A lot of stains for determining live and dead bacterial cells have been described. In literature there is some controversy about them. For example DAPI rapidly arrests the growth of bacteria when illuminated with UV. It is an indicator for live cells but if you measure it bacteria stop growing. All of the stains we found sofar have to be washed away, incubated shortly, or the cells have to be monitored in a non growing medium which is not a natural situation. Others degrade in water or medium. I am searching for substances which are not degraded by culturing conditions (eg can be used in LB medium or another defined medium) and which can be used to monitor on a long term basis (eg 24 hours) the growth and/or the death of bacteria.
Any help, protocol, own experience will be highly appreciated
quantifying the Dead and live cells/bacteria can be differentiated by metabolic activity quantification.
The metabolic activity can be measured by Alamar Blue, the oxidation-reduction indicator, that changes color (blue to red) in response to the chemical reduction of growth medium resulting from metabolic activity.
Based on your explanation alamarBlue assay may be suitable for you, more information can be obtained from the following link
http://tools.thermofisher.com/content/sfs/manuals/PI-DAL1025-1100_TI%20alamarBlue%20Rev%201.1.pdf
Best
Hello Sunagar Raju ,
I used already Alamarblue but it is not stable in normal growth conditions, in LB medium. I already got the recommendation to use defined media. But it would be nice to know why the Alamar gets processed without metabolites. Most stainings for live cell monitoring are actually short term measurements. So I'll try defined media with Alamarblue. It would be nice though to have chemicals useable in natural conditions.
Dear Stefan,
you can use FDA (fluoresceine diacetate) for live bacterial cells staining and PI (propidium iodide) for staining the dead bacteria cells.
For more info see
1) Article Limits of propidium iodide as a cell viability indicator for...
2) Article Optimisation of the Protocol for the LIVE/DEAD® BacLightTM B...
3) https://ibidi.com/img/cms/support/AN/AN33_Live_Dead_staining_with_FDA_and_PI.pdf
Dear Ivan,
Thank you for the interesting articles.
I already used fluorescein diacetate but it is highly unstable in water solutions and rapidly hydrolyses so it is unsuitable for long term culturing. I'll give a try with PI.
In my manuscript https://academic.oup.com/jac/article/74/9/2617/5498604 I used several protocols: CFUs quantification, SYTO9/To-Pro-3-iodide (this is a end point assay, is not suitable for long term culturing) and PrestoBlue (it is the same assay of alamarBlue, resazurin). With the PrestoBlue assay I follow different kinetics (up to 4h) in MHB. Also I alredy aptimized this assay for bacterial cultures that growth in LB and at least for incubation up to 6h I had good results.
Dear Sandra N Pinto I had no succesfull results as my Alamarblue readily converted in sterile LB medium without bacteria. I figured that out when I diluted the Alamarblue in LB medium in stead of fysiologic buffer. I will try your suggestion of the Mueller–Hinton broth. Are there any special steps you took? Thank you very much.
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