Media Prep

Media Prep

October 3, 2019 47 By Ewald Bahringer


In order to grow, cells need very
specific environmental conditions food, energy, proper temperature and humidity. When growing cells in the lab we have to
create these conditions using culture media. Solid culture media is a mixture of agar
and nutrients poured into petri dishes. In this exercise we’ll demonstrate the
steps for preparing brain heart infusion agar. Always check the lot number and
expiration date. The right panel lists the composition of
the media and the final pH. First, add 100 mL of deionized
water to a graduated cylinder. Place a stir bar in a
250 mL flask. For the next steps we need an analytical
balance. Place a weigh boat onto the balance pan. Press the tare key to zero out the
weigh boat. Use a lab scoop to add 5.2g
of brain heart infusion agar to the weigh boat. Now add the powder to the flask. Add about 75 mL of
deionized water. Place the flask onto a stir plate. If you’re using a combination hot plate
and stir plate, be sure that the heat is turned off. After about 2 minutes, add the
remaining 25 mL of water from the graduated cylinder. Stir the solution until all of the
visible clumps have been broken down. Cover the flask with aluminum foil. We’re using foil cupcake wrappers
because they’re the perfect size. Place a piece of autoclave tape over the
foil and label it with the media name. Next use the autoclave to sterilize the media. The heat from the autoclave will also
help the agar to properly dissolve in the water. Power on the autoclave and make sure the
drain valve is closed. Add deionized water to the level
indicator line. Place the flask of culture media into
the basket, insert the basket, close the lid, and turn the handle to
create and airtight seal. Use the control panel to set the mode to
“sterilize,” the temperature to 121 degrees celsius. We’ll run this cycle for
17 minutes. While it’s running enter the date and time and the
operation details in the log and initial. Once the cycle ends and the pressure
gauge reads 0 PSI, use heat resistant gloves to slowly open the lid
and remove the sterilized flask. Back at the bench, place the flask onto the stir plate and stir the media very gently. Allow it to cool to about
45-degrees celsius. Pour the media into the petri dishes. The amounts don’t have to be exact, but be sure to cover the entire bottom
surface which is about 20 mL for standard size
petri dishes. Once the media has cooled it will
solidify and turn opaque. Here you can see the difference. The petri dish on the left has set up
properly. Turn the plates upside down to prevent
moisture from condensing on the agar surface, and store them in a
plastic bag until you’re ready to use them.