CN110257234A - A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio - Google Patents
A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio Download PDFInfo
- Publication number
- CN110257234A CN110257234A CN201910437406.9A CN201910437406A CN110257234A CN 110257234 A CN110257234 A CN 110257234A CN 201910437406 A CN201910437406 A CN 201910437406A CN 110257234 A CN110257234 A CN 110257234A
- Authority
- CN
- China
- Prior art keywords
- yeast
- budding
- height
- ratio
- value
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a kind of to detect the method for distinguishing yeast cells form and its ratio of sprouting based on micro-fluidic electrical impedance, it uses electrical impedance flow cytometer detection system acquisition yeast impedance signal, which guarantees that yeast cells is allowed to pass through detection position with the posture that long axis is parallel to pipeline central liquid flow direction.The statistical result of the parameters such as width, height, the width ratio R of typical budding identical to quantity first and the active yeast cell that do not sprout carries out numeric distribution comparison, determines the parameter demarcation threshold of budding yeast Yu non-budding yeast；Tested yeast sample cellular morphology is distinguished and calculates the numerical value of budding yeast ratio according to threshold value when formal detection.This method is simple, flux is high, it can be achieved that Yeast protoplase fast and automatically changes detection and budding ratio calculates, and activity and the proliferative capacity that can be well reflected yeast are horizontal.
Description
Technical field
The present invention relates to microfluid microorganism detection methods, and in particular to one kind is based on electrical impedance flow cytometer detection Yeast protoplase
And the method for budding ratio.
Background technique
The budding ratio of yeast is to embody an important parameter of Yeast proliferation ability.When yeast sprouts, in almost spherical
Cell on grow a raised sprout and gradually grow up, cell shape gradually becomes two spheres to link together,
To a closer rod-shaped form.In the research of screening quality yeast, morphological analysis is an important means, conventional method
Eye-observation need to be leaned on, this method large labor intensity lacks quantitative analysis means, and relies on artificial experience counting and be easy
Mistake causes inefficiency.Micro-fluidic electrical impedance detection technique is mode means more commonly used in Single cell analysis field.The party
Method is unmarked to cell, not damaged in the detection process, therefore the volume for being usually used in cell compares, identification and counting anyway.Benefit
Cellular morphology detection is carried out with micro-fluidic electrical impedance technology and rarely has research, and M.Shaker uses the technology of dielectrophoresis, realizes particle
Directional focusing allows shaft-like particle to hang down by detection position, while in detection position to mutual perpendicular to the posture of flow direction
Directly the both direction of (flow direction and vertical grain direction) detects electric current respectively, using the difference of impedance in two directions,
In both direction current ratio with 1 degree of closeness, to be distinguished to the rod-shaped, spherical of particle.This method is not introducing it
The differentiation to different shape particle is realized in the case where his equipment, but it needs dielectric power and liquid flow to the process of particle manipulation
Precisely cooperation is just able to achieve good differentiation detection between dynamic pressure, and flow velocity cannot be too high, and flux is low, it is difficult to carry out a large amount of particles
Quick detection, this method has identified budding yeast and single yeast, but does not carry out the budding ratio detection of a large amount of yeast
Experiment.
It is combined Deng by impedance detection and CCD shooting, when each particle passes through detection position, carries out impedance inspection
Micro-imaging under white light is carried out while survey, and impedance data and imaging data are subjected to convergence analysis, what realization sprouted to yeast
Detection.This method impedance detection mainly characterizes the volume of yeast, passes through image segmentation, shape recognition to image data
Deng analysis Yeast protoplase, finally the two is combined, and realizes (individually, to go out different yeast using the method for pivot analysis
It is bud, multiple agglomerating) differentiation.The identification of Yeast protoplase may be implemented in this method.But this method needs accurate image and resistance
Anti- matching is built more complicated system and is just able to achieve, and analysis method is also more complicated cumbersome.This article is also without further dividing
Analyse the detection case of a large amount of yeast budding ratios.
Application No. is a kind of micro-fluidic electrical impedance detection differentiation fine particle is disclosed in 201810930070.5 patent
The system and method for shape.The method for distinguishing fine particle shape is detected based on micro-fluidic electrical impedance passes through micro-fluidic chip pipeline
Design and flow velocity lever for adjusting oil shape particle are allowed rod-shaped by the posture of detection position using electrical impedance detector test impedance
Particle obtains relatively wide detection pulse signal width, host computer and signal processing software acquisition impedance signal and handles
It obtains pulse signal height, width and the ratio of width to height and their statistical result is realized to rod-shaped and spherical particle and big gauge rod
The differentiation of shape, spherical particle group.This patent further provides the differentiating method of budding yeast, yeast group stream on this basis
The calculation method of selection rule and yeast the budding ratio of threshold value, finally realizes accurate detection yeast shape in formula scatter plot
State and budding ratio.The advantage of this set system and yeast analysis method, the proportion computing technology that sprouts is to realize budding yeast shape
The method that state is distinguished is simple, flux is high, it can be achieved that quickly detection and budding ratio calculating, can also be based on thin for further progress
The sorting of born of the same parents' form lays the foundation.
