2.8. Primary screening of isolates
During the primary screening, isolates were screened against selected bacterial strains by using perpendicular streak and agar overlay methods. Antagonistic activity of isolates against Gram negative and Gram positive bacteria was primarily screened by using perpendicular streak method and later evaluated by agar overlay method[28],[30]. In perpendicular streak method, nutrient agar media was used and each plate was streaked with individual isolates at the center/diameter of the plate and incubated at 30 °C for 7 d. Later, 24 h fresh sub-cultured test bacteria were prepared and streaked perpendicular to the isolates and incubated at 37 °C for 24 h[30]. In agar overlay method, 5-7 d old colony of actinomycetes was spot inoculated on nutrient agar plates and incubated for 7 d at 30 °C. Then overlaid with 5 mL of semisolid nutrient agar with standardized suspension of test bacteria was inoculated and incubated at 37 °C for 24 h. The zone of inhibition (in mm) around the colonies was measured