Adding Antibiotics and other Additives to Agar Media

Bacterial Culturing

Why add antibiotics?

Antibiotics are added to agar media to select for microorganisms carrying specific resistance genes, most commonly plasmids in bacterial cloning. Only cells that retain the plasmid (and its resistance marker) will survive and form colonies. This ensures:

  • maintenance of plasmids after transformation
  • elimination of non-transformed cells
  • reduced background growth
  • reliable downstream screening

Antibiotics are not sterilizing agents—they are selective pressures.

 
When are antibiotics added?

Antibiotics are heat-sensitive and must be added after autoclaving, once the molten agar has cooled to ~50–60 °C. Adding them too hot will degrade the antibiotic; too cool and the agar will begin to solidify unevenly. Stock solutions are typically prepared at 1000× concentration, filter-sterilized, aliquoted, and stored frozen.

 
Key practical notes

  • Mix gently after adding antibiotics to avoid bubbles
  • Pour plates promptly for even distribution
  • Store plates at 4 °C, protected from light when applicable
  • Always label plates with antibiotic and concentration
Stock and working concentration of antibiotics we typically use in our lab.
Antibiotic Stock Concentration (1000x) Working Concentration
Ampicillin 100 mg/mL 100 µg/mL
Chloramphenicol 25 mg/mL
(dissolve in EtOH)		 25 µg/mL
Kanamycin 50 mg/mL 50 µg/mL

Prerequisites

Prerequisite Protocols
Making LB Plates
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Procedure

Step 1. Prepare a water bath at 60 °C with sufficient water to submerge ~75% of the bottle containing your molten media.
Step 2. Prepare your agar media as normal (protocol below). After the media has been autoclaved, cool the media in the water bath prior to adding the antibiotic. 60 °C is a good temperature because the molten media will remain liquid but most antibiotics will not break down at this temperature.
Step 3. Cooled agar should be warm to the touch; as a rule of thµmb, if you cannot take the molten agar out of the water bath wearing only lab gloves, it’s not likely cool enough. 
Step 4. Once cool, light the Bunsen burner at your workstation.
Step 5. Add the antibiotics to the flask, and swirl to mix. 
Step 6. Pour the plates as normal, cool until they set up, bag them up, label the bag, and store at 4 °C.

How much to add: Because antibiotic stock is prepered at 1000x, you want to add 1 ml of stock for every 1 L of media.

Required recipes

Ampicillan Stock

Recipe calculator · Scales ingredients to target volume
Step 1. Prepare ingredients.
Ampicillin
100 mg
Step 2. Bring final volume to 1 mL with water.

Chloramphenicol Stock

Recipe calculator · Scales ingredients to target volume
Step 1. Prepare ingredients.
Chloramphenicol
250 mg
Step 2. Bring final volume to 10 mL with ethanol.