Summary of the invention
The method based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio that the purpose of the present invention is to propose to a kind of,
It can be well reflected the activity and proliferative capacity level of yeast.
The technical scheme is that
A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio, in which: this method includes following step
It is rapid:
Step 101, culture yeasts bacterium；
Step 102, the yeast cells liquid cultivated with buffer dilution；
Step 103, in the basic parameters phase such as detected sample flow, sample liquid conductivity, exciting signal frequency and amplitude
With in situation, using electrical impedance flow cytometer detection system acquisition yeast sample impedance signal, which guarantees to allow budding yeast
Cell passes through detection position with the posture that long axis is parallel to pipeline central liquid flow direction one by one, to obtain and same volume
The non-budding yeast longer time by detection position, showing on impedance detection pulse signal has broader pulse wide
Degree；
Step 104, electrical impedance flow cytometer detection system are to impedance magnitude, phase, resistance, capacitor, real part, voidPortion's isopulse letter
It number is identified, calculates and store the pulse height, pulse width and the ratio of width to height of each signal, i.e. width ratio, abridged
R；
Typical budding yeast is selected under step 105, microscope, select on mother cell have macroscopic sprout not yet with mother
Cell separates and does not regard as typical budding yeast with the individual cells of other cytoadherences, passes through step 103 and step 104
The value of the parameters such as pulse width, height, the R of the typical budding active yeast cell of acquisition is counted；
Typical non-budding yeast is selected under step 106, microscope, select the sprout being visible by naked eyes on cell and not with
Other cytoadherences, approximate spheres individual cells regard as typical non-budding yeast, pass through step 103 and step 104 acquisition
The value of the parameters such as pulse width, height, the R of the typical active yeast cell that do not sprout is counted；
The statistics of the parameters such as width, height, the R of step 107, typical budding identical to quantity and non-budding yeast cell
As a result numeric distribution comparison is carried out, determines the parameter demarcation threshold of budding yeast Yu non-budding yeast；
Step 108 repeats step 101 to step 104, acquires pulse width, the height, width of yeast cells in tested sample
The information such as height ratio R are counted；
The parameter demarcation threshold of step 109, the budding yeast and non-budding yeast that are obtained according to step 105 to step 107,
The distribution of the parameters such as pulse width, height, the ratio of width to height to tested sample yeast cells is for statistical analysis, determines budding yeast
Yeast cells form and budding yeast, non-budding yeast are assert accordingly and distinguished to the parameter area of non-budding yeast simultaneously
Quantity；
The budding yeast quantity that step 110, calculating step 109 determine accounts for all yeast quantity of the tested sample of acquisition
The ratio is determined as " budding ratio " by ratio.
The beneficial effects of the present invention are: the method for differentiating forms of budding yeast Yu non-budding yeast is provided, in yeast group
It distinguishes the selection rule of the detection parameters threshold value of budding yeast and the calculation method of yeast budding ratio, final realization is more accurate
Budding ratio calculate.Method is simple, flux is high, it can be achieved that quickly automatic detection and budding ratio calculate.It is verified, this
The growth rate of budding ratio and yeast that method calculates is closely related, can be well reflected the activity and proliferative capacity water of yeast
It is flat.
Detailed description of the invention
Fig. 1 is the differentiation schematic diagram of budding yeast provided by the present invention and non-budding yeast；
Fig. 2 is that pulse width histogram provided by the present invention identifies yeast budding and calculates budding ratio chart；
Fig. 3 is that provided by the present invention sprouted using pulse height, R value scatter plot to yeast is identified and calculate budding
Ratio chart；
Fig. 4 is budding yeast pulse width distribution histogram (a) provided by the present invention and includes budding and the ferment that do not sprout
Female actual sample pulse width distribution histogram (b) comparison.
Specific embodiment
The present invention will be described in further detail with reference to the accompanying drawings and examples, it is clear that described embodiment is
Section Example of the invention, instead of all the embodiments.The specific embodiments are only for explaining the present invention for description, and does not have to
It is of the invention in limiting.
Illustrate the working principle of the invention and stream by taking the differentiation of typical budding yeast and non-budding yeast form as an example
Journey.
