wikiHow to Make Tryptic Soy Agar (TSA)

Tryptic Soy Agar (TSA) is a complex medium; it is used to cultivate fastidious or non fastidious microorganisms.

Definitions

  • Agar: A sulfated polysaccharide used as a thickening agent.
  • Autoclave: Is a chamber filled with steam under pressure. The purpose of autoclaving is to sterilize liquids and some solids. It uses wet heat primarily because it kills bacteria faster than dry heat.
  • Buffer: A solution with an exceedingly stable pH.
  • Complex medium: is a medium full of rich amino acids and vitamins that help microbial growth, TSB is an example of a complex medium.
  • Cultivate/ Culture: The process of growing microorganisms.
  • Fastidious: Is an organism that requires a nutrient rich environment to cultivate.
  • Media/ Medium: A liquid or solid gel used to cultivate microorganisms
  • Trypticase Soy Broth (TSB): TSB is rich in amino acids, vitamins and contains several substrates for microbial growth such as enzymatic digest of casein, sodium chloride for osmotic equilibrium, dipotassium phosphate which serves as a buffering agent to maintain pH, papaic digest of soybean meal, and water.

Materials

  • Agar
  • Trypticase Soy Broth (TSB)
  • 1 L Erlenmeyer flask- The vessel must be twice the volume of the liquid to be autoclaved.
  • Metal cap for the Erlenmeyer flask
  • Large magnetic stir-bar
  • Hot stir plate
  • 500ml of distilled water
  • Clean plastic weighing boat
  • Top-loading balance
  • Graduated cylinder
  • Hot pad or mitt
  • Long Teflon magnetic tip rod
  • Autoclave
  • Autoclave Tape
  • Tape
  • Marker
  • Timer

Steps

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    Using the marker and tape label the flask with your name, the date and the name of the solution (TSA).
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    Carefully slide the large magnetic stir-bar into the Erlenmeyer flask.
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    Measure 500 mL of distilled water with the graduated cylinder.
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    Pour 500 mL of distilled water from the graduated cylinder into the Erlenmeyer flask.
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    Place metal cap on the flask.
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    Place flask on the hot stir plate, turn the plate to high temperature setting and bring to a boil.
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    Place the clean plastic weighing boat on the top-loading balance and tare (zero).
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    Weigh approximately 6 grams of agar.
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    With your hot mitt remove the flask from the hot stir plate and pour agar into the flask.
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    Place the metal cap back on the flask and place the flask back on the hot stir plate.
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    Turn on the stir plate selection and start mixing at medium speed, then gradually increase the mixing rate to medium-high over a period of one minute. Continue at high heat and medium high stir rate until all agar is dissolved, approximately 15 minutes.
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    Place the clean plastic weighing boat on the top-loading balance and tare (zero).
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    Weigh out approximately 15 grams of TSB.
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    With your hot mitt remove the flask from the hot stir plate and pour TSB into the flask.
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    Place the metal cap back on the flask and continue to heat the solution on high temperature setting and medium-high stir rate until it is no longer cloudy. (Tip: to determine if the solution is ready pick it up with your hot mitt and hold the flask to the light. If you see any crystals then the solution is not ready.)
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    When the TSA solution is clear, remove it from the hot stir plate using your hot mitt and place it on a clean surface that doesn’t receive much airflow.
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    Turn off the hot stir plate.
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    Use the long teflon magnetic tip rod to remove the stir-bar in the flask. This can be done by placing the rod in the flask and touching the stir-bar.
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    Cover the flask with the metal cap.
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    Cut off a strip of autoclave tape that is approximately 3 inches (7.6 cm) long and place the tape vertically on the side of the metal cap down to the flask. The tape should hold the cap and flask together.
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    Take your solution to the autoclave and follow autoclaving directions.

Tips

  • A 500 mL solution of TSA yields approximately 10-15 plates.
  • Ingredient ratios may vary depending on the product manufacture.
  • Follow the expiration date on the TSB and agar containers.
  • TSA is yellow-amber in color.

Warnings

  • Do not leave TSA broth on the hot stir plate unattended.
  • TSA is an irritant to the eyes, skin, and respiratory system. Use with caution.
  • Follow storage protocol on the TSB and agar containers.
  • Follow proper disposal protocol on the TSB and agar containers.

Sources and Citations

  1. Fluca Analytical. (Unknown). 57994 Tryptic Soy Agar Plates. Available: http://www.sigmaaldrich.com/etc/medialib/docs/Fluka/Datasheet/57994dat.Par.0001.File.tmp/57994dat.pdf
  2. Kutter B, Raya R, Guttman B, Brabban A, & Neitzel J (2005). Procedures: Lab of Phage Biology at the Evergreen State College. Olympia, WA: 13.
  3. Mott P, Stephen B, Gunhold R, & Kessler E. (2002). Artificial environments for growing bacteria.

Article Info

Categories: Chemistry | Science