Microbial Metabolite Production Screening

Indole acetic acid (IAA) production by bacterial isolates was determined in LB broth supplemented with L-Tryptophan (500 μg/mL) at 24, 48, and 72 h as described by Patten and Glick [19 (link)]. For this, bacterial cells were removed by centrifugation at 10,000 rpm for 5 min at 4°C. One mL of the supernatant was mixed with 4 mL of Salkowski's reagent in the ratio of 1 : 4 and incubated at room temperature for 20 min. Development of a pink colour indicated indoles. The absorbance of supernatant mixture (supernatant + Salkowski's reagent) for indole production was measured at 530 nm and quantity of indoles was determined by comparison with a standard curve using an IAA standard graph. Siderophore production was determined by using blue indicator dye and chrome azurol S agar [20 (link)]. Bacterial isolates exhibiting orange halo zone on chrome azurol S agar after 5 d of incubation at 28°C were considered positive for the production of siderophores. For hydrogen cyanide (HCN) production the methodology described by Bakker and Schippers [21 ] was used. Isolates were grown on plates of tryptic soy agar (10%), amended with glycine (4.4 g L⁻¹), and FeCl₃·H₂O (0.3 mM). A change from yellow to orange, red, brown, or reddish brown was recorded as an indication of weak, moderate, or strongly cyanogenic potential, respectively. Organic acid production potential of different isolates was analyzed using thin layer chromatography (TLC) on Silica-G (Merck chemicals) gel plates using different solvent systems. Finally, ammonia production test was performed by growing selected isolated in peptone water for 72 h at 30°C. Change in colour after addition of 1 mL Nessler's reagent (K₂HgI₄; 1.4%) in each tube was observed. The presence of faint yellow colour indicates small amount of ammonia and deep yellow to brownish colour indicates maximum ammonia production.