Computational protocol: Coliform Bacteria for Bioremediation of Waste Hydrocarbons

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Protocol publication

[…] Three local raw domestic sewage samples were collected from Al-Jahra station and used to count and isolate their indigenous coliforms. The conventional dilution-plating method was adopted, and the coliform-selective medium, Eosin Methylene Blue Agar (EMB) [, ], was used. The coliform colonies were counted, subcultured, and purified on the above selective medium. Individual coliforms were also counted after they had been differentiated according to their characteristic morphologies on this medium; Enterobacter colonies were pink with dark centers, Klebsiella colonies mucoid pink, and Escherichia coli colonies black with green metallic sheen The isolates were further characterized according to their potential for acid and gas production from lactose fermentation at 37°C and 44°C, following established routine procedures. Representative strains were isolated, purified, and characterized by comparing the sequences of their 16S rRNA-genes with sequences of type strains in the GenBank database. The method was essentially as follows: The total genomic DNA was extracted with PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, USA) following the manufacturer's description. The 16S rRNA-genes were amplified by polymerase chain reaction (PCR) using a Veriti Thermal Cycler (Applied Biosystems, USA) and a standard protocol with initial denaturation at 95°C for 5 min and 32 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and primer extension at 72°C for 1 min. The mixture PCR contained puReTaq Ready-To-Go PCR Beads (Amersham Biosciences, UK), 25 ng of DNA template, 1 μL of each of the universal primer combinations GM5F (5′-CCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCAATTCMTTTGAGTTT-3′) []. The reaction volume was completed to 25 μL with molecular water (Sigma, UK). Partial sequencing of the 16S rRNA-genes was done using a BigDye version Terminator Kit (Applied Biosystems, USA). The mixture contained 20 ng of the DNA template, 2 µl of the BigDye version 3.1 terminator, 2 µl of the 5x sequencing buffer, l μL of either 907R, or GM5F and the final volume was brought up to 10 μL with molecular water. Labeling was completed in the Veriti Thermal Cycler (Applied Biosystems, USA) using one cycle of 96°C for l min then 25 cycles of l min at 96°C, 5 s at 50°C, and 4 min at 60°C. The pure template DNA samples were processed in a 3130xl genetic analyzer (Applied Biosystems, USA). Sequencing analysis version 5.2 software (Applied Biosystems, USA) was used to analyze the results. Sequences were subjected to basic local alignment search tool analysis with the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) GenBank database []. A phylogenetic tree was constructed using neighbor joining including bootstrap analysis using PAUP∗ v.4 []. Bootstrap proportions were used on 2000 replicates. […]

Pipeline specifications

Software tools BLASTN, PAUP*
Application Phylogenetics
Organisms Escherichia coli
Diseases Klebsiella Infections
Chemicals Acetyl Coenzyme A, Alcohols, Aldehydes, Carbon, Fatty Acids, Hydrocarbons, Hydrocarbons, Aromatic, Toluene