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