CN102967545A - Method for detecting content of active bacteria - Google Patents
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- CN102967545A CN102967545A CN2012104388430A CN201210438843A CN102967545A CN 102967545 A CN102967545 A CN 102967545A CN 2012104388430 A CN2012104388430 A CN 2012104388430A CN 201210438843 A CN201210438843 A CN 201210438843A CN 102967545 A CN102967545 A CN 102967545A
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Abstract
The invention discloses a method for detecting the content of active bacteria and in particular relates to a method for detecting the content of active bacteria through combined use of CTC dye (5-cyano-2,3-ditolyl tetrazoliumchloride) dyeing and a flow cytometry, and the method is called as CTC-flow cytometry method or CTC-FCM method for short. The first method for detecting the content of the active bacteria comprises the following steps of: co-incubating CTC dyes and a liquid sample to be detected, and detecting the content of the active bacteria in the liquid sample to be detected through the flow cytometry. The sensitive detection instrument (namely the flow cytometry) is used for detecting faint labeled cells and determining optimal experiment conditions, thereby greatly improving the accuracy of the detected active bacteria, and fulfilling the aim of accurately and rapidly detecting active pathogenic bacteria. The method is rapid, easy and convenient, has a broad-spectrum recognition property, can accurately and rapidly detect the active pathogenic bacteria and can be used for detecting the active pathogenic bacteria in an environmental sample.
Description
Technical field
The present invention relates to a kind of method of detection of active bacteria content, particularly a kind of by uniting the method for coming the detection of active bacteria content with CTC dyeing and flow cytometer.
Background technology
Total number of bacteria refers to aerobic bacteria, facultative anaerobe and heterotroph in the 1mL water sample in nutrient agar, in 37 ℃ cultivate 24h after, the sum of the bacterial clump of growing (" water and effluent monitoring analytical approach " (third edition)).One of water quality conventional index that total number of bacteria (GB5749-2006) is stipulated as China's " drinking water sanitary standard " has very large reference value aspect evaluating water quality.
The nutrient agar panel cultivation has certain limitation, only has a fraction of bacterium to be counted at flat board in the physical environment, and the research surface is arranged, and can only be accounted for 0.8% of total number of bacteria by the bacterium that cultivation detects in the seawater.In addition, a lot of anaerobions and amphimicrobe can't utilize plating method to detect.Different bacterium is very responsive to condition of culture such as temperature, incubation time, nutrient culture media, and the nutrient agar panel cultivation is difficult to various bacterium are effectively detected simultaneously.And, the detection time that the nutrient agar panel cultivation needs usually long (needing 24h).Simultaneously, studies show that in a large number, a lot of bacteriums are under ambient pressure environment (such as high temperature), can enter " have activity but can't cultivate " (Viablebut not culturable, VBNC) state, cause some pathogen to form bacterium colony at plating medium, but still keep certain metabolic capability and pathogenic, VBNC state pathogen can recover its infectivity under optimum conditions.In addition, the bacterium in flat board can all not generate bacterium colony with identical speed, and the bacterium colony size of formation is also different, if generated some small bacterium colonies, may be missed in counting process.Therefore, plating method can not accurately reflect activated bacterial total amount in the water.
(a kind of oxidation-reduction quality dyestuff, English name are 5-cyano-2 to the CTC dyestuff, and 3-ditolyl tetrazoliumchloride, Chinese are 5-cyano group-2, and 3-xylyl tetrazolium chloride, structural formula are seen formula (I).
The CTC dyestuff is a kind of material of colourless permeable cell membrane, can be reduced by electron transport chain by bacterial cell, in cell, form red fluorescence deposit CTF (CTC-Formazan, under 450/630nm, be excited), the content that generates CTF and CTC concentration, incubation time are relevant.The cell that can reduce CTC and produce fluorescence namely has the cell (CTC+) of respiratory activity.Simultaneously, the CTC dyestuff is compared the advantage that also has a uniqueness with other redox dye, and namely it can react with aerobic bacteria, anaerobion and facultative bacteria.
