Incubator shaker

In this study we used methicillin-resistant Staphylococcus aureus (MRSA) USA 300 as Gram-positive bacteria; Escherichia coli K-12 (ATCC 33780) as Gram-negative bacteria and a luciferase-expressing Candida albicans strain (CEC 749) as fungal yeast. A colony of bacteria or fungal yeast was suspended in 20 mL of brain heart infusion (BHI) broth for bacteria or yeast extract-peptone-dextrose (YPD) broth for C. albicans and grown overnight in a shaker incubator (New Brunswick Scientific, Edison, NJ) at 120 rpm under aerobic conditions at 37 °C for bacteria, at 30 °C for C. albicans. For bacteria, an aliquot of 1 mL from an overnight bacterial suspension was refreshed in fresh BHI for 2–3 h at 37 °C to mid-log phase. Cell concentration was estimated by measuring the optical density (OD) at 600 nm (OD of 0.6 = 10^s cells/mL). The C. albicans cell number was assessed with a hemocytometer and was generally between 10⁷ and 10^s cells/mL. The suspensions were centrifuged (5 min, 4000 rpm) and the pellet was suspended in sterile phosphate-buffered saline (PBS) at concentrations of 10⁷⁻⁸CFU/mL for phototoxicity experiments.

The following microbial strains were used: Gram-positive bacterium, methicillin-resistant Staphylococcus aureus (MRSA) US300; Gram-negative bacteria, Escherichia coli K-12 (ATCC 33780), Proteus mirabilis ATCC 51393 (Xen 44), Klebsiella pneumoniae, Acinetobacter baumannii ATCC BAA 747, and Pseudomonas aeruginosa ATCC 19660 (Xen 5P); fungal yeast, a luciferase-expressing Candida albicans strain (CEC 749). A colony of bacteria or fungal yeast was suspended in 20 mL of brain–heart infusion (BHI) broth for bacteria or yeast extract–peptone–dextrose (YPD) broth for C. albicans and grown overnight in a shaker incubator (New Brunswick Scientific, Edison, NJ, USA) at 120 rpm under aerobic conditions at 37 °C for bacteria and at 30 °C for C. albicans. For bacteria, an aliquot of 1 mL from an overnight bacterial suspension was refreshed in fresh BHI for 2–3 h at 37 °C to mid log phase. Cell concentration was estimated by measuring the optical density (OD) at 600 nm (OD of 0.6 = 10⁸ CFU cells/mL). The C. albicans cell number was assessed with a hemocytometer.

The following microbial strains were used: Gram-positive bacterium: methicillin-resistant Staphylococcus aureus (MRSA) USA 300; Gram-negative bacteria: Escherichia coli (E.coli) K-12 (ATCC 33780), Pseudomonas aeruginosa (P. aeruginosa) ATCC 19660 (Xen 5P), Proteus mirabilis (P. mirabilis) ATCC 51393 (Xen 44), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii) ATCC BAA 747 and E. coli UTI 89, UTI89 was a generous gift from Dr. Patrick Seed’s laboratory, which is a clinical cystitis isolate strain from humans previously described by Mulvey et al [20 (link)]. For a fungal yeast: strain CEC 749 of the luciferase-expressing fungal yeast Candida albicans (C. albicans). A colony of bacteria was suspended in 5 mL of brain heart infusion (BHI) broth and grown overnight in a shaker incubator (New Brunswick Scientific, Edison, NJ) at 120 rpm under aerobic conditions at 37 °C. An aliquot of 1 mL from an overnight bacterial suspension was refreshed in fresh BHI for 2–3 h at 37 °C to mid-log phase. Cell concentration was estimated by measuring the optical density (OD) at 600 nm (OD of 0.6 = 10⁸ cells/mL). A colony of C. albicans was suspended in 10 mL of yeast extract-peptone-dextrose (YPD) and grown overnight in a shaker incubator at 30 °C. The C. albicans cell number was assessed with a hemocytometer and was generally between 10⁷ and 10⁸ cells/mL.

The bacterial strains were: methicillin-resistant Staphylococcus aureus
(MRSA) USA 300, Escherichia coli (E. coli) K-12 (ATCC 33780) A
single colony of bacteria was grown overnight in 5 mL of brain heart infusion
(BHI) broth in a shaker incubator (New Brunswick Scientific) then refreshed in
BHI for 2-3 h at 37 °C to mid-log phase. Cell concentration was estimated
by measuring the optical density (OD) at 600 nm (OD of 0.6 = 10⁸cells/mL). The bacterial suspension was centrifuged, washed, and resuspended in
pH7.4 PBS to arrest microbial growth and 10⁸ cells/mL used for
experiments.

Enterococcus faecalis was obtained from American Type Culture Collection (ATCC, 19433, Manassas, VA). Escherichia coli K12 strain C3000 was graciously provided by Professor Joan B. Rose (Water Quality, Environmental, and Molecular Microbiology Laboratory, Michigan State University). Frozen stocks of E. coli and E. faecalis were re-suspended in sterile test tubes containing Trypticase Soy Broth (TSB, 211768, BD, Sparks, MD). To revive the cells, these tubes were incubated at 37 °C in a shaker-incubator (New Brunswick Scientific, Edison, NJ) at 150 rpm for 12 h. Next, cultures were inoculated into 25 ml of TSB and incubated at 37 °C for about 8-12 h to achieve mid-exponential phase. Bacterial cultures were placed overnight in the refrigerator at 4 °C. Cultures were serially diluted in TSB and colony forming units of E. coli and E. faecalis were enumerated by plating 0.1 ml of culture dilutions on Trypticase Soy Agar (TSA, 211043, BD, Sparks, MD) plates, which were incubated at 37 °C for 12 h. Cell concentrations were about 1×10⁸ CFU/ml for both the bacterial species. Genomic DNA of E. coli strain K-12 (700926D) and E. faecalis strain V583 (700802D) were purchased from ATCC. Before use, dried genomic DNA was re-suspended in nuclease-free sterile water (Fischer Scientific, Pittsburgh, PA).