Journal of Nature and Science, Vol.1, No.

6, e116, 2015

Microbiology

Effect of Water Washing Purification on the Surface Morphology of

Bacillus cereus Spores
1
2
1
2
1,*
Jessica M. Goss , Eric J. McCullough , Cristina Stanciu , Vamsi K. Yadavalli , Christopher J. Ehrhardt
1

Department of Forensic Science, Virginia Commonwealth University, Richmond, VA 23284, USA. Department of Chemical and Life Science
Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA

and the nanoscale texture of individual Bacillus spores (9), this has
not been explicitly tested across multiple strains and a relevant
range of wash steps.
Therefore, the goal of this study is to investigate how the surface
morphology of Bacillus cereus spore surfaces is affected by the
number of water washing steps used to clean the spore culture after
harvesting. We use atomic force microscopy (AFM) as a tool to
characterize the nano- and microscale topography of spores that
have undergone different processing histories after laboratory
culturing. AFM is particularly well suited for analyzing spore
surfaces because it allows for high precision imaging and real-time
measurement of cells without complex or destructive sample
preparation steps (10, 11). Additionally, in comparison to other
types of high resolution microscopic techniques (e.g., electron
microscopy), morphological imaging with the AFM can be
performed in conjunction with nanomechanical and biochemical
analysis of the cell surface (12, 13). In this study, the
nanomechanical properties of single spore cells were characterized
before and after washing to further elucidate physical changes that
occur with water washing purification of Bacillus spore cultures.

The architecture of spore surfaces plays an important role in the

pathogenesis and natural ecology of many Bacillus organisms.
However, changes to the laboratory preparation method for
Bacillus spores may be a source of phenotypic variability in cell
surfaces that can affect our understanding of in situ morphology
and make cross-study comparisons more difficult. In this work,
we examined how variation in the number of water washing
steps during spore purification influenced the three-dimensional
morphology of Bacillus cereus spore surfaces. Two strains of B.
cereus, str. 14579 and T-strain, were cultured in liquid medium
and purified using 0, 1, 3, or 5 water washing steps. Results
showed that the nanoscale morphology of the spore surface is
affected by the number of water washes such that early wash
step samples had higher levels of roughness, quantified with a
Root Mean Square (RMS) algorithm, which decreased with
successive washes. The presence of large (>200 nm) surficial
features, representing cell fragments and/or debris from the
culture, was also inversely correlated with the number of
washes. The presence of cellular debris in unwashed spore
samples also changed the nanomechanical signatures of the
outermost surface but did not affect the properties of the
underlying cell. This suggests that variation in purification
method can affect the nanoscale morphology and mechanics
and should be considered when analyzing micro- to nano-scale
surface properties of Bacillus cereus spores. Journal of Nature
and Science, 1(6):e116, 2015

Experimental
Spore culture and preparation
Two different strains of Bacillus cereus were used for all
experiments: Bacillus cereus T-strain (BcT) which was donated by
the FBI Laboratorys Counterterrorism and Forensic Science
Research Unit (Quantico, VA) and Bacillus cereus 14579
(ATCC#14579, Manassas, VA). B. cereus was chosen because it is
biochemically and structurally similar to Bacillus anthracis (14, 15)
and can be manipulated at Biosafety Level 1. All cultures of
Bacillus cereus were maintained at 30C on Trypticase Soy Agar
(TSA) (30 g Trypticase soy broth (Becton Dickinson, Franklin
Lakes, NJ), 15 g agar (American BioAnalytical, Natick, MA)).
Broth starter cultures were made by picking single colonies of
Bacillus cereus from TSA medium and inoculating into 125 mL of
Trypticase Soy Broth (TSB). Starter cultures were incubated for
~16-18 hours at 30C and 225 rpm. To induce sporulation, either 1
mL or 20 mL of starter culture (Bc14579 and BcT, respectively)
were added to 250 mL of sporulation medium. Preliminary
experiments were initially conducted with Bacillus cereus spores
prepared in four different medium recipes, G Medium (16),
GPep (G Medium supplemented with 8g-1 of meat Peptone),
GTryp (G Medium supplemented with 8gL-1 Tryptone), and
GBHI (G Medium supplemented with 8 gL-1 Brain Heart
Infusions). AFM analysis of various individual spores from each
medium showed that variation in the complex nutrient source
(peptone, tryptone, brain-heart, etc.) had no discernible effect on
the surface morphology of the spore (data not shown). Therefore,
only GTryp medium formulation was used for this study.
Sporulation cultures were incubated at 30 C and 300 rpm in an
orbital shaker. The cultures were monitored throughout the
incubation period and were harvested when the proportion of
spores in the cell population reached 90 %. Cells were typically
harvested between 24 and 48 hours depending on the strain.