Summary of the invention
The method that the purpose of this invention is to provide a kind of detection of active bacteria content, particularly a kind of by uniting the method (being called for short CTC-flow cytometer method or CTC-FCM method) of coming the detection of active bacteria content with CTC dyeing and flow cytometer.
The method of the first detection of active bacteria content provided by the invention is that CTC dyestuff and testing liquid sample are hatched altogether, then detects activated bacterial content in the described testing liquid sample by flow cytometer.
The implementation method of described " detecting activated bacterial content in the described testing liquid sample (such as water sample) by flow cytometer " is as follows: adopt flow cytometer, counting can send the material of red fluorescence under the exciting light of 450/630nm, and each material that can send red fluorescence represents an activated bacterial.
In the described method, described initial system of hatching altogether specifically can be comprised of CTC aqueous solution, testing liquid sample (or dilution of testing liquid sample) and NaCl aqueous solution.In the described method, described initial system of hatching altogether specifically can be comprised of 20 μ L CTC aqueous solution, 10 μ L testing liquid samples (or dilution of testing liquid sample) and 70 μ L0.85g/100mL NaCl aqueous solution.In the described method, in the described initial system of hatching altogether, the concentration of described CTC dyestuff specifically can be 2mmol/L.
The described time of hatching altogether specifically can be 3 hours.
Described bacterium specifically can be Escherichia coli.
In the said method, the setting of fluidic cell instrument parameter can be: FL1:4.11; FL2:6.29; FL3:5.47; Each volume of measuring is can 50 μ L.
The method of another kind of detection of active bacteria content provided by the invention is that CTC dyestuff and solid sample to be measured are hatched in liquid-phase system altogether, then detects activated bacterial content in the described solid sample to be measured by flow cytometer.
The implementation method of described " detecting activated bacterial content in the described solid sample to be measured by flow cytometer " is as follows: adopt flow cytometer, counting can send the material of red fluorescence under the exciting light of 450/630nm, and each material that can send red fluorescence represents an activated bacterial.
In the described method, described initial system of hatching altogether specifically can be comprised of suspending liquid and the NaCl aqueous solution of CTC aqueous solution, solid sample to be measured.In the described method, described initial system of hatching altogether specifically can be comprised of suspending liquid and the 70 μ L 0.85g/100mL NaCl aqueous solution of 20 μ L CTC aqueous solution, 10 μ L solid sample to be measured.In the described method, in the described initial system of hatching altogether, the concentration of described CTC dyestuff specifically can be 2mmol/L.
The described time of hatching altogether specifically can be 3 hours.
Described bacterium specifically can be Escherichia coli.
In the said method, the setting of fluidic cell instrument parameter can be: FL1:4.11; FL2:6.29; FL3:5.47; Each volume of measuring is can 50 μ L.
According to different research purposes, the CTC dyestuff can combine with other technology, as the CTC dyeing is combined with scanning tunneling microscope, the CTC dyeing is combined with the confocal laser electron microscope etc.Be combined to detect living cells if adopt the CTC dyeing with the confocal laser electron microscope, can there be following limitation: because the metabolic characteristic of cell self, so that some cells can only be at the thin a small amount of CTF of intracellular accumulation, do not reach the minimum that fluorescent microscope detects, and so that testing result is lower than substantial activity bacterial population in the sample; The difference of CTC dyeing method (mainly comprising the final concentration of CTC dyestuff and the incubation time after the sample mix) also has larger impact to experimental result.
