If I incubate bacteria with FITC for 30 minutes at RT to make them fluorescent, how do they actually become fluorescent?
Is FITC internalized in a certain degree, does it coat the capsule /cell wall/ membrane or due to something completely different?
Fluorescein isothiocyanate (FITC) reacts covalently with unprotonated primary amino (NH2) groups. This includes exposed lysine residues of surface proteins and their amino termini, if the pH is high enough. It also includes the exposed lysine residues of peptidoglycan in Gram positive bacteria, and perhaps the m-DAP resides in the peptidoglycan of some Gram negative bacteria if it gets past the outer membrane. I don't know whether any of it gets into the bacterial cytoplasm, but if it does, it will react with the proteins inside the bacteria.
Thank you for your answer Adam, this is really helpful. Do you know any papers by heart that describe this, allowing me to use it as a reference?
In addition, would you say that if a 'labeling-buffer' is used around pH 8-9, the labeling of bacteria with FITC will be more optimal/efficient in some extend?
Here is a paper that might interest you:
http://aem.asm.org/content/14/1/119.full.pdf
The selection of buffer is very important. You should use a non-amino buffer because the FITC will react with amino buffers. You can use a fresh solution of 0.1 M sodium bicarbonate to get the right pH. You should probably also wash the cells with this buffer to remove residual growth medium. If you want to keep the cells alive, you will also have to maintain a physiological ionic strength.
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