02 OCT 2026 (RECONSTRUCTED)

To prepare agar selective plates containing antibiotics (IP and MP) and perform isolation streaking to screen wastewater samples alongside control strains (DH5α with pAT64 and empty HB101) . 

• Agar broth base (LB or equivalent solid media matrix)

• IP (imipenem) antibiotic stock

• MP (meropenem) antibiotic stock

• Wastewater samples

• Control cultures: E. coli containing plasmid pAT64, E. coli strain HB101

• Sterile empty Petri dishes (20 total)

• Sterile inoculation sticks

Media Melting & Plate Pouring

• Melted the 200 mL of solid agar broth using a steamer for 30 mins until completely liquefied.

• Divided the melted broth into two equal batches for antibiotic supplementation:

  • Batch 1 (IP): Added 1ug/mL IP antibiotic stock solution to the melted agar.

  • Batch 2 (MP): Added 1ug/mL MP antibiotic stock solution to the melted agar.

• Aseptically poured 10 plates containing IP and 10 plates containing MP (20 plates total) and allowed them to solidify.

Isolation Streaking 

Performed isolation streaking from previously prepared cultures/samples under aseptic conditions onto the 20 prepared plates according to the following layout:

• IP Antibiotic Series (10 Plates Total):

  • Plates 1–8: Streaked with individual wastewater samples

  • Plate 9 (Control 1): Streaked with pAT64 culture

  • Plate 10 (Control 2): Streaked with HB101 culture

• MP Antibiotic Series (10 Plates Total):

  • Plates 1–8: Streaked with individual wastewater samples

  • Plate 9 (Control 1): Streaked with pAT64 culture

  • Plate 10 (Control 2): Streaked with HB101 culture

• Incubated all 20 plates upside down at the 37°C growth temperature for colony development.

Autoclaving

• Packed laboratory consumables and glassware intended for the next day's work into autoclave-safe bins.

• Affixed autoclave indicator tape to the loads.

• Ran a standard gravity sterilization cycle 30 minutes.

• Verified that the autoclave indicator tape successfully changed colour, ensuring appropriate heat and pressure parameters were met. Materials were removed and stored cleanly.

Why this step?

This was the first screen: streaking environmental isolates straight onto plates containing imipenem (IP) or meropenem (MP) at 1 µg/mL tests whether any of them can grow in the presence of a carbapenem.

Why these controls?

• DH5α + pAT64 carries only the tetracycline-resistance plasmid. If it grew on IP or MP, the plasmid itself would be giving resistance, and any result from the library would be meaningless.

• HB101 has no plasmid. It should not grow on IP or MP, which confirms the antibiotic in that batch of plates is active.

• If either control grew, the whole plate set would have to be treated as unreliable.

Next step

Read the plates after overnight incubation (Oct 05).