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Bacteria were cultured in 400 ml M9 medium (6 g Na 2 HPO 4 , 3 g KH 2 PO 4 , 0.5 g NaCl, 1 g NH 4 Cl, 1 ml of 1 M MgSO 4 , 5.6 ml of 2 M glucose, 1 ml of 1% thiamine, 0.1 ml of 1 M CaCl 2 , and 100 g/ml ampicillin in 1 l distilled water) at 20 C for 24 hr in a shaker (Takasaki Scientific Instruments Corp

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Its recommended to wait forty-five minutes before washing it off because at forty-five minutes, peak levels are seen in the blood