Optimisation of the Protocol for the LIVE/DEAD® BacLightTM Bacterial Viability Kit for Rapid Determination of Bacterial Load

Rapid antimicrobial susceptibility testing is needed to reduce prescription of inappropriate antibiotics. A rapid alternative to standard culture-based testing is to determine reductions in cell viability using the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We optimised the kit protocol for this application, focusing on simplifying the process by minimising the steps involved and on determining the optimal analytical parameters for fluorescence measurements from the dyes SYTO 9 and propidium iodide (PI). We demonstrate that for our experimental system, the intensity of emissions should be integrated from 505–515 nm for SYTO 9 and 600–610 nm for PI, and the proportion of live cells calculated from a new dye ratio formula, termed the adjusted dye ratio. We show that the pre-staining washing step is not necessary if a non-fluorescent growth media is used; however, staining must be done for each sampling as prolonged exposure to the dyes negatively impacts cell viability. The optimised methodology was able to reproducibly detect reductions in culture viability when the proportion of live cells in a sample of 1 × 108 cells/ml fell below ∼50% live in a media that supports the growth required for detecting antibiotic killing. Finally, we show that the interaction of fluorescence emission spectra from SYTO 9 and PI stained Escherichia coli cells is influenced by the proportion of dead cells in a sample. The excitation of PI by SYTO 9 was found to occur in populations containing sufficient numbers of dead cells (>25%), whereas in populations with low numbers of dead cells the dye interaction was additive in regard to red emissions, indicating that these dye interactions may offer another dimension to live/dead analysis. Fluorescence measurements from samples established according to the optimised protocol can be taken using a flow cytometer, spectrofluorometer, microplate reader, and the Optrode, a fibre-based spectroscopic system developed at the University of Auckland

Application of new insoluble dietary fibres from triticale as supplement in yoghurt - effects on physico-chemical, rheological and quality properties

BACKGROUND: The need to increase the daily intake of dietary fibres opens a new chapter in the research of functional foods enriched with fibres. The potential application of an innovative product - insoluble dietary fibres from triticale in yoghurts - was deployed by characterising their food application and evaluating physico-chemical, rheological and sensory properties and was the aim of this research. RESULTS: Detailed characterisations of these fibres are presented for the first time and showed very good hydration properties, optimal pH ( slightly acidic), optimal chemical composition, high antioxidant capacity which was proven by phenolics contents. Besides, these fibres showed negligible calorific value, with no phytates and high antioxidant capacity, mainly from ferulic acid. Therefore they could be successfully added to yoghurt. Enrichment of yoghurt having different milk fat content (1.5 and 2.8% w/w) with triticale insoluble fibre (1.5% and 3.0% w/w) significantly influenced the syneresis level, its apparent viscosity, yield stress and thixotropic behaviour. The overall sensory quality scores indicated that yoghurt enriched with 1.5% triticale insoluble fibres was recognised as 'excellent' and had enhanced antioxidant activity. CONCLUSIONS: Insoluble triticale fibre could therefore be used as a supplement to produce functional yoghurt

Classification of maize kernel hardness using near infrared hyperspectral imaging

AgriwetenskappeVoedselwetenskapPlease help us populate SUNScholar with the post print version of this article. It can be e-mailed to: [email protected]

Raman mapping of pharmaceuticals

Raman spectroscopy may be implemented through a microscope to provide fine scale axial and lateral chemical maps. The molecular structure of many drugs makes Raman spectroscopy particularly well suited to the investigation of pharmaceutical systems. Chemometric methods currently used to assess bulk Raman spectroscopic data are typically applied to Raman mapping data from pharmaceuticals; few reports exist where the spatial information inherent to a mapped dataset is used for the calculation of chemical maps. Both univariate and multivariate methods have been applied to Raman mapping data to determine the distribution of active pharmaceutical ingredients (APIs) in tablets, solid dispersions for increased solubility and controlled release devices. The ability to axially (depth) profile using Raman mapping has been used in studies of API penetration through membranes, cellular uptake of drug delivery liposomes, and initial API distribution and subsequent elution from coatings of medical devices. New instrumental developments will increase the efficiency of Raman mapping and lead to greater utilisation of Raman mapping for analyses of pharmaceutical systems. © 2011 Elsevier B.V.Articl

Characterisation of non-viable whole barley, wheat and sorghum grains using near-infrared hyperspectral data and chemometrics

Undesired germination of cereal grains diminishes process utility and economic return. Pre-germination, the term used to describe untimely germination, leads to reduced viability of a grain sample. Accurate and rapid identification of non-viable grain is necessary to reduce losses associated with pre-germination. Viability of barley, wheat and sorghum grains was investigated with near-infrared hyperspectral imaging. Principal component analyses applied to cleaned hyperspectral images were able to differentiate between viable and non-viable classes in principal component (PC) five for barley and sorghum and in PC6 for wheat. An OH stretching and deformation combination mode (1,920-1,940 nm) featured in the loading line plots of these PCs; this water-based vibrational mode was a major contributor to the viable/non-viable differentiation. Viable and non-viable classes for partial least squares-discriminant analysis (PLS-DA) were assigned from PC scores that correlated with incubation time. The PLS-DA predictions of the viable proportion correlated well with the viable proportion observed using the tetrazolium test. Partial least squares regression analysis could not be used as a source of contrast in the hyperspectral images due to sampling issues. [Figure not available: see fulltext.] © 2011 Springer-Verlag.Article in Pres

Near infrared analysis of fossil bone from the Western Cape of South Africa

The burial environment of a fossil bone may be poorly suited to long-term preservation of important chemical constituents. Studies of fossil bone chemistry consequently benefit from knowledge of burial environmental change (burial history). Several techniques have been developed to describe alteration of fossil bone, including measurements of crystallinity using infrared spectroscopy. The utility of near infrared (NIR) spectroscopy was investigated for describing the burial history of fossil bone. Spectra from large sample areas (bulk NIR) identified secondary calcite and clay within fossil bone, and were useful for distinguishing fossils from different localities. Spectra from small sample areas (hyperspectral NIR) were used to map constituents within fossil bones. Calcite and clay pervaded the internal cavities of fossil horn cores, indicating saturation with groundwater. The identification and distribution of secondary minerals is relevant for studies of taphonomic process, hence NIR is a useful supplement to existing techniques for assessing the burial history of fossil bone. © 2011 IM Publications LLP. All right reserved.Articl