International Journal of Food Microbiology 263 (2017) 17–25

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Bacteria, mould and yeast spore inactivation studies by scanning electron MARK
microscope observations
Siti N.M. Rozalia, Elham A. Milania, Rebecca C. Deedb, Filipa V.M. Silvaa,⁎
a
Chemical and Materials Engineering Department, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b
School of Biological Sciences, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: Spores are the most resistant form of microbial cells, thus difficult to inactivate. The pathogenic or food spoilage
Fungi effects of certain spore-forming microorganisms have been the primary basis of sterilization and pasteurization
Spore-former processes. Thermal sterilization is the most common method to inactivate spores present on medical equipment
Heat and foods. High pressure processing (HPP) is an emerging and commercial non-thermal food pasteurization
Thermal inactivation
technique. Although previous studies demonstrated the effectiveness of thermal and non-thermal spore in-
High pressure processing
activation, the in-depth mechanisms of spore inactivation are as yet unclear. Live and dead forms of two food
Scanning electron microscopy
spoilage bacteria, a mould and a yeast were examined using scanning electron microscopy before and after the
inactivation treatment. Alicyclobacillus acidoterrestris and Geobacillus stearothermophilus bacteria are indicators of
acidic foods pasteurization and sterilization processes, respectively. Neosartorya fischeri is a phyto-pathogenic
mould attacking fruits. Saccharomyces cerevisiae is a yeast with various applications for winemaking, brewing,
baking and the production of biofuel from crops (e.g. sugar cane). Spores of the four microbial species were
thermally inactivated. Spores of S. cerevisiae were observed in the ascus and free form after thermal and HPP
treatments.
Different forms of damage and cell destruction were observed for each microbial spore. Thermal treatment
inactivated bacterial spores of A. acidoterrestris and G. stearothermophilus by attacking the inner core of the spore.
The heat first altered the membrane permeability allowing the release of intracellular components.
Subsequently, hydration of spores, physicochemical modifications of proteins, flattening and formation of in-
dentations occurred, with subsequent spore death. Regarding N. fischeri, thermal inactivation caused cell de-
struction and leakage of intracellular components. Both thermal and HPP treatments of S. cerevisiae free spores
attacked the inner membrane, altering its permeability, and allowing in final stages the transfer of intracellular
components to the outside. The spore destruction caused by thermal treatment was more severe than HPP, as
HPP had less effect on the spore core. All injured spores have undergone irreversible volume and shape changes.
While some of the leakage of spore contents is visible around the deformed but fully shaped spore, other spores
exhibited large indentations and were completely deformed, apparently without any contents inside. This cur-
rent study contributed to the understanding of spore inactivation by thermal and non-thermal processes.

1. Introduction
Undesirable microorganisms can spoil foods and cause human dis-
eases. Therefore, several techniques are used to control or destroy mi-
crobial cells. Certain microorganisms can be present in the vegetative
but also in the spore form and these matured spores are difficult to
inactivate due to their concrete multilayer membranes, which can
withstand multiple environmental conditions. Therefore, spores are
commonly used as a target in sterilization and pasteurization processes.
Alicyclobacillus acidoterrestris is an aerobic, non-pathogenic and
spore-forming bacteria that has been detected in several spoiled com-
mercial pasteurized acidic fruit juices. Therefore, this organism has
been suggested as an ideal target of acidic fruit juice pasteurization
(Silva and Gibbs, 2001, 2004; Silva et al., 2014). A. acidoterrestris ve-
getative cells are rod shaped with terminal or sub-terminal endospores
(Goto et al., 2007). This bacterium produces compounds that are re-
sponsible for juice off-odours. Geobacillus stearothermophilus is a non-
pathogenic bacteria, which can produce spores under harsh conditions
when nutrient concentrations are low. Presence of G. stearothermophilus
usually results in flat sour spoilage of canned liquid foods (Watanabe

⁎
Corresponding author.
E-mail address: filipavinagresilva@gmail.com (F.V.M. Silva).

