Disk Diffusion Method | Kirby-Bauer Susceptibility Testing PDF

## Chapter 2: Disk Diffusion Method | Kirby-Bauer / Susceptibility Testing ### Principle - This method is based on antibiotic-impregnated disks placed on agar previously inoculated with a bacterium. - The antibiotic diffuses radially outward through the agar medium, producing an antibiotic concentr...

## Chapter 2: Disk Diffusion Method | Kirby-Bauer / Susceptibility Testing ### Principle - This method is based on antibiotic-impregnated disks placed on agar previously inoculated with a bacterium. - The antibiotic diffuses radially outward through the agar medium, producing an antibiotic concentration gradient. - Concentration of antibiotic is high at the edge of the disk and gradually diminishes as distance from the disk increases. - At some point, the antibiotic is no longer inhibitory and bacteria will grow freely. - A clear zone or ring is formed around an antibiotic disk after incubation if the agent inhibits bacterial growth. ### Media - Mueller-Hinton Agar (MHA) is the best medium for routine susceptibility tests. - MHA has good reproducibility, low in sulfonamide, trimethoprim, and tetracycline inhibitors and gives satisfactory growth of most bacterial pathogens. - Disk diffusion method inoculum is prepared using a suitable broth, such as tryptic soy broth. - Sterile 0.9% salt solution may also be used. - Media is supplemented with 1-2% sodium chloride (NaCl) if intended for marine organisms. #### Preparation of Agar Medium 1. Prepare MHA from the dehydrated medium according to the manufacturer's instructions. Media should be prepared using distilled water or deionized water. 2. Heat with frequent agitation and boil to dissolve the medium completely. 3. Sterilize by autoclaving at 121°C for 15 min. 4. Cool the agar medium to 40-50°C. Pour the agar into sterile glass or plastic petri dish on a flat surface to a uniform depth of 4 mm. 5. Allow to solidify. 6. Check the pH of each preparation after it is sterilized, which should be between 7.2 and 7.4 at room temperature. - This is done by macerating a small amount of medium in a little distilled water or by allowing a little amount of medium to gel around a pH meter electrode. #### Storage - If plates are not to be immediately used, they may be stored in the refrigerator inside airtight plastic bags at 2-8°C for up to 4 weeks. - Prior to use, dry plates at 30-37°C in an incubator, with lids partly ajar, for not more than 30 minutes or until excess surface moisture has evaporated. - Media must be moist but free of water droplets on the surface. - Presence of water droplets may result to swarming bacterial growth, which could give inaccurate results. - Water droplets are also easily contaminated. - Unpoured media may be stored in airtight screw-capped bottles under the conditions specified by the manufacturer. ### Inoculum #### Preparation 1. From a pure bacterial culture (not more than 48 hours old except for slow-growing organisms), take four or five colonies with a wire loop. 2. Transfer colonies to 5 ml of Trypticase soy broth or 0.9% saline. 3. Incubate the broth at 30°C or at an optimum growth temperature until it achieves or exceeds the turbidity of 0.5 MacFarland standard (prepared by adding 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.36 NH2SO4; commercially available). 4. Compare the turbidity of the test bacterial suspension with that of 0.5 MacFarland (vigorously shaken before use) against a white background with contrasting black line under adequate light. 5. Reduce turbidity by adding sterile saline or broth. - Note: Standardized inoculum has a concentration of 1-2x10^8 cfu/ml ### Inoculation of Plates 1. Dip a sterile cotton swab into the standardized bacterial suspension. 2. Remove excess inoculum by lightly pressing the swab against the tube wall at a level above that of the liquid. 3. Inoculate the agar by streaking with the swab containing the inoculum. 