Holographic sensors for the detection of Bacillus species spore germination and vegetative growth are described. Reflection holograms were fabricated using a diffusion method for the distribution of ultra-fine silver bromide grains into pre-formed polymer films, followed by holographic recording using a frequency doubled Nd:YAG (532 nm) laser. Changes in holographic replay wavelength or diffraction intensity were used to characterise the swelling behaviour or structural integrity of a range of holographic matrices in response to various extracellular products of bacterial spore germination and vegetative metabolism. Divalent metal ion-sensitive holograms containing a methacrylated analogue of nitrilotriacetic acid (NTA) as the chelating monomer were successfully used to monitor Ca2+ ions released during B. subtilis spore germination in real-time, which was within minutes of sample addition; the holographic response manifested as a 16 nm blue-shift in diffraction wavelength over the progress of germination. Similarly, pH-sensitive holograms comprising methacrylic acid (MAA) as the ionisable monomer were responsive to changes in pH associated with early vegetative metabolism following germination of B. megaterium spores; a visually perceptible blue-shift in holographic replay wavelength of 75 nm was observed. Casein and starch-based holographic matrices, prepared by co-polymerisation of the appropriate substrate with acrylamide, were used to detect exo-enzymes released during later stages of B. megaterium and B. subtilis vegetative cell growth; holographic responses of both matrices were visible as a reduction in diffraction intensity due to progressive fringe disruption caused by enzymatic cleavage. The combined monitoring of various germination and growth events using the range of aforementioned holographic sensors provides a novel, comprehensive means for the detection of viable bacterial spores.
