Joerg Ueckert wrote- >We're holding us busy by applying CFDASE for ratiometric determination of >internal pH in bacteria and yeast. Apart from strain- and pre-history - >dependent difficulties in dye uptake the generation of a 'useful' calibration >curve is the biggest challenge. The well-known combination >valinomycin/nigericin results in a high degree of variability and curve shifts >upon repetition. Also, apart from these, other agents (e.g. alcohols) or >methods (e.g. electroporation) to facilitate equilibration of internal and >external pH give stunning differences compared to mentioned 'standard' method. >All remarks, suggestions and experiences with agents which provide reliable >calibration curves for pHi in microorganisms are welcome. Chitarra et al, in Wageningen, described a ratiometric CFDASE (CFSE) pH measurement as a viability indicator in Clavibacter michiganensis. The distributions of ratios they published were pretty broad; this may be due to photoelectron statistics if there aren't that many indicator molecules per cell. This wouldn't be the only source of variability, of course, but it would be good to get a ball park estimate of the number of dye molecules by comparing fluorescence of bacteria with a fluorescein standard such as Quantum beads. -Howard
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