Chloramphenicol Plate Preparation

Chloramphenicol is an broad-spectrum antibiotic that kills bacterial microbes by interfering with their ability to replicate. This antibiotic was developed initially to fight infections in humans, but, according to Drug.com, it is only used now, as of this publication, as a last resort when other drugs are not working due to serious side effects such as aplastic anemia, hypoplastic anemia, thrombocytopenia and granulocytopenia.



Chloramphenicol is useful in a research laboratory for molecular biology experiments. When chloramphenicol is prepared on a plate, it can be used to detect bacterial concentration or to isolate specific strains of bacteria for further experiments.

Things You'll Need

• Distilled water
• Chloramphenicol
• 1.0 percent tryptone
• 0.5 percent yeast extract
• 1.0 percent sodium chloride (NaCl) pH 7.0
• 95 percent ethanol
• Agar
• 2-liter flask
• Magnetic stirring plate
• Medium sized magnetic stirbar
• Latex or nitrile gloves
• Laboratory scale
• Autoclave
• 30 sterile 10 cm plates
• Blue marking pen
• Parafilm
• Aluminum foil
• Autoclave tape

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Instructions

• 1

  Put on a pair of latex or nitrile gloves to prepare the LB broth. LB broth is a common abbreviation for Lysogeny broth, or Luria broth, and it is as used as a growth media for culturing bacteria. It can be made from scratch in the laboratory, or to save time it can be purchased from any molecular biology stock supplier.

• 2

  Place a sheet of weighing paper on the scale. Press the zeroing button on the scale so that the weight of the paper is not included in the final weight. Add small amounts of tryptone at a time, until the you reach 10 g. Then fold the weigh paper in half with the tryptone in the center. Pick up the weigh paper and carefully use the folded paper as a funnel to add the tryptone to the 2 L flask. Follow this procedure to weigh 5 g yeast extract, 10 g NaCl, and 15 g agar.

• 3

  Drop the stir bar into the flask and center the flask on the magnetic stir plate. Add enough distilled water to bring the solution up to 1 liter.

• 4

  Turn the stir plate on, and watch the stir bar. The plate should be set at the right speed to allow the stir bar to move at a moderate pace without getting stuck on the sides of the flask. Adjust the settings if necessary. Allow the solution to sit on the plate until all of the reagents have dissolved.

• 5

  Turn off the stir plate, and stretch a 3-inch square piece of parafilm over the top of the flask, to cover and protect the flask from any contaminants or from spilling. Then cover the parafilm with a piece of aluminum foil and add a piece of marking autoclave tape on top of the foil.

• 6

  Place the solution in an autoclave on liquid cycle for 20 minutes at 15 psi to sterilize it.

• 7

  Remove the flask from the autoclave. It will be very hot. Allow the solution to cool to around 55-degrees celsius, by letting it sit at room temperature, or by placing the flask in a 65-degrees celsius water bath.

• 8

  Spray and wipe down the work surface with 95 percent ethanol to prevent contamination. It's also a good idea to change your gloves before handling the plates.

• 9

  Mark the side and lid of each plate with vertical blue band using the marking pen. This indicates the plates contain LB broth and chloramphenicol.

• 10

  Add enough chloramphenicol antibiotic to the solution so that the concentration reaches the desired amount, depending on the bacteria you will be working with. A common final concentration is 100ug/ml. For this concentration, add 1mL chloramphenicol stock (at 25mg/ml). Swirl the flask in a circular motion, or place it back on the magnet stir plate for 1 to 2 minutes to make sure it is well mixed.

• 11

  Wipe down the mouth of the flask with ethanol, and then pour the solution into the plates, until they are filled halfway with the solution. Let the plates harden, cover each plate with a lid, and store them upside-down at 4-degree celsius to prevent them from drying out.