Untreated and scCO2-PAA-treated spores were incubated in LB medium plus 10 mmol l−1 L-valine as described above, but the spores were heat shocked (60 min; 70°C) prior to initiating germination. After 45 min, samples were incubated with the BacLight reagent (Thermo Fisher, Waltham, MA USA) to determine if the germinated spores were likely alive or dead, as live cells and live germinated spores stain green while dead cells and dead germinated spores stain red (Zhang et al. 2007 ). After incubation for ~ 15 min with the BacLight reagent spores were photographed on a fluorescence microscope (Murray et al. 1998 (link); Coleman et al. 2007 (link)). Dormant untreated or scCO2-PAA-treated spores were also stained with the BacLight reagent and photographed as described above. In some experiments, dilutions of spores were spotted on LB medium plates containing either 150 mmol l−1 NaCl, 1 mol l−1 NaCl or 1 mol l−1 NaCl plus 10 mmol l−1 D-glucose, plates incubated at 30-37°C for 24-48 h, and colonies were counted.
Baclight reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The BacLight reagent is a fluorescent dye-based solution used for the detection and enumeration of live and dead bacterial cells. It contains a mixture of nucleic acid-binding dyes that emit different fluorescent signals when bound to cells with intact or compromised membranes, allowing for the differentiation between live and dead bacterial populations.
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Lab products found in correlation
2 protocols using baclight reagent
1
Germination of Bacillus Spores: Viability Assay
2
Imaging Ace-K Effects on A. baumannii Biofilms
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To image the effect of ace‐k treatment on A. baumannii, biofilms were grown on glass microscope slides. Sterile glass slides were placed in sterile tubes. Cultures of AB5075 (OD600 0.5) were added to the tubes so that half of the slide was covered (20 ml total volume) and samples were incubated statically for 18 h at 37°C. Following incubation, slides were removed and biofilms were washed by gently submerging them in sterile deionised water 3 times. Biofilms were then air‐dried in a sterile environment before staining with 200 μl of BacLight reagent (ThermoFisher, UK), prepared as per the manufacturer's guidance. Biofilms were stained for 30 min in a dark, sterile environment. Biofilms were imaged using a Lieca HF14 DM4000 microscope using L5 and CY3 filters. The native LAS AF software was used for image capture. During imaging, five fields of view were visualised and the exact coordinates and imaging parameters of each were noted. The same biofilm was then treated with an 8.85% ace‐K soaked gauze for 1 h before being washed and stained as previously detailed. The same five fields of view were imaged again using the same imaging parameters so that a pre‐ and post‐treatment comparison could be made. Heatmaps and 3‐dimensional representations were prepared as previously described by Pandian et al (2021 (link)) using the ImageJ “3D Interactive Surface Plot” plugin.
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