Vital fluorescence double staining
The vital fluorescence double staining is used to differentiate between living and dead cells and thus to determine cell viability with the help of a flow cytometer , a fluorescence microscope or a fluorescence scanner.
Two substances are added to a cell culture , which are absorbed or metabolized differently by living and dead cells and in which fluorescence can be excited. The first substance is usually a dye that is metabolized in the cell ( fluorescein diacetate , SYT09 or resazurin ) and then makes living cells fluoresce green or blue. The second substance, propidium iodide , ethidium bromide or DAPI, is stored in the DNA and RNA with an accompanying increase in fluorescence in the red or blue color spectrum. These are only taken up very slowly by living cells. The membrane of dead cells, on the other hand, is full of holes so that the dye can enter and color the nucleic acids in the cell red.
The method also allows archaea , eubacteria and eukaryotic cells to be stained and, in the latter case, to differentiate between apoptosis and necrosis .
literature
- L. Boulos, M. Prévost, B. Barbeau, J. Coallier, R. Desjardins: LIVE / DEAD BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. In: J Microbiol Methods. 37 (1), 1999 Jul, pp. 77-86. PMID 10395466
- Michael Berney, Frederik Hammes et al: Assessment and Interpretation of Bacterial Viability by Using the LIVE / DEAD BacLight Kit in Combination with Flow Cytometry. In: Appl Environ Microbiol. 73 (10), 2007 May, pp. 3283-3290. PMC 1907116 (free full text)