CN102051404A - Method for detecting survival rate and activity of microorganism strain - Google Patents
Method for detecting survival rate and activity of microorganism strain Download PDFInfo
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- CN102051404A CN102051404A CN2009101749308A CN200910174930A CN102051404A CN 102051404 A CN102051404 A CN 102051404A CN 2009101749308 A CN2009101749308 A CN 2009101749308A CN 200910174930 A CN200910174930 A CN 200910174930A CN 102051404 A CN102051404 A CN 102051404A
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Abstract
The invention discloses a method for detecting the survival rate and the activity of a microorganism strain. The method comprises the following steps of: A, dissolving the microorganism strain in culture solution containing methylthiazolyldiphenyl-tetrazolium bromide, and diluting to obtain bacterial suspension; B. adding the bacterial suspension with different dilution gradients into all the holes of a culture plate; and C. scanning and determining in real time, calculating the survival rate of the strain and determining the activity of the strain according to a scanning result. By the detection method, the survival rate and the activity of the stored microorganism strain can be conveniently, fast, efficiently and periodically detected.
Description
Technical field
The invention belongs to biological technical field, relate to survival rate and the active detection method of a kind of microbial strains, particularly relate to and a kind ofly adopt the real time scan microorganism growth and the survival rate and the active method of rapid determination bacterial classification.
Background technology
The preservation of microbial strains is a very important job, has the microbial strains of researching value all to need under the preservation in scientific research and testing process, and also has a lot of professional institutions to specialize in the preservation work of microbial strains.Microbial strains is adopted different protective materials, preserves through different modes.After preserving, just need sample at set intervals, and measure survival rate and the activity that extracts bacterial classification, determine whether to continue to preserve according to survival rate and active situation then, or need to preserve again after the rejuvenation bacterial classification.This mensuration work, workload is big, and requires experimental repeatability good.
The microbial strains survival rate is measured, and after soon microbial strains is brought back to life, counts; The microbial strains determination of activity after normally bacterial classification being brought back to life, is cultivated for some time, and regularly the sampling counting is measured its growth curve.Two kinds of mensuration all need to carry out microorganism count, and method of counting also has nephelometry and tetrazolium bromide (MTT) method except methods such as traditional viable bacteria dyeing microscopic counting, plate count at present.Preceding two kinds of traditional methods, length consuming time, complex operation seems very inconvenient in extensive, repeatability in measuring more.Nephelometry, its principle is a bacterial growth, turbidity increases.But because the resolving power of turbidity is lower, and different microorganisms quantity is also different with the relation of turbidity, and therefore following two problems usually take place: the one,, the bacteria suspension turbidity is very low, but microbe population increment in a large number in the actual bacteria suspension; The 2nd,, though two kinds of bacterium turbidity numerical value are very close, but the quantity of two kinds of bacterium differs 3 more than the order of magnitude in the actual bacteria suspension, thereby occurs turbidity and the inconsistent situation of microbe population when causing nephelometry counting microorganism, therefore generally makes to be used less in extensive, repeatability mensuration.
Mtt assay has overcome the shortcoming of nephelometry, and its principle is after for some time, to add MTT and dimethyl sulfoxide (DMSO) at microorganism growth, and termination reaction produces blueness, measures the 520nm place and absorbs, and its absorption value is directly proportional with microbe population.But the mtt assay that adopts all adopts end-point method to detect at present, and is undertaken quantitatively by the typical curve of setting up, and experimental implementation is loaded down with trivial details, and can't realize detecting in real time fast.
Therefore, this area need a kind of more easy, measure bacterial classification survival rate and active method fast.
Summary of the invention
The object of the present invention is to provide a kind of microbial strains survival rate and active detection method, use survival rate and activity that this method can detect bacterial classification fast, easily, according to detected result, if bacterial classification survival rate height, active good, then this bacterial classification can continue to preserve, if the bacterial classification survival rate is low or active low, then can improve its survival rate and activity by the rejuvenation measure, preserve again.
