The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

In this study, a Müller cell culture preparation from young rats was used to investigate the regulation of GLAST transport activity in native cells. Immunohistochemical analysis confirmed GLAST to be the predominant glutamate transporter expressed by the cells through five passages. [3H]-glutamate uptake assays showed the typical Na+-dependent glutamate transport which was blocked by L-(-)-threo-3-hydroxyaspartate (L-THA), a competitive inhibitor. Glutamate transport was decreased significantly in Müller cells exposed to phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. A similar effect on [3H]-D-aspartate (nonmetabolizable glutamate analog) uptake ruled out the possibility that the decrease was a consequence of altered metabolism. However, PMA did not affect Na+-dependent [3H]-glycine transport, indicating the absence of a nonspecific change in the electrochemical gradients. The PMA effect on glutamate uptake was evidenced by partial blocking with a specific PKC inhibitor, bisindolymaleimide II (Bis II). Activation of PKC did not change the Km, but the Vmax was significantly reduced. Image analysis of Müller cells with biotinylated cell membranes immunolabeled with GLAST shows a reduction of GLAST in the plasma membrane. In conclusion, these data show that rat Müller cells in primary cultures express GLAST and that PKC activation affects GLAST transport activity by decreasing cell surface expression.