Trypticase soy broth tsb

H. pylori 26695 (ATCC 700392), isolated from a United Kingdom patient with gastritis, was obtained from the American Type Culture Collection (Manassas, VA, USA). Frozen stocks of H. pylori were recovered and routinely grown for 48 h at 37°C, 5.5% CO₂, and 70 to 80% relative humidity on Trypticase soy agar plates (TSA) from Becton Dickinson (Sparks, MD 21152, USA) supplemented with 0.4% H. pylori selective supplement Dent (Oxoid Basingstoke, Hampshire, England), 0.3% IsoVitalex (Oxoid), and 5% horse serum from Thermo Fisher Scientific HyClone (Utah 84321, USA) [38 (link), 39 (link)]. For liquid growth experiments, cells were grown in Trypticase soy broth (TSB) (Becton Dickinson) with 5% horse serum, supplemented with IsoVitalex and Dent (Oxoid). Bacteria were first grown to an optical density of 0.6 to 1.0 at 600 nm (OD₆₀₀) at pH 7.0 and subsequently diluted to a starting OD₆₀₀ of 0.05. To measure the growth of H. pylori in liquid medium, a serial dilution was prepared, aliquots of the various dilutions were plated on Trypticase soy agar plates, and the number colony-forming units (CFU) was determined [40 (link)].

Human gingival fibroblast – HGF-1 (ATCC^® CRL-2014) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza, Walkersville, MD, United States) with 10% Fetal Bovine Serum (FBS) (Lonza, Walkersville, MD, United States). Human gingival epithelial cells (keratinocyte OBA-9) were cultured in specific medium for keratinocytes, Defined Keratinocyte-SFM (Life Technologies, Carlsbad, CA, United States) (Oda and Watson, 1990 (link); Kusumoto et al., 2004 (link)).
Aggregatibacter actinomycetemcomitans (strain D7S-1) was cultivated from a subgingival plaque from an African-American female patient diagnosed with generalized aggressive periodontitis (Chen et al., 2010 (link)). The bacteria were grown in Trypticase Soy Broth (TSB) (Becton Dickinson, Franklin Lakes, NJ, United States).

Bone infection in this study was induced by the bacterium Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 49,230™) – a strain isolated from a patient with chronic osteomyelitis and effectively used in the induction of bone infections before [20 (link)]. The needed amount of 10³ colony-forming units (CFU) had already been determined in a previous study [19 (link)]. Bacterial cultivation was performed according to the official product instruction, using Trypticase Soy Broth (TSB) for liquid cultures and blood agar plates for plating (Becton, Dickinson and Company, New Jersey, USA). All cultures were handled under sterile conditions and incubated under permanent oxygen supply at a temperature of 37 °C. Bacterial CFU counts on blood agar plates and spectrophotometric measurements of liquid cultures were performed to establish a calibration formula. An overnight culture of S-. aureus in TSB, containing 10³ CFU in 10 μl was used for each surgery.

The bacterial strains used in this study are listed in Table 1. S. aureus and Escherichia coli XL‐II were grown in Trypticase soy broth (TSB; Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) and Luria Bertani (LB) broth, respectively. Tetracycline (5 µg/ml) and chloramphenicol (5 µg/ml) were used to select for S. aureus, and ampicillin (100 µg/ml) was used to select for E. coli when necessary.

Strains were grown in Trypticase Soy Broth (TSB) (Becton Dickinson, U.S.A.) overnight at 37°C. Bacterial cells were harvested from 1 mL TSB by centrifugation at 8,000×g for 3 min and the supernatant was removed. Total genomic DNA was extracted using NucleoSpin Tissue (Macherey Nagel, Germany) according to the manufacturer’s protocol.

A total of nine STEC, four spoilage bacteria (SP) and six lactic acid bacteria (LAB) strains were included in this study (Table 1). The bacterial cultures were maintained in Trypticase Soy Broth (TSB; Becton, Dickinson and Company, MD, United States) supplemented with 15% glycerol and stored at −80°C. Before the experiments, each stock culture was plated on trypticase soy agar (TSA; Difco Becton, Dickinson and Company, MD, United States) since all tested strains can grow well in this media, STEC strains were cultured on MacConkey agar plates (Hardy Diagnostics Inc., Santa Maria, CA, United States). A single colony of each bacterial strain was transferred from each plate into 5 mL of TSB and incubated at 37°C for 18 to 24 h. Cells were harvested by centrifugation (4500xG) for 5 min at room temperature. After centrifugation, the supernatant was decanted, and the pellet was resuspended in 5 mL sterile Butterfield’s Phosphate Buffer (BPB; Hardy Diagnostics Inc., Santa Maria, CA, United States). This procedure was repeated three times for three wash steps in BPB. Each bacterial suspension was adjusted to a final concentration of 10⁸ colony-forming units (CFU/mL) using a 0.5 McFarland Standard and further diluted in Lennox Broth no-salt LB-NS broth (LB-NS; Tryptone 10 g/L and yeast extract 5 g/L) to achieve a concentration of 10⁶ CFU/mL.

The host range of the bacteriophages was determined on the 26 clinical K. pneumoniae strains listed in Table S2 [14 ]. Bacteria were stored at −70 °C in Trypticase Soy Broth (TSB) (Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 20% glycerol (Avantor Performance Materials Poland S.A., Gliwice, Poland). Spot testing was used as a rapid and efficient method for determining the host range in large collections of bacteria [14 ]. The phage titer used for spot testing was 10⁵ PFU/mL with final 5 × 10² PFU per spot. A majority of the clinical isolates used in the host range analysis were selected from ESBL-positive K. pneumoniae strains isolated in Sweden during the year of 2007, individually picked for the largest variation of PFGE patterns (using XbaI) by the Public Health Agency of Sweden (Folkhälsomyndigheten) (Solna, Sweden).