Prepare and autoclave all media in containers that are at least 2 times the volume of the
prepared media volume. This allows room for boiling.
Purpose
To prepare LB agar and LB broth for growing E. coli cultures.
Materials
Balance, tabletop milligram
Weigh boat, 3.5”×3.5”
Lab scoops
LB agar base
Beakers, 400 mL
Glass rods
Magnetic stir bars
Hot plate stirrer, 7”×7”
Hot hands protector
Media bottle, 250 mL
Permanent lab marker pens
LB broth base
Beaker, 250 mL
Media bottle, 125 mL
Sterilizer/autoclave
Autoclave tape
Procedure
Note: Recipes can be adjusted for any desired volume of media.
Part I: Preparation of 125 mL of LB agar
• 125 mL of agar is the maximum that can be prepared in the 250-mL media bottles.
• Use Mass1/Volume1= Mass2/Volume2 to determine the proper amounts of media base to use.
1. Wash a 250-mL media bottle and black cap. Label it with sample name, date, and your
initials. (This is for sterilizing the media.)
2. Wash a 400-mL glass beaker. (This is for mixing the media.)
3. Measure out the amount of LB agar base required for the volume of agar desired. Use the
Mass1/Volume1= Mass2/Volume2 equation
to calculate the mass needed for the desired
volume. Record that mass (M2) in your notebook
along with the calculations. Pour the LB agar
base into the clean 400-mL beaker.
4. Very slowly, add 90 mL of distilled water, stirring
as it is added. The water should, at first, make a
thick paste. As you add more water and stir the
agar mixture, the LB agar base will eventually
become suspended.
Note: Some agar recipes require the addition of
NaOH. If necessary, determine the amount of
1 M of NaOH to add to the suspended agar.
5. Add more water until a total volume of 125 mL
of suspended agar is achieved.
6. Gently add a magnetic stirrer to the beaker by
sliding it down the side of the beaker. Move
the beaker of agar suspension onto a stirring,
hot plate.
7. Heat on medium high, gently stirring the entire
time, until just before it boils. Do not let it boil.
The agar suspension should become clear.
8. Using hot hand protectors (like the Bel-Art
Hot Hand® Protector), carefully remove the
beaker from the hot plate and pour the hot agar
suspension into the labeled, clean, 250-mL
Figure 4.6. A pressure cooker can be used to
media bottle. Very loosely cap the bottle