Microorganism
Microorganisms used in this study, Achromobacter agilis, Pseudomonas fluorescens, Bacillus thuringiensis, Staphylococcus lentus were sourced from rhizobacterial flora of weeds harvested from aged crude oil impacted soil in Bodo, Gokana, Ogoni land of Rivers state, Nigeria (36° 4’N and 15° 7’E). The strains were isolated in the environmental biotechnology laboratory of the Department of Microbiology of the University of Port Harcourt, Rivers State, Nigeria. The total bacterial population in the oil-polluted rhizosphere soil sample was enumerated and isolated adopting serial dilution and the standard plate count technique using the pour plate method ( Ajayi & Abiola, 2018). Ten grams of the soil sample was measured into a conical flask and 90ml of sterile normal saline was mixed with the sample. The suspension was subjected to a shaker for three hours to homogenize the solution and this served as the stock solution. Ten-fold serial dilution of all the homogenized mixture was carried out using sterile normal saline as diluents. Seven test tubes containing 9ml of normal saline were used for the serial dilution. Aliquots of 1ml from 10 -5 and 10 -7 dilutions were introduced into duplicate sterile petri dishes and 20ml of molten nutrient agar incorporated with nystatin (N6261, Sigma-Aldrich) to suppress fungal growth was poured into the plates and swirled to allow homogenization. The plates were incubated at 37°C for 24 hours after which colonies on the plates were enumerated and subculturing of bacterial isolates was done to obtain a pure culture. Bacterial colonies were picked with a sterile inoculating loop and streaked on freshly prepared nutrient agar plates ( Olukunle, 2013). The plates were incubated at 37 °C for 24 hrs. Aliquots of 1ml from dilutions of 10 -5 and 10 -7 were also plated in duplicates on Bushnell Haas Agar (Lab M, China), using the spread plate technique; 100 µgml -1 Nystatin (N6261, Sigma-Aldrich) were added to the Bushnell Haas Agar (Lab M, China) to suppress fungal growth. A filter paper saturated with sterile crude oil was aseptically placed on the inside of the inverted Petri dishes and the culture plates were incubated for 14 days at 37°C. Plates containing colonies were afterwards enumerated for the bacterial load ( Ajayi & Abiola, 2018).