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].