Streptomyces sp. CMB-CS038, CMB-CS145, CMB-CS143, CMB-CS132, CMB-CS138 were cultivated for approximately 15 days in a petri dish (10 cm) containing ISP-4 agar (Burlington, NC, United States). The ISP-4 agar was extracted with 3:1 EtOAc:MeOH (30 mL) and the organic phase concentrated in vacuo to yield approximately 10 mg of crude extract. A solution of crude extract prepared in MeOH (1 mg/mL) was subjected to HPLC-DAD-ESI(±)MS analysis (Zorbax SB-C8 column, 150 mm × 4.6 mm column, 5 μm, 1 mL/min gradient elution from 90% H2O/MeCN to 100% MeCN over 15 min, with constant 0.05% formic acid modifier). Streptomyces sp. CMB-CS038 was also cultivated in other media: Marine agar (Becton Dickinson, Franklin Lakes, NJ, United States), Nutrient Agar (Becton Dickinson, Franklin Lakes, NJ, United States) and R2A agar (Becton Dickinson, Franklin Lakes, NJ, United States) to explore the secondary metabolite production capability of this strain.
Nutrient agar
Manufactured by BD
Sourced in United States
Nutrient Agar is a solid culture medium used for the growth and isolation of a wide range of microorganisms, including bacteria and fungi. It provides essential nutrients and a gelled substrate for microbial cultivation.
Automatically generated - may contain errors
Lab products found in correlation
Nutrient broth, by BD (8 mentions)
Marine agar, by BD (2 mentions)
Tryptic soy agar, by BD (2 mentions)
Tetracycline, by Thermo Fisher Scientific (2 mentions)
Ampicillin, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
R2a agar, by BD (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Tryptic soy agar, by Merck Group (1 mentions)
Nalidixic acid sodium salt, by Thermo Fisher Scientific (1 mentions)
Na2hpo4 12h2o, by Merck Group (1 mentions)
Nah2po4 2h2o, by Merck Group (1 mentions)
Tryptic soy broth tsb, by BD (1 mentions)
Difco, by BD (1 mentions)
Alamarblue cell viability reagent kit, by Thermo Fisher Scientific (1 mentions)
Lb medium, by BD (1 mentions)
Dh5α strain of escherichia coli, by Thermo Fisher Scientific (1 mentions)
Wizard plus miniprep dna purification system, by Promega (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Methyl alcohol, by Merck Group (1 mentions)
43 protocols using nutrient agar
1
Streptomyces Secondary Metabolite Profiling
2
Bacterial Culture Preparation and Characterization
3
Antibacterial Hydantoin Acrylamide Polymer Synthesis
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N-(2-methyl-1-(4-methyl-2,5-dioxo-imidazolidin-4 yl)propan-2-yl)acrylamide (hydantoin acrylamide, HA) was synthesized by using N-(1,1-Dimethyl-3-oxobutyl)acrylamide (DA, Aldrich), ammonium carbonate (AC, Sigma-Aldrich), potassium cyanide (KCN, Merck) and potassium persulfate (PPS, Acros) as an initiator for polymerizing HA. The woven polyethylene terephthalate (PET) fabric (120 g/m2, Tg = 75.8 °C, Tm = 256.3 °C, ΔHm (J/g) 50.8, 72.34 % crystallinity) as polyester was used for this study. The contaminants on the fabric were removed by washing it in a domestic laundry machine at 60 °C for 30 min by using a standard detergent. For antibacterial tests, Tryptic soy broth (TSB), Nutrient Broth (NB), and Nutrient Agar (NA) for supporting growth and cultivation of bacteria were supplied by Becton Dickinson. For preparing a buffer solution, NaH2PO4.2H2O (sodium dihydrogen phosphate) and Na2HPO4.12H2O (disodium hydrogen phosphate dodecahydrate) were purchased from Sigma Aldrich. Escherichia coli (E. coli ATCC 25922) was only used as test strains according to ASTM E2149 because Gram-negative bacteria are usually more resistant than Gram- positive. The 99.5 % purity CO2 used in all experiments was purchased from AGA Industrial gases (Lidingo, Sweden).
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N-(2-methyl-1-(4-methyl-2,5-dioxo-imidazolidin-4 yl)propan-2-yl)acrylamide (hydantoin acrylamide, HA) was synthesized by using N-(1,1-Dimethyl-3-oxobutyl)acrylamide (DA, Aldrich), ammonium carbonate (AC, Sigma-Aldrich), potassium cyanide (KCN, Merck) and potassium persulfate (PPS, Acros) as an initiator for polymerizing HA. The woven polyethylene terephthalate (PET) fabric (120 g/m2, Tg = 75.8 °C, Tm = 256.3 °C, ΔHm (J/g) 50.8, 72.34 % crystallinity) as polyester was used for this study. The contaminants on the fabric were removed by washing it in a domestic laundry machine at 60 °C for 30 min by using a standard detergent. For antibacterial tests, Tryptic soy broth (TSB), Nutrient Broth (NB), and Nutrient Agar (NA) for supporting growth and cultivation of bacteria were supplied by Becton Dickinson. For preparing a buffer solution, NaH2PO4.2H2O (sodium dihydrogen phosphate) and Na2HPO4.12H2O (disodium hydrogen phosphate dodecahydrate) were purchased from Sigma Aldrich. Escherichia coli (E. coli ATCC 25922) was only used as test strains according to ASTM E2149 because Gram-negative bacteria are usually more resistant than Gram- positive. The 99.5 % purity CO2 used in all experiments was purchased from AGA Industrial gases (Lidingo, Sweden).
