An improved method for qualitative and quantitative sampling of bacterial endospores using Fourier transform infrared (FT-IR) microscopy on gold-coated porous alumina membranes is presented. Bacillus subtilis endospores were filtered onto gold-coated alumina membranes serving as substrates. Studies in the mid-infrared (MIR) region revealed the characteristic bacterial absorption spectrum at low surface concentration, while scanning electron microscopy (SEM) images of the same samples provided precise calculation of the surface concentration of the bacterial endospores. Under the conditions of study, the average concentration of endospores was determined to be 1356 ± 35 spores in a 100 × 100 μm2 area, with a relative standard deviation of 0.0260. Examination of ten random spots on multiple substrates with FT-IR microscopy apertured to the same area gave an average relative standard deviation of 0.0482 in the signal strength of the amide A band at 3278 cm−1. An extinction cross-section in reflection of σext = (7.8 ± 0.6) × 10−9 cm2/endospore was calculated for the amide A band at the frequency of its peak absorbance, 3278 cm−1. The absorption cross-section of the amide A band in reflection is estimated to be σabs ≈ (2.10 ± 0.12) × 10−9 cm2/endospore.
Maria V. Schiza, David L. Perkins, Ryan J. Priore, B. Setlow, P. Setlow, Burt V. Bronk, Diane M. Wong, and M. L. Myrick, "Improved Dispersion of Bacterial Endospores for Quantitative Infrared Sampling on Gold Coated Porous Alumina Membranes," Appl. Spectrosc. 59, 1068-1074 (2005)