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The ethanol and acetic acid are sterilized separately by filtration (use filter resistant to these solvents), and added aseptically to the sterile medium. Acetobacter europaeus will not grow on plates containing 1-2% agar, and a double layer system has to be used. A layer of growth medium containing 0.5% agar (about 35 ml) is poured into a plate and allowed to solidify. A thin layer (5-10 ml) of the growth medium containing 1% agar is then poured on top. Carefully streak the plates, which are best incubated in closed containers (e.g. plastic boxes) at 30°C.
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