Agar diffusion test

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In Kirby-Bauer testing, white wafers containing antibiotics are placed on a plate of bacteria. Circles of poor bacterial growth surround some wafers indicating susceptibility to the antibiotic.

Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotics, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition).

The bacteria in question is swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. This along with the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity to that particular antibiotic. In general, larger zones correlate with smaller minimum inhibitory concentration (MIC) of antibiotic for that bacteria. Inhibition produced by the test is compared with that produced by known concentration of a reference compound. This information can be used to choose appropriate antibiotics to combat a particular infection. [1]

Standardization[edit]

Petridishes showing zone of inhibition by various extracts like Pet. ether, Chloroform, Ethanol and acetone done by Pharmacist Bijaya Kumar Sahu[2]

Preparation[edit]

All aspects of the Kirby-Bauer procedure are standardized to ensure consistent and accurate results. Because of this, a laboratory must adhere to these standards. The media used in Kirby-Bauer testing must be Mueller-Hinton agar at only 4 mm deep, poured into either 100m or 150mm Petri dishes. The pH level of the agar must be between 7.2 and 7.4.

Inoculation is made with a broth culture diluted to match a 0.5 McFarland turbidity standard, which is roughly equivalent to 150 million cells per mL.

Incubation Procedure[edit]

  1. Using an aseptic technique, place a sterile swab into the broth culture of a specific organism and then gently remove the excess liquid by gently pressing or rotating the swab against the inside of the tube.
  2. Using the swab, streak the Mueller-Hinton agar plate to form a bacterial lawn.
    • To obtain uniform growth, streak the plate with the swab in one direction, rotate the plate 90° and streak the plate again in that direction.
    • Repeat this rotation 3 times.
  3. Allow the plate to dry for approximately 5 minutes.
  4. Use an Antibiotic Disc Dispenser to dispense disks containing specific antibiotics onto the plate.
  5. Using a flame-sterilized forceps, gently press each disc to the agar to ensure that the disc is attached to the agar.
  6. Plates should be incubated overnight at an incubation temperature of 37°C (98.6°F).[3]

References[edit]

  1. ^ Mohanty A et al, Phusiochemical and Antimicrobial Study of polyherbal Pharmacieglobal, 2010 vol 4 (04), page 1-3.
  2. ^ Sahu B K, Antimicrobial properties of Aerial Part of Sesbania grandiflora(Linn.), The Pharmaceutical college Barpali,India,*th semester Project 2013
  3. ^ http://aminj.myweb.uga.edu/KIRBY-BAUER.html