http://dx.doi.org/10.1016/j.ijfoodmicro.2017.10.008
Received 20 March 2017; Received in revised form 7 September 2017; Accepted 3 October 2017
Available online 04 October 2017
0168-1605/ © 2017 Elsevier B.V. All rights reserved.
S.N.M. Rozali et al. International Journal of Food Microbiology 263 (2017) 17–25

Fig. 1. Structure of a bacterial spore (not drawn to scale).

inner membrane Redrawn from Setlow (2006).

germ cell wall

cortex

outer membrane

core
coat

exosporium

Fig. 2. Structure of a yeast ascospore (not drawn to scale).

Modified from Neiman (2005).

plasma membrane

mannan

beta-glucan
cytoplasm
chitosan

dityrosine

et al., 2003). Fig. 1 shows the typical structure of a bacterial endospore.
The interior compartment of a bacterial spore is called the core. The
spore core is dehydrated with only 10–25% water content, making it
very resistant to heat and chemicals. Pyridine-2,6-dicarboxylic acid or
dipicolinic acid (DPA) is another component found in the inner core of
bacterial spores but not in vegetative cells. This molecule comprises
5–15% of the dry weight of the spore (Molva and Baysal, 2014; Setlow,
2006). Its function is to prevent the denaturation of protein by lowering
the core water content (Molva and Baysal, 2014). The outside of the
spore core is comprised of a thick, loosely cross-linked peptidoglycan
layer called the spore cortex, which prevents hydration of the spore
core. The germ cell wall under the cortex will become the cell wall of
the bacterium after the endospore germinates. The inner membrane on
the other hand, acts as a major permeability barrier against chemicals.
The completed spore is encased in a multi-layered protein shell known
as the coat which protects the spore (Aronson and Fitz-James, 1976).
The spore coat also acts as a permeability barrier (Zhang et al., 2006).
Neosartorya fischeri is a spore-forming phyto-pathogenic mould that
is commonly associated with diseases in fruits growing at ground level.
It is a ubiquitous fungus which usually proliferates in damp environ-
ments, such as soil. It is an extremely rare human pathogenic fungus
that can cause keratitis and in some cases pulmonary aspergillosis in
transplant patients (Lonial et al., 1997). The information on the struc-
tural details of N. fischeri spores is very limited. However, it is known
that the ascospore (spores produced or contained inside an ascus) walls
of N. fisheri are usually ornamented with anastomosing ridges
(Peterson, 1992). N. fischeri spores are known to be extraordinarily
resistant to heat (Beuchat, 1986; Conner et al., 1987; Evelyn et al.,
2016; Evelyn and Silva, 2017; Girardin et al., 1995) compared to bac-
teria, yeasts and other moulds (Silva and Gibbs, 2004, 2009; Silva et al.,
2014).
Saccharomyces cerevisiae is a spherical shaped non-pathogenic yeast
which is well known for its ability to ferment sugars to ethanol, re-
sulting in the production of food and beverages such as beer, wine and
baked products. It is also used to produce alcohol for biofuel from sugar
cane. S. cerevisiae can also produce spores although these spores have a
lower resistance to damaging environments compared to bacterial and
mould spores (Milani et al., 2015a, 2015b, 2016; Milani and Silva,
2016, 2017). Once S. cerevisiae spores are fully formed, the remains of
the mother cell collapses around the four mature spores. This event
results in the formation of the ascus, consisting of four ascospores en-
closed together inside an ascal membrane and wall (Fig. 6), which are

