Name: collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment
Uploaded: 2012-11-15T11:55:00
Description: Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

This work was supported by grants from the U.S. National Science Foundation (EAR-0920299 and EAR-0745808); U.S. National Science Foundation East Asian and Pacific Summer Institutes; the Geological Society of America Research Grant Program and the Alumni Grants for Graduate Research and Scholarship from The Ohio State University. We would like to thank the editor and two anonymous reviewers for their insightful comments.

1. MTB Collection
<ol>
	<li>When deciding on a freshwater site to collect magnetotactic bacteria (MTB), it is often best to start with a pond or slow-moving stream that has a soft muddy sediment layer. In this demonstration we collected a sample at the edge of the Olentangy River on the campus of The Ohio State University (OSU) in Columbus, Ohio (USA). While this was a convenient location for our demonstration, the protocol described here is applicable to any aquatic location. The materials used in this protocol can be...

<ol>
	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. T., Menguy, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cultured+Greigite-Producing+Magnetotactic+Bacterium+in+a+Novel+Group+of+Sulfate-Reducing+Bacteria.">A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria.</a> Science. 334, 1720-1723 (2011).</li><li>Simmons, S. L., Bazylinski, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

Magnetotactic bacteria are not necessarily found in every aquatic environment8 but when they do occur, they can be found on the order of 100 - 1,000 cells per milliliter2. In order to observe the MTB using optical microscopy, you will need approximately 50 bacteria/ml in your sample8. If there are no or few MTB in your sample, then you will either need to select a new environmental site to collect your samples or you will need to try one or more of the techniques discussed in the next sec...

<table border="1">
 <tbody>
 <tr>
 <td>Item Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Glass slides</td>
 <td>Fisher Scientific</td>
 <td>S95933 </td>
 <td></td>
 </tr>
 <tr>
 <td>Glass Pasteur pipets</td>
 <td>Fisher Scientific</td>
 <td>13-678-6A</td>
 <td></td>
 </tr>
 <tr>
 <td>O-ring</td>
 <td>Hardware store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Cover slips</td>
 <td>Fisher Scientific</td>
 <td>12-542B</td>
 <td></td>
 </tr>
 <tr>
 <td>Bar magnet</td>
 <td>Fisher Scientific</td>
 <td>S95957</td>
 <td></td>
 </tr>
 <tr>
 <td>Container</td>
 <td>Any</td>
 <td></td>
 <td>Any plastic or glass container that can hold at least 0.5 L and can be sealed</td>
 </tr>
 <tr>
 <td>Cotton</td>
 <td>Any</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope with 60X dry lens</td>
 <td>Zeiss</td>
 <td></td>
 <td>A 60X dry lens is not absolutely necessary, but this gives a high NA without using oil</td>
 </tr>
 <tr>
 <td>Diamond pen</td>
 <td>Fisher Scientific</td>
 <td>08-675</td>
 <td></td>
 </tr>
 <tr>
 <td>0.22 mm filter</td>
 <td>Fisher Scientific</td>
 <td>09-719C</td>
 <td></td>
 </tr>
 <tr>
 <td>1 ml syringe</td>
 <td>Fisher Scientific</td>
 <td>NC9788564</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge tubes</td>
 <td>Fisher Scientific</td>
 <td>02-681-320</td>
 <td></td>
 </tr>
 <tr>
 <td>Formvar/Carbon 200 mesh, copper grids</td>
 <td>Ted Pella Inc.</td>
 <td>01800</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Ted Pella Inc.</td>
 <td>19481</td>
 <td></td>
 </tr>
 <tr>
 <td>Tecnai Spirit TEM</td>
 <td>FEI</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tecnai F20 S/TEM</td>
 <td>FEI</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

A magnet is an effective tool that can be used to isolate magnetotactic bacteria (MTB) contained in environmental samples (Figure 1A). A capillary racetrack (Figure 1B) uses the magnetic properties of MTB to attract them through a cotton plug where they can be separated from non-magnetotactic microorganisms also contained within the environmental sample.
<img alt="Figure 1" src="/files/ftp_upload/50123/50123fig1.jpg" /><b...

Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Research

JoVE Journal

Biology

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...