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 (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 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 ~400 nm and the
height was reduced by ~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 (Figure 4 (d) ), and
some of the spores were obviously cracked (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.
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°C,
and bright field images were recorded every 30 s for 6 h (Figure
6 (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 (Figure 2
(a) ), the time of germination was much later (Figure 7(a)–(b) ), and the growth rate of spores was much slower
(Figure 7 (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].