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Preparing LB Agar Plates with Antibiotic Selection

Producing sterile agar plates with an antibiotic selection marker. The foundational solid-phase culture medium for selecting plasmid-bearing bacterial colonies after transformation. Every recombinant-DNA workflow (including procedure 59 OpenTaq) depends on this. Written for a novice audience; covers the critical timing of antibiotic addition after autoclaving.

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Version History
Version 0.1.1 Viewing Latest
Effective: 2026-04-20

All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0
Effective: 2026-04-20

Initial release. Atomic technique supporting procedure 59 and any transformation workflow. Standard microbiology practice; procedural content is field convention.

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Procedure Details
Safety & Hazards
  • Autoclave burns. Freshly autoclaved liquid is ~95°C and under slight positive pressure. Always wait for the autoclave's "cycle complete" indicator AND for the pressure to drop to zero before opening. Use heat-resistant gloves. Never open a still-pressurised autoclave.
  • Molten agar burns. Agar just out of the autoclave is hot enough to cause serious burns. Always cool to ≤55°C (touchable on the wrist, though hot) before pouring.
  • Superheated liquids. Microwave-reheated agar can flash-boil when disturbed. Use caution; swirl gently after microwave.
  • Antibiotic handling. Wear gloves. Ampicillin sensitisation in prolonged handlers can cause allergy. Autoclave all antibiotic-contaminated waste.
  • Sterile technique. Every surface that touches cooled agar must be sterile, or your plates will grow contaminants rather than your transformants.
Preparation Notes

What you need ready:

  • LB broth powder (pre-mixed formula, usually 25 g/L: 10 g tryptone, 5 g yeast extract, 10 g NaCl — or buy premixed like Miller LB from any lab supplier).
  • Bacteriological agar (15 g/L target; do not substitute food-grade agar — inconsistent gelling).
  • Deionised water (tap water is not clean enough — minerals inhibit growth).
  • Autoclave (121°C × 20 min liquid cycle).
  • 1 L heat-resistant bottle (Schott / Duran) with a loose-fitting cap or foil cover for autoclaving. Cap must not be sealed tight — pressure builds up and the bottle explodes.
  • Petri dishes (standard 100 mm × 15 mm, sterile, pre-sleeved).
  • Water bath or insulated container set to 50–55°C.
  • Antibiotic stock solution at a known concentration, sterile-filtered (0.22 µm). Standard stocks: ampicillin 100 mg/mL in water, kanamycin 50 mg/mL in water, chloramphenicol 34 mg/mL in ethanol.
  • Biosafety cabinet for the pouring step.
  • Sterile serological pipette + pipettor or a sterile graduated cylinder for pouring 20 mL per plate.

Critical timing concept: antibiotics are mostly heat-labile. If you add ampicillin to 70°C agar, much of it will degrade before the plate sets. The agar MUST cool to 55°C (warm but not hot — you can comfortably hold the bottle for 3-4 seconds) before adding antibiotic. This is the #1 novice mistake: plates that look fine but don't actually select.

Timing
  • Dissolving + autoclaving (1 h): 15 min prep, 20 min autoclave cycle, 20 min cooldown in autoclave.
  • Cooling to 55°C (45 min): hands-off, waterbath or insulated container.
  • Pouring (30 min): ~25–30 plates from 1 L batch in a biosafety cabinet.
  • Setting (30–60 min): hands-off at room temperature.
  • Total active time: ~90 min spread across ~3 h.
  • Makes: ~25–30 × 100 mm plates per 1 L batch.
Equipment (Catalog) 5
  • Microwave
    Specs: Standard kitchen microwave
    Optional — for re-melting stored agar bottles
  • Micropipette Liquid handling
    Specs: P1000 + P200
    For antibiotic stock addition
  • Biosafety cabinet Safety
    Specs: Class II Type A2 or equivalent
    All pouring and antibiotic addition
  • Benchtop incubator Thermal regulation
    Specs: 37°C static
    Optional — for QC sterility test of 1 plate
  • Water bath Thermal regulation
    Specs: 50–55°C, 1 L capacity minimum
    For cooling agar to antibiotic-safe temperature
Materials (Catalog) 2
  • Ampicillin Antibiotic
    Qty: 100 µg/mL final
    1 mL of 100 mg/mL filter-sterilised stock per 1 L agar. Substitute: kanamycin 30 µg/mL, chloramphenicol 17 µg/mL
  • Superior Broth (Athena Enzyme Systems) 0105 Growth media
    Qty: 25 g per L
    LB broth powder (Miller or Lennox formulation); Superior Broth is optional alternative
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
Name Type Required Default Unit Description
agar_concentration_g_per_L number — 15 — Bacteriological agar concentration. 15 g/L standard. Higher (20 g/L) makes firmer plates; lower (10 g/L) makes softer plates for motility assays.
lb_concentration_g_per_L number — 25 — LB broth powder concentration. Standard Miller formulation; Lennox LB uses 20 g/L.
autoclave_time_min number — 20 minute (UO:0000031) Autoclaving duration at 121°C, liquid cycle. 20 min sufficient for 1 L bottles; scale up for larger volumes.
cooldown_temp_c_before_antibiotic number — 55 degree Celsius (UO:0000027) Target agar temperature at time of antibiotic addition. Critical — above 60°C degrades most antibiotics; below 45°C the agar solidifies.
antibiotic_final_conc_ug_per_mL number — 100 — Final antibiotic concentration in the plate. Ampicillin: 100 µg/mL. Kanamycin: 30 µg/mL. Chloramphenicol: 17 µg/mL.
plate_volume_mL number — 20 milliliter (UO:0000098) Volume of agar per standard 100 mm petri dish. 20 mL gives ~5 mm depth — optimal for most applications.
Procedure Steps (Version 0.1.1)

Weigh out LB broth powder and bacteriological agar into a clean 1 L Schott bottle. For 1 L batch: 25 g LB broth powder + 15 g bacteriological agar (the standard Luria-Bertani + 1.5% agar formulation).

