Materials Required

Before beginning, assemble the following:

• LB Agar powder (Aladdin Scientific, SKU: L1528380)
• Distilled or deionised water
• Appropriate glass bottles or Erlenmeyer flasks (autoclavable, at least 2× the liquid volume)
• Magnetic stir bar and hot plate/stirrer
• Autoclave
• Thermometer or temperature probe
• Sterile 90 mm (or required size) disposable Petri dishes
• Bunsen burner or biosafety cabinet (laminar flow hood)
• Parafilm or plate bags for storage
• Personal protective equipment: lab coat, heat-resistant gloves, safety goggles

Optional: Antibiotic stock solutions (e.g., ampicillin, kanamycin) if selective plates are required. Prepare filter-sterilised antibiotic stocks in advance.

Step-by-Step Preparation Protocol

Step 1 – Weigh and Dissolve

Consult the product label for SKU L1528380 to confirm the recommended mass per litre. Typically, weigh the appropriate amount of LB Agar powder and transfer to a clean, autoclavable glass bottle or flask. Add approximately 80% of the final target volume of distilled or deionised water. Stir with a magnetic stir bar on a hot plate until the powder is completely dissolved and the solution appears uniform. Add remaining water to reach the final volume.

Step 2 – Autoclave

Loosely cap or plug the flask with a foam stopper and autoclave tape. Sterilise by autoclaving at 121 °C, 15 psi for 15–20 minutes using a liquid cycle. Allow pressure to release naturally. Carefully remove the flask using heat-resistant gloves — the liquid will be very hot.

Step 3 – Cool to Pouring Temperature

Place the flask in a 50–55 °C water bath or allow it to cool on the bench, swirling occasionally to equilibrate temperature. Monitor with a thermometer. Do not allow the agar to solidify. If selective plates are required, add the appropriate antibiotic at this stage (at 50–55 °C to prevent thermal inactivation of antibiotics).

Step 4 – Pour Plates

In a biosafety cabinet or near a Bunsen burner (to maintain aseptic conditions), briefly flame the flask mouth and pour approximately 25–30 mL of molten LB Agar into each 90 mm Petri dish. Replace lid immediately. Allow plates to solidify on a level surface for 20–30 minutes at room temperature. Surface should be smooth and free of bubbles. If bubbles appear, briefly pass the flame of a Bunsen burner across the surface before the agar sets.

Step 5 – Dry the Plates (Optional but Recommended)

Once solidified, invert the plates and leave them slightly ajar in a biosafety cabinet or in an incubator at 37 °C for 20–30 minutes. This step removes excess surface moisture and reduces satellite colony formation.

Storage and Shelf Life

Poured plates: Stack inverted plates (agar side up) in sealed plastic bags to prevent dehydration and contamination. Store at 2–8 °C protected from light. Use within 4 weeks of preparation. Allow plates to equilibrate to room temperature (or 37 °C) before use to prevent condensation on the agar surface.

Unopened LB Agar powder (L1528380): Store in a cool, dry place, tightly sealed, away from moisture and direct light. Refer to the product label and Certificate of Analysis for the specific expiry date and storage conditions.

Molten agar: If not used immediately after autoclaving, LB Agar can be kept molten in a 50–55 °C water bath for several hours. Prolonged holding at elevated temperature may degrade heat-labile components; re-sterilisation of previously autoclaved media is not recommended.

Quality Control and Troubleshooting

Quality control checks:

• Sterility: Incubate 2–3 uninoculated plates at 37 °C for 24–48 hours. No colony growth should be observed.
• Growth promotion: Inoculate a plate with a known E. coli strain (e.g., DH5α) and incubate at 37 °C overnight. Expected result: robust, well-isolated colonies.
• pH: Verify dissolved agar solution pH is 7.0 ± 0.2 at 25 °C before autoclaving using a calibrated pH meter.

Common problems and solutions: