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\begin{document}

\title{Single-cell analysis reveals microbial spore responses to sodium
hypochlorite}



\author[1]{Weiming Yang}%
\author[1]{Yufeng Yuan}%
\author[1]{Lin He}%
\author[2]{Haihua Fan}%
\affil[1]{Dongguan University of Technology}%
\affil[2]{South China Normal University School of Information and Optoelectronic Science and Engineering}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Pollution from toxic spores has caused us a lot of problems, because
spores are extremely resistant and can survive most disinfectants.
Therefore, the detection of spore response to disinfectant is of great
significance for the development of effective decontamination
strategies. In this work, we investigated the effect of 0.5\% sodium
hypochlorite on the molecular and morphological properties of single
spores of \emph{Bacillus subtilis} using single-cell techniques. Laser
tweezers Raman spectroscopy showed that sodium hypochlorite resulted in
Ca \textsuperscript{2+}-dipicolinic acid release and nucleic acid
denaturation. Atomic force microscopy showed that the surface of treated
spores changed from rough to smooth, protein shells were degraded at 10
min, and the permeability barrier was destroyed at 15 min. The spore
volume decreased gradually over time. Live-cell imaging showed that the
germination and growth rates decreased with increasing treatment time.
These results provide new insight into the response of spores to sodium
hypochlorite.%
\end{abstract}%



\sloppy


\textbf{Single-cell analysis reveals microbial spore responses to sodium
hypochlorite}

Weiming Yang\textsuperscript{1,2}, Yufeng Yuan\textsuperscript{1}, Lin
He11\textsuperscript{1}School of Electrical Engineering and
Intelligentization, Dongguan University of Technology, Dongguan,
Guangdong, China\textsuperscript{2}School of Information and
Optoelectronic Science and Engineering, South China Normal University,
Guangzhou, Guangdong, China *Correspondence Lin He, School of Electrical
Engineering and Intelligentization, Dongguan University of Technology,
Dongguan 523808, Guangdong, China E-mail: \emph{helin@dgut.edu.cn}Haihua
Fan, School of Information and Optoelectronic Science and Engineering,
South China Normal University, Guangzhou 510006, Guangdong, China
E-mail: \emph{fanhh@scnu.edu.cn}\textsuperscript{,*}, Haihua
Fan\textsuperscript{2,*}

\textbf{Abstract:} Pollution from toxic spores has caused us a lot of
problems, because spores are extremely resistant and can survive most
disinfectants. Therefore, the detection of spore response to
disinfectant is of great significance for the development of effective
decontamination strategies. In this work, we investigated the effect of
0.5\% sodium hypochlorite on the molecular and morphological properties
of single spores of \emph{Bacillus subtilis} using single-cell
techniques. Laser tweezers Raman spectroscopy showed that sodium
hypochlorite resulted in Ca\textsuperscript{2+}-dipicolinic acid release
and nucleic acid denaturation. Atomic force microscopy showed that the
surface of treated spores changed from rough to smooth, protein shells
were degraded at 10 min, and the permeability barrier was destroyed at
15 min. The spore volume decreased gradually over time. Live-cell
imaging showed that the germination and growth rates decreased with
increasing treatment time. These results provide new insight into the
response of spores to sodium hypochlorite.

\textbf{Key words} : atomic force microscopy; \emph{Bacillus subtilis} ;
laser tweezers Raman spectroscopy; morphological structure; single-cell
analysis

