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 (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-1 as an example, its relative intensity decreased
dramatically as the treatment timeincreased (Figure 2(c) ). The percentages of spores releasing CaDPA increased
rapidly with an increase in treatment time (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-1, we
detected a slight shift after sodium hypochlorite treatment, although
the relative Raman intensity was largely unchanged (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
Figure 2 (a) Survival of spores after sodium hypochlorite
treatment obtained using plate culture and live cell imaging; (b)
average Raman spectra of 30 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-1. a.u., arbitrary units.
worth noting that the 1004 cm-1 attributed to
phenylalanine decreased after treatment, because sodium hypochlorite
reacts with amino acids in a neutralizing manner, damaging spore
proteins [24].