Because of the lack of fluorescence microscopy I would like to use the trypan blue staining to assess the viability of a bacillus-like strain. Contrary to methylene blue staining on yeast, I cannot discriminate dead and living cells with this staining method.
I mix a 4g/l trypan blue solution in 1:1 ratio with a diluted living cell stock. Under the microscope using a Thoma counting chamber I cannot see any difference between cells so I am questioning wether this method is suitable at all for microbes.
Any suggestions or remarks?
What is your point?
Viability can be reached by culture and colony count.
My point is I want to see how many % of cells are alive while running my process. I can do a colony count but this only gives the total viable cell count and not the ratio. Aside from that this method is too slow. I was wondering why none of the trypan blue staining protocols seem to work while I can find microscopy images of successfull coloration so maybe I am overseeing something.
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.researchgate.net/post/Does_anyone_have_knowledge_on_using_Trypan_blue_for_E_coli_staining/amp&ved=2ahUKEwih6sacpMfcAhVBblAKHT3LC3oQFjAAegQIAhAB&usg=AOvVaw3Kzb3nbTFmX2uzMQ6KtZxC&cf=1
https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.protocol-online.org/biology-forums-2/posts/7281.html&ved=2ahUKEwih6sacpMfcAhVBblAKHT3LC3oQFjALegQICRAB&usg=AOvVaw1Q3gu5A5MyL6Ftu_o2eT25
https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.lonzabio.jp/catalog/pdf/ri/T204.pdf&ved=2ahUKEwih6sacpMfcAhVBblAKHT3LC3oQFjAJegQIBBAB&usg=AOvVaw3KVh5dDuFb5v14YYaEWIEn
The thread mentioned above does not really give me any answers. I am going to persue the option of serum protein interference that is mentioned in this paper:
Article Appendix 3B Trypan Blue Exclusion Test of Cell Viability
Hopefully the medium exchange by centrifugation before staining gives me some visible difference between living and dead cells.
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