Antibiotic Susceptibility Testing: Disc Diffusion & Dilution Methods

DIFFERENT METHODS OF ANTIBIOTIC SUSCEPTIBILITY TESTING

DISC DIFFUSION TYPE OF ANTIBIOTIC SUSCEPTIBILITY TESTING

• Kirby-bauer disc diffusion method
• Stokes method

DILUTION TYPE OF ANTIBIOTIC SUSCEPTIBILITY TESTING

• Tube dilution method
• Plate dilution method

KIRBY-BAUER DISC DIFFUSION METHOD

MEDIA

• Muller Hinton agar (MHA) (pH 7.2-7.4) is poured in plates of 9-10 cm, depth of agar is 3-4 cm.

METHOD OF KIRBY-BAUER DISC DIFFUSION

• Five colonies of the test organism are transferred to 5 ml of trypticase soy broth or nutrient broth, incubated at 37°C for 2-4 hours. 

• Inoculum standardized by matching the turbidity with 0.5 McFarland standard [0.5 ml of 1.175% barium chloride dehydrate solution added to 99.5 ml N/36 (1%) sulfuric acid]. 

• With a sterile cotton swab the test culture is spread evenly over the plate successively in 3 directions, to obtain an even inoculum. 

• The plate is allowed to dry for 3-5 min. antibiotic discs (diameter 6.35 mm) of correct potency are placed on the surface, 15 mm from the edge of the plate with a distance of 24 mm between discs.  

• Not more than 6 discs should be used per plate of 9 cm diameter.

INCUBATION OF KIRBY-BAUER DISC DIFFUSION

• The plate is incubated at 37°C for 18 hours.

INTERPRETATION/ZONES OF INHIBITION OF KIRBY-BAUER DISC DIFFUSION

• The zone of inhibition is measured by a scale to the nearest mm, including disc diameter.  

• Zone size is interpreted as Sensitive, Intermediate or Resistant by referring to a table of critical diameters. 

CONTROLS FOR OF KIRBY-BAUER DISC DIFFUSION

• Standard control strains should be used.
• When new batch of discs is put into use.
• When new batch of medium is put into use.
• Once in a week in parallel with routine antibiogram.

STOKES DISC DIFFUSION METHOD

MEDIA

• Muller Hinton agar (MHA) (pH 7.2-7.4) is poured in plates of 9-10 cm, depth of agar is 3-4 mm.

 

METHOD OF STOKES DISC DIFFUSION

• Test culture is inoculated in middle portion of the plate and control cultures in upper and lower portions of the plate, leaving a zone of not more than 5 mm in between. Maximum 4 discs are put on a plate, two on either side.

INCUBATION

• The plate is incubated at 37°C for 18 hours.

INTERPRETATION OF STOKES DISC DIFFUSION

• Radii or zone of inhibition of test and control organisms are measured with a scale from the edge of the disc to edge of zone of inhibition. 

• Sensitive – Zone equal to or larger or not more than 3 mm smaller than that of the control. 

• Moderately sensitive – Zone radii more than 3 mm, and is also smaller than that of the control by more than 3 mm. 

• Resistant – No zone or less than 3 mm radii.

ADVANTAGES OF STOKES DISC DIFFUSION

• Advantage of this method is that reading of the test strain is always taken in comparison to that of the control.   

• So all the variables of Kirby-Bauer method like quality of agar, concentration of agar, strength of antibiotic discs are taken care of, as they affect both control and test strains equally. 

• Blank discs can also be easily detected by this method.

TUBE DILUTION METHOD

MEDIA

• Muller Hinton broth

 

METHOD

• Two sets of doubling dilutions of antibiotics are prepared in Muller Hinton broth, so that each tube contains 1 ml of diluted antibiotic.  

• One set of tubes is inoculated with 0.5 ml of overnight broth culture of test organism diluted 1/100 and the other set of tubes with control organisms.

INCUBATION OF TUBE DILUTION METHOD

• They are incubated overnight at 37°C.

OBSERVATION OF TUBE DILUTION METHOD

• The last tube showing no growth is taken as the end point.

MIC

• Minimum inhibitory concentration is taken as the lowest concentration of the drug showing no growth.

MBC

• All tubes showing no growth are subcultured onto solid media. 

• Minimum bactericidal concentration is taken as the lowest concentration of antibiotic yielding no growth on plate subculture and represents a kill of more than 99.99%.

PLATE DILUTION METHOD

METHOD

• This is done for MIC of organisms which grow poorly in broth.   

• One ml of each strength of antibiotic is added to 19 ml of melted agar, cooled to 50°C.   

• One loopful of overnight broth culture (diluted 1/100) is added to each concentration of antibiotic in agar.   

• The lowest concentration of antibiotic completely inhibiting growth is the end point.

ADVANTAGE OF PLATE DILUTION METHOD

• Many strains may be tested on one plate.

FACTORS AFFECTING IN-VITRO ANTIBIOTIC SUSCEPTIBILITY TESTING

• Constituents of media
• pH of media
• Atmospheric conditions
• Thickness of medium
• Inoculum size
• Temperature
• Size of antibiotic molecules
• Concentration of discs.

ZONE SIZE INTERPRETATIVE CHARTS

• Zone size interpretative charts are charts depicting list of antibiotics along with their disc content (in mcg, IU etc.), diameters of zones of inhibition for Sensitive, Intermediate and Resistant findings.

• They are used as reference charts for interpretation of antibiotic sensitivity testing results.