EM Viewing of Magnetotactic Bacteria

Introduction

1. Magnetotactic Bacteria

Magnetotactic Bacteria (MTB) belong to a lineage of prokaryotic bacteria which are able to passively orient themselves with magnetic field lines. This ability is called magnetotaxis. [1] The reason that MTB are able to exhibit magnetotaxis is because they synthesize magnetic particles, called magnetosomes, which align in chains. These crystals are usually composed of either magnetite or greigite. [2]

Besides observing magnetotaxis through light microscopy, a diagnostic way of determining whether or not a bacterium is an MTB is to observe magnetosomes in situ. In order to do this Transmission Electron Microscopy (TEM) is utilized. By using TEM magnetosomes can be identified and crystal characteristics such as morphology, composition, crystallographic structure, etc. can be determined regarding the magnetosomes. [3,4,5].

Sample material was previously fixed within solid resin blocks following a routine method for microtomy. These blocks were then sliced via an ultramicrotome to approximately 100 nanometer thicknesses. Resulting slices were distrbuted onto TEM grids for observation.

Selected sample material was preserved and prepared for TEM observation via HMDS techniques. To begin the process the sample was first fixed, after distributing onto TEM grids, using gluteraldehyde. Water in the sample was then replaced through a series of graded ethanol washes (95 and 100 percent ethanol). After the water was replaced, the samples were then placed under a fumehood and dried using HMDS.

3. Bright Field

Figure 1: Bright Field TEM micrographs, A-F (Clockwise from top left image). (A) Four nanoparticles prepared via microtome. (B) Two nanoparticles prepared via microtome. (C) Dipplococcus MTB preserved and prepared via HMDS, reference to image D. (D) Higher magnification micrograph of the magnetosome chain from image C. (E) Dipplococcus MTB preserved and prepared via HMDS, reference to image F. (F) Higher magnification micrograph of the magnetosome chain from image E.

4.Dark Field

Figure 2: Bright field micrograph (left, A) and Dark Field micrograph (right, B) of the same vibrio MTB.

5.EDS

Figure 3: Bright field micrograph (A) highlighting a few of the magnetosome crystals from Figure 1E which were selected for elemental analysis (B).

6.X-ray Diffraction

Figure 4: Bright Field micrograph highlighting a few of the magnetosome crystals from Figure 1E which were selected for x-ray diffraction.

7.ImageJ

Figure 5: Dark Field micrograph (A) highlighting the magnetosome chain from Figure 2. The chain was selected for particle analysis via ImageJ. (B) Threshold and watershed adjusted image. (C) Count map of particles within the chain.

Samples prepared by microtomy (Figures 1A and 1B) were unstable and difficult to image. Samples prepared via HMDS proved successful for imaging (Figures 1C-1F). Specimens such as in Figures 1E and 2 exhibit in-tact bacterial cells. Figure 1C shows that some samples may not have preserved well during the drying process, however.
Magnetosomes were imaged in situ for three MTB cells (Figures 1C, 1E, and 2) through bright field imaging. Dark field imaging was also utilized on selected specimens (Figure 2B). EDS was successful on targeted particles within one specimen (Figure 3). The spectra indicate high levels of Fe and Ca. A few other elements are also present (such as C, Cu, O, P, Mg, and S) likely due to preparation techniques and the presence of a biotic organism.
Further analysis was performed on the magnetosome chains. X-ray diffraction was performed on a selected area of one chain in particular (Figure 4). The diffraction pattern is indicative of crystals with a uniform composition. Magnetosome particles were also analyzed with ImageJ in which a particle count was performed. Total number of magnetosomes were determined (n=11) for one specimen (Figure 5).

I would like to acknowledge Brian McIntyre. He has taught me many techniques involving electron microscopy and has served as an excellent troubleshooting partner for this project. Also, I would like to acknowledge my senior thesis advisor, Dr. John Tarduno, for allowing me to work on this amazing project.

[1]Bazylinski, D and Frankel R. 2004. Magnetosome formation in prokaryotes. Nat. Rev. Microbiol, 2, 217-230.

[2]Pan, Y et al.. 2005. Rock magnetic properties of uncultured magnetotactic bacteria. Earth and Planetary Science Letters, 237, 311-325.

[3]Lefevre, C et al.. 2011. A bacterial backbone: magnetosomes in magnetotactic bacteria. In: Metal nanoparticles in microbiology, Springer-Verlag. Berlin, 75-102.
[4]Pósfai, M et al.. 2013. Biominerals at the nanoscale: transmission electron microscopy methods for studying the special properties of biominerals, in Minerals at the Nanoscale, EMU Notes in Mineralogy, Nieto, F and K Livi (Ed), (London: European Mineralogical Union and Mineralogical Society of Great Britain and Ireland), 377435.
[5]Devouard, B et al.. 1998. Magnetite from Magnetotactic Bacteria: Size Distributions and Twinning. American Mineralogist, 83, 1387-1398.