CN103175768B - Fast detecting biological cell is fluorescent staining kit and the application thereof of state anyway - Google Patents
Fast detecting biological cell is fluorescent staining kit and the application thereof of state anyway Download PDFInfo
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- CN103175768B CN103175768B CN201310060140.3A CN201310060140A CN103175768B CN 103175768 B CN103175768 B CN 103175768B CN 201310060140 A CN201310060140 A CN 201310060140A CN 103175768 B CN103175768 B CN 103175768B
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- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 claims abstract description 18
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- 239000000725 suspension Substances 0.000 claims description 8
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- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 2
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Abstract
The present invention relates to fluorescent staining kit and the application thereof of state anyway of a kind of fast detecting biological cell, kit comprises anthocyanidin dyestuff and propidium iodide dyestuff. Detecting step is as follows: in cell suspension, add anthocyanidin dyestuff and propidium iodide in kit, utilize fluorescence microscope counting or utilize porous fluorescent to read 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 present invention can observe or detect the living cells of various cells and the quantity of dead cell in sample fast in real time, intuitively easy, interference and the long shortcoming of cultivation cycle of other material are got rid of, be not only applicable to zooblast, and be applicable to most gramnegative bacterium and gram-positive bacterium.
Description
Technical field
The invention belongs to cell count field, particularly a kind of fast detecting biological cell fluorescent staining kit of state anywayAnd application.
Background technology
The method of measuring at present viable count has light microscope reading counting method, dull and stereotyped coating to cultivate colony counting method, photoelectricity ratioTurbid method, maximum MPN method, and membrane filter method etc.
Ascites method: the suspension of a small amount of testing sample is placed in to a kind of carrying of definite area and volume that have especiallyOn slide, (claim again meter bacterium device), under microscope, directly observe counting. The method is easy, quick, directly perceived, can for yeast,The counting of the suspensions such as bacterium, mycotic spore and zooblast. Shortcoming is accurately to identify the life or death state of cell, measuredResult is the summation of dead cell and living cells normally.
The method of plate culture count: through suitably dilution, microbe colony or zooblast wherein are fully dispersed into testing sampleIndividual cells state, gets a certain amount of dilution sample liquid and is inoculated on plating medium, through cultivating, numerous by each unicellular growthGrow and form macroscopic bacterium colony or population of cells, single bacterium colony should represent one in raw sample unicellular. This sideMethod is widely used in biological products inspection (as active bacteria formulation), and food, beverage and water (comprising water source) etc. containing bacterium indexOr the detection of pollution level. Shortcoming is, because testing sample is often difficult for being dispersed into completely individual cells, so after cultivating shapeThe single bacterium colony becoming may be from the 2-3 in sample or more cells, or due to the growth cycle difference of thalline, sameIn kind culture medium, due to adaptive difference, some thalline need can grow bacterium colony in 3 days, and some thalline needed for 1 week veryTo the longer time, therefore the result of plate count is often on the low side, conventionally uses CFU (colony-formingUnits, cfu) and do not represent the viable bacteria content of sample with absolute clump count.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of fast detecting biological cell anyway state fluorescent staining kit andIts application, the living cells of various cells and the quantity of dead cell in sample can be observed or detect to this kit fast in real time,Intuitively easy, get rid of the interference of other material and the shortcoming of cultivation cycle length, be not only applicable to zooblast, and be suitable forIn most gramnegative bacterium and gram-positive bacterium.
A kind of fast detecting biological cell of the present invention fluorescent staining kit of state anyway, described kit comprises cyanine uniformly dyeingMaterial and propidium iodide dyestuff, the storage concentration of anthocyanidin dyestuff is 5-6mM, the storage concentration of propidium iodide dyestuff is 100μg/mL。
The dilution buffer liquid that described anthocyanidin dyestuff uses is that 1 × TEbuffer[is containing 1mMEDTA(ethylenediamine tetra-acetic acid)10mMTris-HCl(trishydroxymethylaminomethane-HCl) buffer solution, pH8.0]. This dyestuff can be penetrated under all statesCell, it tends in conjunction with double-stranded DNA in cell, less for the combination of single stranded DNA and RNA, to the core of cellAcid is dyeed, and makes cell send green fluorescence.
Described anthocyanidin dyestuff dilutes 1000-10000 doubly while use.
