CN107326063A - A kind of method that fixation of bacteria observation is counted - Google Patents
A kind of method that fixation of bacteria observation is counted Download PDFInfo
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- CN107326063A CN107326063A CN201710636196.7A CN201710636196A CN107326063A CN 107326063 A CN107326063 A CN 107326063A CN 201710636196 A CN201710636196 A CN 201710636196A CN 107326063 A CN107326063 A CN 107326063A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The invention provides the method that a kind of observation of fixation of bacteria is counted, it is characterised in that methods described comprises the following steps：(1) after tested bacteria sample is dyed, it is resuspended in reagent A；(2) according to volume ratio 1:2~3 ratio, step (1) gains are mixed with fixer；(3) step (2) gains are placed in observation carrier, in progress observation counting under microscope；Solute in the reagent A, every 1L is 8gNaCl, 0.2gKCl, 2.1gK2HPO4, 0.25gTween 80 and 0.5gNa2S, solution is water；The fixer is the agarose solution that weight concentration is 0.8 1%, and solvent is TAE.The present invention can disposably obtain the high count of bacteria effect of accuracy, it is to avoid time-consuming, the laborious and high technical disadvantages of cost that conventional method has.
Description
Technical field
The invention belongs to bacterium observation counted fields, and in particular to a kind of method that fixation of bacteria observation is counted.
Background technology
At present, the method for counting of bacterium is mainly viable plate counts, but the method is time-consuming longer, and need to be according to difference
Bacterium makes the standard curve of bacterial growth, relatively complicated, while the method is not suitable for counting dead bacterium, it is difficult to directly use
To some specific scientific research fields.
Bacterium is dyed, then observed using microscope, is a kind of very easily technology, it is utilized
Dyestuff has the ability combined with other materials in DNA or bacterium, realizes the visual observation counting to bacterium.But, exist
In real work, usually then bacterial suspension is dropped in load fragment, then the fragmentation that closes the lid, be finally placed in PBS or water
Observation counting is carried out under microscope, this causes bacterium to be easy to ceaselessly float in the ken under mirror, cause the inaccurate of counting.
In addition, during film-making, also usually because the excessive, force application angle that in lid cover glass, exerts a force is not pair or because of suspension table
The reasons such as face tension force, cause the Multiple drug resistance on slide extremely uneven, and the bacterium that can not truly reflect in testing sample
Number.
For these reasons, in some need the research work to bacterium progress degree of precision counting, it is often necessary to enter
The sample preparation of row high-volume and repeat count, then carry out data processing, could obtain more real count value, this is not only resulted in
The waste of research reagents (such as dyestuff), also quite time-consuming effort causes the loss of a large amount of scientific research time and efforts.
The content of the invention
In summary, in view of the shortcomings of the prior art, counted it is an object of the invention to provide a kind of observation of fixation of bacteria
Method, this method need not carry out high-volume sample preparation and repeat count, can disposably obtain high-precision measurement result.This method
Comprise the following steps：
(1) after tested bacteria sample is dyed, it is resuspended in reagent A；
(2) according to volume ratio 1:2~3 ratio, step (1) gains are mixed with fixer；
(3) step (2) gains are placed in observation carrier, in progress observation counting under microscope；
Solute in the reagent A, every 1L is 8g NaCl, 0.2g KCl, 2.1g K2HPO4, 0.25g Tween-80 and
0.5gNa2S, solution is water；
The fixer is the agarose solution that weight concentration is 0.8-1%, and solvent is TAE.
The microscope is light microscope or other microscopes based on light microscope；
As shown in the embodiment of the present invention, it is a discovery of the invention that when the above-described method is used, bacterium is carried out using microscope
Observation is counted, and can disposably obtain very high accuracy, counts what is caused to eliminate various factors without being repeated several times
Error.
