ABSTRACTThe Gram-positive spore-forming anaerobeClostridium difficileis a leading cause of nosocomial diarrhea. Spores ofC. difficileinitiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individualC. difficilespores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar toBacillusspores, individualC. difficilespores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca2+and dipicolinic acid (CaDPA) at a heterogeneous timeT1, rapidly released CaDPA atTlag, completed CaDPA release atTrelease, and finished peptidoglycan cortex hydrolysis atTlysis.T1andTlagvalues for individual spores were heterogeneous, but ΔTreleaseperiods (Trelease−Tlag) were relatively constant. In contrast toBacillusspores, heat treatment did not stimulate spore germination in the twoC. difficilestrains tested.C. difficilespores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate.C. difficilespores did not germinate with CaDPA, in contrast toB. subtilisandC. perfringensspores. However, the detergent dodecylamine inducedC. difficilespore germination, and rates were increased by spore coat removal although cortex hydrolysis did not followTrelease, in contrast withB. subtilis.C. difficilespores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the fewsleCspores that partially germinated. Overall, these findings indicate thatC. difficileandB. subtilisspore germination exhibit key differences.IMPORTANCESpores of the Gram-positive anaerobeClostridium difficileare responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts,C. difficilespores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individualC. difficilespores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events inC. difficilespores differs from that inBacillusspores and provide new insights intoC. difficilespore germination.