The detection principle of method provided by the invention is as follows: with the respiratory metabolism activity of the cell judgment criteria as activated bacterial; The permeable bacterial cell membrane of CTC dyestuff, the electron transport chain reaction with in the bacterium with respiratory metabolism activity forms the red fluorescence precipitation, and then can detect by flow cytometer in cell; Flow cytometer can record side scattered light (the side light scatter of individual particle in the sample, SS), green fluorescence (green fluorescence, FL1,53015nm), orange fluorescence (orange fluorescence, FL2,58521nm) and red fluorescence (redfluorescence, FL3,650nm); Among the present invention, when adopting FL3 to be detecting device, the red fluorescent material that activated bacterial and the reaction of CTC dyestuff produce is excited, thereby realizes count of bacteria.The detection principle of method provided by the invention and flow process are as shown in Figure 1.
Adopt sensitive detecting instrument (flow cytometer) to detect cell and definite optimum experimental condition of faint mark among the present invention, the accuracy that can greatly improve the activated bacterial of measuring is to reach purpose accurate, the active pathogen of fast detecting.The method applied in the present invention is quick, easy, has the wide spectrum identity, can be accurately, fast detecting is to active pathogen, can be used for the detection of the active pathogen of environmental sample.
Description of drawings
Fig. 1 is CTC-flow cytometer method detection of active bacterium process flow diagram.
Fig. 2 is the result under different CTC dye strengths and the incubation time.
Fig. 3 is that colibacillary plating method and CTC-flow cytometer method count results compare.
Fig. 4 is that plating method and the CTC-flow cytometer method analysis result of actual water sample compares.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Used PBS damping fluid is the PBS damping fluid of pH 7.2,0.1M among the embodiment.Used Escherichia coli among the embodiment claim again bacillus coli (Escherichia coli): Chinese common micro-organisms strain preservative tube reason center (network address is http://www.cgmcc.net/), CGMCC is numbered 1.2385.The CTC dyestuff: available from Molecular Probes, article No. is B34956.
The optium concentration of embodiment 1, CTC-FCM method and the optimization of incubation time
1, Escherichia coli bacteria liquid is inoculated in nutrient broth medium, 37 ℃, 160rpm shaken cultivation 16h.
2, get the bacterium liquid that 100 μ L steps 1 obtain, be diluted to 1mL with the PBS damping fluid, then 4 ℃, the centrifugal 5min of 12000rpm collect bacterial sediment and are resuspended in 100mL PBS damping fluid, and obtaining viable bacteria concentration is 10
5The bacteria suspension of CFU/mL.
3, with sterilized water the CTC dyestuff is configured to the CTC stock solution that concentration is 10mmol/L.
4, be formulated as follows respectively reaction system:
Reaction system first: formed by the CTC stock solution of 10 μ L steps 3 preparation, bacteria suspension and the 80 μ L 0.85g/100mL NaCl aqueous solution of 10 μ L steps 2 preparation;
Reaction system second: formed by the CTC stock solution of 20 μ L steps 3 preparation, bacteria suspension and the 70 μ L 0.85g/100mL NaCl aqueous solution of 10 μ L steps 2 preparation;
Reaction system third: formed by the CTC stock solution of 50 μ L steps 3 preparation, bacteria suspension and the 40 μ L 0.85g/100mL NaCl aqueous solution of 10 μ L steps 2 preparation;
37 ℃ of lucifuges of above-mentioned each reaction system (being dark condition) are hatched, after hatching 0.1h, 0.5h, 1h, 2h, 3h, 4h, 6h, 10h and 24h, take a sample, utilize Flow cytometry can send the material of red fluorescence, each material that can send red fluorescence represents an activated bacterial.
When calculating bacterial concentration, can send first the quantity of material of red fluorescence divided by the volume (0.05mL) of the solution that is used for Flow cytometry, and then multiply by 10(because the bacteria suspension of step 2 and the volume ratio of reaction system are 1:10), obtain the activated bacterial concentration in the bacteria suspension that step 2 obtains, unit is " individual/mL ".
Carry out repeated experiments three times, results averaged.
The results are shown in Table 1 and Fig. 2.Among Fig. 2, horizontal ordinate is incubation time, and ordinate is the activated bacterial concentration in the bacteria suspension.
