Luria Bertani Broth + Antibiotic (LB/A) Plates
Reagents (for 1L):
- Tryptone (Bacto, 0123-01-1) – 10g
- Yeast Extract (Fluka, 70161) – 5g
- NaCl (Fisher, BP358-212) – 10g
- Agar (Sigma, A7002-500G) – 17g
- Ampicilin (100g/ml) - 1ml
- DI. Water – 1L
- Start by adding approximately half of the volume of DI water and a stir bar to an appropriately sized Erlenmeyer flask.
- Add the yeast extract, NaCl, and tryptone to the water, stirring the entire time. Make sure to add the tryptone slowly, as this tends to clump.
- Once those reagents have been completely dissolved, add the agar and the remaining volume of DI water to the solution.
- Cover the top of the flask with foil and autoclave for 30-60 minutes using a liquid cycle.
- Keep the top covered with foil to maintain sterility. (Figure 1)
- Once the autoclave has finished, cool the solution to about 56-58°C, with constant stirring. There are 2 ways to accomplish this:
- Place the flask in a 56°C water bath and let sit for an hour, or
- Perform a "Quick Water Immersion Cooling (QWIC)." NOTE: This technique works well at quickly cooling the solution uniformly and is utilized in our lab. It is not "scientifically proper," but it works well. Consult your professor first to see if this technique is appropriate for your lab.
- Once the solution has cooled, add 1ml of the antibiotic to the solution. (This will create a final concentration of 100ul/ml for the antibiotic.) The solution should be stirring constantly. Make sure to practice sterile technique with each addition. Open the top and add the antibiotic; do not completely remove the foil. (Figure 2) Immediately recap the top with foil once the addition is complete. (Figure 3)
- Let the solution stir for a minute or two, allowing for even dispersion of the antibiotic in the solution.
- Dispense approximately 30mls per plate into 100x15mm plates. This is most easily accomplished by setting up all the plates to be poured along the edge of the lab bench. (Figure 4 ) Only uncover the single plate you are pouring at that moment, and immediately recover it before moving onto the next plate. This helps maintain the sterility of the plates. (Figure 5 )
- Allow the plates to cool and harden overnight. This should yield approximately 35-40 plates.
- Store plates that will not be immediately used lid side down in their original sleeve at 4°C. (Figure 6 )
Keep the flask covered after removing from the autoclave.
Remove the foil cap only when adding the antibiotic.
Re-cover the top once the antibiotic has been added. Let it stir and enter solution uniformly.
Set up the plates to be poured along the edge of the lab bench before pouring. This make the process easier.
There should only be one uncovered plate at a single moment during the entire process. Only uncover the plate when you pour it. Immediately replace the lid when you are finished.
Once dry, store the plates lid side down at 4°C in their original sleeve.