Bacillus cereus | spore | atomic force microscopy | nanomechanical

Phenotypic profiling of organisms within the Bacillus ACT group

(anthracis, cereus, thuringiensis) can be a valuable tool for
investigating the natural ecology and pathogenic properties of
virulent species/strains. Specific morphological features on
Bacillus spore surfaces correlate well with taxonomic divisions and
can differentiate closely related species (1, 2), an important goal for
public health and biodefense agencies. Surface phenotypes may
also indicate key aspects of the production method for Bacillus
spores relevant to a microbial forensics investigation (3). Although
many of the surface characteristics reported in previous studies
(Table 1) are linked to intrinsic properties of the cell, spore surfaces
are also dynamic features that can be affected by culturing
environment and/or preparation method in the laboratory (2, 4, 5).
Since the culturing protocol for spores often differs between studies
and across laboratories, this may create artificial variability in
surface textures that is unrelated to the underlying biology of the
cell. This may complicate interpretation of surface phenotypes and
make cross-study comparisons more difficult.
One particular aspect of spore preparation that varies across
studies is the purification method. After culturing Bacillus in the
appropriate sporulation medium, cells are typically harvested and
subjected to procedures designed to remove cell debris, vegetative
cells, and residual growth medium from the intact spores (6, 7).
There are many established purification methods and one of the
most common is washing the spores several times in ultrapure
water (e.g., Table 1 and references therein). The number of
successive water wash steps can range anywhere from one to more
than ten. Because the overall purity of the spore culture (i.e., the
proportion of vegetative cells and/or cell debris in solution or
physically associated with cell surface) changes with each wash
step (6, 8), the number of water wash steps has the potential to
influence surface textures of individual spore cells. While previous
work has indicated a correlation between the extent of purification
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_________
Conflict of interest: No conflicts declared.
*Corresponding Author. Department of Forensic Science, Virginia
Commonwealth University, 1015 Harris Hall South, Richmond, VA
23284, USA. (804)828-8420. Email: cehrhardt@vcu.edu
2015 by the Journal of Nature and Science (JNSCI).
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After culturing, spores were harvested after 0, 1, 3, and 5 wash

steps. Each wash step consisted of centrifugation of spore biomass
at 3,000 x g for 15 minutes at 4 C, decanting the supernatant and
replacing it with 50 mL of cold (4 C), ultrapure water (18 M).
For the zero wash step samples, a 10 mL aliquot was taken directly
from the culture broth and stored at 4 C until analysis.

trivial matter of using inbuilt software or simply removing the

background, but an active choice of parameters to obtain an
accurate representation of the surface. We describe isolating the
relevant area of the surface, determining a best fit reference plane,
and utilizing it for calculating the RRMS. Subtraction of the
underlying curvature due to the spherical shape of the spores then
allows small surface features to be observed and accounted for in
estimating the true morphology of the surface (23, 25). The
techniques described will have an impact in accurately quantifying
variations in surface properties and roughness at the nanoscale for
highly curved cellular systems. These analyses were performed in
Igor Pro 6 (WaveMetrics, Oswego, OR). Data was exported into
PAST software program (26) for univariate statistical analyses.
Significant differences between RRMS values at each wash step were
assessed with a Students t-statistic.