Each reaction system of table 1 hatch activated bacterial concentration in the bacteria suspension that detects after each time (individual/mL)
| Incubation time | The reaction system first | Reaction system second | Reaction system third |
| 0.1h | 16800 | 34200 | 33800 |
| 0.5h | 22000 | 109200 | 34800 |
| 1h | 68200 | 233400 | 130000 |
| 2h | ? | 379000 | ? |
| 3h | ? | 412400 | ? |
| 4h | 140000 | 322000 | 149000 |
| 6h | ? | 232000 | ? |
| 10h | 62800 | 87600 | 63800 |
| 24h | 31000 | 6800 | 3000 |
Annotate: "-" represents without related data
The result shows: when incubation time is identical, and can be by the detected competent cell quantity of flow cytometer much smaller than reaction system second (initial concentration of CTC dyestuff be 2mmol/L) in reaction system first (initial concentration of CTC dyestuff is 1mmol/L) and the reaction system third (initial concentration of CTC dyestuff is 5mmol/L).The analysis reason is as follows: when the CTC dye strength is too small, can not be combined with the activated bacterial cell of whole tools; When the CTC dye strength is too high, bacterial cell is produced certain toxicity.Thus, the best working concentration of the CTC dyestuff determined of the present invention is 2mmol/L.
The result shows: when the CTC dye strength is identical, along with the increase of incubation time, can increase first rear minimizing by the detected cell quantity of flow cytometer, testing result reaches maximal value when incubation time is 3h.The analysis reason is as follows: when the CTC dyestuff had just been hatched with bacterial cell, respiratory metabolism was carried out in the change that need to conform certain laundering period afterwards; When CTC dyestuff and cell effect overlong time, cell metabolic activity descends, and reaction makes the CTC dyestuff produce toxic action to cell for a long time.Thus, the CTC dyestuff determined of the present invention is 3h to the incubation time of the best of activated bacterial dyeing.
The detection effect of embodiment 2, employing CTC-FCM method detection of active bacteria content
One, CTC-FCM method
1, Escherichia coli bacteria liquid is inoculated in nutrient broth medium, 37 ℃, 160rpm shaken cultivation 16h.
2, get the bacterium liquid that 100 μ L steps 1 obtain, be diluted to 1mL with the PBS damping fluid, then 4 ℃, the centrifugal 5min of 12000rpm collect bacterial sediment and are resuspended in 100mL PBS damping fluid, and obtaining viable bacteria concentration is 10
5The bacteria suspension of CFU/mL (bacteria suspension first).
3, with sterilized water the CTC dyestuff is configured to the CTC stock solution that concentration is 10mmol/L.
4, be hot deactivation bacterium with the bacteria suspension first at 70 ℃, the purpose of this step of 300rpm vibration deactivation 10min(), the bacteria suspension called after bacteria suspension second that obtains.
5, be formulated as follows respectively sample liquid:
Sample liquid I: bacteria suspension second;
Sample liquid II: 2 parts by volume bacteria suspension first+8 parts by volume bacteria suspension second;
Sample liquid III: 4 parts by volume bacteria suspension first+6 parts by volume bacteria suspension second;
Sample liquid IV: 6 parts by volume bacteria suspension first+4 parts by volume bacteria suspension second;
Sample liquid V: 8 parts by volume bacteria suspension first+2 parts by volume bacteria suspension second;
Sample is VI also: the bacteria suspension first.
6, prepare respectively the reaction system of each sample liquid: formed by the CTC stock solution of 20 μ L steps 3 preparation, sample liquid and the 70 μ L 0.85g/100mL NaCl aqueous solution of 10 μ L steps 5 preparation.
Take a sample after 37 ℃ of lucifuges of reaction system (being dark condition) are hatched 3h, utilize Flow cytometry can send the material of red fluorescence, each material that can send red fluorescence represents an activated bacterial.
