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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Abstract
The invention relates to an observing and counting method of germs in a viable but non-culture state with a fluorescent microscope, which is characterized in that the method comprises the following concrete steps: (1) an observing sample of the VBNC germs is preprocessed; (2) the hydrophobic nature of a microporous filtering film is examined and processed; (3) the microporous filtering film is dyed; (4) the sample of the VBNC germs is fluorescently dyed; (5) a fluorescent sample of the VBNC germs is prepared; and (6) the counting of the VBNC germs is implemented with the fluorescent microscope. The observing and counting method of the germs in the viable but non-culture state with the fluorescent microscope simplifies operation processes, overcomes the disadvantages of narrow application range, difficult material preparation and inaccurate observing result in the prior art, solves the technical difficulty in the research field of the VBNC germs, and raises the practical operation level of practitioners.
Description
Technical field:
But the present invention relates to a kind of non-cultivation conditions fluorescence microscope and method of counting of bacteria in viable, belong to biological technical field.
Background technology:
But fluorescence microscope is non-cultivation conditions (Viable butNon-culturable, VBNC) the most basic technological means used of research institute of bacteria in viable with counting.At present, being used for the VBNC bacterial fluorescence both at home and abroad observes prior art with counting and mainly stems from one piece of article that is entitled as " utilizing nuclear pore filter film to carry out the bacterial fluorescence counting " that people such as Hobbie delivered on " using and environmental microbiology " in 1977.One spotlight of this technology is utilized polycarbonate nuclear pore filter film instead of cellulose miillpore filter exactly, is used for the mensuration of bacterium total bacteria count.Also utilize acridine orange and nalidixic acid that bacterium nuclear is dyed simultaneously, in order to " extremely " and " work " of distinguishing bacterium.In addition, the related material of this technology comprise also that Iraq is black, formaldehyde, interchangeable film needle-based filter etc.Its concrete technical measures are as follows.At first, the adding final concentration is 0.001% nalidixic acid and 0.025% yeast extract in the dilution of debita spissitudo, cultivates 16h-24h for 37 ℃, adds 2% formaldehyde fixed (final concentration) subsequently, then adds 0.1% acridine orange solution-dyed 3min again.Bacterium in the dilution is filled on the nuclear pore filter film of diameter 25mm aperture 0.2um.For eliminating the fluorescence of filter membrane itself, filter membrane is earlier with the black 2% acetum dyeing 24h of 0.2% Iraq.At last filter membrane is placed on the cedar oil that drips no autofluorescence on the microslide,, on cover glass, drips the cedar oil of a no autofluorescence again, under epifluorescence microscope, observe counting at cover glass of oily loam cake.By said method, total number of bacteria can be come out by counting objectively, and viable bacteria also can be made a distinction significantly with bacterium extremely.Therefore, this technology is widely used in VBNC bacterial studies field.But along with the continuous expansion of VBNC research range, the bacterial species with VBNC character is also more and more, has been not limited to 30 various bacteria of 16 kinds finding originally.Not only there is the VBNC state in the Gram-negative pathogen, and probio also exists Gram-positive; Not only bacterium exists in the water, and bacterium also exists in soil, air, the animal and plant body.So this technology in this area research, also shown the storage how unfavorable aspect, exist certain limitation.It is as follows to conclude its technical disadvantages: (1) complex operation, expend time in, and be unfavorable for the fast detecting of VBNC bacterium.Usually, when observing the VBNC bacterium, need with nalidixic acid bacteria growing inhibiting 16h-24h.(2) narrow application range only is suitable for Gram-negative bacteria, and is then inapplicable to Gram-positive.(3) can not truly reflect number of viable.Through acridine orange dyeing, the thalline that presents orange or fluorescent red-orange is difficult to effectively determine that viable bacteria still is dead bacterium.(4) the material therefor kind is many, and the inconvenience of drawing materials.For example, Iraq is black to be employed a kind of black dyes in printing and dyeing industry, but this type of material is seldom sold by each big biochemistry and biotech firm at home.
Summary of the invention:
But the object of the present invention is to provide a kind of non-cultivation conditions fluorescence microscope and method of counting of bacteria in viable, it substitutes acridine orange and nalidixic acid with SYTO-9 and PI, it is black to substitute Iraq with commercially available black fountain pen ink, set up a cover VBNC bacterial fluorescence microscopic examination and a method of counting voluntarily, simplified operating process, shortcoming such as the scope of application that has overcome prior art is narrow, the difficulty of drawing materials, observations are inaccurate, solved the technical barrier in the bacterium VBNC research field, improved practical operation level with the dealer.
