Differentiation of live-viable versus dead bacterial endospores by calibrated hyperspectral reflectance microscopy

This paper describes the use of hyperspectral imaging microscopy (HIM) for the characterization and differentiation of live viable versus dead/non-viable bacterial endospores for two species of Bacillus. To accomplish this, endospore-forming Bacillus were cultured and differentiated into endospores. Non-viable endospores were produced using sporicidal methods representing standard decontamination procedures incorporating chlorine and peroxide. Finally, endospore samples were lyophilized to prepare them for spectral analysis. Prior to HIM, baseline spectral reflectance characterizing the endospores was measured using an ASD (400-900 nm) reflectance spectrometer. These data were used to calibrate the resulting spectral image data. HIM data comprising 32 images ranging from 400 to 720 nm (visible to near infrared) were recorded using a C-mounted VariSpec hyperspectral camera attached to an epifluorescent microscope. The images produced by the system record the reflectance and absorption features of endospores based on the structure of the outer coat. Analysis of the HIM data was performed using accepted image and spectral processing routines. Where peroxide was the sporicide, changes in the outer endospore coat contributed to structurally significant visible and near infrared signature differences between live-viable versus dead, non-viable endospores. A statistical test for divergence, a method for scoring spectral structural diversity, also showed the difference between viable and non-viable peroxide killed endospores to be statistically significant. These findings may lead to an improved optical procedure to rapidly identify viable and non-viable endospores in situations of decontamination.