\section*{INTRODUCTION}

{\label{introduction}}

Spores are dormant bodies formed by spore-producing bacteria under
nutrient-poor conditions {[}1{]}. Spore formation is an extremely
complex process involving multiple changes in morphology, structure,
chemical composition, and other aspects. During sporulation, trophoblast
cells divide asymmetrically to form the prespore and mother cell
compartments, where the smaller prespore compartment is used for spore
formation and the larger mother cell compartment is used to cultivate
the spores and synthesize the external proteolipid layer structure.
Until the prespore is fully developed, the mother cell compartment
engulfs and fuses with the prespore compartment and forms a multilayered
protective layer around the prespore; when the spore is formed, the
mother cell ruptures to release the spore into the environment {[}2{]}.
The spore contains the complete genome of the species and is capable of
resuming growth to form a trophoblast under nutritive conditions. In
addition, spores have a special multi-layered structure and extremely
low water content, giving them the ability to survive for long periods
of time, even in extreme environments, making them extremely resilient.
For example, spores can survive in low and high temperatures, including
space environments, and can be exposed to strong acids and bases,
ethanol, etc {[}3-7{]}. The resilience of spores is mainly dependent on
two types of wall structures, an external protein wall, the spore shell,
and a peptidoglycan wall, the spore cortex. The spore shell is resistant
to a wide range of chemical and enzymatic treatments and acts as a
permeable barrier, limiting the entry of macromolecules into the spore.
The cortex, on the other hand, is a special peptidoglycan-rich structure
deposited between the inner and outer membranes of the spore,
responsible for maintaining the core in a highly dehydrated state, thus
contributing to the extreme dormancy and heat resistance {[}2{]}. In
addition, maintaining the dehydration of the spore core is dependent on
the inner membrane, which is relatively permeable and limits the entry
and exit of small molecules and chemicals {[}6{]}. Degradation of a
spore's cortex and damage to the inner membrane can result in the rapid
rehydration of the spore core and consequent loss of resistance {[}8{]}.
Notably, a specific molecule within the spore core,
pyridine-2,6-dicarboxylic acid (DPA), chelated 1:1 with
Ca\textsuperscript{2+} (CaDPA), plays an important role in spore
resistance and stability {[}9{]}. Although the exact function of CaDPA
in spores is not yet known, a lack of, or low levels of, CaDPA can
result in very unstable spores that germinate spontaneously {[}10{]}.

Currently, chlorination is a popular method for sterilization and sodium
hypochlorite (bleach) is widely used by virtue of its cheapness and
availability {[}11{]}. Many studies have evaluated the effects of sodium
hypochlorite on spores in terms of (i) spore viability, (ii) release of
budding molecules and (iii) electron microscopic capture of budding
structures. These studies have shown that at suitable concentrations and
exposure times, sodium hypochlorite is able to kill spores by damaging
their shells, germination proteins and DNA {[}12, 13{]}. However, there
is controversy as to whether CaDPA is released from spores after sodium
hypochlorite treatment. It has been shown that sodium hypochlorite
destroys the outer shell of spores and damages proteins on the inner
membrane, thus disrupting the permeability barrier, and leading to the
release of CaDPA from the core and DNA denaturation {[}14{]}. However,
in another study, spores did not release CaDPA after hypochlorite
treatment but released CaDPA at germination and were unable to grow
further {[}15{]}. The ability of hypochlorite to inactivate spores is
not disputed; however, its mechanism of action is not yet fully
understood. There are still many unresolved issues, including (i)
whether sodium hypochlorite-treated spores release substances, such as
DNA, at the time of CaDPA release and (ii) whether the degradation of
spore shells by sodium hypochlorite is localized or global. In addition,
methods used in previous studies, such as transmission electron
microscopy and scanning electron microscopy, require the special
treatment of individual organisms, which may cover up some information
about treated spores {[}16{]}. It should also be noted that the overuse
of chemicals to treat spores results in a series of problems, such as
strain resistance and contamination of the environment {[}11, 17{]}.

In the present study, we comprehensively evaluated the response of
spores to sodium hypochlorite by using single-cell techniques. We used
laser tweezer Raman spectroscopy (LTRS) to detect molecular changes in
spores after sodium hypochlorite treatment, evaluated spore morphology
before and after treatment using atomic force microscopy (AFM), and
observed the germination and growth process of single spores before and
after treatment using live-cell dynamic imaging. The results of this
study deepen our understanding of the response of spores to sodium
hypochlorite, including internal molecular and morphological structural
changes, and provide new insights into the effects of sodium
hypochlorite on spores at the single-cell level.

\section*{MATERIALS AND METHODS}

{\label{materials-and-methods}}

\subsection*{Strains, media, and spore
preparation}

{\label{strains-media-and-spore-preparation}}

\emph{Bacillus subtilis} CMCC(B)63501 was purchased from the Chinese
Medical Bacteria Preservation and Management Centre. Spores were
incubated on 30\% oligonutrient LB medium agar plates, and the ambient
temperature was controlled at about 37\selectlanguage{ngerman}°C. After 5 days of incubation,
the spores were collected and purified. The purified spores were stored
in distilled water at 4°C protected from light, and \selectlanguage{english}[?]95\% of the spores
were observed by phase contrast microscopy.

