Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers

摘要

We present a methodology that combines external phasencontrast microscopy, Raman spectroscopy, and opticalntweezers to monitor a variety of changes during thengermination of single Bacillus cereus spores in bothnnutrient (L-alanine) and non-nutrient (Ca-dipicolinic acidn(DPA)) germinants with a temporal resolution of ∼2s.nPhase contrast microscopy assesses changes in refractilitynof individual spores during germination, while Ramannspectroscopy gives information on changes in spore-nspecific molecules. The results obtained include (1) thenbrightness of the phase contrast image of an individualndormant spore is proportional to the level of CaDPA innthat spore; (2) the end of the first Stage of germination,nrevealed as the end of the rapid drop in spore refractilitynby phase contrast microscopy, precisely corresponds tonthe completion of the release of CaDPA as revealed bynRaman spectroscopy; and (3) the correspondence be-ntween the rapid drop in spore refractility and completenCaDPA release was observed not only for spores germi-nnating in the well-controlled environment of an optical trapnbut also for spores germinating when adhered on anmicroscope coverslip. Using this latter method, we alsonsimultaneously characterized the distribution of the time-nto-complete-CaDPA release (Trelease) of hundreds ofnindividual B. cereus spores germinating with bothnsaturating and subsaturating concentrations of L-ala-nnine and with CaDPA.