Tryptic soy broth (tsb)
Manufactured by Scharlab
Sourced in Spain, Germany
TSB (Tryptic Soy Broth) is a general-purpose microbiological growth medium. It provides the essential nutrients and growth factors required for the cultivation of a wide range of microorganisms, including bacteria, yeasts, and fungi.
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Lab products found in correlation
Tryptic soy agar tsa, by Scharlab (3 mentions)
Activated charcoal, by Merck Group (1 mentions)
Skimmed milk, by Scharlab (1 mentions)
Blood agar base bab, by Thermo Fisher Scientific (1 mentions)
Diaminopimelic acid dap, by Merck Group (1 mentions)
Kanamycin km, by Merck Group (1 mentions)
Ampicillin amp, by Merck Group (1 mentions)
Chloramphenicol cm, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Plate count agar, by Scharlab (1 mentions)
Chocolate agar, by Statens Serum Institut (1 mentions)
Tryptic soya agar, by Thermo Fisher Scientific (1 mentions)
Mueller hinton broth (mhb), by Scharlab (1 mentions)
Amorphous pla 4060d, by NatureWorks (1 mentions)
Cinnamic acid, by Merck Group (1 mentions)
Tryptic soy agar, by Scharlab (1 mentions)
Baird parker agar plates, by AppliChem (1 mentions)
Milli q integral 5 system, by Merck Group (1 mentions)
Potato dextrose agar (pda), by Scharlab (1 mentions)
Low molecular weight chitosan, by Merck Group (1 mentions)
29 protocols using tryptic soy broth (tsb)
1
Culturing S. aureus Strains
2
Cultivation and Quantification of E. faecalis
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E. faecalis DSM 20478T was cultivated on blood agar plates (SSI, Copenhagen, Denmark) at 35°C under aerobic conditions and grown in tryptic soy broth (TSB, Scharlab, Barcelona, Spain) for 18 h. Prior to experimental use, cells were washed with fresh TSB and adjusted to an optical density of 0.7 (550 nm) for adhesion experiments (2×108 cells/mL) and of 0.4 (550 nm) for biofilm experiments (108 cells/mL).
3
Brucella suis bv2 CITA 198 Cultivation
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The bacterial strains and plasmids used are listed in Additional file 1 . We used B. suis bv2 CITA 198 (herein Bs2WT) because, although Bs2WT and the B. suis bv2 reference strain (B. suis bv2 Thomsen) have the same PCR-RFLP pattern [3 (link)], the former shows a virulence pattern in mice typical of B. suis bv2 field strains and the latter is attenuated (Additional file 2 ).
Bacteria were routinely grown either in standard tryptic soy broth (TSB; Scharlau, Barcelona, Spain) or TSA (TSB supplemented with agar [Pronadisa, Laboratorios Conda, Spain]) at 37 °C. For the studies in mice, vaccines and challenge strains were grown on Blood Agar Base (BAB; Oxoid, UK). When needed, media were supplemented with 5% sucrose, diaminopimelic acid (DAP [Sigma]; 1 mM), 0.2% activated charcoal, kanamycin (Km) at 50 µg/mL or at 35 µg/mL, ampicillin (Amp) at 100 µg/mL and/or chloramphenicol (Cm) at 20 µg/mL (all from Sigma). The lactate-glutamate-glycerol-vitamins synthetic medium of Gerhardt’s [35 (link)] was supplemented with 1 mM methionine (mGSM) (this amino acid is required for growth of some Brucella strains in synthetic media [32 (link)] including Bs2WT (Zúñiga-Ripa, unpublished observations). All strains were stored at − 80 °C in skimmed milk (Scharlau, Barcelona, Spain) or in TSB supplemented with 0.5% yeast extract (Pronadisa, Laboratorios Conda, Spain) (TYSB) and 7% dimethylsulfoxide.
Bacteria were routinely grown either in standard tryptic soy broth (TSB; Scharlau, Barcelona, Spain) or TSA (TSB supplemented with agar [Pronadisa, Laboratorios Conda, Spain]) at 37 °C. For the studies in mice, vaccines and challenge strains were grown on Blood Agar Base (BAB; Oxoid, UK). When needed, media were supplemented with 5% sucrose, diaminopimelic acid (DAP [Sigma]; 1 mM), 0.2% activated charcoal, kanamycin (Km) at 50 µg/mL or at 35 µg/mL, ampicillin (Amp) at 100 µg/mL and/or chloramphenicol (Cm) at 20 µg/mL (all from Sigma). The lactate-glutamate-glycerol-vitamins synthetic medium of Gerhardt’s [35 (link)] was supplemented with 1 mM methionine (mGSM) (this amino acid is required for growth of some Brucella strains in synthetic media [32 (link)] including Bs2WT (Zúñiga-Ripa, unpublished observations). All strains were stored at − 80 °C in skimmed milk (Scharlau, Barcelona, Spain) or in TSB supplemented with 0.5% yeast extract (Pronadisa, Laboratorios Conda, Spain) (TYSB) and 7% dimethylsulfoxide.
