Agar Overlay (double layer agar) technique

This technique allows you to produce a homogeneous lawn of bacteria within a thin layer of agar across the surface of a plate. Bacteria are added to a soft top agar (0.75% agar, as opposed to the usual 1.5% for agar plates) which has been melted at 100°C and cooled to 45°C. This is warm enough so the agar remains liquid, but cool enough so that the bacteria are not killed (for a period of time). The melted agar/bacterial suspension is mixed and poured evenly across the top of an agar plate and allowed to solidify.

The bacteria distributed through the top agar will grow to produce a homogeneously turbid lawn. If the freshly seeded lawn is exposed to various antibacterial agents and then incubated at 37°C, any inhibition of bacterial growth will cause a reduction in the turbidity of the lawn near the agent: the greater the antibacterial action, the wider the zone of inhibition. Thus, the antibacterial strength of the agent may be judged by the width of the zone of inhibition around it.

Illustrations of the experimental plan for the prep of the inoculated melted agar, and of the pouring of the inoculated agar onto the pre-warmed plate

sterile capped 13×100 mm tubes
sterile pipettes (0.1, 1.0 mL) or
sterile tips for Displacement pipetter
hot block, 45°C, warmed up
Bunsen Burner

melted top agar, about 60°C
fresh overnight culture of indicator bacteria
(such as E. coli B or Staphylococcus aureus)
pre-warmed nutrient agar plates
(or tryptone soy agar plates, etc)
dishpan with diluted Lysol into which to discard used tubes


  1. THE PREVIOUS NIGHT:   Inoculate about 2-3 mL of nutrient broth or tryptone soy broth with the desired indicator bacterium (often E. coli B). Grow as a stationary culture ON at 37°C in the hot block.
  2. THE DAY OF USE:   Pipet about 2 mL of hot melted top agar into sterile capped 13×100 mm tubes in a 45°C hot block. Allow cooling to 45°C (several minutes).
  3. Pipet about 0.1 mL of an ON culture of indicator bacteria into the melted agar, here using an Eppendorf Repeat Pipetter. Down the inside of the tube is OK.
  4. Overlay technique:

    a. Vortex to mix the bacteria into the melted top agar
    b. Immediately pour out onto a pre-warmed agar plate to empty the tube.
    c. Immediately tilt back and forth, shake gently to evenly distribute. Avoid bubbles, and stop agitating before agar begins to gel. Let set undisturbed to gel fully, (several minutes.)
  5. When fully gelled, assign and label positions on the plate bottom where agents will be applied or operations performed such as application of antibacterial agents, antibiotics, exposure to UV, etc. Apply liquids to 5 mm sterile filter discs placed on top of the top agar. Do not let run.
  6. Invert, incubate overnight at 37°C.
  7. THE NEXT DAY:  Next AM, read the plates. Where growth is thickest, there was the least antibacterial action. Where the thinnest, the greatest. Illustrate the plate, measure the zones of inhibition (width of the zone from the edge of agent to the edge of a zone), record data.

The Double-Layer Agar (DLA) technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages.


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