MATERIALS AND METHODS

Strains, media, and spore preparation

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°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 ≥95% of the spores were observed by phase contrast microscopy.

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/cm3 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℃, 10 min, 105 rpm) twice, and the supernatant was discarded. The untreated control was centrifuged in the same manner for consistency. The centrifuged spores were diluted to ~107 spores/ml, stored in distilled water at 4°C, 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