Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 56
Figure 3-3. Step 3b: Add the LB Agar powder to boiling water.
Step 3. Create molten LB agar powder
Get your bottle of sterile water and the tube of LB agar powder from your “Day 1” bag in your Canvas Kit and go
to your microwave. Remove the lid of the sterile water bottle and set it loosely on top of the bottle. This prevents
the boiling vapor from causing the bottle to explode. Heat the water in the microwave until it is boiling. A typical
microwave will require about 45 seconds to boil the 50 mL bottle of water. Keep your eyes on your bottle and your
nger on the stop button so that if you see it boiling over, you can press stop. You’ll know you are ready to move
onto the next step when you see bubbles actively bubbling up. If you don’t see boiling, microwave in 5 seconds
interval until you do see boiling.
Once the sterile water is boiling, remove it from the microwave and add the LB agar powder to the bottle. The
bottle will be hot! Careful! A small amount of powder may stick to the lip of the LB agar tube, and that is OK and
accounted for.
Once you have added the LB agar powder, partly screw on the lid and gently swirl to mix in the powder for 15
seconds. Swirling gently will help you avoid introducing bubbles in the agar. The powder may not fully dissolve
because the water will have cooled down as the cold powder was added. Keep the lid loose and put the bottle
back in the microwave. Heat in intervals of no more than 4 seconds. When it has boiled again, which you will
see since it boils into a foam after LB agar is added, then take the bottle out of the microwave.
If the LB agar has boiled and is well mixed, it should look very clear with a yellow tint since LB agar is slightly
yellow. If it looks like there are still some particles oating about, or if it looks cloudy, microwave for another 4
seconds to help dissolve the remaining powder. Be careful! the liquid boils over quickly once the LB agar powder
has been added. Keep your nger on the button to stop the microwave if you see any liquid coming out of the
bottle, or if the lid pops off due to bubbling. Move to the next step in less than one minute once your agar is
boiled and gently swirl-mixed for 15 seconds.
LB agar Going Deeper 3-1
LB agar is a mixture of ‘LB’ and agar: LB is an abbreviation for Lysogeny Broth (sometimes also said to stand
for Luria Broth, Lennox Broth or Luria-Bertani medium, after its inventor Guiseppi Bertani).
In all instances, LB refer to a “chicken noodle soup”-like mixture of sugars, salts, and minerals that E. coli
bacteria love to eat. When in LB agar form, the agar is a gelling agent that will make the mixture solidify into
a gel so that you can grow E. coli bacteria on its surface.
Figure 3-2. Step 3a: Boil your sterile water in the microwave.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 57
Step 4. Adding antibiotic
Antibiotics are frequently used in genetic engineering experiments. These allow you to “select” for the bacteria
you want to grow while minimizing the chance for contamination. Antibiotics for selection will be discussed in
Chapter 4. For now, follow the manufacturer’s instructions on adding the antibiotics to your LB Agar. You must
do this quickly before your LB agar sets.
Typically, these instructions will be along the lines of: Add the pill with antibiotics to your bottle of hot molten LB
agar immediately, as soon as it has cooled down enough for you to hold the bottle for 5 seconds without burning
your hands but before it gets cool. Swirl the bottle in a circular moti until the contents of the pill are dissolved.
You may see some of the gel capsule oating around, and that is OK. You can move onto the next step.
If you see any of the powder that was inside the capsule remaining, you need to continue swirling. Try not re-heat
the solution when mixing here because this will degrade the antibiotics. If you absolutely need to re-heat, go
ahead, but know that your experiment might not work out perfectly.
Note that if you have the EU-compatible Canvas Kit with naturally colorful bacteria, you will not be adding anti-
biotics. You will learn why in Chapter 4.
Mixing Pro-tip
Mixing sounds easy and trivial, but mastering the art of mixing is a really important skill to learn on your
journey to becoming a Genetic Engineering Hero. The rst thing to remember is that you should always
mix thoroughly when you add one tube of something to another, or if you add a powder into a liquid.
