Antibiotic stocks and dilutions
How much stock to add to a culture, and what to weigh to make the stock.
Solvent: Water. Degraded by β-lactamase secreted into the plate, so satellite colonies appear around a resistant one on older plates. Carbenicillin is the more stable substitute.
Stock to add
A 1 in 1000 dilution, delivering 50 mg.
V_stock = C_working × V_culture ÷ C_stock- Working and stock concentrations for bacterial selection — Addgene molecular biology reference
The reference concentrations are the usual ones for selecting plasmids in E. coli. A low-copy plasmid, a rich medium or a different organism all change them. Add antibiotics to molten agar only once it has cooled to about 50 °C — poured hot, several of them are destroyed before the plate sets.
When to use this
Use this to work out how much antibiotic stock to add to a volume of medium, or what to weigh to make the stock in the first place. The reference concentrations are the usual ones for plasmid selection in E. coli and every field is editable for anything else.
Worked example
Adding ampicillin to 500 mL of LB from a 100 mg/mL stock.
- Antibiotic
- Ampicillin
- Stock concentration
- 100 mg/mL
- Working concentration
- 100 µg/mL
- Medium to treat
- 500 mL
Result
A thousandfold dilution, delivering 50 mg of ampicillin into the flask.
What people get wrong
- Confusing mg/mL with µg/mL. Stocks are conventionally a thousandfold above the working concentration, so the two numbers on the page are identical and only the unit differs.
- Adding antibiotic to agar that is still near boiling. Let it cool to about 50 °C first — several antibiotics are destroyed before the plate sets.
- Autoclaving the stock. Filter sterilise instead; autoclaving destroys most antibiotics.
Questions
+Are these concentrations right for my organism?
They are the usual figures for selecting plasmids in E. coli. A low-copy plasmid, a rich medium or a different species all change them, so treat them as starting points.
+Why are there satellite colonies on my ampicillin plate?
Ampicillin is degraded by β-lactamase secreted from resistant colonies, leaving pockets where sensitive cells grow. Carbenicillin is more stable and largely avoids it.
+How long do plates keep?
Weeks in the dark at 4 °C for most, less for light-sensitive or unstable antibiotics such as tetracycline. If selection looks leaky, suspect the plates before the construct.
Related tools
- Dilution calculator — Work out how much stock and diluent to combine for a target concentration.
- Recipe scaler — Scale a recipe to a different batch size and keep every concentration the same.
- Molarity calculator — Convert between mass, molar concentration and volume for a solution.
Science last reviewed .