Kirby Bauer Antibiotic Filter Paper
The Kirby-Bauer Antimicrobial Susceptibility Test Read section II of chapter 26 in your lecture textbook for background on chemical antimicrobial control (for background)—pages 762-763.
Introduction Today you will perform a frequently used procedure called the Kirby-Bauer Antimicrobial Susceptibility test (disc diffusion technique). Each group will inoculate his/her own plate of Mueller-Hinton agar with an assigned culture. To that inoculated plate, you will then aseptically add sterile filter paper discs (using a disc dispenser), which contain a known concentration of antibiotics. As soon as the antibiotic discs touch the agar, the antibiotic will begin to diffuse into the surrounding agar. During incubation the bacteria you inoculated onto the agar …show more content…
will begin to grow. As the antibiotic diffuses, and the bacteria grow, organisms which are able to be inhibited by the antibiotic will fail to grow where the antibiotic is highest in concentration. This will leave a clear area, called the Zone of Inhibition, around the discs. During the next lab period, you will observe your plates and measure the diameter of the zone of inhibition, and use a standard interpretation table to interpret your results. Organisms may be susceptible, resistant or intermediate in resistance to the antibiotic. After you obtain results from your own plate, you will join the rest of your class to pool your data and discuss the results. Day one. - Materials and Protocol 1. Your instructor will assign you a number. Look at table 1 for your assigned culture. Record the culture you have selected here: ______Group 1______________ Table 1. Number | Organism | 1 | S. aureus (SA) | 2 | E. coli (EC) | 3 | P. aeruginosa (PA) |
2. Your group should obtain the following materials before you can proceed: * A container of sterile cotton swabs * A Petri dish containing Mueller-Hinton agar (one per group; labeled MH) * Your culture * A bottle of sterile media. * Vortex Mixer * A McFarland Standard (#2) 3. Re-suspend the bacteria in your culture tube by gentle flicking of the tube. 4. Compare your culture to a McFarland Standard (#2). Use the 2 mL pipette to add sterile media to your culture (aseptically) to dilute it to the same level as the McFarland Standard. Make sure you votex your culture after each addition of fresh media. 5. Dip a sterile dry cotton swab into the culture, and remove the excess liquid by rolling the swab tip around the test tube above the level of the broth. Use the swab to smear the culture all over the entire plate (see figure 2 below). 6. Your Mueller-Hinton agar plate will be inoculated in all directions to ensure even spreading of bacteria over the surface. This type of inoculation results in an even, confluent growth of bacteria called a ‘lawn’. 7. Allow the plate to absorb the liquid for 2-3 minutes. 8. Use the disc dispenser to dispense five different antibiotic discs on the surface of the plate by placing the dispenser over the plate and pressing the handle down (then release it). 9. Cover the plate and incubate (lid up) at 37 degrees C.
Day Two - Materials and Protocol 1.
Obtain your Petri dish and observe the bacterial growth on the plate. 2. If you have clear areas where bacteria did not grow around your antibiotic discs, these are called 'zones of inhibition.' Turn the plate so that you can place a ruler against the back of the Petri dish and measure the diameter of the zone of inhibition in millimeters. Record your own data in Table 1 below. 3. Using the interpretative key for that antibiotic, determine how susceptible your organism is to the antibiotics (S=Sensitive, R=Resistant, and I=Intermediate). Table 1. Individual Culture Antibiotic Susceptibility S. aureus (SA) Antibiotic Code (on disc) | Antibiotic Name and concentration | Zone of inhibition | Sensitive (S), Intermediate (I) or Resistant ® | GM | Gentamicin 10 | 23 mm | Sensitive | TE | Tetracyclin 30 | 30 mm | ≥19(Sensitive) | E | Erythromycins 15 | 26 mm | ≥23 Sensitive | PB | Polymyxin 300 | 0 mm | Resistance | CZ | Cefazolin 30 | 36 mm | ≥18 Sensitive | NB | Novobiocin 30 | 34 mm | ≥17 Sensitive | Bring your data to the blackboard and enter your data into the tables drawn there.
