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From 2010.igem.org

Jeremy's Notebook

Growing Broth for Pseudomonas

Composition of Medium

• 100 mg Naphthenic acids /L

(Modified Bushnell-Has medium)

• 1000 mg KH2PO4 /L
• 1000 mg Na2HPO4 /L
• 500 mg NH4NO3 /L
• 500 mg (NH4)2SO4 /L
• 200 mg MgSO4 ∙ 7H2O /L
• 20 mg CaCl2 ∙ 2H2O /L
• 2 mg FeCl3 /L
• 2 mg MnSO4 ∙ 2H2O /L

pH of Bushnell-Has Medium: 7.0

Protocol

Prepare naphthenic acids stock

• Mix 1 g naphthenic acid /L in dilute NaOH.
• Adjust pH of the solution to 10-11 to dissolve naphthenic acids into the solution as sodium salts

Making the Growing Broth

• Mix 20 ml naphthenic acid stock with 170 ml sterilized water in 500 ml erlenmeyer flask.
• Prepare 10 ml Bushnell-Has medium in vial
• Mix arbitrary amount of pseudomonas strain in B-H medium
• Transfer medium to erlenmeyer flask
• Incubation (aerobic conditions)
• Incubate stock at room temperature (20 °C) on a shaker at 200 rpm.