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Lab 2 disk diffusion testing

The document discusses the disk diffusion method for testing bacterial susceptibility to antimicrobial drugs. Key points: 1. The disk diffusion method involves placing disks containing known amounts of antimicrobial drugs onto agar plates inoculated with bacteria. As the drugs diffuse out, a gradient is established and zones of inhibition form where growth is inhibited. 2. The size of the zones of inhibition depends on factors like the drug quantity, bacteria susceptibility, agar depth and bacteria concentration. 3. By measuring zone sizes and comparing to standards, bacteria can be classified as susceptible, intermediate or resistant to each drug tested. This helps determine appropriate antibiotic treatment.

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DISK DIFFUSION TESTING Dr. Samira Fattah PhD in Medical Bacteriology College of Health Sciences-HMU
Diffusion methods 1. Disk diffusion method 2. Well diffusion method
Disk diffusion method(or Kirby–Baur method ) Disk diffusion refers to the diffusion of an antimicrobial agent of a specified concentration from disks or strips, into the solid culture medium that has been seeded with the selected inoculum isolated in a pure culture.
Disk diffusion method • The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the antimicrobial. • When the concentration of the antimicrobial becomes so diluted that it can no longer inhibit the growth of the test bacterium, the zone of inhibition is demarcated. • The diameter of this zone of inhibition around the antimicrobial disk is related to minimum inhibitory concentration (MIC) for that particular bacterium/antimicrobial combination. Disk sensitivity test. A – agar; B – antibiotic disc; C – antibiotic diffuses into agar along concentration gradient; D – bacterial growth on surface of agar after 18 hours of incubation; E – zone (diameter) of inhibition.
Disk diffusion method The diameter of the zone depends on a number of factors including: 1. the quantity of antibiotic within the disk 2. the degree of susceptibility of the bacteria to the antibiotic 3. The depth (in mm) of the agar plate 4. The concentration of bacteria in the inoculum.
Disk diffusion method Factors that affecting results of the sensitivity test: 1. The pH : agar medium should have a pH between 7.2 and 7.4 at room temperature, If the pH is too low, certain drugs will appear to lose potency (e.g., aminoglycosides, quinolones, and macrolides), while other agents may appear to have excessive activity (e.g., tetracyclines). 2. Moisture : The surface should be moist, but no droplets of moisture should be apparent on the surface of the medium or on the Petri dish covers when the plates are inoculated.
Disk diffusion method Factors that affecting results of the sensitivity test: 3. Inoculum density : Usually optimal results were obtained with an inoculum size that produced by comparing with McFarland standard . 4. Timing of disc application : If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.
Disk diffusion method Factors that affecting results of the sensitivity test: 5. Temperature of incubation : Susceptibility tests are normally incubated at 35-37 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. At higher temperatures, the entire culture appears to be susceptible. 6. Incubation Time : Most techniques adopt an incubation period of between 16 and 18 hours. 7. Spacing of the antibiotic discs : If larger numbers of antibiotics have to be tested, two plates, or one 14- cm diameter plate, is to be preferred.
Disk diffusion method Materials Required • Mueller- Hinton agar • Antibiotic discs • Cotton swabs • Petri dishes • 0.5 McFarland Turbidity standard • Pure bacterial culture (24 hrs) • Forceps • Metric ruler
Procedure: 1. Take several isolated colonies from 24 hours old culture of bacteria to be tested and immerse the loop in 0.85% saline then compare the turbidity of the inoculated saline tube with 0.5 McFarland standard. 2. Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level. 3. The swab is streaked in three directions over the surface of the Mueller-Hinton agar to obtain uniform growth. 4. Allow the plates to dry for five minutes. 5. Using sterile forceps or a suitable disk dispenser, place paper disks on the surface agar plates at equal distance. 6. Incubate the plates upside down at 37oC for 24 hours. 7. Following overnight incubation, measure the diameter of the zone of inhibition in millimeter (mm) around each disk.
Result and Interpretation • use a metric ruler to measure the zone of inhibition and include the diameter of the disk in the measurement. • Compare the result with CLSI guidelines to report the result. • The results are reported as Susceptible (S), Intermediate (I), or Resistant (R).
Advantages • This test is used in determining the proper antibiotics to treat an infection. • It doesn’t require special equipment to perform and can be interpreted by all medical personnel. • It costs less to perform this test. Limitations • Not all slow or fastidious organisms can be tested accurately. • It is not considered a gold standard test as it is only for screening the susceptibility pattern of the organisms according to the CLSI guideline.
Disk Diffusion Troubleshooting Guide - Zones too small……….. Inoculum too heavy Agar too thick Disk expired or inactive Inoculated plates left too long prior to application of disks Wrong medium for organism - Zones too large……......... Inoculum too light Agar too thin Poor growth (too fastidious, wrong media, not fresh isolate) - Colonies within zone………Mixed population - Deformation of zone………. Disks too close to each other