Atomic Force Microscopy imaging and nanomechanics

The surface morphologies of the spores were imaged using an
Asylum Research MFP-3D Atomic Force Microscope (AFM)
(Asylum Research, Santa Barbara, CA) in non-contact mode. This
is particularly suited for softer and more fragile cell surfaces and
can reveal more surface detail (17). For the AFM study, glass slides
were used as the imaging substrate. 10 L of the spore suspension
were deposited onto a glass slide cleaned with 50 mL of nanopure
water, 50 mL of 100% ethanol, and dried at room temperature in a
biological safety cabinet prior to imaging. Spores were imaged in
air using TR-800PSA silicon nitride tips (Olympus, Japan) with a
nominal force constant of 0.57 N/m and resonance frequency of 73
kHz. Height, amplitude, and phase images were collected
simultaneously. Three-dimensional height and amplitude images
were predominantly used for analysis and presentation. For the
wash step experiments, efforts were made to image spores that
were positioned on the slide with the same orientation (i.e. long
axis parallel to slide) as well as individual spores that were not
physically associated with other cells. Because of the variability
across spore cultures in these two parameters, the number of spores
imaged from each sample group varied from 8-25 cells. In order to
distinguish spore samples that had been processed through a
different number of wash steps, the spore surfaces were
quantitatively compared on the basis of their surface topography.
AFM-based nanoindentation experiments were carried out using
PPP-ZEIHR cantilevers (k~ 20 N/m) (Nanosensors, Neuchatel,
Switzerland). Spring constants of cantilevers were measured prior
to each experiment using the thermal fluctuation method (18).
Stiffer cantilevers were used owing to the well-known high rigidity
of bacterial spores (19). A strategy of elasticity mapping was used
for these experiments. Regions containing cells were identified and
indent curves were obtained by collecting a series of sequential
indent curves in an mn grid. Each indent curve was obtained at
the same loading rate (300 nN/s) by indenting the cantilever to the
samples until a constant load force (150 nN) was reached. All the
procedures including analysis of the indent curves on the basis of
the Hertz model, (20, 21) generating elasticity maps as well as the
overlays of height maps and elasticity maps were carried out with
Igor Pro 6.32 software. Elasticity maps were obtained by collecting
5050 indent curves over a defined area (~33 m) and estimating
the Youngs modulus values.

Results and Discussion

The roughness of cells is an important metric that can be correlated
to the purification and washing of Bacillus spores as well as other
properties such as cell-cell and cell-substrate adhesion (22) or the
germination state of bacteria (27). The AFM is a valuable tool in
this regard owing to the ability to directly investigate the cell
surface morphology in both dry and aqueous (physiological)
environments (28). A brief summary of previously reported work
on AFM-based characterizations of different bacterial spore
systems is shown in Table 1. However, the variation of surface
roughness of Bacillus spores as a function of the processing
conditions has not been systematically investigated. Here, we
demonstrate a flattening algorithm for highly curved spore surfaces
first and then use that to obtain accurate values of RRMS values of
two strains of B.cereus: T-strain (BcT) and 14579 (Bc14579). Next,
we observe changes due to water washing. Finally, we determine
the mechanical properties of the spores before and after washing
and present 3D maps of the surfaces.
Table 1. High Resolution Surface Studies of Bacillus spores.
Surface analysis
Organism
B. thuringiensis
Morphology and imaging
B. thuringiensis
B. atropaeus
B. anthracis
B. atropaeus
B. anthracis
B. cereus
B. subtilis
B. anthracis
B. cereus
B. thuringiensis
B. golibii
B. cereus
B. atrophaeus
B. thuringiensis
B. subtilis
B. cereus
B. anthracis
B. cereus
B. subtilis
B. anthracis
Nanomechanical and Structural
B. anthracis
B. anthracis
Chemical and Biological
B. atropaeus
B. cereus

Analysis and flattening of AFM images

The most often used parameters used to describe a surface are the
average roughness (Ra) and root mean square roughness (RRMS),
providing a measure of the deviation of the height values across a
surface. Typically, for flat samples, estimation of the roughness
from the x, y and Z data is relatively straightforward, using the
surface function Z = f(x, y) to calculate the roughness. However,
the curvature of the spore surfaces presents a complex parameter in
this calculation (22). When a sample has a large curvature (for
example, highly curved architectures that are spherical or fibrillar
in nature), it tends to distort roughness measurements whereby
small feature present on the top of the spores are masked or hidden
by the large range of values (vast minimum and maximum values).
This results in inaccurate RMS values and representation of the
surface. To analyze the nanoscale roughness of the spore surface,
we developed a methodology wherein each image was analyzed by
estimating reference lines and planes (23, 24). To the best of our
knowledge their application to, and effect on, micro- and nanoscale
images collected by AFM for the purpose of surface roughness
analysis have not been well established. Here, we describe the
important considerations to be accounted for when determining the
surface roughness of a curved surface. In most cases, it is not a
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Reference
(35)
(36)
(37)
(2)
(1)