4. Rotate the plate by 60° and repeat the rubbing procedure. Repeat two times. This will ensure an even distribution of the inoculum. 5. Allow the surface of the medium to dry for 3-5 minutes but not longer than 15 minutes to allow for absorption of excess moisture. ### Antimicrobial Disks #### Selection - The number of antimicrobial agents to be tested should be limited. - Only include one representative of each group of related drugs. - Include those indicated for veterinary use to control or prevent disease and those that can be useful for epidemiological or research purposes. - Use antibiotic disks purchased from a reputable manufacturer. - The disk diameter is approximately 6 mm. - Disks should be properly stored in a tightly sealed container with desiccant at 2-8°C. - Expired disks should not be used. #### Application 1. Using sterile forceps or disk dispenser, place antibiotic disk on the surface of the inoculated and dried plate. 2. Immediately press it down lightly with the instrument to ensure complete contact between the disk and the agar surface. 3. Do not move a disk once it has come into contact with the agar surface since some diffusion of the drug spontaneously occurs instantaneously. 4. Position disks such that the center-to-center distance is 24 mm and no closer than 10 to 15 mm from the edge of the petri dish. - A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150 mm plate. - Reduce the number of disks applied per plate if overlapping zones of inhibition are encountered. ### Control Plate - Include one plate inoculated with a control strain (Appendix 2.1) for every set of plates and incubate together. ### Incubation 1. Incubate plates in an inverted position at 30°C or at an optimum growth temperature. ### Reading and Measurement of Zones of Inhibition 1. Observe for the zone of inhibition after 16 to 18 hours. Slow-growing organisms may require longer incubation period. 2. Record the presence of individual colonies (arrow) within zones of inhibition. 3. Read and record the diameter of the zones of inhibition using a ruler graduated to 0.5 mm. 4. Record occurrence of fuzzy zones (arrow). - In measuring the zone diameter, the fuzzy portion of the zone should be ignored - The zone limit is the inner limit of the zone of normal growth. - Round up the zone measurement to the nearest millimeter. ### Interpretation of Results 1. Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standard for veterinary pathogens (Appendix 2.2). 2. Report result as Resistant (R), Intermediate (I) or Susceptible (S). 3. Susceptibility test results using agents other than those listed in the chart are interpreted on the basis of the presence or absence of a definite zone of inhibition and is considered only as qualitative until such time as interpretative zones have been established. ### Rejection Criteria 1. Do not read plates on which growth of test bacteria have isolated colonies or less than semi-confluent growth. 2. Do not read zones of inhibition of two adjacent disks that overlap to the extent that measurement of the zone diameter cannot be made. 3. Do not read zones showing distortion from circular. 4. Reject all data collected in a particular set if the zones of inhibition produced on plate inoculated with a control strain are not within the tolerance limits set. ### Appendix 2.1: Acceptable Inhibitory Zone Diameter (mm) Limit of Control Strains This table provides a guide to acceptable zone diameter limits for several control strains commonly used in antimicrobial sensitivity testing. | Antimicrobial Agent | Disk Content | ATCC 25922 | ATCC 25923 | ATCC 27853 | ATCC 49619 | |---|---|---|---|---|---| | Amikacin | 30 µg | 19-26 | 20-26 | 18-26 | | | Amoxicillin-clavulanic acid* | 20/10 µg | 18-24 | 28-36 | 30-36 | | | Ampicillin | 20/10 µg | >20 | <19 | | | | Cefazolin | 30 µg | 21-27 | 29-35 | 26-32 | | | Cefoxitin | 30 µg | 23-29 | 23-29 | | | | Cephalothin | 30 µg | 15-21 | 29-37 | | | | Chloramphenicol | 30 µg | 21-27 | 19-26 | 26-32 | | | Clindamycin | 2 µg | 