Characteristics

Source

Isolation

Screening

Identification

For the enrichment phase, the method used was soil enrichment method.

1. The cells from overnight culture had been collected for centrifugation.
   

1) 200 ml of sterile mineral salt media, MSM was added to a 500 ml Erlenmeyer flask along with 10 g soil/10 ml water samples that are oily polluted. Next, 2 ml (1%) v/v of crude oil was put into the mixture.

2) The mixture was then incubated for at 30 °C for seven days in an orbital shaker operating at 200 rpm. After that, several subculturings was carried out on the same media with 1% crude oil each for 7 days.

2. Next, the genomic DNA was isolated by a modified method of Sambrook

3. Amplification was carried out using prokaryotes 16S rRNA with a specific forward primer: 50-AGAGTTTGATCMTGGCTCAG-30 and reverse primer: 50-TACGGYACCTTGTTACGACTT-30.

4. The product from PCR was sequenced using the similar PCR primers.

Biotechnological Importance of microbe

5.The DNA similarities, multiple sequence alignment and molecular phylogeny were assessed using Blast program such as ncbi.

3) The cultures' serial dilutions were prepared. A sample was then spread onto two sets of duplicate MSM oil agar plates with 1% (v/v) crude oil as a substrate. NaCl with concentration of 2% was used for the growth of marine bacteria

1. Gravimetric analysis was done in order to find out individual quantitative estimation of oil degradation by collected isolates.
   

2. In each flask, 2% (v/v) of MSM that was supplemented with 1% (v/v) of initially sterilized crude oil was added to pre-culture inoculate suspensions (24 h growth)

3. The control experiment was carried out without the addition of isolates and the flasks were then incubated at temperature of 30 °C for six days. The incubation step took place in an orbital shaker that operated at 200 rpm.

4) The oil agar plated were the incubated at 30 °C for three days.

5) The colonies formed on the oil agar plates were randomly picked and underwent repeated sub-culturing on nutrient agar. This is in order to obtain pure isolates.