Described anthocyanidin dye solvent is dimethyl sulfoxide (DMSO), and (working concentration refers to cell and dyes work ultimate density when useActual concentrations when look) be 5-6 μ M.
Anthocyanidin dyestuff, its structural formula is:
The solvent of described propidium iodide dyestuff and diluent are ultra-pure water. This dyestuff can only be specifically by the cell membrane of dead cell,Be attached to DNA or RNA upper, make 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 ultra-pure water.
A kind of fast detecting biological cell of the present invention application of the fluorescent staining kit of state anyway, detecting step is as follows:In cell suspension, add anthocyanidin dyestuff and propidium iodide in kit, utilize fluorescence microscope counting or utilize microporeFluorescence is read plate device and is detected under wavelength of transmitted light, according to cell in the power reflection biological sample liquid of different colours fluorescent valueExisting state.
The described concrete steps of utilizing fluorescence microscope counting comprise:
(1) draw cell suspension in being surrounded by the centrifuge tube of masking foil;
(2) proportioning taking volume ratio as 7:3(cell suspension and mixed dye) ratio add the anthocyanidin dyestuff of volume ratio as 1-2:1Mixed dye with propidium iodide, fluctuates, and is placed in dark 10-30 minute; Or adding volume ratio is that 4:1(cell is outstandingThe proportioning of liquid and dyestuff) anthocyanidin dyestuff, fluctuate, be placed in dark 10-30 minute, survey live body; Or add bodyLong-pending than being 9:1(cell suspension and the proportioning of dyestuff) PI dyestuff, fluctuate, be placed in dark 10-30 minute, survey extremelyBody;
(3) the cell sample liquid that dyed look is filtered by the black filter membrane (Whatman) of 0.2 μ m, make cell retention blackOn chromogenic filter film, filter membrane is transferred on slide, at film, central authorities drip without fluorescence essential oil, covered;
(4) slide is placed under fluorescence microscope, at cover glass, central authorities drip without fluorescope oil, with the oily sem observation of 100 times, pointNot at the suitableeest exciting light of anthocyanidin dyestuff and PI dyestuff, (the suitableeest exciting light of anthocyanidin dyestuff is not 488nm, PI dyestuff the suitableeestExciting light is 522nm) under detect, in eyepiece, greeny cell is live body, the cell taking on a red color is dead volume.
Describedly utilize porous fluorescent to read the concrete steps that plate device detects under wavelength of transmitted light to comprise:
(1) culture medium that contains cell is filtered through the filter membrane of 0.2 μ m, the cell of collecting is resuspended with physiological saline;
(2) in microwell plate, add cell suspension, the proportioning taking volume ratio as 7:3(cell suspension and mixed dye) ratio micro-In hole, adding volume ratio is the anthocyanidin dyestuff of 1-2:1 and the mixed dye of propidium iodide, fluctuates, and is placed in dark 10-30Minute; Or adding volume ratio is the proportioning of 4:1(cell suspension and dyestuff) anthocyanidin dyestuff, fluctuate, be placed in darkMiddle 10-30 minute, surveys live body; Or adding volume ratio is the proportioning of 9:1(cell suspension and dyestuff) PI dyestuff, upper and lowerShake, is placed in dark 10-30 minute, surveys dead volume; In micropore, cumulative volume is no more than 300 μ L, dyeing 10-30 minute,Exciting light is 460nm-500nm, detects its fluorescence intensity level, the power of fluorescence under the condition that utilizing emitted light is 500nm-530nmHaving reflected the quantity of live body in this cell suspension, is 460nm-530nm at exciting light, the bar that utilizing emitted light is 600nm-640nmUnder part, detect its fluorescence intensity level, 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, according to cell in incubationBioactive difference, cell can send different fluorescence, has reflected different bioactive thin according to the difference of fluorescence intensityThe ratio of born of the same parents in culture medium. This technology be established as further investigation various biological cells existing state provide universal method andTechnology. Particularly the biological characteristics to the less microbial cell of acetobacter xylinum equidimension and the building-up process of bacteria cellulose provideA kind of simple and feasible method.
