User:Sim Huay Ping/Notebook/CBE/08/150/2008/09/26

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Laboratory basics Procedure for preparing culture agar media 1. Weigh out 14 g of the DifcoTM LB Agar, Lennox powder on trace paper on the weighing machine. 2. Suspend 14 g of the powder in 400 mL of purified water. (Written on the container is 35g of the powder in 1L of purified water but we only have 400 mL flasks). 3. Mix thoroughly. 4. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. 5. Autoclave at 121 deg C for 15 minutes. 6. At room temperatures the agar will solidify. Hence, agar is reheated in the microwave to become liquid form again before use. Note that the agar has to be cooled to around 50 deg C before it can be poured into the petri dish. Procedure for preparing nutrient broth 1. Weigh out 3.2 g of the DifcoTM Nutrient Broth powder on trace paper on the weighing machine. 2. Suspend 3.2 g of the powder in 400 mL of purified water. (Written on the container is 8g of the powder in 1L of purified water but we only have 400 mL flasks). 3. Mix thoroughly. 4. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. 5. Autoclave at 121 deg C for 15 minutes. Procedure for preparing Mueller Hinton broth 1. Weigh out 8.8 g of the BBLTM Mueller Hinton Broth powder on trace paper on the weighing machine. 2. Suspend 8.8 g of the powder in 400 mL of purified water. (Written on the container is 22g of the powder in 1L of purified water but we only have 400 mL flasks). 3. Mix thoroughly. 4. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. 5. Autoclave at 116 to 121 deg C for 10 minutes. DO NOT OVERHEAT. Note that the nutrient broth has to be cooled to around 50 deg C before it can be poured into test tubes. Never use a nutrient broth that has been kept for one week or longer after preparation. Never use a nutrient agar that has been kept for one month or longer after preparation.