Tryptic soy broth tsb

A total of 6 LAB strains, including Lactobacillus paracasei SGL 04 (isolated from dairy product), Lactobacillus plantarum SGL 07 (isolated from dairy product), Lactobacillus fermentum SGL 10 (isolated from dairy product), Lactobacillus brevis SGL 12 (isolated from local fermented cheese), Lactobacillus casei SGL 15 (isolated from dairy product) and Lactobacillus salivarius SGL 19 (isolated from aged cheese), were comprised in the present study. Before experiment, the strains stored at − 80 °C in MRS broth (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) plus 15% (v/v) glycerol, were propagated twice in MRS broth at 37 °C for 16 h.
S. aureus (ATCC 25923) and S. pyogenes (ATCC 19615) were grown in nutrient broth (NB) (Liofilchem, Teramo, Italy) and tryptic soy broth (TSB) (Liofilchem, Teramo, Italy) respectively, for 24 h at 37 °C with shaking, and washed twice in phosphate buffered saline (PBS). They were adjusted to a final optical density (OD) of 1.0 at 660 nm, followed by calculations of their colony forming units (CFUs) which corresponded to 1.5 × 10⁹ cells/ml. Both, S. aureus and S. pyogenes were heat-inactivated for 1 h at 70 °C (confirming lack of viability by cultures for 2 days), and then stored in aliquots at − 20 °C until experimental use.

Stocks of the bacterium were stored at −80 °C in 10% glycerol. Before each assay, a bacterial glycerol stock was inoculated in 30 mL of fresh Tryptic Soy Broth (TSB; Liofilchem, Roseto degli Abruzzi, Italy) medium, and grown overnight at 37 °C, with orbital shaking (120 rpm). An aliquot of the grown bacterial culture was inoculated on fresh TSB medium and incubated as described above until the stationary phase (the less susceptible growth phase where the bacterial metabolic processes are slower [34 (link),35 (link),36 (link)]) of the bacterial culture was reached; at this stage, the bacterial concentration presented an optical density (OD) of ca. 1.6 at 600 nm, corresponding to a bacterial cell concentration of ca. 10⁹ colony-forming units per mL (CFU/mL). This procedure was performed before each aPDT assay.

Forty-nine Aeromonas strains were used in this study: 46 Georgian strains, majority collected from sick fish and water in 5 fish farms in Central Georgia, including 39 strains of A. hydrophila, 5 strains of A. caviae, and 2 strains A. sobria (collection of the Eliava Institute. 3 Gotua street, 0160 Tbilisi, Georgia); 3 reference strains: A. hydrophila CIP103770, A. salmonicida achromogenes CIP 104001T, and A. salmonicida salmonicida CIP 103209T, obtained from Collection of Institute Pasteur (Collection De L’Institut Pasteur (CIP), 28 rue du Docteur Roux 75724 Paris CEDEX 15). For the detailed list of strains, see Tables S1 and S2.
For propagation of bacterial strains and for testing of phage activity, Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) (Liofilchem, Italy) were used. The characteristic cultural properties of Aeromonas strains were checked on Aeromonas Selective Agar (ASA) (Liofilchem, Italy).

Triplicated samples (20 mL each), for each storage day, were aseptically packed in UV-light sterilized low-permeability polyamide–polyethylene (PA/PE) bags and manually heat sealed prior to HPP, excluding as much air as possible.
Cultures of E. coli (ATCC 25922) and L. innocua (ATCC 33090) were grown in Tryptic Soy Broth (TSB; Liofilchem, Roseto degli Abruzzi, Italy) at 37 °C for 24 h to reach the stationary phase and then inoculated into raw cream to a final concentration of 108 cells/mL.

P. aeruginosa PAO1 and C. albicans SC5314, two model reference strains with known sequenced whole genome, were used throughout this work. Both strains were stored at– 80 ± 2°C in broth medium with 20% (v/v) glycerol. Prior to each assay, P. aeruginosa and C. albicans strains were subcultured from the frozen stock preparations onto Tryptic Soy agar (TSA) and Sabouraud Dextrose agar (SDA) plates, respectively. TSA and SDA were prepared from Tryptic Soy Broth (TSB; Liofilchem, Italy) or Sabouraud Dextrose Broth (SDB; Liofilchem) supplemented with 1.2% w/v agar (Liofilchem). The plates were then incubated aerobically at 37°C for 18–24 h.
Pure liquid cultures (pre-inocula) of P. aeruginosa were grown overnight in TSB whereas C. albicans was maintained in SDB. For planktonic and biofilm assays, 0.22 μm filter-sterilized RPMI 1640 medium (Gibco® by Life Technologies ^TM, Grand Island, NY, USA) at pH 7.0 was used. Unless otherwise stated, all rinse steps were performed either by using 0.9% (w/v) saline solution (NaCl; J.T. Baker, The Netherlands) or ultrapure (UP) sterile water.

The bacterial strains recombinant bioluminescent E. coli [39 (link)], E. coli FDA strain Seattle 1946 [DSM 1103, NCIB 12210] (ATCC 25922) and S. Typhimurium NCTC 74 (ATCC 13311) were used in this study as phage hosts. All bacteria were grown in Tryptic Soy Broth (TSB; Liofilchem, Roseto degli Abruzzi, Italy). The fresh plate bacterial cultures were maintained in Tryptic Soy Agar medium (TSA; Liofilchem, Roseto degli Abruzzi, Italy) at 4 °C. Before each assay, one isolated colony was aseptically transferred to 30 mL of TSB and grown overnight at 25 °C at 120 rpm stirring. An aliquot of this culture (100 µL) was transferred to 10 mL of fresh TSB medium and grown overnight at 25 °C to reach an optical density (O.D. 600 nm) of 0.8 (HaloDB-20; DynamicaScientific, Livingston, UK), corresponding to about 10⁹ cells/mL.

The Pseudomonas syringae pv. actinidiae strain CRA-FRU 8.43 (Psa 3, also referred to as Psa V or Biovar 3) isolated in Lazio, Italy, in 2008, and obtained from the Culture Collection of the Centro di Ricerca per la Frutticoltura (Rome, Italy) [28 (link),29 (link)] was used. The bacterial strain was grown in Luria–Bertani Agar (LA, Liofilchem, Roseto TE, Italy) at 25 °C for 48 h and then kept at 4 °C. Before each assay, a colony of the bacteria was aseptically inoculated in 30 mL of Tryptic Soy Broth (TSB, Liofilchem, Roseto TE, Italy) and grown aerobically for 24 h at 25 °C under stirring (120 rpm). The viable cell density was approximately 10⁸ colony-forming units per mL (CFU mL⁻¹).