Detect principle: when add two kinds of dyestuffs in cell suspension simultaneously, anthocyanidin dyestuff can enter all cells, makes cellSend green fluorescence. When cell is in existing state, cell membrane only allows anthocyanidin dyestuff to enter cell, and PI dyestuff can not enterEnter cell, the cell of existing state only shows green fluorescence. When cell is in moribund condition, the permeability of cell membrane increases,PI can partly enter into cell, is embedded into the nucleic acid moiety that dyes the cell that has green fluorescence, overrides part green fluorescence,Make cell send the red fluorescence of part, what detect under the microscope is that cell sends fluorescent orange. When cell is in dead shapeState, PI can pass through cell membrane in large quantities, is embedded on the nucleic acid of cell, overrides the green fluorescence of anthocyanidin, deadCell can entirety show red fluorescence.
Beneficial effect
The present invention can observe or detect the living cells of various cells and the quantity of dead cell in sample fast in real time, intuitively letterJust. And anthocyanidin dyestuff derives from nature, and toxicity is low, easy to use. Because dyestuff can only be combined in two strands in specific mannerOn nucleic acid, send fluorescence, got rid of interference and the long shortcoming of cultivation cycle of other material. This colouring method is not only applicable to animalCell, and be applicable to most gramnegative bacterium and gram-positive bacterium.
Brief description of the drawings
Fig. 1 is the acetobacter xylinum that detects same system by fluorescence microscopy microscopy and painting flat band method, the contrast of testing result;
Fig. 2 is the optimum dye concentration of anthocyanidin dyeing thalline, the blank fluorescence-dyestuff of RFU=dyeing thalline total fluorescence intensity-thallineBlank fluorescence.
Fig. 3 is the optimum dye concentration of PI dyeing thalline, the blank fluorescence-dyestuff blank of RFU=dyeing thalline total fluorescence intensity-thallineFluorescence;
Fig. 4 detects with kit the staphylococcus aureus suspension mixing in the ratio of different dead bacterium and viable bacteria, and its RFU accumulative total is strongThe ratio of degree changes, the fluorescence of the blank fluorescence-dyestuff blank of RFU=dyeing thalline total fluorescence intensity-thalline;
Fig. 5 detects with kit the Escherichia coli suspension mixing in the ratio of different dead bacterium and viable bacteria, the ratio of its RFU accumulative total intensityValue changes, the fluorescence of the blank fluorescence-dyestuff blank of RFU=dyeing thalline total fluorescence intensity-thalline;
Fig. 6 detects with kit the acetobacter xylinum suspension mixing in the ratio of different dead bacterium and viable bacteria, the ratio of its RFU accumulative total intensityValue changes, the fluorescence of the blank fluorescence-dyestuff blank of RFU=dyeing thalline total fluorescence intensity-thalline;
Fig. 7 is the calibration curve that detects acetobacter xylinum viable bacteria with kit;
Fig. 8 is the calibration curve that detects the dead bacterium of acetobacter xylinum with kit.
Detailed description of the invention
Below in conjunction with specific embodiment, further set forth the present invention. Should be understood that these embodiment are not only for the present invention is describedBe used for limiting the scope of the invention. In addition should be understood that those skilled in the art can after having read the content of the present invention's instructionSo that the present invention is made various changes or modifications, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
(1) anthocyanidin for dyestuff methyl-sulfoxide (DMSO) be dissolved to 5-6mM concentration, this for preparation anthocyanidin dyestuff store denseSolution.
(2) PI dyestuff is dissolved to 100 μ g/mL with ultra-pure water, and lucifuge stores
(3) the acetobacter xylinum bacterium liquid of drawing 1mL is in being surrounded by the 2mL centrifuge tube of masking foil.
(4) add the anthocyanidin dyestuff of 300 μ L after 1000 times of buffer solution dilutions and the 100 μ g/mLPI dyestuffs of 200 μ L, onLower shake repeatedly, is placed in dark 10-30 minute, and dilution buffer liquid is that 1 × TEbuffer is (containing 1mMEDTA's10mMTris-HCl buffer solution, pH8.0).
(5) the bacterium liquid that dyed look is filtered by the black filter membrane (Whatman) of 0.2 μ m, thalline is trapped in to black and filtersOn film, filter membrane is transferred on slide, at film, central authorities drip without fluorescence essential oil, covered.