(38)
(5)

(29)
(39)
(40)
(37)
(13)

Flattening of images for analysis

In order to obtain accurate quantitative metrics of spores, it is first
necessary to develop suitable algorithms to analyze the surfaces.
Spore surfaces are typically curved with a height difference
between the highest and lowest points on the order of several
micrometers, necessitating the use of flattening algorithms. In the
absence of accurate flattening, a roughness value that is near the
difference between the highest and lowest point would be obtained.
However, the roughness of the cell surface if it were flattened out,
would actually be different. This is because the extrema are not
2

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Figure 1. Flattening of highly curved bacterial cells: (a) A roughness value is calculated using the differences between the highest and lowest point in
reference to the line of best fit. A sample of large curvature tends to distort roughness measurements masking small feature present on the top of the spores (b)
In analysis, the spore top is isolated from the background substrate and flattened using a least squares polynomial plane fitting. Flattening reduces the
calculated RRMS of the spore surface by about one order of magnitude.

necessarily due to the large curvature of the cell but could arise
from objects on the cell surface such as adhered debris or simply
the nature of the membrane including surface receptors. In our
procedure, shown in Figure 1, the spore top was initially isolated
from the background substrate and the edges of the spore that could
contain AFM-tip imaging artifacts. Flattening is performed using a
least squares polynomial plane fitting, widely used for calculating
reference planes (24). The order of the plane is determined by the
underlying nature of the sample image and deduced mathematically
by selecting the plane equation that best fits the surface. The
procedure to find the best fit plane of a curved surface has been
shown previously (23), but is generally applicable. The equation
for the fitting plane is of the form of a three variable
, where
polynomial

Morphology of Bacillus cereus spore surfaces

In this study, the surface morphology of Bacillus cereus spores
were compared at four different points in the purification procedure
corresponding to zero washes, one wash, three washes, and five
washes. In general, unwashed BcT and Bc14579 spore cells
sampled directly from the culturing medium (zero wash step, W0)
were irregular in shape and had several features ranging from ~20
nm to ~200 nm in size distributed over the surface (Figures 2a, 3a).
Such spore samples also had poorly defined edges which prevented
systematic measurements of the overall dimensions of the spore. In
contrast, spore preparations washed the maximum number of times
(W5) had fewer features and appeared ellipsoidal in shape with
well-defined boundaries (Figure 2d, 3d). Laterally traversing ridges
characteristic of Bacillus spores (2, 29) were observed on many of
the spores washed five times and occasionally on spores after the
third wash step (Figure 2c, 3c). Importantly, ridges were not
apparent on any of the W0 spores. Spores from the first or the third
wash step generally showed a morphology that was intermediate to
spores sampled from W0 or W5. The abundance of 20-200 nm
surface features noticeably decreased after the first wash step
(Figure 2a and 3a compared to subsequent wash step images).
Similarly, well-defined cell boundaries and ridge structures were
only observed on spores in W1, W3, and W5 samples. These
results were consistent across both BcT and Bc14579.
To quantify the effect of the number of wash steps on the spore
morphology, RRMS measurements were made on the spore surfaces
sampled from each wash steps following the flattening protocol
discussed above. Overall, both Bc14579 and BcT spores showed
the highest average RRMS values in W0 samples (17.4 nm and 13.1
nm, respectively). Average values then decreased incrementally
after each successive wash step (Table 2). For Bc14579 spores,
RRMS variance remained high (>20 nm) through the first three
washes then decreased to 5.6 nm in spores after the fifth wash.
RRMS variance in BcT spores was also high in zero and one wash
samples (22.0 nm, 19.0 nm) but decreased to 12.4 nm and 10.8 nm
in the third and fifth washes, respectively.