\subsection*{Measurement of sodium hypochlorite treatment and internal
molecules in
spores}

{\label{measurement-of-sodium-hypochlorite-treatment-and-internal-molecules-in-spores}}

The sodium hypochlorite solution was purchased from Shanghai Aladdin
Biochemical Science and Technology Co. Ltd. (product no. S298682), with
a density of 1.2 g/cm\textsuperscript{3} and an effective chlorine
content of 100--140 g/l. Anhydrous sodium thiosulphate was also
purchased from Shanghai Aladdin Biochemical Science and Technology Co.
Ltd. (product no. S100824). The spores were incubated in 0.5\% sodium
hypochlorite solution for 5, 10, 15 and 20 min, followed by addition of
5\% sodium thiosulphate solution. Samples were left for 10 min for
sufficient reaction, centrifuged (4\selectlanguage{english}, 10 min, 10\textsuperscript{5} rpm)
twice, and the supernatant was discarded. The untreated control was
centrifuged in the same manner for consistency. The centrifuged spores
were diluted to \textasciitilde{}10\textsuperscript{7} spores/ml, stored
in distilled water at 4degC, protected from light, and subsequently
analyzed for viability and assayed by LTRS.

Individual spores suspended in water were randomly trapped by the laser
beam, and Raman spectroscopy was used to assess internal changes in the
molecule. The scheme\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} Illustration of the LTRS setup used to acquire Raman
spectra of individual spores. The trapping laser is reflected by a
planar dichroic mirror and focused through an objective lens to form an
optical potential well in the sample cavity. The captured spore Raman
scattering excited by the captured laser is returned along the original
path and focused into the spectrometer. To illuminate a sample, we used
a lamp and acquired images using a CMOS camera. When the trapping laser
is turned on, the trapped spore is imprisoned. When the trapping laser
is turned off, the spore can move freely.

of the LTRS system is shown in \textbf{Figure 1} . A diode laser of 532
nm wavelength is used for trapping and Raman excitation of the
individual spores. The combination of two biconvex lens
(L\textsubscript{1} (f\textsubscript{1}=100mm) and
L\textsubscript{2}(f\textsubscript{2}=150mm)) is used to expand the beam
to overfill the inverted microscope objective (100\selectlanguage{ngerman}×, 1.40 NA), which
acts as a trapping and Raman excitation and collection objective. The
spectrometer (HR Evolution, Horiba Jobin 7 Yvon, Japan) has an 1800
gr/mm grating blazed at 500 nm and a liquid nitrogen-cooled charge
coupled device (CCD). The laser power was 2.5 mW and integration time
was 30s. The characteristic peaks of CaDPA are located at 660, 825, and
1017 cm\textsuperscript{-1}, the peak at 782 cm\textsuperscript{-1} is
attributed to DNA, and the peak at 1004 cm\textsuperscript{-1} to
phenylalanine {[}18, 19{]}. The content of each type of substance can be
determined based on the relative intensities of the characteristic peaks
{[}20{]}.

\subsection*{Phase contrast
micrography}

{\label{phase-contrast-micrography}}

To investigate CaDPA release from the core of the spores before and
after sodium hypochlorite treatment, the state of spores was observed
using phase contrast microscopy. Briefly, 2 \selectlanguage{greek}μ\selectlanguage{english}l of the budding suspension
was added dropwise to a slide and dried in a vacuum desiccator for 10
min. Approximately 300 \selectlanguage{greek}μ\selectlanguage{english}l of sterile water was added to the sample
chamber and a cover glass was added to form an airtight space. The
sample chamber was fixed on an inverted microscope (Ti2; Nikon, Tokyo,
Japan) with a 100\selectlanguage{ngerman}× oil lens (Nikon). Images were obtained with a CMOS
camera (2048×2048).

\subsection*{Cell viability analysis}

{\label{cell-viability-analysis}}

Untreated and sodium hypochlorite-treated spore samples were diluted to
a suitable concentration of spore suspension, coated on nutrient-rich LB
agar medium, and placed in a constant temperature incubator at 37°C for
48 h, after which the colonies were counted and the survival of each
group of spore samples was calculated.