But technical scheme of the present invention is achieved in that the non-cultivation conditions fluorescence microscope and the method for counting of bacteria in viable, and it is characterized in that: concrete steps are
1, the VBNC bacterium is observed the pre-treatment of sample
Bacterial 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 handled
At 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, and miillpore filter preferably directly uses hydrophilic filter membrane;
3, miillpore filter dyeing
The 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 an amount of " heroic board " 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 dye
SYTO-9 and PI dye ligand are made working concentration, deposit standby for-20 ℃; VBNC bacterial sediment in step poly-1 is dissolved in the 50 μ l sterile salines, and each 25 μ l 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 sample
To 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 counting
At 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.
Good effect of the present invention is that SYTO-9 and two kinds of nucleotide fluorescent dyes of PI of selecting for use are critical materials that the VBNC bacterial fluorescence is observed, wherein, SYTO-9 is that a kind of can the infiltration has the interior micromolecule of intact cell theca cell, be green fluorescence, PI then is in a kind of thalline that only can be seeped into the cell membrane breakage and the big molecule of the fluorescence that takes on a red color, and with SYTO-9 competition nucleic acid and to dye the site; Behind this reagent dyeing, send the viable bacteria that is of green fluorescence, and send the dead bacterium that is of red fluorescence.These two kinds of dyestuffs also all are suitable for Gram-negative and gram-positive bacteria, all are beneficial to the differentiation of living cells and dead cell; Thereby SYTO-9 and PI not only can shorten the VBNC cell observation phase, but also are widely used.It is convenient but also cheap to adopt the black fountain pen ink not only to draw materials; Employing does not only produce autoluminescence by the heroic board black fountain pen ink that Shanghai company limited of Ying Xiong Pen Factory produces, and can also remove the light of filter membrane; Not only can deepen visual field background depth, and can also strengthening membrane to the absorption affinity of slide, thereby this board ink is best to the Fluirescence observation effect of VBNC bacterium.Adopt the physical fixation mode, promptly utilize of suction-operated and the Manual pressure effect of moistening filter membrane to microslide and cover glass, this fixed form does not influence the existing state of bacterium, thereby when measuring total bacteria count, can in the lump viable count be measured out yet.
Embodiment:
Embodiment 1:
1, at first the VBNC bacterium is observed sample and carry out pre-treatment, the bacterial concentration before the VBNC bacteria-induction is 10
6Individual/mL, get and can cultivate bacterium several 1 * 10
6The VBNC bacterium liquid 1mL of individual/mL with 8000r/min centrifugal treating 2min, abandons supernatant to aseptic centrifuge tube; Fully wash thalline with sterile saline then and precipitate 2 times, each all 8000r/min, centrifugal 2min abandons supernatant; During the washing thalline, should eliminate inoculum, 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;
2, next carrying out the miillpore filter hydrophobicity checks and processing, at first the miillpore filter with diameter 25mm aperture 0.2um immerses 2min in the sterile saline fully, again miillpore filter is taken out and place plate, 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, and miillpore filter preferably directly uses hydrophilic filter membrane;
3, the miillpore filter light that disposes is faced up be tiled in the plate, can not be overlapping between film and the film; Then draw an amount of " heroic board " 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, the VBNC bacteria sample is carried out fluorescent dye, SYTO-9 and PI dye ligand are made working concentration, and deposit standby at-20 ℃; VBNC bacterial sediment in step poly-1 is dissolved in the 50 μ l sterile salines, and each 25 μ l 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 sample, to 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 the OLYMPUSBX-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 counting select 10 different visuals field at random, and the bacterial population in each visual field is 30, 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.
Embodiment 2:
1, at first the VBNC bacterium is observed sample and carry out pre-treatment, the bacterial concentration before the VBNC bacteria-induction is 10
5Individual/mL, get and can cultivate bacterium several 1 * 10
5The VBNC bacterium liquid 1mL of individual/mL with 8000r/min centrifugal treating 2min, abandons supernatant to aseptic centrifuge tube; Fully wash thalline with sterile saline then and precipitate 2 times, each all 8000r/min, centrifugal 2min abandons supernatant; Thalline is stored in 4 ℃, standby in 2 days;
2, next carrying out the miillpore filter hydrophobicity checks and processing, at first the miillpore filter with diameter 25mm aperture 0.2um immerses 2min in the sterile saline fully, again miillpore filter is taken out and place plate, 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, and miillpore filter preferably directly uses hydrophilic filter membrane;
3, the miillpore filter light that disposes is faced up be tiled in the plate, can not be overlapping between film and the film; Then draw an amount of " heroic board " 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, the VBNC bacteria sample is carried out fluorescent dye, SYTO-9 and PI dye ligand are made working concentration, and deposit standby at-20 ℃; VBNC bacterial sediment in step poly-1 is dissolved in the 50 μ l sterile salines, and each 25 μ l 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 sample, to 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 the OLYMPUSBX-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 counting select 10 different visuals field at random, and the bacterial population in each visual field is 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.
Claims (3)
- 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 |
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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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| CN101344476B (en) | 2011-03-16 |
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