Toward absolute viability measurements for bacteria

We aim to develop a quantitative viability method that distinguishes individual quiescent from dead cells and is measured in time (ns) as a referenceable, comparable quantity. We demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for Streptococcus mutans. A random forest machine-learning model assesses whether individual S. mutans can be correctly classified into their original populations: stationary phase (quiescent), heat killed and inactivated via chemical fixation. We compare the results to intensity using three models: lifetime variables (τ1, τ2 and p1), phasor variables (G, S) or all five variables, with the five variable models having the most accurate classification. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species and fluorophores will help refine this approach

Examining the Initiation of the Polymerization Mechanism and Network Development in Aromatic Polybenzoxazines

[Image: see text] Three bis-benzoxazine monomers based on the aniline derivatives of bisphenol A (BA-a), bisphenol F (BF-a), and 3,3′-thiodiphenol (BT-a) are examined using a variety of spectroscopic, chromatographic, and thermomechanical techniques. The effect on the polymerization of the monomers is compared using two common compounds, 3,3′-thiodiphenol (TDP) and 3,3′-thiodipropionic acid (TDA), at a variety of loadings. It is found that the diacid has a greater effect on reducing the onset of polymerization and increasing cross-link density and T(g) for a given benzoxazine. However, the addition of >5 wt % of the diacid had a detrimental effect on the cross-link density, T(g), and thermal stability of the polymer. The kinetics of the polymerization of BA-a were found to be well described using an autocatalytic model for which values of n = 1.64 and m = 2.31 were obtained for the early and later stages of reaction (activation energy = 81 kJ/mol). Following recrystallization the same monomer yielded values n = 1.89, m = 0.89, and E(a) = 94 kJ/mol (confirming the influence of higher oligomers on reactivity). The choice of additive (in particular the magnitude of its pK(a)) appears to influence the nature of the network formation from a linear toward a more clusterlike growth mechanism