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How to Make Luria-Bertani (LB) Agar Plates

Nutrient agar has been a useful tool for microbiologists since 1882 when German microbiologist
Walther Hesse, an assistant in the famed Robert Kochs laboratory, attempted to use it as a medium
for growing microorganisms after it was suggested by his wife Fannie. Koch had previously been
using gelatin, which was limited in that it was difficult to solidify and would sometimes revert to a
liquid state. Agar turned out to be ideal because it remains solid and is indigestible to many
microorganisms. This ensures that the medium stays stable as the microorganisms grow, allowing for
a better controlled experiment.
There are many subtypes of nutrient agar, one of the most common being Luria-Bertani (LB) agar.
This is the general medium used by microbiology students for routine cultivation of
microorganisms. Its use is widespread due to its ease of preparation and lack of preference for
growing any one type of microorganism. Chemically, agar is a polymer of the sugar galactose. It is a
component of the cell walls of red algae which is often harvested from Eastern Asia and California.

MATERIALS
This recipe will enable preparation of one liter of Luria-Bertani agar. This is equivalent to ~50 small
petri dishes or ~25 large petri dishes. Some suppliers to consider obtaining materials from are
Science Kit & Boreal Laboratories, Carolina Biological Supplies, Bio-Rad Laboratories, and Sigma.
Tryptone
Yeast Extract
Sodium Chloride (NaCl)
Agar
Deionized (DI) Water
Sterile Petri Dishes
Scale
Stir Bar
Hot Plate
Autoclave
2 L Beaker
2 L Flask
Sharpie

INSTRUCTIONS
NOTE: Sterile technique applies throughout this entire process
Step 1: Using the scale, separately weigh out 9.1 grams tryptone, 4.6 grams yeast extract, 4.6 grams
NaCl, and 13.7 grams agar.
Step 2: In a beaker containing the stir bar, add the tryptone, yeast extract, NaCl, and agar.
Figure 1: "Glass Beaker 2000ml Bomex 2 Liter." N.p., n.d. Web. 22 June 2014.
Figure 2: "How Does an Autoclave Work?" Anatometal -. N.p., n.d. Web. 22 June 2014.
Figure 3: "Pour LB-Agar Plates." BenchFly. N.p., n.d. Web. 22 June 2014.

Step 3: Add a liter of deionized water to the beaker and
immediately place the beaker on the hot plate.
Step 4: Turn the hot plate to 280
o
C and 350 RPM.
NOTE: It is important to continuously stir the broth to
ensure it doesnt harden in the beaker
Step 5: Allow the agar to cook for 10 minutes.
Step 6: Pour the agar into a separate flask and place the
flask in an autoclave at 121
o
C.
Step 7: Label the plates with sharpie around the
edge so as to not obstruct the view of cell
colonies and fill approximately 1/3 to 1/2 with
agar solution.
Step 8: Once hardened, store in a refrigerator
stacked upside down.
NOTE: Do not freeze, as this will compromise
the agar


GLOSSARY
Agar A medium on which cells are grown
Autoclave A device used for sterilization
by subjecting objects to intense heat and
pressure
Deionized water Water purified so that
ions which are normally present are removed
Polymer a complex chain of simpler
molecules (monomers)
RPM Rotations Per Minute
Tryptone a source of amino acids for the growing bacteria
Yeast Extract The components of yeast cells (with the cell walls removed)
Figure 2: An autoclave
Figure 1: Agar solution in a beaker
Figure 3: Finished plates

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