The differentiation principle of budding yeast and non-budding yeast is shown in attached drawing 1.Budding yeast is presented close to rod-shaped shape, is not gone out
Budding yeast is substantially spherical in shape.When budding yeast is flowed through in specific pipeline with specified flow velocity, posture can be adjusted to
The parallel direction of pipeline long axis is by detection position, so that the time of impedance pulse signal is more same when it passes through detection position
The non-budding yeast signal of volume increases.I.e. the more non-budding yeast pulse width (W1) of budding yeast pulse width (W2) is big；Together
When due to its volume usually, impedance signal pulse height (H2) more non-budding yeast pulse height big compared with non-budding yeast volume
(H1) also bigger；Since the increased amplitude of budding yeast pulse width is smaller and the increased amplitude of pulse height is bigger, and pulse is wide
Height ratio R value (R2) then becomes smaller compared to non-budding yeast R value (R1).It can be according to this series of difference to budding yeast and not
Budding yeast is effectively distinguished.
Embodiment 1: yeast budding is identified using pulse width histogram and calculates budding ratio
Implement according to the following steps:
Step 101, culture yeasts bacterium.For inoculation yeast bacterium to YPD culture medium, 30 DEG C of stationary cultures are multiple for 24 hours under aseptic condition
Soviet Union's strain.Aseptically take 100 μ L of recovery bacteria culture fluid be added 100mL YPD fluid nutrient medium in 30 DEG C,
Under the conditions of 180r/min culture 6~for 24 hours.
Step 102 takes a small amount of saccharomycete sample, dilutes the yeast cells liquid of culture with phosphate buffer (PBS), makes
The concentration of yeast cells is obtained 105/ mL or so；
Step 103, the impedance signal that yeast is acquired using electrical impedance streaming systems.It is required that the system can allow shaft-like particle
Pass through detection position one by one with the posture that long axis is parallel to pipeline central liquid flow direction, to obtain spherical with same volume
The particle longer time by detection position, showing has broader pulse width on impedance detection pulse signal.Specifically
Way can be with are as follows: after micro-fluidic chip connect and is powered with impedance detection instrument, draws detection sample with liquid-transfering gun and injects chip
Entrance pool makes to detect sample full of pipeline；Amplitude (200mV), the frequency of pumping signal are set by the control software in PC machine
Whether parameters such as (1MHz) are normal in software interface inspection acquisition signal.Connection auxiliary aspiration pump, adjusts and takes out at chip outlet bath
Air pump speed is 4 μ L/min, and yeast cells can be flowed by certain speed in the duct at this time, flows through central detection area one by one
Domain, each particle generate a pulse signal.
Step 104, system identify that the pulse height of each signal, width value are extracted storage to pulse signal.It adopts
The signal of collection is low-pass filtered, baseline tracking, pulse event touching method identification, pulse characteristics identification, to pulse signal into
Row identification, the pulse height of each signal, width value are identified, calculate and store.
The main thought (see attached drawing 1) of pulse recognition algorithm is:
1, it determines data baseline and tracks baseline.Current data point ordinate is compared with base-line data, in data
Fluctuation range, which is no more than under certain threshold condition, is considered baseline noise, takes the baseline number that a certain number of current data points are neighbouring
It is averaging together with current data according to (1000 points before such as current data) and is used as current baseline value (U1), and baseline value is carried out
It updates；
2, pulse starting point is identified.Current data point ordinate has been more than the threshold value set relative to the variation of baseline, it is believed that
It is a pulse event, starting point of the current point as a pulse signal records its horizontal, ordinate into pulse recognition state,
And current baseline value is set as equal with a upper baseline value and baseline value is updated；
3, pulse terminal is identified.When current data point ordinate is less than pulse terminal threshold value, it is believed that current PRF reaches eventually
Point.Cross, the ordinate for recording terminal, the feature searched element, calculate pulse, such as peak value of pulse, pulse width, will be all interested
Pulse information store in pulse event storage region (matrix).
4, baseline tracking mode is come back to.After recording complete pulse event, pulse recognition state is jumped out, base is returned to
Line tracking mode.It is equal when baseline value continues to be set as starting with pulse, terminate pulse recognition state, reenters step 1
Baseline more new state；
5, pulse parameter knows method for distinguishing.
5.1, peak value of pulse and pulse height are identified.One pulse ordinate maximum (small) from origin-to-destination all the points
Ordinate value be peak value of pulse, it is pulse height (U that peak value of pulse, which subtracts current baseline value,2-U1)；
5.2, pulse width is identified.The difference of abscissa value between one pulse origin-to-destination is pulse width (t2-
t1), usually as unit of the time；
5.3, pulse R value is calculated.By the width of a pulse divided by its height (R=(t2-t1)/(U2-U1)), obtain R
Value.
The determination of step 105, budding yeast recognition threshold.
