Exercise 15: Antimicrobial Susceptibility

Introduction

Antibiotics are naturally occurring chemical agents produced by microorganisms that inhibit or kill other microorganisms. Penicillin G is one example produced by the mold Penicillium notatum. Naturally occurring forms can be chemically altered to improve their effectiveness. These improved versions are called semi-synthetics. Today, there are many agents used to treat bacterial infections that are fully synthetic – made from scratch in a lab. The generic term antimicrobial is used to describe any of these inhibitory substances. Some antimicrobial agents kill the organism and are termed bactericidal. Other drugs inhibit growth by preventing the bacteria from dividing, but do not kill them. These agents are termed bacteriostatic. The Kirby-Bauer method for evaluating antimicrobial agents was first published in 1966. It is a standardized disk diffusion method for testing multiple antimicrobial chemicals at once.

Mueller-Hinton plates are inoculated with a cell suspension to form a bacterial lawn. Paper disks containing a specified concentration of agent are placed on the agar plate. Then the plates are incubated to allow for bacterial growth and time for the agent to diffuse into the agar. As the agent diffuses through the agar, it establishes a concentration gradient around the disk. If the organism is susceptible to the agent, growth will be inhibited and a clear zone will appear around the disk – the zone of inhibition. If the organism is resistant to the agent, growth may occur up to the edge of the disk. Statistical averages are used to standardize the zones for each antibiotic. Some results may end up between the two and are indicated as intermediate.

Each disk is labeled with a code for the chemical agent and a number indicating the concentration (i.e.; P10 for penicillin at 10μg). Disks are dispensed onto the inoculated plates and incubated at 35°C for 24 to 48 hours. After incubation, the zone of inhibition for each agent is measured in millimeters and can be compared to a standardized table of results. Students must use the table to determine if the result for that particular organism and agent is susceptible (S), resistant (R) or intermediate (I).

Course Intended Outcomes

Explain the basis for antibiotic susceptibility and interpret results obtained when performing antibiotic susceptibility testing.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Sterile swabs, 2/pk
• Test tube rack
• Additional materials and cultures will be assigned by your instructor

Procedure, Day 1

Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Prepare a suspension of your assigned bacterial culture. Your suspension should closely match the turbidity of the 0.5 McFarland standard (Figure 15.1).
3. If your suspension is too clear, then add more bacterial culture. If your suspension is too turbid, add more water to the suspension.
4. Use a sterile swab to inoculate your suspension onto the surface of one Mueller-Hinton plate. Streak the entire surface of the plate in one direction.
5. Turn the plate 90 degrees and streak the entire surface again in that direction.
6. Leave the plate right side up with the lid on for 2-3 minutes so that the suspension can be absorbed into the medium.
7. Repeat steps 1 through 6 for the second culture assigned.
8. Bring inoculated plates to the front bench for application of the antibiotic discs.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Clearly label plates (on the bottom side) with your group name or initials, organism name(s), and section day/time.
4. Place inoculated plates in the rack labeled “to be incubated” on the back bench. Always place the plates with lids down and bottoms up.

Procedure, Day 2

1. Collect your group’s plates from the back bench area labeled “last week’s lab”.
2. Examine your plates for growth and for “zones of inhibition” around the antibiotic discs where there is no growth of the bacteria (Figure 15.2).
3. Flip your plates so that the bottom side is up and use a plastic ruler to measure diameter of the zone of inhibition in mm (Figure 15.3).
4. If the bacteria grows all the way to the edge of the disc, then enter “0” for the zone of inhibition.
5. Compare the measured value to the table provided to determine if the bacteria is susceptible, resistant, or intermediate to the antibiotic.
6. Record your results in your lab notebook.

Lab Clean-up

Return bacteria cultures to the front bench after inoculations are complete.

Remove labels from tubes and place in the tube rack ABOVE the biohazard container.

Plates are disposable and should be thrown away in the biohazard container when everyone in the group has completed the exercise.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Complete the table below.

Organism 1: __________________ Organism 2: __________________

Critical Thinking Questions

What properties of an antibiotic may affect its diffusion rate in the disc diffusion test?

Why are Gram negative organisms susceptible to different antibiotics than Gram positive organisms?

If the zone of inhibition for Ampicillin at 10 ug/ml is measured at 12 mm, what would be the expected zone of inhibition if dosage was increased to 20 ug/ml?