For achieving the above object, the invention provides survival rate and the active detection method of a kind of microbial strains, it comprises the steps:
A. microbial strains is dissolved in the nutrient solution that contains tetrazolium bromide, and dilutes and obtain bacteria suspension;
B. the bacteria suspension that difference is diluted gradient joins in each hole of culture plate; With
C. real time scan is measured, and calculates the survival rate of bacterial classification, the activity of judgement bacterial classification according to scanning result.
As long as owing to there is viable bacteria to exist, just can make tetrazolium bromide produce blue material through cultivating, therefore can be according to the situation in the hole that absorption peak occurs, the hole count of determining that minimum 3 extent of dilution of growth take place and growth taking place, for example can determine number of viable in the bacterial classification by with reference to coliform most probable number MPN (MPN) key.Number of viable in the preservation bacterial classification that obtains divided by the original number of viable of bacterial classification, can be calculated survival rate.
Because absorption value is directly proportional with the growth situation, therefore can according to real time scan obtain time-absorption curve shape judgement microbial strains growth activity.Growth curve can be divided into three types: precipitous type, osculant, high order smooth pattern.Precipitous type, lag phase is short, and logarithmic phase almost linearly rises, and maintains higher absorption value stationary phase, and longer duration; Osculant, lag phase is long, and logarithmic phase slowly rises, and maintains lower absorption value stationary phase, and the time length is short, enters decline phase very soon; High order smooth pattern, entire curve are parabolic type or press close to X-axis linearly, keep lower absorption value always.Active good bacterial classification is precipitous sigmoid growth curve; The bacterial classification of poor activity is osculant or high order smooth pattern growth curve.The growth curve in different dilution each hole of same bacterial classification, lag phase length has difference, but after this growth slope of logarithmic phase and the absorption value basically identical of stationary phase, thereby can determine the activity of bacterial classification according to the type of growth curve.
The principle of present method counting is the bacteria suspension that adds a plurality of (as 8) gradient dilution (as 10 times of gradient dilutions) in containing the substratum of MTT, each bacterial strain repeats (as 3 times) for several times, cultivate after 24 hours, the hole count (hole that absorption peak occurs represents that this extent of dilution has growth) of determining that minimum several (as 3) extent of dilution of growth takes place and growth taking place, with reference to coliform most probable number (MPN) key, determine number of viable in the bacterial classification.Number of viable in the preservation bacterial classification that present method is obtained divided by the original number of viable of bacterial classification, can calculate survival rate.
Present method is measured active principle, and bacterial classification inoculation was cultivated 24 hours after arriving in each hole (as 96 holes), along with the increase of number of viable in the hole, blueness is deepened gradually, therebetween real time scan, measure the absorption value in each hole, do time-absorption curve, be used to characterize the growing state of bacterial classification.In the higher animal cell, MTT is reduced into blue Fomazan particle by the desaturase in the plastosome, therefore when carrying out higher animal cell quantity and determination of activity, must add dimethyl sulfoxide (DMSO), the Fomazan particle of formation can be solved homogeneously in the cell suspension.And in bacterial cell, succinodehydrogenase is present in plasma membrane and the mesosome, MTT produces the Fomazan particle after by the succinodehydrogenase utilization of bacteria in viable cell, be easy to be diffused in the bacteria suspension, and within the specific limits, the granuloplastic amount of Fomazan is also relevant with the bacterial cell dehydrogenase activity, also is directly proportional with bacterial cell quantity.Therefore, when bacterial growth is measured, can take the real time scan method, METHOD FOR CONTINUOUS DETERMINATION time-absorption curve, therefore the height of absorption value is directly proportional with bacterial number, can be with the growing state of this curve representation bacterium.
According to survival rate and the active detection method of microbial strains of the present invention, the nutrient solution in the described steps A is preferably the brain heart infusion nutrient solution.The dilution gradient of the bacteria suspension among the described step B is 10 times of gradients.Survival rate among the described step C is with reference to coliform most probable number key, determines to preserve the number of viable in the bacterial classification, the number of viable when again this number of viable being begun to preserve divided by bacterial classification, thereby the survival rate of obtaining.