n is the degree of the polynomial and Ax are the coefficients that

need to be determined. The fit of the polynomial is checked by
calculating the coefficient of determination, R2. To fit a polynomial
plane to the raw data, a series of equations, one for each height
point, is solved simultaneously to determine the coefficients (23,
24). Once the underlying least squares plane (or line) equation is
determined, the surface roughness can be correctly calculated. The
plane is subtracted from the original data to produce a flattened
representation of the data, and RRMS is calculated by
, where m is the number of points
collected in the x direction, n is the number of data points collected
in the y direction and Z is the height value for each point. A second
order plane was fit to the surface using the least squares method,
and then subtracted from the original height data to obtain a
flattened spore surface for analysis (Figure 1b). The effect on the
calculated RRMS value is clear, as the original section image has an
RRMS of 49.6 nm while the flattened image 6.1 nm, a difference of
about one order of magnitude. The surface topology is also clearer,
making it easy to distinguish specific features on the surface. Here,
we used this technique to analyze all the spores imaged under
different conditions.
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Figure 2. Three dimensional height AFM images of BcT spores grown in Tryptone supplemented G medium at different washing steps. (a) Spore without any
washing; (b) Spore after one wash in water; (c) Spore after three washes in water; (d) Spore after five washes in water. All images 2.5 x 2.5 m and presented
to the same z-scale.

Figure 3. Three dimensional height images of Bc14579 spores across five wash steps. (a) unwashed spores, (b) one wash, (c) three washes, (d) five washes.
The scale bars on each images = 0.5 m. All spores are presented to the same z-scale.
Table 2. Statistical analysis of RMS surface values for each wash step (RMS values are in nm, variance values are in nm2).
Bc14579
0 Washes (n=10)
1 Wash (n=10)
3 Washes (n=6)
5 Washes (n=12)
8.8
7.6
9.2
9.2
Min
24.7
26.1
25.0
17.1
Max
17.4
15.3
13.7
12.6
Mean
21.8
34.8
21.9
5.6
Variance
BcT
Min
Max
Mean
Variance

0 Washes (n=22)
4.3
23.0
13.1
22.0

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1 Wash (n=18)
5.8
26.3
12.2
19.0

3 Washes (n=18)
6.1
22.4
11.0
12.4

5 Washes (n=10)
4.0
13.3
8.5
10.8

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Figure 4. Three-dimensional nanomechanical maps of the Bacillus spores as a function of washing. The Youngs modulus (GPa) is overlaid on the 3D
morphology. (top) Zero wash samples and (bottom) three wash samples. All images have been presented to the same z-scale for clarity. Scale bars = 500 nm.

variation in roughness was observed between spore preparations

from early wash steps (W0, 1) and later steps (W3, 5) indicate a
clear relationship between cell surface texture and the purification
procedure on harvested spore cultures. Overall, this shows that
variation across the first three wash steps can be an important
source of phenotypic variability for Bacillus cereus spore surfaces
at the micrometer to nanometer scale.

Comparisons of RRMS values taken from each wash step using the
Students t-test indicated that the differences between W0 and W5
were statistically significant (p< 0.001). Significant differences in
average RRMS were also observed when images of all spores from
early washes (W0 and W1) were combined into one group and
compared to a second group containing spores images from later
washes (W3 and W5). Similarly, average RRMS from W0 samples
was significantly different from pooled data from W3 and W5 (p<
0.001). The overall trend of decreasing RMS across successive
wash steps indicates changes to the nanoscale morphology during
purification. However, surface roughness of individual spores did
exhibit heterogeneity throughout the wash steps as indicated by
high levels of RRMS variance. For example, variance in Bc14579
spores was above 20 nm2 through the first three wash steps and
decreased to 5.6 nm2 only after the fifth wash. Variance in BcT
spores was also high in the first two wash steps (W0 - 22.0 nm2,
W1 - 19.0 nm2) but decreased to 12.4 nm2 and 10.8 nm2 in the third
and fifth washes, respectively. This suggests that some surface
features are retained after the first wash step for both Bc14579 and
BcT spores but are mostly removed by the fifth wash.
Although the specific chemical properties of the nanoscale
surface features observed with AFM are unknown, their size and
decreasing abundance through successive water washes is
consistent with biological debris and fragments that are generated
from cell lysis during culturing. This can occur when spores are
released from the mother cell or during breakdown of dead
vegetative cells or spores (8, 30). The persistence of cell debris in
batch spore preparations and their subsequent removal through
purification steps is well established (6, 31). However, these results
suggest that this process significantly affects the nanoscale
morphology of the spore surface through the third wash step.
The results also indicate that the abundance of small surface
features (< 200 nm) and the associated RRMS values may be useful
indicators for the number of wash steps used during spore
preparation. Because there is significant overlap in the observed
surface textures and distribution of RRMS values across each wash
step, it is difficult to extract distinct ranges for each wash step.
However, obvious visual differences and statistically significant
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Nanomechanics of Bacillus cereus spore surfaces

