CN103525896B - High-activity yeast cell quantitative screening method based on TTC staining method - Google Patents
High-activity yeast cell quantitative screening method based on TTC staining method Download PDFInfo
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Abstract
The invention discloses a high-activity yeast cell quantitative screening method based on a TTC staining method, which comprises the steps of coating yeast cell bacterial liquid to be detected on a flat plate, and culturing until a single bacterial colony grows out; then inoculating the strain in culture solution of each well of a 96-well cell culture plate, culturing until the OD value of the obtained thallus is 1-3, centrifuging and removing supernatant; adding TTF extractant into each hole of a 96-hole cell culture plate, shaking for 10-15S, and centrifuging; and absorbing the supernatant subjected to oscillation centrifugation into a 96-well enzyme label plate, measuring the TTF absorption value by using an enzyme label instrument at the wavelength of 486nm, and calculating the TTF content in each well according to the TTF standard curve and the measured cell OD value so as to determine the cell activity. The screening result of the method quantitatively and reliably reflects the cell activity directly, so that the screening time can be obviously shortened, and human errors generated by visual judgment in the operation process can 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, color reaction can be there is to various kinds of cell (yeast, bacterium, vegetable cell etc.), TTC, originally as colourless, takes on a red color when the dehydrogenase reaction in itself and normal cell, and its principle is that TTC substitutes 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 in respiratory chain-nucleotide structure enzyme system, can be reduced to red TTF by the respiratory chain dehydrogenase in viable cell by colourless oxidation state TTC, so, TTC staining is usually used to the vigor measuring cell
TTC was used in 1894 detecting the viability of seed first, within 1958, was once used to the cellular form, infarct size etc. of staining examine mammalian brain under ischemic infraction, reflected the ischemic infraction severity of cerebral tissue.Current TTC dyeing appearance method is widely used in the detection of seed cell, bacterial respiratory activity etc., is also applied to the screening of high-yield ethanol yeast.Liu Hua, the plum people such as to make the country prosperous utilizes TTC method to measure yew cell vigor; The people such as Yang Tianyou utilize TTC-ldh assay method research low-temperature vacuum environment on the impact of bacterial respiratory activity; The people such as Dong Boyu are by the shehatae candida of high-yield ethanol after TTC staining screening ultraviolet mutagenesis.The TTF produced in color reaction is in cell, and water insoluble, and TTF through organic solvent extraction, need measure its light absorption value under certain wavelength, and what its absorption value size reflected cytolemma electron transport chain passs hydrogen capacity of water.The depth that in cell, TTC reacts the redness presented reflects the power of its vitality, and it is stronger that color more deeply feels its vigor bright.In yeast cell, the main path of ethanol fermentation comprises the anaerobic degradation of glycolytic and pyruvic acid, sugar in the process-its Main Function of ethanol conversion enzyme, TTC can produce color reaction to the meta-bolites of yeast, the size of invertase activity in its born of the same parents can be judged by the depth of its color, it is stronger that color more deeply feels its cytoactive bright, and producing and ethanol ability is also stronger.
In these researchs existing, also there is a lot of defect or deficiency.First is that flat panel production containing TTC substratum is complicated, and after inoculation yeast, growth fraction is comparatively slow, and during the darker yeast of follow-up picking color, operates also more loaded down with trivial details, take longer; Second is the color reaction by TTC staining, utilize the depth of human eye observation's reaction color to judge cell viability thus the height of prediction producing Yeast ethanol, because cell to be screened is various, its defect is: 1. cannot be quantitative, judge cannot accurately judge resultant content by naked eyes, and be easy to produce tired and mistake, and screening gained height vigor cell from different culture plates can because the limitation of human eyesight causes erroneous judgement, and making finally to screen the cell obtained may not be required high vigor high-yield ethanol cell; When 2. screening a large amount of cell, workload is large, takes time and effort, affects cell screening progress.
