P. aeruginosa and S. aureus were grown separately under shear (130 rpm) at 35 °C on 24 removable polycarbonate coupons in a CDC biofilm reactor (BioSurface Technologies Corp., Bozeman, MT, USA). S. aureus biofilm was grown in 15 g/L (50%) tryptone soya broth (TSB) (Sigma Aldrich, St. Louis, MO, USA) in batch phase for 24 h and then replaced with fresh media 6 g/L (20% TSB) flowing through the chamber at 80 mL/h for a further 48 h. P. aeruginosa was grown in 600 mg/L (2%) TSB in batch phase for 24 h and then with fresh media (TSB 2%) flowing through the chamber at 80 mL/h for a further 48 h. Coupons were harvested by washing gently, three times, in phosphate buffered saline (PBS) to remove loosely attached and planktonic bacteria. The number of bacteria per coupon was 3.52 × 107 and 2.3 × 107 for S. aureus and P. aeruginosa, respectively. Bacterial biofilm was gently scraped off from the outer side of each coupon using a 12.5% sodium hypochlorite-soaked paper towel, and then again washed three times in TSB to remove residual chlorine.
Tryptic soy broth (tsb)
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom, Spain, France, Canada, Poland, Italy, Switzerland, Saudi Arabia
Tryptic soy broth is a general-purpose, nutrient-rich culture medium used for the growth and maintenance of a wide variety of microorganisms, including bacteria, fungi, and yeasts. It provides a balanced source of amino acids, carbohydrates, and other essential nutrients to support microbial growth and proliferation.
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Lab products found in correlation
Tryptic soy agar, by Merck Group (35 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Glycerol, by Merck Group (11 mentions)
Sodium chloride (nacl), by Merck Group (11 mentions)
Phosphate buffered saline (pbs), by Merck Group (10 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Crystal violet, by Merck Group (8 mentions)
Yeast extract, by Merck Group (7 mentions)
Methanol, by Merck Group (7 mentions)
Luria bertani broth, by Merck Group (7 mentions)
Triton x 100, by Merck Group (6 mentions)
Mueller hinton agar (mha), by Merck Group (6 mentions)
Streptomycin, by Merck Group (5 mentions)
Vancomycin, by Merck Group (5 mentions)
Macconkey agar, by Merck Group (5 mentions)
Sodium hydroxide (naoh), by Merck Group (4 mentions)
Acetic acid, by Merck Group (4 mentions)
Sucrose, by Merck Group (4 mentions)
M ller hinton agar, by Merck Group (4 mentions)
Nutrient broth, by Merck Group (4 mentions)
459 protocols using tryptic soy broth (tsb)
1
Biofilm Formation of P. aeruginosa and S. aureus
2
Microcosm Establishment for Diverse Ecosystems
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Microcosms were established in 100 mL glass vials with a plastic screw cap lid (S2 Fig. ) containing sterile acid washed sea sand with pore size fractions ranging from 0.075 to 0.425 mm (Honeywell Specialty Chemicals Seelze GmbH, Germany). The amount of sand was either 25 g (Microcosms supplemented with 1.5 mL 1/10th strength Tryptic Soy Broth (nutrient rich media) (5.0 gL−1 NaCl (Merck), 1.0 gL−1 KH2PO4; 3 gL−1 Tryptic Soy Broth (OXOID)) or 30 g (Microcosms supplemented with 1.5 mL nutrient poor media (5.0 gL−1 NaCl (Merck), 1.0 gL−1 KH2PO4; 0.1 gL− (NH4)2SO4; 0.5 gL−1 Tryptic Soy Broth (OXOID)). The sand was weighed directly into the glass vials and afterwards sterilized by autoclaving for 20 minutes. The sterilized microcosms were dried overnight in a 60°C oven prior to inoculation. All treatments were performed in triplicates over a time of 6 days. A detailed overview of all treatments and controls is given in Table 2 .
3
Photoinactivation of Non-Pathogenic Bacteria
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Bacteria investigated in this study were obtained from DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig, Germany). Due to safety regulations in the available laboratory, it is not possible to cultivate pathogenic strains there. However, it was previously demonstrated that non-pathogenic representatives reacted similar or even less susceptible towards visible light photoinactivation as their pathogenic relatives [48 ]. Pseudomonas fluorescens (DSM4358) were cultivated in 535 medium (30 g tryptic soy broth (Sigma-Aldrich Chemie GmbH, München, Germany) per liter) at 30 °C and Staphylococcus carnosus (DSM20501) were cultivated in M92 medium (30 g tryptic soy broth (Sigma-Aldrich Chemie GmbH, München, Germany), 3 g yeast extract (Merck KGaA, Darmstadt, Germany) per liter) at 37 °C at rotary conditions of 170 rpm from an overnight preculture in 30 mL fresh medium until mid-exponential phase (optical density at 600 nm OD600 = 0.35) was reached. Bacterial cultures were centrifuged at 7000× g for 5 min and the resultant pellet resuspended in PBS, washed in PBS and diluted to 1–5 × 107 CFU/mL for experimental use.
4
Storing and Culturing Bacterial and Fungal Samples
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Bacteria were streaked from glycerol stocks (25% glycerol) on TSA plates (15 g/L Tryptic Soy Broth, Sigma Aldrich; with 10 g/l Bacto Agar, Duchefa Biochemie) and grown at 25 °C. Single colonies were inoculated in liquid 50% TSB (15 g/L Tryptic Soy Broth, Sigma Aldrich) and grown until dense at 25 °C with 180 rpm agitation. Dense cultures were then stored at 4 °C and diluted 1 to 10 in TSB the day before the experiment and cultured at 25 °C with 180 rpm agitation overnight to ensure sufficient cell densities for slow- and rapidly-growing bacteria. Glycerol stocks were stored at -80 °C and kept on dry ice when transported. Individual pieces of fungal mycelium were transferred to potato dextrose agar (PDA; Sigma-Aldrich) Petri dishes from glycerol stocks (approx. 30 pieces of fungal mycelium in 25% sterile glycerol, stored at -80 °C). Fungi were grown at 25 °C in the dark for 14 days.
