Bacterial strains and culture media
Aeromonas hydrophila and S. mutans were obtained from the Research Institute of LG Household a Health Care Co., Ltd. (Daejeon, Korea). X. oryzae (KACC 10331) and Y. enterocolitica (KCCM 41657) were obtained from the Genebank Information Center in the Rural Development Administration (Jeonju, Korea) and Korean Culture Center of Microorganisms (Seoul, Korea), respectively.
YGC agar plates were made with 50 g/l of glucose, 5 g/l of yeast extract, 12.5 g/l of CaCO3, and 15 g/l of agar. M210 medium and XOM2 medium were prepared as previously described by Zhang et al. (2013) and Tsuge et al. (2002), respectively. LB agar plates were made with 25 g/l of LB broth (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) and 15 g/l of agar. TYE medium was made with 10 g/l of tryptone and 5 g/l of yeast extract. M63G medium (100 ml) was made with 20 ml of 5× minimal M63 medium (15 g/l of KH2PO4, 35 g/l of K2HPO4, 10 g/l of (NH4)2SO4, and 2.5 mg/l of FeSO4), 1 ml of 20 % glucose, 0.1 ml of 20 % MgSO4, and filled with water. BHI medium was made with 37 g/l of brain heart infusion broth (Becton, Dickinson, and Company). To make BHI agar plates, 15 g/l of agar was additionally added to BHI medium. To make BHI-S medium, 10 g/l of sucrose was additionally added to BHI medium (Beckloff et al. 2007; Coenye et al. 2007). Tryptone soy broth with yeast extract (TSBY) was prepared as previously described (Kirov et al. 2004).
Monocaprylin (1-octanoyl-rac-glycerol, catalog number: M2265) and monocaprin (1-decanoyl-rac-glycerol, catalog number: M2140) were purchased from Sigma-Aldrich Korea Co. (Seoul, Korea). Monolaurin (1-lauroyl-rac-glycerol, catalog number: G0081) was purchased from Tokyo Chemical Industry Co., LTD (Tokyo, Japan). Monomyristin (1-myristoyl-rac-glycerol, catalog number: O-1335) and monopalmitin (1-palmitoyl-rac-glycerol, catalog number: O-1420) were purchased from Bachem AG (Bubendorf, Switzerland). Monostearin (1-stearoyl-rac-glycerol, catalog number: 43883) was purchased from Alfa Aesar (Seoul, Korea). Monoarachidin (1-arachidoyl-rac-glycerol, catalog number: 31-2000) and monobehenin (1-behenoyl-rac-glycerol, catalog number: 31-2200) were purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ, USA).
Biofilm formation in 96-well microplates
Xanthomonas oryzae stored at −80 °C was streaked on YGC agar plates and incubated at 28 °C for 2 days. A single colony was inoculated in 5 ml of M210 and incubated at 28 °C and 250 rpm for 2 days. The cell density of the culture was measured at A600. The cultured cells were inoculated in 100 μl of XOM2 media in 96-well polyvinyl chloride (PVC) microplates with 0.05 of A600 as the final cell concentration. The cells in the microplates were incubated at 28 °C for 24 h and the amount of biofilm was quantified using crystal violet.
Yersinia enterocolitica biofilm was produced according to a previously described method (Kim et al. 2008).
Streptococcus mutans biofilm was produced according to a method previously described for X. oryzae with some modifications for S. mutans. All cultures were cultivated at 37 °C. BHI agar plates were used to obtain a single colony from the stored strain and BHI medium was used to prepare a pre-culture for 1 day. BHI-S medium was used for biofilm formation in 96-well PVC microplates.
Aeromonas hydrophila biofilm was also produced according to a method previously described for X. oryzae with some modifications for A. hydrophila. All cultures were cultivated at 30 °C. LB agar plates were used to obtain a single colony from the stored strain for 1 day and LB medium was used to prepare a pre-culture for 1 day. TSBY medium was used for biofilm formation in 96-well PVC microplates.
Cell density and bacterial biofilm quantification using crystal violet
After 24 h of biofilm formation in 96-well PVC microplates, optical density of each well at 595 nm was measured before quantitative analysis of biofilm using an Opsys MR microplate reader (Dynex Technologies, Chantilly, VA, USA). The cell density was correlated with the optical density at 595 nm. Quantitative analyses of biofilms were performed using the crystal violet assay as described in a previous study (Kim et al. 2008) with a change of all incubation times from 20 to 15 min. The optical density at 595 nm of each well was also measured using an Opsys MR microplate reader.
Biofilm formation in continuous-culture flow cells
Biofilm formation of S. mutans in a flow cell system was observed as reported in a previous experiment with some modifications (Kim et al. 2008). The flow cells and the bubble traps were purchased from the Center for Biomedical Microbiology (Technical University of Denmark, Lyngby, Denmark). The flow cells were covered with microscope coverslips made from polyvinyl chloride. Media were fed using a peristaltic pump (Masterflex L/S Digital Drive EW-07523-90, Masterflex L/S 12-channel, 8-roller cartridge pump head EW-07519-25, Masterflex L/S small cartridges EW-07519-85, Cole-Parmer Instrument Company, LLC., Vernon Hills, IL USA).
First, monolaurin was dissolved in methanol and mixed with media in order to facilitate dissolution. The final concentration of monolaurin in BHI-S medium was 95 or 190 mg/l with 0.48 % methanol. S. mutans was incubated in 5 ml of BHI medium for 24 h at 37 °C without shaking. The cells were diluted to an OD600 of 0.15 with BHI-S media and inoculated in a 350 μl volume. The flow cells were incubated for 1 h with the coverslip side down. The flow cells were turned over and fed with media for 24 h at a speed of 13 ml/h according to a previous study (Wen et al. 2006). Biofilm formed on the coverslips was observed after 1 and 24 h of incubation using an Axio Scope.A1 microscope (Carl Zeiss Co., LTD., Seoul, Korea) with ZEN microscope software (Carl Zeiss Co., LTD.).
Measuring the sterilization effect of monolaurin on S. mutans in a planktonic growth
The inhibitory activity of monolaurin on the growth of S. mutans was measured by the colony forming unit (CFU) method. The monolaurin was dissolved in 10 % methanol immediately before treatment. Monolaurin was tested with various concentrations ranging up to 190 mg/l. The cells were incubated in BHI broth at 37 °C for 24 h as a pre-culture. The cultured cells with 0.05 of A600 as a final cell concentration were inoculated into 2 ml BHI media with 1 % sucrose and the test concentration of monolaurin. Cells were additionally incubated at 37 °C for 24 h. The homogeneous culture solution (100 µl) was spread onto BHI agar plates after serial dilution. The spread plates were incubated at 37 °C for 48 h and the number of colonies was counted. All experiments were repeated three times at each concentration.