Multi-drug resistant (MDR) P. aeruginosa strain PAO1 (ATCC 47085) and MDR E. coli O157:H7 strain SMS-3-5 (ATCC BAA-1743), both isolates from humans or the environment, and not produce associated, were revived from freezer stocks stored at -80°C by streaking onto Tryptic Soy Agar (TSA, Becton Dickinson, Franklin Lakes, NJ, United States) and incubating for 24 h at 37°C to obtain isolated colonies. An isolated colony of P. aeruginosa was streaked onto Pseudomonas Isolation Agar (PIA, Becton Dickinson, Franklin Lakes, NJ, United States) and an isolated colony of E. coli O157:H7 was streaked on to Eosin Methylene Blue Agar (EMB, Becton Dickinson, Franklin Lakes, NJ, United States) followed by incubation for 24 h at 37°C. Separate single colonies from PIA and EMB were incubated separately in Tryptic Soy Broth (TSB, Becton Dickinson, Franklin Lakes, NJ, United States) at 180 rpm for 24 h at 37°C. Cells were washed two times in 0.1% (wt/vol) peptone (Becton Dickinson, Franklin Lakes, NJ, United States) and were separately suspended in 9 ml of 0.1% (wt/vol) peptone to prepare the inoculation solution.
Peptone
Manufactured by BD
Sourced in United States, Germany, Japan, United Kingdom, Austria, Poland
Peptone is a complex mixture of amino acids and peptides derived from the enzymatic digestion of animal or plant proteins. It is commonly used as a growth supplement in microbiological culture media to support the cultivation of a wide range of microorganisms, including bacteria, yeast, and fungi.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by BD (95 mentions)
Dextrose, by BD (13 mentions)
Malt extract, by BD (10 mentions)
Tryptone, by BD (9 mentions)
Yeast nitrogen base, by BD (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Yeast nitrogen base without amino acids, by BD (6 mentions)
Bacto agar, by BD (5 mentions)
Dextrose, by Merck Group (5 mentions)
Beef extract, by BD (5 mentions)
Sodium chloride (nacl), by Merck Group (5 mentions)
Dextrose, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Glycerol, by Merck Group (4 mentions)
Yeast extract, by Merck Group (4 mentions)
Meat extract, by BD (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Yeast extract, by Thermo Fisher Scientific (4 mentions)
Hydrogen peroxide, by Duksan Pure Chemicals (3 mentions)
150 protocols using peptone
1
Reviving and Culturing MDR Pathogens
2
Bacterial Strain Culture Media Protocols
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The bacterial strains used in this study are listed in Table 1 . Brain Heart Infusion (BHI) medium (Becton Dickinson, Franklin Lakes, NJ), BHI medium supplemented with 1% (w/v) glucose (Wako, Osaka, Japan) (BHIG), BHI medium supplemented with 3% (w/v) NaCl (Wako) (BHIN), tryptic soy broth (TSB) (Becton Dickinson), or peptone–NaCl–glucose (PNG) [0.33% (w/v) peptone (Becton Dickinson), 0.26% (w/v) sodium chloride, and 0.33% glucose (w/v)] were autoclaved at 121 °C for 15 min. If required, BHIN was mixed with 10% (w/v) activated charcoal and filtered using a 0.2-µm pore filter (Kanto Chemical, Tokyo, Japan), and the filtrate BHINC was used to culture the biofilms. RPMI medium (Thermo Fisher Scientific, Carlsbad, CA) was supplemented with 1% glucose (w/v) (RPMIG) and passed through a 0.2-µm pore filter. Lysogeny-broth (LB) medium (Millipore, Bedford, MA) was used to culture Escherichia coli. All the bacterial strains were grown in the appropriate media at 37 °C.
3
Plasmid Construction and Yeast Cultivation
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Escherichia coli NovaBlue (Merck Millipore, Darmstadt, Germany) was used for plasmid construction and amplification. The microorganisms were routinely cultured at 37 °C and 200 rpm in LB medium [10 g/L tryptone (Nacalai Tesque, Kyoto, Japan), 5
g/L yeast extract (Nacalai Tesque), and 5 g/L NaCl] supplemented with 100 μg/mL ampicillin.
The yeast strains used in this study are listed in Table 1. P. pastoris CBS7435 Δdnl4 Δhis4 was used as a parent strain since it has improved gene targeting efficiency for homologous recombination 37 (link) . P. pastoris strains were cultivated in YPG medium [10 g/L yeast extract, 20 g/L peptone (BD Biosciences, San Jose, CA, USA), and 20 g/L glycerol], YPD medium [10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose], or SD medium [6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 20 mg/L histidine and appropriate antibiotics including 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany).
g/L yeast extract (Nacalai Tesque), and 5 g/L NaCl] supplemented with 100 μg/mL ampicillin.