A, according to abovementioned steps 103-104, the value for acquiring the pulse width of 100 typical budding active yeast cells is carried out
Statistics, obtains average value, standard deviation, the Poisson distribution curve of fitting, pulse width distribution histogram etc.；The budding yeast side of selecting
Method are as follows: observe under the microscope, typical budding yeast is considered if occurring apparent gemma on yeast cells；Such as yeast cells
It is upper without apparent gemma, cell is rounded, then it is assumed that is typical non-budding yeast.
The value of b, the pulse width of the typical single active yeast cell that do not sprout of same acquisition 100 are counted, and are obtained
Average value, standard deviation, the Poisson distribution curve of fitting, pulse width distribution histogram etc.；
C carries out numerical value to the pulse width statistical result of typical budding yeast and the typical active yeast cell that do not sprout
Profiles versus determines the parameter demarcation threshold of different type yeast.It can be by the signal pulse width of 200 yeast of all acquisitions
According to sequence arrangement from small to large, then making histogram, (ordinate is the quantity of the yeast in certain section, and abscissa is
Pulse width), it sees the distributed number situation in different pulse widths, two peaks, peak in general can be presented on the histogram
Indicate that the region yeast quantity is more.Usual of a sort cell histogram distribution shows the feature of normal distribution.It is two kinds thin
The setting method of born of the same parents' pulse width demarcation threshold with different according to specifically needing, for example can be set between two peaks
Demarcation threshold of the minimum as two primary yeast cells, greater than the threshold value, it is believed that be early stage budding yeast, less than threshold value
For non-budding yeast.Can also two peaks distance farther out when take budding yeast more stringent identification standard, with budding
The pulse width distribution mean value of yeast subtracts a standard deviation as threshold value, can just regard as budding yeast greater than the threshold value.
It can even determine and just regard as budding yeast greater than budding yeast pulse width average value, threshold value is the typical case of statistics at this time
Budding yeast pulse width average value.
Step 106, have been completed step 105 threshold value setting in the case where, pass through step 101-104 acquisition 10000
The pulse width information of a tested yeast cells is counted.
The pulse width demarcation threshold of step 107, the budding yeast that basis obtains and non-budding yeast is thin to tested yeast
The pulse width statistic histogram of born of the same parents is analyzed, and determines that pulse width is greater than the quantity of the budding yeast of threshold value；
Step 108 calculates the budding yeast quantity (M that step 107 determines0) account for the tested yeast quantity (M) of all acquisitions
Ratio, which is determined as " budding ratio " (calculation formula: )。
As shown in Fig. 2, pulse width histogram is identified and is calculated budding ratio to yeast budding, and S is not sprout
Yeast, L are budding yeast, and dotted line is distribution histogram and the matched curve of non-budding yeast, and solid line is advanced stage budding yeast point
Cloth histogram and matched curve, upper right illustration are the ROC curve of budding yeast and non-budding yeast, and ordinate TPR is kidney-Yang
Property rate, abscissa FPR be false positive rate.Fig. 2 is the differentiation figure of budding yeast and non-budding yeast.Fig. 2 shows that two primary yeasts are thin
False positive rate is very low (<5%) while the differentiation of born of the same parents can obtain very high budding yeast identification true positive rate (>95%).
The pulse width (62.2 ± 17.7ms) of budding yeast and typical non-budding yeast pulse width (27.7 ± 7.1ms) have significantly
It distinguishes, from histogram it can be seen that threshold value can be set in 40ms or so, it is budding yeast that pulse width, which is greater than 40ms, is less than
40ms is non-budding yeast.And threshold value setting can according to actual needs, such as guaranteeing true positive rate, the higher the better (such as > 99%),
Then threshold value will move to left, and be set to smaller (such as 30ms)；Guaranteeing that the lower false positive rate the better (such as < 1%), then threshold value will move to right,
It is set to bigger (such as 50ms).It is possible thereby to find, impedance pulse width can be distinguished the yeast of different shape.It is similar
Method, impedance magnitude, R value etc. can also distinguish the yeast of different shape.
Budding ratio can be total divided by yeast according to the budding yeast quantity that threshold value determines on the basis of differentiating forms,
Obtain budding ratio.
The yeast analysis method of this patent, the advantage for the proportion computing technology that sprouts are the method for realizing that budding yeast is distinguished
Simply, flux is high, it can be achieved that quickly automatic detection and budding ratio calculate.
Embodiment 2: yeast budding is identified using pulse height, R value scatter plot and calculates budding ratio under
Column step is implemented:
Step 101-104, it is same as Example 1.
The determination of step 105, budding yeast recognition threshold.If it have been determined that threshold value, then skip this step.
