Compound 1 was isolated from bacterial cultures started from a non-axenic glycerol stock. The bacterial glycerol stock originally contained a Leifsonia sp. isolate. The stock was found to be contaminated with both Shewanella sp. and Serratia sp. after several steps of cultivation and production of new glycerol stock solutions. The non-axenic glycerol stock was inoculated onto three different agar plates, in order to gather information of the different isolates present. Originally, the Leifsonia sp. isolate was provided as an axenic culture by The Norwegian Marine Biobank (Marbank, Tromsø, Norway) (Reference number: M10B719). The bacterium was isolated from the intestine/stomach of an Atlantic hagfish (Myxine glutinosa) collected by benthic trawl in Hadselfjorden (Norwegian Sea, 16th of April, 2010). The bacterium was grown in liquid FMAP medium (15 g Difco Marine Broth (Becton Dickinson and Company, Franklin Lakes, NJ, USA), 5 g peptone from casein, enzymatic digest (Sigma, St. Louis, MS, USA), 700 mL ddH2O, and 300 mL filtrated sea water) until sufficient turbidity, and cryo-conserved at −80 °C with 30% glycerol (Sigma). Filtration of sea water was done through a Millidisk® 40 Cartridge with a Durapore® 0.22-µm filter membrane (Millipore, Burlington, MA, USA).
Peptone from casein
Manufactured by Merck Group
Sourced in Germany
Peptone from casein is a laboratory-grade nutrient source derived from the enzymatic hydrolysis of casein, a milk-based protein. It provides a complex mixture of amino acids, peptides, and other nutrients that can support the growth and cultivation of various microorganisms in cell culture and microbiology applications.
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Lab products found in correlation
Glycerol, by Merck Group (2 mentions)
Yeast extract, by Merck Group (2 mentions)
Chloroform, by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Malt extract, by AppliChem (2 mentions)
Sodium chloride (nacl), by Avantor (2 mentions)
Tween 20, by Merck Group (2 mentions)
Enzymatic digest, by Merck Group (1 mentions)
Difco marine broth, by BD (1 mentions)
Agar agar, by Merck Group (1 mentions)
Saccharose, by Merck Group (1 mentions)
Montanide gel 01, by Seppic (1 mentions)
Serotonin hydrochloride, by Merck Group (1 mentions)
Powerwave xs2 microplate spectrophotometer, by Agilent Technologies (1 mentions)
48 well plate, by Greiner (1 mentions)
Peptone from casein, by Carl Roth (1 mentions)
L 1 nacl, by Carl Roth (1 mentions)
L 1 yeast extract, by Carl Roth (1 mentions)
15 protocols using peptone from casein
1
Isolation and Characterization of Leifsonia sp. from Atlantic Hagfish
2
Fungal Growth on Ionic Liquids
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Malt extract media (MEM) agar was prepared as 3% malt extract (Roth) and 0.5% peptone from casein (Sigma) in 1.5% agar-agar (Sigma). Minimal medium (MM) agar for fungal spore generation was prepared as 1x Vogel’s salts [28 ] and 2% sucrose in 1.5% agar-agar (Sigma). MM agar with 2% carboxymethyl-cellulose (CMC low viscosity, Sigma Aldrich) instead of sucrose was used for ionic liquid dependent growth experiments for all fungal strains and was supplemented with varying amounts of different ILs provided by Nitrochemie Aschau GmbH (1-butyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, 1-allyl-3-methylimidazolium-chloride, 1-hexyl-3-methylimidazolium-chloride) for some experiments as stated in the main text. Paper samples treated in the aforementioned paper reinforcement process [19 ] with 1-butyl-3-methylimidazolium chloride or 1-butyl-3-methylimidazolium acetate as cellulose solving ionic liquids were provided by Nitrochemie Aschau GmbH. 1-Butyl-3-Methylimidazolium chloride was applied as a solution in DMSO as it was solid at room temperature.