High flux screening (High throughput screening, HTS) technology is based on the experimental technique of molecular level and cell levels, utilize 96 orifice plates and microplate reader as experimental tool carrier, gather experimental result data with sensitive detecting instrument fast, quantification can detect a large amount of sample at one time.It has the features such as quantitative, quick, sensitive and accurate.Utilize High Throughput Screening Assay to be widely used in the fields such as drug screening at present, and achieve obvious progress.In microbial strains seed selection, also Many researchers is had to utilize 96 orifice plates to carry out seed selection work in bibliographical information, 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 is many weak points, namely these bibliographical informations are just undertaken transferring to inside 96 orifice plates with the detection of thalline number in analytical unit volume in cuvette in microorganism culturing, all need in follow-up study to coat slat chain conveyor basal growth by growing the higher bacterial strain of OD value in Tissue Culture Plate 96 hole, recycle other screening methods and carry out high-yield ethanol bacterial strain screening, take length, as Cai Wanlin meets follow-up yeast screening assay needs growth 7-9 days in screening high yield pipe capsule yeast utilizing 96 orifice plates and yeast saccharomyces cerevisiae growth circle.So, though these methods make use of 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 is only quantitative to cell colony density, what reflect is the proliferative conditions of cell in the certain growth cycle, not the 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 provided based on TTC staining and microplate reader and 96 orifice plates is provided, the method is quantitative by microplate reader, rapid detection, effectively can improve screening efficiency and the screening accuracy of high reactivity cell, Large-scale Screening cell work amount is improved large with this, progress is slow, the deficiency that accuracy is low.
For achieving the above object, the technical solution used in the present invention is:
Based on a high vigor yeast cell quantitative screening method for TTC staining, comprise following steps:
1) yeast cell to be detected is cultivated: yeast cell bacterium liquid to be detected coats flat board, cultivates and treats that it grows single bacterium colony; The incubation time of different strains of Yeast differs, and usually cultivates 48-72h;
2) cell list colony lift to 96 porocyte culture plate.By single colony inoculation of cultivating in step 1) in 96 porocyte culture plate each hole nutrient solutions, be added with TTC in nutrient solution, be cultured to mensuration and obtain thalline OD value between 1 and 3, centrifugally abandon supernatant; Then in the 96 each holes of porocyte culture plate, TTF extraction agent is added, centrifugal after concussion 10-15S; Select nutrient solution according to different strains of Yeast, yeast cell is cultivated in the nutrient solution used and is added TTC to finite concentration under normal conditions.
3) by step 2) in concussion centrifugal after supernatant liquor be drawn in 96 hole enzyme plates, microplate reader is utilized to measure TTF absorption value under 486nm, according to TTF typical curve and step 2) the middle cell OD value measured, calculate TTF content in each hole, determine cell viability size with this.TTF content is higher, shows that cellular respiration vigor is stronger.The bacterial strain that screening TTF content is high is the yeast cell of high vigor.
Step 2) in nutrient solution TTC concentration be 0.3%-0.5%(W(g)/V (mL)), pH is 6-8.
Step 2) described in TTF extraction agent be dimethyl sulphoxide aqueous solution, concentration range (dimethyl sulfoxide (DMSO) and water mixed volume than) is: 85%-100%(V/V).
Step 2) in TTF extraction agent and cell culture fluid volume than being 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, such as 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, has the following advantages compared with common detection methods:
1) accuracy: because the TTF generated is at temperature 20-40 DEG C, pH6-8, equal energy stable existence in 24h, and can by microplate reader accurately, rapid detection, the quantitative reliably direct reaction cell viability of the selection result, obviously can not only reduce screening time, and the mistake of the visual determination generation in operating process can be reduced;
2) high efficiency: based on 96 orifice plates, can high-throughput rapid detection, only its OD value and TTF growing amount need be measured carry out cell cultures in Tissue Culture Plate after, whole mensuration process only needs 1-2h, without the need to the at substantial time, and the method is not only confined to 96 orifice plates, also other Tissue Culture Plates such as 384 holes can be developed into;
3) present method operational condition is not harsh, TTC concentration in operating process, pH controls, dimethyl sulfoxide concentration, in follow-up 96 orifice plates, cell OD value etc. have certain operating restraint, without the need to preparing 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. comparatively conventional sense are more saved.The dimethyl sulfoxide (DMSO) used in present method is cheap, and effect of extracting is good, saves testing cost, and present method can as a kind of quick, effective, reliable, the economic means detecting cell viability.