\subsection*{AFM imaging of spores}

{\label{afm-imaging-of-spores}}

Spore suspensions were added dropwise to Ploy-coated slides, allowed to
stand in air, and dried for 30 min. Slides were fixed to sample holders
and images were obtained in air. Images were acquired using a Bioscience
AFM (NanoWizer4; Bruker, Wissembourg, France) operated in AC mode. The
AFM probe used for imaging consisted of a silicon tip on a silicon
nitride cantilever beam with a resonance frequency of --320 kHz and an
elasticity coefficient of 42 N/m. All images were scanned at 512 pixels
per line with a scanning frequency of 0.5--1.0 Hz.

\subsection*{Dynamic live-cell imaging}

{\label{dynamic-live-cell-imaging}}

To analyze the sprouting and growth of untreated and sodium
hypochlorite-treated spores, dynamic imaging of live cells dynamic
imaging (N-storm; Nikon) was used to observe and record growth in real
time. A small drop of budding solution was spread on a slide and dried
naturally at 25°C for half an hour in order to distribute the spores
evenly. A drop of melted 100\% enriched LB agar medium was increased by
approximately 400 \selectlanguage{greek}μ\selectlanguage{english}l to the top of the sample spores to form an agar pad
with a thickness of about 3 mm. The agar on one side of the pad was
removed to form some small holes for air flow, and was subsequently
covered with a coverslip to form an airtight space above the agar. The
slides were visualized under an inverted microscope (Ti2; Nikon) and the
growth of the spores was recorded with a CCD camera (12bits, 2044 \selectlanguage{ngerman}×
2048) at 30 s/frame for 6h at a constant temperature of 37°C.

\section*{RESULTS AND DISCUSSION}

{\label{results-and-discussion}}

\subsection*{Survival and Raman spectroscopy analysis of spores after
sodium hypochlorite
treatment}

{\label{survival-and-raman-spectroscopy-analysis-of-spores-after-sodium-hypochlorite-treatment}}

Sodium hypochlorite (bleach) is an inexpensive and common decontaminant,
and a concentration of 0.5\% has been reported to kill spores {[}21{]}.
In addition, sodium hypochlorite degrades organic matter through a
variety of reactions such as saponification of fatty acids,
neutralization of amino acids, and ammoniation {[}22{]}. Therefore, the
treatment of spores with sodium hypochlorite can cause damage to lipids,
proteins, and DNA.

In this study, two methods were used to record spore survival, plate
culture and live-cell imaging techniques. The survival of spores after
sodium hypochlorite treatment was significantly lower than that of
untreated spores and showed a decreasing trend with increasing treatment
time (\textbf{Figure 2 (a)} ). In addition, the maximum difference (5
min) in spore survival obtained using these two methods was
\textasciitilde{}3\%, indicating that the results obtained using dynamic
live-cell imaging were consistent with the actual status of spores
before and after treatment.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

To observe the effects of sodium hypochlorite on spore properties, LTRS
was used to compare the Raman spectra of spores treated with sodium
hypochlorite for 0, 5, 10, 15, and 20 min (\textbf{Figure 2 (b)} ). We
found substantial changes in the Raman spectra of treated spores, with
the most obvious change in the characteristic peak of CaDPA. Taking 1017
cm\textsuperscript{-1} as an example, its relative intensity decreased
dramatically as the treatment timeincreased (\textbf{Figure 2(c)} ). The
percentages of spores releasing CaDPA increased rapidly with an increase
in treatment time (\textbf{Figure 3} ). These two results indicate that
sodium hypochlorite treatment caused the spores to release CaDPA and had
greater effects on spores over time. It is worth noting that CaDPA is
located in the core of spores, and the inner membrane controls the entry
and exit of materials from the core. Therefore, the release of CaDPA
from spores after sodium hypochlorite treatment is sufficient evidence
that sodium hypochlorite disrupts the permeability barrier of the
spores, including the inner membrane {[}14, 23{]}. The mechanism by
which sodium hypochlorite promotes the release of CaDPA from the spores
is not fully understood; however, Raman spectroscopy and phase contrast
micrography results suggest that CaDPA is released from spores after
treatment and that the number of individuals releasing CaDPA increases
with the duration of the treatment are in agreement with the results of
a previous report {[}14{]}. By comparing the Raman peaks of DNA at 782
cm\textsuperscript{-1}, we detected a slight shift after sodium
hypochlorite treatment, although the relative Raman intensity was
largely unchanged (\textbf{Figure 2(d)} ). These results indicated that
sodium hypochlorite may damage the DNA of spores, without affecting the
DNA content. Among the 30 spectra for single spores measured in each
group at 0, 5, 10, 15, and 20min, the spectra of 0, 0, 2, 6, and 15
spores had shifted peaks, respectively, indicating that DNA denaturation
occurred and intensified as the treatment time increase {[}14{]}. It can
be speculated that sodium hypochlorite entered the spores after
destroying the permeability barrier, resulting in damage to the DNA but
not the loss of DNA. It is