Specifically includes the following steps:
A, according to abovementioned steps 101-104, pulse height, the R value for acquiring 100 typical budding active yeast cells are carried out
Statistics；
B, same acquisition 100 typical do not sprout the pulse height of single active yeast cell, R value are counted；
C counts the pulse statistical result scatter plot of typical budding yeast and the typical active yeast cell that do not sprout
Distribution value comparison, determines the parameter demarcation threshold of budding yeast Yu non-budding yeast.It can be by the letter of 200 yeast of all acquisitions
Number respectively sequence arrangement by pulse height, R from small to large.Using pulse height, R as horizontal, ordinate, two of each particle
Parameter value describes the scatter plot of all yeast cells as its coordinate value in a coordinate system.With the pulse height of budding yeast
Distribution mean value subtracts a standard deviation as threshold value, can just regard as budding yeast greater than the threshold value；Simultaneously with budding yeast
R Distribution value mean value plus a standard deviation as threshold value, can just regard as budding yeast, two above item less than the threshold value
Part, which need to be provided simultaneously with, can just be considered budding yeast, and the lower right field that two dotted lines as shown in Fig. 3 intersect is only budding ferment
It is female.
Step 106-108, it is same as Example 1.
Attached drawing 3 is using pulse width > T0、R<R0(i.e. pulse height is greater than typical budding yeast statistical result lower limit
(such as average value-standard deviation), pulse the ratio of width to height are less than typical budding yeast statistical result lower limit (such as average value+standard deviation)) make
The density gradient scatter plot dividing method obtained for threshold value.In four regions that two threshold lines are constituted, the region in the lower right corner
For budding yeast (M0: R < R0And T > T0Cell number), quantity accounts for the ratio between all yeast total (M) as budding ratio
Embodiment 3: the culture of yeast is instructed with the budding ratio measured
The budding ratio of detection can be used to instruct the culture and fermentation industry of yeast.If following experimental result is with regard to table
Bright, yeast has different values in the budding ratio of different cultivation stages, reflects the proliferative capacity and vigor state of yeast,
Show whether the culture environment locating for it is suitable for quickly breeding.
Step 1, culture yeasts bacterium.Inoculation yeast bacterium to YPD culture medium, 30 DEG C of stationary cultures is recovered for 24 hours under aseptic condition
Strain.100 μ L of recovery bacteria culture fluid is aseptically taken to be added in the YPD fluid nutrient medium of 100mL in 30 DEG C, 180r/
48h is cultivated under the conditions of min.
Step 2, using by recovery bacterium solution injection culture medium as starting point, choose 0,6,12,18,24,30,48h as detection
Time point carries out the detection of bacterium solution absorbance value to the yeast of different time nodes,
Step 3 and step 2 simultaneously, the unicellular stream signal of yeast, actual conditions are acquired using electrical impedance streaming systems
Are as follows: a small amount of saccharomycete sample is taken, is diluted the yeast cells liquid of culture with phosphate buffer (PBS), so that yeast cells
Concentration is 105/ mL or so.
After step 4, micro-fluidic chip and impedance detection instrument are connected and be powered, detection sample is drawn with liquid-transfering gun and injects core
Piece entrance pool makes to detect sample full of pipeline；
The ginseng such as the amplitude (200mV) of pumping signal, frequency (1MHz) is arranged in step 5, the DDS control software opened in PC machine
Whether normal number observes the signal acquired in software interface.
Connection auxiliary aspiration pump at step 6, chip outlet bath, adjusting pumping pump speed is 4 μ L/min, at this time yeast cells
It can be flowed in the duct by certain speed, flow through central detection area domain one by one, each particle generates a pulse signal.
Step 7 identifies that the pulse height and pulse width and R value of each signal are extracted storage to pulse signal.
Step 8, each time point repeat step 3-7, finally draw different time nodes and dissipate in relation to the density of yeast cells
Point diagram determines budding ratio, and the absorbance detected in conjunction with step 2 according to the budding yeast parameter threshold that embodiment 2 determines
Be worth draw Yeast proliferation curve and budding ratio change curve, sum up yeast sprout in proliferating cycle ratio the case where.
Budding yeast bound of parameter described in 2 in conjunction with the embodiments, we are selected by the statistical result of actual sample detection data
Pulse height=0.0758 V, R=4140 is taken to be divided to the boundary value of two class cell colonys as group, pulse height>0.0758, R<
4140 be considered as budding yeast cell.Yeast is calculated by the identification of the impedance pulse data scatter plot to acquisition to exist
Proliferation each stage (0,6,12,24,30,48h) yeast budding ratio (42.57%, 54.06%, 71.99%,
21.75%, 26.27%, 4.83%).