Add deionised water to ~900 mL total volume. Swirl to suspend the powder (it won't fully dissolve until heating).

Cap the bottle LOOSELY (do not seal tight — pressure will build up during autoclaving). Alternatively cover with aluminium foil secured with autoclave tape.

Autoclave at 121°C for 20 minutes on a liquid cycle. The liquid cycle has a slower pressure release to prevent the agar from boiling over when depressurised.

When the cycle is complete AND the pressure indicator has returned to zero, remove the bottle using heat-resistant gloves. The bottle is at ~95°C and contents near boiling.

Place the bottle in a water bath or insulated cooler set to 50–55°C. Allow to cool for 45 minutes, checking periodically. The correct temperature is reached when you can hold the bottle comfortably for 3–4 seconds against your wrist — warm but not painful.

While waiting, set up the biosafety cabinet: wipe with 70% ethanol, arrange sterile petri dishes in stacks of 5, have your sterile pipette and antibiotic stock ready on ice.

When the agar is at ~55°C, move the bottle to the biosafety cabinet. Verify temperature one more time by touch — this is the most common failure point.

Add antibiotic stock to the final working concentration for your selection (ampicillin: 100 µg/mL final, i.e. 1 mL of 100 mg/mL stock into 1 L agar; kanamycin: 30 µg/mL final; chloramphenicol: 17 µg/mL final).

Swirl the bottle gently to mix the antibiotic evenly. Do NOT shake vigorously — bubbles on the plate surface are impossible to remove later.

Pour approximately 20 mL per plate. Gauge visually: the agar should cover 1/3 to 1/2 of the plate depth with room to spare for lid clearance. A 1 L batch yields 25–30 plates at this volume.

As you pour, tilt the plate slightly if air bubbles form; pop them with a flame loop, briefly pass a gas flame over the surface, or leave them to pop during setting.

After pouring, close lids and leave plates on a level surface to set at room temperature. Allow 30–60 minutes undisturbed.

Once solid, invert plates (agar side up) and transfer to the biosafety cabinet for drying. Leave lids ajar for 30 min at room temperature, or overnight at 4°C.

Once dried, stack plates in sleeves of 5–10, seal with Parafilm, label with: LB + antibiotic name + final concentration + date + your initials.

Store at 4°C, inverted, in the dark. Record the batch in LibreBiotech: one Sample record per batch of plates with annotations for lot number, antibiotic, concentration, quantity, sterility QC result, and storage location.

Before using plates for a critical experiment, perform a QC test: streak a negative-control strain (no plasmid) on 1 plate; should yield zero colonies. Discard batch if selection fails.

Completion Notes

Expected outcome. 25–30 even-thickness (~5 mm depth) agar plates with smooth surfaces, no bubbles, no visible cracks. The colour should be straw/amber, slightly translucent. Antibiotic is incorporated uniformly throughout.

Drying. Fresh plates contain excess moisture. After setting, leave them inverted, lids ajar, in the biosafety cabinet for 30–60 min or overnight at room temperature. This dries the surface enough that colonies grow discrete rather than swimming.

Quality control.

  • Sterility check: Incubate 1 plate from the batch at 37°C overnight without any inoculation. Should stay clean. Any contamination means the batch must be discarded.
  • Selection check: Streak a known-non-resistant E. coli (e.g. fresh DH5α without plasmid) on 1 plate; should yield zero colonies after overnight at 37°C. Any colonies indicate antibiotic failure (too cool when added? stock degraded?).

Storage. Seal stacks of dried plates with Parafilm, invert, store at 4°C in the dark (especially important for light-sensitive antibiotics like tetracycline). Shelf life:

  • Ampicillin plates: 4–6 weeks
  • Kanamycin plates: 4–6 weeks
  • Chloramphenicol plates: 6–8 weeks
  • Tetracycline plates: 2–3 weeks, wrap in foil

Troubleshooting.

Symptom Likely cause Fix
Plates don't solidify Insufficient agar, not autoclaved long enough, or autoclave issue Verify agar concentration (15 g/L); autoclave longer (25 min); check autoclave calibration
Bacteria grow everywhere including negative controls Antibiotic added to agar that was too hot Recheck cooldown timing; remake batch
Condensation on plate surface Poured before agar fully cooled / agar still gassing Invert plates earlier; let bottle sit longer before pouring
Uneven agar depth Shaky pouring technique Practice; use a graduated cylinder or serological pipette; pour more gently
Mould colonies appearing after 2–3 days Contamination during pouring Use a fresher biosafety cabinet; flame loops between streaks; remake batch
References
  1. Bertani G (2004). Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186(3):595–600. (Historical review of the LB medium formulation). DOI paper
  2. Standard microbiology practice; see standard molecular biology references (e.g. Green & Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed, CSHL Press 2012). book