4
Antibacterial Evaluation of Novel Compounds
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Nutrient Agar (DIFCO, Becton Dickinson) and Nutrient Broth (DIFCO, Becton Dickinson) were used for disc diffusion as well as MIC and MBC. Muller Hinton II broth cation adjusted (Becton Dickson) and Muller Hinton Agar was used for time killing assay. AlamarBlue cell viability reagent kit (alamarBlue, Invitrogen) was used for bacteria viability assay. The 96-well microplates (NEST, Nest Biotech Co. Ltd) were used for MIC, MBC and time kill curve. SEM specimen stubs (SPI Supplies, Structure Probe, Inc., PA, USA) were used for the embedding of the bacteria sample for the SEM processing. Sodium Phosphate Monobasic (Na2HPO4, anhydrous, Fisher Scientific) and Glutaraldehyde reagent (E.M. Grade, SPI Supplies, Structure Probe, Inc., USA) were used for SEM and TEM.
5
Cultivation of Diverse Bacterial Strains
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Escherichia coli (E. coli, KCTC 2571), Escherichia hermannii (E. hermannii, KCTC 22526), Enterobacter aerogenes (E. aerogenes, KCTC 2190), Enterobacter cloacae (E. cloacae, KCTC 1685), Klebsiella pneumoniae (K. pneumoniae, KCTC 2208), Klebsiella alba (K. alba, KCTC 12878), Citrobacter freundii (C. freundii, KCTC 2006), Citrobacter braakii (C. braakii, KCTC 2006), Bacillus subtilis (B. subtilis, KCTC 1022), Bacillus cereus (B. cereus, KCTC 3674), Staphylococcus epidermidis (S. epidermidis, KCTC 1917), Staphylococcus aureus (S. aureus, KCTC 1621), Pseudomonas putida (P. putida, KCTC 1639), Sphingomonas insulae (S. insulae, KCTC 12872) and Shigella sonnei (S. sonnei, KCTC 2518) were obtained from the Korean Collection for Type Culture (KCTC, Jeongeup-si, Korea). Nutrient broth and nutrient agar were purchased from Becton & Dickinson, Co. (Franklin Lakes, NJ, USA). E. coli, S. epidermidis, K. pneumoniae, K. alba S. sonnei, E. hermannii, C. braakii, and S. aureus were cultivated at 37 °C, E. aerogenes, E. cloacae, C. freundii, B. subtilis, and B. cereus were cultivated at 30 °C, and P. putida and S. insulae were cultivated at 25 °C. All bacterial cells were grown in Nutrient broth medium (5.0 g of peptone and 3.0 g of beef extract in 1 L of distilled water) under aerobic conditions.
6
Bacterial Strain Maintenance and DNA Isolation
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The DH5α strain of Escherichia coli (Invitrogen) was used for cloning and amplifying plasmid DNA. The strains of crystalliferous B. thuringiensis subsp. israelensis (Bti) 4Q5, acrystalliferous Bti 4Q7, and L. sphaericus (Ls) 2363 were obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus, OH). Erythromycin-resistant recombinants 4Q7/pWF45 and 4Q7/p45S1, producing, respectively, Cyt1Aa and BinA/BinB (42 kDa/51 kDa) parasporal bodies have been described previously13 (link), 30 (link)–32 (link). All strains were maintained on Nutrient agar (Becton Dickinson, Sparks, MD) throughout the study. LB medium (Becton Dickinson, Sparks, MD) was used for growing E. coli and extracting plasmid DNA using the Wizard Plus Mini-prep DNA Purification system (Promega). Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen).
7
Culturing Pseudomonas protegens Pf-5 Strains
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The bacterial strains used in this study are listed in Table 1 . Pseudomonas protegens Pf-5 and mutant strains were cultured at 27°C on King's Medium B agar (King et al., 1954 (link)), Nutrient Agar (Becton, Dickinson and Company, Sparks, MD) supplemented with 1% glycerol (NAGly) or Nutrient Broth (Becton, Dickenson and Company) supplemented with 1% glycerol (NBGly). Liquid cultures were grown with shaking at 200 r.p.m.
8
Antioxidant and Antimicrobial Assays
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1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS), phosphate buffered saline, butylated hydroxytoluene (BHT), ferrozine, EDTA-2Na, and methyl alcohol were purchased from Sigma-Aldrich Co. (St. Louis, MO). Trichloroacetic acid was purchased from Acros Co. Thermo Fisher Scientific (Geel, Belgium). CuSO4, K3Fe(SCN)6, FeSO4·7H2O, and FeCl3 were purchased from Showa Chemical Industry Company Ltd. (Tokyo, Japan). Nutrient Broth, Nutrient Agar, Tryptic Soy Broth, and Tryptic Soy Agar were purchased from Becton, Dickinson and Company (Sparks, MD).
9
Bacterial Growth Media Protocols
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All media used for bacterial growth and chemotaxonomy analysis—Bennett’s agar, ISP2, ISP4, Luria-Bertani (LB), marine agar (MA), nutrient agar (NA), potato dextrose agar (PDA), Reasoner’s 2A agar (R2A) and trypticase soy agar (TSA)—were provided by BD Difco (Becton, Dickinson and Co., Sparks, MD, USA). Other than those mentioned specifically, all chemicals were purchased as analytical grade from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).
10
Bacterial Colony Counting on Agar Plates
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Bacterial suspensions were serially diluted and spread onto the surface of nutrient agar (Becton Dickinson) in a petri dish, followed by incubation at 37°C for 24 hours. The resulting colonies were counted manually.
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