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Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a reliable and rapid technique applied widely in the identification and classification of microbes. MALDI-TOF MS has been used to identify many endospore-forming  Bacillus  species; however, endospores affect the identification accuracy when using MALDI-TOF MS because they change the protein composition of samples. Since culture conditions directly influence endospore formation and  Bacillus  growth, in this study we clarified how culture conditions influence the classification of  Bacillus  species by using MALDI-TOF MS. We analyzed members of the  Bacillus subtilis  group and  Bacillus cereus  group using different incubation periods, temperatures and media. Incubation period was found to affect mass spectra due to endospores which were observed mixing with vegetative cells after 24 hours. Culture temperature also resulted in different mass spectra profiles depending on the temperature best suited growth and sporulation. Conversely, the four common media for  Bacillus  incubation, Luria-Bertani agar, nutrient agar, plate count agar and brain-heart infusion agar did not result in any significant differences in mass spectra profiles. Profiles in the range  m/z  1000–3000 were found to provide additional data to the standard ribosomal peptide/protein region  m/z  3000–15000 profiles to enable easier differentiation of some highly similar species and the identification of new strains under fresh culture conditions. In summary, control of culture conditions is vital for  Bacillus  identification and classification by MALDI-TOF MS.

Bacillus Classification Based on Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry—Effects of Culture Conditions