CN101344476A - Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state - Google Patents
Fluorescence microscope viewing and counting method for bacteria in viable but non-culturable state Download PDFInfo
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- CN101344476A CN101344476A CNA2008100511069A CN200810051106A CN101344476A CN 101344476 A CN101344476 A CN 101344476A CN A2008100511069 A CNA2008100511069 A CN A2008100511069A CN 200810051106 A CN200810051106 A CN 200810051106A CN 101344476 A CN101344476 A CN 101344476A
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- But 1, the non-cultivation conditions fluorescence microscope and the method for counting of bacteria in viable, it is characterized in that: concrete steps are:(1), the VBNC bacterium is observed the pre-treatment of sampleBacterial concentration before the VBNC bacteria-induction is 10 5-10 6Individual/mL, getting and can cultivating the bacterium number is 1 * 10 6The VBNC bacterium liquid 1mL of individual/mL is to aseptic centrifuge tube, and the centrifugal 2min of 8000r/min abandons supernatant; Fully wash bacterial sediment 2 times with sterile saline then, each all 8000r/min, centrifugal 2min abandons supernatant; During the washing thalline, should eliminate inoculum as much as possible, in order to avoid nutrient solution produces autoluminescence and disturbs observations; The precipitation that needs at last washing is finished places room temperature, is used for observing immediately; Perhaps thalline is stored in 4 ℃, standby in 2 days;(2), the miillpore filter hydrophobicity is checked and is handledAt first the miillpore filter with diameter 25mm aperture 0.2um immerses 2min in the sterile saline fully, miillpore filter is taken out place plate again, and light faces up, examine microporous membrane surface, if microporous membrane surface has the part area not have washmarking, represent that then this filter membrane is hydrophobic, should change again; Perhaps in the Tween-80 with hydrophobic filter membrane immersion 0.5%, soak 1min, then miillpore filter is changed in the interchangeable film needle-based filter, in needle tubing, inject sterile saline 2mL, in the lump Tween-80 and salt solution are leached; Filter membrane after the handling with Tween-80 after the checking all light faces up and is tiled in the plate, 37 ℃ of oven dry, and room temperature is deposited standby;(3), miillpore filter dyeingThe above-mentioned miillpore filter light that disposes faced up to be tiled in the plate, can not be overlapping between film and the film; Then draw the black fountain pen ink with suction pipe, drip at the filter membrane circle centre position, allow ink contaminate diffusion voluntarily to all around from center membrane, after treating that ink all soaks into film, plate is moved in the xeothermic case, and 60 ℃ of roasting film 20min need to pick up filter membrane gently with tweezers therebetween and stir 2 times, cooling then, room temperature is deposited standby;(4), VBNC bacteria sample fluorescent dyeSYTO-9 and PI dye ligand are made working concentration, deposit standby for-20 ℃; VBNC bacterial sediment in step poly-1 is dissolved in 50 μ, 1 sterile saline, and each 25 μ 1 of the SYTO-9 of adherent adding working concentration and PI are to centrifuge tube, and moment is centrifugal, pressure-vaccum mixing, room temperature lucifuge dyeing 15min;(5), the preparation of VBNC bacterial fluorescence sampleTo dye the microslide center that black miillpore filter places no autofluorescence through ink, draw the VBNC bacterium liquid that 10 μ l dyeing finishes, drop on the miillpore filter center of circle, when treating that bacterium liquid is just blotted by filter membrane, covered thereon, and, eliminate air blanketing with the hand compressing tablet of exerting oneself; The last oil that drips no autofluorescence on cover glass is observed in the darkroom and is counted with OLYMPUS BX-51 epifluorescence microscope; The excitation wavelength 480nm of fluorescent microscope, wavelength of transmitted light are 610nm; Mark sheet to be checked can place in the warm box, at 4 ℃ of low-temp storage 1d;(6), VBNC bacterial fluorescence microscopic countingAt least at random select 10 different visuals field during counting, and the bacterial population in each visual field is not less than 30-50, then averages; When observing counting, finish within 1min in each visual field; When seeing the viable bacteria that is that presents green fluorescence, and present the dead bacterium of being of red fluorescence; The concrete counting of VBNC bacterial population can be with reference to following formula:Wherein E be total number of bacteria in the sample (individual/mL); X is the mean value (individual) of each counting visual field total number of bacteria; S 1Be filter membrane area (mm); S2 is the visual field area (mm) of microscope oil mirror; V is the volume (mL) of VBNC bacterium liquid to be checked.
- But 2, the non-cultivation conditions fluorescence microscope and the method for counting of the described bacteria in viable of claim 1, the fixed form that it is characterized in that described fluorescent microscope specimen preparation is the physical fixation mode, promptly utilizes suction-operated and the Manual pressure effect of moistening filter membrane to microslide and cover glass.
