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). RECIPE HERE 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) at 0%, 1%, 3%, 5%, 8%, 10%, 12%, 15%, and from 0% to  20% w/v NaCl. pH tolerance was measured in liquid media (25C) at from  pH 3.4, 4.0, 4.9, 5.5, 6.3, 7.9, 8.2, 8.8, and 9.1. 3.4 to pH 8.0.  Temperature preference was measured in liquid culture across the range 4C to 30C. 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.