18
S.N.M. Rozali et al.
both derived from the mother cell. These asci are also termed tetrads
due to the presence of four ascospores. Because of this process, the
intact ascus has a similar surface texture to vegetative cells (Coluccio
and Neiman, 2004). Extraction of a single ascospore from an ascus can
be performed using the zymolyase enzyme. Fig. 2 shows the detailed
structure of a yeast ascospore, consisting of four multi-laminar coats
surrounding an individual spore (mannan, beta-glucan, chitosan and
dityrosine as the outermost layer). These layers provide the spore with
protection from environmental stresses (Briza et al., 1994; Neiman,
2005). The dityrosine is a major component of the spore which is re-
sponsible for the enhanced resistance of S. cerevisiae spores towards
many stresses (Briza et al., 1990).
For food products, many studies have been conducted to investigate
the performance and efficiency of conventional thermal treatments and
innovative techniques including high pressure processing (HPP), pulsed
electric field (PEF) and ultrasound in terms of spore inactivation
(Evelyn et al., 2016; Evelyn and Silva, 2016; Evelyn et al., 2017; Milani
et al., 2015a, 2015b, 2016; Milani and Silva, 2016). Although there are
comprehensive studies regarding the thermal and non-thermal in-
activation techniques, the in-depth mechanisms regarding how dif-
ferent spores are killed by different treatment processes are not well
explained. The main aim of this study was to contribute for the un-
derstanding of microbial spore inactivation by thermal and non-thermal
HPP, through the examination of the morphology of live and dead
spores under an electron microscope. The specific objectives were to
observe live and dead spores of (i) Alicyclobacillus acidoterrestris and
Geobacillus stearthermophilus bacteria (ii) Neosartorya fischeri mould,
and (iii) Saccharomyces cerevisiae yeast asci and free spores.
2. Material and methods
2.1. Microscopes
A Philips XL30S FEG (FEG = Field Emission Gun) unit was used
during scanning electron microscopy (SEM) analysis while FEI Quanta
200 FEG unit was used for environmental scanning electron microscopy
(eSEM) analysis. The sputter coater used for standard electron micro-
scopy samples is a Quorum Q150RS. It is designed to give a thin,
minimal metal coating suitable for electron microscopy viewing. All
spore samples were sputter coated with platinum (Pt). Both XL30 and
Quanta eSEM can be used to analyze all spore samples prepared by any
sample preparation method listed in the next sections. All spore images
were collected at 10 kV.
2.2. Production of bacterial and mould spores
2.2.1. A. acidoterrestris and G. stearothermophilus bacteria
A strain of A. acidoterrestris NZRM 4447 (New Zealand Reference
Culture Collection, Medical Section) was acquired from the Institute of
Environmental Science and Research (ESR), New Zealand. The bacterial
culture was grown for 3 days at 45 °C on potato dextrose agar (PDA)
(BD Difco, North Ryde, Australia) adjusted to pH 4.0 (Mettler Toledo,
S20 – SevenEasy, USA), after sterilization, using 10% w/v (0.1 g/mL) of
tartaric acid (Univar, Ajax Finechem, Australia) (Uchida and Silva,
2017). The spores were first centrifuged and washed repeatedly. The
final pellet was resuspended in sterile water and stored at 4 °C until use.
G. stearothermophilus ATCC 7953 spore suspension was purchased
from Merck Millipore (Merck KGaA Darmstadt, Germany).
2.2.2. Production of N. fischeri mould free spores
N. fischeri JCM 1740 was obtained from the Japan Collection of
Microorganisms (JCM). The fungal culture was spread by plating onto
malt extract agar (MEA) and incubated at 30 °C for four weeks until
visible colonies were formed. The spores were collected by flooding the
surface of the culture plates with 5 mL sterile distilled water (SDW), and
gently rubbing the agar surface with a sterile bent glass rod. The spore
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International Journal of Food Microbiology 263 (2017) 17–25
