Making Agar Plates

Agar plates are the standard solid support material for growing microorganisms. Microbial growth media contains nutrients and an energy source to fuel the microbes as they grow, and agar to keep the media in a semi-solid, gel-like state.

On solid media, a single microbe will grow and divide to produce a "colony," a spot of identical descendants. Different types of microbes produce colonies with different characteristics-shape, color, texture-which help microbiologists determine if a culture is pure, or identify the types of microbes in a mixed sample.

A number of biological supply companies sell pre-made plates, but making your own is much less expensive. With a little practice, you will find that it is very easy to make your own plates, and you will have the added flexibility of being able to customize recipes to suit your needs.

Tip: Microbiologists typically use sterile, disposable polystyrene plates, but you can also use glass Petri dishes or even baby food jars. If you use glass dishes or jars, you may find it easier to sterilize the culture media directly in them. Prepare the media as in Step 3, but instead of sterilizing it, boil it until the agar is dissolved, then pour into individual containers. Sterilize the media in the containers in an autoclave or pressure cooker.

_1. Choose a recipe

Choose a recipe from the Media Recipes page or use one of your own.

Decide how many plates you will need. Our recipes will make 1 L (1000 mL) of media, enough to fill approximately forty 100 mm plates, but they can be scaled up or down as needed. Plan on using about 25 mL per 100 mm plate.

_2. Gather supplies

  • Media Recipe ingredients (see the Media Recipes page)
  • sterile, polystyrene Petri dishes. 100 x 15 mm is the most common size, but 60 and 35 mm sizes also work
  • glass container that will hold at least twice the volume of your media
  • aluminum foil for covering your media container, or plastic wrap if you use a microwave
  • autoclave, pressure cooker, microwave, or hot plate for sterilizing your media (see the Sterilizing Liquids page)
  • heat-resistant gloves, hothands, or potholders for handling hot containers
  • 70% ethyl or isopropyl (rubbing) alcohol
  • household cleaner, 10% bleach, or disinfectant wipes for cleaning your work area
  • graduated cylinder for measuring water
  • balance for weighing solid ingredients
  • tools for handling solid ingredients (such as weigh boats and scoopulas, or paper plates and spoons)

_3. Prepare media

Use a glass container (ideally an Erlenmeyer flask) that will hold at least twice the volume of your media.

  1. Assemble the ingredients according to the recipe of your choice.
  2. Cover with aluminum foil, or plastic wrap if you use a microwave.

_4. Sterilize

Sterilize using one of the methods described on the Sterilizing Liquids page.

One advantage of high-salt media is that typical contaminating microbes won't grow on it, so media with a salt concentration of at least 10% can be sterilized by boiling.

Make sure the agar dissolves completely. In media with 15% or more salt, the agar may be slow to dissolve. The media may look cloudy, or you may see small, translucent lens-like objects floating in it. Continue boiling until the media is completely clear; this may take longer than 15 minutes. Incompletely dissolved agar will leave your media squishy or fragile.

_5. Pour into plates

Tip: If you don't have a long uninterrupted chunk of time, you can prepare solid media in bottles, sterilize it, and let it cool completely in the bottle. At a later time, you can re-heat the media in a hot water bath or microwave until it melts. Let it cool until it is comfortable to the touch, then pour it into plates.
  1. Prepare a suitable work area. (see the Sterile Technique page for details).
  2. Label the plates with the type of media you will pour into them.
  3. Swirl the hot media vigorously to mix.
  4. Cool the media until it is just cool enough to handle, about 20-30 minutes. You should be able to hold your hand agains the container reasonably comfortably for a few seconds. If the media is too cool, it will start to solidify in the container. If it is too hot, it will leave excess condensation on the lids.
  5. Swirl the media again to mix just before pouring; be careful not to incorporate bubbles.
  6. Pour into plate until it covers the bottom, approximately 25 mL (see video below).
  7. After several hours to overnight, return the plates to the plastic sleeve they came in or place them in a plastic bag. Label the bag with the media type and the date, and store upside down in a refrigerator.

Plates will keep refrigerated for 4-6 weeks.