(6) slide is placed in to 100 times of thing Microscopic observations of fluorescence microscope, because different dyestuffs has the suitableeest different wave band that excites,Therefore need under the suitableeest exciting light of anthocyanidin dyestuff and PI dyestuff, detect, in eyepiece, greeny thalline is aliveThalline, the thalline taking on a red color is dead thalline. From same system, take out the acetobacter xylinum of 1mL, diluted 10 times,100 times, 1000 times, 10000 times, the thalline having diluted is coated with respectively to flat board, bacterium colony to be grown, will calculate gainedColony counts and the experimental result of fluorescence microscope contrast (experimental result is as Fig. 1). Experimental result shows, makesThe thalline quantity result ratio testing result of the method for plate culture count gained measured with fluorescence microscope on average exceeds1-2 the order of magnitude, this with in document, report identical, owing to using testing sample when colony counting method to be often difficult for completelyBe dispersed into individual cells, so the single bacterium colony forming after cultivating may be from the 2-3 in sample or more cells,Therefore the result of plate count is often on the low side. But the result linear relationship that itself and fluorescence microscopy detect is good,R2=0.9872。
Embodiment 2
(1) anthocyanidin dyestuff is stored to 1000 times to 20000 times of concentrated solution dilutions, with volume is identical but the dyestuff that extension rate is differentBy the thalline that dyes of the step 1-3 in embodiment 1, use MolecularDevicesFlexstationII type calcium current fluorescenceInstrument detects, and finds that the fluorescence intensity of thalline declines with the increase of the extension rate of dyestuff, in the time diluting 5000 times,Its Color is better and dye dosage is relatively low, both economical. If extension rate increases, detected fluorescenceValue approaches limits of error in-scope, and the error of detected value increases (experimental result is as Fig. 2).
(2) concentration of dyestuff PI is mixed with from 1 μ g/mL to 100 μ g/mL, with the identical but dyestuff that extension rate is different of volumeDyeing thalline, its Color best (experimental result is as Fig. 3) when discovery concentration is 10 μ g/mL.
Embodiment 3
(1) staphylococcus aureus being cultivated to the turbidity OD value that bacterium is dense is 0.7, gets 25mL bacterium liquid in 12000r/min conditionLower 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 20mL70% isopropyl alcohol;
(4) within every 15 minutes, stir once incubated at room 1 hour;
(5) centrifugal 10-15 minute under 12000r/min condition;
(6) be resuspended in respectively the physiological saline of 20mL, then centrifuge washing, operating procedure 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 (mL) of viable bacteria | 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 adds the 5mM anthocyanidin dyestuff of 40 μ L under dark room conditionsWith the 100 μ g/mLPI dyestuffs of 20 μ L, be to excite under 488nm at exciting light, respectively in 522nm and 622Under the detection light of nm, detect, by the fluorescence intensity recording under 522nm divided by the fluorescence intensity recording under 622nm,Obtain the ratio of RFU. Then with the ratio of viable bacteria, RFU ratio is mapped, return and obtain regression equation and R2Value,Experimental result is as Fig. 4. Result shows that the linearity of regression equation is fine, R2=0.9599, the dyestuff in this kit is describedAfter staining cell, the fluorescence intensity exciting under relevant excitation wavelength can be corresponding well with the life or death state of cell.
Embodiment 4
(1) Escherichia coli being cultivated to the turbidity OD value that bacterium is dense is 1.2, gets 25mL bacterium liquid centrifugal under 12000r/min condition10-15 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 20mL70% isopropyl alcohol;
(4) within every 15 minutes, stir once, room temperature insulation is hatched 1 hour;
(5) centrifugal 10-15 minute under 12000r/min condition;
(6) be resuspended in respectively the physiological saline of 20mL, then centrifuge washing, operating procedure 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 adds the 5mM anthocyanidin dyestuff of 40 μ L under dark room conditionsWith the 100 μ g/mLPI dyestuffs of 20 μ L, be to excite under 488nm at exciting light, at 522nm and 622nmDetection light under detect, by the fluorescence intensity recording under 522nm divided by the fluorescence intensity recording under 622nm,Obtain the ratio of RFU. Then with the ratio of viable bacteria, RFU ratio is mapped, return and obtain regression equation and R2Value,Experimental result is as Fig. 5. Result shows that the linearity of regression equation is fine, R2=0.9804, the dyestuff in this kit is describedAfter staining cell, the fluorescence intensity exciting under relevant excitation wavelength can be corresponding well with the life or death state of cell.