The yeast strains used in this study are listed in Table 1. P. pastoris CBS7435 Δdnl4 Δhis4 was used as a parent strain since it has improved gene targeting efficiency for homologous recombination 37 (link) . P. pastoris strains were cultivated in YPG medium [10 g/L yeast extract, 20 g/L peptone (BD Biosciences, San Jose, CA, USA), and 20 g/L glycerol], YPD medium [10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose], or SD medium [6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 20 mg/L histidine and appropriate antibiotics including 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany).
4
Diverse Media for Yeast, Worm, and Cell Culture
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The yeast strains used in this study are listed in Table S1 . Strains were grown in SC media containing 2% glucose (Sigma-Aldrich), 0.67% nitrogen base (BD), and an appropriate amino acid drop-out mix (US Biological); or YPD media containing 1% yeast extract (BD), 2% peptone (BD), and 2% glucose; or YPG media containing 1% yeast extract (BD), 2% peptone (BD), and 3% glycerol; or nitrogen starvation medium (SD-N) media containing 2% glucose (Sigma-Aldrich) and 0.67% nitrogen base (BD). Where indicated, Etn (Sigma-Aldrich) was added to media to a final concentration of 1 mM from a 1M stock.
The C. elegans strains used in this study were N2 (Bristol, WT) and AG645 (lpd-3[av254]V. CRISPR/Cas9 Edit, deletion of exons 1–6 and intervening introns).
HEK-293 cells were cultured with DMEM (4.5 g/liter D-glucose + L Glutamine; Gibco), 10% FBS heat-inactivated (Quality Biological), and penicillin and streptomycin (Gibco) in incubator at 37°C 5% CO2.
The C. elegans strains used in this study were N2 (Bristol, WT) and AG645 (lpd-3[av254]V. CRISPR/Cas9 Edit, deletion of exons 1–6 and intervening introns).
HEK-293 cells were cultured with DMEM (4.5 g/liter D-glucose + L Glutamine; Gibco), 10% FBS heat-inactivated (Quality Biological), and penicillin and streptomycin (Gibco) in incubator at 37°C 5% CO2.
5
Bacterial and Yeast Cultivation Protocols
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The strains used in this study are shown in Table 2. Escherichia coli DH5α (New-England-Biolabs (NEB)) was used for cloning and plasmid amplification. Bacteria were propagated in low-salt lysogeny broth (LS-LB) medium, consisting of 0.5% (w/v) sodium chloride (Merck), 0.5% (w/v) yeast extract (Lab M) and 1.0% (w/v) tryptone (Lab M) with or without 1.5% agar (BD) and with the required antibiotics. The Pichia pastoris (syn. Komagataella phaffii)
NRRL-Y-11430 strain (kindly obtained from Prof. Nico Callewaert (VIB, Ghent University)) was used for recombinant protein production. Yeast strains were plated on yeast extract peptone dextrose (YPD) plates (1% (w/v) yeast extract, 2% (w/v) peptone (BD), 2% (w/v) dextrose (Merck), 1.5% agar (Difco)) with the required antibiotics. All antibiotics were purchased from Thermo Fischer Scientific with the exception of carbenicillin, which was obtained from Gold Biotechnology. For recombinant protein expression, strains were grown in buffered glycerol-complex medium (BMGY) and induction was performed in buffered methanol-complex medium (BMMY). Both BMGY and BMMY consist of 1% (w/v) yeast extract, 2% (w/v) peptone (BD), 100 mM phosphate buffer (Sigma-Aldrich) at pH 6.0 and 1.34% (w/v) yeast nitrogen base (YNB, Formedium) with 1% glycerol (v/v Sigma) or 1% methanol (v/v, VWR) as sole carbon source respectively.
NRRL-Y-11430 strain (kindly obtained from Prof. Nico Callewaert (VIB, Ghent University)) was used for recombinant protein production. Yeast strains were plated on yeast extract peptone dextrose (YPD) plates (1% (w/v) yeast extract, 2% (w/v) peptone (BD), 2% (w/v) dextrose (Merck), 1.5% agar (Difco)) with the required antibiotics. All antibiotics were purchased from Thermo Fischer Scientific with the exception of carbenicillin, which was obtained from Gold Biotechnology. For recombinant protein expression, strains were grown in buffered glycerol-complex medium (BMGY) and induction was performed in buffered methanol-complex medium (BMMY). Both BMGY and BMMY consist of 1% (w/v) yeast extract, 2% (w/v) peptone (BD), 100 mM phosphate buffer (Sigma-Aldrich) at pH 6.0 and 1.34% (w/v) yeast nitrogen base (YNB, Formedium) with 1% glycerol (v/v Sigma) or 1% methanol (v/v, VWR) as sole carbon source respectively.
