A source of error is with FITC-labeled bacteria that are taken up into
phagosomes where the pH may drop. Low pH will also quench fluorescence. It's
a start. Perhaps another dye that is less pH sensitive but is quenched by
crystal violet may be a good alternative.
Another source of error that I noticed when I did bacterial-cell surface
binding studies a dozen years ago was the labeling process affected binding.
This could be due to the requirement of pH 9+ to directly label bacterial
surface proteins with FITC, or a modification of the structures/molecules
that effected binding--pili in the case that I studied. ( Scatchard plots
showed that labeling affected bacterial binding. Scatchard plots can be done
by looking at the cellular mean fluorescence (related to numbers of bound
FITC-labled bacteria) as labled bacteria are competed away by unlabeled
bacteria.) If any structures/molecules that effect binding could be modified
by labeling, proper controls are needed to ensure that you are reporting
binding and not reducing or ehancing binding.
Dave Coder
dcoder@u.washington.edu