OBJECTIVE: We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and surveillance.

Multiple fluorochromes are extensively used to investigate different microalgal aspects, such as viability and physiology. Some of them can be used to stain nucleic acids (DNA). Well-known examples are SYBR Green I and SYTO 9, the latter of which offers several advantages, especially when combined with flow cytometry (FCM)—a powerful method for studying microalgal population heterogeneity and analyzing their cell cycles. However, the effects of these dyes on the microalgae cell physiology have not been fully elucidated yet. A statistical experimental design, using response surface methodology (RSM) with FCM was applied in this study to optimize the DNA staining of a non-conventional microalgae, Chromochloris zofingiensis, with SYBR Green I and SYTO 9, and to optimize the variables affecting staining efficiency, i.e., the dye concentration, incubation time and staining temperature. We found that none of these factors affects the staining efficiency, which was not less than 99.65%. However, for both dyes, the dye concentration was shown to be the most significant factor causing cell damage (p-values: 0.0003; <0.0001) for SYBR Green I and SYTO 9, respectively. The staining temperature was only significant for SYTO 9 (p-value: 0.0082), and no significant effect was observed regarding the incubation time for both dyes. The values of the optimized parameters (0.5 µM, 05 min and 25 °C) for SYTO 9 and (0.5 X, 5 min and 25 °C) for SYBR Green I resulted in the maximum staining efficiency (99.8%; 99.6%), and the minimum damaging effects (12.86%; 13.75%) for SYTO 9 and SYBR Green I, respectively. These results offer new perspectives for improving the use of DNA staining fluorochromes and provides insights into their possible side effects on microalgae.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire\'s disease.

Measuring viability is an important and necessary assessment in studying microorganisms. Several methods can be applied to Leptospira spp., each with advantages and inconveniencies. Here, we describe the traditional colony-forming unit method, together with two other methods based, respectively, on the reducing capacity of live cells (Alamar Blue® Assay) and differential staining of live and dead cells (LIVE/DEAD BacLight®). The Alamar Blue® Assay uses the blue reagent resazurin, which can be reduced into the pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance or fluorescence reading. The LIVE/DEAD BacLight® assay uses a mixture of two nucleic acid dyes (Syto9 and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The two other methods are not laborious and well-adapted to high-throughput studies, but the range of detection and linearity are limited.

OBJECTIVE: Biofilm microorganisms are known to have a much higher tolerance to antimicrobials compared to their planktonic equivalents. Therefore, traditional antimicrobial susceptibility testing may not extrapolate to clinical treatment of infections of biofilm origin, and as a result, there is a need to not only develop antimicrobials with antibiofilm activity, but also suitable in vitro testing methods for their evaluation. In this study, we report on a novel method of antibiofilm testing using a thermo-reversible matrix (poloxamer 407), coupled with live/dead staining of bacteria cultured from the matrix.
METHODS: Pseudomonas aeruginosa (NCIMB 8626) was cultured in medium containing poloxamer 407 at 37°C for 24 hours to generate biofilms. The preparation was cooled to liquefy the poloxamer and allow recovery of the biofilm cells, which were then stained with SYTO9 to determine viability following exposure to four antimicrobials: polyhexanide, octenadine dihydrochloride, povidone-iodine and silver carbonate. Over an 8-minute time period, fluorescence levels were spectrophotometrically measured and compared with bacterial controls, cultured in the absence of poloxamer and without antimicrobial.
RESULTS: Untreated cells showed no reduction in viability over this period. Importantly, planktonic cells were more susceptible to test agents compared with those of a \'biofilm\' phenotype cultured in poloxamer. Antibiofilm activity was evident for all of the test agents, with highest relative activity seen with octenadine dihydrochloride.
CONCLUSIONS: In summary, a novel and relatively rapid approach to screen compounds for antibiofilm activity has been described. The method uses standard laboratory equipment and can be readily adapted to test a wide range of microorganisms and other antibiofilm compounds.
BACKGROUND: This research was, in part, supported by Advanced Medical Solutions in the form of a Knowledge Transfer Project. Mr J. Nosworthy was employed by Advanced Medical Solutions. There are no other conflicts of interests to declare.