Tl. Romick et G. Tharrington, AN AUTOMATED-METHOD FOR QUANTIFYING THE L-ALANINE TRIGGER OF BACILLUS-SUBTILIS SPORE GERMINATION AND COMPETITIVE-INHIBITION BY D-ALANINE, Journal of rapid methods and automation in microbiology, 5(3), 1997, pp. 215-221
We developed an automated method for studying the germination kinetics
of Bacillus subtilis spores using a microtiter plate (MP) reader. Pho
sphate buffer supplemented with L-alanine was used to isolate the germ
ination phase as determined by decrease in optical density (OD630). Us
ing a standard 96-well MP, L-alanine triggered germination kinetics we
re measured by automatic OD measurement every 3 min until the maximum
OD630 change (Delta OD630) was determined. When Delta OD630 values wer
e plotted against L-alanine concentration on a double reciprocal plot,
a straight line (R-2 = 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 in
hibitors to germination can be studied. Since 96 Delta OD630 determina
tions can be done simultaneously in small volumes (200 mu L) extensive
data can be generated about inhibitors using relatively small spore c
rops in a single, short (1.4 h) incubation.