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2.3.2. Agar plug diffusion methodAgar plug diffusion method is often used to high light the antagonism between microorganisms [34,35], and the procedure is similar to that used in the disk-diffusion method.It involves making an agar culture of the strain of interest on its appropriate culture medium by tight streaks on the plate surface.During their growth , microbial cells secrete molecules which diffuse in the agar medium. After incubation,an agar-plot or cylinder is cut aseptically with a sterile cork borer and deposited on the agar surface of another plate previously inoculated by the test microorganism. The substances diffuse from the plug to the agar medium.Then, the antimicrobial activity of the microbial secreted molecules is detected by the appearance of the inhibition zone around the agar plug (Fig. 1C).2.3.3. Cross streak methodCross streak method is used to rapidly screen microorganisms for antagonism [36]. The microbial strain of interest is seeded by a single streak in the center of the agar plate.After an incubation period depending upon the microbial strain,the plate is seeded with the microorganisms tested by single streak perpendicular to the central streak . After further incubation,the antimicrobial interactions are analyzed by observing the inhibition zone size.2.3.4. Poisoned food methodPoisoned food method is mostly used to evaluate the antifungal effect against molds [37–39]. The anti fungal agent or th eextract is in corporated into the moltenagar at a desired final concentration and mixed well.Then,the medium is poured into Petridishes. After over night preincubation,the inoculation can be done by a my celi a disc ranging from 2 to 5 mm , which is deposited in the center of the plate. After further incubation under suitable conditions for the fungal strain tested,the diameter sof fungal growth in control and sample plates are measured ,and the anti fungal effect is estimated by the following formula:
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