24-33 | 19-25 | | | | Erythromycin | 15 µg | 22-30 | 25-30 | 25-30 | | | Gentamicin | 10 µg | 19-26 | 19-27 | 16-21 | | | Imipenem | 10 µg | 26-32 | 20-28 | | | | Kanamycin | 30 µg | 17-25 | 19-26 | | | | Oxacillin | 1 µg | 18-24 | <12 | | | | Penicillin* | 10 units | 26-37 | 24-30 | | | | Rifampin | 5 µg | 8-10 | 26-34 | 25-30 | | | Tetracycline | 30 µg | 18-25 | 24-30 | 27-31 | | | Ticarcillin | 75 µg | 24-30 | 21-27 | | | | Ticarcillin-clavulanic acid* | 75/10 µg | 24-30 | 29-37 | | | | Spectinomycin | 100 µg | 21-25 | 13-17 | 10-14 | | | Sulfisoxazole | 250 µg or 300 µg | 15-23 | 24-34 | | | | Trimethoprim-sulfamethoxazole* | 1.25/23.75 µg | 23-29 | 24-32 | 20-28 | | | Vancomycin | 30 µg | 17-2 | 20-27 | | | *These agents have a different mechanism of action and are not susceptible to enzymatic inactivation by bacterial enzymes. ### Appendix 2.2: Zone Diameter Interpretative Standard for Veterinary Pathogens This table provides a guide to interpreting zone diameters for common veterinary pathogens. This information is helpful for determining whether a bacterium is susceptible, intermediate, or resistant to a specific antibiotic. | Antimicrobial Agent | Disk Content | Escherichia coli | Staphylococcus aureus | Pseudomonas aeruginosa | Streptococcus pneumoniae | Zone Diameter (mm) | |---|---|---|---|---|---|---| | Amikacin | 30µg | >17 | 19-26 | 18-26 | | | | Gentamicin | 10µg | >15 | 19-26 | 16-21 | | | | Kanamycin | 30µg | >18 | 17-25 | | | | | Spectinomycin | 100μg | >14 | | | | | | Amikacin | 30µg | 19-26 | 20-26 | 18-26 | | S | | Ampicillin | 20/10µg | 18-24 | 28-36 | 30-36 | | | | Cefazolin | 30µg | 21-27 | 29-35 | 26-32 | | | | Cefoxitin | 30µg | 23-29 | 23-29 | | | | | Cephalothin | 30µg | 15-21 | 29-37 | | | | | Chloramphenicol | 30µg | 21-27 | 19-26 | 26-32 | 21 | S | | Clindamycin | 2µg | 24-33 | 19-25 | | 21 | S | | Erythromycin | 15µg | 22-30 | 25-30 | 25-30 | 21 | S | | Gentamicin | 10µg | 19-26 | 19-27 | 16-21 | | | | Imipenem | 10µg | 26-32 | 20-28 | | | | | Kanamycin | 30µg | 17-25 | 19-26 | | | | | Oxacillin | 1µg | 18-24 | <12 | | | | | Penicillin* | 10 units | 26-37 | 24-30 | | | | | Rifampin | 5µg | 8-10 | 26-34 | 25-30 | >19 | S | | Tetracycline | 30µg | 18-25 | 24-30 | 27-31 | 23 | S | | Ticarcillin | 75µg | 24-30 | 21-27 | | | | | Ticarcillin-clavulanic acid* | 75/10µg | 24-30 | 29-37 | | | | | Spectinomycin | 100µg | 21-25 | 13-17 | 10-14 | | | | Sulfisoxazole | 250 µg or 300 µg | 15-23 | 24-34 | | | | | Trimethoprim-sulfamethoxazole* | 1.25/23.75 µg | 23-29 | 24-32 | 20-28 | 19 | S | | Vancomycin | 30 µg | 17-2 | 20-27 | | 17 | S | | Antimicrobial Agent | Disk Content | Escherichia coli | Staphylococcus aureus | Pseudomonas aeruginosa | Streptococcus pneumoniae | Zone Diameter (mm) | |---|---|---|---|---|---|---| | Amikacin | 30µg | 15-16 | ≤ 14 | | | I | | Gentamicin | 10µg | 13-14 | ≤ 12 | | | I | | Kanamycin | 30µg | 14-17 | < 13 | | | I | | Spectinomycin | 100μg | 11-13 | < 10 | | | I | | Amikacin | 30µg | ≤ 14 | | | | R | | Ampicillin | 20/10µg | < 13 | | < 14 | | | | Cefazolin | 30µg | 14-17 | ≤ 13 | < | Cefoxitin | 30µg | | | | | R | | Cephalothin | 30µg | | | | | R | | Chloramphenicol | 30µg | 13-17 | ≤12 | ≤17 | ≤ 15 | R | | Clindamycin | 2µg | | | | ≤ 14 | R | | Erythromycin | 15µg | | | | ≤ 15 | R | | Gentamicin | 10µg | | | | | R | | Imipenem | 10µg | < 13 | | | | R | | Kanamycin | 30µg | | | | | R | | Oxacillin | 1µg | | | | ≤ 10 | R | | Penicillin* | 10 units | | ≤ 28 | | ≤ 19 | R | | Rifampin | 5µg | | | | ≤ 16 | R | | Tetracycline | 30µg | 15-18 | ≤ 14 | < 18 | | R | | Ticarcillin | 75µg | ≤19 | | | | R | | Ticarcillin-clavulanic acid* | 75/10µg | ≤ 14 | | | | R | | Spectinomycin | 100µg | | | | | R | | Sulfisoxazole | 250 µg or 300 µg | | | | | R | | Trimethoprim-sulfamethoxazole* | 1.25/23.75 µg | < 11 | ≤ 10 | < 10 | ≤ 15 | R | | Vancomycin | 30 µg | ≤ 14 | ≤ 9 | | | R | *These agents have a different mechanism of action and are not susceptible to enzymatic inactivation by bacterial enzymes.