\textbf{Figure 2} (a) Survival of spores after sodium hypochlorite
treatment obtained using plate culture and live cell imaging; (b)
average Raman spectra of 30 \emph{Bacillus subtilis} spores after
different times of sodium hypochlorite treatment; (c) average Raman
spectra in the CaDPA bands and the number of spores releasing CaDPA at
different time; and (d) average Raman spectra in the DNA band and the
offset at 782 cm\textsuperscript{-1}. a.u., arbitrary units.

worth noting that the 1004 cm\textsuperscript{-1} attributed to
phenylalanine decreased after treatment, because sodium hypochlorite
reacts with amino acids in a neutralizing manner, damaging spore
proteins {[}24{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} Phase contrast micrographs of \emph{Bacillus subtilis}
spores at different times of sodium hypochlorite treatment. (a) 0 min;
(b) 5 min; (c) 10 min; (d) 15 min; (e) 20 min; (f) proportion of CaDPA
released at different times. Scale bar of the figure is: 5 \selectlanguage{greek}μ\selectlanguage{english}m. Red
circles are spores releasing CaDPA.

\subsection*{Effect of sodium hypochlorite on the surface morphology and
size of
spores}

{\label{effect-of-sodium-hypochlorite-on-the-surface-morphology-and-size-of-spores}}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

To confirm the effect of sodium hypochlorite on the surface topography
and morphology of spores, groups of spores treated with sodium
hypochlorite for different times were characterized separately using
AFM. Most of the untreated spores had a relatively rough outer surface
(\textbf{Figure 4} \textbf{(a)} ), with some individuals showing shallow
ridges on the surface. After sodium hypochlorite treatment, the external
surface of the spore was obviously changed; the surface of the spore
individual was uneven and had protrusions consistent with shell protein
retention after 5 min. The surface of the spore individual treated for
10 min was extremely smooth, with a few attachments. After treatment for
15 min, obvious ridges appeared on the spore surface, holes of different
sizes were detected, and there were many crystal-like attachments around
the spore. After 20 min, it became smoother, the individual spores
became significantly smaller, and some spores had gaps on the surface
(\textbf{Figure 4(b)--(e)} ). Sodium hypochlorite treatment caused
obvious damage to the surface morphology and morphological

\textbf{Figure 4} AFM images of individual spores treated with sodium
hypochlorite for different times. (a) 0 min; (b) 5 min; (c) 10 min; (d)
and (e) 15 min; (f) 20 min. (Picture size of individual spores: 2.5 \selectlanguage{greek}μ\selectlanguage{english}m,
scale bar: 500 nm; picture size of local details of spores: 500 nm,
scale bar: 100 nm)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\par\null

\textbf{Figure 5} Variation curves of length, width and height
dimensions of spores after different times of treatment.

structure of spores. Previous studies have shown that DPA within spores
exists in the form of CaDPA crystals; accordingly, it is likely that
these crystal-like attachments are CaDPA {[}25{]}. In addition, sodium
hypochlorite degrades the spore shells and cortical peptidoglycan
{[}26{]}. The overall trend in which the length, width, and height of
the spores decreased as the treatment time increased (\textbf{Figure5} )
is consistent with the degradation of the shells by sodium hypochlorite
and core CaDPA leakage.

