Selective Enrichment and Isolation of Oil-Degrading Marine Bacteria from Seawater Samples

Abstract

Source: Villela, H. D. M., et al. Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation. J. Vis. Exp. (2019)

This video demonstrates the selective isolation of oil-degrading marine bacteria by enriching seawater samples on oil-based media, enabling the recovery of pure strains with potential applications in crude oil bioremediation and environmental cleanup.

Protocol

1. Water and coral collection and storage for microbial isolation

NOTE: It is essential to take the coordinates and temperature of the sampling sites. If possible, metadata such as salinity, pH, depth, and light intensity can also help in finding fine-tuned cultivation approaches and future interpretation of data. For reliable results, keep the samples stored for the minimum length of time possible. The water/coral microbiomes may change considerably if the samples are not kept at the right temperature and/or are stored for long periods. If the isolation step is not performed instantly after collection, it is crucial to maintain samples at 4 °C....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
500 mL PYREX Media Storage Bottle	thomas scientific/Corning	1743E20/1395-500	Used to sample water.
500 mL Aspirator Bottles	thomas scientific/Corning	1234B28/1220-2X	Used to separate the oil fractions.
6-inch wire cutter plier	thomas scientific/Restek	1173Y64/23033	Used to cut coral fragments.
Agar	Himedia	PCT0901-1KG	Used to make solid media.
Bushnell Haas Broth	Himedia	M350-500G	Used as minimum media to be supplemented with carbon sources.
Erlenmeyer Flask	thomas scientific/DWK Life Sciences (Kimble)	4882H35/26500-125	Used to incubate coral macerate with glass beads.
Laminar Flow Hood			Needed to work at sterile conditions.
Luria Bertani Broth, Miller (Miller Luria Bertani Broth)	Himedia	M1245-1KG	Used as rich media to grow bacteria.
Marine Agar 2216 (Zobell Marine Agar)	Himedia	M384-500G	Used as rich media to grow bacteria.
Orbital-Shaker Incubator			Used to incubate liquid media and oil.
Plates Incubator			Used to incubate plates.
Porcelain Mortar and Pestle	Thomas scientific/United Scientific Supplies	1201U69/JMD150	Used to macerate coral fragments.
Refrigerated Centrifuge			Used to centrifuge bacterial cultures.
Spectrophotometer			Used to measure optical density of bacterial cultures.
Wizard Genomic DNA Purification kit	Promega	A1120	Used for microbial strains DNA extraction.