In addition to the variation in morphology, measuring the
mechanical properties of the spores is another important parameter
to characterize various cellular systems (32, 33). There have been
previous reports on measuring mechanical properties of bacteria
using AFM based nanoindentation (12, 34). As shown above,
morphology of the spore surface is observed to change on account
of the cellular debris that is removed in sequential washing steps.
We hypothesize that the soft external debris is likely not to affect
the underlying nature of the spore itself. The unique properties of
the AFM allow us to determine this progression via
nanoindentation coupled with a technique of elasticity mapping
to determine the mechanical nature of the entire spore at the
nanoscale. Representative samples of spores at W0, W3, and W5
were collected and their nanomechanical distribution was obtained
using elasticity mapping. The indent curves fit well to a Hertzian
mechanical model, allowing us to obtain spatially resolved
Youngs modulus. In each case, the mechanical elasticity map
could be overlaid on the topography of the cell to show the
variation of properties across the entire spore surface (Figure 4).
Figure 4 shows how the mechanical properties vary across the
3D surface of Bacillus spores. Here, we show measurements of
elasticity over 2500 points over a smaller area of the cell surface in
the form of a 50x50 array. The images bear some analysis as on
first glance it appears as if the mechanical properties change with
the wash step. However, a closer analysis reveals that each spore is
surrounded by debris in the form of a thin layer. This was also seen
by the imaging discussed earlier. As the AFM cantilever collects
mechanical information, the debris in the unwashed samples
presents as higher modulus areas owing to the probing of the
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underlying substrate. Indeed, if we remove the debris and focus on

the spore alone, it is seen that the modulus distributions are very
similar across wash steps which confirms our hypothesis that the
spore itself is unaffected by these washing protocols. The sizes of
the cells are also in agreement with the imaging results presented
above. Taken together, our nanoindentation data further confirm
the morphology observations from the spatially observed
progression of this process. It must be noted here that Bacillus
spores (as indeed several cellular systems) are highly curved in
nature. Therefore, an accurate determination of the exact elasticity
values across the entire cell, especially at the edges is challenging.
In our results therefore, there may be some errors at the edges of
the cells. This may be due to two reasons: the curvature/edge
effects of nanoindentation and the collapsed outer coat of sample
during air drying. However, the flat center areas of the spores
provide reliable elasticity values to confirm our observations.
Future efforts can therefore study the endospore mechanical
properties in liquid. In addition, imaging and indenting biological
samples in liquid, as a unique ability of AFM compared to other
conventional techniques, provides us a powerful toolkit to evaluate

the spore morphology as a function of the environment and

processing conditions.

