Potassium efflux was measured using the K+-sensitive fluorescent dye 1,3- benzenedicarboxylic acid, 4,4’-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5- methoxy-6,2-benzofurandiyl))bis (PBFI, Molecular Probes/Invitrogen). A culture of
Micrococcus luteus was grown to an OD600 of 1.0 and washed five times with assay buffer (5
mM HEPES, 5 mM glucose, pH 7.2). Washed cells were resuspended in one-fifth the volume of assay buffer containing PBFI (final concentration of 10 μM) and aliquots were added to UV- transparent microtiter plates (Nunc). Sample fluorescence (excitation = 346 nm, emission = 505 nm) background data was collected (BioTek Synergy 2 plate reader, excitation filter 360/40, emission filter 508/20) until baseline signal was achieved, followed by the addition of haloduracin (Halα and Halβ in a 1:1 ratio) or nisin at 10 μM, 5 μM, 1 μM, 250 nM, and 0 nM. For pre-incubation studies of the haloduracin peptides, baseline signal was achieved followed by the addition of either Halα or Halβ to wells at the concentrations indicated and incubated for 5 min prior to the addition of Halβ or Halα, respectively.
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2.4.13. Inhibition of Bacillus anthracis spore outgrowth
B. anthracis spores at a concentration of 4.0 x 106 spores mL-1 were incubated in Brain
Heart Infusion (BHI) medium supplemented with haloduracin (0.1, 1, 10, 100 μM at a Halα:Halβ ratio of 1:1) or 0.1 M MOPS (pH 6.8) as a control. For assessment of Halα and Halβ used independently, spores were incubated in BHI medium supplemented with the peptide at 0.1, 1, 10, 100 μM or with 0.1 M MOPS (pH 6.8) as a control. As a positive control, spores were treated with nisin under the same conditions and concentrations as used for Halα and Halβ. Cultures were incubated at 37 °C under aeration conditions and ambient CO2 (0.03% CO2). At 0, 5, and 10 h, samples were removed from the B. anthracis cultures and fixed by incubation in 3% formaldehyde (v/v) for 30 min at 37 °C. Samples were mounted on glass microscope slides in 20% glycerol (v/v). Differential interference contrast (DIC) microscopy images were collected with an Applied Precision assembled DeltaVision epifluorescence microscope equipped with an Olympus Plan Apo x100 oil objective with a numerical aperture of 1.42 and a working distance of 0.15 mm. Images were processed with SoftWoRX Explorer Suite.
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CHAPTER 3: NOVEL METHODOLOGIES IN THE PREPARATION OF