A New Twist to the Kirby-Bauer Antibiotic Susceptibility Test Activity—Increasing Antibiotic Sensitivity of Pseudomonas fluorescens through Thermal Stress

INTRODUCTION Antibiotic sensitivity and the effect of temperature on microbial growth are two standard laboratory activities found in most microbial laboratory manuals. We have found a novel way to combine the two activities to demonstrate how temperature can influence antibiotic sensitivity using a standard incubator in instructional laboratory settings. This activity reinforces the important concepts of microbial growth and temperature along with Kirby-Bauer antibiotic susceptibility testing. We found that Pseudomonas fluorescens can be manipulated to become more sensitive to several antibiotics by simply increasing growth temperature and exposing the organism to various antibiotics. No additional equipment is required beyond a standard incubator. Pseudomonas fluorescens is an excellent choice for this activity since it is a safe alternative to Pseudomonas aerugi-nosa, a biosafety level 2 agent. Pseudomonads are important to explore in the microbiology laboratory since Pseudomonas aeruginosa poses a serious issue in health care settings, as this organism is known to be a multi-drug-resistant pathogen (6). More importantly, P. fluorescens is a good alternative in the laboratory to P. aeruginosa since it is also pigmented (5) and a possible reservoir of antibiotic resistance genes (4). In addition, it grows best at room temperatures and can easily be thermally stressed by placing in a standard 35ºC to 37ºC incubator. PROCEDURE We run the Kirby-Bauer susceptibility test similar to that described in most lab manuals and use an American Type Culture Collection strain of P. fluorescens (Strain 13525). Briefly, P. fluorescens cultures are grown overnight at room temperature (20ºC–25ºC), then suspended in either nutrient broth or sterile saline to match the turbidity of a 0.5 McFarland Standard and swabbed onto Mueller-Hinton (MH) agar plates (3). Antibiotic disks are usually dispensed using a commercial multidisc dispenser and incubated for an additional 24 hours at room temperature. We have found that six antibiotics work well in this activity (chloramphenicol, streptomycin, penicillin, neomycin, tetracycline, and eryth-romycin). Zones of inhibition are measured and compared to standardized tables usually published in the laboratory manual or provided with the antibiotic disks. To thermally stress the organism, the above procedure is slightly modified. After swabbing the MH agar plates, these are placed at 35ºC to 37ºC for 24 hrs. P. fluorescens are inhibited at these temperatures and will not grow. After 24 hours the instructor or students can dispense antibiotic disks and continue incubation at room temperature overnight , where P. fluorescens will grow. The next lab period, control …