent dye kit of state anyway, it is characterized in that: the solvent of described anthocyanidin dyestuff is dimethyl sulfoxide (DMSO), the solvent of propidium iodide dyestuff is ultrapure water.
6. a kind of fast detecting biological cell according to claim 1 fluorescent dye kit of state anyway, it is characterized in that: the work ultimate density of described propidium iodide dyestuff is 10-20 μ g/mL.
7. fast detecting biological cell as claimed in claim 1 application of the fluorescent dye kit of state anyway, it is characterized in that: detecting step is as follows: anthocyanidin dyestuff and propidium iodide add kit in biological sample liquid in, utilize the fluorescence microscope counting or utilize porous fluorescent to read the plate device and detect under wavelength of transmitted light, reflecting the existing state of cells in biological samples according to the power of different colours fluorescent value.
8. a kind of fast detecting biological cell according to claim 7 application of the fluorescent dye kit of state anyway is characterized in that: the described concrete steps of fluorescence microscope counting of utilizing comprise:
(1) draw cell suspension in the centrifuge tube that is surrounded by masking foil;
(2) adding volume ratio is the anthocyanidin dyestuff of 1-2:1 and the mixed dye of propidium iodide, and the volume ratio of cell suspension and mixed dye is 7:3, fluctuates, and is placed in dark 10-30 minute; Perhaps add with cell suspension volume to fluctuate the anthocyanidin dyestuff of 4:1 than being, be placed in dark 10-30 minute, survey live body; Perhaps add with cell suspension volume to fluctuate the PI dyestuff of 9:1 than being, be placed in dark 10-30 minute, survey dead volume;
(3) will dye the biological sample liquid of look by the black filtering membrane filtration of 0.2 μ m, and make cell retention on the black filtering membrane, filtering membrane is transferred on microslide, central authorities drip without fluorescence essential oil, covered at film;
(4) slide is placed under fluorescent microscope, central authorities drip without fluoroscope oil at cover glass, with the oily sem observation of 100 times, detect under the suitableeest exciting light of anthocyanidin dyestuff and PI dyestuff respectively, in eyepiece, greeny cell is live body, and the cell that takes on a red color is dead volume.
9. a kind of fast detecting biological cell according to claim 7 application of the fluorescent dye kit of state anyway is characterized in that: describedly utilize porous fluorescent to read the concrete steps that the plate device detects under wavelength of transmitted light to comprise:
(1) will contain the nutrient culture media of cell through the filtering membrane filtration of 0.2 μ m, the cell of collecting is resuspended with physiological saline;
(2) add cell suspension in microwell plate, adding volume ratio in micropore is the anthocyanidin dyestuff of 1-2:1 and the mixed dye of propidium iodide, and the volume ratio of cell suspension and mixed dye is 7:3, fluctuates, and is placed in dark 10-30 minute; Perhaps add with cell suspension volume to fluctuate the anthocyanidin dyestuff of 4:1 than being, be placed in dark 10-30 minute, survey live body; Perhaps add with cell suspension volume to fluctuate the PI dyestuff of 9:1 than being, be placed in dark 10-30 minute, survey dead volume; In micropore, cumulative volume is no more than 300 μ L, dyeed 10-30 minute, be 460nm-500nm at exciting light, utilizing emitted light is to detect its fluorescence intensity level under the condition of 500nm-530nm, the power of fluorescence has reflected the quantity of live body in this cell suspension, be 460nm-530nm at exciting light, utilizing emitted light is to detect its fluorescence intensity level under the condition of 600nm-640nm, and the power of fluorescence has reflected the quantity of dead volume in this cell suspension.
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| CN113846145A (en) * | 2021-08-20 | 2021-12-28 | 李彦萍 | Fluorescent dye stabilizer, kit containing same and related application |
| CN113687071A (en) * | 2021-09-10 | 2021-11-23 | 佛山墨赛生物技术有限公司 | Salmonella typhimurium viable bacteria quantitative detection test strip, kit and detection method |
| CN114561441A (en) * | 2021-09-30 | 2022-05-31 | 北京威妙生物科技有限公司 | Method for detecting inhibition of antibacterial drugs on fungi |
| CN115406873A (en) * | 2022-08-25 | 2022-11-29 | 云南农业大学 | Method for quantitatively detecting microbial death and activity by utilizing micro-fluidic chip |
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| CN116693891A (en) * | 2023-07-07 | 2023-09-05 | 重庆大学 | Anthocyanin-based antibacterial hydrogel preparation process |
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Inventor after: Hong Feng Inventor after: Zhang Shuo Inventor after: Du Qianwen Inventor after: LAN Shui Inventor after: Chen Lin Inventor after: Li Dengxin Inventor after: Zhang Wenkang Inventor after: Chen Qi Inventor after: Guo Shilin Inventor before: Hong Feng Inventor before: Zhang Shuo Inventor before: Du Qianwen Inventor before: Lan Shui Inventor before: Chen Lin Inventor before: Li Dengxin |
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