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Mechanosensitive (MS) channels act to protect the cytoplasmic membrane (CM) of living cells from environ- mental changes in osmolarity. In this report, we demonstrate the use of time-resolved second-harmonic light scattering (SHS) as a means of experimentally observing the relative state (open versus closed) of MS channels in living bacteria sus- pended in different buffer solutions. Specifically, the state of the MS channels was selectively controlled by changing the composition of the suspension medium, inducing either a transient or persistent osmotic shock. SHS was then used to monitor transport of the SHG-active cation, malachite green, across the bacterial CM. When MS channels were forced open, malachite green cations were able to cross the CM at a rate at least two orders of magnitude faster compared with when the MS channels were closed. These observations were corroborated using both numerical model simulations and complementary fluorescence experiments, in which the propensity for the CM impermeant cation, propidium, to stain cells was shown to be contingent upon the relative state of the MS channels (i.e., cells with open MS channels fluoresced red, cells with closed MS channels did not). Application of time-resolved SHS to experimentally distinguish MS channels opened via osmotic shock versus chemical activation, as well as a general comparison with the patch-clamp method is discussed.