Bacterial strains, S. aureus ATCC 25923, E. coli ATCC 25922, S. Enteritidis ATCC 13076, S. Typhimurium ATCC 13311, and S. Typhimurium UK-1 ATCC 68169, were inoculated in nutrient broth (Acumedia, Lansing, MI, USA, USA), and incubated at 37 °C for 24 h. Cultures reached a concentration of 109 CFU/mL. After the growth, the bacterial strains were serially diluted in a saline 10-fold solution up to 10−7, and dilution was plated in nutrient agar to confirm the total CFUs. A volume of 10 µL of each dilution was placed on the film’s square with and without bio-AgNP and stored in a sterile Petri dish at room temperature for 24 h. The initial cellular density in contact with films ranged from 106 CFU/cm2 to 1 CFU/cm2. After contact with bacteria, film samples were inoculated in nutrient broth and placed at 37 °C for 24 h. Bacterial growth was confirmed by samples plating in agar medium, E. coli in MacConkey Agar (Acumedia, Lansing, MI, USA, USA), S. aureus in Mannitol Salt Agar (Difco®, Tucker, GA, USA), and Salmonella sp. in SS Agar (Difco®, Tucker, GA, USA).
Nutrient broth
Manufactured by Acumedia
Sourced in United States
Nutrient broth is a common laboratory medium used for the cultivation and growth of a wide variety of microorganisms. It provides essential nutrients and growth factors required for the optimal development of microbial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Brilliant green agar plates, by Acumedia (2 mentions)
Nutrient agar, by Acumedia (2 mentions)
Lysogeny broth, by Merck Group (1 mentions)
Pseudomonas isolation agar, by Merck Group (1 mentions)
Triclosan, by Merck Group (1 mentions)
P aeruginosa, by American Type Culture Collection (1 mentions)
S aureus, by American Type Culture Collection (1 mentions)
Milli q water, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Macconkey agar, by Acumedia (1 mentions)
Yeast extract, by Acumedia (1 mentions)
Sodium hydroxide (naoh), by Dinâmica (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Type 1 agarose, by Merck Group (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
6 protocols using nutrient broth
1
Antimicrobial Efficacy of Bio-AgNP Films
2
Isolation of Arctic Pseudomonad Strains
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Approximately 1 g of glacier foreland soil sample was inoculated into 10 mL of Nutrient Broth (Acumedia, Lansing, MI, USA) and 10 mL of lysogeny broth (LB) (Merck KGaA, Darmstadt, Germany)48 (link). The inoculated media were incubated at 20 °C for 24 hr under aerobic condition. The enriched culture was then serially diluted and 100 μl was plated on Pseudomonas Isolation Agar (17208, Sigma-Aldrich, St. Louis, MO, USA) plates (Peptic digest of animal tissue, 20 g/L; Potassium sulfate, 10 g/L; Magnesium chloride, 1.4 g/L; Triclosan (Irgasan), 0.025 g/L; Agar, 13.6 g/L; pH, 7.0 ± 0.2). Agar plates were incubated at 4, 15, 20 and 25 °C for two weeks under aerobic condition. Single pseudomonads colonies were obtained and each colony was repeatedly plated on Pseudomonas Agar (P2102, Sigma-Aldrich, St. Louis, MO, USA) to obtain pure isolated aerobic pseudomonads. Pure cultures were stored at 4 °C and cryopreserved in 20% (v/v) glycerol at −70 °C. Subsequently, Arctic pseudomonad strains (8–6, 6–4, psy1, psy2, and psy3) were deposited into the Korea Polar Research Institute (KPRI) based Polar and Alpine Microbial Collection (PAMC) center under the general deposit category with accession numbers PAMC 28615 to PAMC 28619, respectively.
3
Salmonella Enteritidis Challenge in Poultry
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Salmonella challenge was performed when birds were two days old. Inoculum was prepared using one colony of nalidixic-acid resistant Salmonella Enteritidis (SENal+) into nutrient broth (Acumedia, USA) containing nalidixic (100 μg/mL) acid for 24h at 37°C and then an aliquot (0.1 mL) was cultured again for four hours at 37°C. Inoculum concentration was determined by plating a serial dilution (10-1 to 10-6) onto brilliant green agar plates (Acumedia, USA) with nalidixic acid (100 μg/mL) and incubating at 37°C. Salmonella colonies were counted after 24 hours. All birds in each box were inoculated with 0.5mL of Salmonella (9.5 x 107 UFC/mL) into the crop, except sham-inoculated control birds, which were given only nutrient broth.