3
Influenza Vector Vaccines with Brucella Antigens
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Influenza vector vaccines (IVV) were prepared as described above in 10-day-old embryonated chicken eggs (CE; Lohmann Tierzucht GmbH, Cuxhaven, Germany) at 34°C for 48 h. The titer of the IVV was determined in CE as previously described [17 (link)]. The allantoic suspensions containing IVV H5N1 or H1N1 (titer of approximately 8.0 log10 EID50/ml) inserted with different Brucella antigenic genes were combined in a single pool at 1:1:1:1 ratio to obtain a tetravalent vaccine formulation. The mixtures of IVV (L7/L12, Omp16, Omp19 and SOD) were mixed in a 1:1 ratio with sterile stabilizing medium containing 12% peptone from casein (Sigma-Aldrich) and 6% saccharose (Sigma-Aldrich), mixed, aliquoted in 1 ml ampoules, lyophilized and stored at 2–8°C. Immediately before vaccination the lyophilized vaccine was resuspended (2.5 ml per ampoule) in a 20% solution of the adjuvant Montanide Gel01 (Seppic, Puteaux, France) in PBS.
4
Vaccine Formulation and Lyophilization Protocol
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The KM-40 strain containing suspension with an activity of > 106.0 EID50/ml on CAM of ECE (Supplementary Materials ) was prepared to use as a vaccine candidate. A mixture of 1:1 ratio of attenuated virus suspension with sterile stabilizing medium containing 13% peptone from casein (Sigma-Aldrich, Germany) was aliquoted into 1 ml ampoules (20 doses), lyophilized, and stored at 4°C. Master seed, working seed and the experimental vaccine batches were prepared using ECE according to the World Organization for Animal Health (OIE) principles for the production of veterinary vaccines (1 ). Methods of infection of ECE, titration of virus infectivity, preparation of stabilizing medium, sublimation drying, and preparation of 50% glycerol solution are presented in the Supplementary Materials .
5
Yeast Viability Assay with Serotonin and Quinine
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The yeast strains Saccharomyces cerevisiae BY4743 or Candida albicans SC5314 were cultured in Sabouraud's medium containing 30 g L−1 maltose, 10 g L−1 peptone from casein (Sigma), 3 g L−1 yeast extract (Sigma), adjusted to pH 7.021 . After 24 h growth of starter-cultures, strains were inoculated to the same medium in Erlenmeyer flasks and incubated for 4 h at 34°C with orbital shaking at 120 rev min−1. Cultures were then adjusted to 106 cells mL−1 in the same medium and distributed in 300 μL aliquots per well in 48-well plates (Greiner Bio-One). Wells were supplemented with serotonin hydrochloride (Sigma) or quinine dihydrochloride dihydrate (QN; Sigma) as specified. Growth of the yeasts with shaking at 34°C was monitored with a BioTek Power Wave XS2 Microplate Spectrophotometer.
6
Microbial Expression Systems for 3D Cultivation
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Standard microbial expressions systems were used to evaluate the 3D‐printed cultivation vessel. All media were prepared with deionized water (Arium 661 Ultrapure water system, Sartorius Stedim Biotech S. A., Göttingen, Germany). Escherichia coli BL21 (DE3) as prominent example for gram‐negative bacteria were grown from 20% v/v glycerol stocks stored at −80°C in lysogeny broth (LB) media: 5 g L−1 yeast extract (Carl Roth), 10 g L− 1 peptone from casein (Sigma–Aldrich), 10 g L−1 NaCl (Carl Roth) pH 7.1. Pre‐cultures were carried out in 250 mL SF with baffles at 30°C at 150 rpm overnight. Main‐cultures were conducted at 37°C. As a gram‐positive model microorganism, Bacillus subtilis (DSM 168) was used. Cells were stored as glycerol stock with 20% v/v glycerol at −80°C. Pre‐cultures and main‐cultures were conducted in the same way as the E. coli cultures. Saccharomyces cerevisiae NCYC 1024 a yeast was selected as simple model organism for eucaryotes. For cultivations Yeast‐Peptone‐Dextrose (YPD) media were used: 10 g L−1 yeast extract, 20 g L−1 peptone from casein, and a variant amount of glucose (Carl Roth), pH 6.0. Pre‐cultures were performed in 250 mL SFs with 20% filling volume at 30°C at 150 rpm overnight.
