Method for detecting activities of cells in fermenting process through adopting flow cytometry (FCM)

A flow cytometry, dead cell technique used in microorganism-based methods, biochemical equipment and methods, assay/examination of microorganisms, etc.

Inactive Publication Date: 2013-10-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still relatively few reports on the detection of cell activity in the fermentation process, and it is still in the research stage.

Method used

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  • Method for detecting activities of cells in fermenting process through adopting flow cytometry (FCM)
  • Method for detecting activities of cells in fermenting process through adopting flow cytometry (FCM)

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Torulopsis glabrata was activated by seed medium, inoculated into shake flask fermentation medium, cultured until the logarithmic phase of cell growth for 24 hours, and each took 3 tubes of 1ml fermentation broth. Take one of the tubes and place them in boiling water for 8-10 minutes and then ice-bath, numbered 1, and put the other two tubes at room temperature, numbered 2 and 3 respectively. Take 500ul of fermentation broth from No. 1 and No. 2 tubes to No. 4 tube and mix well. Filter the fermented broth diluted by a certain multiple in tubes 1, 3, and 4 with a 40um pore mesh, centrifuge at 3000g for 5min, discard the supernatant, collect the cells, wash the cells with PBS, add 500ul PBS after centrifugation, and gently resuspend the cells. Add 5ul100ug / ml propidium iodide staining solution, mix gently, react at room temperature in the dark for 5min, then ice-bath, and detect with flow cytometry.

Embodiment 2

[0039] Torulopsis glabrata was activated by the seed medium, inoculated into the fermentation medium supplemented with 70g / L NaCl, cultured until the logarithmic phase of cell growth for 24 hours, and took 2 tubes of 1ml fermentation broth. Filter the diluted fermentation broth with a 40um pore size mesh, centrifuge at 5000g for 5min, discard the supernatant, collect the cells, wash the cells with PBS, add 500ul PBS after centrifugation, and gently resuspend the cells. Add 5ul100ug / ml propidium iodide staining solution to one of the tubes, mix gently, react at room temperature in the dark for 5min, and then ice-bath. Add 450 μL PBS and 50 μL Rh123 working solution to another tube, mix well, react in the dark at room temperature for 10 minutes, centrifuge and collect the cells, wash the cells twice with PBS, stain with PI, mix well, and react in the dark at room temperature for 5 minutes. Finally, it was immediately detected by flow cytometry.

Embodiment 3

[0041] Put the stained sample into the sample tube, use the FL1 and FL3 channels to detect the fluorescence intensity signal, adjust the detection voltage, get the FCM map, adjust the appropriate "cross gate", the Rh123 and PI staining solution can divide the cell population into Three-zone, quadrant analysis yields reliable ratios of dead, apoptotic, and live cells.

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Abstract

The invention relates to a method for detecting the activities of cells in a fermenting process through adopting FCM. In the invention, the activities of microbial cells (yeast or moulds) cultured through Rh123 / PI double staining are detected in a shaking bottle under normal fermentation or high salt stress through the FCM to obtain an FCM map, an appropriate cross door is adjusted, and a reliable ratio of dead cells to living cells to apoptotic cells can be obtained through quadrant analysis. The method is a simple method for detecting the activities of the cells in the industrial production.

Description

technical field [0001] The invention relates to a method for detecting cell activity in a fermentation process by flow cytometry, which belongs to the field of bioengineering, and is particularly convenient and reliable in industrial production. Background technique [0002] Biological fermentation has the advantages of less pollution, generally using glucose as a substrate, and low cost. It is a very promising method. Therefore, people have focused their attention on the research of biological fermentation in recent years. However, various stress factors such as temperature, pH, and starvation will reduce the activity of cells or even autolyze them, affecting the yield. Therefore, the establishment of a simple, rapid and reliable cell viability detection method has practical significance for fermentation production. [0003] At present, conventional methods for detecting microbial cell activity include colony counting method, methylene blue staining method, and specific ox...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12R1/88
Inventor 刘立明俞晓霞杨松鑫陈坚
Owner JIANGNAN UNIV
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