Lg. Chitarra et al., Rapid fluorescence assessment of intracellular pH as a viability indicatorof Clavibacter michiganensis subsp michiganensis, J APPL MICR, 88(5), 2000, pp. 809-816
The viability of Clavibacter michiganensis subsp. michiganensis (Cmm) was d
etermined by measuring the intracellular pH (pH(in)) as a viability paramet
er. This was based on the observation that growth of Cmm was inhibited at p
H 5.5 and below Therefore, viable cells should maintain their pH(in) above
this pH value. The pH(in) of Cmm was determined using the fluorescent probe
5(and 6-)-carboxyfluorescein succinimidyl ester (cFSE). The pH(in) of Cmm
cells exposed to acid treatments was determined using fluorescence spectrof
luorometry, and for cells exposed to elevated temperatures, the pH(in) was
determined using fluorescence spectrofluorometry and flow cytometry (FCM).
A good correlation was found between the presence of a pH gradient and the
number of colony-forming units (cfu) observed in plate counts. However, wit
h the spectrofluorometry technique, the analysis is based on the whole cell
population and the detection sensitivity of this technique is rather low,
i.e., cell numbers of at least 10(7) cfu ml(-1) are needed for the analysis
. Using FCM heated-treated and non-treated Cmm cells could be distinguished
based on the absence and presence of a pH gradient, respectively. The majo
r advantage of FCM is its high sensitivity, allowing analysis of microbial
populations even at low numbers, i.e., 10(2)-10(3) cfu ml(-1).