The increment curve of yeast is the growth curve for being similar to " S " type, and absorbance detection result will meet with this, ferment
Mother belongs to increased logarithmic phase in early period, by 24 hours after be proliferated and gradually slow down.Yeast is opposite in logarithm growth period budding ratio
Higher, with the stationary phase and decline phase for entering Yeast proliferation process, budding ratio is gradually decreased.This may be due to cultivating body
Caused by the toxic side effect that subalimentation and metabolism in system generate.Budding ratio and yeast cells through testing result calculating
Vegetative state is related.Therefore it will can further be somebody's turn to do " budding ratio " and be used to judge the proliferative capacity and growth rate of yeast, with
And the growth conditions of yeast, to instruct adjustment Yeast Cultivation environment.Specific judgment basis can be with are as follows: budding ratio is higher than 40%
As the preferable threshold value of culture environment, ratio of sprouting is lower than 30% threshold value poor as culture environment, and budding ratio is lower than 5%
The threshold value very poor as culture environment.These specific numerical value can be adjusted according to actual needs, to instruct to ferment
The processes such as industry.
Embodiment 4: electrical impedance flow cytometer detection system and its for ratio detection example of sprouting
The detection of yeast bud ratio is carried out using step 101-108 same as Example 1, used in electrical impedance detection
Micro-fluidic chip can be defined by patent ZL201610962524.8.
Micro-fluidic electrical impedance system described in this patent carries out shape and distinguishes detection, can be by number of patent application
201810930070.5 publication defined.
When carrying out step 105 same as Example 1, when on single histogram (pulse height, width or R histogram)
Budding yeast can be formed when being significantly divided to group (two peaks) with other yeast and can be put down with the statistics of typical budding yeast is assert
Group representated by that immediate peak of mean value is budding yeast；When single histogram cannot obviously distinguish typical budding with not
When budding yeast, the mode of 2 parameter combinations or 3 parametric joint given thresholds can be used to determine budding yeast.Specifically
For, can the statistical nature of typical budding yeast be used as benchmark, pulse width is greater than under typical case's budding yeast statistical result
Limit (such as average value-standard deviation), pulse height is greater than typical budding yeast statistical result lower limit (such as average value-standard deviation), R
Threshold value less than typical budding yeast statistical result lower limit (such as average value+standard deviation) as streaming scatter plot, 2 parameter combinations
Mode can be pulse height, width, optional two kinds in R three, while meeting respective parameter threshold condition and regarding as sprouting
Yeast, 3 parametric joints, which can be, on the scatter plot of aforementioned 3 parametric joints while to meet recognizing for 3 parameter threshold conditions
It is set to budding yeast.
The ratio that budding yeast quantity accounts for all yeast quantity is finally calculated, which is determined as " budding ratio ".
Itself of the parameter thresholds such as pulse width, height, R is it is determined that method.The setting of parameter threshold can be according to specific
It needs and is adjusted.To budding yeast identification need to realize with the best differentiation of non-budding yeast, false positive and false negative it
With it is minimum when can use embodiment 1,2 method；When need accomplish assert budding yeast false positive it is lower, true positives compared with
Height, when without considering false negative, the mean parameter for the closer typical budding yeast for needing to set threshold value；, whereas if needing
False negative is lower, and when not considering false positive, threshold value setting can be closer to the mean parameter of typical non-budding yeast.In addition, also
The position that crosses of matched curve of products of typical yeast distribution can be chosen as threshold value, also can choose the comparable two kinds of typical cases of quantity
It sprouts, the minimum point on the fitting of distribution curve of non-budding yeast summation between two peaks is as threshold value.When tested yeast
The condition of test condition and acquirement threshold value is not it is also possible to which the pulse width mean value for choosing typical budding yeast subtracts a mark
Quasi- poor (33.71-6.25=25.46ms) as the pulse-width threshold (attached drawing 4) for determining budding yeast, pulse width is greater than
25.46ms is budding yeast, is non-budding yeast less than 25.46ms.