suspension was subsequently filtered through layers of gauze to remove
any remaining hyphal fragments. Spore pellets were obtained after
centrifugation in SDW at 4000 ×g, 15 min, 4 °C and the procedure was
repeated three times. The final spore suspension was then stored at 2 °C
in SDW containing glass beads until use (Evelyn et al., 2016).
2.3. Production of S. cerevisiae asci and free spores
2.3.1. Production of S. cerevisiae asci
Asci of DSMZ 1848 S. cerevisiae was produced according the method
described by Milani et al. (2015a). The culture was initially streaked
onto yeast extract peptone glucose (YEPG) agar and, after growth, a
fresh single colony was inoculated into the presporulation liquid com-
posed of 0.8% yeast extract, 0.3% peptone, 10% glucose, and 25 mg/L
zinc sulfate with overnight incubation at 28 °C. Then 1.5 mL of the
presporulation medium was added to the sporulation broth comprised
of potassium acetate 1% (w/v), bacto yeast extract 0.1% (w/v), glucose
0.05% (w/v), and zinc sulfate 25 mg/L, and incubated at 18 °C for
14 days. After that period the spores were extracted by adding zymo-
lyase buffer (1.2 M sorbitol, 0.1 M KH2PO4, pH 7.2), zymolyase solution
(50 mg/mL), spheroblasting buffer (2.2 M sorbitol), and softening
buffer (100 mM Tris-SO4, pH 9.4, 10 mM dithiothreitol solution) and
centrifuged. Finally, the spores were suspended in salt triton dithio-
threitol (STD) solution (0.1 g NaCl in 10 mL of 0.05% Triton X-100) to
avoid spore clustering during storage at 4 °C.
2.3.2. Production of S. cerevisiae free single spores
The extraction of single ascospores from the ascus was performed
according to Milani et al. (2015a). The spore solution (before the stage
of STD addition) was split into 1 mL Eppendorf tubes. The spores were
extracted from the vegetative parental cells by adding 100 μL zymo-
lyase solution (5 mg/mL solid zymolyase in pH 7.2 buffer containing
1.2 M sorbitol and 0.1 M KH2PO4), 900 μL spheroblasting buffer (2.2 M
sorbitol), and 800 μL softening buffer (100 mM Tris-SO4, pH 9.4,
10 mM dithiothreitol (DTT) solution). Then, the mixture was incubated
at 30 °C in a water bath for 2 h with gently inversion of Eppendorf tubes
every 20 min to accelerate the breakup of tetrads into single ascospores.
The spores were harvested by centrifuging three times at 9700 g (rotor
F-45-12-11) for 1 min and resuspending in 200 μL of 0.5% Triton X-100
to ensure total removal of the zymolyase enzyme. After the last re-
suspension, 4 μL DTT was added to the Eppendorf tubes containing the
spore solution. Then, the Eppendorf tubes were sonicated three times at
6 Hz for 2 min, both to break up tetrads into single ascospores and to
kill any vegetative cells remaining in the medium. Finally, 1 mL of STD
solution (0.1 g NaCl in 10 mL of 0.05% Triton X-100) was added to the
spore solution to avoid spore aggregation. The spores were stored at
4 °C until use.
2.4. Spore thermal and non-thermal HPP inactivation treatments
For each spore solution, 10 μL of sample was inoculated inside
0.99 mL of sterile distilled water and then vacuum packed into
5 cm × 5 cm transparent plastic film pouches (CasePak plastic vacuum
pouch, New Zealand). Air was removed from within the pouch and then
it was thermosealed using vacuum (Multivac C200, Germany). The
plastic film was made of cast polypropylene for excellent transparency
and heat sealing qualities which can withstand temperatures of up to
125 °C. All microbial spore species were thermally processed, while
HPP was only applied to S. cerevisiae for comparing the damages caused
by non-thermal (HPP) and thermal treatments. The thermal and HPP
conditions used were set based on previous studies and are detailed in
the following sections.
2.4.1. Thermal treatments
With regards to spore thermal inactivation, the following conditions
were selected to result in ≥ 5.0 log reductions, based on previous
S.N.M. Rozali et al.