- But 3, the non-cultivation conditions fluorescence microscope and the method for counting of the described bacteria in viable of claim 1 is characterized in that described black fountain pen ink is " heroic board " black fountain pen ink.
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102618464A (en) * | 2012-03-26 | 2012-08-01 | 浙江师范大学 | Resuscitation medium capable of promoting growth of VBNC (viable but non-culture) bacteria and improving separation abundance, and preparation method and application thereof |
CN103063633A (en) * | 2012-12-25 | 2013-04-24 | 南昌大学 | System capable of automatically detecting bacteria in water |
CN104662425A (en) * | 2012-05-02 | 2015-05-27 | 查尔斯河实验室公司 | Viability staining method |
CN104677809A (en) * | 2013-11-29 | 2015-06-03 | 中国人民解放军第二军医大学 | Detection kit for activity of cryptococcus and detection method |
CN106153591A (en) * | 2016-08-15 | 2016-11-23 | 吴江华衍水务有限公司 | A kind of measure the method for bacterial density in water sample |
CN106636299A (en) * | 2016-12-27 | 2017-05-10 | 扬州大学 | Bacterial pollution detection method for edible fungus liquid strain |
CN106834197A (en) * | 2017-04-17 | 2017-06-13 | 内蒙古农业大学 | A kind of abductive approach of lactobacillus VBNC states |
US9709500B2 (en) | 2012-05-02 | 2017-07-18 | Charles River Laboratories, Inc. | Optical method for detecting viable microorganisms in a cell sample |
CN108254238A (en) * | 2017-12-29 | 2018-07-06 | 乔治洛德方法研究和开发液化空气有限公司 | A kind of colouring method of filamentous microorganism and application thereof |
CN108982179A (en) * | 2018-07-25 | 2018-12-11 | 湖南省天骑医学新技术股份有限公司 | A kind of method and apparatus of fast transfer miillpore filter |
CN111088314A (en) * | 2019-12-31 | 2020-05-01 | 湖南景翌湘台环保高新技术开发有限公司 | Improved viable bacteria counting method |
CN115094017A (en) * | 2022-06-30 | 2022-09-23 | 江南大学 | Method for inducing yeast to enter VBNC state |
-
2008
- 2008-08-20 CN CN2008100511069A patent/CN101344476B/en not_active Expired - Fee Related
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102618464A (en) * | 2012-03-26 | 2012-08-01 | 浙江师范大学 | Resuscitation medium capable of promoting growth of VBNC (viable but non-culture) bacteria and improving separation abundance, and preparation method and application thereof |
CN104662425A (en) * | 2012-05-02 | 2015-05-27 | 查尔斯河实验室公司 | Viability staining method |
US9709500B2 (en) | 2012-05-02 | 2017-07-18 | Charles River Laboratories, Inc. | Optical method for detecting viable microorganisms in a cell sample |
CN103063633A (en) * | 2012-12-25 | 2013-04-24 | 南昌大学 | System capable of automatically detecting bacteria in water |
CN104677809A (en) * | 2013-11-29 | 2015-06-03 | 中国人民解放军第二军医大学 | Detection kit for activity of cryptococcus and detection method |
CN106153591A (en) * | 2016-08-15 | 2016-11-23 | 吴江华衍水务有限公司 | A kind of measure the method for bacterial density in water sample |
CN106636299B (en) * | 2016-12-27 | 2021-04-27 | 扬州大学 | Method for detecting bacterial contamination of edible fungus liquid strain |
CN106636299A (en) * | 2016-12-27 | 2017-05-10 | 扬州大学 | Bacterial pollution detection method for edible fungus liquid strain |
CN106834197A (en) * | 2017-04-17 | 2017-06-13 | 内蒙古农业大学 | A kind of abductive approach of lactobacillus VBNC states |
CN108254238A (en) * | 2017-12-29 | 2018-07-06 | 乔治洛德方法研究和开发液化空气有限公司 | A kind of colouring method of filamentous microorganism and application thereof |
CN108982179A (en) * | 2018-07-25 | 2018-12-11 | 湖南省天骑医学新技术股份有限公司 | A kind of method and apparatus of fast transfer miillpore filter |
CN108982179B (en) * | 2018-07-25 | 2023-11-07 | 湖南省天骑医学新技术股份有限公司 | Method and equipment for rapidly transferring microporous filter membrane |
CN111088314A (en) * | 2019-12-31 | 2020-05-01 | 湖南景翌湘台环保高新技术开发有限公司 | Improved viable bacteria counting method |
CN115094017A (en) * | 2022-06-30 | 2022-09-23 | 江南大学 | Method for inducing yeast to enter VBNC state |
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