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A. Making Media Purpose: To cultivate microbes Types: Agar & broth
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A. Making Media Materials: Per pair of students: 1-500mL Erlenmeyer flask Media bottles & caps Hotplate 16x125 Test tubes & caps 1-5mL, 1-10mL pipettes Pipetting bulb 2 Aluminum foil squares 1 Stir bar Gloves Trypticase Soy Agar Tryptic Soy Broth Weigh boats Spoons/scoopula Balance Graduated cylinder Stir bar remover General Supplies (to be shared):
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Steps (TSB): A. Making Media Record media name, manufacturer, lot number, expiry date and directions on log book. Weigh the specified amount of TSB on a balance using the spatula and weigh boat. Suspend the powder in the specified amount of distilled water in the Erlenmeyer flask, place the stir bar in and cover with aluminum foil. Place the flask on the hotplate to mix the solution well with the magnetic stir bar and warm until fully dissolved. Dispense 7.0mL portions into test tubes using the pipetting bulb and appropriate pipette. Cap loosely and label the rack with your name, course & section, date and media type. Sterilize the medium by autoclaving for 15 minutes at 121°C. Incubate the tubes at 35°C for 24-48 hours.
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Record media name, manufacturer, lot number, expiry date and directions on log book. Weigh the specified amount of TSA on a balance using the spatula and weigh boat. Ensure you use clean materials to avoid contamination. Suspend the powder in the specified amount of distilled water in the Erlenmeyer flask, place the stir bar in and cover with aluminum foil. Place the flask on the hotplate to mix the solution well with the magnetic stir bar and boil until fully dissolved. The solution will clear up immediately after boiling. Allow the media to cool down a bit before dispensing equal amounts into media bottles. Cap loosely and label the bottles with your name, course & section, date and media type. Sterilize the medium by autoclaving for 15 minutes at 121°C. Steps (TSA): A. Making Media
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B. Autoclaving Purpose: To sterilize It has been determined that microbes can be controlled by steam under 15 psi (pressure) that yields 121°C. The autoclave is superior in sterilizing many heat-resistant materials, but is ineffective on substances that repel moisture.
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Place labeled item in an autoclave bucket found in the Prep Room. Label the bucket with your Instructor’s name, course & section and place a small strip of autoclave tape on the bucket (not necessary if it is only garbage). Wear oven mitts before placing the bucket into the autoclave. Close the autoclave door tightly. Press the appropriate button twice (“1” for gravity or empty glassware sterilization, “2” for liquid sterilization and “4” for garbage). You should hear a hissing sound that indicates the start of the cycle. After the cycle is completed, carefully open the autoclave door. Make sure you stand beside the autoclave to avoid getting burned by the steam coming out from the door. Steps: B. Autoclaving
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For glassware, wait 10 minutes to allow the contents to cool down. Glass may explode when they clink while extremely hot. Wear oven mitts when taking out the autoclave bucket. For sterilized media and clean glassware, store them in their appropriate places. For sterilized dirty glassware, place the bucket onto the cart next to the autoclave for washing. For autoclaved garbage bags, throw them into the garbage bins next to the autoclaves. Throw the “Broken Glass” bag in the Broken Glass container. Return empty autoclave buckets to their appropriate shelves in the Prep Room. Steps (cont’d): B. Autoclaving