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 aeruginosa, 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.
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 (20°C–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 erythromycin). 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 plates (dispensed with antibiotic after swabbing the bacterium) are compared with the thermally stressed plates. These comparisons, using the Kirby-Bauer antibiotic sensitivity method, show measurable differences in zones of inhibition when using several antibiotics.
Another variation that we found to work well is to perform the experiment setting the incubators to 30ºC or 32ºC. We have found that P. fluorescens will tolerate these temperatures better than 35ºC or 37ºC. With this variation, both control plates (P. fluorescens grown with antibiotic disks at 25ºC) and thermal stress plates can have antibiotic disks dispensed at the same time. The plates are placed at their appropriate temperature and the results can be read the next day or placed at 4ºC until the next laboratory period. Typical antibiotic zones of inhibition with the various stress temperatures and their interpretations are found in Table 1.
Zones of inhibition of P. fluorescens (ATCC 13525 strain) grown at various temperatures.
The P. fluorescens cultures (ATCC Strain 13525) used are considered biosafety level 1 agents (www.attc.org) and should be handled accordingly.
By simply thermally stressing a P. fluorescens culture plate prepared for the Kirby-Bauer test and comparing it to a similar plate placed at optimal temperature, students can easily see how temperature affects microbial metabolism and antibiotic sensitivity. We typically present this laboratory as an open ended activity, informing students that we are investigating the effect of elevated temperature on antibiotic sensitivity. We use the ATCC strain 13525 to avoid antibiotic sensitivity variations observed in laboratory strains of P. fluorescens. This strain is hypersensitive to tetracycline, neomycin, and streptomycin upon thermal stress, as indicated in Table 1. In addition to the increase in antibiotic sensitivity, we noted that P. fluorescens grown at elevated temperatures of 30ºC and 32ºC do not have the bright green pigment when grown at room temperature, thus adding another dimension of inquiry as to what could be happening at the elevated temperatures. Undoubtedly, a search of the literature will uncover a number of papers that explain what effect elevated temperature can have on membrane, efflux pumps, and gene regulation in this bacterium (1, 2). This is an excellent activity to demonstrate how environmental factors such as thermal stress can affect bacterial metabolic processes and lead to phenotypic changes. The results are easily discernable, as noted by the differences in pigment coloration and antibiotic sensitivity. Students are excited to see and measure changes in P. fluorescens that can be easily manipulated in the laboratory by varying temperature.
1. Adebusuyi AA, Foght JM. An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a. BMC Microbiol. 2011;11:252. doi: 10.1186/1471-2180-11-252. [Online.] http://www.biomedcentral.com/1471-2180/11/252. [PMC free article][PubMed][Cross Ref]
2. Burger M, RG Woods RG, McCarthy C, Beacham IR. Temperature regulation of protease in Pseudomonas fluorescens LS107d2 by an ECF sigma factor and a transmembrane activator. Microbiol. 2000;146:3149–3155.[PubMed]
3. Hudzicki J. Kirby-Bauer disk diffusion susceptibility test protocol. American Society for Microbiology; Washington, DC: Dec 8, 2009. posting date. [Online.] http://www.microbelibrary.org/component/resource/laboratory-test/3189-kirby-bauer-disk-diffusion-susceptibility-test-protocol.
4. Maravic A, Skocibusic M, Samanic I, Puizina J. Antibiotic susceptibility profiles and first report of TEM extended-spectrum beta-lactamase in Pseudomonas fluorescens from coastal waters of Kastela Bay, Croatia. World J Microbiol Biotechnol. 2012;28:2039–2045. doi: 10.1007/s11274-012-1006-5.[PubMed][Cross Ref]
5. Meyer JM, Abdallah MA. The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification, and physicochemical properties. J Gen Microboiol. 1978;107:319–328. doi: 10.1099/00221287-107-2-319.[Cross Ref]
6. Pool K. Pseudomonas aeruginosa: resistance to the max. Front. in Microbiol. 2011;2:65. [Online.] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128976/?report=classic. [PMC free article][PubMed]
Purpose and Scope:
The Kirby-Bauer test, known as the disk-diffusion method, is the most widely used antibiotic susceptibility test in determining what choice of antibiotics should be used when treating an infection. This method relies on the inhibition of bacterial growth measured under standard conditions. For this test, a culture medium, specifically the Mueller-Hinton agar, is uniformly and aseptically inoculated with the test organism and then filter paper discs, which are impregnated with a specific concentration of a particular antibiotic, are placed on the medium. The organism will grow on the agar plate while the antibiotic “works” to inhibit the growth. If the organism is susceptible to a specific antibiotic, there will be no growth around the disc containing the antibiotic. Thus, a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism. The measurement is compared to the criteria set by the National Committee for Clinical Laboratory Studies (NCCLS). Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible (S).