Our AFM images confirmed that sodium hypochlorite degrades the tissue
structure of spores. As the treatment time increased, sodium
hypochlorite destroyed and degraded the morphology and structure of the
spores from the outside in. At 0--10 min, substantial alterations in
surface morphology were detected, and the rough structure of the outer
layer gradually disappeared, revealing a relatively smooth structure.
However, electron microscopy released that the thicknesses of the outer
shell protein layer and cortex of \emph{Bacillus subtilis} were about
200 nm and 70--200 nm, respectively {[}2, 27{]}. At 10 min, the length
and width of the buds were reduced by \textasciitilde{}400 nm and the
height was reduced by \textasciitilde{}100 nm, indicating that at this
time, the rough outer shell protein layer of the buds was almost
completely degraded and the smooth cortex layer was revealed. Sodium
hypochlorite continued to destroy the cortical structure of the spores;
at 15 min, many holes were distributed on the surface of the spores,
some of the spores swelled locally to form irregular ridges
(\textbf{Figure 4 (d)} ), and some of the spores were obviously cracked
(\textbf{Figure 4 (e)} ). At this time, a large number of spores
released CaDPA, indicating that sodium hypochlorite can continue to
degrade the cortical structure of the spores, destroying the osmotic
pressure barrier of the spores. The enabled outside substances to enter
the spores, and the spores swelled to form ridges. When the osmotic
barrier was destroyed to a certain extent, the internal substances were
released, such as CaDPA. In addition, according to Raman spectra of DNA,
as the contact time with sodium hypochlorite increased, more spore
individuals showed deviations in characteristic peaks. Approximately
50\% of the individuals had deviations by 20min, indicating that sodium
hypochlorite is highly likely to enter into the spore and damage DNA.
Furthermore, large gaps appeared on the surface of the spores at 20 min,
indicating that the degree of damage increased with the time. By
comparing the AFM images of spores at different time points, we can see
that spore damage cause by sodium hypochlorite occurs from the outside
to the inside, and prolonging the treatment time will aggravate damage.

\subsection*{Effects of sodium chlorate on the sprouting and growth of
spores}

{\label{effects-of-sodium-chlorate-on-the-sprouting-and-growth-of-spores}}

To analyze the effect of sodium hypochlorite on germination and growth,
the spores were cultured on 100\% enriched LB agar Petri dishes at 37\selectlanguage{ngerman}°C,
and bright field images were recorded every 30 s for 6 h (\textbf{Figure
6} \textbf{(a)--(e)} ). Some of the spores treated with sodium
hypochlorite were still able to germinate and grow. However, compared
with those of untreated spores, the germination rate of sodium
hypochlorite-treated spores was substantially lower (\textbf{Figure 2
(a)} ), the time of germination was much later (\textbf{Figure
7(a)--(b)} ), and the growth rate of spores was much slower
(\textbf{Figure 7} \textbf{(c)} ), indicating that sodium hypochlorite
had an extremely strong inhibitory effect on spore germination and
growth, and the inhibitory effect was more obvious over longer time
periods. The inhibitory effect of sodium hypochlorite on spores' growth
can be explained in two ways. (i) Proteins related to sprout growth were
damaged. The spore shell has a multilayered structure with a variety of
proteins; it serves as a permeability barrier, restricting
macromolecules from entering the interior, and is able to sense changes
in the external environment, which plays an important role in the
process of germination and growth {[}6, 28, 29{]}. Sodium hypochlorite
degraded the shell of the spores, destroying proteins attached to the
shell, making spores unable to receive and transmit the signals for
germination quickly. Therefore, there was a lag in the germination and
growth of spores and, eventually, failure to germinate. Previous studies
have also shown that hypochlorite can cause significant damage to a
variety of germination proteins {[}15{]}. (ii) It can be explained by
DNA damage. Nucleic acids store genetic information for survival and
reproduction {[}6{]}. Raman spectroscopy and AFM images showed that
sodium hypochlorite disrupts the permeability barrier of the spores;
while the spores release CaDPA from the core, external sodium
hypochlorite enters the interior of the spores and may cause damage to
substances, such as DNA and proteins. As a result of DNA damage, genomic
instability and the loss of some growth-related functions may arise,
leading to abnormal spore growth {[}30{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6} Images of germination and growth of spores after
different times of sodium hypochlorite treatment. (a) 0 min; (b) 5 min;
(c) 10 min; (d) 15 min; (e) 20 min. Black, green and orange arrows
labelled are the sprouted individuals incubated at 37\selectlanguage{ngerman}°C for 1h, 2h and
3h respectively. (Scale bar in the figure: 10 \selectlanguage{greek}μ\selectlanguage{english}m.)\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
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\textbf{Figure 7} (a) Mean curves of germination rate versus time for
untreated spores; (b) mean curves of germination rate versus time for
spores after different times of sodium hypochlorite treatment; (c) mean
curves of growth length versus time for spores after sodium hypochlorite
treatment; (d)--(h) curves of cell length versus time for single spores
treated with sodium hypochlorite for 0--20 min.

