To begin, obtain the extruded agarose segments from gradient syringes, inoculated with either wild type or mutant methylomona species, LW13. Add 0.75 milliliters of 0.85%sodium chloride in water to the extruded agarose sample, and homogenize by vortexing. Further, dilute the sample 1 to 10 in a new micro centrifuge tube with 900 microliters of salt solution.
Incubate the samples with three microliters of a one to one mixture of SYTO 9 and propidium iodide stains in the dark at room temperature for 15 minutes. To determine the cells per milliliter of agarose, sonicate the microsphere counting bead suspension in a water bath for five minutes. Then add 10 microliters of the suspension to the sample before flow cytometry analysis.
Analyze samples using a flow cytometer. Compare side scatter versus forward scattered op plots between cell-free control samples and inoculated agarose samples to draw bacterial event voltage gates that exclude background agarose particles. To count colony forming units within the gradient syringe, add 800 microliters of nitrate mineral salts medium to the extruded agarose segment and vortex for 10 seconds to aid in pipetting.
Prepare a sterile 96-well plate with 180 microliters of nitrate mineral salts medium in each well. Add 20 microliters of diluted agarose sample to each well in the first column and mix. Using a multi-channel pipette, serially dilute 20 microliters of samples tenfold, starting from the first row of wells.
Label square grid plate containing nitrate mineral salts auger or media of choice. Using a multichannel pipette, spot five microliters from a column of the 96-well plate onto the auger plate. Flow cytometry using the extruded agarose samples confirmed that the OAT deletion mutant of LW13 showed reduced cell growth as compared to the wild type strain.
Complementation of the mutant with the OAT gene restored cell numbers and band formation to levels similar to the wild type, indicating the gene's specific role in band formation.