Embodiment 5
(1) 25mL acetobacter xylinum is filtered with the filter membrane of 0.22 μ m, resuspended with 2mL physiological saline;
(2) get 1mL bacterium liquid and be dissolved in the physiological saline of 1mL, separately get 1mL bacterium liquid 1mL5% glutaraldehyde and fix;
(3) within every 15 minutes, stir once, under room temperature, place 1 hour;
(4) filter with the filter membrane of 0.22 μ m, by the thalline physiological saline washed twice being retained on filter 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 by viable bacteriaMix with dead bacterium.
(6) the bacterium liquid that adds 140 μ L to mix in microwell plate adds 5mM anthocyanidin dyestuff and 20 μ L under dark room conditions100 μ g/mLPI dyestuffs, be to excite under 488nm at exciting light, in the detection of 522nm and 622nmUnder light, detect, the fluorescence intensity recording under 522nm, divided by the fluorescence intensity recording under 622nm, is obtainedThe ratio of RFU. Then with the ratio of viable bacteria, RFU ratio is mapped, return and obtain equation and R2Value, experimental resultAs Fig. 6. Result shows that the linearity of regression equation is fine, R2=0.977, the dyeing cell in this kit is describedAfter, the fluorescence intensity exciting 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 (mL) of viable bacteria | 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 |
Embodiment 6
(1) acetobacter xylinum is filtered with the filter membrane of 0.22 μ m, with the resuspended bacteria suspension of making of physiological saline. In bacteria suspension, addAnthocyanidin dyestuff and PI dyestuff take out a part and detect for fluorescence microscope from same system, and a part is for glimmeringLight is read plate device and is detected, and the Bacterial stain number in fluorescence microscope and the fluorescence intensity level comparison of reading to read in plate device are doneScheme, obtain the corresponding relation (experimental result is as Fig. 7) of acetobacter xylinum viable count and fluorescence intensity, and acetobacter xylinum is deadThe corresponding relation (see figure 8) of the fluorescence intensity that bacterium number and PI dyeing obtain.
Fig. 7 shows the dense coefficient R with fluorescence intensity RFU of the bacterium of anthocyanidin dyeing acetobacter xylinum2=0.9379，Illustrate linear fine.
Fig. 8 shows the coefficient R of dead bacteria concentration and the fluorescence intensity RFU of PI dyeing acetobacter xylinum2=0.9723，Illustrate that the linear relationship between fluorescence intensity and the dead bacteria concentration generating after PI dyeing is also fine.
(2) Sensitivity of anthocyanidin dyestuff and PI dyeing be bacterium dense be 105, highest detection is limited to bacterium dense 109。
(3) there is error in the detection that fluorescence is read plate device, the registration of fluorescence microscope is in the situation that bacterium amount is too much, and count results can be depositedIn error, also can there is error in the dilution of bacterium liquid, often can not be exactly according to order of magnitude stepwise dilution.
Claims (9)
1. a fast detecting biological cell fluorescent staining kit for state anyway, is characterized in that: described kit comprises flowerBlue or green uniformly dyeing material and propidium iodide dyestuff, the storage concentration of anthocyanidin dyestuff is 5-6mM, the storage concentration of propidium iodide dyestuff is100 μ g/mL; Anthocyanidin dye structure formula is:
2. a kind of fast detecting biological cell according to claim 1 fluorescent staining kit of state anyway, is characterized in that:The dilution buffer liquid that described anthocyanidin dyestuff uses is 1 × TEbuffer, and the diluent that propidium iodide dyestuff uses is ultrapureWater.
3. a kind of fast detecting biological cell according to claim 1 fluorescent staining kit of state anyway, is characterized in that:Described anthocyanidin dyestuff dilutes 1000-10000 doubly while use.
4. a kind of fast detecting biological cell according to claim 1 fluorescent staining kit of state anyway, is characterized in that: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 staining kit of state anyway, is characterized in that:The solvent of described anthocyanidin dyestuff is dimethyl sulfoxide (DMSO), and the solvent of propidium iodide dyestuff is ultra-pure water.
6. a kind of fast detecting biological cell according to claim 1 fluorescent staining kit of state anyway, is characterized in that:The work ultimate density of described propidium iodide dyestuff is 10-20 μ g/mL.
7. a fast detecting biological cell as claimed in claim 1 application for the fluorescent staining kit of state anyway, its feature existsIn: detecting step is as follows: in biological sample liquid, add anthocyanidin dyestuff and propidium iodide in kit, utilize fluorescence microscopySem observation is counted or is utilized porous fluorescent to read plate device and detect under wavelength of transmitted light, according to the power of different colours fluorescent valueThe existing state of reflection cells in biological samples.
