AN AUTOMATED METHOD FOR QUANTIFYING THE L-ALANINE TRIGGER OF BACILLUS SUBTILIS SPORE GERMINATION AND COMPETITIVE INHIBITION BY D-ALANINE

We developed an automated method for studying the germination kinetics of Bacillus subtilis spores using a microtiter plate (MP) reader. Phosphate buffer supplemented with L-alanine was used to isolate the germination phase as determined by decrease in optical density (OD₆₃₀). Using a standard 96-well MP, L-alanine triggered germination kinetics were measured by automatic OD measurement every 3 min until the maximum OD₆₃₀ change (OD₆₃₀) was determined. When OD₆₃₀ values were plotted against L-alanine concentration on a double reciprocal plot, a straight line (R²= 0.98) was produced. The addition of D-alanine to the medium demonstrated classical competitive inhibition on double reciprocal plots. A 3-dimensional representation of the untransformed data showed the response surface nature of competitive inhibition. The method automates the tedious task of determining loss of refractility associated with spore germination under defined conditions so that inhibitors to germination can be studied. Since 96 OD₆₃₀ determinations can be done simultaneously in small volumes (200 μL) extensive data can be generated about inhibitors using relatively small spore crops in a single, short (1.4 h) incubation.