CN103525896A - Quantitative high-vitality yeast cell screening method based on TTC (2,3,5-triphenyltetrazolium chloride) staining method - Google Patents
Quantitative high-vitality yeast cell screening method based on TTC (2,3,5-triphenyltetrazolium chloride) staining method Download PDFInfo
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Abstract
The invention discloses a quantitative high-vitality yeast cell screening method based on a TTC staining method. The quantitative high-vitality yeast cell screening method comprises the steps as follows: coating a plate with a yeast cell liquid to be detected, and culturing the yeast cell liquid until single colonies grow; then inoculating the yeast cell liquid in a culture solution in each hole of a 96-hole cell culture plate, culturing the yeast cell liquid until OD values of thalli obtained through measurement are in a range of 1-3, centrifuging the liquid and abandoning supernatant; adding a TTF extracting agent to each hole of the 96-hole cell culture plate, and oscillating the liquid for 10-15S before centrifugation; and absorbing oscillated and centrifuged supernatant in a 96-hole EKISA plate, measuring a TTF absorption value under the wavelength of 486nm through an EKLSA reader, and calculating the content of TTF in each hole according to a TTF standard curve and the measured cell OD values to determine the vitality of cells. Screening results reflect the vitality of the cells directly and reliably, the screening time can be reduced obviously, and human errors caused by visual judgments during an operation process can also be reduced.
Description
Technical field
The present invention is applicable to biotechnology and medical science, pharmaceutical field, is specifically related to a kind of high vigor yeast quantitative screening method based on TTC staining.
Background technology
2,3,5-triphenyltetrazolium chloride (TTC) is a kind of developer, can there is color reaction to various kinds of cell (yeast, bacterium, vegetable cell etc.), TTC this as colourless, when its with normal cell in dehydrogenase reaction and take on a red color, its principle is TTC alternative O in cellular respiration
2accept H
+, TTC is reduced to red TTF(TF) and present redness.Reaction equation is as follows:
Due to above color reaction, TTC, as the proton acceptor of pyridine-nucleosides structure enzyme system in respiratory chain, can be reduced to red TTF by colourless oxidation state TTC by the respiratory chain dehydrogenase in viable cell, so, TTC staining is usually used to measure the vigor of cell
TTC was used for detecting the viability of seed first in 1894, within 1958, be once used to staining examine mammal brain and be organized in the lower cellular form of ischemic infraction, infarct size etc., reflected that the ischemic of cerebral tissue blocks severity.TTC dyeing appearance method is widely used in the detection of seed cell, bacterial respiratory activity etc. at present, is also applied to the screening of high-yield ethanol yeast.Liu Hua, the plum people such as make the country prosperous utilizes TTC method to measure yew cell vigor; The people such as Yang Tianyou utilize the impact of TTC-ldh assay method research cryogenic vacuum environment on bacterial respiratory activity; The people such as Dong Boyu by TTC staining screening ultraviolet mutagenesis after the shehatae candida of high-yield ethanol.The TTF producing in color reaction is in cell, and water insoluble, and TTF need measure its light absorption value through organic solvent extraction under certain wavelength, and what its absorption value size had reflected cytolemma electron transport chain passs hydrogen capacity of water.The red depth that in cell, TTC reaction presents has reflected the power of its vitality, and it is stronger that color is more deeply felt bright its vigor.In yeast cell, the main path of ethanol fermentation comprises the anaerobic degradation of glucose glycolysis and pyruvic acid, its Main Function of sugar-ethanol conversion enzyme in this process, TTC can produce color reaction to the meta-bolites of yeast, by the depth of its color, can judge the size of invertase activity in its born of the same parents, it is stronger that color is more deeply felt bright its cytoactive, and producing and ethanol ability is also stronger.
In existing these researchs, also there is a lot of defects or deficiency.The firstth, the flat panel production that contains TTC substratum is complicated, and after inoculation yeast, growth fraction is slower, and during the darker yeast of follow-up picking color, operates also more loaded down with trivial detailsly, takes longer; The secondth, by the color reaction of TTC staining, thereby the depth of utilizing human eye observation reaction color judges the height of cell viability prediction producing Yeast ethanol, because cell to be screened is various, its defect is: 1. cannot be quantitative, by naked eyes, judge and cannot accurately judge resultant content, and be easy to produce fatigue and mistake, and from different culture plates, screening the high vigor cell of gained can be because the limitation of human eyesight causes erroneous judgement, and the cell that final screening is obtained may not be required high vigor high-yield ethanol cell; While 2. screening a large amount of cell, workload is large, takes time and effort, and affects cell screening progress.