8. a kind of fast detecting biological cell according to claim 7 application of the fluorescent staining kit of state anyway, its featureBe: the described concrete steps of utilizing fluorescence microscope counting comprise:
(1) draw cell suspension in being surrounded by the centrifuge tube of masking foil;
(2) adding volume ratio is the anthocyanidin dyestuff of 1-2:1 and the mixed dye of propidium iodide, the volume of cell suspension and mixed dyeThan for 7:3, fluctuate, be placed in dark 10-30 minute; Or add with cell suspension volume than the cyanine uniformly dyeing for 4:1Material, fluctuates, and is placed in dark 10-30 minute, surveys live body; Or add with cell suspension volume than dying for the PI of 9:1Material, fluctuates, and is placed in dark 10-30 minute, surveys dead volume;
(3) the biological sample liquid that dyed look is filtered by the black filter membrane of 0.2 μ m, makes cell retention on black filter membrane,Filter membrane is transferred on slide, and at film, central authorities drip without fluorescence essential oil, covered;
(4) slide is placed under fluorescence microscope, at cover glass, central authorities drip without fluorescope oil, with the oily sem observation of 100 times, pointUnder the suitableeest exciting light of anthocyanidin dyestuff and PI dyestuff, do not detect, in eyepiece, greeny cell is live body, takes on a red colorCell is dead volume.
9. a kind of fast detecting biological cell according to claim 7 application of the fluorescent staining kit of state anyway, its featureBe: describedly utilize porous fluorescent to read the concrete steps that plate device detects under wavelength of transmitted light to comprise:
(1) culture medium that contains cell is filtered through the filter membrane of 0.2 μ m, the cell of collecting is resuspended with physiological saline;
(2) in microwell plate, add cell suspension, in micropore, adding volume ratio is the anthocyanidin dyestuff of 1-2:1 and mixing of propidium iodideClose dyestuff, the volume ratio of cell suspension and mixed dye is 7:3, fluctuates, and is placed in dark 10-30 minute; Or add, fluctuate than the anthocyanidin dyestuff for 4:1 with cell suspension volume, be placed in dark 10-30 minute, survey live body; Or addEnter with cell suspension volume and compare the PI dyestuff for 9:1, fluctuate, be placed in dark 10-30 minute, survey dead volume; In microporeCumulative volume is no more than 300 μ L, and dyeing 10-30 minute, is 460nm-500nm at exciting light, and utilizing emitted light is 500nm-530nmCondition under detect its fluorescence intensity level, the power of fluorescence has reflected the quantity of live body in this cell suspension, is 460 at exciting lightNm-530nm, detects its fluorescence intensity level under the condition that utilizing emitted light is 600nm-640nm, the power of fluorescence has reflected this cellThe quantity of dead volume in suspension.
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| CN104977237B (en) * | 2015-07-01 | 2018-02-23 | 北京理工大学 | CO in a kind of in situ detection single living cell inner cell organ2The method of generating rate |
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| CN108254237A (en) * | 2017-12-12 | 2018-07-06 | 浙江海洋大学 | A kind of live cell fluorescent colouring method |
| CN108254238B (en) * | 2017-12-29 | 2021-06-22 | 乔治洛德方法研究和开发液化空气有限公司 | Dyeing method of filamentous microorganisms and application thereof |
| CN109496230A (en) * | 2018-02-09 | 2019-03-19 | 柴崎株式会社 | Bacteria detecting apparatus and method of detecting bacterium |
| EP3830547B1 (en) * | 2018-08-01 | 2023-11-29 | Sartorius BioAnalytical Instruments, Inc. | Methods, kits and stain compositions for flow cytometry evaluation of unassociated virus-size particles using multiple fluorogenic dyes |
| CN113846145A (en) * | 2021-08-20 | 2021-12-28 | 李彦萍 | Fluorescent dye stabilizer, kit containing same and related application |
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| 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 |
| CN115406873B (en) * | 2022-08-25 | 2024-05-03 | 云南农业大学 | Method for quantitatively detecting dead or alive microorganisms by utilizing microfluidic chip |
| CN116693891A (en) * | 2023-07-07 | 2023-09-05 | 重庆大学 | Anthocyanin-based antibacterial hydrogel preparation process |
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