Preparation of LB Broth and LB Agar:
LB broth and
LB agar:
LB broth (Luria-Bertani broth) is a
nutrient-rich liquid medium commonly used to grow bacteria, especially
Escherichia coli (E. coli), in microbiology and molecular biology labs.
LB Broth Composition (per 1 liter):
-
Tryptone 10 g Source of peptides and amino
acids
-
Yeast extract 5 g Source of vitamins, minerals,
and growth factors
-
NaCl (salt) 10 g Maintains osmotic balance
LB Agar (solid): LB broth + 1.5% agar for
growing colonies on plates.
Antibiotic: Ampicillin 100 µg/mL
Steps:
For LB Agar
Plates:
1.
Weigh LB agar powder according
to label
2.
Add ~900 mL of distilled water
in a large flask
3.
Stir and heat if necessary to
dissolve
4.
Top up to 1 L with distilled
water
5.
Autoclave at 121°C for 20
minutes
6.
Cool to ~50–55°C
7.
Add antibiotics (e.g., 1 mL of
100 mg/mL ampicillin per 1 L)
8.
Pour into Petri dishes (~20 mL
per plate) in a sterile area
9.
Let solidify (~30–60 minutes)
10. Store plates inverted at 4°C in sealed bags or containers
For LB Broth:
1.
Weigh LB broth powder as per
label.
2.
Dissolve powder in ~900 mL of
distilled water in a flask or bottle
3.
Stir until fully dissolved
4.
Top up to 1 L with distilled
water
5.
Autoclave at 121°C for 20
minutes
6.
Cool to ~50°C, then add
antibiotic (e.g., ampicillin) Add 1 mL of 100 mg/mL stock per 1 L of LB
7.
Store at 4°C if antibiotic is
added; otherwise, room temperature is fine
8.
Dispense into bottles or
culture tubes
Common Uses:
-
Growing E. coli for
cloning or protein expression
-
Selecting transformed bacteria
(when antibiotics are added)
-
Making glycerol stocks or
plasmid prep cultures