High flux screening (High throughput screening, HTS) technology be take the experimental technique of molecular level and cell levels and is basis, utilize 96 orifice plates and microplate reader as experimental tool carrier, with sensitive detecting instrument fast, gather experimental result data, at one time the interior a large amount of samples of detection by quantitative.It has the features such as quantitative, quick, sensitive and accurate.Utilize High Throughput Screening Assay to be widely used at present the fields such as drug screening, and obtained obvious progress.Aspect microbial strains seed selection, in bibliographical information, also there is Many researchers to utilize 96 orifice plates to carry out seed selection work, as the people such as Zhang Jilin utilize 96 orifice plate breeding high-yield ethanol yeasts, the people such as Cai Wanlin utilize 96 orifice plates and yeast saccharomyces cerevisiae growth circle to meet screening high yield pipe capsule yeast etc.But in these researchs, also there are many weak points, be that these bibliographical informations are just undertaken transferring to 96 orifice plate the insides with the detection of thalline number in analytical unit volume in cuvette in microorganism culturing, in follow-up study, all need the higher bacterial strain of OD value of growing in Tissue Culture Plate 96 holes to coat plate culture medium growth, recycle other screening methods and carry out high-yield ethanol bacterial strain screening, take length, as Cai Wanlin utilize 96 orifice plates and yeast saccharomyces cerevisiae growth circle meet screening high yield pipe capsule yeast in the follow-up yeast screening assay needs 7-9 days that grows.So, though these methods have been utilized 96 orifice plates, but obviously do not reduce working hour and workload, key is also, the result that these methods are surveyed when utilizing 96 orifice plate be only to cell colony density quantitatively, reflection be cell in certain growth the propagation situation in the cycle, the not vitality of individual cells.Therefore, need to improve rapidity, accuracy and the simplicity in cell screening process.
Summary of the invention
The high vigor yeast high-throughput screening method providing based on TTC staining and microplate reader and 96 orifice plates is provided, the method is quantitative by microplate reader, rapid detection, can effectively improve screening efficiency and the screening accuracy of high reactivity cell, with this, improve Large-scale Screening cell work amount large, progress is slow, the deficiency that accuracy is low.
For achieving the above object, the technical solution used in the present invention is:
A high vigor yeast cell quantitative screening method for TTC staining, comprises following steps:
1) yeast cell to be detected is cultivated: yeast cell bacterium liquid to be detected is coated flat board, cultivates and treats that it grows single bacterium colony; The incubation time of different strains of Yeast differs, and conventionally cultivates 48-72h;
2) cell list colony lift to 96 porocyte culture plate.Single colony inoculation of cultivating in step 1), in each hole nutrient solution of 96 porocyte culture plates, is added with to TTC in nutrient solution, is cultured to mensuration and obtains thalline OD value between 1-3, the centrifugal supernatant of abandoning; Then in 96 each holes of porocyte culture plate, add TTF extraction agent, centrifugal after concussion 10-15S; According to different strains of Yeast, select nutrient solution, yeast cell is cultivated in the nutrient solution using and is added TTC to finite concentration under normal conditions.
3) by step 2) in the supernatant liquor of concussion after centrifugal be drawn in 96 hole enzyme plates, utilize microplate reader under 486nm, to measure TTF absorption value, according to TTF typical curve and step 2) the middle cell OD value of measuring, calculate TTF content in each hole, with this, determine cell viability size.TTF content is higher, shows that cellular respiration vigor is stronger.The high bacterial strain of screening TTF content is the yeast cell of high vigor.
Step 2) in nutrient solution, TTC concentration is 0.3%-0.5%(W(g)/V (mL)), pH is 6-8.
Step 2) extraction agent of TTF described in is dimethyl sulphoxide aqueous solution, and concentration range (dimethyl sulfoxide (DMSO) and water mixed volume ratio) is: 85%-100%(V/V).
Step 2) in, TTF extraction agent and cell culture fluid volume ratio are 1:1.
This method, including, but not limited to 96 orifice plate Tissue Culture Plates, is also applicable to other porous culture plates and cultivates, for example 384 porocyte culture plates; Present method is not limited to the screening of yeast cell, is equally applicable to the vigor screening of other microorganisms, vegetable cell, zooblast etc.
