19 OCT 2026 

To prepare a large batch of LB agar and pour 52 selective plates containing tetracycline (20 µg/mL).

• LB Agar ingredients

  • Agar

  • Yeast Extract

  • NaCl 

  • Tryptone

  • DI Water

• Media storage vessels (20 bottles total)

• Tetracycline (tet) stock solution

• Sterile empty Petri dishes (52 plates total)

Aliquoting & Media Preparation

• Suspended the required mass of LB Agar powder in deionised water following the recipe, scaled up for the full batch.

• Distributed the liquid around 200mL evenly across 20 individual laboratory bottles.

• Secured bottle caps loosely and ran a standard liquid sterilization cycle using the laboratory autoclave (121°C for 20 minutes) to sterilize the media.

Selective Formulation & Plate Pouring

• Transferred the 20 sterile LBA bottles to a fumehood to cool down cleanly to approximately 50–55°C.

• Supplemented the molten media bottles with tetracycline (tet) stock solution to a final concentration of 20 µg/mL.

• Aseptically poured the antibiotic-supplemented agar solution into sterile Petri dishes, making 52 selective plates.

• Allowed the plate set to cool undisturbed until fully solidified, then labelled and stored them.

Why tetracycline plates?

pAT64 carries a tetracycline-resistance gene, so growth on tet shows that a clone still has the plasmid. An isolate that grows on IP or MP and also on tet fits with the carbapenem resistance coming from the DNA fragment on the plasmid. One that grows on IP or MP but not on tet may have lost the plasmid, in which case the resistance could be coming from somewhere else.

Why cool the agar first?

Tetracycline breaks down at high temperatures, so it is added only once the agar has cooled to about 50–55°C. Making all 52 plates from one batch also means every plate has the same antibiotic concentration.

Next step

Streak the purified isolates onto these plates (Oct 20).