Gerhard Nebe-von Caron notes, re Elaine Kunze's question about pH measurement in E. coli- >Due to the dye transporter systems the only working system for me has been >carboxy-fluorescein-diacetate-succinimidylester. However in the absence of >dual >excitation ratio (as described in Howard Shapiro's book) your measurements are >severely limited due to variations in the dye loading and cell volume => width >of the distribution. Thus you only get a rough estimate. Unfortunately you can >also not work in the exponential growth phase as that won't allow you dye >loading in the first place. All a bit limited, the whole method unless you >have >a non-pumping strain.. I agree with Gerhard that the dye loading is the major problem, especially with Gram-negative organisms such as E. coli. A recent paper (Chitarra LG, Breeuwer P, van den Bulk RW, Abee T: Rapid fluorescence assessment of intracellular pH as a viability indicator of Clavibacter michiganensis subsp. michiganensis. J Appl Microbiol 2000; 88:809-816) describes ratiometric flow cytometry of pH in a Gram-positive organism using carboxyfluorescein succinimidyl ester (CFSE), taking the ratio of emissions in the 520-540 nm and 560-590 nm range. Even with the ratiometric method, the "pH" curves observed were relatively broad; however, while they did not completely discriminate heat-killed (nonviable) and untreated (viable) cells, they did provide a usable, if crude, indication of pH. This area needs some more work. -Howard
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