Quantitative high-vitality yeast cell screening method based on TTC (2,3,5-triphenyltetrazolium chloride) staining method

Abstract

The invention discloses a method for quantitative screening of highly active yeast cells based on TTC staining method. The yeast cell liquid to be detected is coated on a flat plate, cultivated until a single colony grows; and then inoculated in each well of a 96-hole cell culture plate for culture. cultured in the culture medium until the measured OD value of the bacteria was between 1-3, then centrifuged and discarded the supernatant; added TTF extractant to each well of the 96-well cell culture plate, and centrifuged after shaking for 10-15S; the supernatant after shaking and centrifuging The supernatant was drawn into a 96-well microtiter plate, and the TTF absorption value was measured at a wavelength of 486nm by a microplate reader. According to the TTF standard curve and the measured cell OD value, the TTF content in each well was calculated to determine the cell viability. The screening results of the method can quantitatively and reliably directly reflect the cell viability, not only can obviously reduce the screening time, but also can reduce the human error caused by the visual judgment in the operation process.

Description

technical field [0001] The invention is applicable to the fields of bioengineering technology, medicine and pharmacy, and specifically relates to a method for quantitatively screening high-activity yeast based on a TTC staining method. Background technique [0002] 2,3,5-Triphenyltetrazolium chloride (TTC) is a chromogenic agent that can produce color reactions to various cells (yeast, bacteria, plant cells, etc.), TTC itself is colorless, when It reacts with dehydrogenase in normal cells and turns red. The principle is that TTC replaces O in cellular respiration. 2 Accept H + , TTC is reduced to red TTF (triphenylmethyl month replacement) and appears red. The reaction equation is as follows: [0003] [0004] Due to the above color reaction, TTC, as the proton acceptor of the pyridine-nucleoside structural enzyme system in the respiratory chain, can be reduced from the colorless oxidized TTC to red TTF by the respiratory chain dehydrogenase in living cells. Therefore,...

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Problems solved by technology

The first is that the preparation of the plate containing TTC medium is complicated, and the growth of the yeast after inoculation is relatively slow, and the subsequent operation of picking darker yeast is cumbersome and takes a long time; the second is the color development by TTC staining. Reaction, using the human eye to observe the color of the reaction to judge the cell viability and predict the level of ethanol production by yeast. Due to the large number of cells to be screened, the disadvantages are: ①It cannot be quantified, and the content of the product cannot be accurately judged by naked eyes, and it is easy to produce Fatigue and mistakes, and the high-viability cells screened from different culture plates will lead to misjudgment due to the limitation of human eyesight, so that the cells finally screened may not be the required high-viability and high-ethanol-producing cells; ②The workload of screening a large number of cells Large, time-consuming and labor-intensive, affecting the progress of cell screening

However, in these studies, there are also many deficiencies, that is, these literature reports only transfer the detection of the number of bacteria per unit volume in a cuvette to a 96-well plate for microbial culture, and all follow-up studies need to transfer The strains with higher OD value in the 96-well cell culture plate are spread on the plate medium for growth, and then other screening methods are used to screen high-yield ethanol-producing strains, which takes a long time. For example, Cai Wanlin used a 96-well plate and S. Subsequent yeast selection in Pachysomyces requires 7-9 days of growth

Therefore, although these methods use 96-well plates, they do not significantly reduce the working time and workload. The key point is that the results measured by these methods when using 96-well plates are only quantification of the density of the cell population, which reflects the density of the cells. Proliferation in a certain growth cycle is not the vitality of individual cells

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