Preparing antibiotic-containing media.

Materials

  • Antibiotics - use either (1) Pre-made stock solution of antibiotic (ideal if you lack the immediate means to filter-sterilize solutions), or (2) Prepare your own stocks -- see table below; many can be ordered from Sigma Chemical or Carolina Biological Supply. Research-grade antibiotics should never be ingested or used in people or pets. These antibiotics may contain toxic impurities that medical-grade antibiotics lack.
  • Solvent for the antibiotics: distilled water, antibiotics such as tetracycline and chloramphenicol dissolve better in alcohols such as 70% ethanol or isopropyl alcohol (available from a drugstore).
  • Scale and weighing paper to measure the amount of dry antibiotics for preparing stock solutions.
  • Pipettor or sterile eyedropper to add antibiotics to media.
  • Tempered agar media.

    Antibiotic Ampicillin Chloramphenicol Erythromycin Kanamycin Tetracycline
    Amount to add for a 10mL Stock solution 500mg (1/2gram) 300 mg 100mg 500 mg 100 mg>
    Final Concentration in agar media 50ug/mL 30ug/mL 10ug/mL 50ug/mL 10ug/mL
    Solvent water Ethanol or isopropyl alcohol water water Ethanol or isopropyl alcohol

    Method

    1. Prepare and autoclave/sterilze stock media as desired, such as LB Agar or minimal media.
    2. The hot agar solution must cool before adding antibiotics as the heat may inactive the drugs. Have an adult help with this part. Let the flask sit at room temperature, swirling every 15 minutes or so, until you can touch your fingers to the side of the flask for 2-3 seconds without removing them. At this point the agar should be warm enough that it won't soon solidify, but cool enough that it won't inactive the antibiotics.
    3. The above stock solutions are at a 1000X concentration, used to select for common Gram-negative or Gram-positive organisms that might carry resistance plasmids or other resistance genes (as opposed to susceptible strains that lack these genes). Add 1 milliliter of stock antibiotic per liter (=1000mL) of agar. If you prepared 1/2 L (500 mL) of media, add 1/2 mL of antibiotic stock solution, etc. Use a sterile pipette to add the solution. If nothing else is available, wash an eydropper with 70% ethanol, let dry, and add 50 drops of antibiotic stock solution (1 drop is approximately 20 ul (microliters) of solution for most droppers; 20 X 50 = 1000uL = 1mL).
    4. Swirl the agar media + antibiotic to mix it thorough. Try not to introduce air bubbles.
    5. Pour the plates and let solidify. Refrigerate in a clean plastic bag until use. If your refrigerator keeps a light source on, or the plates will be stored in ambient light for some period prior to use, cover the stack of plates with tinfoil, as light inactivates many antibiotics.


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