EXERCISE 9

ANTIBIOTIC SENSITIVITY TESTING

I. INTRODUCTION

Antibiotics are chemical compounds that selectively interfere with the growth of microorganisms while causing little or no damage to the animal or human host. Because of occasional adverse side effects and the increasing emergence of antibiotic resistant organisms, chemotherapeutic agents must be used properly and judiciously to safeguard their clinical usefulness.

A. Kirby-Bauer Method

The Kirby-Bauer method is based on the inhibition of bacterial growth measured under standard conditions. The organism to be tested is grown to a specific turbidity in a standard liquid medium. An inoculum from this culture is spread across the surface of a Mueller-Hinton agar plate to give confluent growth. Paper discs containing specific concentrations of antibiotics are placed on the agar surface. The antibiotic in each disc diffuses outward from the disc, and the concentration of the antibiotic diminishes as the distance from the disc increases. After incubation, the diameter of the zone of growth inhibition is measured and scored according to the size of the zone and the particular antibiotic, as sensitive, intermediate, or resistant.

The size of the zone of inhibition is directly proportional to the sensitivity of the organism to the antibiotic. Infections due to organisms designated as sensitive to a given antibiotic are more likely to yield clinically to that antibiotic than are infections with strains designated as intermediate or resistant.

B. MINIMAL INHIBITORY CONCENTRATION (MIC)/ MINIMAL BACTERICIDAL CONCENTRATION (MBC)

The Minimal Inhibitory Concentration (MIC) is determined for each bacterial isolate-antibiotic combination. Only antibiotics that show inhibitory activity toward the bacterial isolate using the Kirby- Bauer method are tested further. The active antibiotic is serially diluted to make a range of antibiotic concentrations that encompasses the concentration used in the Kirby-Bauer method. A constant, small amount of test bacterium is added to all tubes. After incubation, the MIC is identified as the smallest concentration of antibiotic that inhibits the growth of the test bacterium. This MIC will inhibit growth but not necessarily kill. The killing concentration is the MBC.

The Minimal Bactericidal Concentration (MBC) is determined by subculturing each of the No Growth tubes in the above MIC test to a solid medium that does not contain antibiotic. The antibiotic in the subculture diffuses into the agar, releasing any inhibitory effect it may have had on any living bacteria in the subculture. The MBC is identified as the smallest concentration of antibiotic that prevents any growth of the test bacterium (i.e., kills).

The clinician uses the MIC and MBC data in two ways:

• First, these values, like the zone diameters for Kirby-Bauer, provide a basis for selecting a drug likely to be effective against the infection.
• Second, MIC and MBC data, more so than the Kirby-Bauer data, can be useful in judging the dosage of the drug.

Note that the MBC and MIC provide a value at which there is confidence in strong antibacterial action; there is no need to increase the dosage toward more toxic levels. This balancing of peak drug level between the MIC and toxicity is most important for drugs that are toxic at low levels (e.g., aminoglycosides - cf. the EMIT assay in Exercise 2). For such drugs, drug levels in the patientŐs serum should be monitored to allow for adjustments in the dosage.

II. LAB WORK

A. KIRBY-BAUER METHOD

• 1 BAP of a Gram-negative organism (red dot)
• 1 BAP of a Gram-positive organism (yellow dot)
• 2 Prompt® Inoculation wands
• 2 Prompt® Dilution tubes
• 2 sterile cotton swabs
• 2 Mueller-Hinton agar plates (2 green stripes)
• 1 multi-welled plate containing antibiotic discs

1. One student in each pair will test the Gram-positive organism while the other student will test the Gram-negative organism.
2. Prepare the standardized inoculum for the Kirby-Bauer method as follows:
   • a.Hold the Prompt® Inoculation wand by the handle and touch the bottom of the wand to three colonies on the BAP.
   • b.Snap off the cap of the Prompt® Dilution tube, insert the wand and make a tight seal between the wand and the tube.
   • c. Vortex on a mixer (one for each side of the room) for at least 5 seconds.
   • d. Discard wand in Biohazard bag provided. Replace cap onto tube.
3. Moisten a cotton swab in the dilution tube and allow the excess liquid to flow back into the tube. The swab should be wet but not dripping.
4. "Paint" the swab once over the entire surface of the MH agar, as evenly as possible. Turn the plate 90° and without rewetting the swab, paint the agar again. Discard both swab and dilution tube in the Biohazard bag.
5. Allow the plate to dry for 3-5 minutes. Flame your forceps. Apply antibiotic discs to the agar surface in a rectangular pattern, keeping them as far apart as possible, but at least 3/4 inch from the edges of the plate. The chart on page 41 indicates which antibiotic discs should be placed on which bacterial lawn. Gently tap on each disc with the forceps to ensure contact.
   Incubate the plates inverted at 37°C for 18-24 hours.

B.MINIMAL INHIBITORY CONCENTRATION/MINIMAL BACTERICIDAL CONCENTRATION (MIC/MBC)

• 1 rack of 10 numbered tubes containing broth (a completed MIC)
• 1 TSA plate divided into 6 sections (a completed MBC)

1. The antibiotic, tetracycline was serially diluted (2-fold dilutions), starting with tube #1 (100 µg/ml) and ending with tube #9 (tube #10 = Control).
2. A constant amount of bacteria was added to all tubes and they were incubated at 37ˇC for 18-24 hours.
3. Mix each tube and examine them for growth, comparing each tube to the Control. 4.The MIC is the lowest concentration of antibiotic that still inhibits growth. Fill out the chart below.

5. From each of the NEGATIVE tubes (tubes in which no growth is apparent) and the Control tube, samples were swabbed onto the surface of a TSA plate.
6. The plate was incubated at 37ˇC for 18-24 hours.
7. The MBC is the least concentration of antibiotic that does not permit growth.

THE MBC OF TETRACYCLINE FOR THIS STRAIN OF E. coli IS _____ug/ml.

With which kind of antibiotic (static or cidal) would you expect the MIC and MBC to be about the same concentration? To be vastly Different concentrations?

1. Compare growth inhibition zones on the Mueller-Hinton plate with the appropriate inhibition patterns on the next page.
2. Record the extent of growth inhibition as R (resistant), I (intermediate), or S (sensitive) for each antibiotic.
3. Sample results
4. Measuring the tetracycline inhibition zone