In addition, we found that surviving spores after sodium hypochlorite
treatment were able to germinate and grow to form trophoblasts; however,
inactivated spores were unable to germinate despite incubation for up to
3 h (\textbf{Figure 6} ). Notably, there were differences in the curves
of cell length versus time for eight single spores (treated and
untreated groups) (\textbf{Figure 7 (d)--(h)} ), indicating that there
were individual differences in the effect of sodium hypochlorite on
spores under the same conditions, reflecting the individual
heterogeneity of the spores {[}31{]}.

\section*{CONCLUSIONS}

{\label{conclusions}}

The key contribution of this study was the analysis of the response of
spores to sodium hypochlorite at the single-cell level. Using
single-cell techniques, our results revealed that sodium hypochlorite
inactivates spores by degrading the spore shell, damaging germination
proteins, breaking the permeability barrier, and damaging DNA. Upon
contact with spores, sodium hypochlorite reacted with the shell protein
layer, gradually dissolving the shell until it was completely degraded
and the attached budding proteins were destroyed. Subsequently, it
damaged the cortex, endosperm, and other structures of the spores,
destroying the permeability barrier and causing the spores to release
CaDPA. Sodium hypochlorite entered the interior of the spores, causing
damage to DNA and other substances and ultimately leading to spore
inactivation. In addition, with the prolongation of sodium hypochlorite
treatment, the inhibitory effects on germination and growth increased,
and damage to the structure of spores was more serious. Long-term sodium
hypochlorite treatment will continue to degrade other structures of the
spores and even the entire spore. The volume of the spores may decrease
dramatically after degradation, making the acquisition of single-cell
Raman spectra difficult; therefore, we controlled the treatment time to
less than 20 min. The effects of increasing the treatment time on spores
need to be investigated in the further.

\section*{Funding}

{\label{funding}}

Lin He and Weiming Yang received support from the National Natural
Science Foundation of China (Grant No. 91851210).

\section*{Author contributions}

{\label{author-contributions}}

LH and HF contributed to conception, design of the study and
supervision. WY carried out the experimental design, performed the
experiments and the statistical analysis. YY was responsible for writing
- reviewing and editing. All authors contributed to the article and
approved the submitted version.

\section*{Data availability}

{\label{data-availability}}

Data underlying the results presented in this paper are not publicly
available at this time but may be obtained from the corresponding author
upon reasonable request.

\section*{Conflict of interest}

{\label{conflict-of-interest}}

The authors have no conflicts of interest to declare that are relevant
to the content of this article.

\section*{Consent for publication}

{\label{consent-for-publication}}

The university is aware about the work has consent for publication. The
authors declare consent for publication.

\section*{ORCID}

{\label{orcid}}

Lin He:\emph{https://orcid.org/0009-0009-7479-8557}

\section*{References}

{\label{references}}

{[}1{]} G.C. Stewart, \emph{Microbiol. Mol. Biol. Rev.} \textbf{2015} ,
79, 437.

{[}2{]} A. Driks, \emph{Microbiol. Mol. Biol. Rev.} \textbf{1999} , 63,
1.

{[}3{]} M.C.T. Nguyen, H.Q. Nguyen, H. Jang, S. Noh, S.Y. Lee, K.S.
Jang, J. Lee, Y. Sohn, K. Yee, H. Jung, J. Kim,
\emph{Analyst}\textbf{2021} , 146, 7682.

{[}4{]} C. Camerlingo, G. Di Meo, M. Lepore, M. Lisitskiy, A. Poli, M.
Portaccio, I. Romano, P. Di Donato, \emph{Sensors} \textbf{2020} , 20.

{[}5{]} T. Kobayashi, T. Azuma, D. Yasokawa, S. Yamaki, K.
Yamazaki,\emph{Biocontrol Sci.} \textbf{2021} , 26, 147.

{[}6{]} P. Setlow, \emph{Microbiol. spectr.} \textbf{2014} , 2.

{[}7{]} L. He, S.W. Wang, M. Cortesao, M.Y. Wu, R. Moeller, P. Setlow,
Y.Q. Li, \emph{Npj Microgravity} \textbf{2018} , 4.

