A media is prepared with an antibiotic in that media and then cells that have resistance gene on the plasmid for that antibiotic. Colonies will appear on the media.

1. 24-october-2018
Practical# 3
E.coli culturingon LB agar and LB broth
Reagents
 LB medium
 Kanamycin ( or any other antibiotics)
 Conical flask with cotton plug (autoclaved)
Equipment
 Bunsen burner
 Clean workbench
 Autoclaved toothpick/Pipettetips/Inoculationloop
Method:
1.Culturingon LB agar
 Prepare LB agar as according to the set methods and
after autoclaving add the antibiotic for which E.coli
is resistant.
 Take Bacterial colonyon antibiotic (for which e.coli
is resistant) containingLB-Agar plate
 Touch the surface of a bacterial colony with a sterile
toothpick or pipette tip or inoculating loop.
 Drop it into the antibiotic containingLB agar plates.

2.  Incubate the coloniesat 37◦
C for 12-16 h.
2.Culturingon LB broth
 Take Bacterial colonyon antibiotic (forwhich e.coliis
resistant) containing LB-Agar plate.
 Before starting Set the shaking incubator at 37°C.
 Transfer 3 ml LB medium aseptically to Falcon tubes
or Erelenmeyer flasks.
 Add 3 µl of antibiotics stock solution of kanamycin
(50 mg/ml).
 Touch the surface of a bacterial colony with a sterile
toothpick or pipette tip
 Drop it into the antibiotic containingLB medium.
 Set the culturetubein shaker incubator. Use
appropriateinclined (30° – 45°) angle if you are
using falcons tube to ensures good shaking.
 Set the speed 200 – 300 rpm and start the shaker.
Incubate for 12 – 16 h.