When calculating bacterial concentration, can send first the quantity of material of red fluorescence divided by the volume (0.050mL) of the solution that is used for Flow cytometry, and then multiply by 10(because the bacteria suspension of step 2 and the volume ratio of reaction system are 1:10), obtain the activated bacterial concentration in the sample liquid that step 5 obtains, unit is " individual/mL ".
Two, contrast method (the dull and stereotyped counting method of cultivating)
Simultaneously, each sample liquid that 5 of step 1 obtains is coated respectively on the nutrient broth medium flat board, 37 ℃ of lucifuges were cultivated 48 hours, by the activated bacterial concentration in the counting CFU calculating sample liquid.
Carry out repeated experiments three times, results averaged.
The results are shown in Table 2 and Fig. 3.Among Fig. 3, horizontal ordinate represents the logarithm value of cultivation counting acquired results, and ordinate represents the logarithm value of CTC-FCM detection method acquired results.Table 2 detects the activated bacterial concentration (log in each sample liquid that obtains by flow cytometer
10Activated bacterial concentration)
| ? | The CTC-FCM method | Contrast method |
| Sample liquid I | 2.69 | 0.69 |
| Sample liquid II | 4.65 | 3.45 |
| Sample liquid III | 4.77 | 3.68 |
| Sample liquid IV | 4.88 | 4.39 |
| Sample liquid V | 4.94 | 4.75 |
| Sample is VI also | 5.13 | 5.09 |
The result shows: the CTC-FCM method has good linear dependence (coefficient R with the dull and stereotyped result who cultivates counting method
2=0.9465); The result that the CTC-FCM method obtains cultivates the result of counting method all in 0.5log with flat board, when the deactivation bacterial population is 100%, can identify the nonactive bacterium of the 2.5log order of magnitude, this result is slightly higher than the result of cultivation, this mainly is because under hot conditions, bacterium may enter the VBNC state, namely can't cultivate but has activity, and this activity can react with the CTC dyestuff.Therefore the method sign activated bacterial preferably; In addition, the CTC-FCM method can obtain the result in 3h, and is short than the 48h required time of cultivation, and simple to operate, highly sensitive, and accuracy is good, can be used for the activated bacterial of accurate fast detecting sample.
Actual water sample is taken from Tsing-Hua University's tap water and Gaobeidian City sewage treatment plant, gets respectively the sample of second pond water outlet, coagulating basin water outlet and sand filter water outlet, and sample is got in every kind of water outlet twice.
The measuring method of total clump count in the actual water sample: according to standard GB/T/T5750.2-2006 " the Drinking Water method of inspection-microbiological indicator ", the mensuration of total plate count adopts plate count, and 37 ° of C cultivate 48h under aerobic conditions on the nutrient agar.
The total clump count measuring method of CTC-flow cytometer: with the CTC aqueous solution of 200 μ L water samples and 8 μ L50mmol/L, hatch 3h in 37 ° of C lucifuges, under flow cytometer, analyze afterwards.
When calculating the bacterial concentration in the water sample, can send first the quantity of material of red fluorescence divided by the volume (0.050mL) of the solution that is used for Flow cytometry, and then multiply by 25(because the bacteria suspension of step 2 and the volume ratio of reaction system are 1:25), obtain the activated bacterial concentration in the water sample to be measured, unit is " individual/mL ".
Analysis result and the plate count result of flow cytometer are compared, the results are shown in Table 3.
Activated bacterial concentration (LOG in each water sample of table 3
10Activated bacterial concentration)
| ? | The CTC-FCM method | Contrast method |
| Sample liquid I | 2.609 | 2.544 |
| Sample liquid II | 3.139 | 3.079 |
| Sample liquid III | 3.900 | 3.538 |
| Sample liquid IV | 2.307 | 2.708 |
| Sample liquid V | 2.721 | 2.618 |
| Sample liquid VI | 1.690 | 1.176 |
Acquired results is done linear dependence, and the result as shown in Figure 4.
