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Furman University

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A START-UP MANUAL FOR UNDERGRADUATE RESEARCH STUDENTS IN MICROBIOLOGY
Active Learning from the Very Beginning
Keri Law, Rachel Lamb, and Min-Ken Liao

POURING PLATES

A common way of culturing bacteria and isolating specific strains is by growing the cells on a plate of solid medium. Below is a description of how to prepare such a plate.

1. Prepare a liquid medium containing agar by following the protocol in Section 5 and using the appropriate recipe from the Appendix.

2. After the medium has been autoclaved, place it in a 45 water bath to cool it to a working temperature (approx. 30 min.)

3. Meanwhile prepare your plates by removing them from the packaging (if necessary) and labeling the bottoms with the type of medium.

4. Once the medium has cooled, aseptically add any desired "heat-labile" ingredients, such as an antibiotic, if the plates are to contain a selective medium. (i.e. Only bacteria containing an antibiotic-resistance gene will grow on the plate).

5. Flame the open mouth of the flask containing the liquid medium before pouring it into a plate. Remove the lid from the plate just before pouring the liquid medium.

6. Pour approx. 15 mL of medium into the plate (a few centimeters below the top of the plate). Be careful that any medium that runs down the outside of the flask does not drip into the plate and cause contamination.

7. Replace the lid on the plate. Flame the flask again, holding it constantly at a 45 angle. (WHY should you hold it at a 45 angle? Hint: Bacteria FALL they DON'T FLY!)

8. Repeat this until you have all the plates you need or until the medium begins to solidify in the flask. Occasionally swirl the medium in the flask to insure even distribution of the contents.

9. Allow the plates to harden for 20 to 60 minutes.

10. Store plates in refrigerator.

To practice this technique, ask your instructor what plates your lab needs next and pour them for everybody!