{[}8{]} D.L. Popham, J. Helin, C.E. Costello, P. Setlow, \emph{Proc.
Natl. Acad. Sci. USA} \textbf{1996} , 93, 15405.

{[}9{]} B. Setlow, S. Atluri, R. Kitchel, K. Koziol-Dube, P.
Setlow,\emph{J. Bacteriol.} \textbf{2006} , 188, 3740.

{[}10{]} P. Setlow, \emph{Curr. Opin. Microbiol.} \textbf{2003} , 6,
550.

{[}11{]} I.A. Jones, L.T. Joshi, \emph{Molecules} \textbf{2021} , 26.

{[}12{]} J.H. Yim, K.Y. Song, H. Kim, D. Bae, J.W. Chon, K.H.
Seo,\emph{J. Veterin. Sci.} \textbf{2021} , 22.

{[}13{]} D. Malyshev, I.A. Jones, M. McKracken, R. \selectlanguage{ngerman}Öberg, G.M. Harper,
L.T. Joshi, M. Andersson, \emph{Bmc Microbiol.} \textbf{2023} , 23.

{[}14{]} D. Malyshev, T. Dahlberg, K. Wiklund, P.O. Andersson, S.
Henriksson, M. Andersson, \emph{Analyt. Chem.} \textbf{2021} , 93, 3146.

{[}15{]} B. Setlow, J. Yu, Y.Q. Li, P. Setlow, \emph{Letters in Applied
Microbiology} , \textbf{2013} , 57, 259.

{[}16{]} R.F. Egerton, P. Li, M. Malac, \emph{Micron} \textbf{2004} ,
35, 399.

{[}17{]} R.A. Wu, J.S. Feng, M. Yue, D.H. Liu, T. Ding, \emph{Crit. Rev.
Food Sci. Nutri.} \textbf{2023} .

{[}18{]} L.B. Kong, P. Setlow, Y.Q. Li, \emph{Analyst} \textbf{2012} ,
137, 3683.

{[}19{]} S.W. Wang, C.J. Doona, P. Setlow, Y.Q. Li, \emph{Appl. Environ.
Microbiol.} \textbf{2016} , 82, 5775.

{[}20{]} K. McCann, J. Laane, \emph{J. Mol. Struct.} \textbf{2008} ,
890, 346.

{[}21{]} F. Barbut, D. Menuet, M. Verachten, E. Girou, \emph{Infect.
Control Hosp. Epidem.} \textbf{2009} , 30, 507.

{[}22{]} C. Estrela, C.R.A. Estrela, E.L. Barbin, J.C.E. Spano, M.A.
Marchesan, J.D. Pecora, \emph{Brazil. Dent. J.} \textbf{2002} , 13, 113.

{[}23{]} D.E. Cortezzo, K. Koziol-Dube, B. Setlow, P. Setlow, \emph{J.
Appl. Microbiol.} \textbf{2004} , 97, 838.

{[}24{]} K. Gallandat, R.C. Kolus, T.R. Julian, D.S. Lantagne, \emph{Am.
J. Infect. Control} \textbf{2021} , 49, 90.

{[}25{]} R.S. Huang, X. Huang, L.L. Xu, Y.Q. Li, S.S.
Huang,\emph{Spectrosc. Spectr. Analys.} \textbf{2011} , 31, 681.

{[}26{]} S.F. Bloomfield, M. Arthur, \emph{The Journal of applied
bacteriology} , \textbf{1992} , 72, 166.

{[}27{]} D.L. Popham, \emph{Cell. Mol. Life Sci.} \textbf{2002} , 59,
426.

{[}28{]} D. Imamura, R. Kuwana, H. Takamatsu, K. Watabe, \emph{J.
Bacteriol.} \textbf{2010} , 192, 518.

{[}29{]} D. Krajcíková, V. Forgác, A. Szabo, I. Barák, \emph{Microbiol.
Res.} \textbf{2017} , 204, 72.

{[}30{]} S.Y. Qiu, H.H. Fan, L. He, \emph{J. Innov. Opti. Health
Sci.}\textbf{2023} , 16.

{[}31{]} P. Setlow, J. Liu, J.R. Faeder, \emph{Heterogeneity in
Bacterial Spore Populations} , \textbf{2012.}\selectlanguage{english}
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