In the lab, bacteria are often grown on an all-purpose bacterial growth medium such as tryptic soy agar (TSY). TSY provides nutrients, moisture and the proper pH for most bacteria to grow. Agar is a Jello-like substance that is liquid when heated and solid when cooled, and it must be sterilized before use.
For sterilization, freshly mixed agar is poured into loosely capped bottles and placed in an autoclave, where high temperatures and pressure are used to kill microbes currently in the agar. After the agar is sterilized, it is stored in a warming oven, so that it remains liquid, and ready for use.
Pouring Agar to Create Sterile Plates
Agar is often ultimately poured into sterile Petri dishes (plates). If aseptic technique is not used, bacteria from the environment will be introduced to the media, and the media will become contaminated and useless.
The supplies needed to pour sterile plates are:
- A Bunson burner
- Sterile Petri dishes (glass or plastic)
- Matches
- A bottle of warm sterilized agar
Step 1: Before pouring the agar, make sure that everything is properly set up. The Bunsen burner should be hooked up to the gas supply, matches handy, and the sterile Petri dishes positioned close to the Bunsen burner. The farther the Petri dishes are from the burner, the more opportunity there will be for contamination to occur while pouring the plates.
Step 2: Light the Bunsen burner and adjust the flame so that there is a small, bright blue cone in the center. Extinguish the match and set it on the bench, making sure not to throw the match in the trash until it has cooled.
Step 3: Begin by removing the cap from the bottle of agar. The cap cannot be set down on the bench or held open-side-up. Doing either of these things can introduce bacteria into the cap; so one hand should be holding the bottle of agar, and the other holding the bottle cap, open side down.
Step 4: Slowly move the open neck of the agar bottle through the flame a couple of times. This ensures that the neck of the bottle is not contaminated.
Step 5: To pour a plate, lift one side of the Petri dish top only far enough to pour agar into the bottom half. Do not take the top of the Petri dish off and place it on the bench. This will result in contamination.
Step 6: Only pour enough agar to fill ¼ inch of the bottom Petri dish. If too little is poured, the agar may dry out later when bacterial cultures are incubated. If too much agar is poured, the agar will take a long time to solidify. Two or three plates can be poured before that neck of the agar bottle needs to be flamed again.
Step 7: When finished, flame the neck of agar bottle immediately before recapping, and return the bottle to the warming oven.
Step 8: Do not move or disturb the poured plates for at least 15 minutes, or until the poured agar has completely cooled and solidified.
Step 9: If the plates are not to be used right away, place them in a lab refrigerator.
For more information, see the SPO Virtual Microbiology Classroom or Todar's Online Textbook of Bacteriology.
Sources
Schauer Cynthia (2007) Lab Manual to Microbiology for the Health Sciences, Kalamazoo Valley Community College.
Bauman, R. (2005) Microbiology. Pearson Benjamin Cummings.
Tami Port - Tami Port is a college professor of cell and microbiology and creator of ScienceProfOnline.com, a free science education website.
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