4
Antimicrobial Activity of Bacterial Hydrolysates
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Antimicrobial activity was determined by comparing the bacterial growth curves following the method of Hill et al. (2013) [29 (link)], with some modifications described by Bueno-Gavilá et al. (2019) [30 (link)]. The bacterial species studied were Enterococcus faecalis (NCIMB 775), Escherichia coli (NCIMB 9484), Listeria innocua (CCUG 15531), and Pseudomonas fluorescens (NCIMB 9046). All of these species were acquired from the Spanish Type Culture Collection. The culture media used were peptone water (Pareac-Cultimed, Barcelona, Spain), tryptic soy broth (Scharlau, Sentmenat, Spain), and plate count agar (Scharlau). As a positive inhibition control, gentamicin (Sigma-Aldrich) of ≥95% purity was used. The curve data were processed using DMFit 3.5 according to the model of Baranyi and Roberts (1994), obtaining the most representative kinetic parameters (latency phase and maximum growth rate) and the maximum growth. The experiment was carried out in triplicate for each type of hydrolysate.
5
ESBL Phenotypic Confirmation by Double-Disk Synergy
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The double-disk synergy test was used for phenotypic confirmation of ESBL producers, as previously described [30 (link)]. E. coli ATCC 25922 and E. coli ATCC 35218 (American Type Culture Collection) were used as negative and positive controls, respectively. Commercially available disks (HiMedia) of cefotaxime (30 μg) and ceftazidime (30 μg) together with amoxicillin-clavulanic acid (30 μg) were placed 25 mm apart from each other on the surface of inoculated Mueller-Hinton agar plates and incubated at 37°C for 24 h. The test was regarded as positive when the decreased susceptibility to cefotaxime and/or ceftazidime was joined with a clear-cut augmentation of the inhibition zone of cefotaxime and/or ceftazidime in front of the amoxicillin-clavulanic acid antibiotic disk, forming a characteristic “champagne-cork” or “keyhole” shape [30 (link)]; otherwise, the results were regarded as negative. The bacterial samples were stored in tryptic soy broth (Scharlab SL, Barcelona, Spain) containing 20% glycerol at −70°C for long-term preservation.
6
Cultivation of Oral Bacterial Strains
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Organisms included in the study were Aggregatibacter actinomycetemcomitans (HK915), Candida albicans (NCO 09001; = ATCC 11775), Enterococcus faecalis (DSM 20478), Escherichia coli (ATCC 11775), Lactobacillus paracasei (DSM 5622), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedia (CCUG 24041) and Propionibacterium acnes (DSM 1897).
A. actinomycetemcomitans was cultivated on chocolate agar (Statens Serum Institut, Copenhagen, Denmark) in air enriched with 5% CO2; C. albicans, E. faecalis, E. coli and L. paracasei were grown on blood agar (Statens Serum Institut, Copenhagen, Denmark) under aerobic conditions; P. acnes was grown on blood agar under anaerobic conditions; P. gingivalis and P. intermedia were cultivated anaerobically on modified Columbia blood agar (Statens Serum Institut, Copenhagen, Denmark). All microorganisms were grown at 36.5°C.
Prior to experimental use, all organisms were grown in liquid culture until late exponential phase. A. actinomycetemcomitans, P. gingivalis, P. intermedia and P. acnes were cultivated in plaque medium [38 (link)]; C. albicans, E. faecalis, E. coli and L. paracasei were grown in tryptic soy broth (Scharlab, Barcelona, Spain). SeeS1 Table for details.
A. actinomycetemcomitans was cultivated on chocolate agar (Statens Serum Institut, Copenhagen, Denmark) in air enriched with 5% CO2; C. albicans, E. faecalis, E. coli and L. paracasei were grown on blood agar (Statens Serum Institut, Copenhagen, Denmark) under aerobic conditions; P. acnes was grown on blood agar under anaerobic conditions; P. gingivalis and P. intermedia were cultivated anaerobically on modified Columbia blood agar (Statens Serum Institut, Copenhagen, Denmark). All microorganisms were grown at 36.5°C.
Prior to experimental use, all organisms were grown in liquid culture until late exponential phase. A. actinomycetemcomitans, P. gingivalis, P. intermedia and P. acnes were cultivated in plaque medium [38 (link)]; C. albicans, E. faecalis, E. coli and L. paracasei were grown in tryptic soy broth (Scharlab, Barcelona, Spain). See
7
Inhibitory Effects of PRP-Ag Clusters on Microbial Biofilms
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The inhibitory activity of PRP-Ag clusters against Gram-positive S. aureus ATCC 25923, S. epidermidis ATCC 35984, or yeast C. albicans ATCC 10231 biofilm formation was evaluated by the MTT-based assay. Biofilm formation was carried out as described in Ceresa et al. [84 (link),85 (link)].