In microorganism scientific research field, agarose is generally used for the culture (such as flat board culture) of bacterium or consolidating for bacterium
Fixedization (such as prepares gel micro-ball).It is general to go back because the intensity of Ago-Gel is smaller in the immobilization research field of bacterium
The modification of material need to be carried out.However, bacterium is embedded in microballoon, a kind of unsuitable this area count of bacteria is had proved to be
Method.This is due to that bacterium in microballoon can not be co-located on same plane so that it is real that microscope is difficult to display comprehensively
Bacterial population.And utilize laser confocal microscope, then it can not adjust specific step-length.
Therefore, first bacterium is fixed using gel rubber material and counted again, be not suitable for solving above-mentioned this area
The technical problem faced.
In the use of the present invention, be not relying on agarose can into gel property, be only dissolved in TAE solution, and
After bacterium is resuspended using inventive formulation solution, mix, be just used directly for microscopical with agarose solution of the present invention
Observation.
As shown in the comparative example of the present invention, when using some viscosity solutions, (such as 50% glycerite or PVA are water-soluble
Liquid) substitute agarose solution of the present invention when, gained count results are unsatisfactory.In addition, will be replaced for the solution that bacterium is resuspended
For common PBS, acquired results are also poor.
Bacterium of the present invention includes at least one of eubacteria and archeobacteria in gram- bacteria.
In step (1), when being dyed, vital stain used includes AO, DAPI/PI, SYTO 9/PI, SybrGreen I
One kind in.
The present invention is not any limitation as dyestuff used, as long as the dyestuff that the micro- sem observation that can be used for bacterium is counted is equal
It is suitable for the present invention.The several of the example above are only relatively conventional dyestuff.
Preferably, in step (2), according to volume ratio 1:2.5 ratio, by step (1) gains and fixer
Mixing.
The observation carrier is slide.In general, when observation counting is carried out to bacterium using fluorescence microscope, it is used
Observation carrier is slide (being made up of slide and cover glass).
When implementing the present invention, the pH of rate-determining steps (1) and step (2) gains is 5.8~7.2.
Preferably, the weight concentration of the agarose solution is 0.8%.
Beneficial effects of the present invention：
The present invention can disposably obtain the high count of bacteria effect of accuracy, it is to avoid the expense that conventional method has
When, the laborious and high technical disadvantages of cost.
Brief description of the drawings
Fig. 1 counts the figure of taking pictures during bacterium for micro- Microscopic observation of the invention.
Embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be following examples be use
It is further detailed in the present invention, it is impossible to be interpreted as limiting the scope of the invention, the field is skilled in technique
Some nonessential modifications and adaptations that personnel are made according to foregoing invention content, still fall within protection scope of the present invention.
Embodiment 1
The present embodiment is using Escherichia coli as experimental bacteria, and dense bacterium is 1 × 106CFU/mL, is respectively placed in the water that pH is 4.5
Solution 3, after 5 and 8 minutes, calculate motility rate.During dyeing, using Live/DeadBacLight BacterialViability Kit
Dyed, specification built in method reference reagent box.Sample preparation is carried out as follows and observes counting：
(1) after dyeing is finished, it is resuspended in reagent A；
(2) according to volume ratio 1:2.5 ratio, step (1) gains are mixed with fixer；
(3) step (2) gains are placed on slide, and covered, in progress observation meter under fluorescence microscope
Number；
Solute in the reagent A, every 1L is 8g NaCl, 0.2g KCl, 2.1g K2HPO4, 0.25g Tween-80 and
0.5gNa2S, solution is water；
The fixer is the agarose solution that weight concentration is 0.8%, and solvent is TAE.
Checking test：The number (10 groups of Duplicate Samples are set) of bacterium after being handled using viable plate counts calculating, with
This calculates motility rate.
The result is determined：
Method：It is compared with the result of the result of calculation of the present embodiment and checking test, calculation error.
As a result：For the experimental group through three kinds of different disposal time, (disposable sample preparation, is only calculated the method for the present embodiment
Bacterial population under one ken) error of acquired results and checking test result is no more than 3%, with a high credibility.
Embodiment 2
Except in step (2), volume ratio is 1:2, and agarose solution weight concentration be 1.0% outside, remaining
It is consistent with embodiment 1.
