Has anyone used stains like DiO or a derivative of this or Mitotracker probes to label the membrane of bacteria and had any success? I know you can use FM4-64 or similar but would like to find an alternative.
For E. coli we used a voltage-sensitive dye for staining the cell membrane - Di-SC3-5 carbocyanine iodide (AnaSpec) with an excitation wavelength of 642 nm and emission wavelength 670 nm. Dye was added at a concentration of 0.4 µM. E. coli OD was 0.6. Relatively easy to work with.
We had used successfully the lipophilic fluorescent dye FM1-43 to stain the cell membrane and also inner membranes of uncultivated bacteria. See images and the protocol in our paper:
Albuquerque, J. P., Keim, C. N., Lins, U. (2010). Comparative analysis of Beggiatoa from hypersaline and marine environments. Micron 41: 507–517.
Hi Christa:
I haven´t worked in bacteria but FM dyes (2-10; 4-64; 1-43) are excellent membrane reporters. However I can say that FM1-43 labels bacteria really well (observed in contamination in neuronal cultures). Mitotracker is also considered an FM dye, so, why do you have to find an alternative?
However, there is a series of compounds called CellMask (TM) that might be of your interest.
Cheers
Dear Carolina N Keim ,
As you mentioned here I followed the same protocol with FM 1-43 DYE for E.coli.
Briefly, cells were grown for 5 hrs after subculturing , media were drained and added 1X PBS containing 1ug/ml of FM 1-43 and went for microscopy
There was no fluroscence in any range of spectra.
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