1. Zolock RA, Li GM, Bleckmann C, Burggraf L, Fuller DC. Atomic

force microscopy of Bacillus spore surface morphology. Micron.
2006;37(4):363-9.
2. Wang R, Krishnamurthy SN, Jeong JS, Driks A, Mehta M, Gingras BA.
Fingerprinting species and strains of Bacilli spores by distinctive coat
surface morphology. Langmuir. 2007 Sep 25;23(20):10230-4.
3. Review of the Scientific Approaches Used During the FBI's
Investigation of the 2001 Anthrax Letters: The National Academies
Press, 2011.
4. Rose R, Setlow B, Monroe A, Mallozzi M, Driks A, Setlow P.
Comparison of the properties of Bacillus subtilis spores made in liquid
or on agar plates. J Appl Microbiol. 2007 Sep;103(3):691-9.
5. Plomp M, Leighton TJ, Wheeler KE, Malkin AJ. The high-resolution
architecture and structural dynamics of Bacillus spores. Biophysical
Journal. 2005 Jan;88(1):603-8.
6. Harwood CR, Cutting SM. Molecular Biological Methods for Bacillus
(Modern Microbiological Methods) 1990.
7. Whiteaker J, Fenselau C. Quantitative determination of heme for
forensic characterization of Bacillus spores using matrix assisted laser
desorption/ionization time-of-flight mass spectrometry. Anal Chem.
2004;76:2836-41.
8. Tavares MB, Souza RD, Luiz WB, Cavalcante RCM, Casaroli C,
Martins EG, et al. Bacillus subtilis Endospores at High Purity and
Recovery Yields: Optimization of Growth Conditions and Purification
Method. Curr Microbiol. 2013 Mar;66(3):279-85.
9. Wang D-B, Yang R, Zhang Z-P, Bi L-J, You X-Y, Wei H-P, et al.
Detection of B. anthracis Spores and Vegetative Cells with the Same
Monoclonal Antibodies. PLoS One. 2009;4(11):e7810.
10. Dufrene YF. Towards nanomicrobiology using atomic force
microscopy. Nature Reviews Microbiology. 2008;6(9):674-80.
11. Alsteens D, Dufrene YF. Atomic Force Microscopy of Living Cells. In:
Fornasiero EF, Rizzoli SO, editors. Super-Resolution Microscopy
Techniques in the Neurosciences; 2014;225-55.
12. Wang C, Stanciu C, Ehrhardt CJ, Yadavalli VK. Morphological and
mechanical imaging of Bacillus cereus spore formation at the nanoscale.
Journal of microscopy. 2015;258(1):49-58.
13. Wang CZ, Ehrhardt CJ, Yadavalli VK. Single cell profiling of surface
carbohydrates on Bacillus cereus. J R Soc Interface. 2015;12(103).
14. Kaneda T. Fatty Acids in the genus Bacillus II. Similarity in the fatty
acid compositions of Bacillus thuringiensis, Bacillus anthracis, and
Bacillus cereus. Journal of Bacteriology. 1968;95:2210-6.
15. Carrera M, Zandomeni RO, Fitzgibbon J, Sagripanti JL. Differences
between spore sizes of Bacillus anthracis and other Bacillus species. J
Appl Microbiol. 2007;102:303-12.
16. Hashimoto T, Black SH, Gerhardt P. Development of fine structure,
thermostability, and dipicolinate during sporogenesis in a bacillus.
Canadian Jour Microbiol. 1960 1960;6((2)):203-12.
17. Zolock A. Characterization of the Surface Morphology of Bacillus
Spores by Atomic Force Microscopy: Air Force Institute of Technology,
2002.
18. Hutter JL, Bechhoefer J. Calibration of atomic force microscope tips.
Rev Sci Instrum. 1993 Jul;64(7):1868-73.
19. Fernandes JC, Eaton P, Gomes AM, Pintado ME, Malcata FX. Study of
the antibacterial effects of chitosans on Bacillus cereus (and its spores)

by atomic force microscopy imaging and nanoindentation.

