Antibiotic Sensitivity Disks

Introduction

Antibiotic sensitivity disks are paper disks treated with antimicrobial compounds (antibiotics). They are used to evaluate the action of specific antibiotics against various microorganisms.

Concepts

• Antibiotic sensitivity
• Microorganisms

Background

The disks are made of highly absorbent paper, similar to filter paper or chromatography paper. The manufacturer impregnates each disk with a precisely known quantity of a specific antibiotic. The disks are applied to the agar surface of freshly inoculated culture dishes. Once in contact with the moist agar medium, the antibiotic diffuses outward from the disk into the surrounding medium. After a standard incubation period, the growth of the bacterial culture is examined. If the bacteria is sensitive to the test antibiotic, its growth will be slowed or prohibited, and a clear, circular zone of inhibition will be evident around the disk. If the bacteria is resistant, no clear zone (bacteria growing right up to the edge of the disk), or a clear zone of less than a specified diameter, will be seen. 

Safety Precautions

Wear chemical splash goggles and chemical-resistant gloves when working with the bacterial cultures. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Contaminated plates, swabs and implements should be sterilized before disposal. Best methods are autoclaving or disinfecting by soaking materials for at least one hour in a 5 to 10% bleach solution. Biohazard bags, in which materials can be collected for treatment and disposal, are recommended. Dispose of bleached plates according to Flinn Suggested Disposal Method #26b.

Procedure

1. Sterile Petri dishes should be poured with a sterile, prepared culture medium to a depth of 4 mm, and allowed to harden. In clinical applications, Mueller-Hinton agar is used. Mueller-Hinton agar is formulated to reduce or eliminate compounds with the potential to inhibit or inactivate certain antibiotics. Other media can be used, as long as they are used consistently—so that valid plate-to-plate comparisons can be made.
2. Inoculate the entire agar surface of the dish with the bacterial culture being tested (sterile cotton swabs and broth cultures work best). Inoculate by streaking the surface in three different directions—rotating the plate 60° each time. Cover and let stand 10 minutes.
3. Place the disks directly on the agar surface. Avoid touching or handling the disks with any non-sterile implements. Gently press each disk to the agar surface with a sterile swab or forceps. Do not attempt to reposition the disks as diffusion of the antibiotic begins immediately. Disks should be at least 20 mm apart (edge to edge) and 15 mm from the inside edge of the plate. Follow the pattern/template in Figure 1.
   {13008_Procedure_Figure_1_Disk placement pattern}
4. Incubate the dishes for 16 to 24 hours at a temperature appropriate to the test culture.
5. Examine the plates against a dark background. Measure the clear zones of inhibition around each disk to the nearest millimeter.
6. Compare measurements to the ranges listed in the table below. For a given antibiotic, the bacteria tested should be designated resistant, intermediate or sensitive. 
   {13008_Procedure_Table_1}

Teacher Tips

• Disks should be stored under refrigeration. One to two hours before use, remove the disks from refrigeration and allow them to come to room temperature. If desired, blank (control) disks can be punched from filter or chromatography paper using a standard hole punch. They must be sterilized (autoclaved) before use. Latex gloves and safety eyewear are recommended throughout the procedure.

References

Difco Manual, 10th ed.; Difco Laboratories: Detroit. 1984; pp 844–850.