Escherichia coli ATCC 25922(=NBRC15034) was purchased from the National Institute of Technology and Evaluation Biological Resource Center (NRBC). The bacteria were stored in 10% glycerol solution at −85˚C. Prior to experiment, they were thawed and pre-incubated in nutrient broth (Eiken Chemicals Co. Ltd., Tokyo, Japan) at 37˚C for 24 h. The pre-incubated E. coli ATCC 25922 cells were washed and resuspended in 10 mL sterile saline. A portion (0.1 mL) of the bacterial suspension was added to a 100-mL plastic vial (IWAKI, Asahi Glass Co. Ltd.) containing 40 mL of Todd-Hewitt Broth (Oxoid, Cambridge, UK). A sterilized slide glass plate (76 × 26 mm, Matsunami Glass Ind. Ltd., Tokyo, Japan) was immersed in the broth and incubated at 37˚C for 48 h (110 strokes/min). Following incubation, the plate was removed from the broth and gently washed twice with 10 mL sterile deionized water (hereafter, this plate is called the BF plate). For the conductimetric assay, a small piece (10 mm × 26 mm) of the prepared biofilm plate was cut with a diamond cutter.

Powder from scallop-shells (Patinopecten yessoensis) was obtained from Soycom Co. Ltd. (Atsugi, Kanagawa, Japan). The powder was heated at 1000˚C in air for 1 h and ground in a ball mill. The approximate mean size of the HSSP particles was 5 µm. The calcium and magnesium content of scallop shell powder heated at 1000˚C for 1 h was 70.8 wt% and 0.16 wt%, respectively. Trace amounts of phosphorus (0.073 wt%), sodium (0.014 wt%), and iron (0.003 wt%) were also present. Silver and copper, which possess antibiotic properties, were not detected (<0.01 mg/kg). At a calcium concentration of 70.8 wt%, the shell contains 99% CaO by weight (Sawai et al., 2001). The powder was mixed with sterile saline (0.85 w/v%) to produce a slurry. The prepared biofilm plate was immersed in a vial containing the prepared HSSP slurry (100 mL) and incubated at 37˚C (110 strokes/min).

The above procedure was repeated for sodium hydroxide (NaOH) and sodium hypochlorite (Wako Pure Chemical Industries) treatments. NaOH and sodium hypochlorite were solubilized in sterile deionized water.

Electric conductivity was measured by a RABIT™ (Rapid Automated Bacterial Impedance Technique) system (Don Whitley Scientific Ltd., UK). Conductivity was measured in a capped sample tube containing paired electrodes at the bottom. Conductimetric assays detect microbial growth and metabolism as changes in the electrical conductivity or impedance of the growth medium [24]. Such conductivity changes indicate the presence of electrolytes produced by metabolic activity, such as the conversion of glucose to lactic acid and/or increased mobility caused by the cleavage of large charged molecules (such as proteins) into smaller, more mobile charged molecules (such as amino acids). When the difference between two successive electrical conductivity measurements of the medium exceeds 5 µS, the RABIT™ system detects the change. The time required to reach the threshold concentration is called the TTD (time to detection); the TTD values may be used as criteria for determining bacterial growth [24].

The BF plates exposed to HSSP or other treatments were gently washed twice with 10 mL sterile deionized water, and immersed in the RABIT sample tubes containing 5 mL Whitley Impedance Broth (WIB: Don Whitley Scientific Ltd.). An untreated BF plate was used as a control. The tubes were set in the incubator module, and the conductivity changes in the growth medium, induced by metabolism and growth of E. coli, were monitored at 37˚C at 6 min intervals for 48 h.

Growth of E. coli ATCC 25922 was calibrated from the planktonic cells and the cells attached to the glass plate. To assess planktonic cell growth, serial ten-fold dilutions of E. coli suspensions were prepared with sterile saline. A 0.1 mL aliquot of each diluted suspension was added to the RABIT sample tube containing 5 mL WIB, and the conductivity change was measured at 37˚C for 48 h at 6 min intervals.

For assessing the growth of cells attached to the glass plate, serial ten-fold dilutions of suspended cells were also prepared with sterile saline. A 5 mL aliquot of E. coli suspension was spread onto the glass plate (10 mm × 26 mm) and dried. The dried plate was immersed in the RABIT sample tube containing 5 mL of WIB, and the conductivity change was measured at 37˚C for 48 h at 6 min intervals.

The BF plates exposed to HSSP and other treatments were stained with LIVE/DEAD BacLight™ Bacterial Viability Kit (Invitrogen Co., CA, USA). This assay uses SYTO9 and propidium iodide (PI) to discriminate between live cells with intact membranes (green fluorescence) and dead cells with compromised membranes (red fluorescence). The cells were stained according to the manufacturer’s instructions. The stained BF plate was examined by fluorescence microscopy (Olympus FV1000D IX81, Tokyo, Japan).

Active oxygen species generated from the HSSP react with luminol and trigger a chemiluminescent response, as described by Kohtani et al. [25]. A Mithras LB09470 microplate reader (Berthold Technologies, Bad Wildbad, Germany) was used for measurements. Samples of HSSP slurry (100 μL) were pipetted into 96-well microplates, and the chemiluminescence reaction was initiated by adding 50 μL of 0.7 mM luminol (Nacalai Tesque, Kyoto, Japan). Chemiluminescence was then recorded by the microplate reader. To examine the effects of antioxidative enzymes, 25 µL of 0.1 mg/mL superoxide-dismutase (SOD) or 0.1 mg/mL catalase solution (Wako Pure Chemical Industries) was added to the wells from the dispenser prior to luminol addition.

Each experiment was performed in triplicate and the average values are reported in the results.