14 OCT 2026 (RECONSTRUCTED)

To isolate, subculture, and purify specific wastewater for the half of the sample colonies exhibiting positive selection profiles by generating a total of 40 fresh agar plates back-referenced to the screening runs performed on October 06 and October 08.

• Stored experimental screening plates from refrigerator:

  • IP Selection Series (Inoculated Oct 06)

  • MP Selection Series (Inoculated Oct 06)

  • IP Selection Series (Inoculated Oct 08)

  • MP Selection Series (Inoculated Oct 08)

• Freshly prepared/melted LB Agar base supplemented with selective antibiotics:

  • IP Selection Plates (1 µg/mL working concentration)

  • MP Selection Plates (1 µg/mL working concentration)

• Sterile empty Petri dishes (40 total)

• Sterile inoculation loops/sticks

Plate Labeling

• Retrieved the positive wastewater screening plates from the October 06 and October 08 cohorts from cold storage.

• Aseptically poured 20 plates containing IP and 20 plates containing MP (40 plates total) and allowed them to solidify

• Labelled 40 fresh agar plates containing selective antibiotics, grouping them into four distinct 10-plate tracking batches:

  • 10× IP plates for the October 06 pool

  • 10× MP plates for the October 06 pool

  • 10× IP plates for the October 08 pool

  • 10× MP plates for the October 08 pool

Isolation Streaking & Incubation

• Using a fresh sterile inoculation stick for each transfer, picked target candidate colonies from the source plates and performed isolation streaking across the fresh selective surfaces.

• Maintained a strict 1-to-3 stick replacement rule across all 40 individual plates to ensure pure subculture isolate separation.

• Allowed the streaked plates to dry completely before inverting them.

• Transferred all 40 inverted plates into the static incubator at 37°C for overnight incubation.

Why re-streak?

A colony on a screening plate can be a mix of more than one clone, or can sit right next to background growth. Streaking each candidate onto a fresh selective plate separates it into single colonies (a pure isolate) and also checks that the resistance is stable: the clone has to grow on the same antibiotic again.

Why three sticks per plate?

Using a new stick for every plate stops cells from one isolate being carried onto another, which would make every later result impossible to trust. While, using a new stick for every quadrant helps to spread cells effectively.

Keeping the two screens apart

Because the Oct 06 and Oct 08 screens had both used plate numbers 1–20 (see Oct 08), the new plates were grouped and labelled by source date (Oct 06 pool, Oct 08 pool) as well as by antibiotic.

Next step

Check growth on these plates and do a second round of streaking (Oct 15).