6
Yeast Strain Cultivation Protocol
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The S. cerevisiae strains used in this study are derived from BY4742 (MATa his3Δ1 leu2Δ0 ura3Δ0), obtained from Euroscarf. All yeast strains used in this work are listed in Table 1 . YPD medium contained 1% yeast extract (BD, USA), 2% peptone (BD, USA), and 2% dextrose (Amresco, USA). YPG medium contained 1% yeast extract (BD, USA), 2% peptone (BD, USA), and 2% galactose (Amresco, USA). All cultivations were carried out at 30°C.
7
Swarming Motility Assay for P. aeruginosa
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The swarming motility of P. aeruginosa strains was assessed using the previously described methodology with some modifications (75 (link)). Swarming medium was prepared by mixing 0.5 % (wt/vol) Bacto agar (Axil Scientific, 1st Base), 0.5 % (wt/vol) peptone (Becton, Dickinson), and 0.2 % (wt/vol) yeast extract (Axil Scientific, 1st Base). Following agar sterilization, the medium was supplemented with 1.0 % (vol/vol) glucose (Axil Scientific, 1st Base) and left to cool at 45°C in a water bath. Molten agar (20 ml) was poured into 15-cm petri dishes and dried open for 5 min in a fumigated hood. Overnight cultures (2 ml) of P. aeruginosa grown in the presence of ethanol (control), steroids, or colistin were centrifuged (8,000 rpm, 10 min). Pellets were spot inoculated at the center of the agar surface using a sterile toothpick, and plates were then incubated for 16 h at 37°C. Images of swarming colonies were taken using a Gel Doc XR + system (Bio-Rad), and the extent of swarming was determined by measuring the swarming area using ImageJ software (76 (link)). At least, three biological and two technical replicates were performed to ensure the reproducibility of each experiment.
8
Cultivation of Saccharomyces cerevisiae Kyokai No. 701
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The Saccharomyces cerevisiae strain, Kyokai No. 701, was obtained from the Brewing Society of Japan [10 (link)]. Yeast peptone dextrose (YPD) medium (1% yeast extract (Becton, Dickinson (BD) and Company, Sparks, MD, USA), 2% peptone (BD), and 2% glucose) was used for the cultivation of S. cerevisiae.
9
Lipid Formulation Development and Characterization
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1,2-di-palmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipoid P-100 phosphatidylcholine (>97%) from soybean non-(GMO) was kindly supplied by Lipoid GmbH (Ludwigshafen, Germany). Cholesterol, curcumin (cur ≥ 65%), α-tocopherol (α-toc ≥ 96%), malondialdehyde (MDA), and 1,1,3,3-tetraethoxypropane (TEP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Purified water was obtained from an ultrapure water system (Thermo Scientific Barnstead MicroPure ST, Langenselbold, Germany). HPLC grade chloroform, HPLC grade acetonitrile, HPLC grade methanol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 1, 1 diphenyl 2-picryl-hydrazyl (DPPH), thiobarbituric acid (TBA), and Triton X-100 were purchased from Merck. Trolox was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Dulbecco’s Modified Eagle Medium (DMEM), DMEM without phenol red, fetal bovine serum, penicillin, streptomycin, fetal bovine serum (FBS), and Trypsin-EDTA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Peptone, Yeast Extract–Peptone–Dextrose Broth, and Sabouraud Dextrose Agar were obtained from Becton, Dickinson and Company (Sparks, MD, USA).
10
Fermentation of Secondary Metabolites
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Fermentations were carried out in duplicate. Values given are means of the quantities determined in the individual fermentation experiments. The standard deviation did not exceed 5% ± of the total amount measured. All fermentations were carried out in 1 l malt extract medium composed of malt extract (20 g, Lindenmeyer GmbH & Co. KG, Heilbronn, Germany), peptone (1 g, Becton, Dickinson GmbH, Heidelberg, Germany) and glucose (20 g) per 1 l tap H2O in 2 l Erlenmeyer flasks. Agar plugs of well-grown cultures were transferred aseptically as inoculum. Fermentation was carried out at 22 °C on an orbital shaker at 120 rpm. A sample was taken every second day to monitor the production of secondary metabolites and to measure glucose content (Diabur-Test 5000, Roche Diagnostics, Mannheim, Germany) and pH value (pH 209, HANNA® Instruments Deutschland GmbH).
The samples were separated into mycelium and culture filtrate by filtration via a Büchner funnel for HPLC analysis. After extraction of the culture filtrate with an equal volume of EtOAc, the organic solvent was dried over Na2SO4 and evaporated in vacuo to dryness. The mycelium was discarded.
The samples were separated into mycelium and culture filtrate by filtration via a Büchner funnel for HPLC analysis. After extraction of the culture filtrate with an equal volume of EtOAc, the organic solvent was dried over Na2SO4 and evaporated in vacuo to dryness. The mycelium was discarded.
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