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Fluorescent bacteria

Sam Bowser bowser at WADSWORTH.ORG
Wed Mar 1 10:20:58 EST 1995

Regarding the recent discussion of fluorescently-labeling bacteria for 
trophic studies: I never tried the following, I only offer it as another 
possible approach.

Joan Bernhard and I found that "marine bacterial consortia" (i.e., bacterial 
biofilms) could be intensely labeled with Hoechst 33342, a DNA-binding 
fluorochrome (Mar. Ecol. Prog. Ser. 83:263-272, 1992). About 90% of the 
bacteria were labeled by simply incubating coverslips containing the 
biofilms for 30 min in a 10 microgram/ml solution of the dye in sea water. 
Since binding is to DNA, it seems reasonable that the bacterial surface 
remains unchanged (in the short term) and trophic specificity would seem 
less likely affected. As a cautionary note, it is possible that other 
charged biopolymers, like certain proteoglycans, can bind "A-T specific" 
benzimidazole derivatives. 

After feeding your bugs these labeled bacteria, it should be possible to 
wash them well, extract the Hoechst, measure the fluorescence emission, and 
with proper calibration obtain the number of bacteria eaten (similar to cell 
proliferation assays; e.g., Blaheta et al., J. Immunol. Meth. 142:199-206, 
1991). Then again, I can think of several complicating factors (including 
washing difficulties as mentioned by Joe Balczon); still, it might be worth 
trying...I'd be interested if anyone has any experience with such 
non-radioactive approaches.

One final comment: we found huge differences in the feeding efficiencies of 
foraminifera fed suspended bacteria vs. bacterial biofilms. Certain forams 
really dig the 'films; others didn't touch them. I wonder how many important 
trophic interactions are missed because, for the sake of "clean" 
experiments, we typically plop our bugs into a bacterial suspension rather 
than present them bacteria attached to a surface???

"There are things a man must do to remain a man" James T. Kirk

S.S. Bowser
Wadsworth Center for Laboratories and Research
P.O. Box 509
Albany, NY 12201-0509 USA
Phone: (518) 473-3856
FAX: (518) 474-7992
bowser at wadsworth.org

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