Bacteria Lab I: Preparing Agar Plates and Culturing Bacteria
Purpose: To prepare nutrient agar plates and to culture bacteria from the school environment.
Materials and Methods: (wear safety glasses, tie back long hair, never leave flame unattended)
- Obtain the required equipment for this experiment. Wash and dry the Erlenmeyer flask and glass stiring rod. DO NOT REMOVE THE PETRI DISH LIDS. Set up the retort stand.
- Label the bottom of the petri dishes with your name and class day/period (ie. E. Kimmel, D2P1).
- Fill a large beaker (larger than the Erlenmeyer flask but small enough to be secured by the ring clamp) about 1/2 full with tap water. Using a small-to-medium sized Bunsen burner flame (blue), get the water gently boiling and keep it boiling. This is your hot water bath.
- Add _________ mL of distilled water plus _______ g of nutrient agar to the Erlenmeyer flask and stir to mix.
- Carefully lower the Erlenmeyer flask into the hot water bath and hold its neck with tongs. Stir constantly while heating until the agar is fully dissolved. It will look clear yellow with no powder floating.
- Cautiously remove the flask using the tongs and place it on a fire-resistant mat on the lab bench. Constantly stir the agar solution while your partner prepares a cold water bath by filling a large beaker (larger than the flask) about 1/3 full of room-temperature tap water. This is your cold water bath.
- Carefully lower the Erlenmeyer flask into the cold water bath and hold its neck with tongs. Stir constantly as the agar begins to cool for 30-60 seconds. DO NOT LET IT SOLIDIFY.
- Lift the lid of one petri dish slightly on one side and position the flask so that you can pour the agar into the dish. Only add enough agar to just cover the bottom of the dish. Immediately put the lid on and very gently swirl the dish to spread the agar evenly. Repeat quickly with the second petri dish before the agar solidifies in the flask. Leave the petri dishes (also called agar plates) absolutely untouched until the agar is fully solidified.
- Wash all glassware with warm, soapy water. Wash your lab bench. Lastly, wash your hands with soap and warm water.
- When the agar has solidified, inoculate one agar plate with bacteria collected from a single location using a clean swab. Do not inoculate the other plate. DO NOT SWAB ANY PART OF YOUR BODY.
- Once you have swabbed a surface to obtain bacteria, lift the lid of the petri dish slightly on one side and lightly brush the entire surface of the agar with the swab. Do not penetrate the surface of the agar.
- Tape the petri dish lid shut. Label it with the location from which your sample was taken. Write small so as not to obscure the viewing area through the lid. The inoculated plate will be incubated in a warm, dark environment for a few days.
- Put away all hot equipment when it is safe to handle.
- Wash your hands again.
- Why should you not use a swab from your body to inoculate the agar plate?
- Why should the lid of the petri dish only be opened slightly while pouring the agar or inoculating the plate?
- Why should a bacterial colony that appears shiny (indicating bacterial capsules) be treated with particular care?
- Explain why a bandage left on a wound for a number of days can promote the growth of bacteria rather than control it.
- Why should your agar plate not be incubated at 370C?