20 OCT 2026

To subculture wastewater isolates by streaking them onto the 52 tetracycline plates (20 µg/mL) poured on October 19, using growth from the October 14 and October 15 streak plates.

• 52 tetracycline selection plates (20 µg/mL, poured Oct 19)

• Source plates: IP and MP streak plates (Oct 14) and streak plates (Oct 15)

• Sterile inoculation loops/ Sticks

Plate Organization

• Retrieved the 52 tetracycline plates poured on October 19.

• Sorted the October 14 and October 15 source plates.

• Labelled the bottom of each fresh plate to match its source isolate.

Isolation Streaking

• Worked aseptically next to a Bunsen burner flame.

• Using a fresh sterile loop for every transfer, picked an isolated colony from each source plate and streaked it onto a tetracycline plate.

• Let the plates dry on the bench before inverting.

Incubation

• Inverted all 52 plates and incubated them overnight at 37°C.

The October 14 and 15 source plates had plenty of growth for picking colonies.

Why streak onto tetracycline?

Re-streaking each purified isolate onto tet checks whether it still carries pAT64 (see Oct 19). This links the carbapenem resistance seen earlier to the plasmid, and so to the DNA fragment it carries.

What the result will tell us?

Isolates that grow on IP or MP and also on tet are the strongest candidates for carrying a carbapenem-resistance gene on their DNA fragment, and are the ones worth taking forward.

Next step

Check growth on these plates (Oct 21).