14: Antibiotics

Learning Outcomes

By the end of this lab period, you will be able to

• Set up and perform a disk diffusion assay
• Explain the utility of Mueller Hinton media
• Identify and measure zones of inhibition and the location of the minimum inhibitory concentration of each antibiotic on the MH plate.
• Determine if each organism studied is sensitive, intermediate, or resistant to a set of antibiotics.

Introduction

Antibiotics, or antimicrobials are small molecules that inhibit bacterial growth. We have covered the history and mechanisms of antibiotics, as well as resistance to antibiotics in the lecture portion of the course. There is also a great chapter for your review in OpenStax Microbiology (Chapter 14 - Antimicrobial Drugs).

The mechanisms of antibiotic action are typically divided into five groups

1. Cell wall synthesis inhibitors
2. Protein synthesis inhibitors
3. Nucleic acid (DNA/RNA) synthesis inhibitors
4. Metabolism inhibitors (sulfonamides)
5. Plasma membrane disrupters

Some antibiotics are “broad spectrum” and work against a wide variety of bacteria. However, we know that bacteria as a group are incredibly diverse, and differences in cell walls and metabolic features such as enzyme expression mean that not every antibiotic will be effective against every organism. In order to determine which antibiotic is a good choice as therapeutic, potential antibiotics must be tested against that organism.

Antibiotic Effectiveness Testing

Testing antibiotic effectiveness can be done in several ways. One common method is a dilution assay, where the antibiotic is continually diluted to lower and lower concentrations to determine at which point organisms are able to grow. Dilution assays can be done in test tubes or in microwell plates (Figure \(\PageIndex{1}\)).

Figure \(\PageIndex{1}\): Diagram of an antibiotic dilution assay. As the concentration of antibiotic increases, bacterial growth is inhibited.

Today, we will be using a method called a Disk Diffusion Assay, first described by Kirby and Bauer in 1966. The Disk Diffusion Assay uses small antibiotic-impregnated disks which are placed on a newly inoculated Muller-Hinton agar plate. Antibiotics diffuse from the disks outward creating an antibiotic gradient. Where concentrations of antibiotic are high enough, bacterial growth will be inhibited (Figure \(\PageIndex{2}\)).

Figure \(\PageIndex{2}\): Principles of the Disk Diffusion Assay

Mueller Hinton Agar

When the antibiotic disk is placed on inoculated Mueller Hinton (MH) agar, the antibiotic will diffuse outward from the disk. The further from the disk, the lower the antibiotic concentration. In order for these results to be reproducible, MH Agar is a non-selective, non-differential medium that almost all organisms will grow on. It’s been designed to be low in molecules that can inhibit antibiotics and it’s poured to a precise thickness in order for antibiotic diffusion to be uniform between plates. MH plates also contain starch, which absorbs toxins released from bacteria that might interfere with the antibiotics. Lastly, MH agar is a loose medium that better allows for antibiotic diffusion.

ZOI and MIC

The clearing zone around the antibiotic disk is referred to as the “zone of inhibition” (ZOI). The edge of that zone, where the growth of the microorganism begins, represents the “minimum inhibitory concentration” or MIC. This is the minimum concentration of antibiotic that is required to prevent the bacteria from growing. In order to determine antibiotic effectiveness, the zones of inhibition can be measured in millimeters (mm) and compared to standard values (Figure \(\PageIndex{3}\)).

Figure \(\PageIndex{3}\): A. Antibiotic, shown in red, diffuses outward from the disk. The further from the disk, the lower the concentration. B. The zone of inhibition (ZOI) is measured in millimeters (mm). The minimum inhibitory concentration is the concentration of antibiotic at the very perimeter of growth.

Figure \(\PageIndex{4}\): Disk diffusion assay performed on S. aureus. Note that Polymyxin B (PB 300 disk) does not inhibit S. aureus growth.

Materials

Day 1

• Three Mueller-Hinton agar plates
• Broth cultures of S. aureus, E. coli and P. aeruginosa
• Antibiotic disks representative of the five different mechanisms of antimicrobial action
• Antibiotic disk dispenser or forceps for placement of disks
• Alcohol pads or bath to sterilize forceps
• Cell spreaders, alcohol, and beakers for creating bacterial lawns or sterile cotton swabs

Day 2

• Metric ruler for measuring Zones of Inhibition

Experiment

Day 1

1. Work in pairs. Collect 3 Muller-Hinton agar plates.
2. Label plates with sample type, pair names, date, and lab section
3. Using the three different bacteria provided, pairs will create a bacterial lawn on each Mueller-Hinton plate (one species of bacteria per plate). Utilize sterile cotton swabs or plate-spreading technique as demonstrated by your instructor.
4. Sterilize/disinfect forceps using one of two methods:
   1. Swab with an alcohol pad
   2. Use the alcohol dip and flame technique as demonstrated by your instructor
5. Apply eight (8) different antimicrobial disks on each plate using forceps and gently tap into place. Do not push so hard that you force the disk into the agar. Make sure that all disks are evenly spaced to allow maximum growth of bacteria around them. Use the same set of antibiotics on each plate to allow comparisons of their effects on the three organisms. Make note of which antibiotics you used.
6. Replace lids and set plates in the 37oC incubator upside-down until the next lab meeting. Note: for longer incubations, lower the incubator temperature to 30oC.

Day 2

1. Remove the plates from the incubator. Hold each plate dark background to better visualize the zones of inhibition. The edge of a zone is where no growth is visible to the naked eye.
2. Measure the diameter of each zone of inhibition in millimeters (mm) and record it in your lab notebook.
3. Record your data in Table 2 (or in your lab notebook). Use Table 1 to determine if each organism was sensitive (S), intermediate (I), or resistant to each antibiotic.

Data

Determine if each organism is sensitive, intermediate, or resistant to each of the antibiotics you tested. Your instructor will provide a separate table with standard values for your use.

Table 1 - Interpretation of Zone Diameters

Table 1: Student Data Table

		S. aureus		E. coli		P. aeruginosa
Antibiotic Name/Disk ID	Mechanism of Action	Zone diameter	S/I/R	Zone diameter	S/I/R	Zone diameter	S/I/R

Questions

1. Overall, which organism was most resistant to antibiotics? Consider the environment where this organism is found. Does this result make sense?
2. Which antibiotic was the most broad-spectrum in this experiment? What mechanism of action does it represent?
3. Which antibiotic was the most narrow spectrum in this experiment? What mechanism of action does it represent?
4. Define/describe
   1. Mueller Hinton agar
   2. Zones of inhibition
   3. Minimum inhibitory concentration
5. Suppose you perform this test on a hypothetical Staphylococcus species with the antibiotics penicillin and chloramphenicol. You record the zone diameters of 25mm for both the chloramphenicol and penicillin disks. Which antibiotic would be more effective against this organism? What does this tell you about comparing zone diameters to each other, and the importance of using the zone diameter interpretive table?