results with the same strains (Evelyn et al., 2016; Evelyn and Silva,
2016; Milani et al., 2015a; Watanabe et al., 2003): 5 min at 97 °C for A.
acidoterrestris inactivation, 10 min at 120 °C for G. stearothermophilus,
7 min at 95 °C for N. fischeri and 20 min at 65 °C for S. cerevisiae free
spores. Instead of 65 °C, a higher temperature of 75 °C for 20 min was
applied to the asci of S. cerevisiae. The higher temperature was used to
ensure that the spores inside the ascal membrane were destroyed. After
these treatments, plating in appropriate agar (Evelyn et al., 2016;
Milani et al., 2015a; Silva et al., 1999; Silva and Gibbs, 2001; Uchida
and Silva, 2017) did not result in any colony growth, indicating that the
spores were inactivated and possibly killed by the processing conditions
applied. The sealed pouch containing the spore solution with con-
centration ca 107 cfu/mL was completely immersed in a thermostatic
water bath set at the specific temperature and time conditions, to en-
sure spore inactivation. The pouch was then dipped in an ice cooled
water bath for 1 min to immediately stop further reaction. After that,
the pouch containing the spores was immediately stored at 4 °C until
use.
2.4.2. HPP
The HPP unit used was the 2 L-700 Laboratory Food Processing
System (Avure Technologies, Columbus, Ohio, USA). The equipment
consists of a 2-L cylindrical shape pressure treatment chamber, water
circulation, a pumping system and the control system operated through
a computer with software supplied by the manufacturer. Distilled water
was used as the medium in the chamber. The sealed pouch was placed
inside the pressure chamber. Based on previous HPP studies with S.
cerevisiae spores by Milani et al. (2016) and Milani and Silva (2016), a
5 min non-thermal HPP treatment at 600 MPa was performed at am-
bient conditions of 25–30 °C. This treatment achieved at least 7.0 log
reductions in the spores (Milani and Silva, 2016), and no colony has
grown in YEPG agar plates after incubation at 28 °C, showing total in-
activation and non-viable spores. The processing conditions were set at
the control system operated through computer software. After treat-
ment, the pouch was immediately stored at 4 °C until further use.
2.5. Spore preparation for electron microscope observations
2.5.1. Short air drying
A. acidoterrestris, G. stearothermophilus, N. fischeri and S. cerevisiae
free single spores were observed under the microscope after short air
drying. Live and dead spores were directly adhered onto thin cover slips
which were mounted on metal stubs. The attached spores were left to
dry inside a desiccator for a maximum of 1 h before they were sputter
coated with platinum.
2.5.2. Long air drying
S. cerevisiae asci containing spores were observed under microscopy
after long air drying. A similar procedure to short air drying was per-
formed. The spores were allowed to dry for at least 24 h before they
were sputter coated with platinum.
3. Results and discussion
3.1. Basic morphological characteristics of the different type of microbial
spores observed
Based on the photographs observed from SEM and eSEM, it is evi-
dent that bacterial spores possess a shape that is longer than it is wide
(Figs. 3 & 4); mould spores have a cubical shape (Fig. 5) while yeast
spores possess a spherical shape (Figs. 9–10). A. acidoterrestris spores
are approximately 1 μm wide and 1.8–2 μm long (Fig. 3(a)), appearing
as elliptical ovals. In contrast, the G. stearothermophilus spores exhibit a
long cylindrical shape with a full and compact core and a well-defined
outer wall (Fig. 4). The width of the G. stearothermophilus spores is quite
homogeneous with approximately 0.85 μm wide and length varying
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International Journal of Food Microbiology 263 (2017) 17–25
between 1.7 and 2 μm (Fig. 4(b)).
The cubical shape of the N. fischeri spores is made up of approxi-
mately 1.3 μm edges on each side, as shown in Fig. 5(a). The edges look
