Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 58
Step 5. Pour LB agar plates
*If your molten LB agar cools too much, it will solidify. If this happens, you can place it back in the microwave for 5-second
increments until it is liquid again. Remember that heating with antibiotics in the LB agar can degrade the antibiotic!
Within the Canvas Kit, you will nd four petri dishes. It is common in the scientic world to call these “plates.”
When you get your plates, note that they are made of two parts: the top lid is slightly larger in diameter than the
bottom dish and will overlap the bottom when you close it. During this step, and at all times when using plates,
make sure you do not accidentally pour the LB agar into the plate lid!
Place the petri dishes on a clean at surface, take the lids off, and place them next to the petri dish bottoms. The
bottom is the half with the star-shaped border. Get your hot molten LB agar bottle, remove the lid and pour the
molten LB agar into the dishes. Your goal is to ll the plates one half to two thirds full. Make sure the entire plate
bottom is covered by the LB agar. Set the plate lids to cover three-quarters of the plate bottom, allowing heat to
escape. Let the LB agar cool, solidify, and dry slightly. This usually takes between 15 and 30 minutes depending
on the temperature and humidity of your workspace. Humidity and temperature play an important role in the
timing of agar solidifying. For example, in the Canadian prairies during a blizzard, the humidity can be as low as
20%. This means that agar plates can solidify and dry in under 5 minutes! In a humid tropical location like Flor-
ida it can take 30 minutes or more! As noted in Chapter 2, if you are in an environment that might have mold, a
HEPA lter is an excellent tool for minimizing contamination. If you think you’ll have “dirty” air or have had some
contamination in prior experiments, you can also fully cover the plates with their lids while your LB agar cools.
In Figure 3-6, you will see images of LB agar in petri dishes that demonstrate what fully dissolved LB agar should
look like after it is poured in a petri dish and solidied. In Figure 3-6 (right), the powder was not fully dissolved,
and you see particulates, which are making the agar opaque. If your LB agar powder was not fully dissolved, it
will also be less solid and will be easily punctured with an inoculating loop. If this occurs, then the next time
you make plates try microwaving your water-LB agar powder mixture for further 5-second intervals until it is
boiling and fully dissolved.
At the end of this step, the manufacturer of the Canvas Kit suggests that you keep your sterile water bottle to
measure and add bleach to the inactivation bag at the end of this exercise. You can rinse it out with tap water
and screw the lid back on to save it for later. If you have leftover LB agar, you can pour it into your inactivation
bag. If the agar has solidied, shake the bottle vigorously to dislodge the solidied agar and drop it in the inac-
tivation bag. You can wash the bottle out with soap and keep it for future experiments or storage after you use
it in the inactivation procedure.
Figure 3-5. Step 5: Pour the LB Agar into the bottom half of the petri dishes (plates). The bottom is the side with the star-shaped
border. The plates should be one half to two thirds full.
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