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Ideal method for culturing cells: Start an overnight culture from a frozen stock and use that overnight culture for work. Always start from the frozen stock.
Less ideal method but still acceptable in a pinch: Start an overnight culture from a frozen stock, store that overnight culture at 4C, and use that overnight culture for further subdilutions and work. Usually, I use cultures left in the 4C for starting new cultures for about 3-5 days at most.
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