well defined and sharp with the appearance of concave cube faces.
Regarding the spherical-shaped S. cerevisiae spores, the diameter is
approximately 2–2.4 μm (Figs. 9(a) and 10(a)).
3.2. Observation of A. acidoterrestris bacterium live and dead spores
Based on Fig. 3(a), the live bacterial spores are elliptical shaped
with thick, fat edges. Fig. 3(b) shows deep indentations in the dead
spores with thinner edges. The dead spore image suggests that the
spores were split along the longitudinal axis which makes the inner
region now visible. The presence of deep indentations indicates that the
spores have lost their internal contents and volume. These images are in
agreement with others obtained with A. acidoterrestris by Molva and
Baysal (2014). DPA is a major chemical component found in the inner
core of bacterial spores, representing approximately 5–14% of spore dry
weight (Molva and Baysal, 2014), and contributing to the wet heat
resistance of the spores (Kort et al., 2005). Hence, although DPA outside
the dead spores was not determined, it is suggested that the volume loss
of the spores might be due to the release of this component with the
thermal treatment. When the spores were heat treated, DPA was re-
leased, inducing the inactivation process as the spores change from a
heat resistant state to a DPA-free state, which is susceptible to heat
(Mallidis and Scholefield, 1985). Generally, the mechanism for the
thermal inactivation of bacterial spores involves two main stages. The
first stage is the production of injured DPA-free spores and the second
stage is the inactivation of the injured spores by further application of
heat. Firstly, inactivation started with membrane or cortex layer in-
juries that allowed the release of DPA from the spore core through the
opening of DPA channels. In the second stage, DPA is released, trig-
gering spore inactivation as the water content inside the spore core was
increased to replace DPA. The absence of DPA, and the increase of high
temperature water inside the spore, eliminated the protection towards
core proteins which were denatured.
3.3. Observation of G. stearothermophilus bacterium live and dead spores
Fig. 4 shows the differences between live and dead spores. The live
spores are a perfect rod shape with a full and compact core and a well-
defined outer wall (Fig. 4(a) and (b)). The live image is in agreement
with others obtained by Rossi et al. (2009). In contrast, the dead spores
show an imperfect or irregular rod shape and with the absence of a
well-defined outer spore coat (Fig. 4(c) and (d)). It is unknown whether
the treated spores have an empty core or a partially empty core due to
the release of intracellular content. It is clearly visible that some of the
dead spores were completely destroyed from the heat treatment and
some of the spore coat was also torn apart. Besides that, some dead
spores can be seen to be cut in half (Fig. 4(c)). These evidences suggest
that the inactivation of G. stearothermophilus spores includes a complete
break down or tearing apart of the spore body. As mentioned pre-
viously, Mallidis and Scholefield (1985) also found DPA in the inner
core of G. stearothermophilus, and confirmed the significant relationship
between DPA release and stearothermophilus spore death. The destruc-
tion of G. stearothermophilus was more adverse than A. acidoterrestris,
thus the loss of G. stearothermophilus spore volume is suggested due to
total absence of DPA component and other spore components. Mallidis
and Scholefield (1985) demonstrated a 70 to 100% release of DPA after
10 min at 115 °C. As in the present study, a higher temperature was
employed (120 °C–10 min), more adverse death effects were expected
(El-Bisi et al., 1962; Grecz and Tang, 1970; Walker and Matches, 1965).
3.4. Observation of N. fischeri mould live and dead spores
The live mould spore in Fig. 5(a) shows well-defined, sharp edges
S.N.M. Rozali et al. International Journal of Food Microbiology 263 (2017) 17–25

