05 OCT 2026 (RECONSTRUCTED)

To evaluate and record the colony growth from the wastewater screening plates inoculated on October 02, and to inoculate fresh overnight (ON) cultures from selected colonies.

• Incubated selective agar plates (10× IP series, 10× MP series) from October 02

• Lysogeny Broth (LB) liquid medium

• Tetracycline (tet) stock solution 

• Sterile glass test tubes

• Sterile 50 mL Falcon tube 

• Sterile Serological and micropipettes

• Sterile inoculation loops/sticks

Inoculation of Overnight Cultures

• Working under aseptic conditions inside a fumehood, dispensed LB medium into a sterile 50 mL Falcon tube using a sterile serological pipette.

• Aseptically transferred 5 mL of the LB medium into each of the five clean glass test tubes using a fresh sterile serological pipette.

• Labelled the glass test tubes sequentially as follows:

  • Tube 1,2 : DH5α (pAT64) + tet

  • Tube 3,4 : HB101 (Empty)

  • Tube 5: Control (Negative Control)

• Added tetracycline (tet) 20 µg/mL to Tube 1 and 2 to maintain selective pressure for the pAT64 plasmid using a micropipette.

• Using a sterile inoculation loop, picked a single, well-isolated colony from the DH5α + pAT64 plate and submerged it into Tube 1 and then used a fresh loop for Tube 2.

• Using a fresh sterile loop, picked a single, well-isolated colony from the empty HB101 plate and submerged it into Tube 3 (no antibiotic) and then repeated this for Tube 4 with a fresh loop.

• For Tube 5 (Control), dipped an empty, unused sterile inoculation stick directly into the LB broth to check for environmental contamination.

• Placed all five glass tubes into the incubator shaker at 37°C shaking for overnight incubation (~12–16 hours).

What the Oct 02 plates show?

Neither control grew on IP or MP, so the antibiotic plates were working. Growth on some of the isolate plates suggests those isolates can tolerate 1 µg/mL of a carbapenem. On its own, this doesn’t say which gene is responsible; that is what the library screen is designed to answer.

Why overnight cultures?

• The control strains are grown overnight so there is a fresh, dense culture ready to spread on Oct 06.

• Tetracycline is added to the DH5α + pAT64 tubes to keep selection on the plasmid; without it, some cells could lose pAT64.

• Duplicate tubes protect against one tube failing, and the uninoculated tube checks that the broth and technique are sterile.

Next step

Spread the library and controls onto selective plates (Oct 06).