Procedure of Kirby Bauer Disc Diffusion Method
- Sterilize the area with disinfectant and open burner before performing the test.
- A sterile cotton swab is dipped into the inoculum and remove excess medium by pressing the swab onto the wall of the tube.
- Swab the surface area of the plate completely by rotating the plate. ...
- Allow the plates to dry for 5 minutes so that the medium absorbs the inoculum properly.
What is disk diffusion method?
B. Kocsis, inAntibiotic Resistance, 2016 Disk Diffusion Method The disk diffusionmethod (DDM) is classified as an agar diffusion method (ADM) because the plant extract to be tested diffuses from its reservoir through the agar medium seeded with the test microorganism.
What does disk C mean on a disk diffusion test?
In diagnostic laboratories, the disk diffusion test is used to determine the susceptibility of clinical isolates of bacteria to different antibiotics. An effective antibiotic will produce a large zone of inhibition (disk C), while an ineffective antibiotic may not affect bacterial growth at all (disk A).
How do you test for pathogens using disk diffusion?
Disk diffusion by the Kirby-Bauer method is a standardized technique for testing rapidly growing pathogens.89 Briefly, a standardized inoculum (i.e., direct suspension of colonies to yield a standardized inoculum is acceptable) is swabbed onto the surface of MH agar (i.e., 150-mm plate diameter).
What is disk-diffusion agar method?
The disk-diffusion agar method tests the effectiveness of antibiotics on a specific microorganism. An agar plate is first spread with bacteria, then paper disks of antibiotics are added. The bacteria is allowed to grow on the agar media, and then observed.
How do you do disk diffusion method?
The method consists of placing paper disks saturated with antimicrobial agents on a lawn of bacteria seeded on the surface of an agar medium, incubating the plate overnight, and measuring the presence or absence of a zone of inhibition around the disks (Figure 1).
What is the process of setting up the Kirby-Bauer disc diffusion method?
In Kirby-Bauer testing, bacteria are placed on a plate of solid growth medium and wafers of antibiotics (white disks, shown) are added to the plate. After allowing the bacteria to grow overnight, areas of clear media surrounding the disks indicate that the antibiotic inhibits bacterial growth.
What does disc diffusion method test for?
In diagnostic laboratories, the disk diffusion test is used to determine the susceptibility of clinical isolates of bacteria to different antibiotics.
What is the Kirby-Bauer disc diffusion method and what is it used for?
The purpose of the Kirby-Bauer disk diffusion susceptibility test is to determine the sensitivity or resistance of pathogenic aerobic and facultative anaerobic bacteria to various antimicrobial compounds in order to assist a physician in selecting treatment options for his or her patients.
How do you set up a Kirby-Bauer test?
0:394:01Kirby Bauer Disk Diffusion Method - YouTubeYouTubeStart of suggested clipEnd of suggested clipThe test tube and remove the cap and gently flame sterilize dip the swab into the bacterial brothMoreThe test tube and remove the cap and gently flame sterilize dip the swab into the bacterial broth remove from the tube and gently flame sterilize recap the tube.
How do you calculate the zone of inhibition in disc diffusion?
Take a ruler or caliper that measures in millimeters and place the "0" in the center of the antibiotic disk. Measure from the center of the disk to the edge of area with zero growth. Take your measurement in millimeters. This measures the radius of the zone of inhibition.
Can a antibiotic disc wipe out the entire bacterial lawn?
Since it's very unlikely that one antibiotic disk will kill all the bacteria on the plate, I suggest you use a method like what Asyed suggested earlier: increasing the number of antibiotic disks on each plate until all the bacteria are killed.
What are the limitations of the disc diffusion method?
However, the disk diffusion test has additional limitations that should be considered. Training and experience are required both for performing and reading the results. Further, the test is labor-intensive and has similar limitations to other culture-based tests.
What does it mean if the zone of inhibition is large?
A larger zone of inhibition around an antibiotic-containing disk indicates that the bacteria are more sensitive to the antibiotic in the disk.
How is Kirby-Bauer test measured?
0:096:20Interpreting the Results of the Bauer Kirby Method of Antibiotic ...YouTubeStart of suggested clipEnd of suggested clipTo interpret the results of the Bauer biotic susceptibility testing we're going to be measuring theMoreTo interpret the results of the Bauer biotic susceptibility testing we're going to be measuring the diameter of zones of inhibition in millimeters using a metric ruler.
What factor must be carefully controlled in the Kirby-Bauer method?
The factors that are needed to be carefully controlled in the Kirby - Bauer method are the following : Size of inoculum , Distribution of the inoculum , Incubation period , Growth rate of bacterium , Depth of agar , Diffusion rate of antibiotic , and Concentration of antibiotic in the disk .
What is modified Kirby-Bauer technique?
Modified Kirby-Bauer disc diffusion test method is a reference method that could be used as a routine technique to test the susceptibility of a bacterial isolate in a clinical laboratory. The disc diffusion method was originally described in 1966, is well standardized, and has been widely evaluated.
What is disc diffusion method for antimicrobial activity?