According to an embodiment of detection method of the present invention, described detection method may further comprise the steps:
Tetrazolium bromide is configured to 5 ± 0.5% storing solution with PBS, sterilization, refrigerator is preserved; The tetrazolium bromide storing solution is added in the brain heart infusion nutrient solution, make its final concentration reach 0.1 ± 0.05%, sterilization, refrigerator is preserved standby; The brain heart infusion that will contain tetrazolium bromide joins in each hole of culture plate; After the dissolving of bacterial classification usefulness brain heart infusion nutrient solution, carry out 10 times of gradient series dilutions with sterilized water; Each dilution bacteria suspension is added in the micropore; Microwell plate is put into microplate reader, culture temperature is set at 36 ℃, the mensuration wavelength is 520nm, and the working power mode determination is measured once the METHOD FOR CONTINUOUS DETERMINATION certain hour at set intervals; Calculate the survival rate of bacterial classification, the activity of judgement bacterial classification according to scanning result.
According to an embodiment of detection method of the present invention, described detection method may further comprise the steps: tetrazolium bromide is configured to 5 ± 0.5% storing solution with PBS, and sterilization, 4 ℃ of refrigerators are preserved; The tetrazolium bromide storing solution is added in the brain heart infusion nutrient solution, make its final concentration reach 0.1 ± 0.05%, sterilization, 4 ℃ of refrigerators are preserved standby; The brain heart infusion that will contain tetrazolium bromide joins in each hole of 96 well culture plates; After the dissolving of bacterial classification usefulness brain heart infusion nutrient solution, carry out 10 times of gradient series with sterilized water and be diluted to 10
-8With 10
-1-10
-8Dilution bacteria suspension adds in the micropore; Microwell plate is put into microplate reader, culture temperature is set at 36 ℃, the mensuration wavelength is 520nm, and the working power mode determination was measured METHOD FOR CONTINUOUS DETERMINATION 24 hours 1 time every 30 minutes; With the minute is X-axis, is Y-axis with the absorption value, makes the time-absorption curve in every hole; Judge bacterial activity according to curve; With reference to coliform most probable number key, determine to preserve the number of viable in the bacterial classification, the number of viable when again this number of viable being begun to preserve divided by bacterial classification obtains the survival rate of bacterial classification.
For microbial strains survival rate of the present invention and active detection method, when measuring the bacterial classification survival rate, the MPN method can be combined with the tetrazolium bromide colour developing, be used for measuring the number of viable of bacterial classification.Compare with nephelometry, have better specificity; Compare with traditional method, operate easylier, the reagent of use is still less more economical.In addition, can utilize scanner to obtain the microorganism curve, can judge microorganism active, compare with traditional method for testing microbial activity and have easier, more economical advantage by the shape of curve by real time scan.
That is to say that microbial strains survival rate provided by the invention and active measuring method are by the growing state of real time scan monitoring bacterial classification in nutrient solution, thus quantitative assay number of viable wherein, the activity of qualitative judgement viable bacteria.
The present invention especially is suitable for microbial strains survival rate and active rapid determination, and is active good as finding bacterial classification survival rate height, then can continue to preserve, and low as finding the bacterial classification survival rate, poor activity can be passed through the rejuvenation of spawn means, preserves bacterial classification again.