Beneficial effect: the high vigor cell high-throughput screening method based on TTC staining and microplate reader and 96 orifice plates of the present invention, compare and have the following advantages with conventional sense method:
1) accuracy: because the TTF generating is at temperature 20-40 ℃, pH6-8, equal energy stable existence in 24h, and can by microplate reader accurately, rapid detection, the selection result is direct reaction cell viability reliably quantitatively, screening time can not only be obviously reduced, and the mistake that the visual determination in operating process produces can be reduced;
2) high efficiency: based on 96 orifice plates, can high-throughput rapid detection, in Tissue Culture Plate, carry out only needing to measure its OD value and TTF growing amount after cell cultures, whole mensuration process only needs 1-2h, without expending the plenty of time, and the method is not only confined to 96 orifice plates, also can develop into other Tissue Culture Plates such as 384 holes;
3) present method operational condition is not harsh, TTC concentration in operating process, pH controls, dimethyl sulfoxide (DMSO) concentration, in follow-up 96 orifice plates, cell OD value etc. has certain operating restraint, without preparation especially accurately, each step is simple to operate, to instrument, reagent etc. require lower, are convenient to follow-up laboratory of promoting the use of in different condition;
4) economy: microwell plate is measured, and agents useful for same etc. are more saved compared with conventional sense.The dimethyl sulfoxide (DMSO) of using in present method is cheap, and effect of extracting is good, has saved testing cost, and present method can be used as a kind of quick, effective, reliable, the economic means that detect cell viability.
This detection method can further detect the power of its producing and ethanol ability when detecting the active power of yeast cell.
Accompanying drawing explanation
Fig. 1: TTF typical curve.
Embodiment
The following examples elaborate to the present invention, but to not restriction of the present invention.
embodiment 1
The high flux screening of high vigor yeast
The drafting of 1.TTF typical curve
Get respectively 0.005,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1%(W(g)/V (mL)) TTC solution 1ml adds in 10ml centrifuge tube, adds the Tris-HCl(pH8.4 of 2ml), finally add 10%(W(g)/V (mL)) and Na
2s
2o
4solution 1ml, 37 ℃ of all in the dark operations of the above step of water-bath concussion 5min(), make TTC be reduced to TTF completely, 10000r/min is centrifugal, and 10min abandons supernatant, adding 5ml concentration is 85%(v/v) dimethyl sulphoxide solution fully mixes, in 486nm place, survey light absorption value, and to take mixed solution be ordinate zou in 486nm place light absorption value, using TTC concentration as X-coordinate drawing standard curve, the typical curve that drafting obtains is shown in Fig. 1: from typical curve, can find out: when TTC concentration is during at 0.01-0.06%, OD value is in 0.15-0.88 scope, both are good linear relationship, can be for quantitative assay.Typical curve is for the TTF value detecting Hemapoiesis drops in a believable interval, we of this interval, does not think that this value is insincere.
2. yeast cell to be detected is cultivated: get 100 μ L yeast cell bacterium to be detected liquid (Tang Gou Wine Co., Ltd provides by Jiangsu) and coat on YPD solid plate substratum, cultivate 48h, treat that it grows single bacterium colony for 30 ℃; YPD solid culture based formulas wherein: 1% yeast powder, 2% peptone, 2% glucose, 2% agar powder;
3. cell list colony lift to 96 porocyte culture plate: by single colony inoculation of above-mentioned cultivation in each hole nutrient solution of 96 porocyte culture plates, in each hole, nutrient solution volume is 150 μ L, nutrient solution is YPD nutrient solution, (YPD liquid culture based formulas: 1% yeast powder, 2% peptone, 2% glucose), in nutrient solution, TTC concentration is 0.4%(W(g)/V (mL)), nutrient solution pH6-8, alcohol concn is set to respectively 0%, 10%, 14%, add the ethanol of different concns, in order to see the cell viability of bacterial strain under different ethanol concentration, ethanol is toxic to cell, concentration is higher, active just less, according to concentration setting, both can detect the cell viability of same strain bacterium in the situation that having or not ethanol and alcohol concn high or low by this method of TTC, can also detect and compare the vigor of different strains under same alcohol concn, the convenient bacterial strain of selecting resistance to ethanol, culture condition: 30 ℃, 200r/min cultivates 4h, measure its cell OD value, make cell OD value between 1-3, cell OD value in TTF concentration now between 1-3 between 0.15-0.88, light absorption value and its concentration of measuring TTF are good linear relationship, quantitatively more accurate, after the centrifugal 5min of 4000r/min, abandon supernatant, then in 96 each holes of porocyte culture plate, adding 150 μ L concentration is 85%(v/v) dimethyl sulphoxide solution, concussion 15s after the centrifugal 5min of 4000r/min,
4. with the volley of rifle fire, the supernatant liquor shaking in above-mentioned Tissue Culture Plate after centrifugal is drawn to 100 μ L in 96 hole enzyme plates, under 486nm, measure TTF absorption value, according to the cell OD value of measuring in TTF typical curve and step 3, calculate cell TTF content=experiment and record TTF absorption value * (1/ cell OD
600absorption value), with resulting TTF value in the bent scope of mark, determine that cellular respiration power is cell viability size.TTF content is higher, shows that cellular respiration vigor is stronger.Measure the ethanol production of each strain bacterial strain (mensuration of ethanol production adopts gas phase marker method, and GC conditions is, pillar model: SGE 30QC2/AC20-0.25 capillary column simultaneously; Post case temperature: 60 ℃; Sampler temperature: 190 ℃; Detector temperature: 240 ℃.Internal standard substance: n-propyl alcohol.Detect liquid collocation method: (0.5ml 6% n-propyl alcohol solution+0.5ml sample) is settled to 10ml.Sample size: 2ul), observe the relation of TTF value and ethanol production, finally filter out the 6 strain bacterial strains with stronger vigor, by cell viability power, be arranged in order as #5, #1, #8, OY-11, #7, #6, experimental result is in Table 1.