This method is based on the principle that antibiotic-impregnated disk, placed on agar previously inoculated with the test bacterium, pick-up moisture and the antibiotic diffuse radially outward through the agar medium producing an antibiotic concentration gradient.
What is the purpose of the control disc?
What was the purpose of the control disk? The purpose of the control disk was to show what the disk looks like when there is no antibiotic fighting against the bacteria. Propose reasons why an antibiotic may not be as effective at inhibiting E.
What is the use of antibiotic discs?
An antibiotic disc is a small, circular piece of filter paper which has been impregnated with an antibiotic solution and allowed to dry. The discs are used to find out which antibiotics an infective organism is sensitive to so that doctors know what drug to prescribe.
Why is Antimicrobic sensitivity testing done?
Why do I need an antibiotic sensitivity test? You may need this test if you have an infection that has been shown to have antibiotic resistance or is otherwise hard to treat. These include tuberculosis, MRSA, and C.
When will EUCAST disk diffusion be published?
EUCAST disk diffusion of anaerobic bacteria is under development 2021. Reviewed clinical breakpoints and disk diffusion correlates will be published with breakpoint table v 12.0 (1 January, 2022). The method will be valid for 5 species ( Bacteroides spp, Prevotella spp, Fusobacterium necrophorum, Clostridium perfringens and Cutibacterium acnes) and for anaerobic incubation for 16 - 20h (extended incubation not allowed). For anyone who wants to prepare and practice, EUCAST already now publish the methodology, reading guide and QC criteria.
What is the EUCAST disk test?
The EUCAST disk diffusion test is based on MH media and disks of a good quality. It is calibrated to EUCAST clinical breakpoints using broth microdilution for MIC determination. Updates are published regularly.
How long to incubate a disk?
Place a duplicate of each disk on the other inoculated plate, using the same procedures. Invert the plates and incubate them at 35°C for 18 to 24 hours. Results. Observe for the presence or absence of growth around each antimicrobial disk on each plate culture.
What happens if a zone of inhibition surrounds the disk?
If a zone of inhibition surrounds the disk, the organism is not automatically considered susceptible (S) to the drug being tested. The diameter of the zone must first be measured (in millimeters) and compared for size with values listed in a standard chart (Table 15.1).
How to inoculate agar plate?
Dip a swab into the bacterial suspension, express any excess fluid against the side of the tube, and inoculate the surface of an agar plate as follows: first streak the whole surface of the plate closely with the swab; then rotate the plate through a 45° angle and streak the whole surface again; finally rotate the plate another 90° and streak once more. Discard the swab in disinfectant.
What is disk diffusion test?
In diagnostic laboratories, the disk diffusion test is used to determine the susceptibility of clinical isolates of bacteria to different antibiotics. An effective antibiotic will produce a large zone of inhibition (disk C), while an ineffective antibiotic may not affect bacterial growth at all (disk A). Antibiotics to which a bacterial isolate is ...
Where are antibiotic disks placed?
Disks containing increasing antibiotic concentrations are placed on a seeded bacterial lawn on the agar surface and plates are incubated. Zone sizes are measured from the edge of the disk to the end of the clear zone. Interpretation is more complicated in mixed susceptibility populations.
When was agar diffusion first used?
Agar diffusion was first used by Martinus Beijerinck in 1889 to study the effect of auxins on bacterial growth. However, the method has been developed, refined and standardized by many scientists and scientific organizations over the years including George F. Reddish, Norman Heatley, James G. Vincent, Alfred W. Bauer, William M.M. Kirby, John C.
Can disk diffusion test differentiate bacteriostatic and bactericidal activity?
Although the disk diffusion test cannot be used to differentiate bacteriostatic and bactericidal activity, it is less cumbersome than other susceptibility test methods such as broth dilution.
What is disc diffusion test?
As the Broth dilution method are time-consuming, many laboratories in the United States adopted disc diffusion tests in the early 1950s. Kirby Bauer tests also known as the Disc diffusion test is used for antibiotic susceptibility testing. Lacks of standardization creates a problem in the 1960s and later Kirby and Bauer reviewed the description. In 1961, WHO standardized the procedure. Currently, CLSI updates and modifies the original procedure which ensures uniformity worldwide. This test is mainly performed to determine the sensitivity or resistivity of aerobes or facultatively anaerobes against different classes of antibiotics.
How to sterilize forceps before picking up antibiotic discs?
Firstly sterilize the forceps with alcohol before picking up antibiotic discs.
What is the process of swabing the surface area of a plate?
Swab the surface area of the plate completely by rotating the plate. This technique is called lawn culture or carpet culture.
How long to incubate a plate upside down?
Incubate the plate upside down for 24 hours at 37ºC.
Most recent answer
What i think, the concentration that you have loaded on the disk must be lower than Minimum inhibitory concentration. you can load four or five or more disc with increasing known concentration of compound (50ug/ml, 100ug/ml, 200ug/ml,300ug/ml,400ug/ml500ug/ml) along with a standard control.
All Answers (4)
Impregnating your disc with the extract would be my suggestion to you. Directly pipetting to the disc might lead to spilling of extract in the plate and ultimately give you a false result.