Record this data on Table
two.
Table 2. Class Data for Antibiotic Susceptibility (Fill in S, I or R) Antibiotic | PA g- Efflux pump | SA Gram + | EC Gram - | CZ-30 *Less broad | RR | SS | S | Nb 30 *more narrow | RR | SA | RR | PB 300 *more narrow | SS | RR | SS | TE 30 * less broad | RR | SS | SS | GM 10 Broad Spectrum—sensitive to all, work on both G- & G+ | SS | SS | SS | E 15 | R | SS | R |
Resistance to Antibiotics 1. Pump it out- efflux pump 2. Outer membrane with selective porins GRAM NEGATIVE BACTERIA!!! 3. Antibiotic Resistance genes that can a. Change a molecule that is the antibiotic target, e.g pencillin inhibit transpeptidase enzyme b. Change the antibiotic (inactivate it) c. Use another mechanism to activate how the cell will achieve the metabolic process that was inhibited by the antibiotic e.g. sulfur drug inhibit a critical co-enzyme folic acid. Organisms that are inhibit to sulfur drug will develop another mechanism to make folic acid
Questions: 1. Is there one particular bacteria that is more resistant than the others? Which one and why is it more resistant?
Yes, P. aeruginosa (PA) because it is Gram negative with selective porins and efflux pump.
2. Which appears to be more susceptible Gram Positive or Gram Negative?
Gr
3. If we consider a broad spectrum antibiotic being one that works on both Gram positive and Gram negative, do we have a broad spectrum antibiotic? If so, which one or ones are broad spectrum?
4. What is another method that can be used to test the effectiveness of these antibiotics and what advantage does it have over the disk diffusion method?
5. What is the difference between bacteriostatic, bacteriocidal and bacteriolytic?
6. What is the difference between broad and narrow spectrum?
7. Organism Z is resistant to antibiotic A if the zone is greater than 5 mm, but it is resistant to antibiotic B if the zone is greater than 10mm. Why is there a difference in the zone size determination for resistance? (think about what influences the ability of the antibiotic to diffuse through the media).
Introduction Today you will perform a frequently used procedure called the Kirby-Bauer Antimicrobial Susceptibility test (disc diffusion technique). Each group will inoculate his/her own plate of Mueller-Hinton agar with an assigned culture. To that inoculated plate, you will then aseptically add sterile filter paper discs (using a disc dispenser), which contain a known concentration of antibiotics. As soon as the antibiotic discs touch the agar, the antibiotic will begin to diffuse into the surrounding agar. During incubation the bacteria you inoculated onto the agar …show more content…
will begin to grow. As the antibiotic diffuses, and the bacteria grow, organisms which are able to be inhibited by the antibiotic will fail to grow where the antibiotic is highest in concentration. This will leave a clear area, called the Zone of Inhibition, around the discs. During the next lab period, you will observe your plates and measure the diameter of the zone of inhibition, and use a standard interpretation table to interpret your results. Organisms may be susceptible, resistant or intermediate in resistance to the antibiotic. After you obtain results from your own plate, you will join the rest of your class to pool your data and discuss the results. Day one. - Materials and Protocol 1. Your instructor will assign you a number. Look at table 1 for your assigned culture. Record the culture you have selected here: ______Group 1______________ Table 1. Number | Organism | 1 | S. aureus (SA) | 2 | E. coli (EC) | 3 | P. aeruginosa (PA) |
2. Your group should obtain the following materials before you can proceed: * A container of sterile cotton swabs * A Petri dish containing Mueller-Hinton agar (one per group; labeled MH) * Your culture * A bottle of sterile media. * Vortex Mixer * A McFarland Standard (#2) 3. Re-suspend the bacteria in your culture tube by gentle flicking of the tube. 4. Compare your culture to a McFarland Standard (#2). Use the 2 mL pipette to add sterile media to your culture (aseptically) to dilute it to the same level as the McFarland Standard. Make sure you votex your culture after each addition of fresh media. 5. Dip a sterile dry cotton swab into the culture, and remove the excess liquid by rolling the swab tip around the test tube above the level of the broth. Use the swab to smear the culture all over the entire plate (see figure 2 below). 6. Your Mueller-Hinton agar plate will be inoculated in all directions to ensure even spreading of bacteria over the surface. This type of inoculation results in an even, confluent growth of bacteria called a ‘lawn’. 7. Allow the plate to absorb the liquid for 2-3 minutes. 8. Use the disc dispenser to dispense five different antibiotic discs on the surface of the plate by placing the dispenser over the plate and pressing the handle down (then release it). 9. Cover the plate and incubate (lid up) at 37 degrees C.