The present invention is applicable not only to the detection of yeast cells, is equally applicable to for other biological particle of different shapes
The detection and classification of (such as haemocyte, cancer cell, bacterium), and may be implemented 3 kinds or 3 kinds of above approximately it is spherical with it is rod-shaped thin
The differentiation of born of the same parents' form, and lay the foundation to further realize the cell sorting based on cellular morphology.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. it is a kind of based on electrical impedance flow cytometer detection Yeast protoplase and sprout ratio method, it is characterised in that: this method include with
Lower step:
Step 101, culture yeasts bacterium；
Step 102, the yeast cells liquid cultivated with buffer dilution；
Step 103 is mutually sympathized in detected sample flow, sample liquid conductivity, exciting signal frequency and the basic parameters such as amplitude
Under condition, using electrical impedance flow cytometer detection system acquisition yeast sample impedance signal, which guarantees to allow budding yeast cell
With the posture that long axis is parallel to pipeline central liquid flow direction pass through detection position one by one, does not go out to obtain with same volume
The budding yeast longer time by detection position, showing has broader pulse width on impedance detection pulse signal；
Step 104, electrical impedance flow cytometer detection system to impedance magnitude, phase, resistance, capacitor, real part, imaginary part isopulse signal into
Row identification, calculates and stores the pulse height, pulse width and the ratio of width to height of each signal, i.e. width ratio, be abbreviated as R；
Typical budding yeast is selected under step 105, microscope, select on mother cell have macroscopic sprout not yet with mother cell
It separates and does not regard as typical budding yeast with the individual cells of other cytoadherences, pass through step 103 and step 104 acquires
The value of the parameters such as pulse width, height, the R of typical budding active yeast cell is counted；
Typical non-budding yeast is selected under step 106, microscope, select the sprout being visible by naked eyes on cell and not with it is other
Cytoadherence, approximate spheres individual cells regard as typical non-budding yeast, typical case is acquired by step 103 and step 104
The values of the parameters such as pulse width, height, the R of the active yeast cell that do not sprout counted；
The statistical result of the parameters such as width, height, the R of step 107, typical budding identical to quantity and non-budding yeast cell
Numeric distribution comparison is carried out, determines the parameter demarcation threshold of budding yeast Yu non-budding yeast；
Step 108 repeats step 101 to step 104, acquires pulse width, height, the ratio of width to height of yeast cells in tested sample
The information such as R are counted；
The parameter demarcation threshold of step 109, the budding yeast and non-budding yeast that are obtained according to step 105 to step 107, to quilt
The distribution of the parameters such as pulse width, height, the ratio of width to height of this yeast cells of test sample is for statistical analysis, determines budding yeast and not
The parameter area of budding yeast and the quantity for assert and distinguishing yeast cells form and budding yeast, non-budding yeast accordingly；
Step 110 calculates the ratio that the budding yeast quantity that step 109 determines accounts for all yeast quantity of the tested sample of acquisition
The ratio is determined as " budding ratio " by value.
2. a kind of method based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 1,
It is characterized by: the typical budding yeast of the identical quantity with statistical significance and typical non-budding yeast are counted institute respectively
Pulse width, height, between R average value, the smallest pulse width of the sum of two primary yeast quantity, height, the ratio of width to height R value point
It is not determined as pulse width, the height, R threshold value of the boundary of two primary yeast forms.
3. a kind of method based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 1,
It is characterized by: the typical budding yeast of the identical quantity with statistical significance and typical non-budding yeast set are united respectively
Pulse width that meter result is fitted, height, on R Distribution value curve, pulse width, height, the R value between two peak values are minimum
Value is determined as pulse width, the height, R value threshold value of budding yeast and the boundary of non-budding yeast.
4. a kind of side based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 2 or 3
Method, it is characterised in that: the foundation using the pulse width of tested sample yeast cells, height, R Data-Statistics result as analysis,
Width threshold value is greater than with pulse width respectively or pulse height value is greater than height threshold or R value is less than the yeast cells of R threshold value
Budding yeast is regarded as, and counts the quantity of the range endogenous budding yeast, budding yeast is calculated and accounts for tested sample yeast quantity
Ratio, and as " budding ratio ".
5. a kind of side based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 2 or 3
Method, it is characterised in that: the foundation using the pulse width of tested sample yeast cells, height, R Data-Statistics result as analysis,
Respectively with pulse width be greater than width threshold value and pulse height value be greater than height threshold or pulse width be greater than width threshold value and
R value is less than that R threshold value or pulse height are greater than height threshold and R value is less than the yeast cells of R threshold value and regards as budding yeast,
And the quantity of the range endogenous budding yeast is counted, the ratio that budding yeast accounts for tested sample yeast quantity is calculated, and as " budding
Ratio ".
6. a kind of side based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 2 or 3
Method, it is characterised in that: the foundation using the pulse width of tested sample yeast cells, height, R Data-Statistics result as analysis,
It is greater than width threshold value with pulse width and pulse height value is greater than height threshold and R value is regarded as less than the yeast cells of R threshold value
Budding yeast, and the range endogenous budding yeast quantity is counted, it calculates budding yeast and accounts for the ratio of tested sample yeast quantity, and make
For " budding ratio ".
7. a kind of method based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 1,
It is characterized by: being scattered in the concentration of the yeast cells of buffer 103~108/ mL acquires 20~5000 typically respectively
Budding active yeast cell, 20~5000 do not sprout typically active yeast cells, 20~20000 tested sample yeast cells arteries and veins
Rush the information such as width, height, R.