Briefly, bacterial suspension at the concentration of 1 × 107 CFUs/mL was prepared in Tryptic Soy Broth (Scharlab, Barcelona, Spain) supplemented with 1% glucose (Scharlab, Barcelona, Spain). Fungal suspensions at the concentration of 1 × 106 CFUs/mL were prepared in RPMI+2%G; pH 7.0. PRP-Ag were co-incubated with 200 µL of microbial suspensions at 37 °C for 24 h. Negative controls, PRP-PS for bacteria or PRP-AMB for fungal strains, and the positive control (PRP only), were also included.
Biofilms were washed two times with 200 µL of PBS, dipped with 200 µL of an MTT working solution, and incubated at 37 °C for 10 min (Staphylococcus spp.) or 40 min (C. albicans).
Afterwards, formazan crystals formed by metabolic active cells within biofilms were dissolved with 200 µL of LB, and the absorbance of the obtained solutions was measured at 570 nm. Data were normalized to the background and expressed as percentages of formed biofilm, considering the mean value of the positive control as 100% vitality.
Briefly, bacterial suspension at the concentration of 1 × 107 CFUs/mL was prepared in Tryptic Soy Broth (Scharlab, Barcelona, Spain) supplemented with 1% glucose (Scharlab, Barcelona, Spain). Fungal suspensions at the concentration of 1 × 106 CFUs/mL were prepared in RPMI+2%G; pH 7.0. PRP-Ag were co-incubated with 200 µL of microbial suspensions at 37 °C for 24 h. Negative controls, PRP-PS for bacteria or PRP-AMB for fungal strains, and the positive control (PRP only), were also included.
Biofilms were washed two times with 200 µL of PBS, dipped with 200 µL of an MTT working solution, and incubated at 37 °C for 10 min (Staphylococcus spp.) or 40 min (C. albicans).
Afterwards, formazan crystals formed by metabolic active cells within biofilms were dissolved with 200 µL of LB, and the absorbance of the obtained solutions was measured at 570 nm. Data were normalized to the background and expressed as percentages of formed biofilm, considering the mean value of the positive control as 100% vitality.
8
Escherichia coli CECT 434 Cultivation
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Escherichia coli CECT 434 was used in this study. The bacteria cultures were prepared in beads and kept in vials in a freezer at −70 °C. The stock cultures were re-activated by inoculation onto Tryptic Soya Agar (TSA) (Oxoid, UK) plates, which were incubated without shaking for 24 ± 2 h at 37 °C. To prepare the inoculum, a single colony was picked up from stock and incubated in Tryptic Soy Broth (TSB) (Scharlau, Spain) at 37 °C for 24 ± 2 h in a shaking incubator. A subculture was also prepared in TSB at the same temperature and for 18 ± 2 h allowing the bacteria to reach the stationary phase (approximately 108 CFU·mL−1).
9
Isolation and Cryopreservation of A. baumannii
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The current study was conducted on 40 clinical isolates of A. baumannii that were recovered from the patients who were admitted to or attended various medical departments at a large tertiary care hospital in Taif, KSA during the period from October 2016 to May 2017. All isolates were recovered from the routinely investigated clinical specimens sent to the microbiology laboratory as a part of routine hospital laboratory procedures. The clinical isolates were recovered from sputum (n = 20), blood (n = 4), tracheal aspirate (n = 2), urine (n = 4), peritoneal fluid (n = 1), wound swab (n = 6), and catheter tip (n = 3). All strains were cryopreserved at −80 °C in tryptic soy broth (Scharlau, Spain) containing 15% glycerol to be used when needed.
10
Cultivation of Fluorescent Bacterial Strains
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Reference laboratory strains of P. aeruginosa PAO1 (ATCC 15692) and B. cenocepacia J2315 (ATCC BAA-245) were grown in Luria–Bertani (LB; tryptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L) medium (Scharlab, Barcelona, Spain) and in tryptic soy broth (TSB; casein peptone 17 g/L, soy peptone 3 g/L, sodium chloride 5 g/L, dipotassium phosphate 2.5 g/L, and dextrose 2.5 g/L) medium (Scharlab, Barcelona, Spain) at 37 °C and 30 °C, respectively. P. aeruginosa PAO1::eGFP (MK171) [23 (link)] constitutively expressing a green fluorescent protein marker from the chromosome (eGFPmut3) and P. aeruginosa PAO1 chromosomally modified by the integration of a mini-Tn7-lux constitutively expressing the luciferase genes [24 (link)] were also used, supplementing the media with 50 μg/mL gentamicin as the selective pressure. B. cenocepacia was transformed with pETS248-Tc-E2Crimson plasmid (see the plasmid construction and bacterial transformation and its low copy number vector) and growth supplementing media with 80 μg/mL of tetracycline. The strains were preserved in 20% glycerol stocks at −80 °C and were freshly revived for each experiment.
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