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Salmonella challenge was performed when birds were two days old. Inoculum was prepared using one colony of nalidixic-acid resistant Salmonella Enteritidis (SENal+) into nutrient broth (Acumedia, USA) containing nalidixic (100 μg/mL) acid for 24h at 37°C and then an aliquot (0.1 mL) was cultured again for four hours at 37°C. Inoculum concentration was determined by plating a serial dilution (10-1 to 10-6) onto brilliant green agar plates (Acumedia, USA) with nalidixic acid (100 μg/mL) and incubating at 37°C. Salmonella colonies were counted after 24 hours. All birds in each box were inoculated with 0.5mL of Salmonella (9.5 x 107 UFC/mL) into the crop, except sham-inoculated control birds, which were given only nutrient broth.
4
Antimicrobial Efficacy of Dopamine
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Dopamine hydrochloride, ammonium hydroxide (38–40%) and ethanol were purchased from Sigma-Aldrich (Pty. Ltd., Sydney, NSW, Australia). Gentamicin sulphate was purchased from AK Scientific (Union city, CA, USA). Chromatography grade acetonitrile, ammonium formate and formic acid were purchased from Sigma-Aldrich. All the solutions used in the following experiments were prepared from Milli Q water (Millipore Simplicity system) having a resistivity of 18.2 MΩ cm. Nutrient agar and Nutrient broth were supplied by Acumedia (Melbourne, Australia). The microbial species tested were S. aureus (ATCC 25923) and P. aeruginosa (ATCC 9721).
5
Antioxidant and Antimicrobial Evaluation
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Quercetin (≥95 %), tannic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), (+) - 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid - Trolox (≥97 %), type I agarose and anhydrous potassium persulfate (K2S2O8), dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Aldrich Brasil Ltda., Brazil). l -ascorbic acid (≥99.0 %), ethanol PA (99.5 %), and anhydrous sodium carbonate Na2CO3 (≥99.5) were obtained from Synth (LABSYNTH, Brazil), while ultrapure monohydrate gallic acid and aluminum chloride (III) (≥99.5 %) from Vetec (VETEC-Sigma-Aldrich, Brazil). The Folin-Ciocalteu reagent and bovine serum albumin–BSA were acquired from Merck (Merck, USA). Glacial acetic acid PA and sodium hydroxide were obtained from Dinâmica (Dinâmica, Brazil. Nutrient broth, malt extract, yeast extract, potato extract, and agar were purchased from Acumedia (Acumendia, USA). DMSO‑d6 and TMSP-d4 (2,2,3,3-d4-3- sodium trimethylsilylpropionate) were purchased from Cambridge Isotope Laboratories, Inc. (USA). The reagents used in antimicrobial susceptibility tests were ciclopirox olamine [(LOPROX®), 10 mg g−1, Sanofi-Aventis, Brazil)], DMSO [(≥99.0), Sigma-Aldrich (Aldrich Brasil Ltda., Brazil], DMEM Mix F12, penicillin G, gentamicin, amphotericin B, glucose, l -glutamine, and fetal from Gibco (Thermo Fisher Scientific, Brazil).
6
In Ovo Threonine Supplementation in Salmonella Challenge
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The birds were challenged with Salmonella at two days old. Inoculum was prepared using one colony of nalidixic acid-resistant Salmonella Enteritidis (SE NalR) in nutrient broth (Acumedia, USA) containing nalidixic acid (100 μg/mL) for 24 h at 37°C, and then, an aliquot (0.1 mL) was cultured again for four hours at 37°C. Inoculum concentration was determined by plating a serial dilution (10−1 to 10−6) on brilliant green agar plates (Acumedia) with nalidixic acid (100 μg/mL) and incubating at 37°C. Salmonella colonies were counted after 24 hours. All birds in each box were inoculated with 0.5 mL of Salmonella Enteritidis NalR (8.3 × 107 CFU/mL) into the crop, except the sham-inoculated control birds, who received only nutrient broth.
The birds were distributed in a completely randomized design with four treatments after being challenged with Salmonella: no in ovo Thr supplementation and sham-inoculated in the posthatch challenge (NT-SHAM), in ovo Thr supplementation and sham-inoculated (T-SHAM), no in ovo Thr supplementation and SE NalR-challenged (NT-SE), and in ovo Thr supplementation and SE NalR-challenged (T-SE).
The birds were distributed in a completely randomized design with four treatments after being challenged with Salmonella: no in ovo Thr supplementation and sham-inoculated in the posthatch challenge (NT-SHAM), in ovo Thr supplementation and sham-inoculated (T-SHAM), no in ovo Thr supplementation and SE NalR-challenged (NT-SE), and in ovo Thr supplementation and SE NalR-challenged (T-SE).
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