This detection method can detect the power of its producing and ethanol ability further while 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 do not limit the present invention.
embodiment 1
The high flux screening of high vigor yeast
The drafting of 1.TTF typical curve
Get 0.005 respectively, 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 add in 10ml centrifuge tube, add the Tris-HCl(pH8.4 of 2ml), finally add 10%(W(g)/V (mL)) and Na
2s
2o
4solution 1ml, 37 DEG C of water-bath concussion more than 5min(steps all in the dark operate), TTC is made to 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, light absorption value is surveyed in 486nm place, and with mixed solution in 486nm place light absorption value for ordinate zou, using TTC concentration as X-coordinate drawing standard curve, draw the typical curve obtained and see Fig. 1: as can be seen from typical curve: when TTC concentration is at 0.01-0.06%, OD value is in 0.15-0.88 scope, both are in good linear relationship, may be used for quantitative assay.Typical curve is to drop in a believable interval detecting the TTF value of Hemapoiesis, does not think that this value is insincere we of this interval.
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, cultivates 48h, treats that it grows single bacterium colony for 30 DEG C; Wherein YPD solid culture based formulas: 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 96 porocyte culture plate each hole nutrient solutions, 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 0% respectively, 10%, 14%, add the ethanol of different concns, it is the cell viability in order to see bacterial strain under different ethanol concentration, ethanol is toxic to cell, concentration is higher, active less, arrange according to concentration, both can with this method of TTC detect same strain bacterium when with or without ethanol and alcohol concn high or low cell viability, can also detect and compare the vigor of different strains under same alcohol concn, the convenient bacterial strain selecting resistance to ethanol, culture condition: 30 DEG C, 200r/min cultivates 4h, measure its cell OD value, make cell OD value between 1 and 3, cell OD value between 1 and 3 TTF concentration now between 0.15-0.88, the light absorption value and its concentration that measure TTF are good linear relationship, quantitatively more accurate, supernatant is abandoned after the centrifugal 5min of 4000r/min, then in the 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 by shake in above-mentioned Tissue Culture Plate centrifugal after supernatant liquor draw 100 μ L in 96 hole enzyme plates, TTF absorption value is measured under 486nm, according to the cell OD value measured in TTF typical curve and step 3, calculate cell TTF content=experiment and record TTF absorption value * (1/ cell OD
600absorption value), determine cellular respiration power and cell viability size with the obtained TTF value in the bent scope of mark.TTF content is higher, shows that cellular respiration vigor is stronger.(mensuration of ethanol production adopts gas phase marker method to the ethanol production of each strain bacterial strain of Simultaneously test, and GC conditions is, pillar model: SGE 30QC2/AC20-0.25 capillary column; Post case temperature: 60 DEG C; Sampler temperature: 190 DEG C; Detector temperature: 240 DEG C.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, be arranged in order as #5, #1, #8, OY-11, #7, #6 by cell viability power, experimental result is in table 1.
Table 1
As can be seen from Table 1: be 0%, 10%, when 14% at alcohol concn, bacterial strain 5#TTF absorption value is the highest, and 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#.Simultaneously at mensuration bacterial strain ethanol production, the ethanol production of bacterial strain 5# is also the highest, and other bacterial strains are followed successively by 8#, 1#, OY-11,7#, 6# by ethanol production height, and 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 concentration is 85%-100%(V/V) and can, without the need to preparing 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, such as 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 (1)
1., based on a high vigor yeast cell quantitative screening method for TTC staining, it is characterized in that comprising the following steps:
1) yeast cell bacterium liquid to be detected is coated flat board, cultivate and treat that it grows single bacterium colony;
2) by single colony inoculation of cultivating in step 1) in 96 porocyte culture plate each hole nutrient solutions, be cultured to mensuration and obtain thalline OD value between 1 and 3, centrifugally abandon supernatant; Then in the 96 each holes of porocyte culture plate, TTF extraction agent is added, centrifugal after concussion 10-15S;
3) by step 2) in concussion centrifugal after supernatant liquor be drawn in 96 hole enzyme plates, microplate reader is utilized to measure TTF absorption value under 486nm wavelength, according to TTF typical curve and step 2) the middle cell OD value measured, calculate TTF content in each hole, determine cell viability size with this;
Wherein: step 2), in nutrient solution, TTC concentration is 0.3%-0.5%(W(g)/V (mL)), pH is 6-8;
Described TTF extraction agent is dimethyl sulphoxide solution, and concentration range is 85%-100%(V/V); TTF extraction agent and cell culture fluid volume are than being 1:1.
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