David Coil edited Methods.md  over 9 years ago

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**METHODS**  Cells were initially grown on plates containing either Reasoner´s 2A agar (R2A), or lysogeny broth agar (LB). LB was made with 10g tryptone, 10g NaCl, and 5g yeast extract per liter. A clear preference for LB was observed and so was used for all subsequent experiments. Salt tolerance was measured in liquid media (25C) from 0% to 20% w/v NaCl. pH tolerance was measured in liquid media (25C) from pH 3.4 to pH 8.0. Temperature preference was measured in liquid culture across the range 4C 4°C  to 30C. 30°C.  Cell morphology, motility, and presence/absence of flagella was examined by light microscopy (Zeiss Axio Lab.A1) and transmission electron microscopy (TEM). Cell cultures in either exponential or stationary phase were prepared for TEM by the UC Davis Electron Microscopy lab as follows. 400 mesh copper grids with formvar/carbon support film (Ted Pella, Inc., Redding, Ca.) were placed on dental wax. A 10μl drop of fixed of unfixed sample was placed onto the grid and left in a dust-free environment for 10 min. Then excess was wicked off with filter paper. A 10μl drop of 1% PTA pH 5.8 (phosphotungstic acid) or 1% ammonium molybdate in DDH2O was added to the grid and wicked off immediately. Grids were allowed to air-dry completely before viewing in a Philips CM120 (FEI/Philips Inc, Hillsborough, Or.) electron microscope at 80KV.  Catalase and oxidase activity, as well as the hydrolysis of starch and casein were assayed by standard methods. Carbon source utilization was assayed using the Phenotypic MicroArray(TM) services offered by Biolog Inc following standard proceedures for gram-negative bacteria. Colonies were grown on blood agar at room temperature and suspended in IF-0a inoculating fluid (Biolog) to a density of 42% transmittance. The cell suspension was diluted 1:6 in IF-0a plus 1x Dye H (Biolog) and a carbon source utilization MicroPlate (PM1; Biolog) was inoculated with 100μl per well. The PM1 microplate was incubated at 23 degrees C 23°C  and read by the OmniLog instrument every 15 minutes for 96 hours. Duplicate sets of OmniLog data were converted to average read value and a threshold of 78 was required in both replicates for a positive call. **Respiratory quinones, polar lips, and fatty acids**