15 OCT 2026 (RECONSTRUCTED)

To isolate, subculture, and purify specific wastewater for the other half of the sample colonies exhibiting positive selection profiles by generating a total of 72 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 (72 total)

• Sterile inoculation loops/sticks

Plate Labeling

• Retrieved the positive wastewater spread plates from cold storage.

• Aseptically poured 62 plates containing IP and 10 plates containing MP (72 plates total) and allowed them to solidify

• Labelled 72 fresh agar plates containing selective antibiotics, grouped as follows:

  • 62× IP plates 

  • 10× MP plates 

Isolation Streaking & Incubation

• Using a fresh sterile inoculation loop 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-1 loop replacement rule across all 72 individual plates to ensure pure subculture isolate separation.

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

• Transferred all 72 inverted plates into the static incubator at 37°C for overnight incubation.

Why a second streak?

One round of streaking does not always give a pure culture. A second streak, taken from a single well-separated colony, is the standard way to make sure each isolate comes from one cell before it is tested further.

What the results suggest?

Most isolates from both screens grew again under selection, so their resistance held up through re-streaking. A few failed to grow or grew only weakly. That can happen if the resistance is unstable, if the original colony was a mix of clones, or if too few cells were transferred on the loop.

Labelling error

I numbered Oct 14 plates 1–40, the same numbers I had used for the Oct 15 plates, instead of continuing from 41 to 112. Because both sets were stored together, the two days’ plates became easy to confuse when colonies were picked from them later.

What I changed?

• Every plate now gets a unique ID that includes the date and the plate type (for example 08-IP-07), so no two plates can share a label.

• Plates are labelled on the bottom before anything is added, and checked against a written plate map.

• Plates from different days are stored in separate, labelled containers.

Next step

Prepare tetracycline plates (Oct 19) to check which isolates still carry the pAT64 plasmid.