Hello everyone,
We are working with FITC-labelled Yersinia ruckeri and Aeromonas
hydrophila for phagocytosis assays. The trick in these assays is to
remove or quench the fluorescence associated to extracellular bacteria
(those not internalised by the phagocyte). Several chemicals are used
for this objective, trypan blue and coomassie being the most common.
We are attempting to apply these technique without much success so
far. We've been told that the reason is that the bacterial cells must
be made permeable to trypan blue or coomassie before so that this dye
has access to the FITC binding sites.
I guess making bacterial cells permeable will be a difficult task,
depending on many factors. We have made a simple attempt with Tween 20
(1%, 10min) with no success.
The protocol we use is the following:
1.- Bacterial cells are grown 18h @ 22dC in BHIB
2.- Cells are washed and fixed in 1% paraformaldehyde
3.- Cells are Labelled with 1mg/ml FITC in PBS pH 7.4 for 60min
4.- Cells are washed (Tween 20 has been applied here)
5.- Coomassie blue is mixed with the bacterial suspension.
Bacterial cells at poit 4 are fluorescing green intensely. After point
5 they should have lost fluorescence intensity, but they do not.
Any suggestion, change in protocol, etc, which could make the
bacterial membrane permeable would be greatly appreciated.
Replies will be summarised and posted to the newsgroup.
Thanks very much
Natalio Garcia Garbi
Institute of Aquaculture
Stirling University
Scotland, UK
