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The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. After reaching peak fluorescence, the population of live cells decays. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. The neutral probe rapidly permeates the membrane and enters the cytosol. The developed method includes exposure of cells to a weak acid probe at low pH. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms.