Factors such as the volume of extract placed on the paper disk, the thickness of the agar layer and whether a solvent is used vary considerable between studies. On the other hand in agar well test is a quantitative method in which the extract is deposited into well cut into the agar and can be used with large number of extracts and/or large number of bacterial isolates (Dorman and Deans, 2000). To make bacterial growth easier to visualize, triphenyl tetrazolium chloride may also added to the growth medium (Elgayyar et al, 2001; Mourey and Canillac, 2002). Microorganisms are generally termed susceptible, intermediate, or resistance, depending upon the diameter of the inhibition zone. Quantitative results are possible with a high degree of standardization. This method should not be used for anaerobic microorganisms.

The disk diffusion method (also known the zone of inhibition method) is probably the most widely used method because of its simplicity and low cost. It uses only small amounts of the test substance (10-30 цЬ), can be completed by research staff with minimal training (plate-1). The method involves the preparation of a Petridish containing 15-25 mL agar, bacteria at a known concentration are then spread across the agar surface and allowed to establish.

Plate 1: Photograph of disc diffusion method
a. Photograph with different zone of inhibition
b. Zone of inhibition of essential oil against Shigella species
c. Zone of inhibition of ethanol extract (3), positive control antibiotic-ciprofloxacin (1), negative control-ethanol (2) against Pseudomonas aeruginosa