Dilution experiments. To determine the order of germinant binding to B. anthracis spores, germination was triggered upon fast dilution. B. anthracis spores treated with inosine and rap- idly diluted with L-alanine solution germinated normally and were stained red. Germination occurred even though inosine concentration was reduced from an optimal (100 M) to an inactive (4 M) concentration. Conversely, L-alanine-treated B. anthracis spores also germinated normally after dilution with an inosine solution (Fig. 2A). As a positive control, B. anthracis spore aliquots were resuspended in a solution con- taining 100 M inosine–100 M L-alanine. In this case, all spores germinated and were stained red (data not shown). As negative controls, B. anthracis spore aliquots were indepen- dently resuspended in solutions containing either 100 M ino- sine–4 M L-alanine or 4 M inosine–100 M L-alanine. In these cases, spores failed to germinate and were stained green (data not shown). Similar results were observed for dilution experiments of B. anthracis spores using inosine–L-serine and inosine–L-valine.