Protein in vitro digestibility was evaluated according to a method as previously described by Boisen and Fernandez (1995) . Feedstuff sample with 1.0 g (accurate to 0.001 g) was placed in a 100 mL conical flask, and then added 10 mL of 0.01 M hydrochloric acid (pH 2.0) and pepsin solution containing 1.0 mg porcine pepsin (product no. P7-000; Sigma, St. Louis, MO, USA). To prevent bacterial growth, 0.5 mL of chloramphenicol solution consisting of 0.5 g chloramphenicol (product no. 0230; Amresco, Solon, OH, USA) in 100 mL ethanol was added to the mixture, followed by incubation for 4 h at 37°C. After neutralization with 0.2 M sodium hydroxide, 10 mL of 0.2 M phosphate buffer (pH 6.8) was added. The pH was adjusted to 6.8 with 1 M HCl or 1 M NaOH, and the solution was mixed with 1 mL of a freshly prepared pancreatin solution containing 50 mg porcine pancreatin (product no.P7-545-100G; Sigma, USA). The flask was closed with a rubber stopper and incubated with continuous magnetic stirring at 39°C for 4, 8, 12, 16, 20, 24, and 28 h, respectively. After adding 5 mL of 20% sulfosalicylic acid, the sample was centrifuged at 15,000 rpm for 15 min; the supernatant was discarded and the precipitate was heated at 80°C for 24 h. CP digestibility (in %) was calculated as CP content of the original sample −CP content of precipitate/CP content of the original sample×100%.
Chloramphenicol
Manufactured by Avantor
Sourced in United States, Belgium, Germany
Chloramphenicol is a broad-spectrum antibiotic used in laboratory applications. It is effective against a variety of Gram-positive and Gram-negative bacteria. Chloramphenicol functions by inhibiting protein synthesis in bacterial cells.
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Lab products found in correlation
Ampicillin, by Avantor (5 mentions)
Streptomycin, by Avantor (3 mentions)
Kanamycin, by Avantor (3 mentions)
Tetracycline, by Avantor (3 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Peptone, by Reanal (2 mentions)
Sodium nitrate, by Reanal (2 mentions)
Potassium hydrogenphosphate, by Reanal (2 mentions)
Magnesium sulfate, by Reanal (2 mentions)
Potassium chloride (kcl), by Reanal (2 mentions)
Ferrous sulfate, by Reanal (2 mentions)
Allyl cyanide, by Merck Group (2 mentions)
Phenylpropionitrile, by Merck Group (2 mentions)
Dichloran, by Avantor (2 mentions)
Allyl isothiocyanate, by Merck Group (2 mentions)
2 phenylethyl isothiocyanate, by Merck Group (2 mentions)
Tigecycline, by Thermo Fisher Scientific (2 mentions)
Vitek 2, by bioMérieux (2 mentions)
Gentamicin, by Avantor (2 mentions)
21 protocols using chloramphenicol
1
Protein in vitro Digestibility Assay
2
Bromodomains Expression in E. coli
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Plasmid vectors coding bromodomains (BRD2(1), BRD3(1), BRD4(1), BRDT(1) and BRD4(2)) were added into Eppendorf tube (Greiner Bio One, Kremsmünster, Austria) containing chemically competent E. coli BL21 (DE3) cells, which were kept on ice for 30 min. For this solution, heat shock was performed at 42 °C for 40 s, and then it was kept in on ice. Luria-Bertani (LB) Broth (Carl Roth, Karlsruhe, Germany) medium was added (100 µL) and all cells were grown at 37 °C for 1 h. After, cells were selected on plates enriched by agar containing kanamycin (50 µg/mL) (Carl Roth, Karlsruhe, Germany) and chloramphenicol (34 µg/mL) (Amresco, Solon, ME, USA). Then, 50 mL of 2× LB medium concentration with kanamycin (50 µg/mL) (Carl Roth, Karlsruhe, Germany) and chloramphenicol (34 µg/mL) (Amresco, Solon, ME, USA) were inoculated with the selected cells. The cells were grown overnight (200 RPM, 37 °C).
3
Analytical Methods for Glucosinolate Analysis
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Reagents were of analytical purity. Media components (peptone, glucose monohydrate, sodium nitrate, potassium hydrogenphosphate, magnesium sulfate, potassium chloride, ferrous sulfate) were from Reanal (Budapest, Hungary). Allyl isothiocyanate, allyl cyanide, 2-phenylethyl isothiocyanate and phenylpropionitrile and glutathione (reduced form) were from Sigma Aldrich (MO, USA). Pure glucosinolates were from Phytoplan (Germany). LC-MS grade acetonitrile, water and formic acid were purchased from Fisher Scientific (Belgium). Solvents (1-propanol, 2-propanol) and media additives (streptomycin, chloramphenicol and dichloran) were from VWR. Double distilled water was used throughout the study.
4
Antibiotic Screening and Growth Curve
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Cultures for growth curve and antibiotic screening procedures were started from seed culture diluted to 0.02 OD600 or 2.5% (v/v), respectively, and were grown in 10 g/L TSBG. New tubes were opened at each time point to maintain anaerobic conditions throughout and all measurements were made in biological triplicate. Antibiotics used included kanamycin (Teknova, Hollister, CA), tetracycline (Thermo Scientific, Waltham, MA), ampicillin (Research Products International, Mount Prospect, IL), gentamicin (Fisher Scientific, Waltham, MA), spectinomycin (VWR, Radnor, PA), and chloramphenicol (VWR, Radnor, PA) at concentrations listed in Results and Discussion. Growth repression for each sample was calculated using Eq 1 . Each sample OD600 (GS) was divided by average growth in wild type (GWTavg), converted to a percentage and subtracted from 100 to obtain the percent repression. Growth repression was averaged across three sample replicates for each treatment.
