Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Laccase immobilization on bacterial nanocellulose membranes: antimicrobial, kinetic and stability properties

This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline I cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48 kJ mol1 for 2,6-dimethylphenol (DMP), catechol and 2,2 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. The Michaelis-Menten constant (Km) value for the immobilized laccase (0.77 mM) was found to be almost double of that of the free enzyme (0.42 mM). However, the specific activities of immobilized and free laccase are similar suggesting that the cage-like structure of BNC allows entrapped laccase to maintain some flexibility and favour substrate accessibility. The results clearly show the antimicrobial effect of laccase in Gram-positive (92%) and Gram-negative (26%) bacteria and cytotoxicity acceptable for wound dressing applications.

Separation by thermal diffusion in a rotary column

A rotary thermal diffusion column with the inner cylinder rotating and the outer cylinder static was used to separate n-heptane-benzene mixtures at different speeds of rotation. The results show that the column efficiency depends on the speed of rotation. For the optimum speed the increase in efficiency relative to the static column was of the order of 8%. The role of the geometric irregularities in the annulus width on performance of the rotary column is also discussed.

Nonsymmetrical separations in thermal diffusion columns

The influence of the feed composition upon the actual degrees of separation attained at the top and bottom sections of a thermogravitational column is discussed using the classical phenomenological theory of Furry, Jones, and Onsager. It is shown that, except for a feed composition of C 0 = 0.5 (mass fraction), the separation profile is nonsymmetric, i.e., the separations in the top and bottom sections of the column are nonsymmetric with respect to the feed composition, the asymmetry increasing as the feed composition moves away from C 0 = 0.5. An equation for the determination of the optimum feed location as a function of the feed composition is derived.