An embodiment according to detection method of the present invention, this method adopts following simple procedure: microbial strains is dissolved in the brain heart infusion agar, and---bacteria suspension is carried out 10 times of gradient dilutions, and---brain heart infusion agar that will contain MTT joins 96 well culture plates, and---bacteria suspension that difference is diluted gradient joins in each hole---real time scan mensuration---looks into the MPN table according to the situation that absorption peak occurs, calculates the viable count in the bacterial classification; According to time-the absorption curve shape, judge the active situation of bacterial classification.Microbial strains is dissolved in the brain heart infusion nutrient solution (except the special microorganism of small part nutritional requirement, most microbial strainss can both be in nutritious brain heart infusion nutrient solution growth and breeding), along with the microbial growth breeding, produce succinodehydrogenase, decompose MTT, produce blue Fomazan.Under the 520nm wavelength, measure light intensity, make microorganism time-absorption growth curve, situation according to the hole that absorption peak occurs, the hole count of determining that minimum 3 extent of dilution of growth take place and growth taking place, with reference to coliform most probable number (MPN) key, determine number of viable in the bacterial classification, the number of viable in the preservation bacterial classification that present method is obtained, divided by the original number of viable of bacterial classification, can calculate survival rate; According to time-the absorption curve shape judges the microbial strains growth activity.As find the microbial strains survival rate with last time detected result compare rapid drawdown, perhaps survival rate is lower than 50%, or growth curve is osculant and high order smooth pattern, and then microbial strains survival rate and activity go wrong, need to preserve again behind the rejuvenation bacterial classification by the rejuvenation of spawn means; As find that the microbial strains survival rate is higher than 50%, and with last time detected result to compare variation not remarkable, and growth curve is precipitous type, then microbial strains survival rate and active high can continue to preserve.
Though the present invention also adopts MTT indicator microoraganism growth, has been to use the porous plate culturing micro-organisms, and the light absorption value that real time scan is measured the nutrient solution that contains MTT changes, thereby simplified experimental implementation, improved detection efficiency.Use this microbial strains survival rate and active real time scan measuring method, can be convenient, fast and regularly detect the survival rate and the activity of the bacterial classification of preserving efficiently.
Description of drawings
Fig. 1 is the collection of illustrative plates of growing in real time through the Enterobacter sakazakii bacterial classification of preservation in 1 year.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail, but the present invention is not limited to these embodiment.
Embodiment
Present embodiment has carried out real time scan mensuration in illustrational mode to some bacterial classifications, but scope of the present invention is not limited to present embodiment.
Tetrazolium bromide is configured to 5% storing solution with PBS, sterilization, 4 ℃ of refrigerators are preserved.The tetrazolium bromide storing solution is added the brain heart infusion nutrient solution, make its final concentration reach 0.1%, sterilization, 4 ℃ of refrigerators are preserved standby.The brain heart infusion that 180 μ l is contained tetrazolium bromide joins in each hole of 96 well culture plates.Open the bacterial classification pipe, after bacterial classification is dissolved with the brain heart infusion nutrient solution, carry out 10 times of gradient series with sterilized water and be diluted to 10
-8Add 10 by row
-1-10
-8Dilution bacteria suspension 20 μ l are in micropore, and each extent of dilution repeats 3 times, i.e. 1 strain bacterial classification, 3 row; Every microwell plate 12 row can be realized the detection of 4 strain bacterial classifications.Cover the microwell plate lid, microwell plate is put into microplate reader, culture temperature is set at 36 ℃, the mensuration wavelength is 520nm, and the working power mode determination was measured METHOD FOR CONTINUOUS DETERMINATION 24 hours 1 time every 30 minutes.With the minute is X-axis, is Y-axis with the absorption value, makes the time-absorption curve in every hole.If any corresponding software, can directly adopt software, make growth curve.As long as owing to there is viable bacteria to exist, just can make tetrazolium bromide produce blue material through cultivating, therefore can be according to the situation in the hole that absorption peak occurs, the hole count of determining that minimum 3 extent of dilution of growth take place and growth taking place, with reference to coliform most probable number (MPN) key, determine number of viable in the bacterial classification.Number of viable in the preservation bacterial classification that present method is obtained divided by the original number of viable of bacterial classification, can calculate survival rate.Because absorption value is directly proportional with the growth situation, therefore can according to time-absorption curve shape judgement microbial strains growth activity.Growth curve can be divided into three types: precipitous type, osculant, high order smooth pattern.Precipitous type, lag phase is short, and logarithmic phase almost linearly rises, and maintains higher absorption value stationary phase, and longer duration; Osculant, lag phase is long, and logarithmic phase slowly rises, and maintains lower absorption value stationary phase, and the time length is short, enters decline phase very soon; High order smooth pattern, entire curve are parabolic type or press close to X-axis linearly, keep lower absorption value always.Active good bacterial classification is precipitous sigmoid growth curve; The bacterial classification of poor activity is osculant or high order smooth pattern growth curve.The growth curve in different dilution each hole of same bacterial classification, lag phase length has difference, but after this growth slope of logarithmic phase and the absorption value basically identical of stationary phase.Thereby can determine the activity of bacterial classification according to the type of growth curve.As find the microbial strains survival rate with last time detected result compare rapid drawdown, perhaps survival rate is lower than 50%, or growth curve is osculant and high order smooth pattern, and then microbial strains survival rate and activity go wrong, need to preserve again behind the rejuvenation bacterial classification by the rejuvenation of spawn means; As find that the microbial strains survival rate is higher than 50%, and with last time detected result to compare variation not remarkable, and growth curve is precipitous type, then microbial strains survival rate and active high can continue to preserve.