8. a kind of method based on electrical impedance flow cytometer detection Yeast protoplase and yeast budding ratio according to claim 1,
It is characterized by: detecting the budding of other tested yeast samples after determining parameter threshold to step 107 by step 105
Step 105 is skipped when ratio to step 107, is directly carried out step 108 after step 104, is made using fixed parameter threshold
Yeast protoplase, which is assert, for standard and completes budding ratio calculates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910437406.9A CN110257234A (en) | 2019-05-24 | 2019-05-24 | A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910437406.9A CN110257234A (en) | 2019-05-24 | 2019-05-24 | A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110257234A true CN110257234A (en) | 2019-09-20 |
Family
ID=67915321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910437406.9A Pending CN110257234A (en) | 2019-05-24 | 2019-05-24 | A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110257234A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104812910A (en) * | 2012-10-18 | 2015-07-29 | 奈克塞勒姆生物科技有限责任公司 | Automated yeast budding measurement |
| CN105866011A (en) * | 2016-03-31 | 2016-08-17 | 艾康生物技术(杭州)有限公司 | Pulse baseline value calculation method and particle counting method for blood cell analyzer |
| CN206577776U (en) * | 2016-10-28 | 2017-10-24 | 中国人民解放军军事医学科学院卫生装备研究所 | Electrical impedance flow cytometer detection molecule, the micro-fluidic chip of cell |
| CN108872047A (en) * | 2018-08-15 | 2018-11-23 | 军事科学院系统工程研究院卫勤保障技术研究所 | The system and method for fine particle shape is distinguished in a kind of micro-fluidic electrical impedance detection |
-
2019
- 2019-05-24 CN CN201910437406.9A patent/CN110257234A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104812910A (en) * | 2012-10-18 | 2015-07-29 | 奈克塞勒姆生物科技有限责任公司 | Automated yeast budding measurement |
| US20150232906A1 (en) * | 2012-10-18 | 2015-08-20 | Nexcelom Bioscience Llc | Automated yeast budding measurement |
| CN105866011A (en) * | 2016-03-31 | 2016-08-17 | 艾康生物技术(杭州)有限公司 | Pulse baseline value calculation method and particle counting method for blood cell analyzer |
| CN206577776U (en) * | 2016-10-28 | 2017-10-24 | 中国人民解放军军事医学科学院卫生装备研究所 | Electrical impedance flow cytometer detection molecule, the micro-fluidic chip of cell |
| CN108872047A (en) * | 2018-08-15 | 2018-11-23 | 军事科学院系统工程研究院卫勤保障技术研究所 | The system and method for fine particle shape is distinguished in a kind of micro-fluidic electrical impedance detection |
Non-Patent Citations (1)
| Title |
|---|
| 曾杨清: "《食品微生物检验技术》", 31 January 2016, 西南交通大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Paul et al. | Viability testing and characterization of germination of fungal spores by automatic image analysis | |
| US7796815B2 (en) | Image analysis of biological objects | |
| CN102507417B (en) | Method for automatically classifying particles | |
| CN105940301B (en) | A kind of stream type cell analyzer and its multidimensional data sorting technique, device | |
| CN107832801B (en) | Model construction method for cell image classification | |
| CN102324092B (en) | Method for automatically cutting granular object in digital image | |
| CN108052886B (en) | A kind of puccinia striiformis uredospore programming count method of counting | |
| CN109684938A (en) | It is a kind of to be taken photo by plane the sugarcane strain number automatic identifying method of top view based on crop canopies | |
| CN107490672B (en) | method for rapidly analyzing crustacean hemolymph cell group and number and application | |
| Guez et al. | Real time in situ microscopy for animal cell-concentration monitoring during high density culture in bioreactor | |
| CN114723704A (en) | Textile quality evaluation method based on image processing | |
| CN106780347B (en) | Early loquat bruise identification method based on OCT image processing | |
| Wang et al. | Improved rotation kernel transformation directional feature for recognition of wheat stripe rust and powdery mildew | |
| CN109613526A (en) | A kind of point mark filter method based on support vector machines | |
| CN109815870A (en) | The high-throughput functional gene screening technique and system of cell phenotype image quantitative analysis | |
| CN105424560A (en) | Automatic quantitative analysis method for data of flow-type particle instrument | |
| Akkoyun et al. | Rapid characterization of cell and bacteria counts using computer vision | |
| CN106326914B (en) | A kind of more classification methods of pearl based on SVM | |
| CN110257234A (en) | A method of based on electrical impedance flow cytometer detection Yeast protoplase and budding ratio | |
| CN108875541A (en) | A kind of visual fatigue detection algorithm based on virtual reality technology | |
| CN105740918A (en) | Otolith measurement and fish population discriminating method based on polar coordinate | |
| Yumang et al. | Bacterial Leaf Blight Identification of Rice Fields Using Tiny YOLOv3 | |
| CN105624295A (en) | Minutocellus polymorphus fluorescent quantitative PCR detection method | |
| Thomas et al. | A review on white blood cells segmentation | |
| US20180171376A1 (en) | Method for evaluation of grafts |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190920 |
|
| WD01 | Invention patent application deemed withdrawn after publication |