Bio-Rad Microbial Culturing Module - Manual (Page 6)

Preparation of Liquid Media
Preparation of Ampicillin
Ampicillin is an antibiotic used to selectively eliminate bacteria that have not been transformed
with plasmids containing an ampicillin resistance gene. Ampicillin is shipped freeze dried in a
small vial containing 30 mg ampicillin. With a sterile pipet, add 3 ml of sterile water directly to
the vial to rehydrate the antibiotic to make a 10 mg/ml or 200x solution. Ampicillin is used at a
final concentration of 50 µg/ml in both LB-Ampicillin broth and LB-Ampicillin agar. Ampicillin
should be stored at –20° and is good for 1 year.
Preparation of LB Broth
This protocol is used to prepare liquid LB broth for growth of bacteria. LB broth has been supplied
in convenient capsule form; each capsule makes 50 ml of LB broth when reconstituted with
water. To avoid contamination of a common stock of LB broth, it is recommended that each
capsule be reconstituted with 50 ml of water in a separate container. To make 50 ml of sterile
LB broth, label a clean container and add one capsule of LB and 50 ml of distilled water and
cover appropriately. (Note: never completely seal a container that is to be autoclaved.) Allow
the capsule to dissolve. Swirling or stirring with a magnetic stir bar will hasten this process.
Autoclave the container on the wet cycle for 30 min. Allow the broth to cool to room temperature
before use. If an autoclave is not available, LB can be filter sterilized through a 0.2 µm filter, or
can be sterilized in the microwave by heating to boiling at least 3 times. (Note: reduce evaporation
by using a reduced power setting on the microwave so the solution simmers rather than boils.)
Storage of LB broth at 4°C is recommended and can be stored for up to 1 year. LB broth can
also be stored at room temp if desired — however this is not recommended if the microwave
method has been used for sterilization, or if the bottle has been opened after sterilization. LB
broth containing antibiotics should be stored at 4°C and is good for one month.
2
Add water Add capsule
Swirl
Autoclave
or
microwave
to boiling