SEM Visualization of the Lyme Producing Bacterium, Borrelia
burgdorferi
Charles Worley
Department of Biology
East Carolina University
Greenville, North Carolina 27858
[email protected]
By Charles Worley
Spring 2009 Scanning Electron Microscopy Course Project
Summary
RESULTS AND DISCUSSION
Borrelia burgdorferi
Electron microscopy is a powerful tool which has led to numerous
breakthroughs in biological research. While it has contributed to many areas of
research the area of interest to my project was visualization of Borrelia
burgdorferi, the causative organism of Lyme disease, using Scanning Electron
Microscopy (SEM). A significant human pathogen close range visual analysis
of the organism can provide important clues on the roles of certain genes in
motility and pathogenicity. Understanding the role of these genes in turn can
lead to potential improvements in detection and treatment of patients suffering
from Lyme disease. However, the preparation and visualization of
microorganisms pushes the abilities of Transmission Electron Microscopy and
Scanning Electron Microscopy (SEM) to their limit. For this reason well
designed protocols have developed over time for studying such small fragile
specimens.
SEM was successful however a significant number of specimens, were lost or
distorted (loss of spiral structure) by the preparatory procedures. Greater care and
caution reduced this loss dramatically, nonetheless some distorted specimens
remained (Figure 7 distorted, Figures 1-6 normal cells.). Future preparations can
include Environmental SEM (ESEM) in which cells may or may not be preserved and
are not dehydrated. This potentially involves the least amount of specimen
preparation and may result in minimal artifacts. This class project involved SEM which
is quite useful for examining whole spirochetes, visualization of flagella for future
studies might best include TEM negative staining as well as ESEM.
INTRODUCTION
B. burgdorferi is an unusual bacterium responsible for causing
Lyme disease. Unlike most bacteria which generally tend to fall on a
continuum between cocci (spherically shaped) and bacillus (rod shaped)
B. burgdorferi is a spirochete, a bacterium with an unusual wave or spiral
body plan. Other medically relevant spirochetes include Treponema
pallidum, which causes syphilis, and Treponema dentolitica, which is a
cause of periodontal (gum) disease. This unusual body plan is due to a
unique quality of these organisms’ flagella.
Bacteria move and swim, typically, by employing one of two
structures. They may use small hairs called cilia or one or more flagella.
The flagella is a tail like structure, a good example of which is the ‘tail’ of a
sperm cell. In spirochetes, unlike other bacteria, the flagella are internal
and the tension they produce on the outer surface of the cell causes the
distinctive shape of these organisms. Swimming is achieved by using the
flagella to generate a wave like motion which permits movement in very
low viscosity solutions.
Motility is required for pathogenicity of B. burgdorferi. Thus it is
important medically to understand how the various proteins form and
regulate the formation of the flagella. Creating mutations in these genes
is an important means of studying their function. Because mutations
affecting flagella construction could produce visible changes in both the
flagella and the cell structure, electron microscopy is a useful tool in
evaluating the effects of some of these mutants. The purpose of this
study is to develop a reliable method for SEM examination of B.
burgdorferi so that we can routinely and accurately compare wild-type
with mutant strains.
Figure 2.
Figure 3.
Figure 7.
Figure Legend and References
Figure 1. A typical specimen of B. burgdorferi.
Figure 2. A field image showing the rough density of B. burgdorferi after this
preparation.
Figure 3. Two B. burgdorferi intertwined, the lower specimen is at the organism’s
standard length while the top organism is nearly ready to divide
Figure 4. A specimen of B. burgdorferi imaged at a relatively high magnification.
Figure 5. A grouping of B. burgdorferi fixed on top of some debris.
Figure 6. B. burgdorferi at times associate with one another forming collections or
clumps, this is a particularly large example.
Figure 7. A badly distorted specimen.
•Magnifications have been blanked due to the fact that enlargement of the image onto
poster board increased the overall magnification beyond the recorded value.
Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions.
Mohammed Abdul Motaleb, Linda Corum, James L. Bono, Abdallah F. Elias, Patricia
Rosa, D. Scott Samuels, and Nyles W. Charon. PNAS September 26, 2000 vol. 97 no.
20 10899-10904
Figure 1.
ACKNOWLEDGEMENTS
METHODS
Figure 4.
I would like to thank Dr. Thomas Fink and Dr. MD Motaleb for their
insight and assistance in advising me during this project.
Preparation: Poly-l-lysine-coated Thermanox coverslips were
covered in a few drops (approximately 50-150ul) of BSK containing late
log phase B. burgdorferi for one hour. Following a gentle wash with
Sorenson’s phosphate buffer (0.15M, pH 7.3) specimens were fixed with
2.5% glutaraldehyde (in same buffer) for one hour. Following three
washes with buffer, specimens received a secondary fixation with 1%
osmium tetroxide in buffer for one hour, and washed three times in buffer.
Specimens were then dried with increasing concentrations of ethanol up
to 100% concentration and treated three times with
hexamethyldisilazane. Each drying step lasted ten minutes. Remaining
solution was pipetted off and the coverslip was allowed to dry at room
temperature in a fume hood inside a Petri dish (to protect from dust).
Great care was taken at all steps due to the delicacy of the specimen.
Specimens were sputter coated with gold/palladium prior to examination.
Spirochetes were examined at 30KV spot size 4 under high vacuum
conditions. They were detected at 400x-600x magnification and
examined at magnifications beyond 600x.
Figure 5.
Figure 6.