Eo. Puchkov et An. Melkozernov, FLUOROMETRIC ASSESSMENT OF PSEUDOMONAS-FLUORESCENS VIABILITY AFTER FREEZE-THAWING USING ETHIDIUM-BROMIDE, Letters in applied microbiology, 21(6), 1995, pp. 368-372
The relationship between impairment of the Pseudomonas fluorescens eel
envelope's permeability barrier for ethidium cation, the fluorescent
moiety of ethidium bromide, and viability after freeze-thawing was inv
estigated. Ethidium fluorescence in the suspension of intact bacteria
did not change. Disruption of the bacterial permeability barrier by ce
tyltrimethylammonium bromide (CTAB) led to ethidium fluorescence incre
ase due to interaction of the fluorochrome with intracellular nucleic
acids. In the suspension of freeze-thawed cells, ethidium fluorescence
increased and the subsequent treatment by CTAB resulted in further fl
uorescence increase up to the final level corresponding to that in CTA
B-treated intact bacteria. For bacteria exposed to different freeze-th
awing regimes, the relative ethidium fluorescence increase closely cor
related with the relative number of fluorescing cells revealed microsc
opically. In the suspension of freeze-thawed cells, the relative addit
ional ethidium fluorescence increase after CTAB treatment closely corr
elated with viability evaluated by plate counts. It is concluded that
the fluorimetric approach may be used as a means of rapidly evaluating
bacterial viability after freeze-thawing.