Ultramicroscopy. 2009 Jul;109(8):854-60.
Lin DC, Horkay F. Nanomechanics of polymer gels and biological
tissues: A critical review of analytical approaches in the Hertzian
regime and beyond. Soft Matter. 2008;4(4):669-82.
Touhami A, Nysten B, Dufrene YF. Nanoscale mapping of the
elasticity of microbial cells by atomic force microscopy. Langmuir.
2003 May;19(11):4539-43.
Mege JL, Capo C, Benoliel AM, Foa C, Galindo R, Bongrand P.
Quantification of cell-surface roughness - a method for studying cell
mechanical and adhesive properties. Journal of Theoretical Biology.
1986 Mar 21;119(2):147-60.
Hani AFM, Prakasa E, Fitriyah H, Nugroho H, Affandi AM, Hussein
SH. High Order Polynomial Surface Fitting for Measuring Roughness
of Psoriasis Lesion. Visual Informatics: Sustaining Research and
Innovations, Pt I. 2011 2011;7066:341-51.
Dong WP, Mainsah E, Stout KJ. Reference planes for the assessment of
surface roughness in three dimensions. International Journal of
Machine Tools and Manufacture. 1995;35(2):263-71.
Xiaohui X, Yan C. The Method of Spherical Surface Roughness
Measurement. Modern Applied Science 2009;3(12).
Hammer O, Harper DAT, Ryan PD. PAST: Paleontological Statistics
Software Package for Education and Data Analysis. Palaeontologia
Electronica. 2001;4(1).
Pinzon-Arango PA, Scholl G, Nagarajan R, Mello CM, Camesano TA.
Atomic force microscopy study of germination and killing of Bacillus
atrophaeus spores. Journal of Molecular Recognition. 2009
Sep-Oct;22(5):373-9.
Francis LW, Lewis PD, Wright CJ, Conlan RS. Atomic force
microscopy comes of age. Biology of the Cell. 2010
Feb;102(2):133-43.
Chada VGR, Sanstad EA, Wang R, Driks A. Morphogenesis of Bacillus
spore surfaces. Journal of Bacteriology. 2003 Nov;185(21):6255-61.
Wang DB, Yang RF, Zhang ZP, Bi LJ, You XY, Wei HP, et al.
Detection of B. anthracis Spores and Vegetative Cells with the Same
Monoclonal Antibodies. Plos One. 2009 Nov 13;4(11).
Cliff JB, Jarman KH, Valentine NB, Golledge SL, Gaspar DJ,
Wunschel DS, et al. Differentiation of spores of Bacillus subtilis grown
in different media by elemental characterization using time-of-flight
secondary ion mass spectrometry. Applied and Environmental
Microbiology. 2005;71(11):6524-30.
Dufrene YF, Pelling AE. Force nanoscopy of cell mechanics and cell
adhesion. Nanoscale. 2013;5(10):4094-104.
Suresh S. Biomechanics and biophysics of cancer cells. Acta Biomater.
2007 Jul;3(4):413-38.
Cerf A, Cau JC, Vieu C, Dague E. Nanomechanical Properties of Dead
or
Alive
Single-Patterned
Bacteria.
Langmuir.
2009
May;25(10):5731-6.
Gillis A, Dupres V, Delestrait G, Mahillon J, Dufrene YF. Nanoscale
imaging of Bacillus thuringiensis flagella using atomic force
microscopy. Nanoscale. 2012;4(5):1585-91.
Sun W, Romagnoli JA, Palazoglu A, Stroeve P. Characterization of
Surface Coats of Bacterial Spores with Atomic Force Microscopy and
Wavelets. Ind Eng Chem Res. 2011 Mar 2;50(5):2876-82.

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Conclusions
The goal of this study was to determine if variation in Bacillus
spore purification methods produced quantifiable morphological
signatures on the cell surface. Using AFM as a sensitive, single cell
technique, it is possible to investigate the nano and microscale
topographies of these bacterial spores quickly and
non-destructively. Overall, the roughness and nanomechanical
profiles suggest that the spore surface is affected by the purification
procedure through the first five wash steps. Considering the wide
range of washing methods reported for Bacillus spores, our results
suggest that the purification protocol may need to be tested and/or
optimized for surface studies to ensure that taxonomically- or
environmentally-relevant morphological properties are being
characterized. The study can be the foundation for future efforts
that examine the quantitative changes to surface morphology across
either wash steps or other purification procedures relevant to
Bacillus spore production.

20.
21.
22.

23.

24.
25.
26.
27.

28.
29.
30.
31.

32.
33.
34.
35.
36.

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Journal of Nature and Science, Vol.1, No.6, e116, 2015

37. Plomp M, Malkin AJ. Mapping of Proteomic Composition on the
Surfaces of Bacillus Spores by Atomic Force Microscopy-Based
Immunolabeling. Langmuir. 2009 Jan 6;25(1):403-9.
38. Tarasenko O, Nourbakhsh S, Kuo SP, Bakhtina A, Alusta P, Kudasheva
D, et al. Scanning electron and atomic force microscopy to study
plasma torch effects on B. cereus spores. Ieee T Plasma Sci. 2006
Aug;34(4):1281-9.

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39. Pinzon-Arango PA, Nagarajan R, Camesano TA. Effects of L-Alanine

and Inosine Germinants on the Elasticity of Bacillus anthracis Spores.
Langmuir. 2010 May 4;26(9):6535-41.
40. Zaman MS, Goyal A, Dubey GP, Gupta PK, Chandra H, Das TK, et al.
Imaging and analysis of Bacillus anthracis spore germination. Microsc
Res Techniq. 2005 Apr 15;66(6):307-11.

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