The method his-and-hers watches 1 listed bacterial classification of describing according to present embodiment carries out real time scan mensuration, and the survival rate of bacterial classification is shown in Table 1; The real-time growth collection of illustrative plates of some bacterial classifications is shown in the drawings.
Fig. 1 is the collection of illustrative plates changing conditions of growing in real time through the Enterobacter sakazakii bacterial classification of preservation in 1 year, wherein Figure 1A is the (A1 wherein of the real-time growth collection of illustrative plates before preserving, B1, C1～A6, B6, C6, D1, D2, D3 is respectively IQCC10403-20, IQCC10424, IQCC10434, IQCC10439, IQCC10456, IQCC10481, IQCC10486), Figure 1B is for preserving growth collection of illustrative plates after 1 year (A1 wherein, B1, C1～A6, B6, C6, D1, D2, D3 is respectively IQCC10424, IQCC10434, IQCC10439, IQCC10456, IQCC10481, IQCC10486, IQCC10403-20).The growth curve of Enterobacter sakazakii among comparison diagram A and the figure B is found lag time, and logarithmic phase slope and the equal difference of stable growth phase absorption value are not remarkable, judges that therefore this bacterial classification is not affected through the preservation activity in 1 year.
Table 1 is preserved the survival rate of bacterial classification after 1 year
Number of viable when the number of viable in the preservation bacterial classification that present method is obtained begins to preserve divided by bacterial classification can calculate survival rate.Number of viable when general bacterium begins to preserve is generally 10
8CFU/mL～10
9CFU/mL, through after preserving, the bacterial number when beginning to preserve is compared, and when bacterial number still is the same order of magnitude, then thinks survival rate 100%; When reducing by an order of magnitude, bacterial number then thinks survival rate＞10%; When reducing the two or more orders of magnitude, bacterial number then thinks survival＜10%; Dead totally when bacterium, think that then survival rate is 0%.
As seen, compared with prior art, the present invention can be convenient, fast and regularly detect survival rate and the activity of the microbial strains that preserves efficiently.
Claims (6)
1. survival rate of a microbial strains and active detection method, it comprises the steps:
A. microbial strains is dissolved in the nutrient solution that contains tetrazolium bromide, and dilutes and obtain bacteria suspension;
B. the bacteria suspension that difference is diluted gradient joins in each hole of culture plate; With
C. real time scan is measured, and calculates the survival rate of bacterial classification, the activity of judgement bacterial classification according to scanning result.
2. detection method according to claim 1 is characterized in that, the nutrient solution in the described steps A is the brain heart infusion nutrient solution.
3. detection method according to claim 1 is characterized in that, the dilution gradient of the bacteria suspension among the described step B is 10 times of gradients.
4. detection method according to claim 1, it is characterized in that the survival rate among the described step C is with reference to coliform most probable number key, determine to preserve the number of viable in the bacterial classification, number of viable when again this number of viable being begun to preserve divided by bacterial classification, thereby the survival rate of obtaining.