Principle of the method:
The media used in this test has to be the Mueller-Hinton agar because it is an agar that is thoroughly tested for its composition and its pH level. Also, using this agar ensures that zones of inhibitions can be reproduced from the same organism, and this agar does not inhibit sulfonamides. The agar itself must also only be 4mm deep. This further ensures standardization and reproducibility.
The size of the inoculated organism must also be standardized (using the BBD Prompt system). The reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than what it is supposed to be (“the antibiotics will have a distinct advantage”) and if the inoculum is too large, the zone of inhibition will be smaller.
Well isolated colonies from BAP agar plate only.
Perform weekly Quality Control for the antimicrobial disks by setting up the stock culture of E. coli (ATCC 25922) for Kirby Bauer sensitivities. The zones sizes should be as follows:
|E. Coli (ATCC 25922)||S. Aureus (ATCC 25923)|
Ampicillin 10 16-22mm
Cefazolin 29-35 mm
|Cephalothin 30 15-21 mm||Clindamycin 24-30 mm|
Ciprofloxacin 5 30-40 mm
|Oxacillin (Cefoxitin) 23-29 mm|
|Nitrofurantoin 300 20-25 mm||Penicillin 26-37 mm|
Sulfisoxazole 250 15-23 mm
Tetracycline 24-30 mm
|Trimeth/Sulfa 23-29 mm||Trimeth/Sulfa 24-32 mm|
Reagents and Supplies:
Mueller Hinton agar plates
BBL BD Sensi Disks (various antimicrobials as in QC above)
37 C Incubator
Sterile polyester or cotton swabs
Hardy Diagnostic Saline tubes
McFarland Latex 0.5 Standard and Wickerham Card
Calipers, ruler, or template for measuring the diameters of inhibitory zones.
- Preparation of Bacterial Suspension
- Remove a Hardy Diagnostic Saline 0.85%, 1.8mL tube from the box, label with the patient name and place in a test tube rack.
- Using a 1ul loop, pick several isolated colonies from the agar surface.
- Immerse the loop in a labeled saline tube. Vortex.
- Using a Wickerham Card and a vortexed McFarland Latex 0.5 standard, compare the turbidity of the inoculated saline tube with the Standard. If the turbidity is comparable, proceed with the inoculation of the Mueller Hinton Plate. If not, adjust the turbidity by adding more isolated colonies in the same manner if the turbidity is less than the standard or more saline if the turbidity is greater. Once the turbidity is comparable to the standard, proceed with the inoculation of the labeled Mueller Hinton plate.
- The bacterial suspension should be used within 6 h of preparation. If not used immediately after preparation, shake vigorously to resuspend the bacteria just prior to use.
- B. Inoculation of Mueller Hinton Agar
Allow plates to come to room temperature before use.
- Dip a sterile cotton swab into the bacterial suspension. To remove excess liquid, rotate the swab several times with a firm pressure on the inside wall of the tube above the fluid level.
- Using the swab, streak the Mueller-Hinton agar plate to form a bacterial lawn.
- To obtain uniform growth, streak the plate with the swab in one direction, rotate the plate 90° and streak the plate again in that direction.
- Repeat this rotation 3 times.
- Allow the plate to dry for approximately 5 minutes.
- Use an Antibiotic Disc Dispenser to dispense disks containing specific antibiotics onto the plate.
- Using sterile sticks or loops, gently press each disc to the agar to ensure that the disc is attached to the agar.
- Plates should be incubated overnight at an incubation temperature of 37°C.
C. Reading and Interpreting Zone Sizes
- After overnight incubation measure the zone sizes (area of no growth around the disk) in millimeters using a ruler or template.
- Enter the zone sizes into the Kirby Bauer sensitivities log along with the patients information.
- Interpret the results as Resistant, Intermediate or Sensitive for each antimicrobial according to the ranges listed on the log for Enteric gram negative rods.
- Enter the results (R, I or S) into the LIS.
- For Staph species that are Cefoxitin (Oxacillin) Resistant, Penicillin and Cefazolin will be reported as "not effective for MRSA".
This system is only set up for Enteric gram negative rods or a Staph aureus gram positive cocci. Therefore make sure the gram negative organism is lactose fermenting,and oxidase negative before setting up.
Non-enterics, streptococci/enterococci, gram positive rods and gram negative cocci must be sent out to Quest if sensitivities are needed.
BD BBL Prompt Inoculation System Pkg insert #5308-10 Rev 06/2010
Bauer, A.W., W.M.M. Kirby, J.C. Sherris, and M. Turck. 1966. Antibiotic
susceptibility testing by a standardized single disk method. Am. J. Clin.
Pathol. 45:493 496.
National Committee for Clinical Laboratory Standards. 2003. Approved
standard: M2-A8. Performance standards for antimicrobial disk susceptibility
tests, 8th ed. National Committee for Clinical Laboratory Standards, Wayne,