Fig. 3. SEM images of A. acidoterrestris spores (a) untreated

(a) (b) live (25,000 ×) (b) heat treated dead (35,000 × magnifi-
cation).

along the mould spore surface with concavity spaces between the sharp
edges. In contrast to the rod shape possessed by the two bacterial spores
studied, the N. fischeri live spore presents a cubic shape. Fig. 5(b) show
clear evidence of cellular destruction with release of intracellular
components which is well visible around the spore. This study proves
that the 95 °C–7 min heat treatment was enough to kill the ascospores,
resulting in significant changes in the spores' appearance: tearing apart
of the spore membrane, resulted in spore thinning and massive in-
tracellular leakage of internal spore components. In future studies it is
essential to determine the content of the leakage that leads to spore
thinning. There are no studies as yet to explain the thermal inactivation
effect on mould spores using SEM observation, and very limited in-
formation on the inactivation mechanisms of mould spores, especially
N. fischeri.
3.5. Observation of S. cerevisiae yeast live and dead spores inside the ascus
Both the live and treated spores contained inside the S. cerevisiae
ascus were air dried overnight. A longer duration of air drying, which
took a minimum of 24 h, was performed in order to improve the effi-
ciency of air drying and produce sharper and clearer images under the
electron microscope. The resulting images under XL30 are shown in
Figs. 6, 7 and 8. Fig. 6 shows the untreated ascus containing the four
live spores and vegetative cells. As the ascal wall is derived from the
vegetative wall (Coluccio and Neiman, 2004), their surface texture
looks the same. Four live spores are attached together inside the ascus
Fig. 6, circled area of right side image, in the expected tetrad ar-
rangement. Based on the indentations present on the surface of the
circled ascus/tetras, we can visualize the shape of three spores, and the
last spore would be behind these and not visible. The entire circled

Fig. 4. eSEM images of G. stearothermophilus

(a) (b) spores (a & b) untreated live (from left: 12,000 ×,
24,000 × magnification) (c & d) heat treated dead
(from left: 12,000 ×, 24,000 × magnification).

Perfect rod
shape

(c) Destroyed (d)

spores
Spores cut
in half

Intracellular Completely
content destroyed spores

21
S.N.M. Rozali et al.
(a) (b)
Vegetative
cell
without
spores
Destroyed
ascus
ascus is only slightly larger than a vegetative cell, therefore the four
spores inside have to be smaller than a single vegetative cell. The spores
(2–2.4 μm) are at least half of the size of the vegetative cells (~ 5 μm,
also indicated in right side image of Fig. 6) left over from the pre-
sporulation process. The reason for the existence of vegetative cells in
the untreated sample is that the zymolyase enzyme treatment and so-
nication were not performed as previously mentioned.
Figs. 7 and 8 show the treated ascus containing the dead spores by
thermal processing and HPP, respectively. Fig. 7 shows the heat treated
tetrad appearing crumpled within the ascus with a loss of cell structure/
volume. However, the spores still remain attached together in tetrad
form. Regarding the HPP-treated ascus (Fig. 8), single spores appear to
be spread over the thinning and destroyed ascal wall as if the asci have
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International Journal of Food Microbiology 263 (2017) 17–25
Fig. 5. eSEM image of N. fischeri spore (a) un-
treated live (15,000 ×) (b) heat treated dead
(24,000 × magnification).
Fig. 6. SEM image of live S. cerevisiae spores in
tetrad or ascus form (from left: 3,500 ×,
12,000 × magnification).
Spore in
tetrad form
Fig. 7. SEM images of dead S. cerevisiae spores in
Destroyed tetrad or ascus form after being treated by
thermal processing (from left: 8,000 ×, 10,000 ×
ascus magnification).
burst open. Since the spores are inside the ascus in tetrad form in the
thermal treatment, it is difficult to compare them with the HPP-treated
dead spores, which have clearly exploded. The morphology between
live and dead spores contained inside the ascus was also difficult to
observe due to the mixing of vegetative cells and intact asci. Thus, the
observation of free single spores of S. cerevisiae before (live) and after
treatments (dead) was also carried out and results are shown in the
following section.
3.6. Observation of free spores of S. cerevisiae live and dead
Figs. 9(a) and 10(a) show healthy untreated S. cerevisiae spores,
which possess a perfect spherical shape. The S. cerevisiae live spore wall
S.N.M. Rozali et al. International Journal of Food Microbiology 263 (2017) 17–25

Fig. 8. SEM images of dead S. cerevisiae spores in

tetrad or ascus form after being treated by HPP
Escaped spores
(from left: 5,000 ×, 10,000 × magnification).

Destroyed ascal
membrane

Fig. 9. SEM images of S. cerevisiae free spores (a)

(a) untreated live (35,000 ×) (b & c) heat treated
dead (25,000 × magnification).