5. detection method according to claim 1 is characterized in that, described detection method may further comprise the steps:
Tetrazolium bromide is configured to 5 ± 0.5% storing solution with PBS, sterilization, refrigerator is preserved; The tetrazolium bromide storing solution is added in the brain heart infusion nutrient solution, make its final concentration reach 0.1 ± 0.05%, sterilization, refrigerator is preserved standby; The brain heart infusion that will contain tetrazolium bromide joins in each hole of culture plate; After the dissolving of bacterial classification usefulness brain heart infusion nutrient solution, carry out 10 times of gradient series dilutions with sterilized water; Each dilution bacteria suspension is added in the micropore; Microwell plate is put into microplate reader, culture temperature is set at 36 ℃, the mensuration wavelength is 520nm, and the working power mode determination is measured the METHOD FOR CONTINUOUS DETERMINATION certain hour at set intervals 1 time; Calculate the survival rate of bacterial classification, the activity of judgement bacterial classification according to scanning result.
6. detection method according to claim 1 is characterized in that, described detection method may further comprise the steps: tetrazolium bromide is configured to 5 ± 0.5% storing solution with PBS, and sterilization, 4 ℃ of refrigerators are preserved; The tetrazolium bromide storing solution is added in the brain heart infusion nutrient solution, make its final concentration reach 0.1 ± 0.05%, sterilization, 4 ℃ of refrigerators are preserved standby; The brain heart infusion that will contain tetrazolium bromide joins in each hole of 96 well culture plates; After the dissolving of bacterial classification usefulness brain heart infusion nutrient solution, carry out 10 times of gradient series with sterilized water and be diluted to 10
-8With 10
-1-10
-8Dilution bacteria suspension adds in the micropore; Microwell plate is put into microplate reader, culture temperature is set at 36 ℃, the mensuration wavelength is 520nm, and the working power mode determination was measured METHOD FOR CONTINUOUS DETERMINATION 24 hours 1 time every 30 minutes; With the minute is X-axis, is Y-axis with the absorption value, makes the time-absorption curve in every hole; Judge bacterial activity according to curve; With reference to coliform most probable number key, determine to preserve the number of viable in the bacterial classification, the number of viable when again this number of viable being begun to preserve divided by bacterial classification obtains the survival rate of bacterial classification.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103424370A (en) * | 2013-07-19 | 2013-12-04 | 湖南大学 | Method for detection of living cell biomass of phanerochaete chrysosporium under heavy metal stress |
| CN104789636A (en) * | 2015-05-12 | 2015-07-22 | 广西壮族自治区梧州食品药品检验所 | Quantitative dilution method for microorganism detection |
| CN106244668A (en) * | 2016-10-14 | 2016-12-21 | 安徽农业大学 | A kind of method that quick mensuration lactic acid bacteria coerces Strain survival rate |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
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2009
- 2009-11-03 CN CN2009101749308A patent/CN102051404A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103424370A (en) * | 2013-07-19 | 2013-12-04 | 湖南大学 | Method for detection of living cell biomass of phanerochaete chrysosporium under heavy metal stress |
| CN103424370B (en) * | 2013-07-19 | 2015-09-23 | 湖南大学 | The detection method of Phanerochaete chrysosporium viable cell biomass under heavy metal stress |
| CN104789636A (en) * | 2015-05-12 | 2015-07-22 | 广西壮族自治区梧州食品药品检验所 | Quantitative dilution method for microorganism detection |
| CN106244668A (en) * | 2016-10-14 | 2016-12-21 | 安徽农业大学 | A kind of method that quick mensuration lactic acid bacteria coerces Strain survival rate |
| CN106244668B (en) * | 2016-10-14 | 2019-04-30 | 安徽农业大学 | A kind of method of quick measurement lactic acid bacteria stress Strain survival rate |
| CN111562210A (en) * | 2020-06-16 | 2020-08-21 | 北京挑战农业科技有限公司 | Method for detecting viable count in pre-coated forage microecological preparation product |
| CN111562210B (en) * | 2020-06-16 | 2023-01-03 | 北京挑战农业科技有限公司 | Method for detecting number of viable bacteria in pre-coated feed microecological preparation product |
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Application publication date: 20110511 |