The result：Compared with checking test result, error is no more than 3%.
Embodiment 3
Except in step (2), volume ratio is 1:3, and agarose solution weight concentration be 0.9% outside, remaining
It is consistent with embodiment 1.
The result：Compared with checking test result, error is no more than 3%.
Comparative example 1
Except in step (1), reagent A is replaced with outside PBS, remaining is consistent with embodiment 1.
The result：Compared with checking test result, error is up to 12~19%.
Comparative example 2
Except fixer is replaced with into glycerine so that glycerine to outside final concentration of 50%, remaining and embodiment 1 one
Cause.
The result：Compared with checking test result, error is up to 17~23%.
Comparative example 3
In addition to PVA (polyvinyl alcohol) aqueous solution that fixer is replaced with to 1%, remaining is consistent with embodiment 1.
The result：Compared with checking test result, error is up to 20~25%.
Comparative example 4
Only the bacterium after dyeing is resuspended in PBS solution, fluorescence microscope is then carried out.
The result：Compared with checking test result, error is up to 22~46%.
Claims (7)
1. a kind of method that fixation of bacteria observation is counted, it is characterised in that methods described comprises the following steps：
(1) after tested bacteria sample is dyed, it is resuspended in reagent A；
(2) according to volume ratio 1:2~3 ratio, step (1) gains are mixed with fixer；
(3) step (2) gains are placed in observation carrier, in progress observation counting under microscope；
Solute in the reagent A, every 1L is 8g NaCl, 0.2g KCl, 2.1g K2HPO4, 0.25g Tween-80 and
0.5gNa2S, solution is water；
The microscope is light microscope and other microscopes based on light microscope；
The fixer is the agarose solution that weight concentration is 0.8-1%, and solvent is PAE.
2. according to the method described in claim 1, it is characterised in that the bacterium includes eubacteria and archeobacteria in gram- bacteria
At least one.
3. according to the method described in claim 1, it is characterised in that in step (1), when being dyed, vital stain bag used
Include one kind in AO, DAPI/PI, SYTO 9/PI, SybrGreen I etc..
4. according to the method described in claim 1, it is characterised in that in step (2), according to volume ratio 1:2.5 ratio, will be walked
Suddenly (1) gains are mixed with fixer.
5. according to the method described in claim 1, it is characterised in that in step (3), the observation carrier is slide.
6. according to the method described in claim 1, it is characterised in that the pH of rate-determining steps (1) and step (2) gains is 5.8
~7.2.
7. according to the method described in claim 1, it is characterised in that the weight concentration of the agarose solution is 0.8%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101589156A (en) * | 2006-11-10 | 2009-11-25 | 马德里自治大学 | Method for determining DNA fragmentation in microorganisms |
| CN101629136A (en) * | 2009-08-06 | 2010-01-20 | 中国海洋大学 | High-efficient culture and sorting method and culture device of microorganisms |
| CN102321729A (en) * | 2011-07-06 | 2012-01-18 | 华中农业大学 | Fluorescent microscopic counting method for detecting bacterial count in soil and sediment |
| CN106754888A (en) * | 2017-01-21 | 2017-05-31 | 青岛农业大学 | The extracting method and kit of a kind of grand genome DNA of fish enteric microorganism |
-
2017
- 2017-07-31 CN CN201710636196.7A patent/CN107326063A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101589156A (en) * | 2006-11-10 | 2009-11-25 | 马德里自治大学 | Method for determining DNA fragmentation in microorganisms |
| CN101629136A (en) * | 2009-08-06 | 2010-01-20 | 中国海洋大学 | High-efficient culture and sorting method and culture device of microorganisms |
| CN102321729A (en) * | 2011-07-06 | 2012-01-18 | 华中农业大学 | Fluorescent microscopic counting method for detecting bacterial count in soil and sediment |
| CN106754888A (en) * | 2017-01-21 | 2017-05-31 | 青岛农业大学 | The extracting method and kit of a kind of grand genome DNA of fish enteric microorganism |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Application publication date: 20171107 |