Disc diffusion test for Antibiotic Sensitivity testing is carried out to determine the appropriate antibiotic agent to be used for a particular bacterial strain isolated from clinical specimens.
Disc Diffusion tests
The most commonly used methods in a laboratory to determine susceptibility of bacteria isolates to antibiotics. In this method, discs impregnated with known concentrations of antibiotics are placed on agar plate that has been inoculated with a culture of the bacterium to be tested. The plate is incubated at 37°C for 18–24 hours. After diffusion, the concentration of antibiotic usually remains higher near the site of antibiotic disc, but decreases with distance. Susceptibility to the particular antibiotic is determined by measuring the zone of inhibition of bacterial growth around the disc.
Selection of Media
The medium that supports both test and control strains is selected for carrying out antibiotic susceptibility testing of the bacteria. For example, Mueller–Hinton agar is used for testing Gram-negative bacilli and Staphylococcus, blood agar for Streptococcus spp. and Enterococcus spp. species, chocolate agar for Hemophilus influenzae, and Wellcotest medium for sulfonamides and cotrimoxazole. The medium is prepared by pouring onto the flat horizontal surface of Petri dishes of 100 mm to a depth of 4 mm. The pH of the medium is maintained at 7.2–7.4. More alkaline pH increases the activity of tetracyclines, novobiocin, and fusidic acid, whereas an acidic pH reduced the activity of aminoglycosides and macrolides, such as erythromycin. The plates after preparation may be stored at 4°C for up to 1 week.
Preparation of inoculum
The bacteria are first isolated in pure culture on a solid medium. At least three to four morphologically similar colonies of the bacteria to be tested are touched and inoculated into appropriate broth and incubated at 37°C for 4–6 hours. The density of bacterial suspension in the broth is adjusted to 1.5X108 cfu /mL by comparing its turbidity with that of 0.5 McFarland opacity standard tube. The broth is inoculated on the medium by streaking with sterile swabs. A sterile cotton swab is dipped into the broth and excess broth is removed by rotation of the swab against the sides of the tube above the fluid level.
Antibiotic discs
Only the clinically relevant antibiotics are tested in antibiotic susceptibility tests. Antibiotic discs (6-mm filter paper discs) can be prepared from pure antimicrobial agents in laboratories or can be obtained commercially. The discs are applied with sterile forceps, a sharp needle, or a dispenser onto the surface of the medium, streaked with test strains, and the reading is reported after incubating the plate for 18–24 hours at 37°C aerobically.
Types of disc diffusion tests
Disc diffusion tests are of the following types:
- Kirby–Bauer disc diffusion method
2. Stokes disc diffusion method
Kirby–Bauer disc diffusion method:
Most common method used routinely for determination of antibiotic sensitivity of bacteria isolated from clinical specimens. In this method, both the test strains and the control strains are tested in separate plates. The test is performed by inoculating the test organism in a suitable broth solution, followed by incubation at 37°C for 2–4 hours. Then 0.1 mL of the broth is inoculated on the surface of the agar medium by streaking with a sterile swab. In this method, either nutrient agar or Mueller–Hinton agar in Petri dishes is used. The inoculated medium is incubated overnight at 37°C. The susceptibility of drug is determined from the zones of inhibition of bacterial growth surrounding the antibiotic discs. The diameters of the zone of inhibition are calculated with Vernier calipers or a thin transparent millimeter scale to the nearest millimeter. The point of abrupt diminution of the zone is considered as the zone edge. A maximum of six antibiotic discs are tested in a Petri dish of 85 mm size.
Interpretation of the zone size is done as per the interpretation chart. Depending on the zone size, bacteria can be considered sensitive, intermediate or resistant to antibiotics.
Stokes method
This is a disc diffusion method, which makes use of inbuilt controls against many variables. In this method, the Petri dish containing the Mueller–Hinton agar is divided horizontally into three parts. The test strain is inoculated in the central area and the control strains on the upper and lower third of the plate. In modified Stokes method, control strain is inoculated in the central part but test strains are inoculated on the upper and lower third of the plate. The plates are incubated at 37°C and observed for zones of bacterial inhibition around the discs. A maximum of six antibiotic discs can be applied on a 100 mm Petri dish.
Reporting of the result is carried out by comparing the zones of inhibition of test and control bacteria. The zone sizes are measured from the edge of the disc to edge of the zone. It is interpreted as follows:
- Sensitive (S): The zone of test bacterium is equal to or more than that of control strain. The differences between the zone sizes of control and test strains should not be more than 3 mm if the zone size of the test bacterium is smaller than that of control.
- Intermediate (I): The zone size of the test bacterium should be at least 2 mm, and the differences between the zone of test and control strain should be at least 3 mm.
- Resistant (R): The zone size of the test bacterium is 2 mm or less.
Interpretation of disc diffusion tests:
Results of disc diffusion tests, such as Kirby–Bauer and Stokes method, are interpreted as follows:
• Sensitive (S): Infection treatable by the normal dosage of the antibiotic.
• Intermediate (I): Infection may respond to higher dosage.
• Resistant (R): Unlikely to respond to usual dosage of the antibiotics.
However, for certain bacteria and antibiotic discs, the following may be kept in mind while interpreting results of disc diffusion tests:
- Proteus mirabilis, Proteus vulgaris, and other bacteria producing swarming produce a thin film on agar surface often extending into the zones of inhibition. In such situations, the zones of swarming should be ignored and the outer clear margin should be measured to determine the zones of inhibition.
- Many strains of MRSA grow very slowly in the presence of methicillin. They produce growth within the zone of inhibition on incubation for more than 48 hours. This problem can be overcome by incubating the bacteria at 30°C or by using 5% salt agar and incubating at 37°C.
- Penicillinase producing strains of Staphylococcus often fail to secrete enough enzymes to neutralize penicillin close to the antibiotic disc. In such situation, it may show a zone of inhibition, but with the presence of large colonies at the edge of the zone and without any gradual fading away of the growth of the bacteria toward the disc. In such condition, the zones of inhibition should not be considered, and it should be reported resistant irrespective of the zone size.
- Trimethoprim and sulfamethoxazole should be tested separately to know whether the bacterium is sensitive to both or only to one of these. These should never be tested in combination, the way these two drugs are used in combination in clinical practice.
