This study describes a system for identifying Bacillus species using API 20E and API 50CHB biochemical test strips combined with morphological and supplementary tests. The authors analyzed 1,075 Bacillus strains, including type strains for most recognized species, to establish a comprehensive identification matrix. Results are presented as percentage positive reactions for each species across 75 tests (12 from API 20E, 49 from API 50CHB, and morphological characters). The authors discuss species-specific patterns, highlighting challenges in Bacillus taxonomy including species complexes (B. cereus group, B. circulans), spectra arrangements (B. firmus, B. lentus), and heterogeneous species (B. coagulans). Several tests proved uninformative for Bacillus identification. The system provides good discrimination among most species despite considerable within-species variation in several taxa. The results matrix enables both manual identification and computer-assisted identification using similarity coefficients and principal coordinate analysis. The authors note that while some species remain difficult to separate using API tests, the system provides more reproducible results than classical methods and is faster and more practical for routine laboratory use.

Key findings

• API 20E and API 50CHB biochemical strips provide reproducible and discriminatory identification of most Bacillus species, with only a small number of redundant tests despite the systems being originally designed for Enterobacteriaceae
• Significant challenges remain for Bacillus taxonomy, including species complexes (B. cereus/mycoides/thuringiensis), spectra arrangements (B. firmus/lentus), and poorly defined heterogeneous species (B. coagulans), which cannot be clearly separated by API tests alone
• The system enables both manual identification using biochemical patterns and computer-assisted identification through similarity coefficients and principal coordinate analysis
• Eight tests proved uninformative (always negative) and several others had limited discriminatory value, though supportive data aids identification of atypical strains
• Five Bacillus species were excluded from the study due to special growth requirements (fastidious insect pathogens, extreme pH requirements) that are incompatible with API media