2.3. Isolation and Purification

The treated soil samples were suspended in 0.9% sterile saline solution and serially diluted until 10⁻⁵ [19]. Aliquots of 100 µL from the last three dilutions were plated on three isolation media supplemented with nystatin (50 mg/L) as antifungal agent [20]. The media used were actinomycete isolation agar (AIA), Emerson's agar (EA), and soybean casein digest agar (SCDA) [21–23]. The plates were incubated at 28°C for up to 14 d to isolate slow growing actinomycetes. Plates were observed daily for bacterial colonies exhibiting typical features of actinomycetes. Colonies that appeared dry, with or without pigments, and with aerial or substrate mycelium were considered as putative actinomycetes [24]. Selected colonies were purified by three-way streak method and were maintained in agar slants of their respective isolation medium [25].