Table 1
As can be seen from Table 1: at alcohol concn, be 0%, 10%, in the time of 14%, bacterial strain 5#TTF absorption value is the highest, its activity is the strongest in all bacterial strains of test as seen, and other bacterial strains are followed successively by 1# by active power, 8#, OY-11,7#, 6#.Measuring bacterial strain ethanol production, the ethanol production of bacterial strain 5# is also for the highest simultaneously, and other bacterial strains are just followed successively by 8# by ethanol production, 1#, and OY-11,7#, 6#, can find out, the cell viability of bacterial strain is into positively related with ethanol production to a certain extent.
Present method operational condition is not harsh, and in operating process, TTC concentration is at 0.3%-0.5%(W(g)/V (mL)), dimethyl sulfoxide (DMSO) concentration is 85%-100%(V/V) and can, without preparation especially accurately, each step is simple to operate, and to instrument, reagent etc. require lower.
This method, including, but not limited to 96 orifice plate Tissue Culture Plates, is also applicable to other porous culture plates and cultivates, for example 384 porocyte culture plates; Present method is not limited to the screening of yeast cell, is equally applicable to the vigor screening of other microorganisms, vegetable cell, zooblast etc.
Claims (4)
1. the high vigor yeast cell quantitative screening method based on TTC staining, is characterized in that comprising the following steps:
1) yeast cell bacterium liquid to be detected is coated to flat board, cultivate and treat that it grows single bacterium colony;
2) by single colony inoculation of cultivating in step 1) in each hole nutrient solution of 96 porocyte culture plates, be cultured to measure and obtain thalline OD value between 1-3, the centrifugal supernatant of abandoning; Then in 96 each holes of porocyte culture plate, add TTF extraction agent, centrifugal after concussion 10-15S;
3) by step 2) in the supernatant liquor of concussion after centrifugal be drawn in 96 hole enzyme plates, utilize microplate reader under 486nm wavelength, to measure TTF absorption value, according to TTF typical curve and step 2) the middle cell OD value of measuring, calculate TTF content in each hole, with this, determine cell viability size.
2. the high vigor yeast cell quantitative screening method based on TTC staining according to claim 1, is characterized in that: step 2) in nutrient solution TTC concentration be 0.3%-0.5%(W(g)/V (mL)), pH is 6-8.
3. the high vigor yeast cell quantitative screening method based on TTC staining according to claim 1, is characterized in that step 2) described in TTF extraction agent be dimethyl sulphoxide solution, concentration range is 85%-100%(V/V).
4. the high vigor yeast cell quantitative screening method based on TTC staining according to claim 1, is characterized in that step 2) in TTF extraction agent and cell culture fluid volume ratio be 1:1.
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| CN103808678A (en) * | 2014-02-25 | 2014-05-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for measuring cell activity and application of kit |
| CN106442275A (en) * | 2016-09-18 | 2017-02-22 | 中国人民武装警察部队总医院 | Method and kit for qualitatively detecting chyle |
| MD4465C1 (en) * | 2014-08-22 | 2017-08-31 | Государственный Университет Молд0 | Process for determining the dehydrogenase activity in fermentation biomass |
| CN111175407A (en) * | 2020-02-11 | 2020-05-19 | 道道全粮油股份有限公司 | Method for detecting residual quantity of organic solvent in leached oil crop meal |
| CN111693475A (en) * | 2020-07-28 | 2020-09-22 | 扬州大学 | Improved method for measuring activity of plant root system in sections |
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