5
Murine Model of S. aureus Skin Infection
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S. aureus were cultured on tryptic soy agar or tryptic soy broth (Sigma-Aldrich) at 37°C overnight, followed by short-term propagation for 2–3 h in fresh tryptic soy broth to an OD600 of 1.8 (Montgomery et al., 2014 (link); on the day of infection). The backs of mice were shaved, and remaining hair was chemically removed the day before infection. The stratum corneum was removed by performing 20 strokes with 240-grit sandpaper (3M) followed by application of 2 × 107S. aureus particles in 50 µl of sterile PBS (Gibco). Retrospective verification by serial dilution was performed to enumerate CFUs on tryptic soy agar. The drop-plate method was used to enumerate the number of S. aureus on infected mice by excising and homogenizing skin lesions in PBS containing 0.1% Triton X-100, followed by plating (in triplicate) 20 µl of serially diluted skin lesion samples.
6
Bacterial Infection Protocols for In Vitro and In Vivo Studies
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The MRSA strain USA300/LAC53 (link) was provided by M. Otto (NIH). For infection, USA300/LAC was grown overnight (O/N) at 37 °C in tryptic soy broth (TSB, Sigma) at 250 r.p.m. and was subcultured at a 1:100 dilution for 3.5 h in TSB to mid-log phase. K. pneumoniae strain 43816 serotype 2 was purchased from American Type Culture Collection and was grown O/N at 37 °C in TSB at 250 r.p.m. for infection. S. pneumoniae WU-2 strain from R. Malley (Boston Children's Hospital) was grown at 37 °C under 5% CO2 without shaking for 18 h in Todd's Hewitt broth (THB, Sigma) with 0.5% yeast extract and was subcultured at a 1:10 dilution for 8 h in fresh THB with 0.5% yeast extract to reach mid-log phase for infection. P. aeruginosa strain PA01V from G. Pier (Brigham and Women's Hospital) was grown O/N at 37 °C in TSB at 250 r.p.m. and was subcultured at a 1:100 dilution for 4 h in TSB for infection. For all strains, cultures were centrifuged at 5,000 r.p.m. for 5 min, and bacterial pellets were washed and resuspended in phosphate-buffered saline (PBS). The OD600 was measured to estimate bacterial density, and serial plating was performed on tryptic soy agar (TSA) plates to quantify CFU values. For intravital imaging, a GFP–MRSA S. aureus transgenic bacterium was used, whose construction has been previously reported25 (link).
7
Bacterial Culture Preparation with Antibiotics
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Preparation of bacterial cultures was performed as follows. Bacterial stocks frozen at − 80 °C in TSB (Sigma-Aldrich, St. Louis, MO) with 20% glycerol was inoculated into 5 mL of TSB. The culture was aerated by shaking at 120 rpm at 37 °C and grown overnight. Proper concentration of antibiotics was added if bacteria strain contains resistance genes for positive selection.
8
Preparation of Staphylococcus Bacterial Cultures
9
Biofilm Formation Assay for Listeria
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The 15 strains that represented the four clonal complexes that seemed to persist in the factory (CC9 (n = 9), CC204 (n = 2), CC121 (n = 2)), (CC6 (n = 2)) were chosen for biofilm assays, according to the protocol published by Harvey et al. with minor changes [21 (link)]. A single colony was inoculated into 5 mL of tryptone soy broth (TSB, from Fluka, obtained from Sigma-Aldrich, Buchs, Switzerland), incubated for 20 h at 30 °C with shaking at 200 rpm, subcultured 1:250 into fresh TSB, and incubated for an additional 20 h at 30 °C with shaking at 200 rpm. The resulting cultures were adjusted to an OD600 of 1.0, diluted 1:80 in TSB and added to 96-well plates, which were incubated for 96 h at 22 °C, or for 168 h at 8 °C, respectively. Biofilms were then washed three times with distilled water, stained with crystal violet, and washed five times with distilled water. The remaining crystal violet was dissolved in ethanol and the OD600 was measured in a Synergy plate reader (BioTek, Lucern, Switzerland). Control strains that were high and low biofilm formers (Institute for Food Safety and Hygiene, Zurich; unpublished results) (Table 1 ) were included in each experiment.
10
Antibiotic Susceptibility Testing
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Overnight cultures were diluted 1,000-fold into 2 mL of TSB (Sigma 22092) and grown for 24 h. TSB was supplemented with 10 μg/mL chloramphenicol for the maintenance of plasmid pOS1. A total of 100 μL of culture was removed, serially diluted onto TSA plates, and incubated at 37°C for 18 h to determine the initial population. Cultures were challenged with either with 64 μg/mL (16 to 32× MIC) vancomycin, 32 μg/mL daptomycin (16× MIC), 16 μg/mL ceftaroline (32 to 64× MIC), or the combination of 32 μg/mL daptomycin and 16 μg/mL ceftaroline. After 24 h, 48 h, and 72 h, 100 μL of culture was removed, pelleted, and washed three times with 1 mL of 0.9% saline. To quantify the surviving population, pellets were resuspended in 100 μL of 0.9% saline, serially diluted, plated on TSA, and incubated for 24 h at 37°C prior to colonies being counted. Fraction survival was calculated by dividing the CFU counts of the surviving population by the CFU counts of the initial population.
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