Direct Analysis of Water Content and Movement in Single Dormant Bacterial Spores Using Confocal Raman Microspectroscopy and Raman Imaging

Heavy water (D2O) has a distinct molecular vibration spectrum, and this has been used to analyze the water content, distribution, and movement in single dormant Bacillus cereus spores using confocal Raman microspectroscopy and Raman imaging. These methods have been used to measure the kinetics of D2O release from spores suspended in D2O, the spatial distribution of D2O in spores, and the kinetics of D2O release from spores during dehydration in air at room temperature. The results obtained were as follows. (1) The Raman spectrum of single D2O-loaded dormant spores suggests that D2O in spores is in a relatively weak hydrogen-bonded mode, compared to the strong hydrogen-bonded mode in pure D2O. (2) The D2O content of individual spores in a population was somewhat heterogeneous. (3) The spatial distribution of D2O in single dormant spores is uneven, and is less dense in the central core region. Raman images of different molecular components indicate that the water distribution is somewhat different from those of proteins and Ca-dipicolinic acid. (4) Exchange of spore D2O with external H2O took place in less than 1 s. (5) However, release of spore D2O during air dehydration at room temperature was slow and heterogeneous and took 2-3 h for complete D2O release.