Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in a 1:1 chelate with calcium ion (Ca-DPA) comprises 5 to 15% of the dry weight of spores of Bacillus species. Ca-DPA is important in spore resistance to many environmental stresses and in spore stability, and Ca-DPA levels in spore populations can vary with spore species/strains, as well as with sporulation conditions. We have measured levels of Ca-DPA in large numbers of individual spores in populations of a variety of Bacillus species and strains by using microfluidic Raman tweezers, in which a single spore is trapped in a focused laser beam and its Ca-DPA is quantitated from the intensity of the Ca-DPA-specific band at 1,017 cm(-1) in Raman spectroscopy. Conclusions from these measurements include the following: (i) Ca-DPA concentrations in the spore core are >800 mM, well above Ca-DPA solubility; (ii) SpoVA proteins may be involved in Ca-DPA uptake in sporulation; and (iii) Ca-DPA levels differ significantly among individual spores in a population, but much of this variation could be due to variations in the sizes of individual spores.