Antimicrobial susceptibility testing - Disk diffusion method (Kirby- Bauer method): Procedure, Principle, Result Result and Interpretation

 

Scope:  The purpose of this is to give general instructions to the technical staff of the department regarding the manner, in which antibiogram test has to be carried out by Kirby Bauer method.

Kirby- Bauer Method

 Kirby – Bauer Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant bacteria isolates.

Kirby-Bauer antibiotic testing (Disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated paper disks to test whether particular bacteria are susceptible to specific antibiotics.

A known quantity of bacteria is grown on agar plates in the presence of thin filter paper discs containing relevant antibiotics.

If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds, where bacteria are not capable of growing (called a zone of inhibition).

Agar disk diffusion method

Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4

Antibiotic storage -20°C minimum disks temperature

Inoculum McFarland 0.5

Incubator temperature 37°C.

Principle:

The disk diffusion method of susceptibility testing (also known as the Kirby-Bauer method) has been standardized primarily for testing of rapidly growing bacteria.

To perform the test, filter paper disks impregnated with a specific amount of antimicrobial agent are applied to the surface of an agar medium that has been inoculated with a known amount of the test organism. The drug in the disk diffuses through the agar.

As the distance from the disk increases, the concentration of the antimicrobial agent decreases creating a gradient of drug concentrations in the agar medium. 

Concomitant with diffusion of the drug, the bacteria that were inoculated and that are not inhibited by the concentration of the antimicrobial agent continue to multiply until a lawn of growth is visible.

In areas where the concentration of drug is inhibitory, no growth occurs, forming a zone of inhibition around each disk.

Criteria currently recommended for interpreting zone diameters and MIC results for commonly used antimicrobial agents are published by CLSI.

Results are reported categorically as Susceptible (S), Intermediate (I), or Resistant (R).

Material Required:

1. Muller Hinton Agar
2. Inoculation Loop
3. Sterile swab
4. Peptone water
5. De ionized water
6. Petri plates
7. BaCl2
8. H2SO4

Equipment Required:

1. Biological Safety Cabinet.
2. Bunsen burner
3. Disc dispenser
4. Incubator

Turbidity standard for inoculum preparation

To standardize the inoculum density for a susceptibility test, a BaCl2 turbidity standard, equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex particle suspension), should be used. 

A BaCl2 0.5 McFarland standards may be prepared as follows:

Procedure:

1. Label six test tubes of required diameter as standards 0.5,1,2,3,4 and 5.In amounts (ml) Shown in the table below, add a 1% solution of anhydrous barium chloride and a cold 1% (v/v) Solution of pure sulphuric acid.
2. Seal the tubes and keep in a refrigerator. Washington et al., (1972) discuss the stability of the standards.
3. The table shows the approximate number of bacteria/ml equivalent to the opacity standard, but the numbers vary with the size of the bacteria.

McFarland Standards Preparation Chart

McFarland Standard	1% Bacl2 (ml)	1% H2SO4 (ml)	Approximate Cell Count Density (x10ꓥ8 cells)
0.5	0.05	9.95	1.5 x 10ꓥ8
1.0	0.1	9.9	3.0 x 10ꓥ8
2.0	0.2	9.8	6.0 x 10ꓥ8
3.0	0.3	9.7	9.0 x 10ꓥ8
4.0	0.4	9.6	12.0 x 10ꓥ8

Inoculum preparation

1. Prepare a pure culture (18-24 hrs) of the sample on a non-selective medium.
2. Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standards.
3. Aseptic transfer of colonies for propagation of bacteria Select at least 4 to 5 well-isolated colonies of the same morphological type from an agar plate.
4. Touch the top of each colony with a wire loop and transfer the growth to a tube containing 4 to 5 ml of peptone water.
5. Allow the broth culture to incubate at 37°C until it achieves or exceeds the turbidity of 0.5 McFarland standards.

McFarland Turbidity Standards.

Be Cautious

The agar medium should have pH 7.2 to 7.4 at room temperature.  The surface should be moist but without droplet of moisture.

The antibiotic disks should be maintained at 8°C or lower or freeze at -20°C. Allow the disks to warm to room temperature before use. Don't use expired disks.

Getting ideal results depend on right inoculum

To avoid extremes in inoculum density, never use undiluted overnight broth cultures for streaking plates

Uniform inoculum on plates is essential for effective detection

Inoculate the plate with uniformity within 20 minutes after adjusting the turbidity of the inoculum suspension; dip a sterile non-toxic swab on an applicator into the adjusted suspension.

Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab. Inoculate the dried surface of a Muller-Hinton agar plate by streaking the swab over the entire sterile agar surface.

Repeat this procedure two more times, and rotate the plate 60° each time to ensure an even distribution of inoculum.

Replace the plate top and allow 3 to 5 minutes, but no longer than 15 minutes, for any excess surface moisture to be absorbed before applying the antibiotic disks.

Inoculate the plate with uniformity

Procedure:

1. Apply antibiotic impregnated disks on the bacterial lawn. 
2. Important: where the disk drops is where it stays and do not load the plates with too many antibiotic disks.
3. Place the appropriate disks evenly (no closer than 24 mm from center to center) on the surface of the agar plate either by using a sterile forceps or the dispensing apparatus.
4. No more than 6 disks should be placed on a 90 mm plate. A disk is not to be moved once it has come in contact with the agar surface since some of the compound diffuses almost instan­taneously.
5. Incubate for 16-18 hours at 37 ± 2°C unless otherwise instructed.

Proceed with incubation of plates

Invert the plate and place them in an incubator at 35°C within 15 minutes after disks are applied. The plates should be incubated aerobically.

After 16-18 hrs of incubation, examine each plate and measure the diameters of the zones of complete inhibition, including the diameter of the disk.

Large colonies growing within a clear zone of inhibition should be subcultured, re-identified and retested to know whether they are contaminants or mutants.

Antibiotics diffuse out onto the agar concentration of antibiotics decrease as they diffuse further away from the disks.

After incubation, observe for a clearing on the bacterial lawn (zone of inhibition) incubation Bacterial growth Zone of inhibition.

Result and Interpretation

Interpret the zone sizes by the CLSI or EUCAST interpretation of the zone of inhibition is different for each bacteria-antibiotic combination results and report the organism to be susceptible, intermediate or resistant.

Never compare the zone sizes of two different antibiotics and judge their effectiveness accordingly.

Interpret the zone sizes

Quality control procedure

Precision and accuracy ensured through control strains, whenever a new lot is put in-use

Known susceptibility to antimicrobial agents Standard strains include

v Staphylococcus aureus ATCC 25923

v Escherichia coli ATCC 25922

v Pseudomonas aeruginosa ATCC 27853

Factors affecting antimicrobial susceptibility testing

1. Heavy inoculums
2. Mixed isolates
3. Disk not properly applied
4. An agar gel that is too thick leads to smaller zones

Uses of antibiotic sensitivity testing

1. A laboratory test which determines how effective antibiotic therapy is against a bacterial infections.
2. Antibiotic sensitivity testing will control the use of Antibiotics in clinical practice.
3.  Testing will assist the clinicians in the choice of drugs for the treatment of infections.

Antibiotic susceptibility is important in patient care

1. Antibiotic susceptibility testing has become a very essential step for properly treating infectious diseases and monitoring antimicrobial resistance in various pathogens.
2. The choice of antibiotic needs to be made taking into consideration the susceptibility profile of the pathogen, pharmacology of the antibiotic, the need for antibiotic therapy, and its cost effectiveness.