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Procedure
1. Using a permanent marker, write the date and name of the bacterial strain on the bottom of the agar petri dish. 2. Dip the sterile inoculating loop, cotton swab, or toothpick in the bacterial culture and follow the standard lab bacterial streaking protocol. 3. Incubate the streaked agar plate either overnight in a 37C incubator, or at room temperature, for 48 hours. Make sure the plate is resting on its lid to prevent water condensation from falling onto the agar. 4. After incubation, you should see that the bacteria have grown on the agar plate. a. There should be regions of heavy bacterial growth where you first started streaking, and individual colonies where you finished the streaking procedure. b. Examine the colonies carefully. All colonies should have similar morphologies (textures, colors, and border shapes). If there are colonies with different morphologies, then you have a contaminant. If you have a contaminant, you will need to streak out each type of colony on a separate plate to get a pure culture and use other information about the bacteria you are working with (for example colony morphology, media preferences, and antibiotic susceptibility/resistance) to determine which bacteria is the one you want. 5. To prevent the agar plate from dehydrating, which will eventually kill the bacteria, either wrap the plate in Parafilm, or place it in a resealable plastic bag.
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6. Bacterial plates should be stored in a refrigerator. They will be good for up to one month under these conditions. Caution: It is not recommended to store bacteria in a refrigerator that is used to store food. If you absolutely must store your bacteria in the same refrigerator as food, place the bacteria plate inside two layers of Parafilm or resealable bags to minimize the risk of contaminating the food.
Procedure
1. Using a permanent marker, label a sterile microcentrifuge or screw-cap tube with the date and the name of the bacteria. 2. Using a micropipette, add 150 l of sterile glycerol to the tube. 3. With a new tip, use the micropipette to transfer 850 l of the bacterial culture to the same tube. 4. Cap the tube and invert it several times to thoroughly mix the glycerol and bacteria. 5. If you are going to store the bacteria in a special -80C freezer, you should first snap-freeze the bacterial stock by dropping it in a container of liquid nitrogen. If you are storing the bacteria in a regular -20C freezer, the bacterial stock can be placed there with no further treatment. Caution: It is not recommended to store bacteria in a freezer that is used to store food. If you absolutely must store your bacteria in the same freezer as food, place the bacterial stock inside two layers of freezertolerant resealable bags to minimize the risk of contaminating the food.
Credits
Parafilm is a registered trademark of American National Can Company.
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