Horizontal ordinate represents the result that colony counting method obtains among Fig. 4, and ordinate represents the result that the CTC-FCM method obtains.It is done linear dependence, find that linear dependence is good, coefficient R
2=0.81.Yet the slope of fitting a straight line is 1.0323, and this testing result that shows the CTC-FCM method is slightly higher than colony counting method testing result.This mainly is still to have the respiratory metabolism activity owing to as seen form the bacterium of bacterium colony on many again nutrient culture media, may recover under certain conditions its energy for growth, and this has proved that also certain existence is in the microorganism of VBNC state.Therefore, traditional plating method may have been underestimated the quantity of active pathogen in the water, and the CTC-FCM method can selectivity be carried out technology to the bacterium with respiratory metabolism activity, thereby measured more accurately the quantity of active bacterium in the actual water body, for the total number of bacteria in the accurate evaluation water body provides new Data support.
Claims (10)
1. the method for a detection of active bacteria content is that CTC dyestuff and testing liquid sample are hatched altogether, then detects activated bacterial content in the described testing liquid sample by flow cytometer.
2. the method for claim 1, it is characterized in that: the implementation method of described " detecting activated bacterial content in the described testing liquid sample by flow cytometer " is as follows: adopt flow cytometer, counting can send the material of red fluorescence under the exciting light of 450/630nm, and each material that can send red fluorescence represents an activated bacterial.
3. method as claimed in claim 1 or 2, it is characterized in that: the described time of hatching altogether is 3 hours.
4. such as arbitrary described method in the claims 1 to 3, it is characterized in that: the initialization concentration of described CTC dyestuff is 2mmol/L.
5. such as arbitrary described method in the claim 1 to 4, it is characterized in that: described bacterium is Escherichia coli.
6. the method for a detection of active bacteria content is with the hatching altogether in liquid-phase system of CTC dyestuff and solid sample to be measured, then detects activated bacterial content in the described testing liquid solid sample by flow cytometer.
7. method as claimed in claim 6, it is characterized in that: the implementation method of described " detecting activated bacterial content in the described solid sample to be measured by flow cytometer " is as follows: adopt flow cytometer, counting can send the material of red fluorescence under the exciting light of 450/630nm, and each material that can send red fluorescence represents an activated bacterial.
8. such as claim 6 or 7 described methods, it is characterized in that: the described time of hatching altogether is 3 hours.
9. such as arbitrary described method in the claim 6 to 8, it is characterized in that: the initialization concentration of described CTC dyestuff is 2mmol/L.
10. such as arbitrary described method in the claim 6 to 9, it is characterized in that: described bacterium is Escherichia coli.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105158429A (en) * | 2015-09-17 | 2015-12-16 | 哈尔滨工业大学 | Method for measuring content of biodegradable organic carbon (BDOC) in water |
| CN110441223A (en) * | 2019-07-11 | 2019-11-12 | 南京信息职业技术学院 | A method of solid particle toxicity evaluation is carried out using photobacteria |
| CN110607343A (en) * | 2019-09-02 | 2019-12-24 | 重庆三峡学院 | Method for detecting live bacteria by double-dyeing method |
| WO2020088062A1 (en) * | 2018-10-30 | 2020-05-07 | 江南大学 | Method for determining optimal storage temperature aerobic denitrifying bacteria in sewage treatment |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105158429A (en) * | 2015-09-17 | 2015-12-16 | 哈尔滨工业大学 | Method for measuring content of biodegradable organic carbon (BDOC) in water |
| WO2020088062A1 (en) * | 2018-10-30 | 2020-05-07 | 江南大学 | Method for determining optimal storage temperature aerobic denitrifying bacteria in sewage treatment |
| CN110441223A (en) * | 2019-07-11 | 2019-11-12 | 南京信息职业技术学院 | A method of solid particle toxicity evaluation is carried out using photobacteria |
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Application publication date: 20130313 |