(b) Release of (c)

intracellular
content

Spore
bursts and
contracts

Fig. 10. eSEM images of S. cerevisiae free spores

(a) untreated live (b) HPP treated dead
(b) Change on
(a) (16,000 × magnification).
shape and
Perfect spherical size of
or round shape spores

Blurred Release of
area intracellular
content

23
S.N.M. Rozali et al.
is predominantly composed of dityrosine while its vegetative cell wall
primarily consists of mannoproteins (Coluccio and Neiman, 2004).
These two different types of polymers can be distinguished by their
surface appearance, with the spores having a ridged or scalloped ap-
pearance, with a perfect round shape. The image of live spores in the
study by Coluccio and Neiman (2004) are similar to our images in
Figs. 9(a) and 10(a) with a round shape and rough, ridged spore sur-
face. The higher magnification of the spore in Fig. 9(a) reveals the
rough and ridged spore surface. Images in Fig. 9(b) and (c) present the
morphology of the free spore after thermal treatment, appearing totally
deflated and shrunken. Although both lost viability, the cellular de-
struction of the heat-treated spores was more severe than the HPP-
treated spores (Fig. 10(b)), where only an imperfect round shape of the
HPP dead spore was observed. Fig. 9(b) and (c) each presents a deep
and large opening hole at the outermost layer and a deflated ball-like
shape of the dead ascospore. On the other hand, the HPP dead spore
presented in Fig. 10(b) was not as badly damaged, although the in-
tracellular constituents of the ascospore might have leaked out from the
destructed membrane and can be seen in the image. According to Marx
et al. (2011), the pressurized membrane alters the spore's permeability,
allowing for changes in volume, which justifies the imperfect round
shape of the HPP treated spore in Fig. 10(b).
4. Conclusion
Four different types of microbial spores were inactivated and ob-
served by scanning electron microscopy to assess both live and dead
spores: bacterial spores of A. acidoterrestris and G. stearothermophilus, N.
fischeri mould spores, and S. cerevisiae yeast spores. All spores possess
different characteristics and sizes in their live state. While S. cerevisiae
spores are spherical (2–2.4 μm diameter) and N. fisheri spores (1.3 μm
edge) are cubic shaped, the A. acidoterrestris (1.8–2 μm long) and G.
stearothermophilus spores (1.7–2 μm long) are more oval or rectangular
in a rod-like shape. Although S. cerevisiae spores are the largest and
coated with a multi-protection layer, they are the least resistant spores
amongst the species studied.
With thermal and HPP treatments, injured bacterial, mould and
yeast spores had irreversible volume and shape changes. The inactiva-
tion of bacterial spores is suggested to begin with injuries to the spore
membrane, which later causes the release of DPA. The absence of DPA
and increase of high temperature water inside the spore, eliminates the
protective ability of the spore to maintain the core, resulting in spore
death. Regarding the N. fischeri mould ascospore, the thermal proces-
sing treatment tore apart the inner spore membrane resulting in the
leakage of intracellular components. Thermal and non-thermal HPP
treatments of S. cerevisiae spores caused severe spore damage, char-
acterized by the formation of massive openings or holes from where the
leakage of intracellular components from the cytoplasm occurred. The
thermal treatment caused more visible/severe damage than HPP
treatment. Further investigations involving molecular techniques to
analyze the intracellular components of the live/dead spore (e.g. DPA)
are recommended in future studies.
Acknowledgements
Project n. 3708802 “Non-thermal pasteurization of beer and other
alcoholic beverages” funded by Morton Coutts grant, Dominion
Breweries is acknowledged. I am grateful to Rafael Uchida and Evelyn,
for gently providing bacterial and mould spores for this study. I want to
express my sincere thanks to Prof Bryony James and Catherine Hobbis
for support with SEM/eSEM analysis and electron microscopes. The
support of Adrian Turner, Peter Gordon Buchanan, Peter Barclay Martin
and Laura Liang with experimental works in the laboratory at
University of Auckland is appreciated.
24
International Journal of Food Microbiology 263 (2017) 17–25
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