5
Plasmid Transformation in E. coli
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All the chemicals, reagents, bacterial growth media such as H2O2 and NaOCl solution, LB medium, M9 medium, chloramphenicol, β-D-1-thiogalactopyranoside (IPTG) were purchased from VWR International (Radnor, PA, USA). The ASKA library and Keio collection were originally from the Coli Genetic Stock Center at Yale University. The no-insert control of pCA24N was from our previous study [23 (link)]. The plasmids with candidate genes were purified from candidate strains individually by Qiagen Miniprep plasmid purification kits (Hilden, Germany) and transformed into MG1655 cells by Bio-Rad Pulser™ Transformation Apparatus (Hercules, CA, USA).
6
Analytical-Grade Reagents for Biochemical Assays
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Reagents were at least of analytical purity. Media components (malt extract, peptone, sodium nitrate, potassium hydrogenphosphate, magnesium sulfate, potassium chloride, ferrous sulfate) were purchased from Reanal (Budapest, Hungary); glucose-monohydrate, streptomycin, chloramphenicol, and dichloran were from VWR (Radnor, PA, USA). Acetone, allyl isothiocyanate, allyl cyanide, 2-phenylethyl isothiocyanate, phenylpropionitrile, dimethyl sulfide, carbon disulfide, methanol, ethyl acetate, methyl acetate, and methyl formate were purchased from Sigma Aldrich (St. Louis, MO, USA). Pure sinigrin was obtained from Phytoplan (Heidelberg, Germany). Type I (18.2 MΩ cm−1) water purified by a Human Zeneer Power I water purification system (Human corporation, Seoul, Korea) was used throughout the study.
7
Antimicrobial Susceptibility Testing Protocol
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Phenotypic antimicrobial susceptibility testing (AST) was performed using the Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France). The MIC values of CAZ-AVI, CAZ-AVI with ATM, tigecycline (dissolved in dimethyl sulfoxide [DMSO]; Acros Organics, Geel, Belgium), and chloramphenicol (dissolved in 99.8% [vol/vol] ethanol; VWR International, Radnor, PA, USA) were determined by broth microdilution according to ISO standard 20776-1. Additionally, the MIC of colistin was examined using MICRONAUT MIC-Strip colistin (Merlin Diagnostika, Bornheim, Germany) according to the manufacturer’s instructions. All results were interpreted according to the published breakpoints and guidelines of EUCAST (42 ).
8
Antimicrobial Susceptibility Testing Protocol
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Phenotypic antimicrobial susceptibility testing (AST) was performed using the Vitek 2 automated system (bioMérieux, Marcy l’Etoile, France). The MIC values of CAZ-AVI, CAZ-AVI with ATM, tigecycline (dissolved in dimethyl sulfoxide [DMSO]; Acros Organics, Geel, Belgium), and chloramphenicol (dissolved in 99.8% [vol/vol] ethanol; VWR International, Radnor, PA, USA) were determined by broth microdilution according to ISO standard 20776-1. Additionally, the MIC of colistin was examined using MICRONAUT MIC-Strip colistin (Merlin Diagnostika, Bornheim, Germany) according to the manufacturer’s instructions. All results were interpreted according to the published breakpoints and guidelines of EUCAST (42 ).
9
Genetic Manipulation of Shigella flexneri
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Strains used in this study are derivative from the wild-type Shigella flexneri 5 strain M90T-Sm [16 (link)] (Table 1 ). Bacteria were grown in tryptic casein soy broth (TSB, Becton Dickinson, Belgium) at 37 °C. E. coli strain DH5α λpir was used for the propagation of plasmids carrying an oriT origin of replication (pSW23T) [17 (link)], and SM10 λpir was used to transfer derivatives of pSW23T to S. flexneri. E. coli Top10 (Invitrogen, Carlsbad, CA, USA) was used for recombinant proteins production. E. coli strains were grown in Luria–Bertani (LB) medium (Becton Dickinson, Belgium) supplemented with appropriate antibiotics: ampicillin, 100 µg mL−1; kanamycin, 50 µg mL−1; streptomycin, 100 µg mL−1 and chloramphenicol, 25 µg mL−1 (VWR, France).
10
Antibiotic Susceptibility Profiling of E. coli
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Antibiotic susceptibility profiles of all the clinical isolates were tested against a panel of eighteen different antibiotics using the Kirby-Bauer disk diffusion method on Muller-Hinton agar (Difco™) as per standard guidelines of CLSI [32 ]. The antimicrobial agents included ampicillin, amoxicillin/clavulanic acid, cephalexin, cefaclor, cefoxatin, cefotaxime, ceftazidime, cefepime, aztreonam, meropenem, nalidixic acid, chloramphenicol, ciprofloxacin, trimethoprim/sulphamethoxazole, tetracycline, gentamicin, cefotaxime/ clavulanic acid and ceftazidime/clavulanic acid (BD BBL™, Oxoid™). Minimum inhibitory concentrations by broth microdilution method of the selected common antibiotics (ampicillin, cefazolin, cefoxatin, cefotaxime, ceftriaxone, ciprofloxacin, norfloxacin, trimethoprim (BBI Life Sciences Corporation, Shanghai, China), amikacin, chloramphenicol, gentamicin, kanamycin and tetracycline (Amresco, OH, USA)) for the ESBL-producing E. coli were also determined in accordance with the guidelines of the CLSI [32 ]. ESBL negative quality control strain was E. coli ATCC25922, whereas, Klebsiella pneumoniae ATCC700603 was used as ESBL positive control strain [32 ]. Multidrug resistant isolates were those found resistant to at least three or more categories of antimicrobial agents.
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