When mixing, it is important that you do not “half mix” something. Otherwise, your experiment will likely
fail, or you will have inconsistent results. When mixing your agar powder into water, for example, you want
to gently swirl so that the powder mixes into the liquid without any splashing that leaves powder stuck on
the inside of the water bottle. In some instances, you may need to re-heat the sample to ensure that the
powder has fully dissolved. In short, mixing is really important, and your goal is to achieve 100% perfect
mixing without losing ingredients to splashing, spilling or evaporation. Scientists and genetic engineers
get good at mixing over years of experiments, so be patient and be aware of this subtle but important skill.
Figure 3-4. Step 4: Add the antibiotics to the LB Agar and mix by gently swirling (if applicable to your kit).
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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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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 59
Labeling Plates Pro-tip
Labeling LB agar plates is a very important practice. While it has not been mentioned yet for this exercise
to maintain simplicity, in future chapters it will become common practice.
When you label your petri dishes in future experiments, use a permanent marker and write your initials
on the bottom of the plate. It is essential to label plates on the bottom because lids can fall off or get mixed
up during experiments. By labeling the plate bottom (where you poured your agar), the label will never get
separated from your experiment.
Figure 3-6. Fully dissolved LB agar powder in a plate (left) vs. incomplete dissolving of LB agar powder in a plate (right). Hold your
nished plates 10 cm - 30 cm above a stencil to see whether you can see them clearly. Soft and semi-opaque agar means that you
need to heat it more in future experiments. If this happened to you, don’t worry! This is a part of your learning journey.
Sterile Water Going Deeper 3-2
Sterile distilled water is provided within the Canvas Kit
TM
. Sterile distilled water is used because it is purer
than ordinary tap water. When doing scientic experimentation, it is essential to use sterile, pure ingredients
and to do your best preventing environmental factors like spores, fungi, and mold from getting into or on
your samples. Tap water and distilled water obtained in a store may contain bacteria. Tap water is generally
OK for you to drink because your immune system will take care of any microorganisms living in the water.
Your agar plates do not have an immune system, and any microorganisms in the tap water can and will grow
in your experiments.
Research labs use an autoclave to sterilize water and other samples. An autoclave is a large and expensive
pressure cooker that lets you heat the samples at, or above, 120 ˚C for long periods of time without the
sample boiling away thanks to pressurization. For example, the sterile water you are using was autoclaved
for more than 30 minutes at such a temperature.
As you become a Genetic Engineering Hero, you may want to complete your own independent experiments,
and if you do not have an autoclave, you can boil distilled water that you get from a grocery store for at least
10 minutes.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 60
Step 6. Use & storage of LB agar plates
After the plates have solidied, you can immediately use them for your projects. You can also place your LB agar
plates into the resealable bag they came in and store them for later use in a refrigerator or in a cool dark place. If
the air in your environment is clean and no mold or spores have landed on your plates, then you can store your
plates for a month or more in a refrigerator. If you have stored your plates but notice some unexpected growth
on the LB agar, it is recommended that you inactivate those plates. Inactivation is covered in Step 13.
In the next step of this exercise, we will use one of the four plates you poured. Put three of them back in their
resealable bag and refrigerate them. Once you have incubated the next step’s plate for ~24 hours, you will use
the remaining three plates to paint your living art.
Figure 3-7. Store your LB agar plates in the supplied resealable bag in the refrigerator for up to a few months. They can be used as
long as no mold has grown on them. If you see mold, you must inactivate!
In general, you should label the plate to indicate:
•
If the LB agar has antibiotics, and if so, which antibiotic. Labs use color codes or abbreviations to iden-
tify the antibiotic in the plate quickly. Different labs may have different color codes. Table 3-1 identies
the color codes used by IGEM, the International Genetically Engineered Machines Competition. You
can use it for your own Genetic Engineering Hero Headquarters.
• Your name or initials so that you or others will know the owner of the plate
• A descriptor to help tell you about the samples. For example, later on in the book, you will use S (+) to
describe a positive control plate, and S(e) to describe your experimental sample.
Table 3-1 - IGEM Antibiotics Color Codes
Antibiotics Color Code Abbreviation
Ampicillin Orange Amp
Chloramphenicol Green Clr/Chlor
Kanamycin Red Kan
Tetracycline Yellow Tet
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