How to Make LB Agar Plates

Written by michael douglas-llyr
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How to Make LB Agar Plates
Proper handling and preparation are crucial when preparing plates for culture. (Bacteria Colonies image by ggw from Fotolia.com)

Agar is a gelatinous substance, manufactured from red algae and used for a variety of purposes. One of its common uses is as a culturing medium for bacteria. LB agar is nutrient agar that has been combined with lysogeny broth (LB). It is a commonly held fallacy that LB stands for Luria-Bertani. However, its inventor, Giuseppe Bertani, states in his paper "Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems," that the initials were always intended to stand for lysogeny broth, not his name and that of his coresearcher, Salvador Luria. LB agar is versatile and fairly simple to make.

Skill level:
Easy

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Things you need

  • For 1 L of finished medium:
  • Scale and weighing papers or trays
  • 1-L graduated cylinder
  • 1 L distilled or deionised water
  • 2-L Erlenmeyer flask
  • 10g tryptone
  • 5g yeast extract
  • 5g noniodized table salt
  • 15g agar
  • Aluminium foil
  • Waterproof oven gloves
  • Autoclave or pressure cooker
  • Sterile Petri plates

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Instructions

  1. 1

    Place the tryptone, yeast extract and salt into the Erlenmeyer flask.

    How to Make LB Agar Plates
    Use an Erlenmeyer flask that holds twice the volume of the medium you intend to prepare. (laboratory equipment image by Vasiliy Koval from Fotolia.com)
  2. 2

    Add the distilled or deionised water and sir or shake to dissolve most of the dry ingredients.

    How to Make LB Agar Plates
    Pay close attention to the pressure of the autoclave or pressure cooker when sterilising. (pressure meter image by Thor Jorgen Udvang from Fotolia.com)
  3. 3

    Add the agar and continue to shake or stir. All of the agar won't dissolve, but it's important that you get most of it into solution. Avoid large clumps of undissolved agar settling in the bottom of your flask.

  4. 4

    Seal the opening of the flask with foil.

  5. 5

    Place the flask in an autoclave-safe container.

  6. 6

    Pour about 1cm of water into the container.

  7. 7

    Put the container and flask into the autoclave. Set the autoclave for a liquid cycle and sterilise for at least 20 minutes. If you are using a pressure cooker for sterilisation, go to Step 8. Otherwise, skip to Step 10.

  8. 8

    If you are using a pressure cooker, place a small amount of water in the cooker and place the flask in the water. Do not attempt to sterilise more than 250ml at a time using this method.

  9. 9

    Bring the pressure cooker to about 20 psi and sterilise for about 30 minutes.

  10. 10

    Remove the flask from the autoclave or pressure cooker, taking care as the flask will be quite hot.

  11. 11

    Swirl the flask gently once sterilisation is completed. This will distribute the agar more evenly throughout your solution.

  12. 12

    Put the flask on a suitable surface and allow it to cool a bit. If you have prepared 1 L or more of medium, give it a gentle swirl every 10 to 20 minutes. You will be able to tell that the medium has cooled sufficiently when you can hold the back of your fingers against the flask for about 2 seconds without its being uncomfortably hot to the touch. Wait about 15 to 20 minutes before attempting this.

  13. 13

    Place a Petri dish on a flat surface. Gently lift the lid at an angle, so that most of the dish is left covered. Do not lift the lid straight off the plate.

  14. 14

    Pour the medium from the flask into the Petri dish until the dish is about half full.

  15. 15

    Immediately replace the lid, move the plate aside and move on to the next until all desired plates have been filled. The plates will complete their solidification in about 30 minutes.

  16. 16

    Place the Petri dishes in the sterile bag in which they arrived or another suitable, sterile container if you are not inoculating them right away. Seal the container to preserve moisture. Label the plates if desired. If you are storing the plates, they should be kept at a temperature of about 3.89 to 4.44 degrees C.

Tips and warnings

  • The recipe can be scaled up or down, as desired. Always use an Erlenmeyer flask that is at least twice the size of the finished amount of medium you require.
  • If you use a pressure cooker, prepare only 250ml of medium in the Erlenmeyer flask at a time.
  • In the absence of an autoclave or pressure cooker, place the flasked medium in an ovenproof pan and heat it at 177 degrees Celsius for one hour. During this time give the flask a swirl every 20 minutes or so to fully dissolve the agar. This method will destroy or inactivate most bacteria, but may not be proof against certain species that thrive on heat.
  • If you want to add antibiotics or other additives to the medium, do so after the initial cooling period. Be sure to swirl the flask a bit to incorporate the additive throughout your mixture.
  • Handle the heated agar with care and always wear heat-resistant, waterproof mitts when handling it. Hot agar can cause serious burns!
  • Keep your sterile Petri dishes closed until ready to pour your medium to avoid airborne contaminants.
  • If you intend to add antibiotics to the medium, do so immediately before pouring into the plates. Make sure to protect these plates from light, which may degrade the antibiotic and mitigate its effectiveness.

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