I want to label PA with several fluorophores to assay phagocytosis and phagosomal processes. Unfortunately the bacteria aggregate during the labeling process (both live and heat killed @ 80C 30min). I do not have this problem when labeling E.coli.
Method: Grow liquid culture in TSB or scrape from Blood TSA plates. Wash several times in PBS and resuspend to ~ 1010 CFU/mL (same as for e.coli) in PBS pH 7.4. NOTE: resuspending the PA (orange hue) looks like resuspending mucous-like gel.
I then add 10uL of an amine-reactive fluorophore (DCF-SE dissolved in DMSO) and incubate for 1hr room temp. This is done under N2 gas in glass container to limit oxidation of fluorophore. It is during this initial incubation that I notice the PA forms numerous clumps (not a giant clump) that is difficult to disrupt.
If i proceed with the rest of the reaction, it gets worse.
Any ideas on how to prevent the clumping? EDTA or Tween?
I have tried this with both the PAO14 and Boston 41501 strains.
I have the same problem using Mycobacterium for phagosytosis and phagolysome maturation analysis. We labeled bacteria with FITC. but the problem with mycobacteria is always clumping du to the cell wall surface.
To resolve partially this, we add, glycerol or tween to the liquid media and incubate under shaking. After labelling reaction, I did a passage throught an insulin syringe/ needle for at least 20 times (up and down) in eppendorf tube. This will allow the disruption of your clumps. We got a very nice individual labelled bacteria under confocal microscopy.
Hope it helps
Thank you, Hafid.
What concentration of Tween (tween-80 i presume) or glycerol did you use?
You had Tween or glycerol to the culture media while you grow the bacteria, or just the buffer you use for labeling the bacteria? Does it matter if the bacteria are in exponential or stationary phases of growth?
Hi Evan
Mycobacteria can use tween80 (0.1 to 0.5 %) or glycerol (5 %) at high concentration as carbon source. you can use less concentration for Pseudomonas since it's knowm that tween inhibit biofilm formation in this species. (0.01 to 0.1 %).
You have to add tween to liquid media and incubate till exponential phase for your phagocytosis and intracellular processing. Be sure that your amine-reactive fluorophore react well since tween is degraded to oleic acid, if Pseudomonas have some lipase that have an esterase activity this will reduce tween to ester bond and then lower the pH of your supernatant. to avoid this, wash your pellet bacteria, and do your labelling reaction in buffer.
Of course the growth phase influence your experiments it dépends what are you looking for ? in stationary phase, several second factors produced influence the phagolysozome maturation pathway, iNOS, signaling pathways... Preferably use early exponential phase.
Good luck
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