Lennox l broth base

Subgingival plaque was collected from hopeless elements extracted for periodontal reasons and with probing greater than 7 mm. Plaque was incubated in 2 mL of LB Broth (Lennox L-broth base, Invitrogen, MA, USA), a generic culture medium for bacteria, for 24 h at 37 °C in oscillating incubator to obtain the inoculum for biofilm cultivation. Discs were placed into wells of 6-well cell plates (Corning Life Sciences, Woburn, MA, USA), covered with 3 mL of subgingival human plaque suspension + 2 mL of fresh LB broth medium. Bacteria and discs finally were incubated for 7 days, at 37 °C. The LB medium was replaced every 24 h. After 7 days of cultivation, the medium rich in bacteria was removed, and the biofilm-covered discs were transferred into new wells of a sterile 6-well plate. All discs were washed with PBS 1X, then discs used for Cell culture were leaved in PBS 1X, whereas discs used for microscopy were fixed in formalin solution neutral buffered 10% (Sigma Aldrich, MO, USA) for 15 min at room temperature and dehydrated using solutions with an increased concentration of alcohol (50%, 5 min—75%, 5 min—80%, 5 min, 95%, 5 min and 100%, 30 min). Fixed and non-fixed discs were stored at 4 °C.

SA187 was grown overnight in LB broth (Lennox L Broth Base, Invitrogen) at 28°C with 190 rpm until the culture reached the exponential growth phase. Cells were harvested by centrifugation, washed twice with 10 mM MgSO₄, and finally resuspended in 10 mM MgSO₄ to a final OD₆₀₀ of 0.5. Thirty microliters of this cell suspension were inoculated by triplicate in 96-well plates, in 300 μl of LB supplemented with increasing concentrations of NaCl (0–4 M) and ½MS alone or supplemented with 1% of the following carbon sources: arabinose, fructose, glucose, glycerol, lactose, maltose, raffinose, sucrose, acetic acid, citric acid, or lactic acid. Bacterial growth was monitored by using a Varioskan Flash microplate reader (Thermo Scientific), where the 96-well plates were incubated for 2 days at 28°C and 300 rpm.

Individual colonies formed by reporter strains E. coli BWtolC and BWsoxS (Jun et al. 2019 ) were placed into LB broth (Lennox-L-Broth Base, Invitrogen, Auckland (New Zealand)) supplemented with kanamycin and then placed on a shaker platform at 37 °C and grown to exponential phase. 100 µl containing about ~ 10⁷cells was then placed on separate NsARC (test), stainless steel (negative control) and copper (positive control) coupons. Sterile cover slips (24 mm × 24 mm) were used to spread the cultures on the sample surfaces. The samples were placed in petri dishes (60 mm × 15 mm) containing damp filter paper. Replicates were simultaneously exposed to visible light of 2100 lx (450–650 nm), UV light (365 nm), ambient light (650–750 nm) and also kept in the dark for a period of not more than 2 h before washing off with PBS and the cells fixed with paraformaldehyde (Chao and Zhang 2011 (link)). 2 µl of the fixed cells were then smeared onto a gelatine-coated glass slide and allowed to dry at room temperature. 6 µl of 50% (v/v) glycerol was then added to the dried smear and a 25 × 25 mm glass cover slip was used to spread it over the dried smear.

Randomly selected plates from the metagenomic library issued from the intestinal microbiota of obese and lean patients (constructed using CopyControl Fosmid Library Production Kit by Genoscope, France) were used in this study. The library consists of E. coli EPI300T1R clones carrying each one fosmid with a metagenomic inserts of approximately 40 kb. E. coli bearing an inactive fosmid with a human 40 kb-insert (ligation control reaction for CopyControl Fosmid Library Production Kit), referred to as EPI300-Cont, was used as control. Bacteria were grown under static or agitated conditions in 96 well-plates in LB broth (Lennox L broth base, Invitrogen) at 37°C. Bacterial growth was monitored by absorbance measurement at 600 nm, using a microplate reader (Infinite 200, Tecan). We used a metagenomic cloneshowing partially-secreted alkaline phosphatase activity, namely 43C8, as a read-out for bacterial lysate activity. Bacterial lysates were obtained through freezing at −80°C and subsequent thawing at room temperature. Bacterial lysates were filtered by centrifugation through a 0.45 µm microplate filter (Corning) prior to use on reporter cell-lines.

Plant growth promoting (PGP) traits were evaluated by using clearing assays. The ability of SA187 to solubilize phosphate was assessed on Pikovskaya’s (PVK) agar plates (M520, Himedia). The production of siderophores was determined by Blue Agar CAS assay, as described by Louden et al. (2011) (link). Zinc solubilization was assessed on modified PVK agar plates, supplemented with 0.1% ZnCO₃, as described by Bapiri et al. (2013) . Assays were performed by inoculating 30 μl of overnight LB bacterial culture into cavities of ∼0.5 cm in diameter. The production of indole-3-acetic acid was qualitatively determined according to Bric et al. (1991) (link), with the following modifications: LB broth supplemented with 2.5 mM L-tryptophan were used in 96-well plates, which were incubated at 28°C and 190 rpm for 2 days. Tolerance to drought and salt stresses were evaluated by growing SA187 in LB broth (Lennox L Broth Base, Invitrogen) supplemented with 20% polyethylene-glycol (PEG) 6000 and 3% or 5% NaCl, respectively. Tolerance to heat stress was assessed by using LB agar plates incubated at 37°C or 42°C. Assays were performed in triplicates and plates and liquid cultures were incubated at 28°C for 2–5 days.

Isolate JZ38 was regularly grown in LB broth (Lennox L Broth Base, Invitrogen) or on LB agar plates at 28°C. Fresh, pure bacterial cultures were used for total genomic DNA extraction using Sigma’s GenElute bacterial genomic DNA kit (Sigma Aldrich) following the manufacturer’s protocol. DNA quality and quantity were assessed by 0.7% agarose gel electrophoresis (35 V, 12 h), NanoDrop 2000 (Thermo Fisher Scientific) and Qubit dsDNA BR assay kit (Thermo Fisher Scientific).