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

Summary

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.

Abstract

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 both^{3, 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 magnet^3,5. 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.

Protocol

1. MTB Collection

1. 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 found in Table 1. Find a location where the depth of the water is between 10 and 100 cm. At such a location, you should collect the ....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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