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 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.
1. MTB Collection
2. MTB Isolation
3. MTB Racetrack
4. MTB Enrichment
5. MTB Observation by Light Microscopy
6. MTB Observation by Transmission Electron Microscopy (TEM)
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.
Figure 1. A clear plastic bottle containing a sediment and water sample collected from the Olentangy River in Columbus, Ohio (USA). The bottle contains approximately one-half sediment and one-half water. The south end of a magnet is placed approximately 1 cm above the sediment for up to several hours (A). After removing some of the fluid from near the magnet on the inside the container, it is placed inside of a capillary racetrack where the MTB swim through a cotton plug (arrow) towards the south end of a bar magnet (B). A close up view of the capillary racetrack showing the sample, cotton, filtered fluid, sealed end of the capillary tube and south end of a bar magnet (C).
Figure 2. Once the MTB have been enriched from the racetrack, a small drop can be placed on a coverslip, which is then flipped upside down and placed on an o-ring that is resting on a slide. This slide-o-ring-coverslip sandwich can be placed on a light microscope stage and viewed using a 60X dry objective (oil lenses are inconvenient to use with a hanging drop).
Figure 3. Bright field microscope image of MTB swimming (thin long arrows) and gathering at the edge of the hanging drop (short arrows) which is next to the south pole of a bar magnet.
Figure 4. Transmission electron microscope image of a single magnetotactic bacterium enriched from an environmental sediment sample. The morphology of the cell (spirillum) and magnetosomes are clearly visible along with a single flagellum.
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 section.
First, you should try collecting more sediment from the environment using a large plastic tub8. This is especially useful if large numbers of unculturable MTB are needed. Depending on the environmental sample, it may not be possible to isolate MTB samples having a concentration of 50 bacteria/ml immediately after collecting the sample. Therefore, when you bring your environmental sample back to the laboratory, it may be beneficial to wait for the sample to acclimate to laboratory conditions before trying to isolate the MTB using a bar magnet. This acclimation period will allow the bacterial community to mature and repopulate the culture leading to higher concentrations of MTB. Another simple technique that often produces more concentrated MTB samples is to leave the bar magnet on the side of the sample container (Figure 1A) for a longer period of time (e.g., several days). This should allow the MTB more time to migrate to the magnet. One final technique that may be useful, is to use several racetracks (Figure 1B) at once and then combine the MTB from each racetrack into one sample. If you believe there is a problem with a racetrack or if there are too many contaminating microorganisms (i.e., non-MTB) in your enriched sample, you can place the racetrack under a light microscope to observe the MTB as they swim through the cotton plug and into the tip. This will allow you to determine if contaminating microorganisms are also coming through the cotton plug and when to stop the enrichment process.
We should mention that there are more sophisticated ways to isolate MTB, but these methods require the use of more specialized equipment. One example involves the use of a magnetic coil, instead of a bar magnet, and customized glass vessels to isolate MTB from freshwater sediments10, 11. The protocol described here represents an inexpensive and effective method for determining whether an environmental site contains MTB. This isolation and enrichment protocol is straightforward enough that microbiology students can master and easily “fine-tune” so that higher yields of MTB can be achieved. Once the MTB have been isolated, other analyses such as fluorescence in-situ hybridization, 16S rRNA sequencing for community analysis, energy dispersive spectroscopy (EDS), TEM, optical microscopy and magnetic measurements can be conducted on the MTB12, 13, 14.
The authors have nothing to disclose.
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.
Item Name | Company | Catalogue number | Comments (optional) |
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 |