Day Two - Materials and Protocol 1.
Obtain your Petri dish and observe the bacterial growth on the plate. 2. If you have clear areas where bacteria did not grow around your antibiotic discs, these are called 'zones of inhibition.' Turn the plate so that you can place a ruler against the back of the Petri dish and measure the diameter of the zone of inhibition in millimeters. Record your own data in Table 1 below. 3. Using the interpretative key for that antibiotic, determine how susceptible your organism is to the antibiotics (S=Sensitive, R=Resistant, and I=Intermediate). Table 1. Individual Culture Antibiotic Susceptibility S. aureus (SA) Antibiotic Code (on disc) | Antibiotic Name and concentration | Zone of inhibition | Sensitive (S), Intermediate (I) or Resistant ® | GM | Gentamicin 10 | 23 mm | Sensitive | TE | Tetracyclin 30 | 30 mm | ≥19(Sensitive) | E | Erythromycins 15 | 26 mm | ≥23 Sensitive | PB | Polymyxin 300 | 0 mm | Resistance | CZ | Cefazolin 30 | 36 mm | ≥18 Sensitive | NB | Novobiocin 30 | 34 mm | ≥17 Sensitive | Bring your data to the blackboard and enter your data into the tables drawn there.
Record this data on Table
two.
Table 2. Class Data for Antibiotic Susceptibility (Fill in S, I or R) Antibiotic | PA g- Efflux pump | SA Gram + | EC Gram - | CZ-30 *Less broad | RR | SS | S | Nb 30 *more narrow | RR | SA | RR | PB 300 *more narrow | SS | RR | SS | TE 30 * less broad | RR | SS | SS | GM 10 Broad Spectrum—sensitive to all, work on both G- & G+ | SS | SS | SS | E 15 | R | SS | R |
Resistance to Antibiotics 1. Pump it out- efflux pump 2. Outer membrane with selective porins GRAM NEGATIVE BACTERIA!!! 3. Antibiotic Resistance genes that can a. Change a molecule that is the antibiotic target, e.g pencillin inhibit transpeptidase enzyme b. Change the antibiotic (inactivate it) c. Use another mechanism to activate how the cell will achieve the metabolic process that was inhibited by the antibiotic e.g. sulfur drug inhibit a critical co-enzyme folic acid. Organisms that are inhibit to sulfur drug will develop another mechanism to make folic acid
Questions: 1. Is there one particular bacteria that is more resistant than the others? Which one and why is it more resistant?
Yes, P. aeruginosa (PA) because it is Gram negative with selective porins and efflux pump.
2. Which appears to be more susceptible Gram Positive or Gram Negative?
Gr
3. If we consider a broad spectrum antibiotic being one that works on both Gram positive and Gram negative, do we have a broad spectrum antibiotic? If so, which one or ones are broad spectrum?
4. What is another method that can be used to test the effectiveness of these antibiotics and what advantage does it have over the disk diffusion method?
5. What is the difference between bacteriostatic, bacteriocidal and bacteriolytic?
6. What is the difference between broad and narrow spectrum?
7. Organism Z is resistant to antibiotic A if the zone is greater than 5 mm, but it is resistant to antibiotic B if the zone is greater than 10mm. Why is there a difference in the zone size determination for resistance? (think about what influences the ability of the antibiotic to diffuse through the media).