To begin, obtain a culture plate with Methylomonas species LW13 colonies. Inoculate the colonies in six milliliters of nitrate mineral salts medium in a glass tube. Seal the tube with a serum stopper and aluminum crimp seal.
Add methane using a syringe to create a final atmosphere of 50%volume by volume methane in the air. Shake this planktonic liquid culture at 200 revolutions per minute at room temperature until turbid, which takes about one day. Passage the liquid cultures at a one to 10 ratio into fresh media.
Continue growing the liquid cultures of methanotrophs to log phase growth, reaching an optical density at 600 nanometers of approximately 0.5. To prepare the syringe, remove the accompanying plungers and place it in a sterile container. Attach a sterile polytetrafluoroethylene or PTFE filter tip to each syringe and place the syringe in a standard test tube rack with the tip facing down.
Then, mix one milliliter of the cells with five milliliters of nitrate mineral salts medium and four milliliters of molten agarose in a sterile conical tube. Slowly pour the agarose mixture into the syringe up to the eight milliliter mark and let it solidify. After approximately 15 minutes, cap the syringe with a sterile 20 millimeter rubber butyl stopper.
Secure the stoppers with lab tape and label the syringe. Next, fill a large 60-milliliter syringe with 100%methane and attach a PTFE filter tip connected to a sterile 23-gauge needle. Pierce the rubber stopper of the syringe with the agarose mixture with a large syringe, and insert a second sterile needle as a gas outlet.
Depress the plunger of the large syringe to allow 20 milliliters of 100%methane to flush through the head space. Remove the outlet needle when there are one to two milliliters of methane left in the syringe to prevent oxygen backflow. Incubate the syringe with agarose mixture and methane at 18 degrees Celsius.
To extrude the agarose, replace the PTFE filter tip with a sterile 23-gauge needle and the rubber stopper with the supplied syringe plunger. Slowly depress the plunger to dispense one milliliter increments into separate sterile 1.5-milliliter microcentrifuge tubes. The wild type LW13 strain formed a distinct horizontal band at a specific depth in the gradient syringe where both methane and oxygen concentrations were low.
LW13 inoculated in gradient syringes showed a methane-oxygen counter gradient with methane depletion and oxygen consumption corresponding to the depth of the horizontal band. The OAT deletion mutant of LW13 lacked the distinct horizontal band formation observed in the wild type strain, indicating the gene's role in this phenotype.
