A filter disc is impregnated with a measured amount of antibiotic, and the disc is placed on agar surface.

The disk is placed on an agar surface. The disk will absorb moisture from the agar, effecting the diffusion of antibiotic of antibiotic into the agar medium. The distance that the antibiotic moves outward from the disk depends on the solubility, concentration, and other characteristics of antibiotic. As the antibiotic diffuses outward, a concentration gradient is established with the highest level in the immediate area of the disk.

One would inoculate a liquid growth medium by touching an inoculating loop to a colony of bacteria growing on a primary isolation plate and transferring the inoculum to a liquid medium. The inoculated liquid medium would be incubated and when turbidity is evident, an aliquot would be added to melted agar and poured into a Petri plate. After solidification, antibiotic-containing disks are then placed on the surface of the agar and the plate incubated. After a 12-to 18-hour incubation period, the zone of inhibition can be measured.

The susceptibility of an organism to an antibiotic is directly related to the diameter of the zone of inhibition. The zone diameters can be interpreted by employing a table prepared by Kirby-Bauer. This table was developed by determining the minimum inhibitory concentrations (MIC) of antibiotics effective against many pathogenic bacteria and relating the MIC to the diameter of an inhibition. Minimum inhibitory concentration (MIC) values can also be determined as follows: A series of tubes containing an appropriate medium and graded concentration of antibiotic are inoculated with equivalent amounts of suspension of the clinical isolate. The tubes are examined for growth after a suitable incubation period. The lowest concentration of antibiotic that inhibits growth completely is considered MIC. Reference: Microbiology Book by Tortora; Epidemiology and Clinical Microbiology Antibiotic Sensitivity pp. 757-758

Preparation of the test organism or inoculum Sterilization Techniques Sterilize media in an autoclave for 15 to 20 minutes at 15 psi With each set wrapped in paper, sterilize filter paper disc, pipettes, test tubes with cotton plugs, steel cylinders in an oven for 2 hours at 160 to 170 degree Celsius.

Sterilize forceps, inoculating hook, loop, and needle by dipping in 95% alcohol and flaming these before use.

The broth culture Take loopful of bacteria from the culture slant and inoculate the bacteria in 20ml nutrient broth in a sterilized test tube. Incubate the culture broth for 18-24 hours at 35 degree Celsius Observe the culture broth for turbidity, indicative of microbial growth. Aseptically transfer 5ml of the broth in sterile screw-capped test tube; Agitate the bacterial suspension and immediately compare against the 0.5 McFarland standard earlier prepared; If the bacterial suspension does not appear to be of the same density as the McFarland standard, adjust the turbidity by adding sterile saline solution or culture broth and subsequently compare the resulting turbidity to the standard.

Preparation of the Assay Plates Pour approximately 15ml of melted Nutrient agar or Mueller-Hinton agar into dry and sterile Petri dishes. Moisten a sterile cotton swab into the test organism (inoculum) suspension;

Cotton Swabbing Aseptically swab the test organism onto solidified nutrient agar or Mueller-Hinton agar plate by streaking the swab over the entire surface of the agar plates three time, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculum on the surface of the medium Let the swabbed plats for 5 minutes;

The Paper Disc Diffusion Method Flame sterilize a pair of forceps until the blue alcohol flame disappears. Let cool for a few seconds; With the forceps pick out one paper disc and immerse the paper disc into the plant extract assay; Lay the moistened filter disc gently on the seeded agar plate. Gently tap the disc with forceps to ensure maximum full contact of the disc with the agar medium;

Incubate the plates inverted

Reading the Assay Plates, Documenting the Results, and Analyzing Results Look for halo or clearing around the discs. This is known the zone of inhibition Invert the plates and with the Vernier caliper measure the diameter of each inhibition zone, in millimeters; Express the results in millimeters diameter zone of inhibition Record also the size of the paper disc used in the assay in millimeters. For a 6mm diameter paper disc, the diameter of the zones of inhibition observed and corresponding inferences: <10 mm, may be expressed as INACTIVE 10-13 mm, PARTIALLY ACTIVE 14-19 mm, ACTIVE >19 mm, VERY ACTIVE

Preparation of the Plant Extract for Assay Weigh 40 mg of the crude ethanol plant extract in a previously sterilized 50ml beaker; Add to the extract in the beaker 10 ml of Mueller-Hinton broth; Tween 80 can be added to the broth with a concentration of 0.1% v/v (0.1 ml Tween 80 in 100ml broth added before autoclaving) to enhance solubility of water immiscible components of the plant; Swirl the contents in the aluminum capped beaker to effect an even homogenization of the extract and the broth; Transfer the suspension into a sterile regular- sized test tube and vortex the contents for 15 seconds. In the absence of a vortex mixer, roll the tube rapidly between both palms for 30 seconds. A 1.0 ml portion of this suspension/mixture contains 4mg or 4,000 ug of the plant extract.

Preparation of Bacteria Streak the bacterial test organism in nutrient agar plate; Incubate at 35 degree Celsius for 24 hours; Inoculate a loopful of the organism into 5ml Mueller-Hinton Broth; Adjust the concentration corresponding to 0.5 McFarland standardized inoculum to a volume of 20ml with Mueller-Hinton broth; Use this inoculum immediately in the determination of the MIC of the plant extracts against bacteria.

Determination of Minimal Inhibitory Concentration (MIC) Sterilize 13 screw capped test tubes (13mmx100mm) and number each tube accordingly; With a sterile 5 ml serological pipette, introduce 1 ml of Mueller-Hinton broth in tubes #2 to #11. To tube #12, introduce 2ml of the Mueller-Hinton broth; Pipette 1 ml of the plant extract into tube #1 and #2, cap the tubes well; Shake gently the contents of tube #2 for 5 seconds With a sterile 1 ml serological pipette, aseptically withdraw 1 ml from the contents of the tube #2 and transfer this to tube #3, cap the tube Shake the content of tube #3; With another clean sterile 1ml serological pipette, aseptically withdraw 1 ml of the contents of tube #3 and transfer this to tube #4, cap the tube. Shake the contents of tube #4; Continue this process until 1 ml has been withdrawn from tube #9 and subsequently added to tube #10, cap the tube. Shake the contents of tube #10 Pipette off 1 ml from the contents of tube #10 and discards this. Introduce 1ml of the diluted bacterial inoculum into tubes #1 to #11 and to tube #13; To tube #13, add 1 ml antibiotic standard. With all the tube tightly capped, gently shake the contents of the tube; Incubate the tubes at 35 degree Celsius for 18 to 24 hours; After the incubation period, examine the tubes for bacterial growth. This can be visible as turbidity in the tube or whitish pellet at the bottom of the tube; The tube with the lowest concentration of plant extracts at which no growth or turbidity is observed s reported as the MIC of the plant extract against bacterial test organism.

Reference: A Guidebook to Plant Screening: Phytochemical and Biological; Revised Edition 2005 edited by Beatrice Q. Guevarra; pp 66-85