Antibiotics are chemicals that are produced by living organisms which, even in minute amounts, inhibit the growth of or kill another organism. While thousands of them have been discovered since Sir Alexander Fleming observed the inhibitory activity of Penicillium on Staphylococcus in 1929, most are not useful medically because of undesirable toxicity or side effects. A simple method using paper discs saturated with the chemicals to be tested can be used to determine the resistance or sensitivity of an organism to different antibiotics. Following inoculation of an agar medium, discs are placed onto the surface of the medium so that the antibiotic will diffuse into the medium. Inhibition of the organism is evident following incubation as a clear region around the disc, called a "zone of inhibition", in which no growth has occurred.

The size of the zone of inhibition is determined by the type of medium used, the solubility and rate of diffusion of the antibiotic, the amount of inoculum, as well as the effect of the antibiotic. This experiment simulates a commonly used clinical technique called the Kirby-Bauer method in which exact specifications must be followed. Numerous antibiotics are tested for usefulness against the actual organism causing a patient's illness. Medical laboratory personnel select the antibiotic discs tested based upon the site and type of infection. Following incubation and measurement of the zone sizes, a chart is consulted which indicates whether the diameter of the zone of inhibition for an antibiotic indicates that it would be effective for use in treating the patient's infection.

Materials per group of 4 students:

• 2 plates of Trypticase Soy Agar (TSA)
• 2 sterile swabs
• Overnight broth cultures of Staphylococcus aureus (Sa), and Pseudomonas aeruginosa (Pa)

Materials to be shared by the class:

• Dispensers of antibiotic discs
• Beakers of disinfectant
• Sterile toothpicks or applicator sticks

Protocol:

1. Dip one swab into the broth of Pseudomonas.
2. Swab the entire surface of one plate labeled, "Pa". Go over the plate at least twice in each direction. Discard the swab in the beaker of disinfectant provided.
3. Repeat this procedure for the Staphylococcus.
4. Add the antibiotic discs to each plate using the dispenser. Remove the cover of the petri dish and set the dispenser over the plate. If the dispenser is not dispensing discs, consult the instructor.
5. Gently tap each antibiotic disc onto the surface of the agar with a sterile stick or toothpick to assure good contact. Discard toothpicks in the disinfectant.
6. Incubate plates at 37EC until the next lab period.
7. Examine each plate for the presence of zones of inhibition.
8. Measure the diameter of each zone in millimeters using the rulers provided.
9. Compare the diameter of each zone to the chart to determine if the organism is sensitive or resistant to the antibiotic.

Organism______________

Organism______________

The teacher must inoculate tubes of Trypticase Soy broth with Staphylococcus aureus and Pseudomonas aeruginosa 24-48 hours before the class. Remember to practice your best aseptic technique while inoculating the cultures. The cultures will be incubated at about 37*C until needed.

While the cultures can be used for more than one class, the potential for contamination of the culture will increase with use. For this reason, you are provided four tubes of broth per class. Per class, inoculate two tubes with Staphylococcus aureus and two tubes with Pseudomonas aeruginosa.

Procedure:

1. Locate the tubes of sterile Trypticase Soy broth provided. Label the tubes either S. aureus or P. aeruginosa. For each tube, remove the cap and place it back on the tube so that it just rests on top of the tube.
2. Locate the plates with S. aureus and P. aeruginosa. Remove the plastic wrap sealing the plates.
3. Choose one of the plates. Use a sterile disposable inoculation loop to place a visible amount of the bacteria from the surface of the plate into the correctly labeled tube of broth. Do not set the loop down. You will use the same loop to inoculate other tubes of broth with the same bacterium. Discard of the loop in the biohazard bag.
4. Once the broth tubes for one bacterium are done, repeat the process with the second bacterium (S. aureus or P. aeruginosa).
5. Snap all the tube caps down tight. Holding the tube just below the cap, thump the bottom of the tube with your other hand to suspend the bacteria in the broth.
6. Incubate the tubes upright in the rack at approximately 37*C until class. Usually 24-48 hours is adequate for abundant growth. The broth in the tubes should be cloudy if the bacteria grew.