7
Antibacterial Biodegradable Polymer Synthesis
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Succinic acid (98%) and sodium nitrate (99.5%) were purchased from Fluka. Stannous octoate, ε-caprolactone (97%), 1, 3-propanediol (99%), tetrabutyl titanate (TBT, 97%), methanol (99%), chloroform (97%), sodium chloride (NaCl), silver nitrate (AgNO 3 ), yeast extract, peptone from casein, pancreatic digest, ethanol (99.8%), Lipase Pseudomonas cepacia, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. chloroform, nitric acid (HNO 3 ), and sodium borohydride were purchased from Merck. HDF cells and all the microorganisms used in the antibacterial studies were purchased from ATCC (UK). Dulbecco's modified Eagle medium (1X DMEM, 4.5 g l -1 D-Glucose, L-glutamine, sodium pyruvate), fetal bovine serum (FBS), HyClone phosphate-buffered saline (10 X PBS, w/o calcium, magnesium), and Pen-Strep (10 000 Units ml -1 penicillin, 10,000 μg ml -1 streptomycin) were obtained from Gibco (UK). Cell proliferation reagent WST-1 was purchased from Roche. PBS tablets were obtained from MP Biomedicals, LLC (France).
8
Morphology of Strain CBS 554.65
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The morphology of strain CBS 554.65 was inspected on minimal medium [22 ] and malt extract agar (30 g/L malt extract (AppliChem, Darmstadt, Germany) and 5 g/L peptone from casein (Merck KGaA, Darmstadt, Germany)). The strain was 4-point inoculated and incubated at 30 °C for one week.
9
Synthesis and Quantification of Quorum Sensing Signals
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The benzimidazole 1 and the nitrophenylmethanol 2 (Figure 2 ) were synthesized as described in literature (Rahme et al., 2012 ; Storz et al., 2014 (link)). d4-HHQ was synthesized following the procedure of HHQ using d5-aniline (Lu et al., 2012 (link)). The tricyclic 3 (Figure 2 ) and amitriptyline were purchased from ChemDiv (United States) and Alfa Aesar (Germany), respectively.
Water (Th. Geyer, Germany), acetonitrile (VWR, Germany) methanol (Sigma–Aldrich, United States) and formic acid (Fluka, United States) were LC-MS grade and used for HPLC-MS/MS experiments.
Yeast extract (Fluka, Germany), sodium chloride (VWR, Germany) and peptone from casein (Merck, Germany) were used for the preparation of Luria Bertani (LB) broth needed for performing the quantification experiments of pqs-related signal molecules. Ready-made mixture of LB broth (Fisher, United States) and Tryptic Soy Broth (TSB) 1% [w/v] (BD, United States) were used for pqsA-GFPASV and persister cells experiments.
Water (Th. Geyer, Germany), acetonitrile (VWR, Germany) methanol (Sigma–Aldrich, United States) and formic acid (Fluka, United States) were LC-MS grade and used for HPLC-MS/MS experiments.
Yeast extract (Fluka, Germany), sodium chloride (VWR, Germany) and peptone from casein (Merck, Germany) were used for the preparation of Luria Bertani (LB) broth needed for performing the quantification experiments of pqs-related signal molecules. Ready-made mixture of LB broth (Fisher, United States) and Tryptic Soy Broth (TSB) 1% [w/v] (BD, United States) were used for pqsA-GFPASV and persister cells experiments.
10
Microbial Growth Media Preparation
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Ethanol (99.9%), potassium dihydrogen phosphate (KH2PO4, 99%), glycerol (87%), peptone from casein, meat extract, and tryptic soy Agar (TSA) were obtained from Merck (Darmstadt, Germany). Agar was obtained from Liofilchem (Teramo, Italy). Anhydrous sodium carbonate (99.8%), sodium chloride (NaCl, 99%), sodium hydroxide (NaOH, 98%), and potassium chloride (KCl, 99%) were purchased from Panreac (Barcelona, Spain) and sodium dihydrogen phosphate dihydrate (Na2HPO4 2H2O, 99%) was obtained from Fluka (Buchs, Switzerland).
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